CN118302540A - Methods and systems including cassettes for sequencing target polynucleotides - Google Patents

Abstract

Methods of identifying target polynucleotide sequences from a sample using cassette-based systems, and methods of forming arrays of amplified nucleic acid molecules on solid supports are described.

Description

Methods and systems including cassettes for sequencing target polynucleotides

Cross Reference to Related Applications

The present application claims the benefit of priority from U.S. provisional application No. 63/224,116, filed on 7/21 at 2021, the contents of which are incorporated herein in their entirety.

Technical Field

The present disclosure relates generally to sample processing and nucleic acid sequencing.

Background

In recent years, nucleic acid sequencing technology has made great progress. An important contribution to this advancement is the advent of second generation sequencing or NGS (see, e.g., the sequence of sequencers: the history of sequencing DNA (2016) HEATHER AND CHAIN; genomics 107:1-8). For example, as part of the human genome project, the first human genome was sequenced within about 13 years, costing at least billions of dollars. Today, in some cases, the Human Genome can be sequenced within days at a cost of $1000 (see https:// www.genome.gov/about-genomics/face-pieces/Sequencing-Human-Genome-cost). Nucleic acid sequencing is now widely used in many fields, and its value is well assessed.

However, in many applications, the sequencing workflow (the process required to prepare the target polynucleotides contained in a sample for sequencing, perform sequencing, and analyze the resulting data) is tedious, time consuming, complex, and often costly. Many of the steps are still performed manually and require highly skilled personnel. Even if a particular procedure in a workflow is automated, multiple instrumentation and ancillary components are required and skilled human intervention at various points is required to perform the entire workflow. In addition, the time from the sample to the result is several hours to several days or more. Moreover, the maximum allowable amount of sample input is low, which represents a further lower limit.

Thus, the ability and value of sequencing (including second generation sequencing) in practical use may be greatly reduced.

Disclosure of Invention

The system and method of the present invention recognizes the existing need for a sequencing workflow as follows: the sequencing workflow is fully automated (sample to report), requires no user intervention once it is started, is rapid (samples can give operational results within a few hours), is sensitive, accurate, cost-effective, and can be used as needed. As disclosed herein, the previous individual steps for sample preparation, library preparation, and sequencing can be automated in a single cartridge within a single bench-top instrument.

Disclosed herein are embodiments useful for rapid analysis of target polynucleotides, including determination of their nucleotide sequences from a wide range of input sample types and amounts using a fully automated system that includes cartridges, instrumentation, and manipulation and analysis software, without human intervention (e.g., sample-to-report) after initiation of operation. Further embodiments disclose the use of the semiconductor chip for detection and methods for generating nucleic acid clusters on a surface, including the surface of the semiconductor chip. The embodiments may be used in a variety of applications including clinical diagnosis, epidemiology and monitoring, oncology, genetic and genomic analysis, including metagenomics, hospital infection control, basic and application research, food testing, forensics, environmental testing, bio-threat detection, animal health, agricultural testing, and the like.

In various embodiments, any of the reagents (e.g., primers, enzymes, and buffers) and/or systems described herein may be combined and provided in one or more kits (kit). Thus, kits comprising any of the systems, components thereof (e.g., cartridges or instruments), and/or reagents described herein and capable of performing any of the methods described herein are contemplated.

Aspects of the invention may include methods for analyzing a target in a sample, including the steps of introducing the sample into a cartridge and introducing the cartridge into an instrument. The instrument is then capable of manipulating the sample within the cassette to thereby automatically separate the target nucleic acid from the sample; amplifying the isolated target nucleic acid; and sequencing the amplified target nucleic acid using second generation sequencing. The sample may remain within the cassette throughout the isolation step, amplification step, and sequencing step. Furthermore, the entire process can be performed within a single instrument without user intervention. In certain embodiments, the separation step, amplification step, and sequencing step may be performed within 8 hours or less after the sample is introduced into the cartridge.

In certain embodiments, the systems and methods of the present invention can provide excellent sensitivity to isolated, amplified, and sequenced target nucleic acids comprising fungal nucleic acids present in a sample at levels as low as 3 copies. In certain embodiments, the target nucleic acid can comprise a bacterial nucleic acid present in the sample at a level as low as 3 copies or a viral nucleic acid present in the sample at a level as low as a single copy.

Another advantage of the systems and methods described herein is that the cartridge takes up relatively little space, providing the advantage of easier handling for transportation, storage, and handling, while allowing for less space for the analyzer, thereby taking up laboratory space at a lower cost. As described herein, the external volume of a cartridge or instrument may refer to the total three-dimensional volume occupied by the cartridge or instrument (e.g., the height x length x width of a rectangular prismatic cartridge). In certain embodiments, the cartridge may have an external volume of about 3 liters or less. The cartridge may have an external volume of about 2.5 liters or less. In some embodiments, the cartridge may have an external volume of about 2.1 liters or less. Similarly, in addition to a relatively small external volume, the cartridges of the present invention may also have a relatively small longest linear dimension (e.g., the longest height, length, or width of a rectangular prismatic cartridge) to facilitate handling and achieve a compact instrument. In certain embodiments, the cassette may have a longest linear dimension of about 200mm or less. In some embodiments, the cassette may have a longest linear dimension of about 160mm or less.

The systems and methods of the present invention can accommodate various assays and different sample types and volumes by, for example, varying the specific times, sequences, and durations of reagents and/or various steps in the workflow. In various embodiments, a single cartridge may be capable of performing multiple different assays, and a single instrument may accept different cartridges if different assays are desired. The systems and methods of the present invention contemplate that if different cartridge internals are required to perform different assays (e.g., different sample preparation units handling biological samples versus environmental samples), the overall size and shape of the different cartridges are substantially the same to allow interoperability with a single instrument. In various embodiments, the sample may be selected from the group consisting of a biological sample, a clinical sample, an environmental sample, and a food sample. A particular advantage of the present invention is the ability to receive raw or minimally processed samples and automatically perform the required workflow steps of a selected assay to provide sequencing results without any user intervention or removal of samples from a single cassette or single instrument. Thus, in certain embodiments, the sample may be a biological sample obtained from a subject and is untreated prior to introduction into the cassette.

Sample processing (which may include isolating target nucleic acids in a sample) may include digestion of proteins in the sample. Proteinase K may be used to digest proteins. Isolation may include lysing organisms (e.g., bacteria, fungi, or host cells such as human cells) in the sample to release the target nucleic acid. Cleavage may include mechanical cleavage. In various embodiments, mechanically lysing may include flowing the sample into a lysing chamber within the cartridge and rotating a paddle within the lysing chamber. Mechanical lysing may further comprise adding zirconium beads to the lysing chamber prior to rotating the paddles within the lysing chamber. Isolation may include denaturing the target nucleic acid. Denaturation includes thermal denaturation. Isolation may include capturing the target nucleic acid by: annealing the target capture oligonucleotide to the target nucleic acid to form a complex; combining the complex with a solid support; and removing unbound material from the solid support. In certain embodiments, removing unbound material may include washing the solid support-bound complex with a wash reagent.

In some embodiments, the amplification step may be performed on a solid support-bound target nucleic acid. The amplification step may be performed within the sample processing unit of the cartridge and may replace or supplement the amplification step performed as part of library preparation in the library preparation unit of the cartridge (e.g., the introduction of any desired barcodes, tags or adaptors). In certain embodiments, the method can include eluting the target nucleic acid from the washed solid support to produce an isolated target nucleic acid. The amplification step for library preparation can be performed directly on the eluted target nucleic acid without an intervening step.

In certain embodiments, the separation step can automatically separate the target nucleic acid from a sample having a volume between about 1mL and about 25 mL. The separation step may comprise only one purification step. The isolated nucleic acid may be amplified in an unquantified condition.

In some embodiments, the amplifying step can include performing a first amplification on the isolated target nucleic acid using a first primer set to produce a first amplification product; diluting the first amplification product and aliquoting into a plurality of aliquots; performing a second amplification of the target nucleic acid in the plurality of aliquots using a second primer set to produce a plurality of second amplification products; and pooling the second amplification product. In various embodiments, one or more primers in the first primer set may be identical to one or more primers in the second primer set. The amplifying step can further comprise purifying the pooled second amplification products to produce amplified target nucleic acids. One or more of the first amplification and the second amplification may comprise PCR amplification. The plurality of aliquots may comprise at least 10 individual aliquots. The first PCR amplification and the second PCR amplification may be performed in an unquantified condition. In some embodiments, the second primer set may be nested relative to the first primer set. The amplification step can include copy control of the amplified target nucleic acid prior to the sequencing step. The amplification step may comprise only one purification step.

In certain embodiments, amplified target nucleic acids can be sequenced without quantification. The sequencing step may comprise: the amplified target nucleic acid is immobilized above a semiconductor surface comprising an Ion Sensitive Field Effect Transistor (ISFET) sensor within a cassette. All the products of the amplification step flow over the semiconductor surface without intervening steps. In some embodiments, the amplified target nucleic acid may be immobilized by a capture oligomer that binds over the ISFET sensor, wherein the capture oligomer hybridizes to a portion of the target nucleic acid. The surface may include an array of ISFET sensors, each ISFET sensor having an aperture located thereabove. At least one aperture is positioned over a plurality of ISFET sensors in the ISFET sensor array. One or more wells can include a surface-bound forward primer that hybridizes to a portion of a target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of a target nucleic acid, and wherein the sequencing step comprises double-ended sequencing. In certain embodiments, one or more wells or the interpore gaps between one or more wells can include a plurality of bound inert oligomers that do not hybridize to the target nucleic acid. Amplified target nucleic acids can be immobilized by a universal capture oligomer that binds over the ISFET sensor, wherein the universal capture oligomer hybridizes to the universal binding site. In some embodiments, the amplifying step can include amplifying the isolated target nucleic acid using a primer that includes a universal binding site. In certain embodiments, the amplifying step may comprise ligating an adapter to the isolated target nucleic acid, the adapter comprising a universal binding site. The sequencing step may comprise clonal amplification of the immobilized target nucleic acid, and the clonal amplification may comprise recombinase polymerase amplification, rolling circle amplification, bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification.

In certain aspects, the systems of the present disclosure may include a sample cartridge comprising: sample input; a sample preparation unit capable of receiving a sample from a sample input and separating a target nucleic acid from the sample; a library preparation unit capable of receiving the isolated target nucleic acids from the sample preparation unit and amplifying the isolated target nucleic acids; and a sequencing unit capable of receiving the amplified target nucleic acid from the library preparation unit and sequencing the amplified target nucleic acid. The system may also include an instrument that includes a cartridge interface that includes a physical connection and an electronic connection through which the instrument can drive movement of samples and reagents within the cartridge and communicate with the sequencing unit. The one or more reagents required for isolating the target nucleic acid, amplifying the isolated target nucleic acid, and sequencing the amplified nucleic acid may be dry reagents, the instrument being capable of reconstituting the one or more reagents.

The systems of the invention may also include one or more kits comprising isolating the target nucleic acid, amplifying the isolated target nucleic acid, and one or more reagents required for sequencing the amplified nucleic acid. The instrument may be capable of transferring reagents from one or more kits to a sample cartridge. By using the kit, contamination and logistic organization problems when the plate is loaded with reagent wells in the instrument can be avoided. In addition, the kit may be sealed after production, thereby avoiding contamination or user error that may occur in manually refilling the reagent wells. The kit may be assay specific and may include 1 complete assay or reagents required for multiple assays. The user can insert the appropriate kit with the sample cartridge at the beginning of the assay run. In certain embodiments, the instrument can monitor reagent levels within the kit, particularly where a single kit contains reagent amounts for multiple assays, and notify the user when reagent levels are low or insufficient to perform the desired assay. The sample cartridge and/or the one or more kits may include a Sealed Pneumatic Interface (SPI) port, and the instrument may be capable of transferring one or more reagents from the one or more kits to the sample cartridge via the SPI port using the one or more pipettes.

In certain embodiments, the instrument may comprise a 3-degree-of-freedom pipette stage capable of transferring one or more reagents. The system is capable of isolating, amplifying and sequencing target fungal nucleic acids at as low as 3 copy levels in the sample, the system is capable of isolating, amplifying and sequencing target bacterial nucleic acids at as low as 3 copy levels present in the sample, and/or the system is capable of isolating, amplifying and sequencing target viral nucleic acids at as low as a single copy level present in the sample.

The instrument of the present invention may have a total external volume of about 150 liters or less. In some embodiments, the external volume of the instrument may be about 135 liters or less. The instrument may have a longest linear dimension of about 700mm or less. In certain embodiments, the instrument may have a longest linear dimension of about 650mm or less. The sample cartridge may be capable of receiving biological, clinical, environmental, and food samples, including untreated samples. Isolating the target nucleic acid may include digesting the protein in the sample (e.g., by exposing the sample to proteinase K in a sample preparation unit). The sample preparation unit may be capable of lysing organisms to release the target nucleic acid. To this end, the sample preparation unit may comprise a lysis chamber comprising a rotating paddle, the instrument being capable of flowing the sample into the lysis chamber and interfacing with the sample cartridge to rotate the rotating paddle to mechanically lyse organisms in the sample. The lysis chamber may include zirconium beads to aid in lysis.

The instrument is capable of providing thermal energy to the sample preparation unit to denature nucleic acids therein. The instrument may further be capable of performing target capture by exposing the sample to the target capture oligonucleotide and the solid support in the sample preparation unit to anneal the target capture oligonucleotide to the target nucleic acid, thereby forming a complex and binding the complex to the solid support. The instrument may then introduce a wash buffer into the solid support bound complex and separate the solid support bound complex from the unbound sample. The instrument can then transfer the isolated solid support-bound complexes to a library preparation unit and amplify the solid support-bound target nucleic acids. The instrumentation of the present invention is capable of introducing an elution buffer into the separated solid support bound complexes to elute target nucleic acids from the solid support and transferring the eluted target nucleic acids to a library preparation unit for amplification. The instrument is also capable of introducing amplification reagents into the solid support-bound complex and amplifying target nucleic acids within the sample preparation unit.

In various embodiments, the cartridge and instrument may be capable of automatically containing a sample received by a sample input having a volume between about 1mL and about 25 mL. The instrument may be capable of interfacing with a library preparation unit of a sample cartridge to introduce the required reagents and to provide thermal energy to: performing a first amplification on the isolated target nucleic acid using the first primer set to produce a first amplification product; diluting the first amplification product and aliquoting it into a plurality of aliquots; performing a second amplification of the target nucleic acid in the plurality of aliquots using a second primer set to produce a plurality of second amplification products; and pooling the second amplification product. One or more primers in the first primer set may be identical to one or more primers in the second primer set. The apparatus may also be capable of purifying the pooled second amplification products to produce amplified target nucleic acids.

The system of the invention may also be capable of copy control of one or more of the isolated target nucleic acid and amplified target nucleic acid and controlling the number of output copies transferred to the library preparation unit or sequencing unit, respectively. The sequencing unit may include a semiconductor surface including an array of Ion Sensitive Field Effect Transistor (ISFET) sensors, each ISFET sensor having an aperture located thereabove, the instrument being capable of immobilizing amplified target nucleic acid thereabove, the ISFET sensor array in electronic communication with the instrument through electronic connection of the cartridge interface when the sample cartridge is positioned therein. The instrument is capable of allowing all output from the library preparation unit to flow into wells on the semiconductor surface.

The cassette can include a capture oligomer bound over the ISFET sensor array, wherein the capture oligomer is configured to hybridize to a portion of the target nucleic acid. At least one aperture may be positioned over a plurality of ISFET sensors in the ISFET sensor array. In some embodiments, one or more wells can include a surface-bound forward primer that hybridizes to a portion of a target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, the instrument being capable of double-ended sequencing. The one or more wells and the interpore gaps between the wells may include a plurality of bound inert oligomers that do not hybridize to the target nucleic acid. The cartridge may include a universal capture oligomer that binds over the ISFET sensor array, wherein the universal capture oligomer is configured to hybridize to a universal binding site.

The instrument can be interfaced to a library preparation unit to amplify the isolated target nucleic acid using primers that include universal binding sites. In some embodiments, the instrument is capable of ligating an adapter to an isolated target nucleic acid in a sample preparation unit or library preparation unit, the adapter comprising a universal binding site. The instrument can interface with a sequencing unit for clonal amplification of the immobilized target nucleic acid, and the clonal amplification can include recombinase polymerase amplification, rolling circle amplification, bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification. The physical and electrical connections between the instrument and the sample and/or the cartridge may include a pneumatic system for driving the movement of fluid between and/or within the sample cartridge. The cartridge interface may further comprise a physical connection and an electronic connection by which the instrument is able to communicate with one or more of the sample preparation unit and the library preparation unit.

Drawings

Fig. 1A-1C illustrate exemplary capture oligomers according to the present disclosure, including capture sequences, blocking portions, complementary sequences (C') of capture sequences, additional sequences (e.g., third additional sequences or fourth additional sequences) and target hybridization sequences, as well as other molecules. In fig. 1A, the capture oligomer anneals to the target polynucleotide (target), and the 3 'end of the target polynucleotide anneals to the 5' end of the target hybridization sequence. The capture sequence anneals to C'. In FIG. 1B, the 3 'end of the target has been extended to the blocking moiety, and the resulting target extension sequence anneals to the additional sequence and C', while the capture sequence has been displaced and has become single stranded. The 3' end of the capture oligomer also extends along the target polynucleotide. In fig. 1C, the complex of fig. 1B is annealed to a second capture reagent comprising a complementary sequence of the capture sequence, a binding partner or a solid matrix. At the same time, excess capture oligomer remains with the capture sequence that anneals to the complement of the capture sequence and does not interact with the second capture reagent.

Fig. 2A shows an embodiment of the present disclosure, wherein the complex as in fig. 1B is annealed with a second capture reagent comprising a complementary sequence of the capture sequence and associated with a solid matrix (in this case, streptavidin-coated magnetic beads). The solid matrix may be part of the second capture reagent or may be associated with the second capture reagent by interaction with a binding partner of the second capture reagent (e.g., biotin).

Fig. 2B shows an embodiment of the present disclosure, wherein the complex of extended capture oligomer and target from fig. 2A has eluted from the second capture reagent.

Fig. 3 shows an embodiment of a capture oligomer according to the present disclosure, comprising a stabilizing (clip) sequence as a first additional sequence, a capture sequence, a linker, an internal extension blocking sequence, a complement of the capture sequence, a stabilizing (clip) sequence as a third additional sequence, a fourth additional sequence, and a target hybridization sequence.

Fig. 4A illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A target molecule is provided wherein the first strand comprises the sequence Sf at its 5' end and Sr at its 3' end, and the second strand comprises the sequence Sf ' at its 3' end and Sr at its 5' end. Herein and throughout, a sequence name with' means complementarity to a sequence without a name. The target molecule may be, for example, an amplicon from a reaction previously performed using primers having the sequences Sf and Sr. The first cycle of extension (cycle 1) is performed, wherein a capture oligomer according to the present disclosure anneals to the first target strand 1 (+) and the capture oligomer comprises a capture sequence C, an internal extension blocking sequence (filled circle), a complementary sequence C 'of the capture sequence, a fourth additional sequence A4, and a target hybridization sequence THS complementary to Sr'. The reverse amplification oligomer anneals to the second target strand 1 (-), the reverse amplification oligomer comprising the additional sequence A2 and the target hybridization sequence Sf complementary to at least Sf'. Sf may include affinity enhancing modifications and/or additional nucleotides complementary to the second target strand to enhance its affinity to the target and promote binding competition with primers (if present) having the sequence Sf from previous reactions. Extension of the capture oligomer and the reverse amplification oligomer produces products 2 (-) and 2 (+), respectively, while the first strand extends along the capture oligomer to produce product 1 (+) e and the second strand extends along the reverse amplification oligomer to produce product 1 (-) e. The capture sequence in the extended capture oligomer 2 (-) is displaced essentially as described in fig. 1B. A second cycle of reaction (cycle 2) was performed in which 2 (-) was annealed to the reverse amplification oligos, causing extension to yield products 2 (-) e and 3.1 (+). At the same time, 1 (+) e anneals to the capture oligomer and the latter extends to give the product 3.1 (-). Other examples of 1 (-) e and 2 (+) and 2 (-) e and 3.1 (+) are also generated by appropriate hybridization and extension events. The reaction scheme illustrates the inclusion of additional sequences at each end of the target and the capture of the target by introducing C in a form available for binding, e.g., binding to a second capture reagent.

Fig. 4B illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A target molecule is provided wherein the first strand comprises the sequence Sf at its 5' end and Sr ' and A4' at its 3' end, and the second strand comprises the sequence Sf ' at its 3' end and Sr and A4 at its 5' end. The target molecule may be, for example, an amplicon from a reaction previously performed using primers having the sequences Sf and A4-Sr, e.g., where A4 is an additional sequence that was not originally present in the template. A first extension cycle (cycle 1) is performed in which a capture oligomer according to the present disclosure, comprising a capture sequence C, an internal extension blocking sequence (filled circles), a complementary sequence C 'of the capture sequence, a fourth additional sequence A4, and a target hybridization sequence THS complementary to A4', anneals to a first target strand (not shown). The reverse amplification oligomer anneals to a second target strand (not shown), the reverse amplification oligomer comprising an additional sequence A2 and a target hybridization sequence Sf complementary to at least Sf'. Sf may include affinity enhancing modifications and/or additional nucleotides complementary to the second target strand to enhance its affinity to the target and promote binding competition with primers (if present) having the sequence Sf from previous reactions. The extension of these complexes results in an extended capture oligomer 2 (-) and an extended first target strand 1 (+) e, and an extended second target strand 1 (-) e and an extended reverse amplification oligomer 2 (+). The capture sequence in the extended capture oligomer 2 (-) is displaced essentially as described in fig. 1B. A second reaction cycle (cycle 2) was performed in which 2 (-) was annealed to the reverse amplification oligomers, causing extension to produce products 2 (-) e and 3.1 (+). At the same time, 1 (+) e anneals to the capture oligomer and the latter extends to give the product 3.1 (-). Other examples of 1 (-) e and 2 (+) and 2 (-) e and 3.1 (+) are also generated by appropriate hybridization and extension events. The reaction scheme illustrates the inclusion of an additional sequence at the end of the target remote from the binding site of the capture oligomer, and the introduction of C (in a form available for binding) using the capture oligomer that can have universal THS (i.e. binding additional sequence A4') attached to the target in a previous step (e.g. by amplification or ligation) so that the target can be captured.

Fig. 5 illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A capture oligomer is provided that includes a 3 'blocking moiety and a Target Hybridization Sequence (THS) that binds to sequence A1' in the target strand, and the like. A1' may be an additional sequence that is attached to the target in a previous step (e.g., by amplification or ligation). The capture oligomer further comprises a sequence x comprising a complement of the capture sequence of the capture oligomer, and may further comprise a third or fourth additional sequence between the complement of the capture sequence and THS. As discussed elsewhere, the target strand may extend along the capture oligomer to displace the capture sequence from its complement. The capture oligomer may be provided in a limited amount (e.g., 10 ¹² copies) relative to the target (e.g., 10 ¹⁴ copies). Primers are also provided that exceed targets (e.g., 10 ¹⁵ copies), including sequences A2 and Sf. Extension of this primer results in a strand that includes A2 at its 5' end and A1' at its 3' end. The target strand also extends along the primer to include sequence A2'. If a second cycle of extension is performed (down arrow), a mixture of products is formed, including those described above, as well as complexes of the target strand with the capture oligomer, wherein the target strand comprises A2 at its 5' end and A1' near its 3' end. The reaction scheme illustrates that single stranded capturable products are produced, including (when the second extension cycle is performed) single stranded capturable products in which additional sequences have been included in the target strand.

FIG. 6 shows how hybridization of a capture oligomer with an extendable 3 'end (above the dotted line) with another capture oligomer, with the capture sequence displaced from C', yields a dimer after extension. This dimerization is manifested as capturable and may interfere with downstream processes, such as competing with capture of the desired target by occupying a second capture reagent (not shown), subsequent analysis of the interference (e.g., the dimer will become part of the sequencing library, thereby reducing the output and quality of subsequent sequencing runs), and the like. Sx' is the complement of a portion of the target hybridization sequence, and the other elements are as shown in the previous figures. Below the dashed line, a capture oligomer (circled x) with a blocking moiety at its 3' end is shown that can prevent the formation of dimer extension products so that no substitution of C occurs for any dimer.

Fig. 7A shows an embodiment in which a capture oligomer comprising a capture sequence, various intermediate elements (denoted by "…"), a reversibly extendable blocking sequence (filled circles) and a Target Hybridization Sequence (THS) is used. The capture sequence and various intermediate elements, if present, are not templates for extension (e.g., extension of the target strand or amplification oligomer) prior to unblocking the reversible extension blocking sequence. This may be achieved by avoiding the introduction of additional sequences complementary to the capture sequence and various intermediate elements (if present) in the product (e.g., in any error-initiated product that may be formed) throughout the extension or amplification process until the reversible extension blocking sequence is unblocked, thereby facilitating more efficient and specific extension or amplification; after unblocking, a capture sequence and various intermediate elements (if present) may be introduced.

Fig. 7B illustrates an embodiment in which a first amplification oligomer is used, the first amplification oligomer comprising, from 3 'to 5': target hybridization sequence Sr, reversible extension blocking sequence (filled square), additional sequence A1 and optional additional elements (represented by "…"), such as an optional capture sequence. Optionally, a second amplification oligomer is used, the second amplification oligomer comprising, from 3 'to 5': the target hybridization sequence Sf, a reversible extension blocking sequence (open square; this sequence may be the same or different from the reversible extension blocking sequence in the first amplification oligomer), the additional sequence A2, and optionally additional elements (represented by "…" which may be the same or different from those in the first amplification oligomer) (as shown in the figure). The additional sequences and optional additional elements (if present) are not templates for extension (e.g., extension of the target strand or amplification oligomer) prior to unblocking of one or more reversible extension blocking sequences. This may facilitate more efficient and specific extension or amplification by avoiding the introduction of sequences and other various elements (if any) complementary to the additional sequences in the product (e.g., in any false-primed product that may be formed) throughout the initial extension or amplification process. The reversibly extended blocking sequence is unblocked (unblocking can occur simultaneously or separately if two reversibly extended blocking sequences are present) and additional sequences and any other elements present can be introduced at a later stage of the method (e.g., a later cycle of extension).

Fig. 8A illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. Initial targeting strands 1 (+) and 1 (-) are identical to those in FIG. 4A. Below the straight vertical arrow, a capture oligomer is provided comprising the target hybridization sequence THS, and additional elements A4, C', internal extension blocking sequences and C as described for the oligomer of fig. 4A. The THS binds to the internal site of the target strand and is extended to produce the product 2N (-). A substitution oligomer including Sr is provided and extended, thereby substituting 2N (-) from 1 (+) and generating 2 (-). A reverse amplification oligomer as in FIG. 4A is provided, which produces 2.1 (+) along extension of 1 (-) and 1 (-) along extension of the reverse amplification oligomer produces 1 (-) e. Once the 2N (-) is displaced (left curved arrow), the reverse amplification oligomers anneal to the 2N (-) and extend each, producing the products 2N (-) e and 2.2 (+), which now include A2 'and wherein C is displaced from C'. This reaction scheme illustrates the use of a displacement oligomer to promote the production of a capturable product comprising additional sequences (e.g., adaptors) at both ends of the target sequence in cycle 1 alone. Furthermore, the reaction scheme shows an embodiment in which the capture oligomer does not bind to a site including the 3' -end of the target strand.

Fig. 8B illustrates additional exemplary molecules and another exemplary reaction scheme according to the present disclosure. The reaction scheme is substantially similar to that depicted in fig. 4A, except for the following 1) and 2). 1) Initial target strands 1 (+) and 1 (-) include additional sequences including the target hybridization sequence THS, the optional spacer sequence S and the replacement oligomer binding site D. These additional sequences are any user-defined sequences and can be introduced into the target, for example, by using an amplification reaction that includes Sr and sequence tags that include THS, S and D, and an amplification oligomer that includes Sf. 2) The THS of the capture oligomer binds to the user-defined THS site. Otherwise, the reaction is performed as shown in FIG. 8A, and the resulting product is shown in FIG. 8B. An optional spacer sequence may be used to improve extension of the displacement oligomer and subsequent displacement of the capture oligomer. As with the scheme shown in fig. 4A, this reaction scheme illustrates the use of a displacement oligomer to facilitate (e.g., only in cycle 1) the production of a capturable product that includes additional sequences (e.g., adaptors) at both ends of the target sequence. Furthermore, this scheme shows the use of additional, user-defined sequences that can serve as binding sites for both capture and displacement oligomers. Such a design may generalize the method and allow for simpler and more cost-effective means of designing capture and displacement oligomers for different targets, including in multiplex format.

FIG. 9 shows the general principle of how blocking an oligomer prevents hybridization between additional sequences in the oligomer and their complementary sequences in the extension product. The amplification reaction is performed with a forward primer containing sequence f that hybridizes to target strand T (-), and a reverse primer containing sequence A (additional sequence not present in the target) and sequence r that hybridizes to target strand T (+). Extension yields the products 1 (-) and 1 (+). Blocking oligomers comprising sequence a and a 3' blocking moiety are provided. In cycle 2, the forward primer extends along 1 (-) to yield 2 (+) and the reverse primer extends along 1 (+) to yield 2 (-). In cycle 3 and subsequent cycles, the blocking oligomer anneals to 2 (+) meaning that hybridization of r to r' is necessary for the reverse primer to initiate extension along 2+. This is beneficial in the event of any false initiation event that would produce a small amount of byproducts having an incompletely complementary sequence of r but extending to include a'. In the absence of blocking oligomers, binding of the reverse amplification oligomers to erroneously initiated byproducts would be more favored due to the interaction between A and A' of the reverse amplification oligomers, resulting in more byproduct amplification (while the forward primer anneals to 2 (-) and is extended) than with blocking oligomers.

Fig. 10A illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. The following combinations are provided: (i) A capture oligomer comprising first and second portions of a capture sequence (C1 and C2), an internal extension blocking sequence (filled circles), first and second portions of a spacer sequence (S1 and S2), a Target Hybridization Sequence (THS) that binds to a site in a target strand comprising its 3' end, and (ii) a complementary oligomer comprising S1' and C2'. When the capture oligomer hybridizes to the target and the target extends along the capture oligomer until the blocking sequence is extended internally, thereby introducing S' into the target strand, the complementary oligomer is displaced. The capture oligomer also extends along the target (note that in other embodiments described herein, the capture oligomer may be blocked and thus no such extension occurs). A second capture reagent is provided that comprises a binding partner or solid support (circled B) linked by a linker (zigzag curve) to the complementary sequence C' of the capture sequence. The second capture reagent anneals to the capture oligomer bound to the extended target but not to the capture oligomer bound to the complementary oligomer, as the latter occupies C2, which is a sufficient amount of capture sequence to substantially prevent the second capture reagent from annealing to the capture oligomer.

FIG. 10B illustrates an embodiment in which a combination of oligomers can be used to capture a target polynucleotide from a composition that includes an amount (e.g., a limited amount, or an amount less than or equal to a predetermined amount) of the target polynucleotide, if desired. The combination comprises: a capture oligomer comprising, from 5 'to 3', a first portion C1 of a capture sequence, a second portion C2 of a capture sequence, optionally a spacer sequence S, a second portion THS2 of a target hybridization sequence, a first portion THS1 of a target hybridization sequence, and optionally a blocking portion (circled X); separate complementary oligomers, which include, from 5' to 3', THS2', S ' (optional; may or may not be used when S is present in the capture oligomer) and C2' (where the complementary sequences of the elements are denoted by ' ") and an optional blocking moiety (circled X) at the 3' end; and a second capture reagent comprising a complementary sequence of the capture sequence, the complementary sequence comprising, from 5 'to 3', C2', C1' (C1 'or C2' may or may not be complementary to the full length of C1 and C2) and a binding partner (illustrated in this figure by a biotin molecule, denoted as encircled B). In the absence of the target polynucleotide, the complementary oligomer binds to the capture oligomer and blocks accessibility of the complete capture sequence to an extent sufficient to block binding of the complementary sequence of the capture sequence in the second capture reagent (see complex of the complementary oligomer and the capture oligomer at the top of the figure). When the target is present, the THS1 region of the capture oligomer binds to the target, followed by the THS2 region (which is energetically favorable), thus displacing the THS2' region of the separate complementary oligomer from the capture oligomer. When this occurs, the C2' region of the complement sequence alone is no longer stable enough to bind the capture oligomer and thus becomes unbound, resulting in a complete capture sequence available for binding as shown under the first arrow. Then, as shown under the second arrow, the complementary sequence of the capture sequence in the second capture reagent binds to the capture sequence of the capture oligomer. The complex can then be isolated from the mixture, for example, by streptavidin-coated magnetic microspheres (as described elsewhere in this disclosure), thereby capturing and purifying the target polynucleotide. Optionally, the capture oligomer may be present in the combination in a greater amount than the second capture reagent. Such oligomers and combinations can be used to capture an amount (e.g., a limited amount or an amount less than or equal to a predetermined amount) of a target polynucleotide from a composition.

Fig. 11A-11B illustrate exemplary molecules and exemplary reaction schemes according to the present disclosure. In fig. 11A, a capture oligomer is provided that includes elements substantially as described for the capture oligomer of fig. 4A, except that THS binds to a site in a target strand that does not include the 3' end (which may be circular as shown, or linear). A complementary oligomer is provided that includes (i) a target hybridization sequence that anneals to the THS adjacent sequence of the capture oligomer and (ii) a complementary sequence of at least a portion of A4. In the absence of target strand, the complementary sequence of at least a portion of A4 is insufficient to anneal to the capture oligomer. In fig. 11B, the complementary oligomer is extended, which will displace C and make it available for capture using a second capture reagent (not shown). This scheme can be used to capture circular molecules and/or represent another approach to use capture oligomers that do not bind at the 3' end of the target strand.

Fig. 12 illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. The capture oligomer comprising the elements as in the capture oligomer of fig. 4A and having a second additional sequence A2 that may comprise a mixed nucleotide segment between the C 'and the internal extension block sequence anneals to the target strand at a site comprising its 3' end. The target strand also includes at its 5' end sequence A5, which sequence A5 may be any sequence, a primer binding site used in a previous amplification reaction, or a sequence added in a previous step (e.g., amplification or ligation). Extension of the target strand along the capture oligomer the sequences A4', C and A2' are added to the 3' end of the target strand. The presence of A4 in the capture oligomer and A4' in the extended target strand is optional. The extended target strand may then be annealed to a splint oligomer comprising the sequences A5', A2, C' and A4, wherein the 5 'and 3' ends of the target strand are immediately adjacent when the extended target strand is annealed to the splint oligomer. The extended target strand may then be circularized by ligation. The A2 and A2' sequences are used to ensure proper juxtaposition of the 5' and 3' ends of the extended target strand. This may be helpful when C and C' are repeat sequences (e.g., poly-A and poly-T or vice versa) that otherwise may tend to slip, which will inhibit the formation of substrates for ligation. This protocol can be used to capture and subsequently circularize target molecules, for example for rolling circle amplification procedures.

Fig. 13 illustrates an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A capture oligomer is provided that includes a capture sequence C (comprising a first portion C1, a second portion and C2; not shown), an internal extension blocking sequence (filled circles), a spacer sequence S (comprising a first portion S1 and a second portion S2; not shown), and a target hybridization sequence THS, and a reverse amplification oligomer is provided that includes sequence S ₂. THS and S ₂ are used to generate amplified targets (e.g., by PCR). A complementary oligomer is added comprising a complementary sequence S1 'of the first part of the spacer sequence and a complementary sequence C2' of the second part of the capture sequence. When S 'anneals to the other strand S of the amplified target, C2' is insufficient to anneal to C of the amplified target. The complementary oligomer anneals to capture the oligomer that is not annealed to the amplified strand. To capture the amplified target, a second capture reagent comprising C' and a binding partner or solid support (circled B) is added. The second capture reagent binds to the amplified target but not to the capture oligomer that does not anneal to the amplified strand, wherein C2' of the complementary oligomer blocks C to a sufficient extent.

FIG. 14 shows fold differences in output of methods using captured oligomers with or without clip sequences.

FIG. 15-Panel A-solution mediated surface phase recombinase polymerase amplification (SM-RPA) forward primers are present in solution, while reverse primers are immobilized on the surface. Upon initial hybridization of the template to the surface primer, the synergistic action of the solution primer, recombinase, single-stranded DNA binding protein (ssDNA) and polymerase allows complementary strand synthesis. Subsequent recombination and extension of the solution phase primer causes one strand to be displaced into solution. These chains can then be locally reacquired. In contrast, in bridge RPA or ExAmp (for example), both primers are co-immobilized on the surface, allowing only surface phase amplification.

Figure 16 depicts a model of cluster formation in bridge RPA (figure a) and SM-RPA (figures B and C) on a holed chip. In bridge RPA (as in bridge PCR and ExAmp), clusters are small (panel a). In SM-RPA, lateral growth of clusters can be achieved using amplification of primers in solution, the size of the clusters being limited by the spatial exclusion of adjacent clusters. The amplicon generated in solution was recaptured near the seed template molecule, promoting the formation of clonal spots of amplicon that were larger than those generated in bridge amplification (panel B; clusters from 3 independent targets were synthesized by:, and)Representation).

Fig. 17 is a schematic diagram of an exemplary surface (e.g., on-chip) template circulation method of the present invention. First, during PCR2 in library preparation, a linker is added to the target template, with portions of the first and second RCA primer binding sites on each end. In the copy control stage of the workflow, the second RCA primer binding site is extended to include the copy control adapter and a second portion of the first RCA primer binding site. The template hybridizes to a first RCA primer immobilized on the surface, which acts as a splint. This forms a structure with a gap between adjacent 5 'and 3' ends. The addition of ligase fills this gap, forming a loop that can promote in situ RCA reactions.

FIG. 18 depicts one embodiment of cluster formation using Rolling Circle Amplification (RCA). (A) Both ends of the linear RCA template are hybridized to the immobilized first primer molecule such that template cycling is performed directly on the surface by a ligase. (B) Once the gap is blocked by the ligase, an amplification mixture containing a highly processive strand displacement polymerase is added. (C) The polymerase extends the free 3' end of the first primer using the loop as a template. (D) The elongated tandem first strand amplicon comprises a repeat unit of the second primer binding site. These sites hybridize to the immobilized second primer molecule and serve as templates for second strand synthesis. (E) Once the synthesis reaches the next unit, the elongated second strands are displaced from each other by the strand displacement activity of the RCA polymerase. (F) The free second strand hybridizes to the remaining free immobilized first primer molecules, facilitating further synthesis of the tandem first strand. Accordingly, the elongated first strands displace each other and the cycle continues.

FIG. 19 shows a method for introducing primer-specific key sequences according to embodiment #1 in the specification. Truncation of the 3' end of Oligo 2 enables the remaining known sequence of Section 1A (Section 1A) to be used as a key sequence.

FIG. 20 shows another variant of the method for introducing target-specific key sequences according to embodiment #1 of the present description, in which case 3 'truncation and 5' extension are used. Truncation of the 3' -end of Oligo 4 enables the remaining known sequence of segment 3A to be used as a key sequence.

FIG. 21 shows a method for introducing primer-specific key sequences according to embodiment #2 in the specification. In this example, the synthetic, non-specific sequence (segment 6B) at the 5' end of the surface-bound capture oligomer/primer (Oligo 6) can be used as a known universal key sequence. Step (a): the sequencing template (Oligo 5) provided in solution hybridizes (surface binds) to segment 6A of the complementary sequence of Oligo 6. Step (b): upon addition of the enzyme, polymerization can occur from the 3' end of Oligo 5 to segment 6B of Oligo 6. The resulting sequence can be used to set the signal base call parameters. The 3' blocking moiety on Oligo 6 prevents polymerization from this end. Step (c): as described above, the 3' end of Oligo 6 was unblocked. Step (d): after addition of additional polymerase (if needed), a sequencing reaction can now be performed through the unknown region of Oligo 5.

Fig. 22 is a top-down rendering of a preferred embodiment of a sample preparation cartridge. Figures a and B depict two possible reagent/assay configurations for the chambers in the main cartridge.

Fig. 23 is a top-down rendering of a preferred embodiment of a sample preparation cartridge depicting a body plus 2 additional functional fins. Panels a and B depict alternative microfluidic configurations of Mag-Sep fins. "Mag-Sep" refers to magnetic separation.

Fig. 24A is a 3D CAD rendering of a preferred embodiment of a sample preparation cartridge. Fig. 24B is a photograph of an actual prototype box constructed according to the design in fig. a.

FIG. 25 is a top-down rendering of a preferred embodiment of the library preparation cassette depicting the body plus 1 additional functional fin. Figures a and B depict two possible reagent/assay configurations for the chambers in the main cartridge. "Mag-Sep" refers to magnetic separation.

FIG. 26, panel A is a 3D CAD rendering of a preferred embodiment of a library preparation cassette. Figure B is a photograph of an actual prototype box constructed according to the design in figure a.

FIG. 27 shows a top view of a preferred embodiment of a cluster generation/sequencing (CA-Seq) cassette. Figures a and B depict two possible reagent/assay configurations for the chambers in the main cartridge.

Fig. 28. Panel a is a 3D CAD rendering of one preferred embodiment of a cluster generation/sequencing (CA-Seq) box. Figure B is a photograph of an actual prototype box constructed according to the design in figure a.

Fig. 29 shows one example of a flow cell assembly for use in the present invention. The valve (showing one possible arrangement) may be glued or welded to the fins. The film may be solvent bonded, heat welded or laser welded, or by a pressure sensitive adhesive. The flow cell is thermally fused by a PCB (printed circuit board). The silicon gasket between the membrane and the chip is not shown.

FIG. 30 is a 3D rendering of a sequencing kit, shown herein connected to a manifold and cluster generation/sequencing kit for fluid control and other desired functions. One particular configuration of the reagent bottle and its contents is shown, but other configurations are also contemplated.

FIG. 31 is a 3D rendering of an exemplary manifold of sequencing kits for fluid control and other desired functions.

FIG. 32 depicts one example of a sequencing agent delivery system.

FIG. 33 shows two exemplary designs of integrated assay cartridges.

Fig. 34 shows the various components contained within the cartridge shown in the previous figures (not all components are shown or depicted in this figure). The functions shown (e.g., STC, PCR1&2, CC, etc.) are exemplary only. These components may be used for a variety of functions as desired for a particular application. Numbered components are (in this example), 1) sample input ports (2 in this configuration); 2) A mechanical cracking unit; 3) A Specific Target Capture (STC) chamber; 4) PCR1 and PCR2 aliquoting chambers; 5) PCR1 and PCR2 reaction chambers; 6) A Copy Control (CC) chamber; 7) Condensation trap chambers (STC and CC); 8) Cluster generation and sequencing flow cells; 9) Pneumatic connection for fluid control; 10 SPI for fluid connections to different chambers; 11 STC and CC fins (multiple thermal mixing chambers and bead capture areas); 12 Amplification/dilution fins (double sided thermal control).

FIG. 35 depicts the following steps of an exemplary Mechanical Lysis (ML) process performed in an integrated cartridge for a given application (e.g., detecting pathogens in blood): 1) Withdrawing blood from the evacuated blood collection tube through the liquid handler; 2) Transferring blood into a Specific Target Capture (STC) chamber, and then adding a liquid reagent to the blood; 3) The fluid is mixed back and forth between the chambers while incubating through the heated instrument interface; 4) The blood and reagent solutions are transferred to a mechanical lysing chamber where a motor on the instrument causes the paddles to rotate rapidly to effect the lysing. Note that not all components of the cartridge are shown in this figure for ease of viewing the features.

Fig. 36 depicts the following steps of an exemplary Specific Target Capture (STC) process for a given application (e.g., detecting pathogens in blood) performed in an integrated cartridge: 1) Transferring the lysed blood solution from the ML chamber back to the STC chamber; delivering capture beads to the chamber via port 1 using a liquid handler; 2) During the heat incubation step in the STC chamber, the beads were thoroughly mixed; 3) Docking the magnet to contact the serpentine channel to collect the beads as blood is drawn through the liquid handler; wash and elution buffers were introduced through port (1). Note that not all components of the cartridge are shown in this figure for ease of viewing the features.

FIG. 37 depicts the following steps of an exemplary target amplification (e.g., PCR1 and PCR2 for targeted enrichment and tag/adapter addition) process for a given application (e.g., detecting pathogens in blood) performed in an integrated cassette: 1) The liquid processor extracts the target nucleic acid from the STC submodule; 2) A liquid handler loads the target solution into the PCR1 chamber, rehydrating the lyophilized reagents in the fluid path; performing a thermal cycle on the chamber by the instrument; 3) After the PCR1 is finished, extracting a PCR1 product by a liquid processor and diluting the PCR1 product; 4) The liquid handler delivers the diluted PCR1 product to the PCR2 chamber, rehydrating the lyophilized reagents in the fluid path; thermal cycling is performed on the chamber by the instrument. Note that not all components of the cartridge are shown in this figure for ease of viewing the features.

FIG. 38 depicts the following steps of an exemplary Copy Control (CC) process performed in an integrated cartridge for a given application (e.g., detecting pathogens in blood): 1) Pooling the PCR2 products by a liquid processor; 2) A liquid processor loads the PCR2 product into a copy control chamber; beads were prepared and delivered by a liquid processor; 3) Mixing and incubating the fluid and beads through a heated instrument interface; 4) The magnet interfaces with the cartridge serpentine to collect the beads as the fluid is drawn into the liquid handler; wash and elution buffers are introduced into port (2) by a liquid handler. Note that not all components of the cartridge are shown in this figure for ease of viewing the features.

FIG. 39 depicts the following steps of an exemplary cluster generation and sequencing process for a given application (e.g., detecting pathogens in blood) performed in an integrated cassette: 1) Collecting the eluted templates by a liquid handler; 2) Introducing the template and the cluster generating reagent into a cluster generating/sequencing flow cell by a liquid handler; 3) Incubating the flow cell through the heated instrument interface; 4) Sequencing reagents are delivered through the instrument fluid manifold and sequencing is performed. Note that not all components of the cartridge are shown in this figure for ease of viewing the features.

Fig. 40 highlights exemplary features of the instrument shown in fig. 45, shown here for a particular application or set of applications. The features are flexible to accommodate a wide range of applications.

Fig. 41 is a schematic cross-sectional view (left) and a 3D rendering of a Sealed Pneumatic Interface (SPI) port.

FIG. 42 depicts two exemplary designs of kits. The fluid may be accessed and moved in a variety of ways, including through a liquid handler (LH; e.g., a pipette system) and a liquid manifold (LM; e.g., on an instrument). Different valve setting methods may be employed, including sealing a pneumatic interface (SPI) port. In the centre graph, the numbers correspond to 1) foil-sealed lyophilized reagent library (LH channel): 2) Foil-sealed liquid reagent reservoir (LH channel); 3) Waste volume of all sequencing waste (and optional assay cartridge waste); 4) Waste inlet ports (manifold ports and SPI); 5) Nucleotide chambers (each with SPI and manifold ports for fluid and CO ₂ washes); 6) Wash chamber (SPI and manifold ports); 7) Soda lime chamber (for CO ₂ wash). The SPI and manifold ports are covered with, for example, removable seals or pierceable foils.

Fig. 43 shows another exemplary design of a kit. Part a contains liquid reagents, part B contains dry reagents, part C is the interface for sequencing reagents and other bulk reagents, and part W (negative space of the kit) is used for waste storage (IM is the connection to the instrument manifold) as required. The liquid and dry reagent chambers/vials are sealed with foil that is pierced by a pipette tip (PIPETTE TIP) of the liquid handler to reconstitute (dry reagent) and transfer the reagent. The design of the chamber and the bottle allows great flexibility in the reagents stored on the cartridge, allowing a large number of different assays/treatments to be performed on the cartridge. The part B module can be separated in the manufacturing process, independently filled and dried, and can be stored in an isolated manner in a low-humidity environment. In some embodiments, part a may also be separated during manufacturing, filling, and storage. In a preferred embodiment, one or more chambers in the kit contain a magnetic stirring bar (see figure) that interfaces with a magnetic stirring motor in the instrument when the kit is loaded into the instrument (e.g., available for on-board preparation/mixing of reagents).

FIG. 44 provides more details regarding the kit and its function.

FIG. 45 is a 3D rendering of one example instrument architecture for use in the system of the present invention.

Fig. 46 highlights some of the cartridge features of the instrument shown in fig. I-1.

FIG. 47 highlights other exemplary features (viewed from the other side) of the instrument shown in FIG. I-1, here shown for a particular application or set of applications. The features are flexible to accommodate a wide range of applications.

Fig. 48 again highlights other exemplary features (focusing on electronics) of the instrument shown in fig. I-1 (from the back), here shown for a particular application or set of applications. The features are flexible to accommodate a wide range of applications.

FIG. 49 highlights yet other exemplary features of the instrument of FIG. I-1 (focusing on cooling), here shown for a particular application or set of applications. The features are flexible to accommodate a wide range of applications.

FIG. 50 is a graph of qPCR results showing recovery of labeled antimicrobial resistant (AMR) targets directly from blood (DfB) using Specific Target Capture (STC) oligomers.

FIG. 51 is an agarose gel electrophoresis image confirming elution of ssDNA. Lane 1: DNA LADDER, lane 2:5ng dsDNA P3 target and 100ng ssDNA (IDT-ultra), lane 3:5ng dsDNA P31 target and 100ng ssDNA (IDT-ultra), lane 4:5ng dsDNA P48 target and 100ng ssDNA (IDT-ultra), lane 5;68.1ng dsDNA from PCR2 reaction, lane 6:70.3ng of multiplexed (P3, P31, and P48 targets) full capture ssDNA material, multiplex (duplicate) 1; lane 7:71.3ng of multiplexed (P3, P31 and P48 targets) full capture ssDNA material, parallel assay 2; lane 8:78.3ng multiplex (P3, P31 and P48 targets) full capture ssDNA material, run in parallel 3.

Fig. 52 is an agarose gel electrophoresis image demonstrating ssDNA eluted from NaOH eluate. Lane 1: DNA LADDER, lane 2: ssDNA control, lanes 3 and 4: dsDNA control, lanes 5 and 6: naOH ssDNA eluate.

FIG. 53 is a fluorescence microscope image showing in-well amplification of three synthetic DNA templates using SM-RPA. This shows three different initial copies of the synthetic DNA template, with amplified target DNA spots visible through the different intensities seen in the images of the respective targets.

FIG. 54 is a fluorescence microscope image of the products of the in-well clonal amplification of a target nucleic acid using RCA. Two different starting copies of the template were tested, each template input using a separate chip. For a given template input, the same region is imaged, which shows discrete clusters of amplified target DNA products (shown in separate images of the respective fluorophores) distinguishable in non-overlapping regions.

FIG. 55 shows the sequencing results of the synthetic DNA template directly immobilized on the chip surface. The scatter plot shows "length of aligned read" on the x-axis and "length of aligned read-error" on the y-axis, with corresponding histograms on top and right, respectively.

FIG. 56 shows the results of sequencing a synthetic DNA template using a direct hybridization method. The scatter plot shows "length of aligned read" on the x-axis and "length of aligned read-error" on the y-axis, with corresponding histograms on top and right, respectively.

FIG. 57 shows the results of automated sample response sequencing of labeled pathogens in whole blood. Top: the scatter plot shows the "length of aligned read" on the x-axis and the "length of aligned read-error" on the y-axis (labeled "EFFECTIVEREADLEN"), with the corresponding histograms on the top and right, respectively. Bottom analysis: the output indicates that the tagged pathogen was invoked correctly.

Fig. 58A shows a perspective view of an exemplary instrument.

Fig. 58B shows a front view of the instrument of fig. 58A.

Fig. 58C shows a side view of the instrument of fig. 58A.

Fig. 58D shows a cartridge interface assembly within the instrument of fig. 58A.

Fig. 58E shows an exemplary pneumatic pumping subunit within the instrument of fig. 58A.

Fig. 58F illustrates the positioning of an exemplary power subunit within the instrument of fig. 58A.

FIG. 58G shows an exemplary air handling and kit intake subsystem within the instrument of FIG. 58A.

Fig. 58H shows a liquid cooling subsystem within the instrument of fig. 58A.

FIG. 58I illustrates the positioning of an exemplary condensation management subsystem within the instrument of FIG. 58A.

Fig. 59 shows a pneumatic subsystem for the various instruments described herein.

Fig. 60 illustrates an exemplary sample or assay cartridge according to certain embodiments.

Fig. 61 illustrates an exemplary kit according to certain embodiments.

FIG. 62 illustrates an exemplary workflow for performing assays using the instruments and cartridges described herein.

FIG. 63 shows an exemplary sample or assay cartridge with a library preparation unit.

FIG. 64 shows an exemplary sample or assay cartridge with sample input and mechanical lysis subunits.

FIG. 65 shows an exemplary sample or assay cartridge with a specific target capture subunit.

FIG. 66 illustrates an exemplary flow cell and pipette reservoir within an exemplary assay cartridge.

FIG. 67 shows an exemplary SPI port configured for a 1mL pipette tip.

FIG. 68 shows an exemplary SPI port configured for a 5mL pipette tip.

FIG. 69 illustrates an exemplary specific target capture subunit according to certain embodiments.

FIG. 70 shows an exemplary library preparation unit or PCR fin.

FIG. 71 shows PCR results demonstrating successful mechanical cleavage and observation of released target nucleic acids using an exemplary cassette-compatible mechanical cleavage subunit.

FIG. 72 shows PCR results illustrating successful capture of specific targets using an exemplary cassette-compatible subsystem.

FIG. 73 shows an electropherogram overlay of various PCR results from example R.

FIG. 74 shows sequencing results obtained from amplified templates using the direct hybridization method in example S.

Detailed Description

The present disclosure provides oligomers, methods, compositions, and kits for rapid analysis of target polynucleotides, including determination of nucleotide sequences thereof. Analysis can be performed from a wide range of input sample types and amounts using a fully automated system that includes cartridges, instrumentation, and operating and analysis software, without manual intervention (sample to report) after operation begins. In addition, a complete workflow is disclosed, illustrating the chemical and mechanical aspects of the invention, as well as others.

Generally, the workflow includes one or more of the following: 1) sample preparation, 2) library preparation, 3) copy control, 4) cluster generation or 5) sequencing. The design and concept of cartridges and instruments are disclosed in which all chemical workflow steps of the various sections can be performed in an automated fashion. Embodiments are disclosed that include sequencing on the surface of a semiconductor chip. Software has been written to control all functions of the instrument, as well as the software control algorithm from raw data to all data analysis stages of the final report (i.e., what the answer to the question of running the test is).

1. Sample type

It is contemplated that a broad range of sample types ("definitions" section provides an exemplary, non-exhaustive list of sample types) are suitable for use with the disclosed invention. In a preferred embodiment, the sample is in liquid form and the entire sample or a portion thereof is introduced into the cartridge. In another preferred embodiment, the sample is in solid form, but is treated to bring the sample or a portion thereof into liquid form prior to introduction into the cartridge. Or the solids may be processed into a suspension, slurry, emulsion or the like, which is then introduced into the cassette. In another embodiment, the solid sample is introduced directly into the cartridge and processed in the cartridge at or near the beginning of the workflow to present the sample in the form of a liquid, suspension, emulsion, or the like. Alternatively, the target polynucleotide may be extracted directly from the solid sample before or after introduction into the cassette. In another embodiment, the sample is already in the form of a suspension, slurry, emulsion or the like at the time of acquisition and is introduced directly into the cartridge or is processed into liquid, solid or gaseous form prior to introduction into the cartridge. In another embodiment, the sample is a gas. Typically in a gaseous sample, the target polynucleotide is present in the sample in the form of an aerosol or suspension, with or without association with the cells. The gas sample may be introduced directly into the cassette, and polynucleotides may be collected, suspended, or otherwise harvested from the gas sample before introduction into the cassette. It is envisaged that the sample input port will accommodate the wide range of sample types. To achieve this, the sample input port may exist in a variety of forms including, but not limited to, 1) a universal sample port that directly accommodates all sample types, 2) a sample port designed for a particular sample type or group of sample types, where cartridges with different sample input ports may be used for different assay types, 3) a quasi-universal sample port that accommodates adapter accessories designed for a given sample type such that the appropriate adapter is connected to the cartridge depending on the assay to be performed.

Preferred sample types include whole blood, e.g., as used in the preferred embodiments of the disclosed invention, wherein the system is utilized to detect blood flow infections as well as antimicrobial drug resistance genes (see further details below) and plasma, e.g., in the preferred embodiments for detecting cell free DNA (cfDNA), including circulating tumor DNA (ctDNA). Another preferred embodiment of the preferred sample types, including whole blood, is to detect targets in plasma, such as but not limited to cfDNA, ctDNA and various viral infections (e.g. HIV). In such embodiments, the separation of plasma from whole blood may be preferred in the cartridge, but may also be performed in a sample collection tube or other device that desirably interfaces directly with the sample input port.

2. Sample preparation

It is contemplated that a wide variety of sample preparation methods and compositions (an exemplary, non-exhaustive list of sample preparation methods is provided in the "definitions" section) are suitable for use with the disclosed invention.

In a preferred embodiment, whole blood is a sample, and the target polynucleotide is contained in various infectious agents (e.g., bacteria, fungi, viruses) present in whole blood (exemplary applications are detection of blood flow infections and antimicrobial drug resistance genes). An exemplary sample preparation method of this embodiment includes the steps of: 1) Mixing a whole blood sample with a sample preparation reagent (e.g., including reagents for sample homogenization and cell lysis; in one aspect of this feature of the preferred embodiment, vigorous mixing helps to dissolve the sample); 2) Further homogenizing the sample by incubating at an elevated temperature while digesting the protein in the sample with proteinase K; 3) Mixing the homogenized, digested sample with a collision bead (bashing beads); 4) Mixing the beads and sample at a relatively high speed (e.g., 8000 RPM) using, for example, a rotating impeller, thereby lysing the infectious agent and releasing the target polynucleotide contained therein into solution; 5) Heating the sample at an elevated temperature (e.g., 95 ℃) to denature the target polynucleotide in double stranded form (this step also aids in stripping the protein and other components and/or structures surrounding and/or binding the target polynucleotide); 6) Mixing the denatured target polynucleotide sample with a set of Specific Target Capture (STC) oligonucleotides (STC oligomers may be designed to target, for example, specific targets, a set of specific targets, a broad range of targets, etc., as discussed further below); 7) Heating the STC oligomer/target mixture (e.g., 60 ℃) to promote annealing of the STC oligomer to its specific target sequence; 8) Mixing the STC oligomer/target mixture with streptavidin-derived paramagnetic particles; 9) Mixing and then incubating the STC oligomer/target mixture (e.g., 45 ℃) to promote binding of the STC oligomer/target mixture (STC oligomer including pendent (appended) biotin molecules) to the beads; 10 Immobilization of STC oligomer/target mixture/bead complex using a magnet; 11 Washing the STC oligomer/target mixture/bead complex; 12 Eluting the target polynucleotide, which is now ready for further processing in the workflow.

The preferred embodiments described above have many advantages including, but not limited to: 1) The whole process is automatically carried out in a box in a full-automatic system; 2) When sample processing is complete, the workflow continues uninterrupted in the closed cassette, thus no user intervention is required to continue processing the test/assay; 3) The cartridge accommodates a large amount of whole blood, improving the overall test/assay performance including sensitivity; 4) Homogenizing the sample, performing cell lysis and denaturation of the target polynucleotide in a rapid and efficient automation protocol, ready for further processing; 5) The Specific Target Capture (STC) process provides high purification in a rapid protocol; 6) STC processes provide a high degree of specificity, among other parameters, by capturing oligomer design and reaction conditions (mixture composition, temperature, etc.); 7) The STC process provides a high degree of inclusion, again using capture oligomer design, reaction conditions, etc., wherein all DNA sequences along the phylogenetic tree can be captured or excluded as desired; 8) The eluted sample may be further processed without analysis.

In other embodiments, the various features of the preferred sample preparation embodiments described in the paragraphs can be directly replaced with alternative features/methods in various combinations, including, for example, 1) proteinase K can be substituted or enhanced with one or more alternative enzymes, lotions, chaotropes (e.g., guSCN), chemical agents (including reducing agents), and the like; 2) No further sample homogenization may be required after mixing with the lysis reagent; 3) Cell lysis by bead impingement of zirconium beads may be replaced or enhanced with alternative beads, spheres or other milling media, sonication, lotions, chaotropes, reducing agents (DTT, beta-mercaptoethanol) and/or other chemical agents; 4) Homogenization and cell lysis may be performed simultaneously; 5) If the target is also available for further processing, the step of denaturing the double stranded target polynucleotide may be omitted; 6) If a given application does not require target capture/isolation/separation, the sample or portion thereof may be used directly for further processing at this point; 7) STC [ alone ] may be replaced with, for example, a) non-specific target capture (e.g., boom method; see other exemplary methods in the "definition" section), b) a combination of non-specific and specific target capture methods (e.g., the Boom method followed by the STC method), c) capture with an aptamer, d) filtration, e) isoelectric focusing, f) other methods listed in the "definition" section, g) combinations thereof.

In some embodiments, the Target Capture Oligomer (TCO) may perform other functions in addition to target capture alone. For example, the TCO can include one or more tags (e.g., unique Molecular Identifiers (UMIs), universal amplification primer sites, promoters (e.g., T7 RNA polymerase promoters), sequencing adaptors, and the like) that can be used in downstream processes. Furthermore, TCOs can be used as primers for extension and amplification (which can work in concert with one or more tags to achieve a desired function). In some workflows, annealing of the TCO and its extension by the polymerase can be performed simultaneously or at different times (or overlapping times) in the same reaction mixture. In other cases, the annealing of the TCO may occur first, followed by an extension of the TCO, for example, upon addition or combination with another reagent. In other cases, separation of the TCO/target complex may occur first after annealing and then be extended in a subsequent step, for example, while the TCO/target complex is still immobilized on a solid support. In some of these last described workflows, extension can be performed, followed by final washing of the immobilized TCO/target complex, followed by elution. In this case, for example, the target polynucleotide is now ready to move to the next part of the process that has been purified, labeled and extended, saving time, steps, etc. in the overall workflow.

In some embodiments, sample preparation is not required (i.e., the target polynucleotide may be used "directly from the sample" in the first step of the method, e.g., amplified).

The broad range of reaction chambers, storage chambers, fluidic interconnections, valve configurations, reagent delivery options, functions (mixing, stirring, heating, transporting, separating, including magnetic forces, etc.) disclosed or contemplated by the cartridges of the present invention, each of these sample preparation workflow options may be included within a rapid, fully automated workflow framework.

3. Library preparation

A broad range of library preparation methods and compositions suitable for use in the disclosed invention are contemplated (an exemplary, non-exhaustive list of library preparation methods is provided in the "definitions" section). Target polynucleotides may be provided as input to library preparation by a variety of routes, including directly from a sample or in the output form of a variety of sample preparation methods. In a preferred embodiment, amplification (e.g., amplification supporting a targeted sequencing method) is used to selectively enrich one or more portions of the target polynucleotide. Other optional features of this preferred embodiment include the introduction of a tag, optionally including adaptors, two or more rounds of separate amplification, optionally including the use of nested primers for the second and/or (if any) subsequent rounds, and copy control (see elsewhere herein; described as separate features, but also overlapping library preparation). One exemplary application is the detection of blood flow infections and antimicrobial drug resistance genes. An exemplary library preparation method of this embodiment includes the steps of: 1) Mixing the target polynucleotide with a first amplification reagent (as described elsewhere herein, the target polynucleotide may be from a variety of sources; one preferred source is the output of the sample preparation method described above, which utilizes whole blood as the sample type); 2) Performing a first amplification using PCR; 3) Diluting the first amplified product; 4) Mixing an aliquot of the diluted first amplification product with a second amplification reagent; 5) Performing a second amplification using PCR; 6) Combining and mixing the second amplification product with a capture reagent (including magnetic capture beads); 7) Incubating the second amplification product/capture reagent mixture at ambient temperature (about 20-26 ℃) for 10 minutes and mixing continuously; 8) Immobilizing the second amplification product/bead complex using a magnet; 11 Washing the second amplification product/bead complex; 12 The elution reagent is added to the washed second amplification product/bead complex, mixed and incubated at ambient temperature (about 20-26 ℃) for 1 minute. The eluted library molecules can now be further processed in a workflow.

Important features and advantages of the preferred embodiment of the library preparation workflow summarized in the preceding paragraph include: 1) A very wide range of sample input types and amounts can be accepted; 2) The first amplification reaction (PCR 1) is designed to have high sensitivity and fidelity; 3) Primers used in PCR1 were designed to widely amplify bacterial and fungal target profiles that may be contained in the sample, targeting bacterial 16S and 23S, fungal 28S, and specific antimicrobial drug resistance (AMR) genes; 4) The PCR1 amplicon does not need to be purified before entering the second amplification (PCR 2), only a simple [ auto ] dilution is required (unlike prior art schemes that require a purification step); 5) The primer for PCR2 is nested within the primer site for PCR1 and is designed to have high specificity for the target polynucleotide or nucleotide of interest; 6) Aliquots of PCR1 products from the same dilution can be used to perform a variety of different PCR2 reactions (e.g., one embodiment of a cassette design (see the "assay cassette" section below) shows 10 separate chambers dedicated to separate PCR2 reactions), increasing the specificity of each reaction by reducing the complexity of the system (i.e., covering the required number of targets in different 10 reactions, rather than, for example, 1 reaction, thereby reducing the number of primers in any given PCR2 reaction) and/or increasing the multiplexing capacity of the system; 7) In one instance of this preferred embodiment, at least one primer of the primer pair used in PCR2 is equipped with one or more biotin molecules, enabling immobilization of its amplicon product using streptavidin-conjugated microspheres (or the like); 8) One or more tags, including adaptors, can be added to PCR1, PCR2, or a combination thereof, including introducing tags at both ends of the amplicon, if desired; 9) The addition of copy control procedures may be added after the library preparation workflow steps outlined above, or designed to overlap/be incorporated into the PCR2 steps, or even earlier in the workflow (see more detailed elsewhere below). In summary, the method can use a variety of target polynucleotide input types and amounts, is highly sensitive and specific, can be used to easily introduce tags, including adaptors, can easily interface (or overlap) with copy control processes, is simple (e.g., only 1 purification step), is fast and is easily automated.

In other preferred embodiments, the various features of the preferred library preparation embodiments described in the paragraphs can be directly replaced with alternative features/methods in various combinations, including, for example, 1) different nested amplification configurations can be used, e.g., a) primers are nested only at the 1-end of the target region, b) no nesting is used, c) different nesting patterns of different target polynucleotides, 2) PCR can be replaced with another amplification method; 3) One amplification reaction can be performed instead of 2 (when desired properties such as sensitivity, specificity, multiplexing capability can be achieved and, if desired, labels, including adaptors, can still be introduced), making the workflow simpler and faster; 4) Various aspects of library preparation may overlap with sample preparation (discussed in more detail below); 4) Tags, including adaptors, including Unique Molecular Identifiers (UMI), can be attached to the target region using methods other than amplification, such as ligation; 5) The copy control method may be added immediately at the end of the workflow described in the above embodiments, or immediately before the purification step is performed, or starting from the PCR2 (or alternative amplification method) step, or even starting from the last cycle or cycles of the second amplification step, or from the PCR1 (or alternative amplification method) step, or 1 amplification step (if only 1), or even as early as sample preparation (copy control aspects are discussed elsewhere herein). Additional library preparation methods that can be used in the methods disclosed herein are listed in the "definition" section.

As described above, aspects of library preparation may overlap with sample preparation. The following are examples to illustrate this concept. 1) The oligomer, which can be a primer, anneals to the target polynucleotide during sample preparation. The primer may also serve other functions, such as target capture oligomer. The oligomer can anneal to the target polynucleotide at a desired level of specificity, increasing the overall specificity of the assay at this early stage (sample preparation), thereby improving assay performance. If desired, the oligomer may further comprise tags, including adaptors, including UMI, universal primer sites, and the like. At some point, for example, concurrently with annealing, after annealing in the second step (e.g., combining the annealing reaction mixture with the extension reaction mixture), after immobilization of the oligomer/target complex (e.g., while still immobilized), after elution, etc., or essentially as part of the first stage of library preparation, the oligomer is extended. Such extension products can now enter the remainder of the workflow, already provided with a tag, and already reach a certain level of specificity, which may save steps and time for the whole assay. Also in this mode, the first (or sole) amplification step may utilize specific counter primers (of any desired level of specificity, possibly even non-specific by using random primers) and, if desired, universal primers that bind to [ at least a portion ] of the tag sequence of the oligomer. Furthermore, the introduction of tags, including adaptors, including UMI, during sample preparation may bypass library preparation of the [ sample preparation ] product, directly into other steps of the workflow, such as cluster generation and/or sequencing (in applications where sensitivity is sufficient and no additional tags are required, etc.). 2) Following the variant of method 1 described above, the oligomer can be annealed to each strand of a double stranded target polynucleotide, each strand having a desired level of specificity, and one or both strands including a tag (including an adapter, including UMI). This may allow more workflow to be completed at this early stage, saving steps and time, and improving overall specificity. Furthermore, the first step (possibly the only one step) of library preparation may be universal amplification using primers that anneal to tag sequences at both ends of the target. These may be the same or different. Furthermore, during the sample preparation step and/or the universal amplification step (using the universal primer equipped with a tag), other elements required for other steps of the workflow may be introduced into the target. 3) Tags (including adaptors, including UMI) may be ligated to the target polynucleotides during sample preparation.

The broad range of reaction chambers, storage chambers, fluidic interconnections, valve configurations, reagent delivery options, functions (mixing, agitation, heating, transportation, separation, including magnetic action, etc.) disclosed or contemplated by the cartridges of the present invention, each of these library preparation workflow options may be included within a rapid, fully automated workflow framework.

4. Copy control

"Copy control" (CC) refers to compositions and methods that control the copy number of a molecule as an output of a given process in a predetermined manner (e.g., a limited amount, i.e., up to but not exceeding a predetermined amount; or a specific predetermined amount). Furthermore, in certain workflows, it is desirable to introduce additional sequences into the target polynucleotide (e.g., tag), such as introducing adaptors into the sequencing library. This may also be accomplished in CC compositions and methods (see "definitions" and figures and their associated brief description for further details). CC is included in many workflows disclosed herein. Compositions, kits and methods for isolating target polynucleotides (PCT/GB 2021/050098), various CC compositions and methods are disclosed in the "compositions, kits and methods for isolating target polynucleotides," which are incorporated herein by reference in their entirety. All CC compositions and methods disclosed therein are suitable for use in the workflow of the present invention. Furthermore, examples of CC processes are summarized in FIGS. 1-14 included herein (see accompanying description in the "FIG. description").

Preferred CC embodiments include a capture oligomer comprising in the 5 'to 3' direction: a capture sequence, an internal extension blocking sequence, a complement of the capture sequence, and a target hybridization sequence, wherein the complement of the capture sequence is configured to anneal to the capture sequence in the absence of an extended target sequence that anneals to the complement of the target hybridization sequence and the capture sequence. In some such preferred embodiments, the capture oligomer has the formula 5'-A1-C-L-B-A2-C' -A3-RB-A4-THS-X-3', wherein A1 is an optionally first additional sequence, C is a capture sequence, L is an optionally linker, B is an internal extension blocking sequence, A2 is an optionally second additional sequence, C' is a complement of the capture sequence, A3 is an optionally third additional sequence, RB is an optionally reversible extension blocking sequence, A4 is an optionally fourth additional sequence, THS is a target hybridization sequence; and X is an optionally present blocking moiety. In some of these preferred embodiments, the capture sequence comprises a poly a or poly T sequence, and the complement of the capture sequence comprises a poly T or poly a sequence.

Another preferred embodiment comprises a combination comprising a capture oligomer and a complementary oligomer, wherein (a) the capture oligomer comprises in the 5 'to 3' direction: a capture sequence comprising a first and a second portion, an internal extension blocking sequence, a spacer sequence comprising a first and a second portion, and a target hybridization sequence; and (b) the complementary oligomer comprises in the 3 'to 5' direction: the complementary sequence of the second portion of the capture sequence and the complementary sequence of the at least first portion of the spacer sequence are configured to anneal simultaneously with the capture oligomer in the absence of the complementary sequence of the spacer sequence. In some such preferred embodiments, the capture oligomer has the formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3', wherein A1 is an optionally present first additional sequence, C1 is a first portion of a capture sequence, C2 is a second portion of a capture sequence, B is an internal extension blocking sequence, A2 is an optionally present second additional sequence, S1 is a first portion of a spacer sequence, S2 is a second portion of a spacer sequence, A3 is an optionally present third additional sequence, RB is an optionally present reversible extension blocking sequence, A4 is an optionally present fourth additional sequence, THS is a target hybridization sequence, and X is an optionally present blocking portion. In some such preferred embodiments, the complementary oligomer has the formula: 5' -S1' -A2' -L-C2' -X-3', wherein S1' is the complement of at least a first portion of the spacer sequence and A2' is the optionally present complement of a second additional sequence optionally present in the capture oligomer; l is an optional linker, C2' is the complement of the second part of the capture sequence, and X is an optional blocking moiety.

Yet another preferred embodiment comprises a combination comprising a capture oligomer and a complementary oligomer, wherein (a) the capture oligomer comprises in the 5 'to 3' direction: a capture sequence comprising a first and a second portion and a target hybridization sequence comprising a second and a first portion; and (b) the complementary oligomer comprises in the 3 'to 5' direction: and a complementary sequence of the second portion of the capture sequence and a complementary sequence of the second portion of the target hybridization sequence, wherein the complementary sequence of the second portion of the capture sequence and the complementary sequence of the second portion of the target hybridization sequence are configured to anneal simultaneously to the capture oligomer in the absence of the complementary sequence of the target hybridization sequence. In some such preferred embodiments, the capture oligomer has the formula: 5'-A1-C1-C2-A2-S-A3-THS2-THS1-X-3', wherein A1 is an optionally first additional sequence, C1 is a first portion of a capture sequence, C2 is a second portion of a capture sequence, A2 is an optionally second additional sequence, S is an optionally spacer sequence, A3 is an optionally third additional sequence, THS2 is a second portion of a target hybridization sequence, THS1 is a first portion of a target hybridization sequence, and X is an optionally blocking portion. In some such preferred embodiments, the complementary oligomer has the formula: 5' -THS2' -A3' -S ' -A2' -C2' -X-3', wherein THS2' is the complement of the second part of the target hybridization sequence and A3' is the optional complement of the third additional sequence optionally present in the capture oligomer; s 'is an optionally present complement of a spacer sequence optionally present in the capture oligomer, A2' is an optionally present complement of a second additional sequence optionally present in the capture oligomer; c2' is the complement of the second portion of the capture sequence and X is an optional blocking moiety.

Another preferred embodiment is a method of capturing a target polynucleotide from a composition, the method comprising: contacting the target polynucleotide with other capture oligomers as described above and disclosed in PCT/GB2021/050098, wherein the target hybridization sequence of the capture oligomer anneals to the target polynucleotide at a site comprising the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the capture sequence that anneals to the capture oligomer such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a second capture reagent comprising a complementary sequence of the capture sequence and either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the second capture reagent; and separating the complex from the composition, thereby capturing the target polynucleotide.

Another preferred embodiment is a method of capturing a target polynucleotide from a composition, the method comprising: contacting the composition with a combination of a capture oligomer and a complementary oligomer as described above and as disclosed in PCT/GB2021/050098 (any one of claims 28-30 or 34-51), wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site comprising the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of a spacer sequence that anneals to the capture oligomer such that the complement oligomer is displaced to a degree sufficient to make the capture sequence of the capture oligomer available for binding; contacting the capture sequence of the capture oligomer with a second capture reagent comprising a complementary sequence of the capture sequence and either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the second capture reagent; and separating the complex from the composition, thereby capturing the target polynucleotide.

In some other preferred embodiments, a combination is provided comprising a capture oligomer and a complementary oligomer, wherein:

(a) The capture oligomer comprises in the 5 'to 3' direction:

A capture sequence comprising a first portion and a second portion, and

A target hybridization sequence comprising a second portion and a first portion; and

(B) The complementary oligomer comprises in the 3 'to 5' direction:

A complement of the second portion of the capture sequence, and

The complementary sequence of the second portion of the target hybridization sequence, wherein the complementary sequence of the second portion of the capture sequence and the complementary sequence of the second portion of the target hybridization sequence are configured to anneal simultaneously to the capture oligomer in the absence of the complementary sequence of the target hybridization sequence. Fig. 10B provides an illustration of exemplary oligomers according to these embodiments. Optional additional elements may be present as described in the further embodiments listed above and/or as shown in fig. 10B (e.g., any single element in fig. 10B or any combination thereof).

This combination can be used for limited capture because the complementary oligomer can be configured to bind free capture oligomer, but not capture oligomer bound to the target polynucleotide. For example, binding of the target hybridization sequence of the capture oligomer to the target polynucleotide may be energetically more favorable than binding of the complementary sequence of the second portion of the capture sequence to the second portion of the target hybridization sequence. In the absence of the target polynucleotide, the complementary oligomer binds to the capture oligomer and blocks the accessibility of the capture sequence (c1+c2 in fig. 10B) to an extent sufficient to block the binding of the capture sequence through the complementary sequence of the capture sequence in a second capture reagent, which may be any of the second capture reagents described elsewhere herein.

Accordingly, there is also provided a method of capturing a target polynucleotide from a composition, the method comprising:

Contacting the composition with a combination of the above or any other embodiment described herein, wherein the target hybridization sequence of the capture oligomer anneals to the target polynucleotide;

Contacting the capture oligomer with a complementary oligomer prior to or after annealing of the capture oligomer to the target polynucleotide, wherein the complementary oligomer anneals to the free capture oligomer and partially occupies its capture sequence, wherein the complementary oligomer does not anneal to a complex comprising the capture oligomer annealed to the target polynucleotide, and wherein annealing of the target hybridization sequence to the target polynucleotide causes dissociation of the complementary oligomer from the capture oligomer if contact of the capture oligomer with the complementary oligomer occurs prior to annealing of the capture oligomer to the target polynucleotide;

contacting the capture sequence of the capture oligomer complexed with the target polynucleotide with a second capture reagent comprising a complementary sequence of the capture sequence and either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the second capture reagent; and

Isolating the complex from the composition, thereby capturing the target polynucleotide. Optional additional elements may be present as described in the further embodiments listed above and/or as shown in fig. 10B (e.g., any single element in fig. 10B or any combination thereof).

Another exemplary CC method in the presently disclosed invention includes the steps of: 1) mixing the PCR2 amplicon (see detailed elsewhere below) with a CC reagent comprising a CC capture oligonucleotide as described herein and in PCT/GB2021/050098, 2) incubating the mixture at an elevated temperature (e.g., about 95 ℃) for about 3-5 minutes to denature the double stranded target nucleic acid (amplicon) present, 3) incubating the mixture at an intermediate temperature (e.g., about 60 ℃) for about 5-10 minutes to facilitate annealing of the CC capture oligomer to the target, 4) extending the 3 'end of the amplicon along the CC capture oligomer to displace the complementary sequence of the target capture sequence (extension may occur during the annealing step when an appropriate reagent is present in the reaction mixture, or as a subsequent step, in which case the reagent for extension is added separately), 5) mixing the extension reaction mixture with a complementary sequence comprising the capture sequence present in a user-defined amount, and (i) a binding partner or (ii) a second capture reagent of a solid support, 6) incubating the mixture at an intermediate temperature (e.g., about 45 ℃) for about 10 minutes to facilitate annealing of the CC capture oligomer to the target capture oligomer, 4) incubating the 3' -end of the amplicon along the CC capture oligomer to displace the complementary sequence (when an appropriate reagent is present in the reaction mixture) and, when an appropriate reagent is present in the reaction mixture, the reagent is added separately for extension, and the reagent is used for extension is for extension, in the subsequent step, in the complementary step, and (i) the complementary reagent is added to the complementary capture sequence is separated, and (i) and (ii) is added to the target capture nucleic acid is separated, and is washed, and the bead is separated, and the target-capturing nucleic. The eluted copy control molecules can now be further processed in the workflow.

Alternatives to the exemplary CC workflow described above include, but are not limited to, 1) performing the above steps after PCR1 (i.e., when PCR2 is not performed in a given workflow), 2) performing the above steps with target amplicons generated by methods other than PCR, 3) adding universal sequence sites as tags during PCR1 or PCR2 (or alternative amplification methods); the use of this universal tag as a universal binding site for a CC capture oligomer (advantages include, but are not limited to, a) designing only 1 CC capture oligomer for multiple targets, b) higher multiplexing capability, c) consistent annealing properties across target amplicons, etc.), 4) adding a tag (instead of or in addition to the universal sequence tag just described) to an amplicon using a CC-based strategy (as disclosed) during PCR1 and/or PCR2 (or other amplification methods), 5) adding all desired tags, including adaptors, as part of the CC process. Note that the CC method (including tag addition) generally described above and elsewhere therein is disclosed in more detail in GB 2021/050098.

5. Cluster generation

"Cluster" refers to a grouping of molecules bound to a solid support, e.g., a nucleic acid molecule bound to a solid support. The term "cluster generation" refers to a process of generating clusters. Examples of cluster generation processes include amplification-based, e.g., clonal amplification, and non-amplification-based, e.g., hybridization of a target molecule to an oligonucleotide immobilized on a solid support in a known specific region (e.g., spot). Clusters can be monoclonal (generally the preferred configuration in this disclosure) or polyclonal.

One widely accepted method of cluster generation is by performing clonal amplification by emulsion PCR on the beads. In second generation sequencing (NGS) applications, after clonal amplification, beads are arranged on the surface of a flow cell (see US8012690B 2). This technology has been incorporated into a number of NGS platforms, including Ion Torrent (Thermo) systems, ABI SOLiD, and Roche 454. Emulsification encapsulates the beads, amplification reagents and individual template DNA molecules in separate droplets (micelles) to prevent cross-contamination. While emulsion PCR is a proven technique, its workflow is complex, time consuming (many hours), and it is difficult to automate the cartridge format. Other widely accepted methods, such as those developed by Illumina (initially Solexa and Manteia), use bridge amplification to perform clonal amplification directly on the flow cell surface. The first generation of cluster generation method using bridge amplification is based on the form of isothermal polymerase chain reaction promoted by cycling of reagent streams (isothermal bridge PCR; see US10370652B2 and US7972820B 2). In these methods, two PCR primers are immobilized together on the surface of a flow cell and a population of target DNA molecules containing matching adaptors at both ends is hybridized. Next, the denaturing and elongating reagents flow into successive cycles, producing discrete clonal clusters typically less than 1 micron in diameter. These small clusters contain a relatively low number of copies of the target nucleic acid, which makes them unusable for platforms requiring larger amounts of target nucleic acid to achieve the desired sequencing performance. Similar to emulsion PCR, isothermal bridge amplification takes time (typically more than 4 hours) and requires high reagent amounts. The second generation method using bridge amplification is based on Recombinase Polymerase Amplification (RPA) in the form of a method called exclusion amplification (ExAmp) (see US9169513B 2). RPA is an isothermal DNA amplification method using two primers similar to PCR design (see US7270981B 2). In ExAmp, both primers are immobilized on the surface of an arrayed (patterned) flow cell. The region between the amplification sites is free of primers and serves to prevent mixing between clonal populations. Instead, the template DNA molecules are hybridized to the surface prior to the start of the amplification process, and multiple targets are added with the amplification mixture. As a result, template hybridization and amplification occur simultaneously, and cluster clonality is achieved by amplification occurring at a faster rate than hybridization. This process is still relatively time consuming (about 3 hours), but the result is a higher density of clusters, which favors sequencing output. However, these are also small clusters containing a relatively low number of copies of the target nucleic acid.

In addition to emulsion PCR and bridge amplification, another method of creating clonal clusters is by Rolling Circle Amplification (RCA). In this method, the template DNA molecule is circularized prior to hybridization to and extension of the individual amplification primers. This results in a long amplicon molecule containing the concatemer of the target sequence, providing a large amount of DNA for downstream analysis. The sequencing platform developed by BGI and Complete Genomics uses the form of solution phase RCA to create DNA nanospheres which are then hybridized to a patterned array (DOI: 10.1126/science.1181498). Creating and manipulating DNA nanospheres requires precise and appropriate quality control, which complicates the application of fast, cartridge-based formats.

In addition to solution phase processes, QIAGEN invested in developing exponential surface phases RCA for their GENEREADER platforms (US 2018/0105871, US 2018/012251, EP1916311, US9683255B2, US 2018/0087099). The method is based on exponential RCA, wherein two (or more) amplification primers are used instead of one (US 5854033, US6143495, 1101/gr.180501, US 6323009). In this method, multiple targets are modified to include adapter sequences on both ends and then used to ligate templates into loops. The DNA circles are then hybridized to the surface of the flow cell where the two amplification primers are co-immobilized. The amplification primer is complementary to the adaptor present in the circular template, which allows for a "branched" exponential RCA reaction (DOI: 10.1093/biomethods/bpx 007). This protocol involves many steps and can be time consuming. A simpler, faster workflow is needed to be effectively applied to a fast, cartridge-based system.

A. immobilization of oligonucleotides

Cluster generation on a solid support typically requires immobilization of one or more oligonucleotides or polynucleotides to the solid support. For the purposes of this disclosure, any method of immobilization that supports one or more of the steps associated with the disclosed embodiments is acceptable. This includes, but is not limited to, covalent and non-covalent methods; direct and indirect methods; attached to a specific area of the surface (e.g., spot, array, etc.) and substantially immobilized throughout the surface; surfaces (e.g., holes, posts, pads), particles, etc., that adhere to planar surfaces, including features; etc.

In several preferred embodiments disclosed therein, the oligonucleotides are attached to the surface of wells made on top of the semiconductor chip. Common methods for such attachment include CLICK CHEMISTRY (see, e.g., Click Chemistry,a Powerful Tool for Pharmaceutical Sciences(2008)Hein,et al.;Pharm Res 25(10)：2216-2230 and A Hitchhiker's Guide to Click-Chemistry with Nucleic Acids(2021)Fantoni et al.,Chem Rev,121:7122-7154). in certain embodiments, the oligonucleotides to be attached include a 5 '-dbco=5' -end Dibenzocyclooctyl (DBCO) moiety (see, e.g., example N, "sequencing of templates generated using in-well clonal amplification"). In such embodiments, one preferred conjugation method involves activating the chip surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide or oligonucleotides. An exemplary protocol includes the following general steps: 1) Cleaning the surface of the semiconductor chip surface by immersing in a 2% Decon 90 solution (Decon Laboratories LTD) for 5 minutes; 2) The surface was rinsed with 18M Ω water, then incubated in 0.1M HCl for 5 minutes, then rinsed again in 18M Ω water, and dried with nitrogen; 3) The chip surface was incubated for 30 minutes in a 0.6% wt/vol solution of MCP click polymer rinsed in 7% ammonium sulfate (NH ₄)₂SO₄ with 5% azide content 4) in 18mΩ water, blow dried with nitrogen, and then baked at 80 ℃ for 15 minutes; 5) Cooling the chip to room temperature; 6) One or more oligonucleotides are conjugated to a surface by: a) Microarray spots-one or more oligonucleotides deposited on surface spots in 150mM sodium phosphate buffer pH 8.5 (Na ₂HPO₄ and NaH ₂PO₄) and sucrose dodecanoate 0.01% wt/vol; in some embodiments, the spot volume is about 200-300pL and the oligonucleotide concentration is about 40-200. Mu.M; Incubating the spotted sample at 75% relative humidity for at least about 16 hours; b) Overflow filling-the oligonucleotide solution (same buffer composition and oligonucleotide concentration as in method a above) is overflowed on the chip surface in the flow cell; sealing the input and output ports of the flow cell and incubating the chip for at least about 16 hours; 7) The chips (method A or B) were washed in 0.1M Tris buffer ((HOCH ₂)₃CNH₂) (pH 9) at 50℃for 15min, then rinsed in 18 M.OMEGA.water, dried with nitrogen and stored in the dark at 4% relative humidity until required.

Other conjugation methods that have been successfully demonstrated for use with selected embodiments include, but are not limited to: 1) a polyacrylamide-based coating containing succinimide groups (e.g., codeLink from Surmotics IVD company), for conjugation of primary amine-derived oligonucleotides, 2) a physical adsorption polymer coating containing bromoacetamide (reactive monomer N- (5-bromoacetamidopentyl) acrylamide), for conjugation of phosphorothioate-containing oligonucleotides, and 3) a conjugation process using ultraviolet radiation and Poly (T) Poly (C) 5' -labeled oligonucleotides.

B. cluster generation using solution-mediated recombinase polymerase amplification (SM-RPA)

Here we disclose solution-mediated recombinase polymerase amplification (SM-RPA) which can be used to generate cloned nucleic acid clusters from multiple targets directly on a solid support in typically 20-60 minutes. This is faster than the RPA-based methods (and other methods) currently used in the art. Furthermore, the clusters produced thereby produce larger size and higher molecular weight "patches" of clonally amplified nucleic acids than small clusters produced by other methods, which is beneficial for applications requiring larger amounts of immobilization materials.

In a preferred embodiment of SM-RPA, one of the two primers (e.g. the reverse primer) is immobilized on a solid support, while the other primer (e.g. the forward primer) is present in the solution phase. As a result, amplification occurs on the surface and the strands are released into the liquid phase, which can locally re-hybridize. FIG. 15 shows the primer configuration and mechanism of SM-RPA, which is compared to bridge amplification. The resulting clonal population appears as a "patchwork" of immobilized DNA, as shown in fig. 16, in which different patches from clonal expansion of 3 different target molecules are shown. The product "grows" laterally, extending in all directions across the solid surface until it encounters an adjacent "patch". This preferred embodiment has been demonstrated from multiple targets (e.g., slides) on planar surfaces and perforated semiconductor chips. The resulting clusters are analyzed using a variety of methods, including post-amplification hybridization using fluorescent labeled probes and sequencing using ISFET signal detection. Experiments describing SM-RPA are summarized in example J, "in which solution-mediated recombinase polymerase amplification (SM-RPA) is used to amplify target nucleic acids in wells".

C. Cluster generation using branched surface phase Rolling Circle Amplification (RCA)

Here we disclose a method and related compositions of rolling circle amplification that can be used to generate clonal nucleic acid clusters from multiple targets directly on a solid support. In a preferred embodiment, it is apparent that the addition of adaptors required for ligation and amplification is accomplished during other parts of the workflow. Furthermore, the method allows the generation of circular templates directly on the surface of the flow cell, wherein the ligation splint oligomer acts as a first amplification primer. All of these aspects simplify and improve workflow efficiency while shortening the overall workflow duration, yielding advantages over current surface RCA methods.

Figure 17 shows a preferred embodiment in which the creation of circular templates for clonal expansion is obviously performed in across 3 different process steps (see legged illustration in the figure). FIG. 18 shows the actual branched surface phase RCA clonal amplification process (see legged illustration in the figure). This method produces discrete clusters of clonally amplified DNA consisting of multiple repeats of the target sequence, providing a high signal-to-noise ratio. It is also noted that both strands of the target molecule sequence are generated by extension of two amplification primers. Specific experiments illustrating this embodiment are summarized in example K, "in which Rolling Circle Amplification (RCA) is used for in-well amplification of target nucleic acids.

D. Cluster generation using hybridization

Non-amplification based methods may also be used to generate clusters, such as hybridization of target molecules to oligonucleotides immobilized on a solid support in known specific areas (e.g., spots), as described elsewhere herein. In a preferred embodiment of the present disclosure, the target-specific oligonucleotide array is spotted (immobilized) in a well on top of the semiconductor chip. These target-specific oligonucleotides can be used as both capture oligomers and sequencing primers. The target polynucleotide sequences flow into the pores and form monoclonal clusters in which target molecules of a given sequence hybridize specifically to complementary immobilized oligonucleotides. The clusters were then sequenced using the immobilized oligonucleotides as sequencing primers. In a preferred embodiment of the sequencing phase, the semiconductor chip comprises an ISFET sensor array, which is used as a detection mode in the sequencing reaction. Specific experiments illustrating this embodiment are summarized in example M "sequencing synthetic templates using direct hybridization methods".

Where applicable (i.e., where this approach achieves the objectives of analysis/assay/test), the use of cluster generation of hybridization provides some significant advantages. First, no cloning amplification step is required, thus generally saving steps, reagents and time. On the other hand, in many applications there is no need for a copy control step, as steps, reagents and time are generally saved. In other cases, the library preparation step may be simplified, e.g., less amplification is required, little or no tag/adapter step is required, it is possible to eliminate purification steps, etc. In cases where the target polynucleotide is sufficient and little or no inhibition of the sample type occurs, it may even be generated directly from sample to cluster and then analyzed (e.g., sequenced). Furthermore, if sequencing is the analytical method of choice, the steps of denaturing the target and annealing the sequencing primers, which are typically performed after clonal amplification, are not required, since the capture oligomers also serve as sequencing primers.

6. Sequencing

A preferred embodiment of the present invention uses a semiconductor chip comprising an ISFET sensor array for sequencing (see, for example, US 7,686,929 B2). The chip also typically includes an array of holes attached to the chip surface and positioned over the ISFET sensor. The preferred sequencing method generally comprises the following steps (sequencing-by-synthesis): 1) Fixing the nucleic acid template to be sequenced in one or more wells (methods of fixing in wells include clonal amplification and direct hybridization; see other sections for details); 2) Annealing the sequencing primer to the template (in some embodiments, annealing the sequencing primer is part of the immobilization step; 3) Binding a sequencing enzyme (polymerase) to the template/sequencing primer complex (also disclosed below is a novel method of adding sequencing primers and enzyme in the same step); 4) Flooding the wells with a given dNTP (in some cases, multiple dntps may be included together in a single stream); 5) Incubation (in some cases, stopping the flow of dntps, and in other cases, not); if complementary nucleotides are present in the template, then the incorporated dNTPs will be incorporated and protons (1 for each nucleotide incorporation) will be released, and one or more ISFETs under one or more of the pores will detect this release; 6) Washing; repeating steps 4-6 for each other dNTP; this completes one "loop" (other loop configurations may also be used); 7) The cycle is repeated to obtain the sequence of templates.

A. Simultaneous annealing of sequencing primers and binding of sequencing enzymes

As outlined above, annealing of the sequencing primer to the nucleic acid template and binding of the sequencing enzyme to the resulting complex are performed in separate steps. This is because the annealing step requires high temperatures (up to about 95 ℃), which also typically involve denaturation of the template, at which point the template is typically double-stranded, and the sequencing enzymes are not stable at these temperatures. We disclose a method of combining these two steps, including the use of thermostable sequencing polymerases (e.g., tin (exo-) LF DNA polymerase from Optigene). Briefly, all components required for sequencing primers and enzymes are combined with templates in one reaction mixture. The double-stranded template is denatured by increasing the temperature (e.g., (up to about 95 ℃) and then decreasing the temperature (e.g., to about 60 ℃) to support annealing of the sequencing primer and subsequent binding of the sequencing enzyme to the primer/template complex (at least some degree of binding of the sequencing enzyme may also occur later in the process if and when the temperature is lower, e.g., between about 20 ℃ and 45 ℃).

B. sequencing after Cluster Generation by clonal amplification

In a preferred embodiment of a sequencing workflow comprising a semiconductor chip as described above, sequencing is performed after cluster generation by clonal amplification (see section iii.b.5 and elsewhere herein above). Also as described elsewhere herein, cluster generation is followed by sequencing primer hybridization and sequencing enzyme binding, followed by sequencing. In some embodiments, the sequencing primer may be universal to all potential targets, utilizing a universal primer binding site introduced into the template in an early step of the process (e.g., library preparation). Alternatively, sequencing can be initiated from a specific target using multiplexing of sequencing primers. Experiments illustrating this mode are summarized in example N "sequencing of templates generated using in-well clonal amplification".

C. sequencing after Cluster Generation by direct hybridization

In another preferred embodiment of a sequencing workflow comprising a semiconductor chip as described above, sequencing is performed after cluster generation by direct hybridization (see section iii.b.5 and elsewhere herein above). Also as described elsewhere herein, the immobilized capture oligomers are also used as sequencing primers in the direct hybridization method. Thus, the steps of the method after template immobilization (hybridization) include sequencing the enzyme binding followed by sequencing. Experiments illustrating this mode are summarized in example M "sequencing synthetic templates using direct hybridization methods".

D. Key sequence

I. introduction to the invention

For semiconductor sequencing, base call parameters are calibrated using known DNA sequences, such as setting thresholds for zero-introduction events (i.e., "0-mers"), single base introduction events (i.e., "1-mers"), and homopolymer introduction (i.e., "2-mers", "3-mers", etc.), typically by using universal key sequences (see, e.g., Genome sequencing in microfabricated high-density picolitre reactors(2005)Marguilies,et al.,Nature,437:376-380). this known sequence may be introduced into the template at an early stage of the workflow (e.g., tag/adapter addition during library preparation).

Embodiment #1 target-specific key sequence

In the first approach, a portion of the target itself may be used as a calibration key sequence. For example, in the case of using a primer to enrich a specific region in an upstream amplification step (AKA-targeted enrichment), the sequence of the primer itself is generally known. In this case, the threshold and other signal processing parameters may be determined using a portion of the known sequence of the primer as a key sequence. This can be achieved by truncating the oligonucleotides to be used as sequencing primers, i.e. surface immobilized capture oligomers/primers in a direct hybridization method of cluster generation, followed by semiconductor sequencing.

When an array of target-specific, surface-immobilized capture oligomers/primers is used in a direct hybridization method, each capture oligomer/primer may be truncated at its 3' end (i.e., distal to the surface) by x bases. The value of x can be specifically determined for various oligomers based on sequence. The truncated bases will form the critical sequence of each specific primer, hereinafter referred to as target-specific critical sequence. Ideally, the target-specific key sequences will produce outputs of at least 0-mer, 1-mer and 2-mer (see the "introduction" paragraph above). However, it is unlikely that virtually any one primer in a given figure will have a sequence that will produce such an output. Thus, the exact target-specific key sequence must be determined on a specific analytical basis, based on the characteristics considered to be most desirable for a particular signal processing method.

FIG. 19 shows an example of a basic target-specific key sequence. For Oligo 1, paragraphs 1A and 1C describe forward and reverse primer binding sites, respectively, for targeted upstream amplification. Thus, the specific sequence of these portions is known. Segment 1B of Oligo 1 shows the region of interest, i.e. the unknown part of the template to be sequenced. Note that for the purposes of this example, the actual sequence of Oligo 1, segment 1B and segment 1C are uncorrelated and therefore not described. Oligo 2 was designed to be complementary to fragment 1A of Oligo 1, with the exception of a nucleotide truncation at the 3' end being notable. In the embodiments described herein, oligo 2 is immobilized on a solid support. Oligo 1 was provided in solution and fragment 1A of Oligo 1 was hybridized specifically with Oligo 2, except for the 5' end key sequence nucleotide.

Table E below provides an example sequence used in this embodiment. Note that the final base of the known primer sequence, i.e., the final base at the 5' end of segment 1A of Oligo 1, may not be used as part of the key sequence. This is because this base is likely to be identical to the first base of the unknown part to be sequenced (Oligo 1, paragraph 1B). Thus, it is known that there will be an introduction of the base, but whether this will be a 1-mer introduction or a homopolymer introduction event is unknown, so the introduction event may be ineffective for the threshold setting, and incorrect such use may result in errors.

Table E-examples of oligonucleotide sequences suitable for use in the scheme shown in FIG. 19.

After the 3' truncation, the surface phase primer may no longer have the same properties as before the truncation that are important for hybridization to the template, such as nucleotide length, melting temperature (Tm) and GC content percentage (% GC). In particular, the melting temperature will now decrease due to the removal of the base. If a set of primers is truncated to different amounts to provide the optimal target-specific key sequence, the kinetics and thermodynamics of template hybridization may be significantly altered, and thus the overall efficiency of hybridization may be reduced. When multiple templates are designed to hybridize to multiple surface capture oligomers/primers, the altered hybridization characteristics may result in different hybridization efficiencies for the entire set. In this case, it may be necessary to further modify the surface capture oligomer/primer to offset the effect of its 3' truncation. Modification may be accomplished in a variety of ways, including but not limited to: 1) Adding bases at the 5' end, i.e., near the surface, to increase primer length and raise melting temperature; 2) Nucleic acid analogues, such as Locked Nucleic Acids (LNA) or Peptide Nucleic Acids (PNA), are used within the surface-bound capture oligomers/primers to increase the melting temperature.

FIG. 20 provides yet another similar example of a target-specific key sequence approach. Fragments 3A and 3C of Oligo3 describe the forward and reverse primer binding sites, respectively, for targeted upstream amplification. In this example, a tailed primer approach was used in the upstream amplification process to extend Oligo3 and additional synthetic 3D. This moiety is not target specific and therefore, segment 3D can be: (a) universal, or (b) target specific. In this approach, additional segment 3D is specifically designed to offset the effects of the 3' truncation required to generate the target-specific key sequence. Segment 3B of Oligo3 shows the region of interest, i.e. the unknown part of the template to be sequenced.

Oligo 4 was designed to be complementary to Oligo3, including complementarity to the target-specific portion (Oligo 2, segment 3A) and the additional portion (Ologo, segment 3D) of Oligo 3. Truncation of the 3' end enables generation of target-specific key sequences. In the embodiments described herein, oligo 4 is immobilized on a solid support. Ologo 3 is provided in solution, and segment 3A and segment 3D of Ologo 3 specifically hybridize to Oligo 4, except for the 5' terminal key sequence nucleotide.

Table F below provides an example sequence for use in aspects of this embodiment depicted in fig. 20.

Table F-examples of oligonucleotide sequences suitable for use in the scheme shown in FIG. 20.

Embodiment #2. Fixed Universal Key sequence

In a second approach, the additional 5' portion of the surface immobilized capture oligomer/primer can be used to generate a key sequence by enabling polymerization from the 3' end of the hybridization template while temporarily preventing polymerization from the 3' end of the surface immobilized oligomer.

In this embodiment, the 3' end of the surface-bound capture oligomer/primer is not truncated, but is equipped with a reversible blocking chemical group to prevent polymerization. Examples of such blocking groups include, but are not limited to: 3' -O- (2-nitrobenzyl), 3' -hydroxylamine and 3' -O-azidomethyl. At the 5' end, the surface-bound capture oligomer/primer includes additional known sequences associated with use as a key sequence (see discussion of possible key sequence elements above). Since the known sequence is additive, it will have a limited effect on the specificity of the surface-bound sequence-specific part of the capture oligomer/primer at most for template hybridization. Thus, it may be common or universal to all surface-bound capture oligomers/primers in the set. After hybridization of the template, a sequencing polymerase is added. Since the 3' -end of the surface immobilized capture oligomer/primer is blocked, sequencing will not start from this end. However, the 3 'end of the template is unmodified, so the additional 5' portion of the immobilized capture oligomer/primer will be used as a template to initiate the sequencing reaction. Sequencing output from this additional 5' portion of the surface-bound capture oligomer/primer will be used as a universal key sequence to set relevant signal processing and base call parameters.

Once sequencing of the 5 'portion is complete, the 3' blocking portion is reversed/removed using appropriate methods. For example, the 3' -O- (2-nitrobenzyl) group may be photocleaved upon exposure to 340nm light, the 3' -hydroxylamine may be unblocked with aqueous sodium nitrite, and the 3' -O azidomethyl group may be removed by reduction with tris (2-carboxyethyl) phosphine. After removal of the blocking moiety, additional polymerase is added to ensure that all primer-template complexes bind to the polymerase and sequencing resumes from the unblocked 3' end of the surface-bound capture oligomer/primer. The sequencing data thus generated is analyzed using signal processing and base call parameters set using previous sequencing data from the universal 5' portion of the capture oligomer/primer itself.

A schematic of this second key sequence embodiment is shown in fig. 21. In step (a), the sequencing target Oligo 5 specifically hybridizes to the fully complementary segment 6A of Oligo 6. Segment 6A may be identical to the primer used to amplify the target upstream of the workflow. Oligo 6 was reversibly blocked at the 3' end, preventing extension. After addition of the polymerase, the 3' end of Oligo 5 can be polymerized using segment 6B of Oligo 6 as a template. Segment 6B can be designed to serve as a key sequence, reporting 0-mer and 1-mer as well as other nucleotide introduction events as needed as sequencing progresses in that portion. Once sequencing of segment 6B is complete, the blocking portion of the 3' end of Oligo 6 is removed according to step (c). If desired, after further addition of polymerase, sequencing is now performed from the 3' -end of Oligo 6 through the unknown target region of Oligo 5.

7. System and method for controlling a system

Disclosed herein in its entirety are embodiments useful for rapid analysis of target polynucleotides, including determination of nucleotide sequences thereof, from a wide range of input sample types and amounts using an automated system. The system includes an instrument and at least one cartridge removably insertable into the instrument. In a preferred embodiment, the system further comprises at least one kit removably insertable into the instrument. In still further preferred embodiments, the system includes a semiconductor chip, wherein in some embodiments the chip is embedded within the cartridge. In a particularly preferred embodiment, the system further comprises a flow cell mounted on top of the chip (either porous or non-porous), which can be used to deliver fluid to and remove fluid from the chip. In a preferred embodiment, the system comprises software. In a particularly preferred embodiment, the software includes operating software (for the control system) and analysis software (for receiving, processing and analyzing the output of the system).

A. Semiconductor chip

The use of semiconductor chips comprising arrays of Field Effect Transistors (FETs) for detecting chemical and/or biological reactions, including sequencing reactions, is well known in the art (e.g. US 7,686,929 B2;US 8,685,228 B2;US 8,986,525B2;US 2010/0137443 A1). In the present disclosure, preferred embodiments include semiconductor chips comprising an array of Ion Sensitive Field Effect Transistors (ISFETs) that can be used as a detection device for a variety of reactions, including nucleic acid sequencing reactions. In a particularly preferred embodiment, the chip further comprises an array of wells located above and in fluid contact with the array of ISFETs. In these embodiments, the sequencing reaction typically occurs within the well and the release of ions is detected by an ISFET sensor. In a particularly preferred embodiment, the chip further comprises a flow cell mounted on top of the chip (either porous or non-porous) that can be used to deliver fluid to and remove fluid from the chip/ISFET array. In a preferred workflow/system embodiment, the chip is integrated into a cartridge, where the entire workflow is performed.

B. Measuring box

As used herein, the term "cartridge" or "cartridge" refers to a device that performs the steps of a particular test, assay, or portion thereof. Typically, such devices include chambers that are fluidly connected to each other to varying degrees. An exemplary design of a cartridge for performing the entire sequencing workflow from sample input to results is disclosed. A variety of sequencing-based assays, as well as other complex assays, can be performed within the cassettes so disclosed. In one set of embodiments, the different stages of the workflow are performed in a "subsystem" box, while in another set of embodiments, all stages of the workflow are performed in an integrated box. In some cases, the assay may be performed in conjunction with a kit (an exemplary design also packaged). Typically, testing is performed in such cartridges in conjunction with instrumentation, but in some cases testing and cartridge configuration may be performed that does not require automation or a minimum degree of automation.

I. subsystem box

Exemplary designs of sample preparation (see, e.g., fig. 22-24), library preparation (including copy control; see, e.g., fig. 25-26), and cluster generation/sequencing (see, e.g., fig. 27-28) are disclosed. In some cases, these processes (sample preparation, library preparation, cluster generation/sequencing) can be performed alone or in various combinations, depending on the overall objective and requirements of the test being performed. In some cases, where 2 or 3 cartridges are used for a given test, the output of one cartridge may be manually transferred to the next cartridge, or may be transferred automatically with the aid of an instrument.

Key features of sample preparation cartridge design include, but are not limited to: 1) A flexible input sample system that can accommodate a relatively large sample volume; 2) The unique design chamber layout enables relatively large sample and measurement volumes to be accommodated in a relatively compact cartridge; 3) A fully integrated rotary valve that can draw and deliver fluid to a large number of chambers and channels; 4) Independent but connected accessories or "fins" that can perform complex operations but provide flexible cartridge designs between different assay types; 5) Serpentine channels improving heating and magnetic separation characteristics; 6) A chamber and related features to enable storage of liquid and dry reagents in the cartridge; 7) The large number of chambers, also in the case of a relatively compact cartridge, thus enables a large number of different sample preparation schemes.

The main features of the library preparation cassette include, but are not limited to: 1) Two embedded rotary valves supporting complex assay flows, delivering and extracting fluids in a combination of multiple chambers and channels; 2) Multiple amplification chambers/stations enabling multiple reactions to be performed to accommodate complex workflows, diluted samples, high level multiplexing, etc.; 3) A chamber and associated features to accommodate tag/adapter addition; 4) A chamber and features housing copy control; 5) Separate but attached accessories or "fins" that can be used in complex operations but provide flexible cartridge designs between different assay types (one or more additional fins can be easily added); 6) Serpentine channels improving heating and magnetic separation characteristics; 7) A chamber and related features to enable storage of liquid and dry reagents in the cartridge; 8) A large number of chambers, also in the case of a relatively compact cartridge, thus enabling a large number of different library preparation schemes.

Key features of the cluster generation/sequencing cassette include, but are not limited to: 1) A fully integrated rotary valve that can draw and deliver fluid to a large number of chambers and channels; 2) In addition to rotary valves, there are on-board "select" valves that provide greater flexibility and options for fluid delivery; 3) A relatively compact large number of chambers that can support multiple forms of cluster generation and sequencing; 4) A chamber and related features to enable storage of liquid and dry reagents in the cartridge; 5) Full fluid connectivity to the flow cell for transporting and extracting fluid from the solid support (in a preferred embodiment, the solid support comprises a semiconductor chip; see, for example, fig. 29); 6) When selected as an option, full fluid connectivity to the kit (see disclosure elsewhere herein) is used to deliver a larger volume of the desired fluid, e.g., sequencing reagents (e.g., dNTP solutions) (see, e.g., fig. 30, 31, and 32).

A detailed description of one specific assay (in this case detection of one or more pathogens from blood) performed in the entire 3 subsystem cassette described above is given in example O "automated sample-responsive sequencing of whole blood labeled pathogens".

Integrated box

Also disclosed herein are exemplary designs of full-integration cassettes in which all steps of the workflow can be performed, including sample preparation, library preparation, copy control, cluster generation, and sequencing (see, e.g., fig. 33-39). When used in conjunction with kits and instruments (see elsewhere herein), the entire second generation sequencing workflow can be performed starting from the sample, thereby forming a fully automated format after the start of the run, without user intervention. This is done quickly in a relatively small-sized cassette and instrument system, making it useful in a variety of settings. This has not been achieved in the art so far, and thus represents a new and novel system and related workflow.

The integrated cartridge design includes several key features. The following is a brief discussion of some of these features. The three-dimensional design of the cartridge is selected to accommodate a large number of desired features in a relatively small space/volume. In fig. 33, two form factor options for such a cartridge are shown. Both provide good functionality in a relatively small volume. The design shown on the left provides a narrower form factor that reduces the instrument width without increasing the instrument depth. Rotating the STC fins and associated thermal interfaces inward also reduces the width of the instrument. The ergonomics of mounting the cartridge into the instrument are ideal choices for both form factors.

In some preferred embodiments of the cartridge, the fluid handling includes [ direct ] pneumatic pressure and precision pipetting using a compact 3 degree of freedom (DOF) stage located in the relevant instrument (see, e.g., fig. 40). Pipette tips can enter the chamber/channel in the cartridge through a Sealed Pneumatic Interface (SPI) port (see fig. 41). As shown in fig. 67 and 68, the SPI port may be configured with a cap 6701 to align the suction head with the SPI port. The inner diameter of the opening of cap 6701 will determine the tip size specified for the fluid transfer step (e.g., 1mL tip in FIG. 67 and 5mL tip in FIG. 68). The SPI port may include a stepped pipette tip interface 6703 as shown in fig. 67 and 68 to accept and seal pipette tips of different sizes. As shown in the exemplary design in fig. 33, the SPI valves are closely packed together to minimize the range of travel required for the 3-DOF bench, thereby reducing engineering complexity, cost, and use of fixed parts (REAL ESTATE) in the instrument. The pipette tip can enter the SPI valve through an opening in the cartridge directly above the valve (see figure). This opening is covered with a pierceable foil seal prior to use to prevent contaminants from entering the cartridge. Additional round holes are depicted for a temporary bench (holding) of a pipette tip used in the assay. The sample input ports (2 shown in fig. 33) are positioned to enable access on top of the cartridge. There are shown 2 cylinders containing (and other sample tubes) evacuated blood collection tubes, indicating that the system can accommodate a large number of input samples. Furthermore, each sample port includes a separate SPI, enabling access to the sample or combination of samples at any point of the assay. Furthermore, the sample port is located very close to the lysis chamber to minimize the travel path of the sample to the lysis chamber (if so provided) during the chemical workflow.

Fig. 34 depicts the features of the cartridge in more detail. As described elsewhere herein, the functionality associated with each feature in this exemplary diagram is based on a particular assay or assay type, but the functionality is flexible and readily re-usable, re-programmable, or otherwise adjustable to accommodate a wide variety of applications. The cartridge is designed as sub-module sections, each section having a specific function or a combination of functions. This is less complex and more cost-effective for the manufacture and loading of reagents. It also enables simpler improvements to cartridges for different applications (e.g., designing a new cartridge for an application with different sample preparation steps would only involve redesigning and manufacturing the sample preparation sub-module compared to the entire cartridge). The different sub-module portions can then be easily assembled into a single integrated box. It should also be noted that other advantages of using a pipette to deliver reagents include the ability to achieve simple sample dilution in the pipette tip, efficient mixing by moving reagents into and out of the chamber/channel (multiple times if necessary), pressure delivery into the chamber/channel (pipette tip inserted into SPI and "dispensing" air) to drive fluid movement, etc.

Fig. 35 to 39 depict some basic steps of the workflow/assay (e.g. pathogen detection in blood) as they will be performed on the cassette. The legend outlines these basic steps. As shown in the legend, not all of the cartridge components are shown in this figure for ease of viewing the features. Furthermore, not all steps of the workflow are summarized. It should be noted that the assay chemistry/reaction can be performed in the channel as well as in the chamber. It should also be noted here (as in the other parts) that the cartridge is designed to have a high degree of flexibility and to be able to perform all the processing steps of many different workflow configurations. Furthermore, as noted above, in some cases, one or more sub-module fins in the integrated cartridge may be interchanged to accommodate a wider range of workflows and applications.

Fig. 60 and 63-65 illustrate another exemplary assay or sample cartridge highlighting the primary fluid handling subsystem, cell, or "fin". FIG. 63 shows an exemplary library preparation unit 6300 or PCR fin with copy control functionality. Fig. 64 shows an exemplary sample input and mechanical lysis fin 6400, and fig. 65 shows an exemplary Specific Target Capture (STC) fin 6500.STC fin 6500 may include a hot zone for heating as needed during target capture and elution steps. In certain embodiments, thermal energy may be applied to the STC fins 6500 from one side (e.g., from inside the cartridge). Library preparation unit 6300 may include double sided heating for PCR and copy control thermal steps. The STC fin 6500 of fig. 65 is shown in more detail in fig. 69. STC chamber 6901 is used for sample preparation heating and mixing, and in the illustrated embodiment, the volume of the STC chamber may be about 9.3mL and is designed to accommodate volumes up to about 6 mL. Additional fluidic functions of STC fin 6500 are achieved using an elution chamber 6903 for elution heating and an auxiliary chamber 6905 (as applicable) that can be used for PCR 1 dilution, PCR 2 pooling, and copy control dilution. The lyophilized reagent bag 6907 may be positioned so as to be capable of being loaded and sealed with a membrane after fin construction. STC fin 6500 may include a condensate trap 6909 to contain any condensate formed during the heating and mixing steps in the STC fin. STC chamber inlet passage 6911 feeds STC chamber 6901 and maintains the fluid within the heating zone during the mixing and heating steps. STC inlet pneumatic line 6913 enables air to be dispensed through the STC inlet, e.g., pushing fluid back into STC chamber 6901 without the use of a pipette tip. An STC and elution serpentine 6915 is included for the magnetic bead capture step.

The exemplary library preparation unit 6300 or PCR fins of fig. 63 are shown in more detail in fig. 70. Various hot chambers 7001 for PCR thermal cycling and direct hybridization elution steps are included as needed. The hot chamber 7001 is positioned to enable heating from both sides of the cartridge within the instrument for faster thermal warming as required for PCR amplification. The lyophilized reagent bag 7005 may enable loading of the desired reagent after fin construction and then sealing with a membrane. A direct hybridization chamber 7003 may be included for mixing and incubation (at the indicated room temperature) for assays using the direct hybridization method. Direct hybridization magnetic coils 7013 for magnetic bead capture may also be included, as desired. Library preparation unit 6300 may include PCR2 bypass channel 7011 to enable PCR2 channel to be filled directly from a single SPI valve. A PCR optical sensor 7007 and a metering controller 7009 may be included for closed loop control, such as for fluid positioning and metering.

The sample input may include an opening or docking interface 6403 for receiving a sample container, such as a vacuum tube or vial, to enable the cartridge to receive a sample for measurement. In the case of assays using mechanical lysis (e.g., by rotating a paddle), a mechanical interface 6405 may be included that enables the instrument to drive the lysis unit by, for example, a motor and shaft that can be coupled to the interface when the cartridge is inserted into the instrument. The sample input, mechanical lysis, and STC steps may typically be combined in a sample preparation step, and these functions may be part of a sample preparation unit within the sample cartridge. After processing in the library preparation unit (as shown in fig. 63), the amplified nucleic acids may be directed to a sequencing unit comprising, for example, a flow cell as described herein for sequencing and analyzing isolated and amplified target nucleic acids from the original sample. An exemplary sequencing unit/flow cell 6600 is shown positioned within the sample cartridge in fig. 66. The sequencing unit/flow cell 6600 can be heated from below the cassette as required by any sequencing step. Further, fig. 66 shows an exemplary pipetting reservoir 6603 within an assay or sample cartridge. As described herein, various fluid transfer operations within a cartridge between a cartridge or other external source and the cartridge may be automated by the instrument using, for example, a pipetting rack and an SPI port as described below. By including the necessary pipette tips within the sealed box that are compatible with the required volume and SPI ports used in the system, ease of operation is increased while reducing the risk of user error or contamination.

C. Kit for detecting a substance in a sample

In a preferred embodiment, a separate kit is used in combination with the assay kit to perform a given test/assay. Exemplary designs of kits are depicted in fig. 42-44. The fluid may be accessed and moved in a variety of ways, including through a liquid handler (LH; e.g., a pipette system) and a Liquid Manifold (LM). Different valve setting methods may be employed, including sealing a pneumatic interface (SPI) port. The reagents stored in the kit may include liquid and dry reagents, assay specific and universal reagents, bulk reagents (typically requiring large amounts of reagents), and other reagents and components as desired (e.g., CO ₂ washed soda lime for the selected reagent). Furthermore, the body of the kit provides a relatively large volume that can be effectively used for waste solutions generated during the performance of the test/assay. Furthermore, the number of reservoirs/units/wells/chambers may be modified as needed to meet the requirements of a given or set of tests/assays. Furthermore, a given configuration of kit may be filled with various reagents that will support many different tests/assays, even if not all of the reagents will be used for each test.

The use of a separate kit has several advantages, including but not limited to: 1) Storing reagents on separate kits greatly reduces the size and complexity of the assay kit (especially when considering the volume required for bulk reagents (e.g., sequencing reagents)), thereby increasing the manufacturing efficiency of the assay kit and reducing manufacturing costs; 2) The assay and the kit can be manufactured, filled and stored independently, so that the complexity and the cost are reduced, and the efficiency is improved; 3) The preparation and storage of dry reagents is much more efficient when dispensed into the kit than the assay kit, particularly when part B of the storage kit is manufactured, filled, dried and isolated (for dry reagents; see fig. 43 and associated legend); 4) The preparation and storage of liquid reagents is more efficient when dispensed to the kit than to the assay kit, particularly when part a of the kit is separately manufactured, filled and stored (for liquid reagents; see fig. 43 and associated legend); 5) In some embodiments, the same kit may be used with different assay kits; 6) For a given test/assay specific application, the kit may be quickly assembled with the subcomponents (e.g., without limitation, filling, drying (as applicable) and storing separately of parts a and B; individually filled bulk reagents (e.g., for sequencing)) to enable most efficient fabrication and storage of subcomponent parts, as well as fabrication in volumes that meet demand; 7) The dried reagent may be reconstituted directly in the kit prior to transfer to the assay kit; 8) In preferred embodiments, most of the reagents in the kit are in dry form, such that most of the liquid in the kit (in some embodiments all of the liquid) is water; 9) In a preferred embodiment, one or more chambers in the kit include a magnetic stirring bar that interfaces with a magnetic stirring motor in the instrument (e.g., for on-board preparation/mixing of reagents) when the kit is loaded into the instrument. Another exemplary kit is shown in fig. 61.

D. Instrument for measuring and controlling the intensity of light

Various cartridge embodiments are disclosed in section (e) above and elsewhere herein. This section (and elsewhere herein) discloses instrument embodiments that can be used to automatically perform tests/assays within the cartridge. Examples of instrument designs and features are given in figures 40, 45 to 49 and the associated legends. The design is such that the space taken up by the instrument is relatively small and therefore can be used in many settings. Furthermore, it is capable of automatically performing all the features required for a complete, complex workflow from sample input to final reporting, including nucleic acid sequencing in the preferred embodiment, in the form of a cassette, all without user intervention once run has been initiated. In a preferred embodiment, the instrument is equipped with a compact 3 degree of freedom (DOF) pipette stage with which to deliver to the cartridge and perform various functions including mixing, dilution, reconstitution (drying of reagents) and moving fluids/reagents.

In a preferred embodiment, the pipette tips enter the chamber and channel via SPI ports, as described elsewhere herein. In a preferred embodiment of the cartridge, the SPI valves are closely packed together to minimize the range of travel required for the 3-DOF gantry, thereby reducing engineering complexity, cost, and use of fixed parts in the instrument. The instrument design includes one or more of the following functions: heating, cooling (including CPU), magnetic separation, magnetic stirring, lysing impeller rotation (for mechanical lysing in the cartridge), generation and control of use of pressurized gas (pneumatic system), detection (e.g., temperature, pressure, flow, liquid level, etc.). It also includes complete CPU/computer control of functions and features, including collection and analysis of output data, such as sequencing signals from semiconductor chips including ISFET arrays. Other features are highlighted in the figures and associated legends.

Another exemplary instrument and its components are shown in fig. 58A-59. Fig. 58A shows a perspective view of an exemplary instrument having a display/user interface, a bar code scanner, and a cartridge door. The dimensions of an exemplary instrument are shown in front and side views of fig. 58B and 58C. The layout of the various internal subsystems of the exemplary instrument is shown in fig. 58D-58I. A cartridge interface assembly for receiving and interfacing with a sample or assay cartridge and a kit is shown in fig. 58D. The cartridge interface may include doors that can be opened or closed manually or automatically to enable a user to insert the cartridge, but to enable a closed controlled environment for assay processing to be created after insertion. In various embodiments, the cartridge interface may include fluidic and electronic connections to enable the instrument to control fluid movement within the cartridge and to communicate with the cartridge and various units therein (e.g., control sequencing and receive sequencing data for processing). In certain embodiments, the fluid control may be pneumatic. Fig. 58E shows an exemplary pneumatic pumping subunit positioned within the instrument for providing pneumatic pressure to be controlled by the analyzer or instrument to drive fluid movement within the cartridge. Such a pneumatic subsystem is further illustrated in fig. 59, and may include syringes of various sizes (e.g., macroscopic and microscopic) to enable large volume fluid movement and fine control. Fig. 58F illustrates the positioning of an exemplary power subunit for providing power to an instrument. Fig. 58G shows an exemplary air handling and cartridge intake subsystem for controlling and handling any air entering the instrument and cartridge. Fig. 58H illustrates a liquid cooling subsystem for providing thermal management, e.g., to cool a processor or other heat generating unit within an instrument. FIG. 58I illustrates the positioning of an exemplary condensation management subsystem for further controlling the environment within the system.

8. Methods of using the system

As described elsewhere herein, prior art sequencing workflows (the process required to prepare target polynucleotides contained in a sample for sequencing, perform sequencing, and analyze the resulting data) are tedious, time consuming, complex, and costly. Many of the steps are still performed manually and require highly skilled personnel. Even if a particular process in a workflow is automated, multiple instruments and auxiliary components are required and skilled human intervention at various points is required to complete the entire workflow. In addition, the time from the sample to the result is several hours to several days or more. Furthermore, the maximum allowable amount of sample input is low, which represents a further current limitation. Thus, the ability and value of sequencing (including second generation sequencing) may be greatly reduced in practical use. Thus, there is a need for a fully automated (sample-to-report) sequencing workflow that, once started to run, does not require user intervention, is fast (samples give disposable results in a few hours), is sensitive, accurate, cost-effective, and can be used at any time when needed.

The embodiments disclosed throughout can be used to rapidly analyze target polynucleotides, including determining their nucleotide sequences, from a wide range of input sample types and amounts using an automated system. As also disclosed elsewhere herein, the system includes an instrument and at least one cartridge removably insertable into the instrument. In a preferred embodiment, the system further comprises at least one kit removably insertable into the instrument. In still further preferred embodiments, the system includes a semiconductor chip, wherein in some embodiments the chip is embedded within the cartridge. In a particularly preferred embodiment, the system further comprises a flow cell mounted on top of the chip (either porous or non-porous), which can be used to deliver fluid to and remove fluid from the chip. In a preferred embodiment, the system comprises software. In a particularly preferred embodiment, the software includes operating software (for the control system) and analysis software (for receiving, processing and analyzing the output of the system).

A number of methods of using the above system are disclosed in this section. These are exemplary and are not meant to limit the scope of potential methods and related applications. The following "examples" section discloses specific embodiments of some of these methods implemented using the system.

A. exemplary general workflow overview

As described above and elsewhere herein, a workflow refers to the process required to prepare a target polynucleotide contained in a sample for sequencing, perform sequencing, and analyze the resulting data. More specifically, preferred embodiments may include one or more steps (in various combinations); sample processing; preparing a library; copy control; generating clusters; sequencing; collecting data; primary, secondary and tertiary data analysis; metering calls (answer questions tested/tested to answer); report generation. For the challenges that have not been addressed so far, the disclosed cartridge or cartridges and instrument present a new and novel solution to complete end-to-end automation of all steps of a workflow in rapid time, within the confines of the relatively small instrument (including the cartridge) space occupied.

As a first step, a sample containing the target polynucleotide is introduced into the cassette. In a preferred embodiment, this is achieved through a sample input port on the cartridge. Furthermore, the cartridge is equipped with features built into or attached to the cartridge to facilitate transfer of the sample in a safe, efficient and pollution-free manner. For example, in some embodiments, the cartridge is provided with a cylindrical structure into which a tube containing the sample (e.g., a standard vacuum blood collection tube) is inserted. In a preferred embodiment, placed at the bottom of the cylinder is a needle that is in fluid contact with at least one chamber in the cartridge through an input port.

In these embodiments. For example, the sample tube may be placed in a cylinder from top to bottom and pushed onto the needle so that the needle penetrates the cap (forming a tight seal around the needle and maintaining a tight seal between the cap and the tube) and the contents of the tube or a portion thereof is transferred into the cassette. Additional exemplary components that help facilitate sample transfer include, but are not limited to: luer lock (luer lock); an external chamber/container (which is in fluid connection with at least one chamber in the kit through a port), into which a liquid sample can be introduced (e.g., by pipetting), then sealed (e.g., via a cap), and the contents of the chamber/container transferred into the cartridge when the cartridge is inserted into the instrument; a pierceable septum through which a sample may be inserted into the cartridge via a syringe equipped with a needle or the like that pierces the septum; in some cases, untreated samples are loaded into a kit. In some cases, the sample is subjected to a user-selected pretreatment (e.g., treatment performed in a sample collection tube, as a standard practice prior to analysis, or treatment performed conventionally for certain sample types). In some cases, the sample is the output of other methods selected by the user, such as the output of the following methods: cell culture, cloning and expression, amplification, nucleic acid extraction, expression of a swab in a transport medium, liquefaction/homogenization of a solid sample, concentration of a sample medium (including a sample in a gas such as air), cell lysis, separation (e.g., phase separation, precipitation, centrifugation, fractionation (e.g., separating whole blood into plasma or serum, buffy coat and red blood cells), filtration, etc., further non-on-board conventional processing methods include lysis of organisms, e.g., lysis of chlamydia reticulum in a lysing agent-based liquid (e.g., as a transport medium in a collection tube), lysis of organisms by vortexing a sample containing organisms in the presence or absence of beads, lysis by freezing/thawing in a collection tube, the external components (if used)/sample entry port/cartridge can accept a wide range of sample input volumes, such as a few microliters to one milliliter in some cases, 2-10mL in some cases, 4-20mL in some cases, and even higher volumes Dynamic fluid control, "on-the-fly" processing as fluid flows through/circulates/passes through a processing element (e.g., heater, magnet, etc.), excellent mixing capability, etc. This unique ability to accommodate such a wide range of sample volumes (including relatively high volumes) meets the need in the art and distinguishes the present disclosure from the prior art.

In some embodiments, once loaded into the cartridge, the sample is acted upon by various possible processing steps to prepare it for further downstream processing and/or analysis (AKA, sample preparation). In other embodiments, such as where the sample matrix itself is relatively uncomplicated and the target polynucleotide is already in a form that can be further processed and/or detected, the sample may bypass the sample processing step and be transferred to a subsequent step in the overall workflow, such as library preparation. In some tests/assays, the sample type is whole blood and the target polynucleotide is inside a cell, e.g., a pathogenic organism (e.g., sepsis) that infects the blood. In a preferred embodiment, sample preparation comprises the following general steps/procedures: 1) The blood is mixed with reagents that support sample homogenization and cell lysis. 2) The sample is heated (with or without continuous movement and/or mixing). This helps to dissolve the sample (where in another preferred embodiment the method includes turbulent mixing; features in the chambers/channels of the cartridge may optionally be included to improve turbulent mixing, e.g. three dimensional features such as pillars between mixing chambers, narrow junctions etc). In some further preferred embodiments, the reagent comprises an enzyme, such as proteinase-K, which enzymatically breaks down the components of the sample (for proteinase-K, heating helps to activate the enzyme). 3) Cells were lysed. This may be accomplished in a variety of ways (examples listed elsewhere herein). In a particularly preferred embodiment, the lysis is achieved using mechanical lysis, including mixing the sample at a relatively high speed in the presence of beads. The cartridge is uniquely designed to include high capacity mechanical lysing chambers equipped with impellers that interface with motors in the instrument when the cartridge is loaded into the instrument. 4) The nucleic acid is released and denatured. Depending on the cell type and the exact lysis method, the target polynucleotide may still be associated/bound/captured by the cell and/or features in the sample. Furthermore, the target nucleic acid may be in double stranded form and must be rendered single stranded (denatured) in order for the next step of the process to function properly. In a preferred embodiment, both release and denaturation are achieved by heating to a relatively high temperature (e.g., about 95 ℃). These processes may be further aided by reagent compositions, for example, comprising one or more lysing agents, chaotropes, or denaturing agents (or combinations thereof). the substance(s) may be included in the lysis reagent or added after lysis. In addition, the whole lysate may be heated to a relatively high temperature by heating the whole lysate as a whole or by heating a portion of the lysate at one time, for example by flowing it through a heated channel, such as a serpentine channel. 5) Isolating the target polynucleotide. A preferred embodiment is a target capture of one or more specific target capture oligomers (specific target capture or STC). The denatured lysate is mixed with hybridization reagents containing capture oligomers, and the mixture is incubated at an elevated temperature (e.g., at about 60 ℃) during which the capture oligomers anneal to one or more target polynucleotides at a user-defined level of specificity (by the capture oligomer design). In some embodiments, the lysate is mixed with a hybridization reagent containing capture oligomers prior to release/thermal denaturation. The sample is then heated to about 95 ℃, e.g., for release/denaturation, and then the temperature is reduced to about 60 ℃, e.g., for annealing of the capture oligomers. In a preferred embodiment, after annealing, the target polynucleotide/capture oligomer complex is captured onto magnetic microspheres, the microspheres are collected from the mixture using a magnet, and the remaining lysate (which is sent to waste in the cassette) is removed. Exemplary steps of how this can be achieved are included elsewhere herein. The beads are then optionally washed and the target polynucleotide eluted. the target polynucleotide is now ready for further downstream processing and/or analysis.

The sample preparation embodiments described and discussed herein above and elsewhere have a number of advantages that distinguish them from the prior art, including but not limited to: 1) The entire sample preparation workflow, including starting from a relatively large volume of complex biological sample (e.g., blood), is performed in a fully automated form in the form of a cartridge, wherein the cartridge is an integrated cartridge, also for other processes of testing/assaying (i.e., the prepared sample need not be transferred to a different cartridge, a different device, etc. to continue the process); 2) Complete mechanical lysis is completed on the cartridge; 3) Complete release and denaturation of the target polynucleotide, including heating/incubating the sample as it comes into contact with the active heating element through a channel (e.g., serpentine channel), can be achieved on the kit in a short time, even in the case of large volumes, by using the excellent heating techniques of the cartridge-based system; 4) The specific target capture configured in the disclosed cassette compositions provides a number of advantages per se, including high purification efficiency, high volume reduction capability, high specificity, and even more broadly high specificity control (can be captured specifically along the phylogenetic tree, including at the subspecies, species, genus, family, etc.) by user-defined oligomer design; furthermore, the oligomer may be designed to specifically exclude unwanted polynucleotides such as human DNA in the sample; 5) The STC method has high scalability and supports high level multiplexing; furthermore, the STC method is very flexible in that new applications/assays can be easily and quickly developed by simply designing new oligomer sets; 6) Only 1 purification step is included in the overall process (sample preparation for second generation sequencing typically includes multiple purification steps, which increases time, complexity, and cost); 7) The entire elution volume of the isolated target polynucleotide can be passed directly to the next step in the process (e.g., library preparation; for a typical second generation sequencing workflow, quantification of the target polynucleotide is required at this step and only a small fraction of the prepared target enters the library preparation process).

In some embodiments, after sample preparation (or in some other embodiments, the sample or portion thereof is used directly, as described elsewhere herein), the sample is subjected to various possible processing steps to prepare it for further downstream processing and/or analysis (commonly referred to as library preparation). In other embodiments, the sample may bypass the library preparation step and be transferred to a subsequent step in the overall workflow, such as cluster generation or sequencing. As described above, in some tests/assays, the sample type is whole blood and the target polynucleotide is inside the cell, e.g., a pathogenic organism (e.g., sepsis) that infects the blood. In these cases, the input to library preparation is target polynucleotides prepared from cells within a whole blood sample (examples of preferred sample preparation methods outlined above). In a preferred embodiment, library preparation comprises the following general steps/procedures: 1) The input sample is mixed with a first amplification reagent. In a preferred embodiment, the first amplification reagent is stored as a dry reagent on the cartridge (in an assay or kit) and reconstituted on the cartridge (using a liquid, such as water, stored on the cartridge (assay or kit)). 2) Amplifying a region of interest (ROI) in a target polynucleotide in a first amplification reaction. This increases the copy number of the ROI in the sample and increases the relative abundance of the ROI as compared to other polynucleotides and/or polynucleotide regions in the mixture. In a preferred embodiment, the primers are designed to amplify a broad range of pathogenic organisms (if present in the sample). For example, depending on the requirements of the test/assay, the primers are designed to amplify any bacteria or fungi, or a subset of the various bacteria or fungi, in the sample. This enriches these bacterial and/or fungal ROIs with respect to human sequences or other potentially interfering sequences. In some embodiments, one or more tags/adaptors are introduced to one or more amplicon products. One exemplary amplification method is PCR. 3) The products of the first amplification reaction (AKA, PCR1 amplicon) were diluted. 4) An aliquot of the diluted PCR1 amplicon is amplified in a second amplification reaction. An exemplary amplification method is PCR (e.g., PCR2 in this case). in a preferred embodiment, at least one primer is nested compared to the corresponding primer in PCR 1. This adds another layer of specificity. In another preferred embodiment, more than one second amplification reaction is performed (e.g., PCR2.1, PCR2.2, PCR2.3; 2-10 or more second amplification reactions may be performed on a cassette). During the different second amplification reactions, each second amplification reaction may use a different set of primers than in the first amplification reaction (to cover a broad range of target polynucleotides), or use the same set of primers as in the first amplification reaction (e.g., to make more final products), or any combination thereof. It should be noted that the first amplification reaction and/or one or more second amplification reactions may be configured to amplify 2 or more targets (i.e., multiplexed amplification). In some embodiments, one or more tags/adaptors are introduced to the amplicon product of one or more second amplification reactions. 5) All amplicon products of the second amplification reaction were pooled together. 6) In some embodiments, the pooled product from the second amplification reaction is purified directly (while in other embodiments the copy control process is performed first; see below). Mixing the output solution with a target capture reagent, incubating the mixture, binding the desired amplicon to paramagnetic beads, immobilizing the beads using a magnet, washing the beads and eluting the target amplicon. A detailed description of one such procedure is given in example O. In some embodiments, the amplicon is labeled with biotin (e.g., by biotinylated primers). In such embodiments, the target amplicon is bound to streptavidin-coated magnetic beads during the incubation steps listed above. In some embodiments, the amplicon is equipped with a universal tag (common tag) sequence, and a capture oligomer complementary to the universal sequence is designed. In some embodiments, the capture oligomer is biotinylated. In such embodiments, the biotinylated capture oligomer is bound to an amplicon comprising the universal tag sequence in a first incubation step. The reaction mixture is then combined with a second capture reagent comprising streptavidin coated magnetic beads and the resulting mixture is incubated in a second incubation reaction during which the amplicon/capture oligomer complexes bind to the magnetic beads. In some embodiments, all components of the first capture reagent and the second capture reagent are in a single capture reagent, and the first incubation and the second incubation are combined into only 1 incubation.

As described above, in some embodiments, a Copy Control (CC) scheme is performed on pooled PCR2 amplicons. Acceptable CC schemes for use in the disclosed invention include one that may employ any of the CC schemes disclosed in PCT/GB2021/050098 (various combinations of the disclosed features are also contemplated). A detailed description of various CC combinations and methods is disclosed elsewhere herein. In a preferred embodiment, the pooled output from the PCR2 reaction is mixed with CC reagents comprising one or more CC oligomers. The mixture is then heated at about 92-95℃to denature the target amplicon. The mixture is then heated to facilitate annealing of the CC oligomer and complementary sequences (if present) and extension of the CC oligomer and target amplicon (3' end of the strand hybridized to the CC oligomer; examples section, elsewhere herein, and examples of various heating schemes and compositions and reaction schemes are given as described in PCT/GB 2021/050098). The resulting mixture is then mixed with a capture oligomer and incubated to promote hybridization with the capture sequence incorporated into the target amplicon/complex. The resulting mixture was then mixed with magnetic beads, the bead/target complex was immobilized using a magnet, the beads washed and the target eluted. In some embodiments, some, most, or all of the copy control process is performed simultaneously with the first amplification and/or one or more second amplification reactions.

The library preparation embodiments (including copy control) described and discussed above and elsewhere herein have a number of advantages that distinguish them from the prior art, including, but not limited to: 1) In embodiments where library preparation input samples are the output of the sample processing methods disclosed therein, the sample processing output material may be used directly without the need for quantification or other characterization. In some preferred embodiments, the entire volume of the sample processing output is used as input, while in other embodiments a portion of the output is used as input. In some embodiments, an initial sample of a portion thereof is used as an input material for library preparation. 2) The system is capable of quickly and efficiently mixing an initial input sample with a desired reagent and other combinations of reagents throughout the process. 3) The targeted enrichment method provides a high degree of leverage to achieve the selectivity and specificity required for a wide range of test/assay applications (two sequential amplification reactions, nested start-up being one option in the second amplification, and seamless integration of the two reactions (including full-automatic dilution, aliquoting and partitioning of aliquots into separate reaction chambers) as well as flexibility (e.g., in some embodiments, only 1 amplification reaction is performed; in some embodiments, each separate second amplification reaction is configured (e.g., by primer design) to produce a different selectivity, specificity, target set, etc.). 4) High multiplexing capability. 5) Great flexibility and the ability to add tags/adaptors. For example, the tag/adapter may be added by any combination of labeled primers (e.g., in some embodiments, PCR1 and multiple PCR2 reactions are performed simultaneously; each reaction may be multiplexed, and each primer or primer set may include the same or different tag as another primer or primer set). In other embodiments, tags/adaptors can be added by ligation (in a fully automated system). In other embodiments, the tag/adapter is added by ligation and using a combination of tagged primers. In addition, tagging may be combined with the copy control process to a predetermined degree, further simplifying and simplifying the overall process, thereby reducing workflow complexity and processing time. 5) The system (cartridge + instrument) provides a new method of diluting, aliquoting and partitioning a reaction mixture (e.g., an amplification reaction, such as PCR 1) into a plurality of new reaction mixtures (e.g., a second amplification reaction, such as PCR 2). 6) In a preferred embodiment, the entire process utilizes only a single purification step (e.g., "direct purification" as used downstream in the disclosed cluster generation direct hybridization method, purification as part of a copy control process, etc.). Furthermore, there is no need to pre-analyze (e.g., quantify) the input sample (e.g., from various disclosed sample preparation processes) or the first amplification reaction and its dilution. These features distinguish the disclosed methods from the prior art, which requires multiple purification steps and [ often ] pre-analytical steps. 7) The disclosed copy control process is novel and clear and fully automated using the disclosed system. 8) The desired reagents can be stored and ultimately reconstituted (if desired) and mixed with other reagents in assays and kits in various combinations and methods to provide unique and efficient methods/paths for reagent storage and use. 9) The whole process is fully integrated and automated through the system. 10 The process is rapid.

In some embodiments, after library preparation (with or without copy control), the sample is clustered on the surface of the solid support. In other embodiments, the sample may bypass the library preparation step and move directly to cluster generation. In a preferred embodiment, the cluster formation is performed on the surface of the semiconductor chip. In a further embodiment, the semiconductor chip comprises an array of ISFET sensors. In still further embodiments, the surface of the semiconductor chip includes holes. In some embodiments, cluster generation includes direct hybridization of target polynucleotides to specific target capture oligomer arrays on the surface of a solid support. In some embodiments thereof, when the analytical method is sequencing, the capture oligomer may also be used as a sequencing primer. An exemplary method for generating clusters using the direct hybridization method (in this case, followed by sequencing) is outlined in example M. In other methods, cluster generation includes clonal amplification. An exemplary method for clonal amplification using Recombinase Polymerase Amplification (RPA) is outlined in example J. An exemplary method for clonal amplification using Rolling Circle Amplification (RCA) is outlined in example K.

The cluster generation embodiments (including copy control) described and discussed above and elsewhere herein have a number of advantages that distinguish them from the prior art, including, but not limited to: 1) The output of the library preparation step (with or without copy control) can be used directly in the cluster generation method. In some embodiments, the output material (solution) is moved directly into a flow cell that covers the solid support. In this case, the volume of the flow cell will dictate how much output solution is used in the cluster generating step. The output solution does not require any additional operations such as quantification, dilution, aliquoting, etc. 2) In a preferred embodiment, cluster formation occurs directly on the surface of the semiconductor chip, on which surface sequencing is also performed. This was not found in the prior art. 3) As described and discussed elsewhere herein, the disclosed methods of clonal amplification using RPA are novel and distinct from the prior art in the field; 4) As described and discussed elsewhere herein, the disclosed methods of circular template formation (starting from the library preparation stage) and clonal amplification using RCA are novel and distinguished from the prior art in the field; 5) The whole process is fully integrated and automated by the system. 6) The process is rapid.

In some embodiments, the target nucleic acid is analyzed by sequencing after cluster generation. In a preferred embodiment, sequencing is performed on the surface of the semiconductor chip. In a further preferred embodiment, the semiconductor chip comprises an array of ISFET sensors. In a still further preferred embodiment, the surface of the semiconductor chip comprises holes. Exemplary methods of sequencing using semiconductor chips comprising an ISFET sensor array and further comprising wells on the surface are summarized in example L (sequencing of synthetic templates directly immobilized on the chip surface), example M (sequencing of synthetic templates using direct hybridization methods), example N (sequencing of templates using in-well clonal amplification), and example O (automated sample-to-answer sequencing of pathogens added to whole blood; the entire workflow, starting from whole blood sample, to sequencing and end of result analysis).

Many of the advantages of semiconductor sequencing are well understood and demonstrated in the art. These advantages include speed (faster than sequencing for most sequencing-by-synthesis methods), no need for modified/labeled nucleotides, no need for optical detection, fewer nucleotides and wash fluids per given length of sequence, etc. The sequencing embodiments disclosed herein have a number of additional advantages, including, but not limited to: 1) In a system comprising a cassette and an instrument, the entire process including cluster formation on the surface of a semiconductor chip and all sample and library preparation steps (if necessary) prior to cluster generation are fully automated. 2) All reagents required for sequencing are contained in a disposable kit and all waste solutions are contained in the kit after the sequencing run. This provides for simple and efficient use and safe disposal of the used components after operation. 3) The disclosed method of simultaneous sequencing primer annealing and sequencing enzyme binding provides simplicity and reduced run time. 4) The disclosed methods of critical sequence introduction and use provide a new method for calibrating sequencing runs and the like. 5) The system enables on-board pH titration of reagents for sequencing, which is important for ISFET-based sequencing.

Further embodiments of the invention include on-board computers and auxiliary/electronic devices and software. The use of computers and software includes control of the system and collection and analysis of signals/data generated by the system.

Exemplary user interface steps for a generic workflow are shown in fig. 62. First, a user may scan various information into the instrument using a bar code scanner in front of the instrument. For example, the ID identification may be scanned to register the user or grant access to certain assays/functions. User information may be stored with any subsequent assay data to control access and/or for quality control or analysis purposes. The sample or sample container may be scanned to read and record sample data for the assay. For example, patient information related to a blood sample may be encoded on a bar code on a evacuated blood collection tube, and the system may correlate any subsequent measurement data with a particular sample or patient by scanning with the instrument prior to entering the sample or measurement cassette. The user may then scan the kit, which may provide information to the instrument about what assays are to be performed. This may eliminate user errors by automatically generating an assay workflow for the desired assay, or may be used as a quality control check to ensure that the correct suite is used for the user-selected assay entered in the user interface. The user may then remove one or more cartridges from the package and prepare them for insertion. In certain embodiments, the only user operations involved in the assay are opening the cartridge, removing the sterile foil covering any ports, and loading (loading) the sample, after which the cartridge is inserted into the instrument for automated processing of the assay. For example, a user may insert a evacuated blood collection tube into a sample cartridge through a sample input subunit specifically designed to receive a particular size/type of sample container. The cassette may then be loaded into the instrument, for example, through an automatically opened door. The instrument may then run the desired assay automatically and provide the results to other computers through a user interface (e.g., a display on the instrument) or through a network. The instrument can then eject the cartridge containing all the waste therein. Thereby, the risk of user error and any contamination is reduced and the instrument is ready for the next test without cleaning or refilling of the on-board reagents, since all fluids are contained within the cartridge.

B. options in a generic workflow

The design of the system has unprecedented processing power and flexibility for performing tests/assays, including in the field of molecular testing, including sequencing. A single integrated system includes one or more cartridges with many different sized and functional chambers therein, with a high degree of fluidic connectivity, with a large volumetric capacity, with novel and diverse means of moving, transporting, combining, mixing, reconstituting, and otherwise processing liquids (as well as gases and solids, such as when reconstituting dry, vitrified, or other solid reagents or components), with means of heating, magnetic separation, agitation, mixing, sensing, titration (e.g., pH titration), detection, and analysis. All of which are controlled by an on-board computer and which may be subjected to a number of process steps in different sequences, durations, conditions (e.g., temperatures), etc. In this case, a wide variety of different tests/assays may be run, and the different steps of each test/assay may be selected in a wide variety.

Many sample preparation options are possible within the scope of the invention. In some samples, for example, when the sample is too viscous, non-uniform, fibrous, gelatinous, the initial sample loaded into the instrument may require an initial processing step. Examples of methods for initial processing of a sample within the scope of the present invention include, but are not limited to: mixing with reagents, including turbulent mixing, zig-zag mixing (tortuous mixing), stirred mixing, etc., including mixing with reagents such as lysing agents, denaturing agents, chaotropes, organic solvents, buffers, salts, etc., and various combinations thereof; and/or enzymatic digestion, e.g., with proteinase K (as described in the general workflow above) and/or other proteases, nucleases, lipases, etc.; and/or compounds including dithiothreitol, beta-mercaptoethanol, other reducing agents, oxidizing agents, acids, bases, and the like. During the initial processing step, the sample may be mixed, heated, sonicated, etc. In some samples, no initial processing is required and the sample can go directly to the next step. In some whole blood samples, whole blood loaded into the cassette is separated into plasma and other components of whole blood using a separation system that is integrated into the cassette and that works in a seamless manner within the system. It is contemplated that all of the above can be implemented in the context of the disclosed system.

In some samples, the target polynucleotide is contained within the cell, including within the nucleus and/or other structures. Within the scope of the claimed invention, examples of methods for cell lysis/release of target polynucleotides include, but are not limited to: mechanical lysis (as described above), the presence or absence of beads (e.g., bead impingement), sonication, heating, mixing (e.g., turbulent mixing), shearing (e.g., by passing it through a small hole), and the like. Each of these processes may also include the action of reagents mixed with the sample that participate in the lysis mechanism (lysing agents, denaturing agents, solvents, etc.; see above for further options). In the case where the target polynucleotide is included in the nucleus of the cell, gentle lysis of the outer membrane of the cell may be performed first, and the contents of the cell separated from the nucleus, followed by lysis of the nucleus using a more stringent method. In some cases, the sample need not be lysed (e.g., cells are lysed before the sample is loaded into a cassette, cells are lysed in an initial processing step, or target nucleic acid is not in cells in the sample, etc.). In these cases, the sample may go directly to the next step in the process. It is contemplated that all of the above can be implemented in the context of the disclosed system.

Other protocols for use when the target polynucleotide is contained within a cell (including the nucleus) and/or other structure include protocols for capturing intact cells (e.g., affinity capture by use of magnetic beads). The remainder of the sample was washed away and the cells were lysed (example of lysis method given above). It is contemplated that all of the above can be implemented in the context of the disclosed system.

In some embodiments, the target nucleic acid must be denatured (e.g., double-stranded to single-stranded) and/or released from association with other structures (e.g., DNA entangled around the histone). Methods of denaturation include heating, treatment with reagents (chemical and/or biological), mixing, sonication, and the like. In some cases, this is not required and the sample can go directly to the next step in the process. It is contemplated that all of the above can be implemented in the context of the disclosed system.

In some cases, the target polynucleotide is isolated. Examples of methods for isolating a target polynucleotide within the scope of the claimed invention include, but are not limited to: 1) Specific Target Capture (STC) as described elsewhere herein. This includes limited target capture as described elsewhere herein, in PCT/GB 2021/050098; 2) Non-specific capture methods, such as solid phase extraction methods, examples of which include, but are not limited to, solid Phase Reversible Immobilization (SPRI), solid Phase Microextraction (SPME), silica-based methods, including the Boom method, the AMPure method, and the like; 3) A combination of non-specific capture (e.g., solid phase extraction) and specific target capture techniques (as described herein, first the Boom process, then STC); 4) The target enrichment strategy is mixed, as in Agilent SureSelect, which uses an RNA capture probe or "decoy" to pull down the region of interest. There are many different such hybridization-based methods, as well as other strategies for targeted enrichment, including, but not limited to, transposon-mediated fragmentation (labeling), molecular Inversion Probes (MIPs), and focused amplification procedures (focused amplification procedures) (e.g., as described elsewhere herein). It should be noted that many of these procedures utilize a fragment nucleic acid target. It should also be noted that many of these procedures can be used in conjunction with or after library preparation, but are described herein in the context of methods that can be used in the sample preparation stage in the disclosed systems (they will also be mentioned in the library preparation section). It should also be noted that the first or early step of amplification may be performed on target polynucleotides captured on beads (in sample preparation, library preparation, copy control and cluster generation). It is contemplated that all of the above can be implemented in the context of the disclosed system.

In some cases, other processes may be performed during the sample processing stage of the workflow within the scope of the claimed invention, examples include, but are not limited to: 1) Fragmenting a target polynucleotide; 2) The target polynucleotide is tagged with a tag/adapter, for example, by annealing and extension (during sample preparation and/or library preparation) or by ligation to the target polynucleotide. 3) The unique molecular identifier may be incorporated into some or all of the target polynucleotides in the sample. In some cases, the target polynucleotide is already in the form of fragments and is also a very short piece in the sample, such as fragment DNA in urine, circulating tumor DNA (ctDNA) in blood, cell free DNA (various sample types), small RNAs (various sample types), and the like. Methods of processing these polynucleotide samples may be performed on the system, including but not limited to, adding tags/adaptors, amplifying, recombining, capturing, and the like. It is contemplated that all of the above can be implemented in the context of the disclosed system.

Many options for library preparation are possible within the scope of the claimed invention. In some embodiments, the input to the library preparation process is the output of the sample preparation method, and in some embodiments, it is the initial sample itself. Other sections herein describe preferred embodiments of the amplification-based methods of targeted enrichment. Variations of these methods contemplated for use in the disclosed systems include, but are not limited to: 1) One or three (or more) separate amplification reactions; 2) As part of the copy control process, tags/adaptors introduced in a broad context (only in one of the amplification reactions, in a combination of amplification reactions, differentially between ends of the same target polynucleotide and from target polynucleotide to target polynucleotide (as described elsewhere herein and in PCT/GB 2021/050098), as a combination of processes in the sample preparation stage (see above) and library preparation stage), and the like. Amplification can be accomplished using a variety of methods known in the art, including, but not limited to, polymerase Chain Reaction (PCR), reverse transcription PCR, nicking Endonuclease Amplification Reaction (NEAR), transcription Mediated Amplification (TMA); loop-mediated isothermal amplification (LAMP); helicase Dependent Amplification (HDA); clustered Regularly Interspaced Short Palindromic Repeats (CRISPR); strand Displacement Amplification (SDA); recombinase Polymerase Amplification (RPA), ligase Chain Reaction (LCR), and the like. Numerous other library preparation methods are known in the art, and it is contemplated that these methods can be used in the disclosed systems. Several examples of general workflow include, but are not limited to: 1) Fragmentation, adaptor ligation, amplification (typically including additional adaptor addition); 2) Fragmentation, amplification (random, semi-random, specific; may include adapter addition), adapter addition; optional amplification (typically including additional adaptor addition); 3 amplification (which may include the examples listed above as well as whole genome amplification (e.g., picoseq, DOPlify, REPLI-g (multiple displacement amplification based, or MDA) and Ampli-1 WGA), long-range PCR, amplification using semi-random and/or degenerate primers, etc.), fragmentation, adaptor addition, optional amplification (typically including additional adaptor addition); 3) Transposon mediated fragmentation (tagging); 4) Methods based on Molecular Inversion Probes (MIPs); etc. After completion of several of the library preparation techniques described above, targeted enrichment can be performed (some of the techniques listed above, such as SureSelect). As described above, these steps of the library preparation and sample preparation processes may be overlapped and performed on different units of the cassette. Also as described above, UMI may be added at various stages of the sample preparation and/or library preparation process (where the exact protocol is dependent on the process). It is contemplated that all of the above can be implemented in the context of the disclosed system.

Copy control may or may not take place after library preparation, overlapping the library preparation process (depending on the application). In some cases, the tags/adaptors used in the copy control process can even be introduced during sample preparation. Various novel copy control methods have been described. In addition, other copy control methods known in the art may also be performed on the system.

Cluster generation methods have been described herein. Other methods may also be used, including the use of different surfaces and/or surface geometries, different surface fixation chemistries, and different amplification methods. We have demonstrated that other methods of supporting cluster generation in the claimed invention include PCR, HDA, SLAM (a proprietary surface phase amplification procedure; patented) and EM-Seq (a proprietary displacement mediated amplification procedure; patented).

Sequencing on a semiconductor chip can be performed on any type of sequencing library (see examples above) using essentially any targeting strategy, i.e., not just sequencing amplicons from, for example, targeted enrichment. In addition, sequencing can be performed after cluster generation using direct hybridization (as described above) to any number of targets and target sources, not just those used as examples. High density arrays of capture oligomers with varying degrees of specificity can be applied to the surface of the chip and employed in the method.

A variety of data analysis methods have been developed, including analysis by triangulation (triangulation strategies are also used for oligonucleotide design, including STC, targeted enrichment, and key sequence development).

Definition of the definition

It is to be understood that this disclosure is not limited to the particular compositions or method steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," "the," and "the" include plural referents unless the context clearly dictates otherwise, and expressions such as "one or more items" include the singular referents. Thus, for example, reference to "an oligomer" includes a plurality of oligomers, and the like. The conjunction "or" should be interpreted in an inclusive sense, i.e., equivalent to "and/or," unless the inclusive sense is unreasonable in the context. When there is "at least one" member of a class (e.g., oligomer), reference to "the" member (e.g., oligomer) refers to at least one (if more than one) of the member(s) present (if only one) or the member(s) present (e.g., multiple oligomers).

It should be appreciated that there is an implicit "about" in advance of the temperatures, concentrations, amounts, times, etc. discussed in this disclosure so as to include minor and insubstantial deviations within the scope of the teachings herein. Generally, the term "about" means an insubstantial change in the amount of a component of the composition without any significant effect on the activity or stability of the composition, e.g., within 10%, 5%, 2%, or 1%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When there is no exclusive expression, such as "not including an endpoint", all ranges are to be construed as including an endpoint; thus, for example, reference to "within 10 to 15" includes the values 10 and 15 and all intermediate integers and (where appropriate) non-integer values. Furthermore, the use of "comprising (comprise, comprises, comprising)", "including (contain, contains, containing), and" containing (include, includes, including) is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the teachings. The section headings are provided only for the convenience of the reader and are not intended to limit the disclosure. When any material incorporated by reference is inconsistent with the present disclosure, the present disclosure's expressions are subject.

Embodiments in the specification that "comprise" various components are also considered to be "consisting of" or "consisting essentially of" the recited components unless specifically stated otherwise. By "consisting essentially of … …" is meant that one or more additional components, ingredients, or method steps may be included in the compositions or methods described herein without substantially altering the essential and novel features of those compositions and methods. Such features include, for example, the ability to hybridize to a target polynucleotide and undergo further binding and/or extension reactions as described herein, as the case may be.

"Sample" refers to a substance that may comprise a target polynucleotide, including but not limited to biological samples, clinical samples, environmental samples, and food samples. Environmental samples include environmental materials such as surface materials, soil, water, sludge, air, and industrial samples, samples obtained from food and dairy instruments, equipment, vessels, disposable and non-disposable items. "biological" sample or "clinical" sample refers to tissue, fluid or other material from a living or dead human, animal or other organism that may contain target polynucleotides, including, for example, a tissue sample, swab, wash, aspirate, exudate, biopsy or body fluid such as blood, spinal fluid, fecal matter, semen or urine. The sample may be treated to physically or mechanically disrupt tissue or cellular structure, thereby releasing intracellular acids into solutions that may contain enzymes, buffers, salts, lysing agents, etc., to prepare the sample for analysis. The sample may also be aqueous or organic solvent or a combination thereof, with or without other components (e.g., buffers, salts, detergents, emulsifiers, EDTA, etc.) including the target polynucleotide. These examples should not be construed as limiting the types of samples that can be applied to the present disclosure.

Sample preparation refers to a method or combination of methods by which a sample containing a target polynucleotide is manipulated to prepare the target polynucleotide for further downstream processing and/or analysis. These methods include, but are not limited to: methods for releasing, accessing, digesting, removing binding components from a sample, concentrating, enriching, capturing, isolating (separate and/or isolate) target polynucleotides from a sample. Such methods also include methods of removing, neutralizing, or otherwise reducing the potential competitive, interfering, masking, or deleterious effects on downstream analyte substances, components, contaminants, organism organisms (including dead or living and/or fragments of such organisms), or other biological, organic, or inorganic materials from a sample containing the target polynucleotide. Such methods include, but are not limited to: 1) Methods of solubilizing (solubilize, dissolve), homogenizing, digesting, or otherwise altering the physical or chemical properties of a sample to aid in the preparation of a target polynucleotide, including, but not limited to, heating, cooling, freezing, freeze thawing, digestion (including using chemical or biological means, including enzymatic means), sonication, solubilization using solvents, reagents, and/or other chemical or biological means, stirring, shearing, mechanical agitation, and the like; 2) Filtering the sample; 3) Concentrating the sample; 4) Tagging, labeling, capturing, concentrating, isolating, or otherwise treating cells suspected of containing a target polynucleotide, including but not limited to: tagging with cell-specific components including antibodies, lecithins, nucleic acids, proteins, peptides, aptamers, dendrimers, other cells, viruses, macrophages, other biological components, and the like, labeling cells with fluorescent dyes, radiolabels, luminescent labels, mass labels, and the like, capturing cells using precipitation, centrifugation (including using density gradients), filtration, affinity capture (including by at least one of the cell-specific components listed in the tagging section above), concentration or separation, including direct or indirect binding to a solid support, cell sorting, and the like; 5) Lysing, digesting, disrupting, partially lysing, shearing, or otherwise manipulating cells or other structures containing or otherwise associated with a target polynucleotide such that the target polynucleotide can be used or otherwise more readily used for further processing or analysis, including but not limited to: heating, cooling, freezing, freeze thawing, boiling, exposing the cells to osmotic shock, treatment with solvents or chemicals or other agents, including the use of heat in combination, sonication, simultaneous heating, beating/impact, enzymatic treatment, agitation, shearing (including mechanical shearing), and the like; 6) Methods of solubilizing (solubilize, dissolve), homogenizing, digesting, or otherwise altering the physical or chemical properties of a target polynucleotide to aid in further processing and/or analysis of the target polynucleotide, including but not limited to at least one of the methods set forth in section (1) above in this paragraph; 7) Methods of tagging, labeling, concentrating, enriching, capturing, isolating (separate and/or isolate) target polynucleotides, including, but not limited to: by direct or indirect, covalent or non-covalent tagging, binding to, immobilization, coupling, introduction or otherwise attaching to a nucleic acid, nucleic acid segment, multiple nucleic acid segments, protein, enzyme, aptamer, lecithin, dendrimer, element, molecule, or any other substance or moiety, including chemical, biological, organic and/or inorganic substances that aid in the further preparation, processing (including amplification) and/or analysis of a target polynucleotide, including all tagging methods generally known in the art, labeling with fluorescent dyes, radiolabels, luminescent labels, mass labels, and the like, precipitation, extraction (including GuSCN, CTAB, chelex (and other resin types) and basic extraction), chromatography (including column chromatography), filtration, centrifugation (including using density gradients), isoelectric focusing, and other focusing techniques known in the art, direct and indirect capture on solid supports, including magnetic microspheres and other solid support materials commonly known in the art, including the use of non-specific target capture methods such as Solid Phase Extraction (SPE), ion exchange SPE, solid Phase Reversible Immobilization (SPRI), solid Phase Microextraction (SPME), Silica-based methods, including the Boom method, the AMPure method, and methods of capturing various targets in a sample using random or semi-random target capture oligomers, specific target capture methods, including methods utilizing one or more oligonucleotides specific for a target polynucleotide or set of polynucleotides of interest, wherein the oligonucleotides anneal to the target nucleic acid and the complex(s) are immobilized on a solid support, wherein some methods of enriching, capturing, isolating (separate and/or isolate) the target polynucleotide or purifying the target nucleic acid in any other way include one or more wash steps, some such methods include an elution step, And further wherein some of the methods further use target nucleic acids directly from the sample for downstream processing (e.g., amplification) and/or analysis; 8) Removal, neutralization or otherwise reduction of potentially competitive, interfering, masking or other deleterious effects on downstream analytes, components, contaminants, organisms (including dead or living and/or debris from such organisms) or other biological, organic or inorganic materials from a sample, such methods include removal of non-target polynucleotides (including genomic DNA, including human genomic DNA), removal of RNAs (including rRNA), removal of proteins, enzymes, lipids, carbohydrates, biological materials, organic materials, inorganic materials, cells (including whole cells and partially or fully lysed or otherwise degraded cells), and other components that may interfere with downstream processing and/or analysis, Digestion, inactivation and the like. Other methods ：J.Dapprich,et al.,The next generation of target capture technologies-large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity(BMC Genomics(2016),17:486) and N Ali,et al.,Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics(BioMed Research International(2017),Article ID 9306564,13pages), for the preparation of the samples are described in the following documents, which are incorporated herein by reference in their entirety.

Specific Target Capture (STC) methods refer to methods or combinations thereof that can be used to tag, isolate (separate, isolate), or otherwise distinguish specific target polynucleotides in a broader mixture of polynucleotides. This is in contrast to non-specific capture methods, which typically operate on all polynucleotides in a mixture (although some type of discrimination may exist, e.g., based on length). STC methods are typically performed based on nucleotide sequence discrimination of polynucleotides (although other discrimination methods are acceptable if the desired level of specificity is achieved). A preferred method in the present disclosure is to use STC oligomers (oligomers) to distinguish targets by sequence. The oligomer or set of oligomers is designed such that it specifically anneals to a given target, set of targets, collection of targets, etc., rather than annealing to non-target polynucleotides that may be present in the sample mixture at any significant level. By combining STC oligomer design with selected reaction conditions, substantially any level of specificity can be achieved throughout the classification spectrum. For example, the target polynucleotides may be distinguished at subspecies/strain, species, genus, family, order, class, and/or door level, even at the kingdom and domain level. In a preferred embodiment of the disclosed invention, wherein the system is used to detect blood flow infections and antimicrobial drug resistance genes, the STC oligomers are designed to bind and selectively capture a wide range of bacterial and fungal targets as well as specific antimicrobial drug resistance (AMR) genes. Once the STC oligomer is annealed to the intended target, the resulting complex may be processed using a number of different methods, including capture, immobilization, separation (separate, isolate), and the like, a few examples of which are discussed elsewhere in this disclosure.

"Nucleic acid" and "polynucleotide" refer to polymeric compounds comprising nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together to form a polynucleotide, including conventional RNA, DNA, mixed RNA-DNA, and polymers as analogs thereof. The nucleic acid "backbone" may be composed of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of the nucleic acid may be ribose, deoxyribose, or similar compounds having a substituent (e.g., 2 'methoxy or 2' halo substitution). the nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., inosine or otherwise; See The Biochemistry of the Nucleic Acids-36, adams et al, ed.,11th ed, 1992), purine or pyrimidine derivatives (e.g., N ⁴ -methyl deoxyguanosine, deazapurine or azapurine, deazapyrimidine or azapyrimidine, pyrimidine bases having a substituent at the 5 or 6 position (e.g., 5-methylcytosine), purine bases having a substituent at the 2, 6 or 8 position, and combinations thereof, 2-amino-6-methylaminopurine, O ⁶ -methylguanine, 4-thiopyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O ⁴ -alkyl-pyrimidine; U.S. Pat. No.5,378,825 and PCT No. WO 93/13121). The nucleic acid may include one or more "abasic" residues, wherein the backbone includes abasic sites of the polymer (U.S. Pat. No.5,585,481). The nucleic acid may comprise only conventional RNA or DNA sugars, bases, and linkages, or may include conventional components and substitutions (e.g., conventional bases having a 2' methoxy linkage, or polymers containing conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), which are analogs that include one or more LNA nucleotide monomers having a bicyclic furanose unit locked into RNA in a simulated sugar conformation that enhances the hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel,2004,Biochemistry 43 (42): 13233-41). Embodiments of oligomers that can affect the stability of the hybridization complex include PNA oligomers, oligomers comprising 2 '-methoxy or 2' -fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or spatial relationship of the hybridization complex, including oligomers comprising groups with electronic linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates). Unless otherwise indicated, methylated cytosines such as 5-methylcytosine can be used in combination with any of the foregoing backbone/sugar/linkages, including RNA or DNA backbones (or mixtures thereof). RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose), and can differ by the presence of uracil in RNA and thymine in DNA. Differences between RNA and DNA equivalents do not result in homology differences, since equivalents have the same degree of complementarity to a particular sequence. It should be understood that when referring to a range of oligonucleotide, amplicon, or other nucleic acid lengths, the range includes all integers (e.g., 19-25 consecutive nucleotides in length includes 19, 20, 21, 22, 23, 24, and 25). Unless otherwise indicated, T residues are understood to be interchangeable with U residues and vice versa. The orientation of the nucleic acid polymer strands can be described as plus (+) sense (or sense, or simply sense or coding strand) or minus (-) sense (or minus, or simply antisense or non-coding strand).

"Target polynucleotide" refers to a polynucleotide that is prepared, isolated (separate), captured, isolated (isolate), enriched, amplified, detected, identified, and/or sequenced, etc., using the compositions or methods described herein. In some embodiments, the target polynucleotide comprises a sequence of DNA or RNA from an organism (e.g., any virus, prokaryote, eukaryote, protist, plant, fungus, insect, animal, mammal, or other biological entity, which may be living or previously living). Exemplary DNA includes genomic DNA, circulating tumor DNA, episomal or plasmid DNA, and mitochondrial DNA. Exemplary RNAs include messenger RNAs, more typically transcribed RNAs, ribosomal RNAs, transfer RNAs, micronuclear RNAs, regulatory RNAs, transfer-messenger RNAs, nucleolar micrornas, guide RNAs, interfering RNAs, micrornas, other regulatory RNAs, non-coding RNAs and the like (and where applicable genomic RNAs, e.g., in the case of certain viruses). The target polynucleotide may be positive, negative, or both positive and negative (e.g., when both strands of the polynucleotide are targeted). The target polynucleotide also includes one or more copies of the nucleic acids discussed above, where additional sequences (e.g., any of the additional sequences described herein) may be added. In some embodiments, the target polynucleotide comprises a non-naturally occurring sequence, e.g., a sequence produced by in vitro synthesis, ligation, site-directed mutagenesis, recombination, or the like.

"Oligomer" or "oligonucleotide" refers to nucleic acids typically less than 1,000 nucleotides (nt), including those within a size range of about 2nt to 5nt at the lower limit and about 500nt to 900nt at the upper limit. Some embodiments are oligomers in a size range from about 5nt to 35nt in a lower limit and about 50nt to 600nt in an upper limit, and other embodiments are oligomers in a size range from about 5nt to 20nt in a lower limit and about 30nt to 1500nt in an upper limit. The oligomer may be purified from a naturally occurring source, but may be synthesized using any well known enzymatic or chemical method. Oligomers may be expressed by functional names (e.g., capture oligomers, primers, promoter primers, or detection probes), but those skilled in the art will understand that such terms refer to oligomers. Oligomers may form secondary and tertiary structures by self-hybridization or by hybridization with other oligonucleotides or polynucleotides. Such structures may include, but are not limited to, duplex, hairpin, cross, bent, triplex, and quadruplex. The oligomer may contain modifications including those described elsewhere in this disclosure. In some cases, an oligomer may refer to a non-nucleic acid-based polymer, such as some aptamers and non-nucleotide-based binding partners (e.g., see Winnacker,M.,&Kool,E.T.(2013).Artificial Genetic Sets Composed of Size-Expanded Base Pairs.ANGEWANDTE CHEMIE-INTERNATIONAL EDITION,52(48),12498–508). for generation of an oligomer by any means, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof, in some embodiments, an oligomer that forms an invasive cleavage (INVASIVE CLEAVAGE) structure is generated in a reaction (e.g., by extension of a primer in an enzymatic extension reaction).

"Arbitrary sequence" refers to any sequence that results from a user (with or without the aid of a computer program) selecting, determining, designing, etc., typically for providing a desired function or purpose in a downstream process. In a preferred mode, any sequence is designed to be non-complementary to or non-reactive with one or more target sequences under the given process conditions. In some embodiments, any sequence may be a randomly generated sequence or collection of sequences, for example, used as a unique molecular identifier or universal primer.

"Capture oligomer", "capture oligonucleotide", "capture probe", "target capture oligomer" and "capture probe oligomer" are used interchangeably and refer to a nucleic acid oligomer or derivative thereof comprising the following sequences: which includes a Target Binding Sequence (TBS) capable of binding to one or more target sequences in a target nucleic acid. Depending on the design of the system and the desired application and results, the binding may be performed at different, user-selected levels of specificity. One mode of binding involves hybridization to a target nucleic acid, again with a different, user-selected level of specificity, from high specificity to low specificity (including designing capture oligomers to capture the desired target at any point in the sorting order; see discussion elsewhere in this disclosure). Target capture oligomers may also include segments (or whole oligomers) of random or semi-random sequences. The capture oligomer may also include one or more of: (i) an extendable 3' terminus, (ii) a non-extendable (e.g., blocked) 3' terminus, (iii) a ligatable 5' terminus; (iv) A first ligand of a ligand pair (e.g., biotin of the ligand pair biotin/streptavidin) which may have one or more copies, (v) a tag sequence overhanging and/or inserted into THS at the 3 'end, 5' end, or both, (vi) a combination of one or more tag sequences and one or more ligands. Exemplary tag sequences include capture sequences capable of hybridizing to secondary oligomers, e.g., immobilized on a solid support or immobilized on a non-immobilized secondary oligomer linked to a binding partner, to facilitate separation of complexes including capture oligomers, targets, and secondary oligomers from other molecules in the composition. The following "tag" definition section introduces other exemplary tag sequence options. The nucleic acid component of the capture oligomer includes any of its forms described in the definition of "nucleic acids" section above, or a combination thereof. In some cases, the capture oligomer may not only function as a capture reagent, but also include, but are not limited to, the following: primers, amplification oligomers, blocking sequences, a portion of the site for cleavage/digestion, substitution sequences, a portion of the recognition site, and the like. The capture oligomer may also be any capture oligomer described in "COMPOSITIONS, KITS AND METHODS FOR ISOLATING TARGET POLYNUCLEOTIDES" (PCT/GB 2021/050098), the entire contents of which are incorporated herein by reference. This document describes in detail the compositions, kits and methods related to copy control (discussed elsewhere herein).

"Nucleic acid amplification" or "amplification" (in the explicit context of nucleic acids; the term "amplification" may have different meanings in different contexts, e.g., generating 1 or more copies of a non-nucleic acid molecule, detecting an increase in signal, such as fluorescence, an increase in electrical signal, etc.) refers to generating 1 or more copies of a sequence of a target nucleic acid (or target polynucleotide, parent molecule, template molecule, etc.) or its complement or a portion thereof (i.e., an amplified sequence containing less than the entire target nucleic acid). Examples of nucleic acid amplification procedures include transcription related methods such as Transcription Mediated Amplification (TMA), nucleic Acid Sequence Based Amplification (NASBA) and other methods (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105 and 5,124,246), replicase mediated amplification (e.g., U.S. Pat. No.4,786,600), polymerase Chain Reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), and, rolling Circle Amplification (RCA) (e.g., U.S. Pat. nos. 5,854,033 and 6,143,495), recombinase Polymerase Amplification (RPA) (e.g., U.S. Pat. No.7,666,598), ligase Chain Reaction (LCR) (e.g., european patent application 0320308), loop-mediated amplification (e.g., loop-mediated isothermal amplification of DNA (2000) Nucleic Acid Res,28 (12): e 63), and Strand Displacement Amplification (SDA) (e.g., U.S. Pat. No.5,422,252). Replicase-mediated amplification uses self-replicating RNA molecules and replicases such as QB replicase. PCR amplification uses DNA polymerase, primers and a cycling step (typically thermal cycling, but other types of cycling, such as chemical cycling, can also be used) to synthesize multiple copies of two complementary strands of DNA or cDNA. Copies of two complementary strands may be produced at ratios other than 1:1, for example in Asymmetric PCR (e.g., asymmetry PCR. In: capinera J. L. (eds) Encyclopedia of Entomology,2008, springer, dordrecht). LCR amplification uses at least four separate oligonucleotides to amplify the target and its complementary strand through the use of multiple hybridization, ligation, and denaturation cycles. SDA uses a primer that contains a recognition site for a restriction enzyme that will create a nick on one strand of a semi-modified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Specific embodiments use PCR, but it will be apparent to one of ordinary skill in the art that the oligomers disclosed herein can be readily used as primers in other amplification methods, and that other amplification methods and primers can generally be used.

"Amplicon" or "amplification product" refers to a nucleic acid molecule that is generated in a nucleic acid amplification reaction and that is derived from a template nucleic acid. Amplicons or amplification products contain amplified nucleic acid sequences (e.g., target polynucleotides/nucleic acids) that can be in the same direction or opposite direction as the template nucleic acids, including DNA or RNA, and include single-stranded or double-stranded products. In some embodiments, the amplicon is about 100-30,000 nucleotides, about 100-10,000 nucleotides, about 100-5000 nucleotides, 100-2000 nucleotides, about 100-1500 nucleotides, about 100-1000 nucleotides, about 100-800 nucleotides, about 100-700 nucleotides, about 100-600 nucleotides, or about 50-500 nucleotides in length.

"Amplification oligonucleotide" or "amplification oligomer" refers to an oligonucleotide that hybridizes to a target nucleic acid or its complement or tag sequence, etc., and participates in a nucleic acid extension or amplification reaction, e.g., as a primer and/or promoter primer, displacement sequence (with or without extension), blocking sequence (e.g., blocking binding or extension), to help promote cleavage or degradation, disruption of structure, etc. Some capture oligomers may also be used as amplification oligomers (see elsewhere in this specification), and some amplification oligomers may also be used as capture oligomers. The amplification oligomer also includes a promoter-providing sequence that includes a promoter that can initiate transcription but does not have to be extended by a DNA polymerase, and may include a 3' blocking moiety. Specific amplification oligomers comprise a target hybridization sequence, complementary hybridization sequence, or tag hybridization sequence of at least about 5 contiguous bases and optionally at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous bases that is complementary to a region of a target nucleic acid sequence or tag sequence or a complementary strand thereof. Other exemplary lengths or ranges of lengths of target hybridization sequences or tag hybridization sequences are described elsewhere herein, and may be applied to amplification of oligomers. The contiguous bases may be at least about 70%, at least about 80%, at least about 90%, or fully complementary to the target sequence of the binding amplification oligomer. In some embodiments, the amplification oligomer includes an insertion linker or non-complementary sequence between two fragments of the complementary sequence, e.g., wherein the two complementary fragments of the oligomer together comprise at least about 5 complementary bases, and optionally together comprise at least 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 complementary bases. In some embodiments, the amplification oligomer is about 10 to about 80 bases in length, and optionally may include modified nucleotides. The amplification oligomers may optionally be modified, for example, by including a 5' region that is non-complementary to the target sequence. Such modifications may include functional additions, such as tags, promoters, or other sequences or portions that are used or available to manipulate, amplify, capture, immobilize, or otherwise process primers, tags, or target molecules.

"Primer" refers to an oligomer that hybridizes to a template nucleic acid and has a 3' end that extends by polymerization. The primer may optionally be modified, for example by including a 5' region that is non-complementary to the target sequence. Such modifications may include functional additions, such as tags, promoters or other sequences or other portions, all of which may be used or available to manipulate, amplify, capture, isolate, immobilize or otherwise process the primer or target oligonucleotide or its complement. "opposing primers (Opposed primers)" refers to at least one positive (+) sense primer and at least one negative (-) sense primer, each of which is complementary to one strand of the target polynucleotide or a copy of one strand of the target polynucleotide, such that when used together in an amplification reaction they are involved in the production of an amplicon (e.g., a primer pair in a PCR).

In some cases the amplification oligomers or primers may function not only as amplification oligomers and primers, but also as primers including, but not limited to: capture oligomers, blocking sequences, a portion of the site for cleavage, digestion, substitution sequences, a portion of the recognition site, etc. function.

Unless otherwise indicated to the contrary, a first sequence is a "complement" of (or equivalently, is "complementary to") a second sequence, wherein the first sequence has a length and content sufficient to anneal to the second sequence under reasonable binding conditions, which may be, but need not be, stringent hybridization conditions as described herein, and also include annealing conditions used, for example, in standard PCR and other techniques involving primer or probe binding and extension.

"Tag" refers to any other nucleic acid sequence besides the target hybridization sequence, which may be included in an oligomer or added to or inserted into a target polynucleotide. Any arbitrary sequence that is present other than the target hybridization sequence may be used as a tag. In some uses, a tag may also refer to a portion other than a nucleic acid that is attached to, or otherwise included in, an oligomer or target polynucleotide. The tag may be introduced into/into the target polynucleotide or one or more fragments thereof by any means known in the art including, adding, inserting, overhanging, etc., including but not limited to, by extension or amplification, by ligation, by transposition chemistry, using an oligomer containing one or more tags and hybridizing specifically, semi-specifically or non-specifically to the target polynucleic acid or one or more fragments thereof. Tags include, but are not limited to, adaptors (see below). Additional examples of tags are promoters, mixed nucleotide elements described elsewhere herein, elements for sample preparation and target capture, and stabilizing sequences including clips. Other additional examples of labels are given in the "sample preparation" definition section above, and are also described elsewhere herein.

An "adapter" (adaptor, adapter) (the two terms are used interchangeably throughout this disclosure and are defined as equivalent) is a sequence that adapts the molecule to which it is added to provide one or more additional functions. For example, the adapter provides a binding site for another molecule (e.g., an amplification oligomer, a sequencing primer, or a capture oligomer). The binding site may be a universal binding site (e.g., having the same binding site specificity for a plurality of capture oligomers, e.g., the same sequence, multiple forms, or for a universal primer). Other examples of binding sites are binding sites for displacement sequence oligomers, probes or nucleic acid modifying enzymes (e.g. RNA polymerase, priming enzyme, ligase, RNAse (e.g. RNAse H) or restriction enzymes), or for attachment to binding sites comprising a solid phase for clonal amplification (including by solid phase primers or capture oligomers), or other one or more functional elements useful in downstream applications (e.g. enrichment, library preparation, clonal amplification or sequencing). Thus, sample barcodes or index sequences, key or calibration sequences, molecular barcodes (including unique molecular identifiers), sites of downstream cloning, and sites of target molecule circularization are other examples of elements that may be included in an adapter.

"Library preparation" in the most general sense refers to the process of preparing a set of target polynucleotides for further downstream processing and/or analysis. Cluster generation (including by clonal amplification) is one example of a downstream processing step. Sequencing is one example of downstream analysis. Library preparation methods typically include one or more steps of adding an adapter sequence or other tag sequence to some or all of the molecules in the library. However, this is not always the case, as some disclosed embodiments do not require the addition of adaptors to the library molecules. Examples of this include some modes of direct hybridization sequencing methods, which are described elsewhere in this disclosure. Library preparation typically also includes one or more amplification steps, e.g., enriching one or more regions of the target polynucleotide, adding tags (including adaptors) to library molecules, and increasing the number of copies of the target region and/or molecule containing the tags (including adaptors). Adaptors and tags may be added without amplification, for example by ligation. The library preparation step may overlap with the sample preparation step (e.g., a tag (including an adapter) may be added during sample preparation and/or a first extension product may be prepared from the targeted region (see "sample preparation" section herein), or may be associated/overlapped with copy control (see other sections herein, including below).

"Copy control" refers to compositions and methods that control the copy number of molecules that are output as a given process in a predetermined manner. For example, in certain workflows, it is desirable to capture (or amplify and capture) or otherwise isolate a target polynucleotide (e.g., which may be native DNA or RNA or amplicon) that does not exceed a predetermined amount (e.g., a maximum expected value for downstream applications, such as sequencing library preparation; which may be referred to as "limited capture" in some cases, which still falls within the overall definition of copy control). Similarly, in certain workflows, it is desirable to capture (or amplify and capture) or otherwise isolate a predetermined specific amount (e.g., a specific number of molecules or molecular copies) of a target polynucleotide (which may be, for example, native DNA or RNA, an amplicon, or a sequencing library) for downstream applications (e.g., clonal amplification, including in a second generation sequencing workflow). Furthermore, in certain workflows, it is desirable to introduce additional sequences into the target polynucleotide (tag), for example, to introduce adaptors into a sequencing library. This can also be achieved in copy control compositions and methods. The present disclosure provides oligomers, compositions, and kits useful for isolating target polynucleotides and/or attaching tags (e.g., adaptors). Isolation includes isolation in limited amounts (limited capture) and isolation in specific amounts (copy control).

"Clone" or "monoclonal" refers to a population of identical units or copies of (at least one) progenitor cell molecule, nucleic acid, polynucleotide, gene, genetic material, cell, etc. In this disclosure, it most often refers to a population of identical copies made from one or more copies of the same nucleic acid/polynucleotide template. In this disclosure, it may sometimes refer to a collection of identical molecules in a population, e.g., the molecules are clustered together (see below). In a most preferred embodiment, the monoclonal is about 100% (i.e., all or nearly all copies are identical). In other preferred embodiments, the monoclonal is about 90% or greater, about 80% or greater, or 70% or greater. In some embodiments, the monoclonal is between about 50% and 70%. By "polyclonal" is meant a population of non-identical units or copies derived from at least 2 different progenitor cell molecules, etc. By clonally amplified is meant nucleic acid amplification of typically a single progenitor nucleic acid molecule (although in some cases it may be 2 or more identical progenitor molecules) to produce the same copy set. By "cluster" is meant a group of molecules, such as nucleic acid molecules, bound to a solid support. By "cluster generation" is meant the process of generating clusters. Examples of cluster generation processes include amplification-based (e.g., clonal amplification), and non-amplification-based, such as hybridization of a target molecule to an oligonucleotide immobilized on a solid support in a known specific region (e.g., spot). Clusters can be monoclonal (generally the preferred configuration in this disclosure) or polyclonal.

A "linker" is a sequential or non-sequential element or combination thereof that connects one portion of an oligomer to another portion. In some embodiments, sequence linkers include sequences that do not hybridize to the target polynucleotides and/or other oligomers in the combination or composition. In some embodiments, the non-sequence linker comprises an alkyl, alkenyl, amido, or polyethylene glycol group [ (-CH ₂CH₂O-)_n ].

A "stabilizing sequence" is a region of clips, mixed nucleotides or other sequences that function to increase the stability of the double-stranded region and/or control hybridization overlap (e.g., when positioned near a sequence that is prone to sliding, such as a sequence containing repeated nucleotides, such as a poly-dA or poly-dT sequence). "alignment sequences" are stabilizing sequences that control hybridization overlap. In addition to the clamp and mixed nucleotide regions described elsewhere herein, stabilizing sequences include GC-rich sequences and modified sequences containing enhanced affinity.

An "internal extension blocking sequence" is an element located within or bound to a nucleic acid sequence that prevents the complementary strand from extending along the nucleic acid. Examples include a non-nucleotide linker, or one or more abasic sites, non-natural nucleotides or chemically modified natural nucleotides, and reversible extension blocking sequences discussed below.

A "reversible extension blocking sequence" is an internal extension blocking sequence whose blocking function can be reversed, i.e., allowing complementary strand extension. An exemplary reversible extension blocking sequence is a non-natural nucleotide having complementary nucleotides that are suitable for the polymerase and exhibit specificity relative to the natural nucleotide (i.e., the polymerase does not add natural bases across the reversible extension blocking sequence). Providing complementary nucleotides reverses the blocking function. Examples of unnatural base pairs are Iso-dC or Iso-dG; xanthine or 5- (2, 4-diaminopyrimidine); 2-amino-6- (N, N-dimethylamino) purin or pyridin-2-one; 4-methylbenzimidazole or 2, 4-difluorotoluene; 7-azaindole or isoquinolone; dMMO2 or d5SICS; or dF or dQ, wherein either of these unnatural base pairs can act as a reversible extension blocker. Other examples of reversibly extending blocking sequences are chemically modified nucleotides, wherein the modification is attachment by a reversible linkage, and the linkage may be reversed by providing any one or more of the following: chemicals, enzymes, temperature changes, reagent composition changes, etc.; reversible nucleic acid structural features; or a molecule that binds reversibly to the capture oligomer, optionally wherein the reversibly bound molecule is a protein, enzyme, lipid, carbohydrate, or chemical moiety.

By "hybridization" or "hybridize" is meant the ability of two fully or partially complementary nucleic acid strands to be brought together in parallel or antiparallel directions under specific hybridization assay conditions to form a stable structure with a double-stranded region. "hybridization" is synonymous with "annealing". The two component chains of this double-stranded structure (sometimes referred to as a hybrid) are held together by hydrogen bonds. Although these hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (a and T or U) or cytosine and guanine (C and G) on a single nucleic acid strand, base pairing can also form between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art (see, e.g., R.L.P.Adams et al The Biochemistry of the Nucleic Acids (11 th edition, 1992)). In addition, the third strand may also be hybridized to a type B DNA duplex by Hoosteen base pairing to form a triplex region.

As used herein, the term "specifically hybridizes" refers to substantially only hybridization detectably (i.e., little or no hybridization detectably) of a probe, primer, or other oligomer (e.g., a capture oligomer) to one or more target sequences in a sample comprising the one or more target sequences under a given hybridization condition. Notably, the oligomer can be configured to specifically hybridize to any one of a set of targets (e.g., sequences from a particular taxonomic group (e.g., species, genus, or higher order) of organisms). In some embodiments, probes, primers, or other oligomers (e.g., capture oligomers) can hybridize to their target nucleic acids to form stable oligomers that are target hybrids, but do not form sufficient amounts of stable oligomers that are non-target hybrids for amplification or capture, as the case may be. Amplification and capture oligomers that specifically hybridize to a target nucleic acid are useful for amplifying and capturing target nucleic acids, but are not useful for amplifying and capturing non-target nucleic acids, particularly of organisms closely related to the germ line. Thus, the extent to which an oligomer hybridizes to a target nucleic acid is much greater than the extent to which an oligomer hybridizes to a non-target nucleic acid, to enable one of ordinary skill in the art to accurately capture, amplify, and/or detect the presence (or absence) of a nucleic acid derived from a particular target (e.g., a particular pathogen). In general, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence will reduce the degree or rate of hybridization of the oligonucleotide to its target region. However, the inclusion of one or more non-complementary nucleosides or nucleobases can facilitate the ability of the oligonucleotide to distinguish between non-target nucleic acid sequences.

By "stringent hybridization conditions" or "stringent conditions" is meant conditions such that: (1) Allowing the oligomer to hybridize preferentially to the target nucleic acid rather than to a different nucleic acid (e.g., a nucleic acid having a difference of as low as 1 nucleotide from the target nucleic acid) or (2) allowing only an oligomer having a higher affinity target hybridization sequence (relative to an oligomer having a lower affinity target hybridization sequence) to hybridize to the target, e.g., wherein the higher affinity target hybridization sequence is longer than the lower affinity target hybridization sequence and/or the higher affinity target hybridization sequence comprises an affinity enhancing modification and the lower affinity target hybridization sequence does not comprise the affinity enhancing modification. Although the definition of stringent hybridization conditions does not vary, the actual reaction environment available for stringent hybridization may vary depending on factors including GC content and oligomer length, the degree of similarity between the oligomer sequences and target and non-target nucleic acid sequences that may be present in the test sample. Hybridization conditions include temperature and composition of the hybridization reagents or solutions. Exemplary stringent hybridization conditions using the oligomers of the present disclosure correspond to temperatures of about 40 ℃ to 75 ℃, e.g., 40 ℃ to 50 ℃, 50 ℃ to 60 ℃, or 60 ℃ to 75 ℃, when the monovalent cation concentration is in the range of about 0.4M to 1M, the divalent cation concentration is in the range of about 0 to 10mM, and the pH is in the range of about 5 to 9. Additional details of hybridization conditions are set forth in the examples section. Other acceptable stringent hybridization conditions can be readily determined by one of ordinary skill in the art.

"Label" or "detectable label" refers to a moiety or compound that is directly or indirectly attached to an oligomer that is detected or generates a detectable signal. Any detectable moiety may be used, for example radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles imparting a detectable color (e.g., latex or metal beads), luminescent compounds (e.g., bioluminescent, phosphorescent or chemiluminescent compounds), and fluorescent compounds (i.e., fluorophores). Embodiments of fluorophores include those that absorb (e.g., peak absorption wavelength is in the range of about 495nm to 690 nm) and emit (e.g., peak emission wavelength is in the range of about 520nm to 710 nm), including fluorophores known as FAM ^TM、TET^TM、HEX、CAL FLUOR^TM (orange or red), CY, and QUASAR ^TM compounds. The fluorophore may be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore to attenuate background fluorescence. Such quenchers are well known in the art and include, for example BLACK HOLE QUENCHAER ^TM (or BHQ ^TM), blackberry(Or)、Or a TAMRA ^TM compound.

An "inextensible" oligomer or oligomer that includes a "blocking moiety at its 3' end" includes a blocking moiety that is sufficiently close to its 3' end (also referred to as the 3' end) to prevent extension. For purposes of this disclosure, any blocking moiety that is sufficiently close to the 3' terminus to block extension is considered to be "at" the 3' terminus, even if it is not bound to or present in place of the 3' hydroxyl group or oxygen. In some embodiments, the blocking moiety near the 3' end is within five residues of the 3' end and is large enough to limit binding of the polymerase to the oligomer, and other embodiments include a blocking moiety covalently attached to the 3' end. Many different chemical groups may be used to block the 3 'end, such as alkyl, non-nucleotide linkers, alkane-diol dideoxynucleotide residues (e.g., 3' -hexanediol residues) and cordycepin. Other examples of blocking moieties include 3' -deoxynucleotides (e.g., 2',3' -dideoxynucleotides); 3' -phosphorylated nucleotides; fluorophores, quenchers, or other labels that interfere with extension; inverted nucleotides (e.g., linked to the previous nucleotide by a 3' -to-3 ' phosphodiester, optionally with an exposed 5' -OH or phosphate); or a protein or peptide linked to an oligonucleotide to prevent further extension of the nascent nucleic acid strand by the polymerase. The non-extendible oligonucleotides of the present disclosure may be at least 10 bases in length and may be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Non-extendable oligonucleotides including detectable labels may be used as probes.

A "binding partner" is a member of a pair of moieties that can be used to form a non-covalent bond. Exemplary groups of binding partners are biotin and biotin binders. Other examples of binding partners include, but are not limited to, digoxigenin/anti-digoxigenin, and more commonly antibodies and targets thereof.

A "biotin binding agent" is an agent (e.g., a polypeptide) that is capable of specifically binding biotin. Streptavidin, avidin and neutravidin represent examples of biotin binders. An anti-biotin antibody is also considered a biotin binding agent.

The term "antibody" encompasses any polypeptide comprising a functional antigen binding region having complementarity determining regions and framework regions (e.g., VH and VL domains), including but not limited to scFv, fab, and full length antibodies (e.g., igA, igG, igD, igE or IgM antibodies).

The terms "Unique Molecular Identifier (UMI)", "Unique Identifier (UID)", "molecular barcode", "random oligonucleotide", "random molecular tag", "random barcode", "primer ID", "molecular tag", "single molecular barcode" and "Single Molecular Identifier (SMI)" are used interchangeably to refer to a polynucleotide sequence, the sequence of which may be random, non-random, partially degenerate or degenerate. UMI can be used to bar code DNA molecules prior to PCR or sequencing methods so that individual DNA strands can be identified. It is assumed that amplicons containing the same UMI are derived from the same DNA molecule. UMI can identify true errors and errors caused by PCR or sequencing methods. The UMI may be between about 5 and 100 nucleotides in length, or longer as desired, to facilitate distinguishing between a greater number of DNA strands, and may be of variable or uniform length. In some embodiments, UMI may be introduced in the first two PCR cycles, or using methods such as ligation, transposition by polymerase, endonuclease, transposase, or any other method known in the art.

The term "triangulation" refers to combining two or more results or data of independent analyses to determine answers with increased confidence.

As used herein, "combination" of oligomers refers to any of a variety of oligomers in proximity to each other, such as in different containers or the same container in a kit, or in a composition or collection of compositions juxtaposed to each other, such as in a plate, rack, or other container.

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. General definitions can be found in technical books related to the field of molecular biology, for example, the 2nd edition of the dictionary of microbiology and molecular biology (Dictionary of Microbiology and Molecular Biology,2nd ed. (Singleton et al, 1994,John Wiley&Sons,New York,NY)) or the Hamper kolin dictionary of biology (THE HARPER Collins Dictionary of Biology, (Hale & Marham,1991,Harper Perennial,New York,NY)).

Examples

The following examples are provided to illustrate certain disclosed embodiments, but should not be construed to limit the scope of the disclosure in any way.

A. extraction of labeled (Spiked) antimicrobial drug resistance (AMR) targets directly from blood (DfB) using Specific Target Capture (STC) oligomers

Oligomers (both supplied by IDT; 3' invdT and 3' invdC are inverted nucleotides at the 3' end)

The bacterial target organism, klebsiella pneumoniae (Klebsiella pneumoniae), ATCC strain BAA-1898; contains AMR carbapenemase gene (KPC); staphylococcus aureus, ATCC strain BAA-2094; contains the AMR-mecA gene (mecA).

Specific Target Capture (STC) oligomer-KCC STC oligomer was designed to bind to the AMR-KPC gene with the following sequence:

KPC_STC_F (SEQ ID No. 6) 5 '-biotin/AAAAACACCGCGCTGACCAACCTC/3' invdT

KPC_STC_R (SEQ ID No. 7) 5 '-biotin/AAAAACACAGCGGCAGCAAGAAAGC/3' invdT

MecA STC oligomers were designed to bind mecA with the following sequence:

mecA_STC_F-INT(SEQ ID No.8):

5 '-biotin/AAAAAAGGTACTGCTATCCACCCTCAAACAGGT/3' invdT

mecA_STC_R2(SEQ ID No.9):

5 '-Biotin/AAAAATTGAGTTGAACCTGGTGAAGTTGTAATCTGG/3' invdT

STC oligomers are designed to capture targets with the following sequences:

mecA 5 '-biotin/sequence/3' invdC

Quantitative PCR (qPCR) was performed using primers with the following sequences:

PCR1

KPC_P28_PCR1_F(SEQ ID No.10)：5’-AACCATTCGCTAAACTCGAACAGG-3’

KPC_P28_PCR1_R(SEQ ID No.11)：5’-CCTTGAATGAGCTGCACAGTGG-3’

mecA_P40_PCR1_F(SEQ ID No.12)：5’-CATGAAAAATGATTATGGCTCAGGTAC-3’

mecA_P40_PCR1_R(SEQ ID No.13)：5’-TGGAACTTGTTGAGCAGAGGTTC-3’

PCR 2

KPC_P28_PCR2_F(SEQ ID No.14)：5’-CTTTGGCGGCTCCATCGG-3’

KPC_P28_PCR2_R(SEQ ID No.15)：5’-CTCCTCAGCGCGGTAACTTAC-3’

mecA_P40_PCR2_F(SEQ ID No.16)：5’-GCTATCCACCCTCAAACAGGTGAAT-3’

mecA_P40_PCR2_R(SEQ ID No.17)：5’-ATTCTTCGTTACTCATGCCATACATA-3’

Scheme/reaction conditions

To 5mL whole blood 50. Mu.L, 25. Mu.L or 15. Mu.L pathogen addition standard (10, 5 or 3CFU/mL, respectively), 1mg proteinase K (20 mg/mL, promega MC 5008), 30. Mu.L STC oligomer pool (20 pmol/5. Mu.L), 100. Mu.L defoamer Y-30 emulsion (SIGMA ALDRICH A6457-100 ML) and 1.667mL lysis NS4X buffer E (100mM Tris pH 8.0, 16.675% SDS) were added and reacted in 15mL Falcon conical polypropylene tube (Corning 352097).

The reaction mixture was mixed by swirling and inverting each sample for about 15 seconds and spun to 700 Relative Centrifugal Force (RCF) using a swinging bowl centrifuge (centrifuge 5810, eppendorf) pulse for a total duration of 10 seconds.

The proteins were digested and then cell lysed-the reaction mixture was incubated for 15 minutes in a custom laboratory heating block (custom, DNAe) pre-heated to 75 ℃, where the internal temperature of the sample reached about 60 ℃ after 15 minutes incubation.

The mixture was then transferred to an 8mL polypropylene spiral cap tube (FISCHER SCIENTIFIC, NC 9691446) containing pre-measured 4gr 0.1mm VHD ZrO mechanical lysis (mL) beads (GLENMILLS GRINDING MEDIA) and mechanically lysed (mL) using OMNI Bead Ruptor Elite (OMNI International) using a "blood" procedure (3 cycles at 6.6m/s, 90 seconds on (mixing) and 20 seconds off (resting)).

The mechanically lysed samples were cooled at room temperature (about 20-26 ℃) for 5 minutes. The ML tube was then centrifuged at 700RCF for 1 min and the liquid was transferred back into the original 15ML tube, leaving ML beads. The sample tube was pulsed in the centrifuge to 700RCF for a total time of 10 seconds.

Inactivation of proteinase K and denaturation of DNA-tubes were placed on a heating block preheated to 100℃and incubated for 30 min.

Target DNA was captured using biotin-labeled STC oligomers-the tube was then transferred to a heating block preheated to 60 ℃ and incubated for 40 minutes.

Target DNA-STC oligomer was captured with streptavidin beads-the tube was removed from the 60 ℃ heat block, the contents were allowed to cool slightly to ambient temperature (about 20-26 ℃) and 1.2mg of streptavidin beads (custom streptavidin spheres, DNAe) were added. The tube was then incubated in a heated/cooled shaking incubator (Benchmark Scientific, model HC 5000-HC) at 45℃for 10 minutes and mixing was continued (1500 RPM).

Bead separation-samples were pulsed to 700RCF for 10 seconds in a centrifuge, sample tubes were placed in a magnetic rack (Invitrogen, dynaMag-15) for 5 minutes, supernatant aspirated and discarded.

Wash-S buffer Wash beads-Wash beads by addition of 1mLWash-S buffer (50mM Tris pH 8.0,0.1%SDS,150mM NaCl) and stir with magnet. The washed beads and buffer were then transferred from the 15mL tube to a new 1.5mL tube, then magnetized for 2 minutes, and the Wash-S buffer was removed and discarded. The beads were then washed again by adding 1mL of Wash-S buffer, magnetizing for 2 minutes, then removing and discarding the Wash-S buffer.

The beads were washed with Wash-T buffer (10mM Tris pH 8.0,0.01% Tween 20) -the samples were then washed with 1mL Wash-T buffer, magnetized for 2 minutes, and the buffer was removed and discarded. The beads were then washed again with Wash-T buffer.

Target was eluted from the beads-the magnet was removed from the 1.5mL tube and 50 μl of IDTE elution buffer (10mM Tris pH 7.5,0.1mM EDTA;Integrated DNA Technologies) was added. The samples were then mixed, pulsed and incubated at 75 ℃ for 3 minutes to elute the target DNA from the streptavidin beads. The tube was then magnetized for 2 minutes, and then the eluate containing the target DNA was transferred to a new 1.5mL tube (DNA LoBind tube,022431021, eppendorf).

Quantification-using the primers described above, two qPCR reactions were used to confirm capture of mecA and KPC targets from whole blood.

Results and conclusions

Two organisms containing AMR targets (Klebsiella pneumoniae containing KPC and Staphylococcus aureus containing mecA) were labeled (spiked) to 5mL whole blood at 10, 5 or 3CFU/mL each and the protocol described above (steps 6-19 above) was performed. The qPCR data in fig. 50 shows that the desired target was captured from whole blood.

B. Multiple full capture scheme

Oligomer

Each of the specified targets was amplified using the following nested PCR2 primers (both provided by IDT; "52 Bio" for two biotin groups on the 5' end of the oligomer):

16s rRNA targets

P3F(SEQ ID No.18)：

5’-AAAACGAGACATGCCGAGCATCCGCTTTAAGTCCCGCAACGAGCGCAA-3’

P3R(SEQ ID No.19)：

/52-Bio/ACCGTGCTGCCTTGGCTTCATTGTGGTCTTGACGTCATCCCCACCTTCCTC-3’

23S rRNA targets

P31F1(SEQ ID No.20)：

5’-AAAACGAGACATGCCGAGCATCCGCCGCATGTGTAGGATAGGTGGGAG-3’

P31F2(SEQ ID No.21)：

5’-AAAACGAGACATGCCGAGCATCCGCCGCATGTACAGGATAGGTAGGAG-3’

P31R(SEQ ID No.22)：/52-Bio/GAGACCGCCCCAGTCAAACT-3’

CTX-M group 1 targets

P48F(SEQ ID No.23)：

/52-Bio/AAAACGAGACATGCCGAGCATCCGCTGTTAGGAAGTGTGCCGCTG-3’

P48R(SEQ ID No.24)：

5’-ACCGTGCTGCCTTGGCTTCATTGTGGTCTCCCGACTGCYGCTCTAAT-3’

("Y" is a mixture of C and T)

Scheme/reaction conditions

1000 Genomic copies of E.coli (E.Coli) genomic DNA (gDNA) were added to the PCR2 singleplex reactions of P3F and P3R, P F1, P31F2 and P31R, and P48F and P48R. The reactants were prepared in 0.2mL tubes according to the formulations shown in table 1.

TABLE 1

¹ P31F1 and P31F2 were used in 0.75. Mu.M pre-mix, respectively; ² dATP, dCTP, dGTP and dTTP in an equimolar mixture; ³ Concentration of each nucleotide in the mixture

PCR2 amplification was performed using the following thermal procedure, steps b, c and d were repeated in sequence for 40 cycles:

a.98 ℃ for 30 seconds, b.98 ℃ for 5 seconds, c.58 ℃ for 10 seconds, d.72 ℃ for 30 seconds, e.maintained at 4 ℃.

Reaction pool-pool 40. Mu.L of each multiplex PCR2 reaction.

Total capture of PCR products-streptavidin beads (MyOne C1, thermoFisher Scientific) were resuspended in bead-resuspension buffer [1.50M NaCl (Invitrogen), 10mM Tris-HCl (pH 7.5) (Invitroen), 0.10% Tween 20 (ThermoFisher Scientific) ] at 8.33 mg/mL.

The biotin-labeled target DNA was bound to streptavidin beads-equal volumes (120. Mu.L) of the resuspended beads and PCR2 product were combined in a 0.2mL tube by pipetting. The tube was incubated at room temperature (about 20-26 ℃) for 5 minutes, gently mixed by vortexing, and then the beads were collected using a magnetic rack.

The streptavidin beads were washed with binding template-then the beads were washed twice by pipetting with 200 μl wash buffer (1M NaCl, 5mM Tris-HCl (pH 7.5), 0.05% tween 20, 0.5mg/mL BSA) and the supernatant was discarded between each wash.

Elution with NaOH-50. Mu.L of 40mM NaOH was added to a 0.2mL tube, vortexed for 10 seconds, and left to stand for 30 seconds. The beads were collected for two minutes using a magnetic rack and then the eluate was transferred to a 0.2mL tube.

Results and conclusions

The target DNA was enriched using a PCR reaction and the resulting biotin-labeled PCR2 products were pooled and captured using streptavidin beads, followed by elution of ssDNA with NaOH. The elution of the desired ssDNA from the three NaOH eluates was confirmed on a TBE (Tris-Borate-EDTA) gel at 200V until the reference dye reached the bottom of the gel. The gel was then stained in 1x SYBR Gold for at least 20 minutes and observed on a UV workstation as shown in fig. 51.

C. Target polynucleotide enrichment using multiplex Polymerase Chain Reaction (PCR)

Oligomers (supplied by IDT; Y is a mixture of C and T, W is a mixture of A and T, S is a mixture of C and G, and R is a mixture of A and G)

Each designated target was amplified using the following Polymerase Chain Reaction (PCR) 1 primers:

16s rRNA targets

P1(SEQ ID No.25)：5’-TGTAGCGGTGAAATGCGYAGA-3’

P1(SEQ ID No.26)：5’-CGGTCGACTTAACGCGTTAGCT-3’

P1(SEQ ID No.27)：5’-CGGAGTGCTTAATGCGTTWGCT-3’

P2(SEQ ID No.28)：5’-CGCAAGGTTGAAACTCAAAGGAATTG-3’

P2(SEQ ID No.29)：5’-CCGCAAGGTTAAAACTCAAATGAATTG-3’

P2(SEQ ID No.30)：5’-GGGACTTAACCCAACATYTCAC-3’

P29(SEQ ID No.31)：5’-CCTGGCTCAGAATGAACGCT-3’

P29(SEQ ID No.32)：5’-CCTGGCTCAGGACGAACGCT-3’

P29(SEQ ID No.33)：5’-GAGTCTGGACCGTGTCTCAGT-3’

P29(SEQ ID No.34)：5’-GAGTCTGGGCCGTGTCTCAGT-3’

P3(SEQ ID No.35)：5’-CGTGTGTAGCCCAGGTCATAAGG-3’

P3(SEQ ID No.36)：5’-CACGTGTGTAGCCCAAATCATAAGG-3’

P3(SEQ ID No.37)：5’-TGTGTAGCCCTGGTCGTAAGG-3’

P3(SEQ ID No.38)：5’-TCAGCTCGTGTCGTGAGATGTT-3’

P3(SEQ ID No.39)：5’-CGTCAGCTCGTGTTGTGAAATGTT-3’

P30(SEQ ID No.40)：5’-CTCCTACGGGAGGCAGCAGT-3’

P30(SEQ ID No.41)：5’-CCTCCGTATTACCGCGGCTG-3’

23S rRNA targets

P31(SEQ ID No.42)：5’-GAAAGACCCCGTGAACCTTTACT-3’

P31(SEQ ID No.43)：5’-GAAAGACCCCGTGGAGCTTTACT-3’

P31(SEQ ID No.44)：5’-CCTTCGTGCTCCTCCGTTAC-3’

P31(SEQ ID No.45)：5’-CCTTTGAGCGCCTCCGTTAC-3’

P4(SEQ ID No.46)：5’-ACACAGGTCTCTGCTAAACCGTAAG-3’

P4(SEQ ID No.47)：5’-ACACAGGTCTCTGCAAAATCGTAAG-3’

P4(SEQ ID No.48)：5’-ACACAGCACTGTGCAAACACGAAAG-3’

P4(SEQ ID No.49)：5’-TACCCGACAAGGAATTTCGCTACC-3’

Internal control target

P25(SEQ ID No.50)：5’-TGGCAGCTTCACTTTCTCTTGC-3’

P25(SEQ ID No.51)：5’-CCAGCTCCAATCACACCAACA-3’

SHV target

P26(SEQ ID No.52)：5’-CAGCTGCTGCAGTGGATGGT-3’

P26(SEQ ID No.53)：5’-CCGGSGTATCCCGCAGATA-3’

KPC target

P28(SEQ ID No.54)：5’-AACCATTCGCTAAACTCGAACAGG-3’P28(SEQ ID No.55)：5’-CCTTGAATGAGCTGCACAGTGG-3’

MecC target

P32(SEQ ID No.56)：5’-GCCGTAATAGTACCTGGTTTGAA-3’

P32(SEQ ID No.57)：5’-GCCYTTYGGGTGTTTTGTTAGG-3’

MCR-1 targets

P33(SEQ ID No.58)：5’-TCTGCAACACCAATCCTTATAACG-3’

P33(SEQ ID No.59)：5’-CATCATATCGCTTAAAATACGCAGGC-3’

NDM target

P34(SEQ ID No.60)：5’-AGATTGCCGAGCGACTTGGC-3’

P34(SEQ ID No.61)：5’-CAACTTTGGCCCGCTCAAGG-3’

Oxa-23-like targets

P35(SEQ ID No.62)：5’-ACAGAATATGTGCCAGCCTCTACA-3’

P35(SEQ ID No.63)：5’-CATGGCTTCTCCTAGTGTCATGTCT-3’

Oxa-48-like targets

P36(SEQ ID No.64)：5’-GCGGTAGCAAAGGAATGGCA-3’

P36(SEQ ID No.65)：5’-TGCTTGGTTCGCCCGTTTA-3’

Oxa-51-like

P37(SEQ ID No.66)：5’-AACGAAGCACACACTACGGGTGT-3’

P37(SEQ ID No.67)：5’-TGCTCAAGGCCGATCAAAGCATT-3’

GyrA targets

P39(SEQ ID No.68)：5’-GCAATGACTGGAACAAAGCCTA-3’

P39(SEQ ID No.69)：5’-ACCAGCATGTAACGCAGCGA-3’

MecA target

P40(SEQ ID No.70)：5’-CATGAAAAATGATTATGGCTCAGGTAC-3’

P40(SEQ ID No.71)：5’-TGGAACTTGTTGAGCAGAGGTTC-3’

VanA target

P41(SEQ ID No.72)：5’-GGCTGCGATATTCAAAGCTCAG-3’

P41(SEQ ID No.73)：5’-CTGAACGCGCCGGCTTAAC-3’

VanB target

P42(SEQ ID No.74)：5’-GTATGGAAGCTATGCAAGAAGCC-3’

P42(SEQ ID No.75)：5’-CATGCAAAACCGGGAAAGCCA-3’

TEM_E104K target

P45(SEQ ID No.76)：5’-GCGGTATTATCCCGTGTTGACG-3’

P45(SEQ ID No.77)：5’-TCACTCATGGTTATGGCAGCA-3’

TEM_G238S target

P46(SEQ ID No.78)：5’-GATAAAGTTGCAGGACCACTTCTG-3’

P46(SEQ ID No.79)：5’-CCCCGTCRTGTAGATAACTACGA-3’

CTX-M group 1 targets

P48(SEQ ID No.80)：5’-CGGCARCCGTCACGCTGT-3’

P48(SEQ ID No.81)：5’-CATCAGCACGATAAAGTATTTGCGA-3’

CTX-M group 2 targets

P49(SEQ ID No.82)：5’-TGCATGCGCAGRCGAACA-3’

P49(SEQ ID No.83)：5’-CCTTACTGGTACTGCACATCGC-3’

P49(SEQ ID No.84)：5’-TTGCTGGTGCTGCACATCGC-3’

CTX-M group 8-25 targets

P50(SEQ ID No.85)：5’-TACCACCACGCCRTTAGCGA-3’

P50(SEQ ID No.86)：5’-ACAACCCACGATGTGGGTAG-3’

CTX-M group 9 targets

P51(SEQ ID No.87)：5’-GTGCTTTATCGCGGTGATGAAC-3’

P51(SEQ ID No.88)：5’-GTTAACCAGATCGGCAGGCT-3’

28S rRNA H13-20 target

P52/53(SEQ ID No.89)：5’-ACTGTACTTGTGCGCTATCGGT-3’

P52/53(SEQ ID No.90)：5’-TCCTCAGTAACGGCGAGTGAAGC-3’

28S rRNA H26-31

P54(SEQ ID No.91)：5’-CCGTCTTGAAACACGGACCA-3’

P54(SEQ ID No.92)：5’-GTTTCCTCTGGCTTCACCCTATTC-3’

28S rRNA H45-46 target

P56(SEQ ID No.93)：5’-AACAACTCACCGGCCGAATG-3’

P56(SEQ ID No.94)：5’-ATGGAACCTTTCCCCACTTCAGT-3’

28S rRNA H78-79 target

P57(SEQ ID No.95)：5’-CCCTGTTGAGCTTGACTCTAGTTTGA-3’

P57(SEQ ID No.96)：5’-CTGCGTTATGGTTTAACAGATGTGC-3’

IMP group distinguishes Reg 1 targets

P59(SEQ ID No.97)：5’-GACGCCTATCTGATTGAYACTCCA-3’

P59(SEQ ID No.98)：5’-CATTTGTTAATTCAGATGCATAYGTGG-3’

P59(SEQ ID No.99)：5’-GAGGCTTACCTAATTGACACTCCA-3’

P59(SEQ ID No.100)：5’-CTGAAGCTTATCTAATTGACACTCCA-3’

P59(SEQ ID No.101)：5’-CTGATGCCTATATAATTGACACTCCA-3’

P59(SEQ ID No.102)：5’-CATTAGTTAATTCAGACGCATACGTGG-3’

IMP group distinguishes Reg 2 targets

P60(SEQ ID No.103)：5’-GCAAATTTAGAAGCTTGGCCAAAGTCY-3’

P60(SEQ ID No.104)：5’-GCCTTTACTTTCATTTAGCCCTTTAA-3’

P60(SEQ ID No.105)：5’-AAATGTTGAAGCATGGCCACATTCG-3’

P60(SEQ ID No.106)：5’-GCCTTTTGCTTTCATTAAGCCCTTTTA-3’

VIM-group targets

P61(SEQ ID No.107)：5’-GGTGTTTGGTCGCATATCGCAAC-3’

P61(SEQ ID No.108)：5’-GCGATCGTCATGAAAGTGCGT-3’

GyrB targets

P62(SEQ ID No.109)：5’-TCCTATAAAGTGTCCGGCGGTC-3’

P62(SEQ ID No.110)：5’-TCTCGCCGGTAACCGCCA-3’

P63(SEQ ID No.111)：5’-AACCAGGCGATTCTGCCG-3’

P63(SEQ ID No.112)：5’-GCAGCTTGTCCGGGTTGTA-3’

P64(SEQ ID No.113)：5’-GCACCATTTAGTGTGGGAAATTGTCG-3’

P64(SEQ ID No.114)：5’-TAACTTCGACAGCTGGACGT-3’

P65(SEQ ID No.115)：5’-GGCGGTGGCGGATACAAAGTAT-3’

P65(SEQ ID No.116)：5’-ACCTGTCTTATCAGTTGTGCCAAC-3’

Each designated target was amplified using the following nested PCR2 primers:

16S rRNA targets

P1(SEQ ID No.117)：5’-TAGAACACCGATGGCGAAGGC-3’

P1(SEQ ID No.118)：5’-TCGTGGACTACCAGGGTATCTA-3’

P2(SEQ ID No.119)：5’-TTTCGATGCAACGCGAAGAACCT-3’

P2(SEQ ID No.120)：5’-TACGAGCTGACGACAGCCATG-3’

KPC target

P28(SEQ ID No.121)：5’-CTTTGGCGGCTCCATCGG-3’

P28(SEQ ID No.122)：5’-CTCCTCAGCGCGGTAACTTAC-3’

MCR-1 targets

P33(SEQ ID No.123)：5’-CGGTATGCTCGTTGGCTTAGATG-3’

P33(SEQ ID No.124)：5’-GTGATTGCCCATTTGGTGCAG-3’

VanA target

P41(SEQ ID No.125)：5’-TTGTATGGACAAATCGTTGACATACA-3’

P41(SEQ ID No.126)：5’-GTAGCTGCCACCGGCCTAT-3’

28S rRNA H45-46 target

P56(SEQ ID No.127)：5’-AATGGATGGCGCTCAAGCGT-3’

P56(SEQ ID No.128)：5’-ACTGCCACCAAGATCTGCACTAG-3’

Scheme/reaction conditions

100 Genomic copies of each organism were added to the "PCR1 premix" (see Table 2 for formulation).

TABLE 2

PCR1-PCR amplification was performed using the following three-step thermal method, steps b and c were repeated in sequence for 25 cycles:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.65℃for 25 seconds (annealing/extension step).

Sample dilution-the sample was then diluted 40-fold using molecular-grade water (ThermoFisher Scientific) to reduce off-target PCR amplicon levels.

Add PCR2 reagent-10. Mu.L of diluted PCR1 material was added to 40. Mu.L of "PCR2 premix" to give a final dilution of 1:200 (see Table 3 for PCR2 premix formulation). In this example, the PCR2 premix included 1.50. Mu.M of each primer pair (P1, P28, P33 and P56).

TABLE 3 Table 3

PCR 2-PCR 2 amplification was performed using the following three-step thermal procedure, steps b and c were repeated in sequence for 40 cycles:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.65℃for 25 seconds (annealing/extension step).

PCR2 products were confirmed using Bioanalyzer assay and quantification.

Results and conclusions

Two multiplex PCR reactions were used to enrich for target DNA. The resulting PCR2 product was resolved and quantified using a Bioanalyzer quantification. Table 4 shows the resulting concentrations from PCR2 targets after successful PCR amplification.

TABLE 4 Table 4

Organism marking	Target(s)	μM
			Klebsiella pneumoniae (K.Pneumoniae)	P1	1.00
Klebsiella pneumoniae (K.Pneumoniae)	P28	1.26
			Candida albicans (C.Albicans)	P56	1.23
Coli (E.Coli)	P33	1.20
			Coli (E.Coli)	P1	1.15

In summary, the use of multiplex PCR to enrich for 100 genomic copies of each organism resulted in the desired dsDNA.

D. Enrichment of target nucleic acids using multiplex Polymerase Chain Reaction (PCR) and full capture

Oligomers (supplied by IDT; 52 Bio "means two biotin groups at the 5' end of the oligomer; Y is a mixture of C and T; W is a mixture of A and T)

The following PCR1 primers were used to amplify the indicated targets:

16s rRNA targets

P1(SEQ ID No.25)：5’-TGTAGCGGTGAAATGCGYAGA-3’

P1(SEQ ID No.26)：5’-CGGTCGACTTAACGCGTTAGCT-3’

P1(SEQ ID No.27)：5’-CGGAGTGCTTAATGCGTTWGCT-3’

P2(SEQ ID No.28)：5’-CGCAAGGTTGAAACTCAAAGGAATTG-3’

P2(SEQ ID No.29)：5’-CCGCAAGGTTAAAACTCAAATGAATTG-3’

P2(SEQ ID No.30)：5’-GGGACTTAACCCAACATYTCAC-3’

VanA target

P41(SEQ ID No.72)：5’-GGCTGCGATATTCAAAGCTCAG-3’

P41(SEQ ID No.73)：5’-CTGAACGCGCCGGCTTAAC-3’

Each 16s rRNA target was amplified using the following nested PCR2 primers:

P2(SEQ ID No.129)：

5’-AAAACGAGACATGCCGAGCATCCGCTTTCGATGCAACGCGAAGAACCT-3’

P2(SEQ ID No.130)：5’-/52-Bio/TACGAGCTGACGACAGCCATG-3’

Scheme/reaction conditions

100 Genomic copies of each organism were added to the "PCR1 premix". The PCR1 premix was prepared in a 0.2mL tube according to the formulation shown in Table 5.

TABLE 5

¹ An equimolar mixture of the primers as set forth in the above [02 ]; ² Concentration of each primer in the mixture

³ DATP, dCTP, dGTP and dTTP in an equimolar mixture; ⁴ Concentration of each nucleotide in the mixture

PCR1-PCR amplification was performed using the following three-step thermal procedure, steps b and c were repeated in sequence for 25 cycles:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.65℃for 25 seconds (annealing/extension step).

Sample dilution-the sample was then diluted 40-fold using molecular-grade water (ThermoFisher Scientific) to reduce off-target PCR amplicon levels.

Add PCR2 reagent-10. Mu.L of diluted PCR1 material was added to 40. Mu.L of "PCR2 premix" to form a 1:200 final dilution. PCR2 premixes were prepared according to the formulations shown in table 6. In this example, the PCR2 premix includes 1.50. Mu.M primer mix (P2 as shown in [0487 ]).

TABLE 6

PCR 2-PCR 2 amplification was performed using the following three-step thermal procedure, steps b and c were repeated for 45 cycles in sequence:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.65℃for 25 seconds (annealing/extension step).

PCR2 products were confirmed using Bioanalyzer assay and quantification.

Total capture PCR products-streptavidin beads (MyOne C1, thermoFisher Scientific) were resuspended in bead-suspension buffer [1.50M NaCl (Invitrogen), 10mM Tris-HCl (pH 7.5) (Invitroen), 0.10% Tween 20 (ThermoFisher Scientific) ] at a concentration of 8.33 mg/mL.

Binding of biotin-labeled target DNA to streptavidin beads-equal volumes (120. Mu.L each) of resuspended beads and PCR2 product were combined in a 0.2mL tube. The tubes were incubated at room temperature (about 20-26 ℃) for 10 minutes, gently mixed by vortexing, and then bead bound with a magnetic rack.

The streptavidin beads were washed with binding template-then the beads were washed three times with 200. Mu.L of wash buffer [1M NaCl,5mM Tris-HCl (pH 7.5), 0.05% Tween 20, 0.5mg/mL BSA ], and the supernatant was discarded between each wash.

Elution with NaOH-50. Mu.L of 40mM NaOH was added to a 0.2mL tube, vortexed for 10 seconds, and left to stand for 30 seconds. The beads were fixed on a magnet rack for two minutes and then the eluate was transferred to a new 0.2mL tube.

Results and conclusions

Elution of ssDNA from NaOH eluate was confirmed on TBE gel at 200V until the reference dye reached the bottom of the gel. The gel was then stained in 1x SYBR Gold for at least 20 minutes and observed on a UV workstation as shown in fig. 52.

In summary, DNA from three target organisms was amplified in PCR1, and then one target was amplified in PCR 2. Full capture of the PCR2 product is then performed to generate the desired ssDNA for hybridization with primers bound to the surface of the semiconductor chip.

E. targeted enrichment (PCR 1 and nested PCR 2) followed by copy control

Oligomers (both supplied by IDT; the "iSP18" is a Hexaethyleneglycol (HEG) internal spacer; 3BiodT is a 3' biotin molecule attached to the terminal dT nucleotide; 56-FAM/5 ' attachment to 6-FAM (fluorescein); ZEN/is a proprietary IDT ZEN quencher molecule; 3 IABkFQ/is a 3' Iowa Black FQ quencher)

The following Polymerase Chain Reaction (PCR) 1 primers were used for enterococcus faecium (e.fa) target:

P41F(SEQ ID No.131)：5’-GGCTGCGATATTCAAAGCTCAG-3’

P41R(SEQ ID No.132)：5’-CTGAACGCGCCGGCTTAAC-3’

the following nested PCR2 primers were used for EFM target amplicon from PCR 1:

P41F(SEQ ID No.133)：

5’-AAAACGAGACATGCCGAGCATCCGCTTGTATGGACAAATCGTTGACATACA-3’

P41R(SEQ ID No.134)：

5’-ACCGTGCTGCCTTGGCTTCATTGTGGTCGTAGCTGCCACCGGCCTAT-3’

the following hairpin oligomer sequence (SEQ ID No. 135) was used:

5'-CGCGCGAAAAAAAAAAAAAAAAAAAA/iSp18/TTTTTTTTTTTTTTTCGCGCGAAAAACTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC-3'

use of hairpin oligomer and insertion oligomer between beads:

poly dt oligomer sequence (SEQ ID No. 136):

TTTTTTTTTTTTTTTTTTTT/3BiodT/

The following primers were used for quantitative PCR (qPCR):

RPA1F(SEQ ID No.137)：5’-AAAACGAGACATGCCGAGCATC-3’

RPA1 external R (SEQ ID No. 138): 5'-TCGCGCGAAAAACTCCTCTGG-3' A

FAM probe (SEQ ID No. 139):

5’-/56-FAM/TGCTGGGAT/ZEN/AGCTACTCCCGCCTTTTGG/3IABkFQ/-3’

control sequence of standard curve (SEQ ID No. 140):

Scheme/reaction conditions

Sample dilution-EFM genomic DNA (gDNA) dilution to 1000 copies/. Mu.L

Add PCR1 reagent-10. Mu.L of diluted EFM gDNA was added to 40. Mu.L of PCR1 premix. PCR1 premixes were prepared according to the formulations shown in table 7.

TABLE 7

PCR1-PCR amplification was performed using the following three-step thermal procedure, steps b and c were repeated for 30 cycles in sequence:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.65℃for 25 seconds (annealing/extension step).

Sample dilution-2.5. Mu.L of PCR1 reaction was diluted in 97.5. Mu.L of water, dilution 1:40.

Add PCR2 reagent-5. Mu.L of diluted PCR1 material was added to 20. Mu.L of PCR2 premix to give a final dilution of 1:200. PCR2 premixes were prepared according to the formulations shown in table 8.

TABLE 8

PCR 2-PCR 2 amplification was performed using the following thermal procedure, steps b, c and d were repeated in sequence for 45 cycles.

98 ℃ For 30 seconds (initial denaturation step), b.98 ℃ for 5 seconds (denaturation step), c.55 ℃ for 10 seconds (annealing step), d.72 ℃ for 30 seconds (extension step), e.each cycle is warmed up from 65 ℃ to 95 ℃ (inclusive) at a rate of 0.5 ℃ (melting step).

PCR2 products were confirmed using Bioanalyzer assay and quantification. The stock concentration was found to be 800 ng/. Mu.L, which corresponds to 1.88X10 ¹³ copies per 40. Mu.L. The 40 μl sample was used as a "pure (coat)" condition.

PCR2 sample dilution-samples were also diluted 1:10 to obtain a separate "1:10" input, i.e., 1.88×10 ¹² copies per 40. Mu.L.

Add extension premix-60 μl of extension premix was added to 40 μl of "pure" or "1:10" pcr2 output material and mixed by pipette, followed by rapid vortexing and rapid rotation. An extension premix was prepared according to the formulation shown in table 9.

TABLE 9

Extension-extension using a thermal program:

a.92℃for 2 minutes, b.64℃for 2 minutes, c.68℃for 10 minutes.

Add hybridization mixture-50. Mu.L of hybridization mixture was added to the completed extension reaction and mixed with a pipette. Hybridization mixtures were prepared according to the formulations shown in table 10.

Table 10

Preparation and addition of streptavidin coupled magnetic beads-0.2 mg of streptavidin beads (internal preparation) were washed 3 times in 1x wash buffer (table 11) and resuspended in 50 μl of 1x wash buffer in a 0.2mL tube. Then 50. Mu.L of the conjugate beads were added to the extension/hybridization mixture and mixed with a pipette.

TABLE 11

The reaction was incubated at 25℃for 2 minutes, and then the tube was placed on a magnetic rack for 30 seconds.

The supernatant was aspirated, the beads were mixed with 1x wash buffer using a pipette (table 5) and placed back on the magnet rack until clear.

The washing was repeated as described above for a total of 3 times.

After the third wash, the supernatant was removed and the beads were resuspended in 30. Mu.L of ultra pure water.

The tube was incubated at 70℃for 1 min, mixed with gentle agitation, spun and returned to 70℃for 1 min.

The eluate (eluates) was removed and quantified using qPCR, and 2 μl was added to 13 μl of PCR quantification premix prepared according to the formulation shown in table 12. All reactions were run three times. The control sequences of the standard curves were run at 2x10 ⁷、2x10⁶、2x10² and 2x10 ⁴ copies.

Table 12

Reagent(s)	Manufacturer (S)	Concentration of stock solution	Final concentration	Every 13. Mu.L of reaction
					PerfeCTa MM	Quantabio	5x	1x	3.00μL
RPA1F	IDT	100μM	1μM	0.15μL
					RPA1 external R	IDT	100μM	1μM	0.15μL
FAM probe	IDT	100μM	0.3μM	0.045μL
					Water and its preparation method	ThermoFisher	NA	NA	9.66μL

QPCR reactions were performed using the following thermal procedure, steps b, c and d were repeated in sequence for 40 cycles:

a.95℃for 60 seconds, b.95℃for 10 seconds, c.64℃for 25 seconds, d.72℃for 10 seconds.

Results and conclusions

The target DNA was enriched using a PCR reaction. The PCR2 output was quantified using a Bioanalyzer and found to be 80.0 ng/. Mu.L. This corresponds to 1.88x10 ¹³ copies per 40 μl (used as "pure" condition). Samples were also taken as 1:10 dilutions to obtain individual "1:10 "input, i.e. 1.88x10 ¹² copies per 40 μl.

The copy control process normalizes the output of two samples with 10 times different input concentrations to nearly equal values (only 6% difference), demonstrating the ability of the process to normalize a wide range of input concentrations to the desired value, as shown in table 13.

TABLE 13

CC results	Copying
		Pure water	3.22E+09
1∶10	3.42E+09
		Difference value	6％

F. capturing a predetermined amount of amplicon using a capture oligomer comprising a capture sequence and its complement

An oligomer.

PCR was performed to amplify fragments of the uidA gene from e.coli (e.coli) using the following primers:

Ec_uidA_F(SEQ ID No.141)：GTATCAGCGCGAAGTCTTTATACC

Ec_uidA_R(SEQ ID No.142)：GGCAATAACATACGGAGTGACATC

Primers were designed to generate an amplicon (SEQ ID No. 143) with the following sequence:

A capture oligomer designated uida_pa_1.2 is provided, having the following sequence (SEQ ID No. 144):

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTCTA/iSp18/TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGACGCAAGCTACTGGTGATTTGGCAATAACATACGGAGTGACATCGGCTTC(iSp18＝ Hexaethylene glycol (HEG) internal spacer (IDT)

In this oligomer, the 5' poly-A sequence is the capture sequence. CCTCTA is a linker sequence. The iSp18 is an internal extension blocking sequence. The poly-T sequence following iSp18 is the complement of the capture sequence. AGACGCAAGCTACTGGTGATTT is a fourth additional sequence. The Target Hybridizing Sequence (THS) is GGCAATAACATACGGAGTGACATCGGCTTC, which hybridizes specifically to a segment of the uidA gene sequence in the target amplicon. In this example, the THS is longer than the reverse PCR primer (see above sequence) that overlaps it to increase the Tm of the THS and provide it with a competitive advantage over the reverse primer in terms of hybridization to the target.

Using a second capture reagent having the sequence:

dT ₂₀ -biotin (SEQ ID No. 145): TTTTTTTTTTTTTTTTTTTT/3' Biotin

The following primers and probes were used for quantitative PCR (qPCR) analysis of copy control products:

Ec_uidA_F(SEQ ID No.141)：

GTATCAGCGCGAAGTCTTTATACC

uidA_Probe (SEQ ID No. 146)

56-FAM/TAGCCGCCCTGATGCTCCATCACTTCCTG/3’IowaBlack

TQ_R(SEQ ID No.147)：AGACGCAAGCTACTGGTGAT

Scheme/reaction conditions

(1) Generating a PCR amplicon of the target uidA using the above primer; amplicons were purified using AMPure XP (Beckman Coulter) according to the manufacturer's recommended protocol and quantified by qPCR using uidA forward and reverse primers and uidA_probe.

(2) Capture oligo annealing and extension of amplicon strand-the purified uidA amplicon was diluted 2-, 10-or 100-fold. An aliquot of 20. Mu.L of each dilution of amplicon was added to a 30. Mu.L final capture oligomer annealing/extension reaction consisting of 0.07U/. Mu. L SDPol (Bioron), 1X SDPol reaction buffer, 0.17mM dNTP's, 3mM MgCl ₂, 1mg/ml BSA and 5X 10 ¹⁰ copies of capture oligomer. The capture oligomer was annealed to the 3' end of the complementary strand of the uidA amplicon and the amplicon strand was extended using a thermal cycler according to the following temperature control scheme: 92℃for 2 minutes, 54℃for 2 minutes, 68℃for 10 minutes, 54℃for 2 minutes, and then controlled cooling (0.3 ℃/sec) to 20 ℃. In this example, the 3' end of the capture oligomer is also extended.

(3) Hybridization of the complementary sequences of the capture oligomers-the whole capture oligomer/amplicon extension reaction mixture was added to 10 μl of the second capture reagent at a concentration of 4X, resulting in a final concentration of 125mM NaCl, 0.25mg/ml BSA, and 10 ⁷, 10 ⁸, or 10 ⁹ copies of the complementary sequences of the capture sequences (i.e., 3 different amounts were tested). Hybridization was performed by incubating the reaction mixture at room temperature (20 ℃ C. To 24 ℃ C.) for 15 minutes.

(4) Capture of amplicon and Capture oligo extension product/Capture oligo complex-5. Mu.L aliquots (50. Mu.g) of MyOneC < 1 > streptavidin beads (ThermoFisher Scientific) in 250mM NaCl and 1mg/ml BSA were added to the hybridization mixture (step 3 above), the complementary sequence complexes of Capture oligo/amplicon extension product/Capture sequence were captured on the beads and the beads were washed according to manufacturer's recommendations.

(5) Elution-after the final wash was completed and the wash buffer was removed, 10 μl of water was added to the bead mass, the beads were resuspended and incubated at 70 ℃ for 2 minutes. The beads were agglomerated using a magnet and the eluate was removed.

(6) Quantification-the amount of eluted product as well as capture oligomer extension product was quantified by qPCR using primers targeting the uida_f primer site and TQ primer-adaptor site as well as uida_probe (see step 2 above).

Results and conclusions:

Copy control procedures (see steps 2-5 above) were performed on 2-fold, 10-fold and 100-fold dilutions of the amplicons produced in the targeted enrichment step (see step 1 above) using 10 ⁷, 10 ⁸ or 10 ⁹ copies of the capture oligomer. As shown in Table 14, the amount of amplicon recovered was proportional to the amount of capture oligomer added to each PCR dilution (approximately at the desired ratio; see "ratio" column of the table). The differences between the duplicate data for each data point are small (see "standard deviation" column).

TABLE 14

* Average of 3 replicates

* Set the value of 2-fold dilution equal to 1

Furthermore, despite the 50-fold difference in amplicon input, the output varies 2.36-fold, 3.11-fold and 3.44-fold for 10 ⁷, 10 ⁸ and 10 ⁹ copies of the captured oligomer, respectively (table 1). As described above, the difference between the duplicate data is small.

These data indicate that the capture oligomers described herein can be used to produce a predetermined amount of target output from a range of different target inputs.

Additional experiments were performed using capture oligomers substantially as described above, but in multiplex format, wherein the THS region was designed to target the uidA gene of escherichia coli, the nuc gene of staphylococcus aureus (Staphylococcus aureus), the vanA gene of enterococcus faecalis (Enterococcus faecallis) or the rpb7 gene of Candida albicans (i.e. 4 capture oligomers were used per reaction). The uidA, nuc and vanA genes were amplified using the primers shown above for uidA and the other primers designed for nuc and vanA genes, respectively, using PCR as described above. The resulting amplicon was diluted 10-fold or 100-fold and 20 μl aliquots of each dilution of each individual target were added to a different capture oligomer annealing and amplicon chain extension reaction containing 5×10 ¹⁰ copies of each of the 4 capture oligomers described above (i.e. 4-fold capture oligomers, but only 1 target). The reaction conditions were the same as those described, except that the following temperature control scheme was used: 92 ℃ for 2 minutes, 64 ℃ for 2 minutes, 68 ℃ for 10 minutes, 98 ℃ for 2 minutes, 57 ℃ for 2 minutes, followed by controlled cooling (0.3 ℃/sec) to 20 ℃. After the reaction was completed, 5×10 ⁸ copies of the complement of the capture sequence of the capture oligomer were added to each reaction. The steps of capturing, washing, eluting and quantifying as described above are then performed.

The output after amplicon capture with a 10-fold difference in input is shown in table 15, which shows that for each of the 3 individual target amplicons tested in multiplex format, the 10-fold difference in amplicon input levels was reduced to no more than a 1.4-fold difference in average output level after controlled copying. Furthermore, the average output level range after controlled copying spans about 1.6 times for all 3 target amplicons, while their input level spans exceed 270 times.

TABLE 15

* Average of 3 replicates

Additional experiments were performed in a singleplex format, substantially as described above, but using capture oligomers in which THS anneal to universal binding sites in tag sequences introduced into the target of interest during the PCR amplification step. Primers were designed to target the bacterial 23S rRNA gene and PCR amplicons were generated from bacterial genomic DNA. The reverse primer contains a universal sequence tag that is incorporated into the amplicon during PCR. The capture oligomer annealing and extension of the amplicon strands was performed as described above, except that 40 μl of pure aliquots were added to a 100 μl final reaction volume of reaction mixture containing 0.02U/μl SD polymerase (Bioron), 0.4x SD polymerase reaction buffer (Bioron), 0.012mM of 4dntp's, 1.8mM MgCl ₂, 0.6mg/mL BSA, and 5 x 10 ¹⁰ copies of capture oligomer (i.e., No dilution; About 9 x 10 ¹² copies) or 50-fold dilutions (about 2 x 10 ¹¹ copies). The temperature control mode used was 92℃for 2 minutes, 54℃for 2 minutes, 68℃for 10 minutes, 54℃for 2 minutes, followed by controlled cooling (0.3℃per second) to 20 ℃. The entire volume of the reaction was added to 50. Mu.L of a 3 Xannealing mixture containing 1mg/ml BSA, 125mM NaCl and 5X 10 ⁸ copies of the second capture reagent. Annealing was performed by incubating the reaction mixture on a thermal block (thermal block) at 25℃for 10 minutes. A volume of 50. Mu.L (200. Mu.g in this experiment) of MyOneC streptavidin beads (ThermoFisher Scientific) in 4 Xwash buffer consisting of 4M NaCl, 20mM Tris-HCl pH7.5, 2mM EDTA, 0.20% Tween 20, 2mg/mL BSA was added to the 150. Mu.L reaction. Capturing the resulting complex comprising the capture oligomer, the amplicon extension product and the complement of the capture sequence on the beads and washing the beads according to manufacturer's recommendations (in this case, using 1X wash buffer; See 4X formulation above). Elution was performed using a volume of 20 μl of water (other aspects of the protocol are the same as described above). qPCR was performed using specific forward and reverse primers targeting the universal tag.

Despite the 50-fold difference in amplicon input levels, the change in output after the capture oligomer annealing and amplicon chain extension steps was only 2.5-fold, and after contact with the second capture reagent and separation of the resulting complex, the change in output was only 1-fold (i.e., complete normalization) (see table 16). Furthermore, these results demonstrate embodiments of the present disclosure in which THS binds to a universal tag sequence.

Table 16

* Pure/50-fold dilution output

The capture oligomers were used substantially as described immediately above (singleplex, universal THS) but in multiplex form to perform additional experiments, the THS in the capture oligomers annealing to universal tag sequences introduced into the target of interest during the PCR amplification step. In eight separate single reactions, eight different amplicons targeting regions in bacterial 16S rRNA gene, 23S rRNA gene and antibiotic resistance marker KPC were generated from klebsiella pneumoniae (k.pneumaonia) genomic DNA. One amplicon targeting the synthesized Internal Control (IC) DNA was also generated for a total of nine individual amplicons. Equal amounts of all 9 amplicons were pooled and 54. Mu.L of either a pure aliquot (i.e., no dilution; about 1X 10 ¹³ copies) or a 10-fold dilution (about 1X 10 ¹² copies) was added to separate the extension reaction mixtures of the capture oligomer annealing and the amplicon strands (each at 100. Mu.L final volume). The remainder of the workflow was performed essentially the same as described above, except that 5 x 10 ¹¹ copies of the capture oligomer and 5 x 10 ⁹ copies of the second capture reagent were used. qPCR was performed using specific forward and reverse primers targeting the universal tag to quantify each target (9 separate PCRs). The recovery of each individual target is summed to determine the overall recovery of the capture process.

Despite the 10-fold difference in amplicon input levels, the total output after the capture oligomer annealing and amplicon chain extension steps was only 2.5, and after contact with the second capture reagent and separation of the resulting complex was only 1.6 (see table 17). Furthermore, these results demonstrate embodiments of the invention in which THS binds to the universal tag sequence in multiplex form, thereby capturing all target amplicons present in the mixture.

TABLE 17

* Pure/50-fold dilution output

G. capturing a predetermined amount of amplicon using a capture oligomer comprising a capture sequence, its complement and a clamp sequence

Target amplicons were prepared essentially as described above for uidA and used in experiments without or with capture oligomers with the embedded clip sequence (GCGCGC) as the first and third additional sequences (see fig. 3). The capture was performed using undiluted, 10X diluted and 100X diluted amplicons essentially as described above. The amount of captured product was quantified substantially as described above. The results are shown in fig. 14. The use of capture oligomers containing clip sequences improves the ability to normalize output for different amplicon dilution fold spans.

H. capturing a predetermined amount of amplicons using capture oligomers and complementary oligomers

An oligomer.

PCR was performed to amplify segments of vanA gene from enterococcus faecium (e.faecium) using the following primers:

Efm_vanA_F(SEQ ID No.148)：GGCTGCGATATTCAAAGCTCAG

Efm_vanA_R(SEQ ID No.149)：CTGAACGCGCCGGCTTAAC

primers were designed to generate an amplicon (SEQ ID No. 150) with the following sequence:

A capture oligomer designated cc_blo_vana_001 is provided having the following sequence (SEQ ID No. 151):

AAAAAAAAAAAAAAAAAAAA/iSp18/CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTCCTGAACGCGCCGGCTTAAC (iSp18=hexaethylene glycol (HEG) internal spacer (IDT))

The oligomer includes the elements shown in the exemplary capture oligomer of fig. 10A. In the oligomer, the 5' poly-A sequence is a capture sequence having a first portion and a second portion. The iSp18 is an internal extension blocking sequence. CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC is a spacer sequence having a first portion and a second portion. The Target Hybridization Sequence (THS) is CTGAACGCGCCGGCTTAAC, which hybridizes specifically to a segment of the vanA gene sequence in the target amplicon.

A complementary oligomer designated blocking sequence _vana_001 is provided comprising the elements shown in the exemplary complementary oligomer of fig. 10A, having the following sequence (SEQ ID No. 152): CGGTGCCAGAGGAGTTTTTTTTTT/invdt/, wherein invdt is the inverted T nucleotide, which serves as blocking moiety. In this oligomer CGGTGCCAGAGGAG is the complement of the first part of the spacer sequence of the capture oligomer and TTTTTTTTTT is the complement of the second part of the capture sequence

Using a second capture reagent having the sequence:

dT ₂₀ -biotin (SEQ ID No. 145): TTTTTTTTTTTTTTTTTTTT/3' Biotin

The following primers and probes were used for quantitative PCR (qPCR) analysis of copy control products:

vanA_PCR2_Fwd(SEQ ID No.153):TTGTATGGACAAATCGTTGACATACA

Efm_Probe_FAM (SEQ ID No. 154):

5'FAM/TGCTGGGATAGCTACTCCCGCCTTTTGG/3'IowaBlack

CC_Univ_Inner_Rev(SEQ ID No.155):ACCGTGCTGCCTTGGCTTC

Scheme/reaction conditions.

(1) PCR amplicons of target vanA were generated using the Efm_vanA_F and Efm_vanA_R primers shown above; amplicon was quantified by chip-based capillary electrophoresis using Agilent BioAnalyzer.

(2) Capture oligo annealing and extension of amplicon strands-vanA amplicon was used undiluted (neat) or diluted 10-fold (about 2X 10 ¹³ copies and 2X 10 ¹² copies, respectively). mu.L aliquots of each amplicon amount (pure and 10-fold diluted) were combined with 20. Mu.L of the capture oligomer annealing/extension reaction mixture, resulting in a final mixture consisting of 0.02U/. Mu. L DEEP VENT (exo-) Pol (NEB), 0.4x Deep Vent Vent Pol reaction buffer, 0.012mM dNTP's, 1.8mM MgCl ₂、0.6mg/ml BSA、1×10¹¹ copies of capture oligomer (CC_Blo_vanA_001) and 1X 10 ¹² copies of complementary oligomer (blocking sequence_vanA_001), with a final volume of 100. Mu.L. The capture oligomer was annealed to the 3' end of the complementary strand of the vanA amplicon and the amplicon strand was extended using a thermal cycler according to the following temperature control scheme: 92℃for 2 minutes, 64℃for 2 minutes, 68℃for 10 minutes. In this example, the 3' end of the capture oligomer is also extended.

(3) Hybridization of the complementary sequences of the capture oligomers-50. Mu.L of a 3 Xconcentration of the second capture reagent was added to the whole capture oligomer/amplicon extension reaction mixture, thus obtaining a final concentration of 42mM NaCl,0.33mg/ml BSA and 10 ⁹ copies of the complementary sequences of the capture sequences (dT ₂₀ -biotin). Hybridization was performed by incubating the reaction mixture at 30℃for 10 minutes.

(4) Capture of amplicon and Capture oligomer extension product/Capture oligomer complex-50. Mu.L aliquots (200. Mu.g) of streptavidin-coated magnetic beads were added to the entire hybridization mixture (150. Mu.L), yielding a final concentration of 1M NaCl, 5mM TrisHCl (pH 7.5), 0.5mM EDTA, 0.05% Tween 20, and 0.5mg/ml BSA. The complexes were captured on the beads at 25 ℃, and the beads were washed using a wash reagent having the same composition as detailed immediately above.

(5) Elution-after the final wash was completed and the wash buffer was removed, 30 μl of water was added to the bead mass, the beads were resuspended and incubated at 70 ℃ for 2 minutes. The beads were agglomerated using a magnet and the eluate was removed.

(6) Quantification-the amount of eluted product as well as capture oligomer extension product was quantified by qPCR using primers targeting vana_pcr2_fwd primer site and universal primer-adapter site (cc_ Univ _inner_rev) and efm_probe_fam (see step 2 above).

Results and conclusions:

The 0-fold (pure) and 10-fold dilutions of the amplicons produced in step 1 (PCR) above were subjected to a copy control procedure using 10 ⁹ copies of the second capture oligomer (see steps 2 to 5 above). As shown in table 18, the output levels were substantially the same despite the 10-fold difference in target input.

TABLE 18

PCR dilution	Output (#copy)	Fold difference
			Pure and pure	3.24E+08	-
10 Times of	3.14E+08	1.03

These data indicate that the capture oligomers and complementary oligomers described herein can be used to produce a predetermined normalized target output when target input spans a 10-fold difference.

Additional experiments were performed essentially as described above, with the following differences:

(1) The PCR amplicons were purified using QIAGEN QIAquick PCR purification kit according to the manufacturer's instructions and then quantified by chip-based capillary electrophoresis using Agilent BioAnalyzer.

(2) Capture oligo annealing and extension of amplicon strands-vanA amplicon was used undiluted (pure), 10-fold diluted and 100-fold diluted (about 8X 10 ¹¹ copies, 8X 10 ¹⁰ copies and 6X 10 ⁹ copies, respectively). The capture oligomer was used at1×10 ¹² copies/reaction of capture oligomer (cc_blo_vana_001) and the complementary oligomer was used at zero or 1×10 ¹³ copies/reaction (blocking sequence_vana_001). The capture oligomer was annealed to the 3' end of the complementary strand of the vanA amplicon and the amplicon strand was extended using a thermal cycler according to the following temperature control scheme: 95℃for 2 minutes and 64℃for 15 minutes. All other conditions in this step are the same as in step 2 above.

(3 To 6) steps 3 to 6 were performed as described in steps 3 to 6 above, except that in step 4, the complex was captured on the beads at 30℃instead of 25 ℃.

Results and conclusions:

The amplicons produced in step 1 (PCR) above were subjected to copy control procedures on 0-fold (pure), 10-fold and 100-fold dilutions (see steps 2 to 5 above). In this experiment, the amounts of the various nucleic acid components used were approximately 8×10 ¹¹, 8×10 ¹⁰ and 6×10 ⁹ copies of the target, 1×10 ¹² copies of the capture oligomer, 0 or 1×10 ¹³ copies of the complementary oligomer and 1×10 ⁹ copies of the second capture oligomer. Table 19 shows the results with or without complementary oligomers.

TABLE 19

* Target amplicon generated in step 1

In the absence of complementary oligomers, the second capture oligomer (dT ₂₀ -biotin) can bind to any capture oligomer molecule, whether or not the capture oligomer binds to the target. In this experiment, 1×10 ¹² copies of the capture oligomer and 1×10 ⁹ copies of the second capture oligomer were used, i.e., there was a 1000-fold difference in these amounts. At the highest target level (pure), most of the capture oligomers will bind to the target, so most of the second capture oligomers will bind to the capture oligomers associated with the target, and the output copy after capture and elution will be relatively high. This was confirmed in data of pure target input (no complementary oligomer), where a relatively high output (9.3X10 ⁷ copies) was observed. However, for a 10-fold dilution of the target, there will be an excess of capture oligomers, and therefore not all capture oligomers will bind to the target. Some of the second capture oligomers will bind to capture oligomers associated with the target, but some of the second capture oligomers will capture oligomers that do not bind to the target. Thus, as actually observed, the output will drop (output = 2.6x10 ⁷ copies). For a 100-fold dilution of the target, a majority of the capture oligomers will not bind to the target, and thus, as such, a majority of the second oligomers will bind to capture oligomers that do not associate with the target. Thus, the expectation under these conditions is a significantly reduced output, which is actually observed (output=5.0×10 ⁵ copies).

In the presence of the complementary oligomer, the capture oligomer that is not bound to the target will bind to the complementary oligomer, which in turn will block the binding of the second capture reagent. Conversely, the capture oligomer that has bound to the target will not bind to the complementary oligomer (which has been displaced), which in turn will enable the second capture reagent to bind. Thus, at all target input levels tested in this experiment, the output in the presence of the complementary oligomer was expected to be higher than in the absence of the complementary oligomer (the results are as described above). This was what was observed (see table 6). Furthermore, the data demonstrate that normalization occurs with only about a 2-fold difference between the output of the pure target level and the output of the 10-fold diluted target level, and only a slight more than 14-fold difference between the output of the pure target level and the output of the 100-fold diluted target level. The output of the 100-fold target dilution is slightly below theoretical because the binding kinetics are slower due to the low level of target. If the incubation time is longer, the output will be increased and the normalization factor will be improved.

These data indicate that the capture oligomers and complementary oligomers described herein can be used to produce a predetermined normalized target output when target input spans a 100-fold difference.

I. Capture products comprising additional sequences (e.g., adaptors) at both ends of the target sequence are generated using capture oligomers, complementary oligomers, displacement oligomers, and forward primers

An oligomer.

PCR was performed to amplify fragments of the vanA gene from enterococcus faecium using the following primers:

Efm_vanA_F(SEQ ID No.148):GGCTGCGATATTCAAAGCTCAG

Efm_vanA_R(SEQ ID No.149):CTGAACGCGCCGGCTTAAC

primers were designed to generate an amplicon (SEQ ID No. 150) with the following sequence:

a capture oligomer designated PCR2 r_adapter_cc is provided having the following sequence (SEQ ID No. 156):

(iSp18=hexaethylene glycol (HEG) internal spacer (IDT))

The oligomer comprises the elements shown in the exemplary capture oligomer of fig. 10A. In the oligomer, the 5' poly-A sequence is a capture sequence having a first portion and a second portion. The iSp18 is an internal extension blocking sequence. CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC is a spacer sequence having a first portion and a second portion. The Target Hybridization Sequence (THS) is GTAGCTGCCACCGGCCTAT, which hybridizes specifically to a segment of the vanA gene sequence in the target amplicon.

A complementary oligomer designated blocking sequence _vanA_001 is provided comprising the elements shown in the exemplary complementary oligomer of FIG. 10A, having the sequence (SEQ ID No. 152) CGGTGCCAGAGGAGTTTTTTTTTT/invdt/, wherein invdt is a reverse T nucleotide, which serves as the blocking moiety. In this oligomer CGGTGCCAGAGGAG is the complement of the first part of the spacer sequence of the capture oligomer and TTTTTTTTTT is the complement of the second part of the capture sequence.

Efm_vanA_R (sequence above), which contains the elements shown in the exemplary displacement oligomer of FIG. 8A. Also provided are oligomers designated as PCR1 F_adaptors comprising the elements shown in the exemplary forward primer with adaptors of FIG. 8A, PCR1 F_adaptors having the following sequence (SEQ ID No. 157): AAAACGAGACATGCCGAGCATCCGCGGCTGCGATATTCAAAGCTCAG.

Using a second capture reagent having the sequence:

dT ₂₀ -biotin (SEQ ID No. 145): TTTTTTTTTTTTTTTTTTTT/3' biotin

The following primers and probes were used for quantitative PCR (qPCR) analysis of copy control products:

CCRPA_uni_F(SEQ ID No.158):AAAACGAGACATGCCGAGCATC

Efm_Probe_FAM (SEQ ID No. 154) 5'FAM/TGCTGGGATAGCTACTCCCGCCTTTTGG/3' IowaBlack

CC_Univ_Inner_Rev(SEQ ID No.155):ACCGTGCTGCCTTGGCTTC

Scheme/reaction conditions

PCR amplicons of target vanA were generated using the Efm_vanA_F and Efm_vanA_R primers shown above; amplicon was purified using QIAGEN QIAquick PCR purification kit according to manufacturer's instructions and then quantified by chip-based capillary electrophoresis using Agilent BioAnalyzer.

Annealing and extension of Capture oligo, displacement oligo and Forward primer with adapter-aliquots containing about 1X 10 ¹² copies of vanA amplicon were combined with annealing/extension reaction mixture, resulting in a final mixture consisting of 0.02U/. Mu. L DEEP VENT (exo-) Pol (NEB), 0.4X Deep Vent Pol reaction buffer, 0.012mM dNTP's, 1.8mM MgCl ₂、0.6mg/ml BSA、5×10¹³ copies of Capture oligo (PCR 2 R_adaptor_CC), + -1X 10 ¹³ copies of displacement oligo (Efm_vanA_R) and 5X 10X ¹³ copies of forward primer with adapter (PCR 1 F_adaptor) in a final volume of 100. Mu.L. Annealing and extension of the capture and displacement oligomers with the input amplicon and the forward primer with adaptors with the extension product of the capture oligomer all take place in the same annealing/extension reaction, wherein the annealing and extension takes place using a thermal cycler according to the following temperature control scheme: 95℃for 5 minutes and then 64℃for 20 minutes.

Quantification-aliquots of each annealed extension reactant were diluted 100-fold and the amount of product contained in each aliquot was quantified by qPCR using primers CCRPA _uni_f and cc_ Univ _inner_rev (forward and reverse targeting universal adapter regions, respectively) and efm_probe_fam.

Results and conclusions:

Single cycle annealing and extension reactions were performed using the above-described input targets and oligomers (single cycle was defined as only 1 denaturation step, e.g. incubation at 95 ℃; another cycle would start with another thermal denaturation step). As shown in Table 20, products containing universal adaptors at both ends of the molecule were formed as demonstrated by amplification using universal primers.

Table 20

Replacement oligomer	Output (#copy)
		+	1.3E+11
-	2.8E+11

These data demonstrate that the embodiment of the invention depicted in fig. 8A can be used to produce a product with adaptors (or other desired sequences) at both ends of the molecule using single cycle annealing/extension. Furthermore, these data indicate that at least one primer-adaptor oligomer (in this case PCR2 r_adaptor_cc) can bind to internal sites in the target, not just to the ends. These data also indicate that the desired product can be produced without displacement of the oligomer. Without wishing to be bound by any particular theory, it is possible that different mechanisms may work in the disclosed embodiments to produce the desired product. In the presence of the displacement oligomer, a variety of mechanisms may be working to produce the observed results.

Additional experiments were performed essentially as described above, with the following differences.

(2) Annealing and extension of Capture oligo, displacement oligo and Forward primer with adapter-an aliquot containing approximately 1X 10 ¹³ copies of vanA amplicon was mixed with the annealing/extension reaction mixture, resulting in a final mixture consisting of 0.02U/. Mu. L DEEP VENT (exo-) Pol (NEB), 0.4x Deep Vent Pol reaction buffer, 0.012mM dNTP's, 1.8mM MgCl ₂、0.6mg/ml BSA、5×10¹⁴ copies of Capture oligo (PCR 2 R_adapter_CC) and 5X 10 ¹⁴ copies of Forward primer with adapter (PCR 1 F_adapter) in a final volume of 100. Mu.L. Annealing and extension of the mixture was performed using a thermal cycler according to the following temperature control scheme: 95℃for 5 minutes and then 64℃for 15 minutes. At this point, 5×10 ¹⁴ copies of the displaced oligomer (efm_vana_r) were added to some replicates of the reaction mixture, and for some samples, only buffer was added and annealing and extension continued using the following temperature control scheme: 64℃for 5 minutes, 75℃for 5 minutes and 72℃for 15 minutes.

Results and conclusions:

Single cycle annealing and extension reactions were performed using the above-described input targets and oligomers (single cycle was defined as only 1 denaturation step, e.g. incubation at 95 ℃; another cycle would start with another thermal denaturation step). The displacement oligomer is added to the annealing and extension reactions partway through the process to further optimize performance. As shown in Table 21, products containing universal adaptors at both ends of the molecule were formed as demonstrated by amplification using universal primers.

Table 21

Replacement oligomer	Output (#copy)
		+	3.2E+12
-	1.9E+12

As described above, these data demonstrate that the embodiment of the invention depicted in fig. 8A can be used to produce a product with adaptors (or other desired sequences) at both ends of the molecule using single cycle annealing/extension. Also as described above, these data demonstrate that at least one primer-adaptor oligomer (in this case PCR2 r_adaptor_cc) can bind to an internal site in the target, not just to the ends. Furthermore, these data demonstrate that by adjusting the annealing and extension temperature control regime, and in this case by adding the displacement oligomer halfway through the process, overall performance can be improved. It is particularly noteworthy that under these conditions, when the metathesis oligomer was present, the amount of the desired product was produced was greater than when no metathesis oligomer was present, indicating that the metathesis protocol was run as shown in fig. 8A. Also without wishing to be bound by any particular theory, different mechanisms may also work in the disclosed embodiments to produce the desired product.

Scheme/reaction conditions (2)

PCR amplicons of target vanA were generated using the Efm_vanA_F and Efm_vanA_R primers shown above; amplicon was purified using QIAGEN QIAquick PCR purification kit according to the manufacturer's instructions and then quantified by chip-based capillary electrophoresis using Agilent BioAnalyzer.

Annealing and extension of Capture oligo, displacement oligo and Forward primer with adapter-aliquots containing about 1X 10 ¹² copies of vanA amplicon were combined with annealing/extension reaction mixture, resulting in a final mixture consisting of 0.02U/. Mu. L DEEP VENT (exo-) Pol (NEB), 0.4X Deep Vent Pol reaction buffer, 0.012mM dNTP's, 1.8mM MgCl ₂、0.6mg/ml BSA、5×10¹³ copies of Capture oligo (PCR 2 R_adaptor_CC), + -5X 10 ¹² copies of displacement oligo (Efm_vanA_R) and 5X 10X ¹³ copies of forward primer with adapter (PCR 1 F_adaptor) in a final volume of 100. Mu.L. Annealing and extension of the capture and displacement oligomers with the input amplicon and the forward primer with adaptors with the extension product of the capture oligomer all take place in the same annealing/extension reaction, wherein the annealing and extension takes place using a thermal cycler according to the following temperature control scheme: 95℃for 5 minutes and then 64℃for 20 minutes.

Hybridization of the complementary sequences of the capture oligomers-50. Mu.L of a 3 Xconcentration of the second capture reagent was added to the whole extension reaction mixture, thus obtaining a final concentration of 42mM NaCl,0.33mg/ml BSA and 5X 10 ¹⁴ copies of the complementary sequences of the capture sequences (dT ₂₀ -biotin). Hybridization was performed by incubating the reaction mixture at 30℃for 10 minutes.

Capture of amplicon and Capture oligomer extension products/Capture oligomer complexes-50. Mu.L aliquots (200. Mu.g) of streptavidin-coated magnetic beads were added to the entire hybridization mixture (150. Mu.L), yielding final concentrations of 1M NaCl, 5mM TrisHCl (pH 7.5), 0.5mM EDTA, 0.05% Tween 20, and 0.5mg/ml BSA. The complexes were captured on the beads at 30 ℃, and the beads were washed using a wash reagent having the same composition as detailed immediately above.

Elution-after the final wash was completed and the wash buffer was removed, 30 μl of water was added to the bead mass, the beads were resuspended and incubated at 70 ℃ for 2 minutes. The beads were agglomerated using a magnet and the eluate was removed.

Quantification-eluted products were quantified by qPCR using primers CCRPA _uni_f and cc_ Univ _inner_rev (targeting universal adapter regions in forward and reverse directions, respectively) and efm_probe_fam.

Results and conclusions:

the single cycle annealing and extension reactions were performed using the above-described input targets and oligomers, and the products were captured, washed, eluted, and then quantified using qPCR. As shown in Table 22, as demonstrated by amplification using universal primers, a product was formed that contained universal adaptors at both ends of the captured and eluted molecules.

Table 22

Replacement oligomer	Output (#copy)
		+	1.4E+09
-	1.5E+10

These data demonstrate that the embodiment of the invention depicted in fig. 8A can be used to produce a product with adaptors (or other desired sequences) at both ends of the molecule using single cycle annealing/extension, and that the product can be isolated by capturing onto beads, washing and eluting. Furthermore, these data demonstrate that at least one primer-adaptor oligomer (in this case PCR2 r_adaptor_cc) can bind to internal sites in the target, not just to the ends. These data also indicate that when no displacement oligomer is present, the desired product can be produced. Without wishing to be bound by any particular theory, it is possible that different mechanisms may work in the disclosed embodiments to produce the desired product. In the presence of the displacement oligomer, a variety of mechanisms may be working to produce the observed results.

Additional experiments were performed essentially as described above, with the following differences:

The annealing and extension-annealing/extension reaction mixtures of the capture oligomer, the displacement oligomer and the forward primer with the adapter are the same as described above, except that a new sample is added which also contains 5×10 ¹⁴ copies of the complementary oligomer (blocker_vana_001). Annealing and extension of the capture oligomer and the displacement oligomer to the input amplicon, annealing of the complementary oligomer to the capture oligomer, and annealing and extension of the forward primer with the adapter to the extension product of the capture oligomer all take place in the same annealing/extension reaction, wherein the use of a thermal cycler is performed in a temperature controlled manner as shown below:

a.95℃for 5 minutes, b.75℃for 30 seconds, c.74℃for 30 seconds, d.73℃for 30 seconds, e.72℃for 30 seconds, f.71℃for 30 seconds, g.70℃for 30 seconds, h.69℃for 2 minutes, i.68℃for 2 minutes, j.67℃for 2 minutes, k.66℃for 2 minutes, l.65℃for 12 minutes.

Hybridization of the complementary sequences of the capture oligomers-the conditions were the same as above, except that 1X10 ⁹ copies of the complementary sequences of the capture sequences (dT ₂₀ -biotin) were used.

Results and conclusions:

Single cycle annealing and extension reactions were performed using the above-described input targets and oligomers, the product was captured using a predetermined amount of the complement of the capture sequence (dT ₂₀ -biotin), washed, eluted, and then quantified using qPCR. The results are shown in Table 23.

Table 23

PCR dilution	Output (#copy)	Fold difference
			Pure and pure	2.0E+06	-
10 Times of	4.3E+05	4.7

These data demonstrate that the embodiment of the invention depicted in fig. 8A can be used to produce a product with adaptors (or other desired sequences) at both ends of the molecule using single cycle annealing/extension, and that the product can be isolated by capturing onto beads, washing and eluting. Furthermore, the 10-fold difference in input target level was normalized to 4.7-fold difference, which exceeded the 2-fold normalization factor. Furthermore, these data demonstrate that at least one primer-adaptor oligomer (in this case, PCR2 r_adaptor_cc) can bind to internal sites in the target, not just to the ends.

Additional experiments were performed essentially as described above, with the following differences:

(2) The annealing and extension-annealing/extension reaction mixtures of the capture oligomer, the displacement oligomer and the forward primer with the adapter are the same as described above, except that 1e+13 and 1e+12 (10-fold dilution) copies/reacted input targets are used; new samples were added that also contained 5X 10 ¹⁴ copies of the complementary oligomer (blocker_vanA_001). Annealing and extension of the capture oligomer to the input amplicon, annealing of the complementary oligomer to the capture oligomer, and annealing and extension of the forward primer with the adapter to the extension product of the capture oligomer all occur in the same annealing/extension reaction, wherein the annealing and extension reactions are performed using a thermal cycler according to the following temperature control scheme: 95℃for 5 minutes and then 64℃for 15 minutes.

(3) Hybridization of the complementary sequences of the capture oligomers-the conditions were the same as above, except that 1X 10 ⁹ copies of the complementary sequences of the capture sequences (dT ₂₀ -biotin) were used. Hybridization was performed at 30℃for 30 minutes.

Results and conclusions:

Single cycle annealing and extension reactions were performed using the above-described input targets and oligomers, the product was captured using a predetermined amount of the complement of the capture sequence (dT ₂₀ -biotin), washed, eluted, and then quantified using qPCR. The results are shown in Table 24.

Table 24

These data demonstrate that the embodiment of the invention depicted in fig. 8A can be used to produce a product with adaptors (or other desired sequences) at both ends of the molecule using single cycle annealing/extension, and that the product can be isolated by capturing onto beads, washing and eluting. In addition, when complementary oligomers were present, the 10-fold difference in input target levels was normalized to 0.67-fold difference (-1). Without the complementary oligomer, the recovery of the product was reduced by more than 10-fold, as expected, while the normalization was similar. Furthermore, these data demonstrate that at least one primer-adaptor oligomer (in this case PCR2 r_adaptor_cc) can bind to internal sites in the target, not just to the ends.

J. in-well amplification of target nucleic acids using solution-mediated recombinase polymerase amplification (SM-RPA)

Oligomer

In-well amplification was performed using a solution primer (HDA 72F) having the following sequence and a primer (extHDA R) immobilized on the well surface:

HDA72F(SEQ ID No.159):5'-AAAACGAGACATGCCGAGCATCCGC-3'extHDA72R(SEQ ID No.160):5'-AmmC12-AAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCCCCiSp18-iSp18-iSp18-iSp18-iSp18/-AAAAAAACTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC-3'(5'-AmmC12＝5'- Terminal C-12 amine and isp18=hexaethylene glycol (HEG) internal spacer (both from IDT)). Synthetic DNA templates representing the micrococcus nuclease gene (nuc) from staphylococcus aureus, the carbapenemase gene (Kpc) from klebsiella pneumoniae, and the Qin prophage protein YdfU gene (ydfU) from escherichia coli, each having the following sequences (including underlined adaptor sequences):

nuc template (SEQ ID No. 161):

kpc die (SEQ ID No. 162):

ydfU template (SEQ ID No. 163):

The synthetic DNA probes were used to detect amplified products from the above templates, each amplified product having the following sequence:

nuc probe (SEQ ID No. 164):

5'-Cy3-TCGAAAGGGCAATACGCAAAGAG-3' (5 '-Cy3=5' -terminal Cy3 fluorophore)

Kpc probe (SEQ ID No. 165):

5'-Alex488N-TAAACTCGAACAGGACTTTGGCG-3' (5 '-Alex488 n=5' -terminal Alexa488 fluorophore)

YdfU probe (SEQ ID No. 166):

5'-Cy5-TGCGGGTATTACTTAGACCTGTTC-3' (5 '-Cy5=5' -terminal Cy5 fluorophore)

Semiconductor chip

The DNAe semiconductor chips used in this embodiment are fabricated using standard CMOS methods and include an array of Ion Sensitive Field Effect Transistor (ISFET) sensors whose voltage outputs are responsive to changes in pH in the fluid solution residing in the wells above the IC. The holes are micron-sized and are produced by standard photolithographic processes. Custom flow cell devices are mounted on top of the IC and well assembly to facilitate fluid delivery over the chip surface. The oligonucleotides were coupled to the pore surface by first activating the pore surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide using standard click chemistry.

Buffer/reagent preparation

According to the formulation shown in table 25, a "buffer mixture" was prepared in an Eppendorf microcentrifuge tube. 20% Carbowax was heated at 65℃for 5 minutes. Water was added and the mixture was vortexed to homogeneity and then placed in a cooling block for 5 minutes. DTT and UvsX reaction buffer were added and the mixture was vortexed until homogeneous.

Table 25

The "energy mix" was prepared in a separate Eppendorf microcentrifuge tube by adding each of the components reported in table 26 in sequence (in the order shown) and then thoroughly mixing (no vortex).

Table 26

The "core mix" was prepared in a separate Eppendorf microcentrifuge tube by adding each of the components reported in table 27 in sequence (in the order shown) and then thoroughly mixing (no vortex).

Table 27

The buffer mixture, energy mixture and core mixture were all stored at 4 ℃ prior to use.

Scheme/reaction conditions

The flow cell was rinsed twice with 1 XRPA annealing buffer (20mM Tris,pH 7.5,5mM Mg (OAc) ₂, 150mM NaCl,0.01% Tween).

Synthetic DNA templates in 1 XRPA annealing buffer were loaded into the flow cell in a volume of 25. Mu.L. In this example, each target was tested as an equimolar mixture of nuc, kpc and YdfU in 10 ⁶、10⁷ or 10 ⁸ copies for each reaction.

The DNA template was annealed to the primer (extHDA R) immobilized on the well surface using the following thermal procedure:

a.95℃for 2 minutes, b.60℃for 5 minutes, c.25℃for 15 minutes.

The solution was removed and the chip was washed twice with 50 μl RPA wash buffer (0.06 x ssc+0.06 tween 20).

The buffer mixture, enzyme mixture and core mixture were combined on a cooling block in the proportions shown in tables 1-3 (total amount of each reaction per 25. Mu.L) and 25. Mu.L was loaded onto the chip.

Amplification was then performed using the following thermal program:

a.43℃for 25 minutes and b.95℃for 3 minutes.

The chip was then incubated on a cooling block for 5-10 minutes, the reaction mixture was removed and the chip was washed twice with 50 μl RPA wash buffer.

The non-immobilized strand of the resulting amplified product was removed by loading 25. Mu.L of 20mM NaOH into the chip and incubating at ambient temperature (about 20-26 ℃) for 10 minutes. The NaOH solution was removed and the chip was washed twice with 50 μl RPA wash buffer.

Then a probe solution containing 1 XRPA annealing buffer and 2. Mu.M each of the nuc, kpc and ydfU fluorescent-labeled probes described above was prepared. Then 25 μl of this solution was loaded onto the chip and the probes were annealed to their respective targets (if present) using the following thermal procedure:

a.95 ℃ for 2 minutes, b.58 ℃ for 5 minutes, c. passive cooling to ambient temperature (about 20-26 ℃) for 15 minutes.

The reaction mixture was removed and the chip was washed twice with 50 μl RPA wash buffer. The chip was dried using a nitrogen gun and then imaged using a fluorescence microscope equipped with a filter, enabling specific detection of Alexa488, cy3 and Cy5 fluorophores.

Results and conclusions

Fluorescence microscopy images of 3 individual chips with 10 ⁶、10⁷ or 10 ⁸ starting copies of each synthetic DNA template showed plaques of amplified target DNA, distinguishable by three fluorophore colors (fig. 53). In summary, SM-RPA produces cloned, "sequenced" amplification products on the surface of a semiconductor chip in a short 25 minute amplification time.

K. In-well amplification of target nucleic acids using Rolling Circle Amplification (RCA)

Oligomer

In-well amplification was performed using two immobilized primers (both purchased from ATDBio) with the following sequences:

DBCO_A10H1_Li-dev[1]_R/SplintXL(SEQ ID No.167):

5'-DBCO-AAAAAAAAAA-isp18-GCTCGAATCAGTCCTGTCAGTCT*T*T*T-3'

DBCO_A10H1_dev[1]-F(SEQ ID No.168):

5'-DBCO-AAAAAAAAAA-isp18-TCCGCGGGAGCTTCAACAT*C*G*C*G-3'

(for both oligomers, 5 '-dbco=5' -terminal dibenzocyclooctyl, isp18=hexaethylene glycol (HEG) internal spacer and =phosphorothioate protection

Synthetic DNA templates (available from IDT Technologies) representing the D-alanine-D-alanine ligase gene (ddl) from enterococcus faecalis (Enterococcus faecalis) and the Qin prophage protein YdfU gene (ydfU) from escherichia coli (ESCHERICHIA COLI), each having the following sequences (including underlined adaptor sequences):

ddl template (SEQ ID No. 169):

ydfU template (SEQ ID No. 170):

synthetic DNA probes (available from IDT Technologies) were used to detect the amplified products from the above templates, each with the following sequence:

ddl probe (SEQ ID No. 171):

5'-Cy5-CAACGATTGCTCGAGAATCAT-3' (5 '-Cy5=5' -terminal Cy5 fluorophore)

YdfU probe (SEQ ID No. 172):

5'-Cy3-TGCGGGTATTACTTAGACCTGTTC-3' (5 '-Cy3=5' -terminal Cy3 fluorophore)

Semiconductor chip

The DNAe semiconductor chips used in this embodiment were fabricated using standard CMOS methods and included an Ion Sensitive Field Effect Transistor (ISFET) sensor array whose voltage output was responsive to changes in pH in the fluid solution residing in the wells above the IC. The holes are micron-sized and are produced by standard photolithographic processes. Custom flow cell devices are mounted on top of the IC and well assembly to facilitate fluid delivery over the chip surface. The oligonucleotides were coupled to the pore surface by first activating the pore surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide using standard click chemistry.

Buffer/reagent preparation

A "10 XRCA annealing buffer" was prepared according to the formulation shown in Table 28.

Table 28

Reagent(s)	Suppliers (catalog number)	Concentration of stock solution	Final concentration	Every 10mL
					Tris-HCL pH 7.5	ThermoFisher	1000mM	500mM	5mL
MgCl2	Sigma	1000mM	150mM	1.5mL
					NaCl	Sigma	5000mM	1200mM	2.4mL
Tween 20	Sigma	100％	0.1％	0.01mL
					Water and its preparation method	Sigma	-	-	1.09mL

Scheme/reaction conditions

The flow cell was rinsed twice with 1X RCA annealing buffer.

Synthetic DNA templates in 1 XRCA annealing buffer were loaded into the flow cell in a volume of 25. Mu.L. In this example, each target was tested as an equimolar mixture of ddl and YdfU in 5x10 ⁷ and 5x10 ⁸ copies for each reaction.

The DNA template was annealed to the reverse primer (DBCO_A10H2_Li-dev [1] _R/SplintXL) immobilized on the well surface using the following thermal procedure shown in Table 29.

Table 29

The solution was removed and the chip was washed twice with 50. Mu.L of RCA wash buffer (37.5 mM Tris-HCl pH 7.5, 10mM MgCl ₂、50mM KCl、2mM(NH₄)₂SO₄).

According to the formulation shown in table 30, a "ligation mixture" was prepared in an Eppendorf microcentrifuge tube.

Table 30

Reagent(s)	Suppliers (catalog number)	Liquid storage	Final concentration	Every 25. Mu.L of reaction
					Water and its preparation method	Sigma	-	-	21.25μL
T4 ligase buffer	NEB	10X	1X	2.50μL
					T4 ligase	NEB	2000U/μL	100U/μL	1.25μL

25. Mu.L of the ligation mixture was loaded onto the chip and incubated at 22℃for 30 minutes. The solution was removed and the chip was washed twice with 50 μl RCA wash buffer.

According to the formulation shown in table 31, an "amplification mix" was prepared in an Eppendorf microcentrifuge tube.

Table 31

25. Mu.L of the amplification mixture was loaded onto the chip and incubated at 45℃for 2 hours. The solution was removed from the chip and the reaction was inactivated by adding 25. Mu.L of "inactivation mix" (50 mM Tris-HCl pH 7.5, 50mM EDTA) and operating two times up and down using a pipette. The solution was removed and the chip was washed twice with 50 μl RCA wash buffer.

The strand of the amplified material was denatured by loading 25. Mu.L of 20mM NaOH into the chip and incubating for 5 minutes at ambient temperature (about 20 ℃ -26 ℃). The NaOH solution was removed and the chip was washed twice with 50 μl RCA wash buffer.

A probe solution containing 1 XRCA annealing buffer and 1. Mu.M of the ddl and ydfU fluorescence-labeled probes described in 004 above was then prepared. 25 microliters of this solution was then loaded onto the chip and the probes annealed to their respective targets (if present) using the following thermal procedure:

a.95 ℃ for 2 minutes, b.54 ℃ for 15 minutes, c. passive cooling to ambient temperature (about 20-26 ℃) for 15 minutes.

The reaction mixture was removed and the chip was washed twice with 50 μl RCA wash buffer. The chip was dried using a nitrogen gun and then imaged using a fluorescence microscope equipped with a filter capable of specifically detecting Cy3 and Cy5 fluorophores.

Results and conclusions

Fluorescence microscopy images of equimolar double-stranded branched surface phase RCA assays showed that for each of the two targets, discrete clusters of amplified target DNA could be distinguished by two different fluorophores (fig. 54). The density of clusters depends on the target copy number input. In summary, branched surface phase RCA produced cloned, "sequenced" amplification products on the surface of the semiconductor chip within 2 hours of amplification time.

L. sequencing of synthetic templates directly immobilized on chip surface

Oligomer

Sequencing was performed using a synthetic DNA template immobilized on the surface of the well (Oligo 1; ATDBio) and a sequencing primer complementary to the 3' region of the template (Oligo 2; IDT) (underlined nucleotides in Oligo-1) with the following sequence:

Oligo 1(SEQ ID No.173):

( 5 '-dbco=5' -terminal dibenzocyclooctyl; the underlined regions represent sequencing primer binding sites; the non-underlined region is a portion of the N gene of SARS-CoV-2 )

Oligo 2 (SEQ ID No. 174): 5'-GCCGACTAGATCCTCTG-3' (sequencing primer)

Semiconductor chip

The DNAe semiconductor chips used in this embodiment were fabricated using standard CMOS methods and included an Ion Sensitive Field Effect Transistor (ISFET) sensor array whose voltage output was responsive to changes in pH in the fluid solution residing in the wells above the IC. The holes are micron-sized and are produced by standard photolithographic processes. Custom flow cell devices are mounted on top of the IC and well assembly to facilitate fluid delivery over the chip surface. The oligonucleotides were coupled to the pore surface by first activating the pore surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide using standard click chemistry.

Buffer/reagent preparation

"Sequencing solutions" (7.5 mM MgCl ₂, 200mM NaCl, 0.02% TERGITOL NP-9) were prepared in glass flasks using deionized water (18 M.OMEGA.; merck Millipore), 1M magnesium chloride solution, 5M sodium chloride solution, and TERGITOL NP-9 (pure; merck). Briefly, mgCl ₂ and NaCl stock solutions were added to deionized water to give final concentrations of 7.5mM and 200mM, respectively. Add a magnetic stirring bar and mix the solution to homogeneity using a magnetic stirring plate. The solution was bubbled with nitrogen (minimum 99.998% nitrogen; BOC part # 44-W) by placing a nitrogen supply tube in the bottle (ensuring the tube reached the bottom) and nitrogen bubbling through the solution for 10 minutes, ensuring removal of dissolved carbon dioxide. Thereafter, a supply of nitrogen is maintained above the solution to ensure that it remains free of CO ₂. Mu.l TERGITOL NP-9 was added using an external piston pipette to give a final concentration of 0.02%. The mixture (under nitrogen) was stirred for an additional 10 minutes to completely dissolve TERGITOL.

A10. Mu.M solution of each individual natural nucleotide (dGTP, dCTP, dATP, dTTP) was prepared by adding 12.5. Mu.L of a 100mM stock solution of the selected nucleotide (FISHER SCIENTIFIC, 11843933) to 125mL of the sequencing solution. The pH of each dNTP solution was titrated to pH 8.05.+ -. 0.01 using 10mM NaOH (prepared from 10M NaOH stock; merck, 72068), using a pH probe (Sentron SIMicroFET (92270-010)). All dNTP solutions were prepared by mixing using a magnetic stirrer bar in an environment without CO ₂, nitrogen control.

In a CO ₂, nitrogen-free environment, mixing was performed using a magnetic stirring bar, and a "wash solution" was prepared by titrating the pH of the sequencing solution to 8.05.+ -. 0.01 using 10mM NaOH (prepared from a 10M NaOH stock; merck, 72068), using a pH probe (Sentron SIMicroFET (92270-010)).

The 1X annealing buffer was prepared by diluting a 20-fold stock of sodium chloride citrate buffer (Life Technologies) to a final 1X concentration of 150mM NaCl and 15mM sodium citrate using molecular grade water (Sigma).

A working solution of 5. Mu.M Oligo 2 (sequencing primer) was prepared by adding 1.25. Mu.L Oligo 2 (100. Mu.M stock) to 5. Mu.L 1X annealing buffer to give a final composition of 5. Mu.M sequencing primer in 0.8X annealing buffer.

25.4U/. Mu.L of "sequencing enzyme" working stock was prepared by diluting 1. Mu. L IsoPol BST + DNA polymerase (ArcticZymes, custom formulation) at 2 kU/. Mu.L in 79. Mu.L of sequencing solution (see preparation details above). The solution was thoroughly mixed by taking a volume of 20. Mu.L of the solution 10 times up and down.

Scheme/reaction conditions

The flow cell was rinsed twice with 1x thermo pol buffer [4.5mL 10X ThermoPol buffer (NEW ENGLAND Biolabs), 27 μl tween 20 (100% stock, MERCK LIFE SCIENCE, P9416), 40.5mL molecular grade water (Sigma) ].

The sequencing primer (5. Mu.M Oligo 2 in annealing buffer; see details of preparation above) was loaded into the flow cell. The inlet and outlet of the flow cell were sealed with custom plugs and the primers were annealed to the immobilized template (Oligo 1) using the following thermal procedure:

a.95℃for 120 seconds, b.90℃for 30 seconds, c.85℃for 30 seconds, d.80℃for 30 seconds, e.78℃for 120 seconds,

F. cooling from 77 ℃ to 64 ℃ (inclusive) at a rate of 1 ℃ per 30 seconds, g.63 ℃ for 120 seconds, h. cooling from 62 ℃ to 59 ℃ (inclusive) at a rate of 1 ℃ per 30 seconds, i.58 ℃ for 15 minutes, j. passive cooling to ambient temperature (about 20-26 ℃) for 2 minutes.

The flow cell was then washed with 200 μl of 1X thermo pol buffer to remove any excess primer.

The sequencing enzyme (25U/. Mu. L IsoPol BST +; see preparation details above) was loaded into the flow cell and incubated for 10 minutes at ambient temperature (about 20-26 ℃). The flow cell was rinsed with 200 μl 1x thermo pol buffer.

An initiation step (PRIMING STEP) was performed in which the wash solution was flowed through the chip at a rate of 5 mL/min. An electrical response test was performed by biasing the reference electrode with an increasing voltage step. The resulting change in mV output measured by the ISFET on the IC is used to determine the relationship between the reference electrode potential and the corresponding potential seen on the ISFET, which is then used to determine the optimal reference electrode potential for the experiment.

Sequencing is then performed cycle by cycle. In each sequencing cycle, 4 separate dNTP solutions (10. Mu.M each; see preparation details above) were each washed through the chip (15 seconds @5ml/min for each nucleotide) in sequence, and each nucleotide stream was separated with a washing step. The washing step is carried out in two stages: 1) "through-wash" to flush nucleotide solution from the fluid channel and flow cell, and 2) purge wash, wherein the wash channel bypasses the nucleotide valve device (nucleotide valving apparatus) to wash the chip, while the next nucleotide solution is directed to the waste container through the purge channel. When a nucleotide is introduced, subsequent proton release is detected by the integrated circuit as a voltage change.

After sequencing is complete, the data is analyzed using an internal proprietary algorithm for base calling and bioinformatics analysis.

Results and conclusions

The sequencing results obtained from the immobilized synthetic templates are shown in FIG. 55. Individual reads (sequencing fragments) are plotted on the x-axis as alignment read length (ARL; total alignment length from start to end position) versus alignment read length-error (ARL-e; total alignment length from start to end position minus the number of errors on the y-axis). The read without error will lie on the diagonal dashed line, where ARL-e=arl. The position of the perfect full length (56 bp) read on the graph is represented by a crosshair symbol. The histogram portion shown in fig. 55 represents the distribution of reads on different axes. The histogram at the top of the figure shows the distribution of reads along the x-axis, ARL (bp); the histogram on the right of the figure shows the distribution of reads along the y-axis, ARL-e (bp). In this embodiment, the total number of reads is 5899. The median of the ARL-e obtained was 54bp. The alignment length of the total reads was 56 bases and the error rate was 0% (Table 32). These data indicate that the system is able to sequence the immobilized template.

Table 32

Sequencing of synthetic templates Using direct hybridization methods

Oligomer

Direct hybridization was performed using a solution phase template (Oligo 3; IDT) with the following sequence and a primer (Oligo 4; ATDBio) immobilized on the surface of the well, which is complementary to the 3' region of the template (Oligo 3 underlined nucleotides), followed by a sequencing method:

Oligo 3(SEQ ID No.175):

Oligo 4(SEQ ID No.176):

5'-DBCO-CCGTTGACGTCATCCCCACCT-3' (5 '-dbco=5' -terminal dibenzocyclooctyl)

Semiconductor chip

The DNAe semiconductor chips used in this embodiment were fabricated using standard CMOS methods and included an Ion Sensitive Field Effect Transistor (ISFET) sensor array whose voltage output was responsive to changes in pH in the fluid solution residing in the wells above the IC. The holes are micron-sized and are produced by standard photolithographic processes. Custom flow cell devices are mounted on top of the IC and well assembly to facilitate fluid delivery over the chip surface. The oligonucleotides were coupled to the pore surface by first activating the pore surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide using standard click chemistry.

Buffer/reagent preparation

All buffers/reagents were prepared as described in the examples above (Oligo 2 working solution, which was not used in this experiment).

A0.5. Mu.M working solution of synthetic DNA template (Oligo 3) was prepared by adding 0.5. Mu.L Oligo 3 (100. Mu.M stock) to 99.5. Mu.L 1 Xannealing buffer.

Scheme/reaction conditions

Synthetic DNA templates (0.5. Mu.M Oligo 3 in annealing buffer; see details of preparation above) were loaded into the flow cell. The inlet and outlet of the flow cell were sealed with custom plugs and the template was annealed to the immobilized primer (Oligo 4) using the following thermal procedure:

a.95 ℃ for 120 seconds, b.90 ℃ for 30 seconds, c.85 ℃ for 30 seconds, d.80 ℃ for 30 seconds, e.78 ℃ for 120 seconds, f. cooling from 77 ℃ to 64 ℃ (inclusive) at a rate of 1 ℃ per 30 seconds, g.63 ℃ for 120 seconds, h. cooling from 62 ℃ to 59 ℃ (inclusive) at a rate of 1 ℃ per 30 seconds, i.58 ℃ for 15 minutes, j. passive cooling to ambient temperature (20-26 ℃) for 2 minutes.

Subsequent steps (addition of sequencing enzymes, priming steps, electrical response testing, sequencing and analysis) were performed as described in the working examples above.

Results and conclusions

The sequencing results obtained from the synthesis of templates from the solution hybridized to the immobilized primers (i.e., direct hybridization) are shown in FIG. 56. A single ready is plotted as ARL on the x-axis and ARS-e on the y-axis (see definition of ARL and ARL-x in the examples above). The read without error will lie on the diagonal dashed line, where ALR-e=arl. The position of perfect full length (91 bp) reads on the graph is represented by crosshair symbols. The histogram portion shown in fig. 56 represents the distribution of reads on different axes. The histogram at the top of the figure shows the distribution of reads along the x-axis, ARL (bp); the histogram on the right of the figure shows the distribution of reads along the y-axis, ARL-e (bp). In this embodiment, the total number of reads is 3829. The median of the ARL-e obtained was 85bp. The alignment length of the total reads was 97 bases and the error rate was 8.25% (Table 33). These data indicate that the system is capable of sequencing templates hybridized to immobilized primers.

Table 33

The notation between the two sequences represents the following: i=perfect match, > = missing, < = insert, = mismatch.

Sequencing of templates generated using in-well clonal amplification

Oligomer

The following oligomers were used for in-well clonal amplification:

Oligo W (solution phase template to be amplified) (SEQ ID No. 177):

Oligo X (surface phase amplification primer) (SEQ ID No. 178):

(5 '-dbco=5' -terminal dibenzocyclooctyl)

Oligo Y (liquid phase amplification primer) (SEQ ID No. 179):

oligo Z (solution phase sequencing primer) (SEQ ID No. 180):

Oligo Y = solution phase amplification primer; oligo Z = solution phase sequencing primer

Semiconductor chip

The DNAe semiconductor chips used in this embodiment, including the Ion Sensitive Field Effect Transistor (ISFET) sensor array, were fabricated using standard CMOS methods, with their voltage outputs responsive to changes in pH in the fluid solution residing in the wells above the IC. The holes are micron-sized and are produced by standard photolithographic processes. Custom flow cell devices are mounted on top of the IC and well assembly to facilitate fluid delivery over the chip surface. The oligonucleotides were coupled to the pore surface by first activating the pore surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide using standard click chemistry.

Buffer/reagent preparation

All buffers/reagents were prepared as follows.

"Sequencing solutions" (5 mM MgCl ₂, 20mM NaCl, 0.025%TERGITOL NP-9) were prepared in glass flasks using deionized water (18 M.OMEGA.; merck Millipore), 1M magnesium chloride solution, 5M sodium chloride solution, and TERGITOL NP-9 (pure; merck). Briefly, mgCl ₂ and NaCl stock solutions were added to deionized water to give final concentrations of 5mM and 20mM, respectively. Add a magnetic stirring bar and mix the solution to homogeneity using a magnetic stirring plate. The solution was bubbled with nitrogen (minimum 99.998% nitrogen; BOC part # 44-W) by placing a nitrogen supply tube in the bottle (ensuring the tube reached the bottom) and nitrogen bubbling through the solution for 10 minutes, ensuring removal of dissolved carbon dioxide. Thereafter, a supply of nitrogen is maintained above the solution to ensure that it remains free of CO ₂. Using an external piston pipette, 250. Mu.l TERGITOL NP-9 was added, yielding a final concentration of 0.025%. The mixture (under nitrogen) was stirred for an additional 10 minutes to completely dissolve TERGITOL.

A10. Mu.M solution of each individual natural nucleotide (dGTP, dCTP, dATP, dTTP) was prepared by adding 12.5. Mu.L of a 100mM stock solution of the selected nucleotide (FISHER SCIENTIFIC, 11843933) to 125mL of the sequencing solution. The pH of each dNTP solution was titrated to pH 8.00.+ -. 0.01 using a pH probe (Sentron SIMicroFET (92270-010)), using 10mM NaOH (prepared from 10M NaOH stock; merck, 72068). All dNTP solutions were prepared by mixing using a magnetic stirrer bar in an environment without CO ₂, nitrogen control.

The "wash solution" was prepared by titrating the pH of the sequencing solution to 8.00.+ -. 0.01 using a magnetic stir bar in an environment without CO ₂, nitrogen control, using a pH probe (Sentron SIMicroFET (92270-010)), using 10mM NaOH (prepared from a 10M NaOH stock; merck, 72068).

"1X thermo pol buffer" was prepared according to the following formulation: 4.5mL 10X ThermoPol buffer (NEW ENGLAND Biolabs), 27. Mu.L Tween 20 (pure, MERCK LIFE SCIENCE, P9416), 40.5mL molecular grade water (Sigma).

The "5x annealing buffer" was prepared according to the following formulation: in molecular water, 10mM Tris pH 7.5 (Life Technologies, 15567), 25mM magnesium acetate (MERCK LIFE SCIENCES, 63052), 750mM NaCl, 0.05% Tween 20, 25% DMSO (MERCK LIFESCIENCE, D8418).

A5. Mu.M working solution of Oligo Z (sequencing primer) was prepared by adding 1.25. Mu.L Oligo 2 (100. Mu.M stock) to 5. Mu.L 5 Xannealing buffer diluted with 18.75. Mu.L molecular grade water to give a final composition of 5. Mu.M sequencing primer in 1 Xannealing buffer.

25U/. Mu.L of working stock of "sequencing enzyme" was prepared by diluting 1. Mu.L of Bst large fragment DNA polymerase (NEW ENGLAND Biolabs, custom formulation) at 2,000U/. Mu.L in 79. Mu.L of 1X thermo pol buffer. The solution was thoroughly mixed by taking a volume of 20. Mu.L of the solution 10 times up and down.

Scheme/reaction conditions

Cloning and dehybridization of the opposite strand (cloning strand) was performed using the SM-RPA method as described in example F.

The flow cell was rinsed twice with 1x thermo pol buffer.

The sequencing primer (5. Mu.M Oligo Z in annealing buffer; see details of preparation above) was loaded into the flow cell. The inlet and outlet of the flow cell were sealed with custom plugs and primers were annealed to amplified templates using the following thermal procedure:

a.95 ℃ for 2 minutes, b.58 ℃ for 5 minutes, c. passive cooling to ambient temperature (about 20-26 ℃) for 15 minutes.

The flow cell was then washed with 200 μl of 1X thermo pol buffer to remove any excess primer.

The sequencing enzyme (25U/. Mu.L Bst large fragment; see preparation details above) was loaded into a flow cell and incubated for 10 minutes at ambient temperature (about 20-26 ℃). The flow cell was rinsed with 200 μl 1x thermo pol buffer and sequencing was performed essentially as described in example H.

Results and conclusions

The sequencing results obtained from a single ISFET on a chip are shown in table 34. Alignment of reads with Reference Sequences (REFs), the symbols between the two sequences represent the following: i=perfect match, > = missing, < = insert, = mismatch. These data indicate that the system is capable of sequencing templates generated by the disclosed in-well clonal amplification method.

Watch 34

Automated sample-to-response sequencing of pathogens labeled to whole blood

Bacterial target organisms

Enterococcus faecium (Enterococcus faecalis), ATCC strainBAA2318 ^TM; containing the antimicrobial drug resistance (AMR) gene vancomycin A+ (vanA+)

Oligomer

Specific Target Capture (STC) oligomers (IDT Technologies) (see Table 35 below)

Table 35

In the first polymerase chain reaction (PCR 1), the specified targets were amplified using the following primers:

P1(SEQ ID No.25)TGTAGCGGTGAAATGCGYAGA

P1(SEQ ID No.26)CGGTCGACTTAACGCGTTAGCT

P1(SEQ ID No.27)CGGAGTGCTTAATGCGTTWGCT

P2(SEQ ID No.28)CGCAAGGTTGAAACTCAAAGGAATTG

P2(SEQ ID No.29)CCGCAAGGTTAAAACTCAAATGAATTG

P2(SEQ ID No.30)GGGACTTAACCCAACATYTCAC

P3(SEQ ID No.35)CGTGTGTAGCCCAGGTCATAAGG

P3(SEQ ID No.36)CACGTGTGTAGCCCAAATCATAAGG

P3(SEQ ID No.37)TGTGTAGCCCTGGTCGTAAGG

P3(SEQ ID No.38)TCAGCTCGTGTCGTGAGATGTT

P3(SEQ ID No.39)CGTCAGCTCGTGTTGTGAAATGTT

P4(SEQ ID No.46)ACACAGGTCTCTGCTAAACCGTAAG

P4(SEQ ID No.47)ACACAGGTCTCTGCAAAATCGTAAG

P4(SEQ ID No.48)ACACAGCACTGTGCAAACACGAAAG

P4(SEQ ID No.49)TACCCGACAAGGAATTTCGCTACC

P25(SEQ ID No.50)TGGCAGCTTCACTTTCTCTTGC

P25(SEQ ID No.51)CCAGCTCCAATCACACCAACA

P29(SEQ ID No.31)CCTGGCTCAGAATGAACGCT

P29(SEQ ID No.32)CCTGGCTCAGGACGAACGCT

P29(SEQ ID No.33)GAGTCTGGACCGTGTCTCAGT

P29(SEQ ID No.34)GAGTCTGGGCCGTGTCTCAGT

P30(SEQ ID No.40)CTCCTACGGGAGGCAGCAGT

P30(SEQ ID No.41)CCTCCGTATTACCGCGGCTG

P31(SEQ ID No.42)GAAAGACCCCGTGAACCTTTACT

P31(SEQ ID No.43)GAAAGACCCCGTGGAGCTTTACT

P31(SEQ ID No.44)CCTTCGTGCTCCTCCGTTAC

P31(SEQ ID No.45)CCTTTGAGCGCCTCCGTTAC

P28(SEQ ID No.54)AACCATTCGCTAAACTCGAACAGG

P28(SEQ ID No.55)CCTTGAATGAGCTGCACAGTGG

P40(SEQ ID No.70)CATGAAAAATGATTATGGCTCAGGTAC

P40(SEQ ID No.71)TGGAACTTGTTGAGCAGAGGTTC

P41(SEQ ID No.72)GGCTGCGATATTCAAAGCTCAG

P41(SEQ ID No.73)CTGAACGCGCCGGCTTAAC

P48(SEQ ID No.80)CGGCARCCGTCACGCTGT

P48(SEQ ID No.81)CATCAGCACGATAAAGTATTTGCGA

in the second polymerase chain reaction (PCR 2), the specified targets were amplified using the following nested primers:

the chip surface binding primers for these targets were as follows:

P41(vanA)(SEQ ID No.126)：5’-GTAGCTGCCACCGGCCTAT-3’

Buffer/reagent preparation and filling of sample preparation, library preparation and sequencing cassettes the reagents shown in preparation tables 36-38 were then loaded onto the sample preparation, library preparation and sequencing cassettes in the chambers mentioned (see figures 22, 25, 27C and 30), respectively.

Table 36

Table 37

Table 38

Scheme/reaction conditions

500 Enterococcus faecium Colony Forming Units (CFU) containing vanA+ gene were added to 5mL whole blood in 10mL evacuated blood collection tubes. After sealing the artificial, labeled blood sample vacuum vessel, 1mL of air was drawn to create negative pressure. The rubber stopper of the evacuated blood collection tube was pierced in the cassette using an 18G needle (Becton Dickinson).

The spiked whole blood sample was transferred from the VT to the lysis buffer by drawing 5mL of air from the atmosphere and dispensing into a vacuum blood collection tube (VT) and then repeated once with a total pressurized volume of 10mL of air. The pressure reached 7psi. The cartridge was vented to atmosphere for 30 seconds to allow the blood in the VT to naturally vent from the local high pressure created inside the VT to the atmospheric pressure in the sample preparation rotary valve 1 (SP-RV 1). 1ml was drawn from VT to aspirate as much volume as possible, and then all blood volume in SP-RV was dispensed into the lysis buffer. In the case of 5mL of blood in VT, about 3-4mL is expelled from the first pressurization. The VT shift step was performed twice, thereby collecting the remaining blood volume according to the same procedure; pressurized with 10mL of air, passively vented into SP-RV1 and the volume was dispensed into a lysis buffer.

Turbulent mixing is used to homogenize the sample to move blood back and forth between the lysis buffer and the lysis overflow. The whole sample (buffer and blood) was aspirated and dispensed at a rate of 50 mL/min, allowed to equilibrate for 10 seconds, and then the process was repeated four more times, ending with the sample in a lysis buffer.

In the lysis buffer, a cylindrical resistive heater was used to heat the sample and activate proteinase K (Pro K). The heater was set at 110 ℃ for 10 minutes (the sample temperature did not exceed 60 ℃ for the duration of this step).

The sample is moved by pumping it from the lysis buffer chamber into the SP-RV1 and dispensing it into the ML chamber (containing zirconium beads therein) in the ML fins. The sample and beads were mixed for 2.5 minutes using an impeller rotating at 8000RPM, allowing the pathogen cells to lyse.

Using SP-RV1, the samples were moved to a lysis buffer and then into sample preparation Mag-Sep fins (serpentine lines in a box on top of a rectangular resistance heater). The sample was passed through the serpentine line and into the STC-Hyb 1 buffer chamber at a flow rate of 0.45 mL/min. The sample is incubated at 95℃and a heater is set at 110℃to account for heat loss, and passed through the line to denature dsDNA released from the cells during the lysis step described above.

STC oligomer annealing-samples were then transferred to STC Hyb 1 buffer, and rapidly cooled to 25-30℃in STC Hyb 1 buffer. It was then transferred to a lysis buffer (containing STC oligomer; preheated to 60 ℃) and incubated at 60℃for 30 minutes to allow the STC oligomer to anneal to its target DNA site.

1Ml of sample was aspirated from the lysis buffer and dispensed into the STC capture bead chamber (including paramagnetic streptavidin beads). The beads were resuspended and mixed by aspirating/dispensing 1mL of sample fraction from SP-RV1 into the STC capture bead chamber at a rate of 10 mL/min. This is repeated once more.

The homogenized sample in the STC capture bead chamber is transferred to a lysis buffer where it is reintroduced with the whole sample volume by being aspirated and dispensed from one chamber to the other using SP-RV 1.

The entire sample volume (performed at a sample temperature of 45 ℃) was mixed by aspirating and dispensing 4mL of sample back into the lysis buffer (using SP-RV 1) at a rate of 30 mL/min to create turbulent mixing. The replicates were repeated 14 times to ensure that the beads were completely dispersed throughout the sample so that the capture oligomer/target complex was bound to the streptavidin paramagnetic particles.

After incubation, the samples and magnetic beads were aspirated into SP-RV1 and distributed into sample preparation Mag-Sep fins. An array of four N52 grade neodymium magnets (K & J Magnets, inc.; oriented in alternating polarity, perpendicular to the serpentine channels) was pressed with a spring-loaded end effector onto the membrane on the top surface of the cartridge, thereby collecting paramagnetic beads from the suspension.

The beads were washed (thereby removing sample waste from the fin channels) by pumping Wash-S buffer into SP-RV1, then dispensing through the fins at a rate of 2 mL/min with the four magnets still docked. The magnet was then disengaged and the beads were resuspended in 200. Mu.L of Wash-S buffer. 100 microliters of air was aspirated and dispensed 4 times to move the sample back and forth across the bead capture zone to clean any remaining sample waste on the beads that remains adhered to the beads. The magnet was re-docked to recapture the beads and then another 200 μl of Wash-S buffer was pushed through the fins at a rate of 2 mL/min.

The above steps were repeated essentially as described except that the Wash-S buffer was replaced with Wash-T buffer.

The magnet was retracted, 100 μl of elution buffer was withdrawn from the STC elution chamber and dispensed into sample preparation Mag-Sep fins. A small amount of air (150. Mu.L) was aspirated and distributed into sample preparation Mag-Sep fins. This was repeated in the area of the capture beads. After resuspension, the fin heater was turned on and set at a temperature of 110 ℃ for 3 minutes to elute the target DNA from the beads (internal sample temperature reached 75 ℃).

The magnet was re-docked to recapture the beads, leaving the eluted DNA (eluate (eluate)) in solution.

The lyophilized PCR1 reagent was resuspended in PCR1 reagent chamber using about 85. Mu.L of eluate. The resulting sample was aspirated from the chamber and dispensed into a PCR1-lyo chamber. The chamber was then pressurized to 27psi to push the sample into the hot zone (PCR 1 in fig. 25) and PCR amplification (PCR 1) was performed using the following four-step thermal procedure, with steps b, c and d repeated in sequence for 25 cycles:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.58℃for 25 seconds (annealing step), d.72℃for 45 seconds (extension step).

After PCR1 was completed, the sample was aspirated and dispensed into the PCR1 product chamber. Then 20. Mu.l was taken out of the chamber and dispensed into a PCR1 dilution chamber pre-filled with 3.98mL of water (Invitrogen; ultra-pure distilled water, free of DNase and RNase) (200-fold dilution) and BSA (2 mg/mL; calbiochem; albumin, bovine serum, fifth fraction, free of fatty acids, free of nucleases and proteases). The samples were then mixed by aspirating and dispensing 2mL of air back and forth from the PCR1 dilution chamber. After mixing, a total of 250. Mu.L of the diluted material was transferred to the PCR2 reagent chamber.

Mix the sample by aspirating and dispensing 200. Mu.L from the PCR1/CC RV to the PCR2 reagent chamber, then aspirating and dispensing 125. Mu.L to the PCR1-lyo chamber and pressurizing to 27psi, then aspirating and dispensing 165. Mu.L to the adaptor addition chamber and pressurizing to 27psi, and perform PCR amplification (PCR 2) using the following four-step thermal method, where steps f, g and h are repeated in sequence for 40 cycles:

a.98℃for 30 seconds (initial denaturation step), b.98℃for 5 seconds (denaturation step), c.58℃for 25 seconds (annealing step), d.72℃for 45 seconds (extension step).

Samples were aspirated from the PCR1 and adaptor addition chambers and dispensed into Hyb chambers where they were combined with preloaded Sav-MyOne C1 beads. The samples were incubated at ambient temperature (about 20-26 ℃) for 10 minutes and mixed continuously (250. Mu.L to Hyb chamber were aspirated and dispensed back and forth from the PCR1/CC RV). Four N52 grade neodymium magnet arrays were pressed to the top surface of the cassette with spring loaded end effectors. Beads were flowed into library prepared Mag-Sep fins and then the magnets were docked to immobilize streptavidin beads.

The beads were washed by disengaging the magnet, allowing 150 μl of wash buffer to flow through the beads, and re-suspending the beads by pumping/dispensing air back and forth in the channels within the fins. The magnet is then re-docked, the beads immobilized, and the wash buffer pushed to the waste chamber. This process was repeated three times.

After the washing process, the beads were resuspended and bound DNA eluted by disengaging the magnet, pushing 60 μl of 40mM NaOH elution buffer into the fins and waiting 60 seconds. The magnet was re-docked and the beads were immobilized, leaving the eluted DNA in the supernatant.

Defoamer Y-30 was added to 1L S2 wash buffer to give a final concentration of 0.0375%.

Will be four150ML of a conventional polystyrene storage bottle (Corning) with a 45mm cap was filled with 100mL of S2 washing buffer, and the remaining 600mL of S2 washing buffer was poured into a 1L bottle for washing.

50 Microliters of each dNTP (Thermo Scientific) was added to 100mL of S2 wash buffer in each of four 150mL bottles to give a final concentration of 50. Mu.M for each nucleotide.

250 Milliliters of soda lime was added to a soda lime bottle (500 mL) and the metal inlet tube of the soda lime bottle was pushed to the bottom of the bottle to ensure that air circulated throughout the soda lime.

All vials and conical tubes were connected to the BB3b cassette and loaded into the BB3 sequencing subsystem (fig. 30).

Two microliters of BST large fragment polymerase (NEW ENGLAND BioLabs) was premixed with 128 μl of 1x thermo pol buffer and added to the Seq enzyme chamber of BB3a cassette.

25 Microliters of annealing buffer was preloaded into the BB2 output chamber.

25. Mu.l of eluted DNA (see above) was pipetted into the BB2 output chamber and mixed with preloaded annealing buffer. Then, by using sequencing rotary valve 1 and selection valve for aspiration and dispensing, it is moved onto the chip. The chip was then pressurized to 15psi.

Template hybridization of immobilized primers was performed using a thermal program as follows:

a.95 ℃ for 2 minutes, b.chip decompression, c.68 ℃ for 5 minutes, d.63 ℃ for 5 minutes, e.58 ℃ for 5 minutes,

F.53℃for 5 minutes, g.48℃for 5 minutes, and h.43℃for 5 minutes.

Simultaneously with the hybridization step, 150mL bottles and 1L wash bottles containing dNTPs were titrated to pH 8.00.+ -. 0.01 using 10mM NaOH.

Once hybridization was complete, the chip was set to 25 ℃ and the chip surface was washed with 1x thermo pol buffer for 10 seconds.

130 Microliters of reagent in the Seq enzyme chamber was pushed into the chip, and then the chip was pressurized to 12psi to prevent bubble formation. The enzyme was incubated for 2 minutes.

The chip record was opened and the sequencing system was started.

An electrical response test was performed by biasing the reference electrode with an increasing voltage step. The resulting change in mV output measured by the ISFET on the IC is used to determine the relationship between the reference electrode potential and the corresponding potential seen on the ISFET, which is then used to determine the optimal reference electrode potential for the experiment. dNTP lines were pretreated with wash buffer and the chip was washed for 20 seconds to remove any air in the lines.

The solution was sequentially flowed over the chip surface for 25 cycles in the following order:

a. Wash solution 25 seconds, b.dttp solution 10 seconds, c.wash solution 25 seconds, d.dgtp solution 10 seconds, e.wash solution 25 seconds, f.dctp solution 10 seconds, g.wash solution 25 seconds, h.datp solution 10 seconds.

The chip was again washed and the chip record was turned off.

Analysis was performed using an internal proprietary algorithm for base calling and bioinformatics analysis.

Results and conclusions

The sequencing results are shown in FIG. 57. Individual reads are plotted as the read length on the x-axis (total length of the output read) (labeled "readLen") and the effective read length on the y-axis (ERL; number of matches to expected sequence minus number of errors) (labeled "EFFECTIVEREADLEN"). The read without error will lie on the diagonal dashed line, where readLen =erl. The histogram portion shown in fig. 57 represents the distribution of reads on different axes. The histogram at the top of the figure shows the distribution of reads along the x-axis, i.e. read length (bp); the histogram on the right of the figure shows the distribution of reads along the y-axis, namely ERL (bp). In this embodiment, the total number of reads is 1442. In these reads 94.3% ERL >30bp. The lower graph in fig. 57 shows that more than 1300 or 1442 reads are aligned with the expected target, so the final assay call is vanA. Thus, these data demonstrate the following capabilities of the disclosed system: i.e. the ability to process a labeled bacterial sample in blood by performing sample preparation, library preparation, sequencing, and correctly invoking a labeled target in a cartridge-based automated system in a short time.

P. mechanical cleavage

To test a mechanical lysis system compatible with the cartridge described above, a mechanical lysis subsystem was used to lyse target organisms. Multiple replicates of 3mL blood aliquots in 15mL conical tubes were prepared. To some of the tubes (sample positive) were added 15. Mu.L of enterococcus faecium (ATCC BAA-2318) at a concentration of 200 CFU/. Mu.L. No sample was added to some tubes (negative control). Then 1mL of lysis buffer (100 mM Tris-HCl, pH 8.0, 16.7% (w/v) lithium dodecyl sulfate) was added to each tube, followed by 60. Mu.L of 100% antifoam Y30, 30. Mu.L of proteinase K (20 mg/mL) and 10. Mu.L of a Pool of specific target capture oligonucleotides (Oligo Pool 38plex V2.0, DNAe). All tubes were vortexed vigorously for 30 seconds, inverted multiple times, vortexed vigorously for 30 seconds, and inverted multiple times. The tube was centrifuged at 700rpm for 15 seconds and then incubated at 75℃for 15 minutes (proteinase K digestion step).

The control method was then used to perform mechanical lysis on a bench or in a ML tester (coupling) (cartridge compatible mechanical lysis subsystem as described above), briefly described below: 1) Control method on bench. The contents from the above-described multiple sample positive tubes and negative control tubes were transferred to an 8mL mechanical lysis tube containing 4g of 0.1mm yttrium stabilized zirconia impact beads. The tube was then placed in OmniRuptor Elite (SKU 19-042E) and 3 cycles of 90 seconds start +20 seconds stop were performed at a rate of 6.6 m/s. The contents of the lysis tube were then transferred back into their corresponding 15mL conical tube. 2) ML test method. The contents of the above-described multiple sample positive and negative control tubes were transferred using a 10mL syringe into an mL tester containing 4g of 0.1mm yttrium stabilized zirconia impact beads and mechanically lysed using an internal procedure. The contents of the ML pilot were then transferred back into their corresponding 15ML conical tubes.

All samples (bench control and ML pilot) were treated identically. Briefly, all tubes were incubated at 95 ℃ for 30 minutes to denature the target nucleic acid, and then at 60 ℃ for 40 minutes to anneal the specific target capture oligomer to the target. 1200 microgram streptavidin coated magnetic beads (DNAe) were added to each sample and the tubes were vortexed and then incubated with constant, gentle vortexing (1500 rpm) for 10 minutes at 45 ℃. The beads were collected using a magnet and the supernatant discarded. The beads were resuspended in 1mL of wash buffer (10 mM Tris-HCl, pH 8.0,0.01% Tween 20) by vortexing. The mixture was transferred to a 1.5mL tube, the beads were collected using a magnet and the supernatant discarded. This washing process was repeated 3 more times. After discarding the supernatant after final washing, 40 μl of elution buffer (same formulation as the wash buffer) was added to the top of each tube, the tubes were vortexed, and then centrifuged briefly to bring the contents to the bottom of the tube. All tubes were incubated at 75℃for 3 min, vortexed and briefly centrifuged. Beads were collected using magnetic force and the supernatant (i.e., eluent (eluent)) was transferred to a 0.5mL tube.

All samples were then analyzed using quantitative PCR as follows, typically: mu.L aliquots of each eluate were added to 15. Mu.L of the premix (0.6. Mu. L SuperFi polymerase, 0.4. Mu.L dNTP mix, 5. Mu. L SuperFi buffer, 8.2. Mu.L water, 1. Mu.M primer mix (vanA_PC2_Fwd (SEQ ID No. 153): TTGTATGGACAAATCGTTGACATACA and vanA_PCR_2Rev (SEQ ID No. 204): GTAGCTGCCACCGGCCTAT) and SYBR Green) in the PCR microtiter plate. PCR was then performed in a thermal cycler (98 ℃ C. For 30 seconds, then 40 cycles: 98 ℃ C. For 5 seconds and 65 ℃ C. For 25 seconds). Fluorescence was measured during the run and the time of occurrence of each sample was determined.

The results are shown in FIG. 71. The data indicate that the lysis of enterococcus faecium in blood using the ML tester is equivalent to the lysis achieved with the bench gold method. Similar results were obtained for gram negative klebsiella pneumoniae (ATCC BAA-1898) and gram positive staphylococcus aureus (ATCC BAA-2094) using the detailed protocol described above (data not shown).

Automatic capture of specific targets directly from blood

The cassette-compatible Specific Target Capture (STC) subsystem or fin as described above was tested to assess the efficacy of extracting and isolating specific target DNA from whole blood, with the results compared to a bench positive control directly from the blood-specific target capture process.

An STC subunit (as shown in fig. 69) was loaded into the fixture, and then using commands as part of an internal procedure, an automated pipette aspirates 4mL of blood labeled 500 parts of purified gDNA klebsiella pneumoniae (CDC AR 0068) into a pipette tip, which was then docked in a Sealed Pipette Interface (SPI) port and dispensed into fins. All pipetting steps can be automated using, for example, a pipette rack in the instrument of the invention as described above. Next, 1mL of lysis buffer (100 mM Tris-HCl, pH 8.0, 16.7% (w/v) lithium dodecyl sulfate), 80. Mu.L of 100% defoamer B (JT-Baker), 50. Mu.L of proteinase K (20 mg/mL) and 10. Mu.L of specific target capture oligonucleotide pool (pool) were added to the 1.5mL tube, respectively, and then a mixture of all 4 reagents was aspirated into the pipette tip and docked in the SPI port for distribution into the fins. The mixture of blood and reagent is placed in a mixing chamber and the mixture is mixed to homogeneity by going back and forth between the two chambers. The thermoelectric cooler (TEC) was then turned on and the sample incubated at 75 ℃ for 15 minutes (proteinase K digestion step), during which time back and forth mixing was continued to evenly distribute the heat. Samples were aspirated/removed from the fins using SPI and pipette tips and dispensed into 8mL mechanical lysis tubes containing 4g of 0.1mm yttrium stabilized zirconia impact beads. The tube was then placed in OmniRuptor Elite (SKU 19-042E) and 3 cycles of 90 seconds start +20 seconds stop were performed at a rate of 6.6 m/s. The sample was then centrifuged to bring the lysis beads to the bottom of the tube and the sample was transferred through a mesh filter into a 5mL tube. The sample is aspirated using a pipette on the fixture and docked back to the SPI port to transfer the sample back to the mixing chamber. Once in the mixing chamber, the TEC is then opened and the sample is incubated at 95 ℃ for 25 minutes (denaturation step), during which time back and forth mixing is continued to evenly distribute the heat. The samples were then incubated at 60℃for 32 minutes (annealing step), again with continuous mixing.

1200 Micrograms of streptavidin-coated magnetic beads (DNAe) were added to the sample using a fixed pipette and SPI port, and the sample was incubated at 45 ℃ for 15 minutes (combined with the magnetic beads) and mixed again continuously. The sample was expelled by inserting a pipette tip into the SPI port and aspirating. The magnet interfaces with the serpentine region and the sample passes at a flow rate of 0.5ml/min enabling collection of the beads and discarding of the supernatant. The beads were resuspended in 0.5mL of wash buffer (10 mM Tris-HCl, pH 8.0,0.01% Tween 20) by mixing back and forth in a serpentine tube using a pipette tip and SPI port. The beads in the mixture were collected again using a magnet and the supernatant discarded. The washing procedure was repeated 1 more time. At the third and last wash, the resuspended beads were transferred from the mixing chamber serpentine SPI to the elution SPI, the beads were recaptured with the magnet, and the supernatant discarded. Beads resuspended with 115 μl of elution buffer (same formulation as wash buffer) were added to the fins through the elution SPI port, and the samples were then pushed into the elution chamber and incubated for 5 minutes at 75 ℃. TEC was set to 25 ℃ for 3 minutes to allow the liquid to cool before final magnetic capture of streptavidin beads. The magnet was docked to the serpentine tube, the beads were collected, and the supernatant (i.e., eluent) was aspirated using a pipette tip, which was then dispensed into a 0.5mL tube.

60 Microliters of eluate (about 100. Mu.L total) was added to the premix (10. Mu. L SuperFi buffer 10X, 20. Mu.L 39-plex primer pool, 1.6. Mu.L dNTP mix (0.4 mM per nucleotide), 2.5. Mu. L SuperFi Enzyme@0.05U/. Mu.L, 1.8. Mu.L MgSO ₄ @1.75 mM). The mixture was added to a 0.2mL PCR tube and cycled on Applied Biosystems ^TMVeriti^TM, 96Well Thermal Cycler using the following conditions: 95℃for 30 seconds, then 25 cycles: 95℃for 5 seconds (denaturation), 55℃for 10 seconds (annealing) and 72℃for 30 seconds (extension).

After PCR, the sample was removed from the thermal cycler and diluted by aliquoting 6.25. Mu.L of the sample and adding 93.75. Mu.L of water to prepare a 1:80 dilution. 10 μl of the PCR1 diluted product was added to the premix (10 μ L SuperFi buffer 5X,0.8 μl forward primer @1.5nM and 0.8 μl reverse primer @1.5nM,0.8 μl dNTP mix (0.4 mM per nucleotide), 1.3 μ L SuperFi Enzyme@0.05U/. Mu.L, 0.9 μl MgSO ₄ @1.75mM and 0.5 μl SYBR Green @1X and 25.1 μl water). The mixture was added to a 0.2mL PCR tube and cycled on CFX96 Touch Real-Time PCR Detection System using the following conditions: at 95 ℃ for 30 seconds, then 40 cycles are performed: 95℃for 5 seconds (denaturation), 55℃for 10 seconds (annealing) and 72℃for 30 seconds (extension).

As shown in fig. 72, the results are comparable between the cartridge-based STC and the bench-standard control.

Automated nested PCR for R.tape cassette PCR systems

Using the cassette compatible PCR subsystem described above and shown in fig. 63 and 70, the nested PCR was tested with 1000 copies of staphylococcus aureus (s.aureus) gDNA using the PCR system (a 3.0 PCR fixture and PCR fins), the run consisted of: one PCR1 reaction (39-plex), dilution step (1:80) and 10 PCR2 reactions (4-fold each).

The staphylococcus aureus gDNA stock (127,325 copies/. Mu.l) was diluted with water to a final concentration of 10 copies/. Mu.l. In total, 100. Mu.l of the lyophilized premix inserted into the "lyo bag" of PCR fins placed on the A3.0 PCR fixture was used for rehydration. The samples (gDNA and MM) were pushed to the hot zone using the command combination in KeySharp to measure, pressurize and place the samples in the correct area for thermal cycling. All of these steps can be performed by the apparatus of the present invention by pneumatic pressure via the interface with the cartridge as described above. The cycle used was as follows, a hot start 95 ℃ for 60 seconds, followed by 25 cycles: 95℃for 5 seconds (denaturation), 55℃for 10 seconds (annealing) and 72℃for 30 seconds (extension).

After the cycle is completed, the sample is recovered by depressurizing the chamber and aspirating the sample. A1:80 dilution was performed by manually aliquoting 15.6. Mu.l of the sample and diluting it with 1209.4. Mu.l of water and 25. Mu.l of BSA.

650 Μl of diluted PCR1 sample was aspirated through the instrument and 50 μl was metered into each PCR2 channel (n=10). Excess sample was removed from the cartridge and the metered sample was moved through the lyo bag containing Master Mix lyophilization reagents using the command combination in KeySharp to pressurize and place the sample in the hot zone, starting the thermal cycle using the following cycle procedure, hot start 95 ℃ for 60 seconds, followed by 40 cycles: 95℃for 5 seconds (denaturation), 55℃for 10 seconds (annealing) and 72℃for 30 seconds (extension).

At the end of the thermal cycle, the chamber is depressurized and the sample is recovered using a common channel. All 10 reactions were recovered and pooled together to obtain a final PCR2 sample, which was analyzed on a Bioanalyzer and compared to the rack negative and rack positive samples.

Bench positive samples used the same gDNA concentration and premix reagent (lyophilized form) as used in the PCR fins (described above). For negative controls, water was circulated with the premix reagent (lyophilized form). Both PCR1 and PCR2 reactions were performed on a CFX Bio-Rad thermocycler that simulates the same cycle used on the A3 PCR fixture.

As shown by the electropherogram stack of the positive, negative bench control (NTC) and A3.0PCR system (AT-401-04) provided in fig. 73, target amplification of all targets (10/10) was observed in the plot of staphylococcus aureus (BAAC 2094), with the observed peaks matching the expected target peaks in table 39 below:

Table 39

S. sequencing of amplified templates Using direct hybridization method with automatic pH adjustment and CO ₂ removal System

Oligomer

Direct hybridization was performed using single-stranded DNA templates (Oligo 18 and 20; ATDBio) prepared using PCR hybridized with primers immobilized on the well surface (sequencing template 1 and sequencing template 2; internal preparation) followed by a sequencing method. The immobilized primers are complementary to the 3' regions of sequencing templates 1 and 2, respectively (see underlined sections).

Sequencing template 1 (SEQ ID No. 205):

Sequencing template 2 (SEQ ID No. 206):

Oligo 18(SEQ ID No.207)：

5'-DBCO-GCCGTACGAGCTGACGACAG-3' (5 '-dbco=5' -terminal dibenzocyclooctyl)

Oligo 20(SEQ ID No.208)：

5'-DBCO-CCGGGCATGCCTAACACA-3' (5 '-dbco=5' -terminal dibenzocyclooctyl)

Semiconductor chip

The DNAe semiconductor chips used in this embodiment were fabricated using standard CMOS methods and included an Ion Sensitive Field Effect Transistor (ISFET) sensor array whose voltage output was responsive to changes in pH in the fluid solution residing in the wells above the IC. The holes are micron-sized and are produced by standard photolithographic processes. Custom flow cell devices are mounted on top of the IC and well assembly to facilitate fluid delivery over the chip surface. The oligonucleotides were coupled to the pore surface by first activating the pore surface with an acrylamide-based polymer coating, followed by covalent attachment of the 5' modified oligonucleotide using standard click chemistry.

Scheme/reaction conditions

Briefly, the preparation of the amplified sequencing template comprises the steps of: PCR1; purifying the product from PCR1; PCR2; capturing of PCR2 product and elution of single stranded DNA product.

PCR1 protocol

Detailed description of oligonucleotides as shown in Table 40

Table 40

100. Mu.L of PCR1 premix (1X Platinum SuperFi PCR premix (Thermo Fisher);1μM Oligo 1(Integrated DNA Technologies(IDT));1μM Oligo 2(IDT);1μM Oligo 3(IDT);3000 copies of DNA template (IDT); PCR grade water) was thermally cycled (Mastercycler 50S, eppendorf) as follows: 98℃for 5 minutes, 1 cycle; 98℃for 10 seconds, 2 cycles; 68 ℃ for 2 minutes and 72 ℃ for 30 seconds; followed by 72 ℃ for 2 minutes.

PCR1 purification protocol

The PCR1 product was purified as follows using AMPure XP heads (Beckman Coulter). mu.L of PCR1 product was added to 150. Mu.L of AMPure beads. The mixture was vortexed and incubated for 5 minutes at room temperature and repeated. The tube was then placed on DynaMag magnets for 5 minutes and the supernatant discarded. The tube with the beads was retained on DynaMag magnets and washed with 150 μl of 80% ethanol. The washed beads were incubated at room temperature (20-22 ℃) for 30 seconds. The supernatant was discarded again and the ethanol wash repeated as described above. The ethanol supernatant was removed and the tube was then left on DynaMag magnets to air dry the beads for 5 minutes. The tube was removed from the magnet and 54 μl of TE pH 8.0 below EDTA (AppliChem GmbH) was added directly to the particles. The mixture was then vortexed thoroughly to disperse the beads, then incubated for 5 minutes and centrifuged briefly to collect the contents. The tube was placed on a magnetic rack and incubated for an additional 2 minutes. Then 50. Mu.L of the purified PCR product was transferred to a PCR2 reaction tube as described below.

PCR2 protocol

The detailed description of the oligonucleotides is shown in Table 41.

Table 41

The PCR product from PCR1 prepared as described above was used as input template material for PCR 2. For 112. Mu.L of PCR2 premix (1X Platinum SuperFi PCR premix (ThermoFisher); 1.5. Mu.M Oligo 4 (IDT); 1.5. Mu.M Oligo 5 (IDT); 1.5. Mu.M Oligo 6 (IDT); 50. Mu.L of PCR1 product) thermal cycling (Mastercycler 50S, eppendorf) was performed according to the following conditions: 98℃for 5 minutes, 1 cycle; the following 30 cycles were then performed: 98 ℃ for 15 seconds, 68 ℃ for 15 seconds and 72 ℃ for 30 seconds; followed by 72 ℃ for 5 minutes.

Capture and elution protocol

The preparation of the capture beads was performed as follows, followed by capturing the PCR2 product onto the beads and eluting the single stranded DNA product. The capture beads were prepared by the following method: mu.L of bead wash buffer (1M NaCl (Sigma-Aldrich), 5mM Tris-HCl (pH 7.5, panReac), 0.5mM EDTA (Sigma-Aldrich), 0.05% Tween 20 (Sigma-Aldrich), 1mg/mL BSA (Sigma-Aldrich), molecular grade water (Sigma-Aldrich)) was added to 110. Mu.L of MyOne C1 beads (Thermo Fisher) and vortexed for 5 seconds. The tube is capped and placed on the magnetic rack. The bead wash buffer supernatant was removed, discarded, and the tube removed from the magnet rack. This was done 3 times in total. Finally, 132. Mu.L of resuspension buffer (1.5mM NaCl;10mM Tris-HCl (pH 7.5), 1mM EDTA, 0.1% Tween 20, 1mg/mL BSA, molecular grade water (suppliers as described above)) was added to the washed beads. This mixture is called capture beads.

Mu.L of capture beads were combined with 110. Mu.L of PCR2 product in a 1.5mL tube (Eppendorf). The tube was incubated for 10 minutes at room temperature while mixing continuously on a rotator (SB 3, stuart). The PCR product from PCR2 consisted of double-stranded DNA, with the single strand of the duplex being biotin-labeled at the 5' end (as described previously) due to the use of biotin-labeled forward primers in the PCR2 reaction mixture. During incubation of the PCR2 product with the capture beads, the biotin-labeled strand of the DNA duplex was bound to streptavidin-coated MyOne C1 beads. After incubation, the reaction tube was placed on a magnetic rack for 2 minutes. The supernatant was removed and discarded, and the beads were washed by adding 200 μl of 1X wash buffer and vortexing for 5 seconds. The washing process was repeated 3 times. The tube was removed from the magnet holder, 50 μl of 40mM NaOH was pipetted into the tube and vortexed for 5 seconds to resuspend the beads. The resuspended beads were incubated for 1 min at room temperature. The presence of 40mM NaOH denatures the DNA duplex so that the biotin-labeled strand remains bound to the streptavidin-coated MyOne C1 beads, while the non-biotin-labeled strand of the duplex dissociates to become free in solution. The tube was then placed on a magnet rack for 2 minutes. The supernatant containing dissociated ssDNA was transferred to a collection tube and prepared for use as an input template for a sequencing reaction. The single-stranded DNA for sequencing prepared from PCR2 is hereinafter referred to as an amplified sequencing template. The sequencing templates used in this example are sequencing template 1 and sequencing template 2.

Sequencing protocol

The concentration of amplified sequencing templates was quantified using Nanodrop 2000 (thermo fisher) and the total DNA concentration of each sample was adjusted to 1.1 μm by adding water without DNA.

A final concentration of 0.4. Mu.M for each DNA template was prepared by adding 17.3. Mu.L of the amplified sequencing template (1.1. Mu.M) to 6.7. Mu.L of the neutralization buffer (0.4M NaCl;75mM Tris pH 7; molecular-grade water).

The neutralization buffer containing the template is loaded into a flow cell assembled on an IC as described above. The inlet and outlet of the flow cell were sealed with custom plugs, the template annealed to the surface immobilized primer, complementary to the amplified sequencing material (Oligo 18 and 20), using the following thermal procedure: a) 95 ℃ for 120 seconds, b) 90 ℃ for 30 seconds, c) 85 ℃ for 30 seconds, d) 80 ℃ for 30 seconds, e) 78 ℃ for 120 seconds, f) cooling from 77 ℃ to 64 ℃ (inclusive) at a rate of 1 ℃ per 30 seconds, g) 63 ℃ for 120 seconds, h) cooling from 62 ℃ to 59 ℃ (inclusive) at a rate of 1 ℃ per 30 seconds, i) 58 ℃ for 15 minutes, j) passive cooling to ambient temperature (20-26 ℃) for 2 minutes. The sequencing enzymes (IsoPol BST +, arcticZymes) and sequencing solutions were formulated as described in example L.

Chip-based pH adjustment protocol

PH titration of sequencing solutions (including nucleotide-containing solutions) was performed using an automated process using a dedicated chip-based pH adjustor (CBA). The automated CBA module uses the mV output of the IC as a relative measure of pH and employs a feedback loop to feed an appropriate volume of 0.04mM NaOH into the sequencing solution to achieve the target pH (pH 8.+ -. 0.1). Briefly, a reference pH solution (Tris, pH 8.0) was pumped across the surface of the IC and the mV output was recorded. The sequencing wash solution was then pumped through the chip and the mV output was recorded. The difference in mV output between the reference solution and the sequencing wash solution indicates the appropriate NaOH dosage to be added to the total volume of sequencing wash solution. The NaOH dose was added to the total volume of sequencing wash solution using an automatic syringe pump. This process was repeated until the mV output of the sequencing wash solution matched the mV output previously recorded for the reference pH solution. The entire process is then repeated for each individual nucleotide solution.

Subsequent steps (addition of sequencing enzymes, priming steps, electrical response testing, sequencing and analysis) were performed as described in example L, except for the following: a proprietary CO ₂ removal device called a "CO ₂ scrubber" was used to maintain the atmosphere in the sequencing solution free of CO ₂. In short, the CO ₂ scrubber eliminates the need to have a compressed nitrogen source on the sequencing solution that maintains an inert atmosphere, which is necessary to prevent unwanted acidification of the sequencing solution. The CO ₂ scrubber employed a column of soda lime particles onto which air was pumped using standard equipment. The soda lime particles react with CO ₂ in the atmosphere to remove them from the air.

Results and conclusions

The sequencing results obtained from the amplified template using the direct hybridization method are shown in FIG. 74. The individual reads are plotted as ARL on the x-axis and ARS-e on the y-axis (units of measurement are base pairs; ARL and ARL-x are defined in example L above). The read without error will lie on the diagonal dashed line, where ALR-e=arl. The histogram at the top of the figure shows the distribution of reads along the x-axis, ARL (bp); the histogram on the right of the figure shows the distribution of reads along the y-axis, ARL-e (bp). In this embodiment, the total number of reads is 6772. The median of ARL-e was 97bp. The consensus reads shown had an alignment length of 97 bases with an error rate of 3.1% (Table 42). These data indicate that the system is capable of sequencing templates hybridized to immobilized primers.

Table 42

The symbols between the two sequences are represented as follows: i=perfect match, > = missing, < = insert, = mismatch; conc = consensus sequence.

T. target capture of low copy number bacterial genome from sample preparation in 3mL whole blood with shortened incubation protocol

Specific Target Capture (STC) oligomers

Specific target capture from whole blood was performed with a modified oligomer complementary to a target sequence containing biotin linked to the 5 'end through a 5 adenosine (AAAAA) base nucleotide linker and modified 3' with inverted dT. Standard desalted purified STC oligomers were ordered from IDT (INTEGRATED DNA Technologies) and reconstituted to 100 micromolar concentration in IDT buffer (IDT).

KPC target STC oligomers of both strands of KPC gene target region in Klebsiella pneumoniae strain: kpc_stc_f2 (SEQ ID No. 215):

/5BiosG/AAAAAACCTCGTCGCGGAACCATTCGCTAAACTCGAACAGG/3InvdT/KPC_STC_R2(SEQ ID No.216)：

/5BiosG/AAAAACAGCACAGCGGCAGCAAGAAAGCCCTTGAATGAGCT/3InvdT/

mecA target STC oligomer for both strands of mecA gene target region in staphylococcus aureus strains:

mecA_STC_F-INT(SEQ ID No.217)：

/5BiosG/AAAAAAGGTACTGCTATCCACCCTCAAACAGGT/3InvdT/

mecA_STC_R2(SEQ ID No.218)：

/5BiosG/AAAAATTGAGTTGAACCTGGTGAAGTTGTAATCTGG/3InvdT/

Preparation of bacterial target genomic DNA

Target genomic DNA for capture in a sample preparation was isolated from bacterial organism strains obtained from CDC (Klebsiella pneumoniae ATCC BAA-1898, KPC+ strain) and ATCC (Staphylococcus aureus BAA-2094, mecA+ strain). The strain stock was grown, genomic DNA isolated using a commercial bacterial genomic DNA isolation kit, the DNA quantified by digital PCR, and further diluted to a Genome Equivalent (GE) copy.

Streptavidin beads

Streptavidin-conjugated paramagnetic beads manufactured with DNAe capture target DNA from whole blood by biotin-labeling of STC oligomers.

Proteinase K

Cell lysis and protein digestion were performed using a commercially available proteinase K enzyme (Roche/Sigma or Thermo FISHER SCIENTIFIC).

Preparation of lysis buffer

Cell lysis and protein denaturation were performed using lysis buffer containing ionic detergent.

Washing and elution buffers

Streptavidin beads were washed and target DNA was eluted using wash and elution buffers containing mild low concentration of surfactant in the buffer.

Whole blood

Whole blood was obtained from BioIVT suppliers.

Defoaming agent

To reduce foaming on the rack or instrument box during sample preparation, an anti-foam B silicone emulsion (Sigma or j.t.baker) was used.

Mechanical cracking beads

For cell lysis of difficult-to-lyse organisms (such as fungal cells or gram positive cells), the cells are sheared open on OMNI International Bead Ruptor Elite instruments using Very Hard Density (VHD) zirconia beads.

Sample preparation reaction set-up

The sample preparation reaction was prepared by combining the following reagents shown in the following table (table 43) at room temperature. The reaction mixture was thoroughly mixed. For the bench and instrument box procedure, the reaction setup was prepared in a 5mL screw cap conical tube (Eppendorf, 30122348).

Table 43

Sample preparation protocol: cleavage by target/bead Capture

The sample preparation procedure required several heating/cooling and incubation steps, as shown in the following table (table 44). 3mL of blood samples were mixed with the components of Table 1, including the lysis buffer containing ionic detergent and proteinase K in Table 1. The mixture was incubated at 75 ℃ for 5 minutes for proteinase K enzymatic digestion, and then the cells that were difficult to lyse were mechanically lysed using zirconia beads.

After cleavage and protein digestion are completed, the sample is heated to 95-100 ℃ for 10 minutes to denature the proteinase K enzyme and convert double stranded DNA (dsDNA) to single stranded DNA (ssDNA) form.

The sample was then gradually cooled from 95-100 ℃ to 60 ℃ over 5 minutes to hybridize the biotin-labeled STC oligomers to their corresponding ssDNA targets.

Following the hybridization step, streptavidin paramagnetic beads were added to the sample, and the sample was mixed at 1500RPM for 10 minutes at 45 ℃ to capture STC oligomers/target hybrids as well as free STC oligomers via strong biotin-streptavidin interactions between the biotin-labeled STC oligomers and the streptavidin-conjugated paramagnetic beads.

For the bench process, heating/cooling and mixing were performed in a 5mL heating block (Benchmark Scientific, H5000-5 MT) on a thermal shaker (Benchmark Scientific, H5000-HC).

For the instrument process, heating and cooling are performed in the cartridge.

Table 44

Sample preparation protocol: reaction clean-up by elution

Bead washing

After the bead capture step, the beads are cleaned. The reaction tube was removed from the heated incubation apparatus (Benchmark SCIENTIFIC H-HC) and the tube was placed on a 5mL tube magnet rack (Thermo Fisher, dynamag-5) for 5 minutes to attract the streptavidin bead-bound target DNA/STC oligomer complex to the side of the tube. The reaction supernatant was carefully removed and discarded without disturbing the beads. The beads were then washed by resuspending the beads with 1mL of wash and elution buffer. The beads in the reaction tube were resuspended off the magnetic rack. The resuspended beads were transferred to a 1.5mL reaction tube (Eppendorf, 22431021). After resuspension, the reaction tube was returned to the magnet rack (Thermo Fisher, dynamag-2) for 1 minute. The wash solution supernatant was removed and discarded. Three more washes were performed in the same 1.5mL tube in the same manner as the first wash, for a total of 4X 1mL.

Target elution

After removal of the final wash, the beads were resuspended in 100 μl wash and elution buffer. The resuspended beads in 1.5mL tube were then placed on a thermal shaker (Benchmark Scientific, H5000-HC) containing a 1.5mL tube heating block (Benchmark Scientific, H5000-CMB) preheated to 75 ℃. The target DNA was eluted by incubating the tube at 75 ℃ for 3 minutes. After the elution was completed, the 1.5mL reaction tube was moved back to the 1.5mL magnetic rack for 1 minute. The target DNA eluate was then transferred to a new 1.5mL tube.

Detection of target DNA in sample preparation eluate

Target DNA detection and quantification in sample preparation eluate was performed by nested PCR with final PCR being quantitative PCR (qPCR) using SuperFi qPCR pre-mix, SYBR Green dye and PCR primers specific for the target region of interest. First PCR amplification (PCR 1) target was amplified for 25 cycles using external PCR primers. The 1:80 diluted PCR1 product was added to the second quantitative PCR (PCR 2) using internal "nested" PCR primers with 40 amplification cycles. Target detection is reported as Cq value from PCR2, which is compared to the no target negative control sample preparation reaction. In addition, melting curve analysis was performed to confirm the identity of qPCR amplicons and exclude primer dimer products.

Table 44: PCR1 and PCR2 primer sequences:

results and conclusions

Quantitative PCR amplification data indicated that antimicrobial drug resistance gene targets (mecA and KPC) in table 45 were detected, with bold values indicating at least 6 Cts above background signal and italics values indicating 2 to 3 Cts above background signal. For each of the two experiments performed, background signals (underlined values) are noted in the PCR1+2 and PCR2 cells on the right.

Table 45

The data also demonstrate that not only can the sample preparation cassette portion be performed equivalently to the bench top method, but that the sample preparation is robust enough to shorten the incubation process steps (digestion, denaturation, and hybridization) and still be able to recover and detect targets of target organisms present at very low copy levels (9 genome equivalents) in a 3mL whole blood input volume.

U.box measurement/instrument operation

Described herein are exemplary assays performed using the cartridges and instruments exemplified in fig. 58A-61, 63-66, 69 and 70, wherein a pipette tip is stored on a cartridge and actuated by a gantry as described above.

Summary of the measurement procedure for each pipette tip:

tip 1,5ml: sample input was performed by denaturation and hybridization.

Tip 2,5ml: STC bead capture was performed by magnetic bead washing.

Tip 3,1mL: STC elution was performed by copy control extension.

Tip 4,1mL: additional copy control reagents were prepared by loading the copy control eluate.

Tip 5,1mL: the clone amplification reagents were prepared by RCA/RPA wash II.

Tip 6,1mL: amplified particles were prepared by sequencing dehybridization washes.

Tip 7,1mL: sequencing hybridization was performed by sequencing enzyme binding.

Overview of SPI interface:

SPI-V: is connected to the evacuated blood collection chamber.

SPI-ML: is connected to the mechanical cracking chamber.

SPI-STC: is connected to the STC chamber and bead capture serpentine.

SPI-PCR1: was connected to the PCR1 chamber.

SPI-AUX: the auxiliary chamber will be used for several steps (PCR 1 and CC dilution, PCR2 pooling).

SPI-PCR2: PCR2 network connected to channels and chambers.

SPI-CC: is connected to the copy control chamber.

SPI-FC: is connected to the flow cell.

Step #1: enzymolysis and sample recovery (RETRIEVAL)

Pipette pick up tip 1 (volume 1-5 mL).

The tip 1 is moved onto the cartridge, piercing the foil on the lysis buffer.

Aspirate lysis buffer.

The suction head 1 interfaces with SPI-STC (STC chamber).

Dispense buffer into the hot mixing chamber, plus additional air to purge the inlet channel, rehydrate lyophilized reagents located in the channel or chamber.

The tip 1 interfaces with SPI-V (vacuum blood collection tube).

Pressurizing the evacuated blood collection tube by a pipette and confirming that there is sufficient sample.

Aspiration of the sample into the pipette tip.

The suction head 1 interfaces with SPI-STC.

Dispense sample into the chamber, plus additional air to purge the inlet channel.

Mix back and forth between hot mixing chambers or between chambers and pipette tips to ensure complete rehydration of lyophilized reagents and reagent mixing.

Pushing all fluid into the thermal mixing chamber.

Mix back and forth between the hot mixing chambers through the pneumatic manifold port while heating the Pro-K incubation. The SPI pushes on a regular basis, keeping the fluid in the STC chamber.

Step #2: mechanical cracking

Sucking the reaction fluid into the suction head 1.

The tip 1 interfaces with SPI-ML (mechanical lysis chamber).

Pushing the fluid into the ML chamber, plus additional air to purge the inlet channel.

The suction head 1 interfaces with SPI-STC.

Sucking the remaining fluid into the suction head 1.

The tip 1 interfaces with SPI-ML (mechanical lysis chamber).

Pushing the fluid into the ML chamber, plus additional air to purge the inlet channel.

Mechanical cracking motor actuation.

Step #3: denaturation/hybridization

Sucking the reaction fluid into the suction head 1.

The suction head 1 interfaces with SPI-STC.

Fluid is distributed into the hot mixing chamber, plus additional air to purge the inlet channel.

Mix back and forth between the hot mixing chambers through the pneumatic manifold ports while incubating at denaturation temperature.

Continue to mix back and forth while incubating at hybridization temperature. The SPI pushes on a regular basis, keeping the fluid in the STC chamber.

The suction head 1 falls in a fixed position.

Step #4: bead target capture

Pipette loading into tip 2.

The tip 2 is moved into the kit above the wash buffer T and pierces the foil.

Pipette tip 2 aspirates a volume of buffer for rehydrating the magnetic target capture lyophilization reagents.

The tip 2 is moved to the magnetic target to capture the lyophilized reagent and pierce the foil.

Dispense fluid into the lyophilized reagent bag to rehydrate the beads.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

Sucking the magnetic target capture reagent into the tip 2.

The tip 2 interfaces with SPI-STC.

Dispensing a magnetic target capture reagent into the thermal mixing chamber.

The blood solution is mixed back and forth between the chamber and the tip to suspend any beads in the channel that may be lost. The solution is eventually pushed back into the hot mixing chamber.

Mix back and forth between the thermal mixing chambers through the pneumatic manifold ports while incubating at the binding temperature. The SPI pushes on a regular basis, keeping the fluid in the STC chamber.

STC magnet actuated against the box coil.

Sucking the liquid into the suction head 2, the beads are granulated (pelleted) with respect to the STC magnet.

Distribution of the waste liquid to the mechanical lysis chamber.

Step #5-6: magnetic bead washing

Repeat 4 times:

The tip 2 was moved into the wash buffer T.

Wash buffer T was aspirated by tip 2.

The omicron suction head 2 is in butt joint with SPI-STC.

The o STC magnet is disengaged from the cassette.

The omicron wash buffer T was mixed back and forth between the pipette tip and the hot mixing chamber to resuspend the beads.

The o STC magnet is actuated relative to the cassette.

The waste liquid is sucked into the suction head 2 and as the beads pass through the serpentine channel they are pressed against the STC magnet to granulate.

The omicron suction head 2 is docked with SPI-ML.

The waste liquid was distributed to the ML chamber.

Loading into PCR1 dilution chamber

The tip 2 is moved onto the cartridge and pierces the foil on the water.

The dilution of PCR1 was aspirated into the tip 2.

The suction head 2 interfaces with the SPI-AUX (auxiliary chamber).

Dispensing diluent into the chamber.

Step #7: elution

The suction head 2 falls in a fixed position.

Pipette into tip 3.

The pipette tip 3 is moved into the kit above the wash buffer T.

Aspirate wash buffer T.

The tip 3 interfaces with SPI-STC.

The STC magnet is separated from the cartridge.

Mix the eluate back and forth between the pipette tip and the thermal mixing chamber to resuspend the beads.

Pushing the eluate and beads into the thermal mixing chamber.

During incubation at elution temperature, the eluate was mixed back and forth between the hot mixing chambers through the pneumatic manifold port. Periodic pushing of the SPI may be required to keep the fluid in the chamber.

Upon cooling, the eluent was mixed back and forth between the hot mixing chambers through the pneumatic manifold ports.

The STC magnet interfaces with the cartridge.

Sucking the STC eluate into the suction head 3, granulating it against the STC magnet as the beads pass through the serpentine channel.

Step #8: PCR 1 inclusion (Inclusivity)

Tip 3 interfaces with SPI-PCR 1.

The eluate is dispensed through the channel, rehydrating the PCR1 lyophilized reagents while simultaneously opening the PCR1 vent.

Once the fluid enters the reaction chamber, the PCR1 exhaust port is closed.

Pressurizing the chamber by pipette tip.

Thermal cycling of TEM to perform PCR.

Step #9: PCR 1 dilution

The PCR1 chamber was depressurized by a pipette.

Part of the PCR1 product was aspirated by the tip 3.

The tip 3 interfaces with SPI-AUX.

The PCR1 product was dispensed into a dilution chamber.

Mix PCR1 product and diluent back and forth between chamber and pipette tip for uniform dilution.

Step #10: PCR2 exclusivity

Tip 3 aspirates diluted PCR1 product.

Tip 3 interfaces with SPI-PCR 2.

The diluted PCR1 product was dispensed into a common pipeline channel.

With the aid of an optical sensor and valve control on a pneumatic manifold connected to the exhaust line, filling the individual metering channels.

Suction of the remaining fluid to clear the common line (bypass channel is emptied, all reaction chambers are emptied and closed).

The lyophilized reagents are rehydrated by opening the vent valve and pumping fluid from the metering chamber to the reaction chamber for loading into the reaction chamber.

The tip 3 interfaces with SPI-ML.

Dispense waste into ML chamber.

All chamber vents are closed.

The reaction chamber is pressurized by a bypass channel.

Thermal cycling of TEM to perform PCR.

When PCR2 thermal cycling occurs:

the omicron suction head 3 is docked with SPI-AUX.

The unused diluted PCR1 product was taken.

The omicron suction head 3 is docked with SPI-ML.

The waste liquid was distributed into the ML chamber.

Automatic pH titration may occur (possibly requiring a switch back to tip 1 or the use of a new tip).

The PCR2 reaction chamber was depressurized through a bypass channel.

Tip 3 interfaces with SPI-PCR 2.

The PCR2 product was pooled into the tip.

The tip 3 interfaces with SPI-AUX.

The PCR2 product was dispensed into the chamber.

Mix back and forth between chamber and tip to mix PCR2 products thoroughly and combine back into a single fluid slug.

An aliquot of pooled PCR2 product was aspirated into tip 3.

Step #11: copy control extension

The tip 3 interfaces with SPI-CC.

The pooled PCR2 product was dispensed into the channel, plus additional air to push the fluid into the hot mixing chamber and rehydrate the Hairpin lyophilized reagents as the fluid passed through.

Fluid is mixed back and forth between the thermal mixing chambers through the pneumatic manifold ports while the copy control TEM is thermally cycled for extension.

When thermal cycling occurs, the pipettor prepares for additional reagents required for copy control.

The tip 3 interfaces with SPI-AUX.

Tip 3 aspirates the remaining PCR2 product.

The tip 3 interfaces with SPI-ML.

The remaining PCR2 product was dispensed into the ML chamber.

The suction head 3 falls to a fixed point.

Pipette loading into tip 4.

The tip 4 is moved onto the kit and pierces the foil on the bead wash.

Move the suction head 4 to "water".

Aspirate the fluid volume required for rehydration of the hybridization lyophilization reagents and the magnetic target capture lyophilization reagents.

The pipette tip 4 is moved into the kit and pierces the foil on the hybridization lyophilization reagents and the magnetic target capture lyophilization reagents.

Dispense rehydrated fluid into the lyophilization reagents wells to rehydrate the reagents.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

Sucking the rehydrated hybridization lyophilization reagents into the pipette tip 4.

Step #12: copy control hybridization

The tip 4 interfaces with SPI-CC.

Dispense hybridization reagent into the chamber, plus additional air required to clear the inlet channel.

Fluid is mixed back and forth between the hot mixing chambers through the pneumatic manifold ports.

Step #13: copy control bead target capture

The pipette tip 4 is moved onto the magnetic target capture reagent of the kit that was previously rehydrated. Sucking the magnetic target capture reagent into the tip 4.

The tip 4 interfaces with SPI-CC.

Dispense reagent into the chamber, plus additional air required to purge the inlet channel.

Fluid is mixed back and forth between the thermal mixing chambers through the pneumatic manifold ports while TEM is set to the incubation temperature.

The CC magnet interfaces with the cartridge.

Sucking fluid into the suction head 4, granulating it against the CC magnet as the beads pass through the serpentine channel.

The tip 4 interfaces with SPI-ML.

Distribution of the waste liquid to the mechanical lysis chamber.

Step #14: copy control magnetic bead washing

Repeat 3 times:

the tip 4 was moved into the kit and pre-puncture magnetic bead washing was performed.

Wash buffer was aspirated into tip 4.

The omicron suction head 4 is in butt joint with SPI-CC.

The omicron CC magnet was detached from the cassette.

The omicron fluid was mixed back and forth between the pipette tip and the thermal mixing chamber to resuspend the beads.

The omicron CC magnet is actuated relative to the cartridge.

The fluid is sucked into the suction head 4, granulating it with respect to the CC magnet as the beads pass through the serpentine channel

The omicron tip 4 interfaces with SPI-ML.

First of all distribute waste liquid to ML Chamber

Step #15: copy control elution

The tip 4 is moved over the cartridge to the pre-penetration water.

Aspirate eluent volume.

The tip 4 is moved into the cartridge and docked with the SPI-CC.

The CC magnet is disengaged from the cassette.

The eluate is dispensed into the chamber and mixed back and forth between the pipette tip and the CC chamber to resuspend the beads.

The bead solution was then pushed into the CC chamber and mixed back and forth through the pneumatic manifold port while maintaining the TEM at the elution temperature.

When thermal mixing occurs, the pipette prepares the clonal amplification reagents.

The suction head 4 falls in its fixed position on the cartridge.

Pipette loading into tip 5.

The tip 5 is moved into the cartridge and pierces several reagent foils.

Move the suction head 5 to "water".

Aspirate the fluid used to rehydrate the hybridization buffer lyophilization reagents through tip 5.

Dispense fluid into wells containing RCA hybridization buffer lyophilization reagents.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

Aspiration of pre-spiked RCA ligation buffer into the tip 5.

Dispense fluid into RCA-linked lyophilization reagents.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

The hybridization buffer is aspirated into the tip 5.

Step #16: RCA/RPA initiation

The tip 5 is moved onto the cartridge and docked with the SPI-FC (flow cell cartridge).

Dispense buffer through the flow cell.

Step #17: RCA/RPA hybridization (including copy control dilution)

The auxiliary chamber washing process may require multiple iterations.

The tip 5 is moved over the kit to the pre-penetration water.

Suction by suction head 5.

The suction head 5 interfaces with the SPI-AUX.

Water was dispensed into the auxiliary chamber and mixed back and forth between the tip and chamber to dilute and rinse any remaining PCR2 product.

Suction of water into the suction head 5.

The tip 5 interfaces with SPI-ML.

Distribution of waste to the mechanical pyrolysis chamber.

The tip 5 is moved over the kit to the hybridization buffer.

The hybridization buffer is aspirated into the tip 5.

Buffer is dispensed into the auxiliary chamber.

The tip 5 interfaces with SPI-CC.

All the eluate is sucked into the suction head 5.

The suction head 5 interfaces with the SPI-AUX.

The eluate is dispensed into the auxiliary chamber and mixed back and forth between the chamber and the pipette tip to ensure thorough mixing with the diluent.

Sucking the diluted CC eluate into the suction head 5.

Tip 5 interfaces with SPI-FC.

Dispense diluted CC eluate through the flow cell.

Flow cell TEM performs one thermal cycle.

Step #17: RCA I wash (RPA cancel this step)

The tip 5 is moved over the kit to the pre-puncture RCA wash buffer.

The wash buffer is aspirated by the tip 5.

The tip 5 is moved back to the cartridge and interfaced with the SPI-FC.

Dispense wash buffer across flow cell chip

Draw waste through the sequencing manifold to the cartridge waste chamber.

Step #17: RCA II cycle (RPA cancel this step)

Move the tip 5 over the RCA connection from the kit to the previous rehydration.

The RCA ligation reagent is aspirated by the tip 5.

The tip 5 is moved onto the cartridge and docked to the SPI-FC.

Dispense RCA ligation reagent through the flow cell.

Fluid incubate in flow cell.

Step #18: RCA/RPA Wash II

Move tip 5 back to the kit over the pre-lancing RCA wash buffer.

The wash buffer is aspirated by the tip 5.

Move the tip 5 back to the cartridge and interface with SPI-FC.

Dispense wash buffer through flow cell chip.

Draw waste through the sequencing manifold to the cartridge waste chamber.

Step #19: RCA/RPA reaction (this step has multiple lyophilization reagents for RPA)

The suction head 5 falls in a fixed position.

Pipette loading into tip 6.

The tip 6 is moved to the kit and pierces the foil over the RCA amplified freeze-dried reagent.

The RCA wash buffer was aspirated into the tip 6.

The fluid is dispensed into the RCA amplification lyophilization reagents.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

Suction head 6 aspirates rehydrated RCA amplification reagent.

The reagent is aspirated by the tip 6.

Move the tip 6 back to the cartridge and interface with SPI-FC.

Dispense reagent through the flow cell.

Set the chip TEM to isothermal amplification temperature.

Step #20a: RCA/RPA washing

TEM set to Cooling temperature

TEM and flow cell cooling:

the tip 6 was moved over the kit to the pre-puncture RCA wash buffer.

Wash buffer was aspirated by tip 6.

After cooling is complete, the tip 6 is moved back to the cartridge and docked with SPI-FC.

Dispense buffer through the flow cell.

Draw waste through the sequencing manifold to the cartridge waste chamber.

Step #20b: RCA inactivation (RPA cancel this step)

The tip 6 was moved over the kit to the pre-puncture RCA inactivation buffer.

The inactivation buffer was aspirated by tip 6.

The tip 6 was moved into the cartridge and docked with SPI-FC.

The inactivation buffer was allocated across the flow cell.

Temperature of TEM was controlled.

Step #20c: RCA washing II (RPA cancel this step)

The tip 6 was moved over the kit to the pre-puncture RCA wash buffer.

Wash buffer was aspirated by tip 6.

The tip 6 was moved back to the cartridge and docked with SPI-FC.

Wash buffer was allocated across the flow cell chip.

The waste was pulled through the sequencing manifold towards the kit waste chamber.

Step #21: dehybridization

The tip 6 was moved over the kit to pre-puncture NaOH.

The NaOH was aspirated by tip 6.

The tip 6 was moved to the cartridge and docked with SPI-FC.

The omicron distributed NaOH across the flow cell.

The waste was pulled through the sequencing manifold towards the kit waste chamber.

Step #22: washing

The tip 6 was moved over the kit to the pre-puncture pre-wash buffer.

Wash buffer was aspirated by tip 6.

The tip 6 is moved to the cartridge and interfaced with SPI-FC.

The wash buffer was pushed through the flow cell.

The waste was pulled through the sequencing manifold towards the kit waste chamber.

Step #24: sequencing hybridization

The suction head 6 is ejected to its fixed position.

The omicron pipette picks up the tip 7.

The tip 7 was moved to the kit and the foil was pierced on the sequencing primer lyophilization reagents and sequencing hybridization buffer.

The sequencing hybridization buffer was aspirated into tip 7.

The buffer was allocated to sequencing primer lyophilization reagents.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

The omicron tip 7 pierces the foil on the sequencing enzyme.

The o tip 7 was moved over the kit to the pre-puncture pre-wash buffer.

Wash buffer was aspirated by tip 7.

The fluid was allocated to the sequencing enzyme.

The aspiration/dispensing cycle is performed as necessary to ensure complete resuspension of the reagents.

The omicron tip 7 was moved into the previously rehydrated sequencing primer lyophilization reagents.

The sequencing primer was aspirated into tip 7.

The tip 7 was moved to the cartridge and docked with SPI-FC.

The primer solution was distributed across the flow cell.

Additional air was allocated to pressurize the flow cell.

The omicron chip TEMS was subjected to hybridization thermocycling.

The o pipettor was depressurized.

The waste was pulled through the sequencing manifold towards the kit waste chamber.

Step #25: sequencing washes

The tip 7 was moved over the kit to the pre-puncture pre-wash buffer.

Wash buffer was aspirated by tip 7.

The tip 7 was moved to the cartridge and docked with SPI-FC.

The wash buffer was pushed through the flow cell.

The waste was pulled through the sequencing manifold towards the kit waste chamber.

Step #26: enzyme binding

The tip 7 was moved over the kit to the sequencing enzyme that was previously rehydrated.

The sequencing enzyme was aspirated into tip 7.

The tip 7 was moved to the cartridge and docked to SPI-FC.

The omicron tip 7 dispenses the reagent through the flow cell.

The waste was pulled through the sequencing manifold towards the kit waste chamber.

Chip TEM was set to enzyme incubation temperature.

Sequencing is then controlled by the instrument/cassette sequencing manifold and associated pumps and valves.

Additional embodiments

According to certain aspects of the invention, various further embodiments of the apparatus, cartridges, systems, and/or methods are contemplated.

Embodiment 1 is a method of preparing a target polynucleotide in a sample for further downstream processing or analysis, the method comprising:

Providing a sample comprising a target polynucleotide; and

The sample or target polynucleotide is processed to obtain a target nucleotide ready for further downstream processing or analysis.

Embodiment 2 is the method of claim 1, wherein the target polynucleotide comprises at least one of DNA (e.g., genome DNA, cfDNA, ctDNA) and RNA (e.g., mRNA, rRNA, tRNA).

Embodiment 3 is the method of any one of the preceding embodiments, wherein the method comprises filtering, concentrating, solubilizing, dissolving, homogenizing, or digesting the sample, or otherwise altering the physical or chemical properties of the sample to facilitate preparation of the target polynucleotide.

Embodiment 4 is the method of any one of the preceding claims, wherein the sample comprises a biological or clinical sample.

Embodiment 5 is the method of embodiment 4, wherein the sample comprises whole blood, plasma, serum, buffy coat, white blood cells, red blood cells, or platelets.

Embodiment 6 is the method of any one of the preceding embodiments, wherein the sample comprises a cell comprising the target polynucleotide.

Embodiment 7 is the method of embodiment 6, wherein the method further comprises labeling, capturing, concentrating, separating, or otherwise treating one or more cells, cell types, or cell populations within the sample.

Embodiment 8 is the method of embodiment 7, wherein the method further comprises tagging, labeling, capturing, concentrating, isolating, or otherwise treating the cell suspected of containing the target polynucleotide.

Embodiment 9 is the method of embodiments 6 to 8, wherein the method comprises lysing, digesting, disrupting, partially lysing, shearing, or otherwise treating cells containing or associated with the target polynucleotide to make the target polynucleotide available or otherwise more available for further processing or analysis.

Embodiment 10 is the method of any one of the preceding embodiments, wherein the method comprises cleaving, digesting, disrupting, partially dissolving, shearing, or otherwise treating a structure containing or associated with the target polynucleotide to make the target polynucleotide available or otherwise more available for further processing or analysis.

Embodiment 11 is the method of any one of the preceding embodiments, wherein the method comprises tagging, labeling, annealing to an oligonucleotide, concentrating, enriching, capturing, isolating (separating and/or isolating) the target polynucleotide.

Embodiment 12 is the method of embodiment 11, wherein the method further comprises tagging or labeling the single target polynucleotide molecule or the collection of target polynucleotide molecules with a Unique Molecular Identifier (UMI).

Embodiment 13 is the method of embodiment 12, wherein the UMI is introduced by ligation.

Embodiment 14 is the method of embodiment 12, wherein the extension is introduced to the UMI by annealing the UMI-tagged oligomer to the target polynucleotide.

Embodiment 15 is the method of any one of embodiments 11 to 14, wherein the method further comprises annealing the at least one capture oligomer to the at least one target polynucleotide to produce at least one capture oligomer/target complex.

Embodiment 16 is the method of embodiment 15, wherein the at least one capture oligomer anneals to the at least one target polynucleotide at a conserved target locus across a plurality of different species.

Embodiment 17 is the method of embodiment 16, wherein the target locus comprises at least one of 16S, 23S, or 28S genomic DNA or a 5' -untranslated region.

Embodiment 18 is the method of any one of embodiments 11 to 17, wherein the method further comprises extending the 3' end of at least one capture oligomer in the at least one capture oligomer/target complex with a polymerase.

Embodiment 19 is the method of any one of embodiments 11 to 18, wherein the at least one or more capture oligomers comprises any one or more capture oligomers described herein.

Embodiment 20 is the method of any one of embodiments 11 to 19, wherein the method further comprises isolating at least one capture oligomer/target complex, thereby isolating the target polynucleotide.

Embodiment 21 is the method of embodiment 20, wherein the method further comprises immobilizing the at least one capture oligomer/target complex on a solid substrate.

Embodiment 22 is the method of any one of embodiments 11 to 21, wherein the at least one capture oligomer comprises a first ligand of a ligand pair conjugated thereto, the ligand pair comprising a second ligand bound to a solid matrix, and isolating the at least one capture oligomer-target complex comprises binding the first ligand to the second ligand to produce an immobilized ligand complex.

Embodiment 23 is the method of embodiment 22, wherein the ligand pair comprises biotin/avidin or biotin/streptavidin.

Embodiment 24 is the method of embodiment 22, wherein the ligand pair comprises a nucleic acid sequence and its complement.

Embodiment 25 is the method of embodiments 12 to 24, wherein the at least one capture oligomer comprises a tag sequence comprising a nucleic acid sequence that does not anneal to the target polynucleotide under a defined set of conditions.

Embodiment 26 is the method of embodiment 25, wherein at least one additional oligomer is provided, and wherein the at least one additional oligomer comprises a nucleic acid sequence complementary to the tag sequence of the at least one capture oligomer, and further comprising a first ligand of a ligand pair that comprises a second ligand bound to the solid matrix, annealing the additional oligomer to the tag of the capture oligomer/target complex, and isolating the at least one capture oligomer-target complex comprises binding the first ligand to the second ligand to produce the immobilized ligand complex.

Embodiment 27 is the method of embodiment 26, wherein the capture oligomer binds to the target polynucleotide and the additional oligomer binds simultaneously to the tag of the capture oligomer.

Embodiment 28 is the method of embodiments 26 to 27, wherein the additional oligomer is provided in a defined and limited amount.

Embodiment 29 is the method of embodiments 26 to 28, wherein the ligand pair comprises biotin/avidin or biotin/streptavidin.

Embodiment 30 is the method of embodiments 26 to 28, wherein the ligand pair comprises a nucleic acid sequence and its complement.

Embodiment 31 is the method of any one of embodiments 21 to 30, wherein the method further comprises washing the solid substrate after immobilization of the complex comprising the target polynucleotide.

Embodiment 32 is the method of any one of embodiments 21 to 31, wherein the method further comprises eluting the target polynucleotide from the solid matrix.

Embodiment 31 is the method of any one of embodiments 11 to 32, wherein the method further comprises annealing one or more primers, blocking sequence oligomers, replacement sequence oligomers, or cleavage site specific oligomers.

Embodiment 34 is the method of any one of embodiments 1 to 11, wherein the target polynucleotide is used for downstream processing of analysis directly from the sample without prior capture, isolation (separating and/or isolating).

Embodiment 35 is a method of making a nucleic acid library, comprising amplifying a portion of a target polynucleotide to obtain a first amplicon.

Embodiment 36 is the method of embodiment 35, wherein the target polynucleotide is the product of the method of any one of embodiments 1 to 33.

Embodiment 37 is the method of embodiment 35, wherein the target polynucleotide is used directly from the sample.

Embodiment 38 is the method of any one of embodiments 35 to 37, wherein amplifying comprises amplifying at least a portion of the target polynucleotide with at least one first primer to generate at least one first amplicon.

Embodiment 39 is the method of embodiment 38, wherein the at least one first primer comprises at least one capture oligomer or other oligomer type of any one of embodiments 14 to 33.

Embodiment 40 is the method of embodiment 38, wherein at least one first primer hybridizes to at least one tag sequence introduced into the target polynucleotide or one or more fragments thereof.

Embodiment 41 is the method of any one of embodiments 38-40, wherein the at least one first primer comprises at least one tag.

Embodiment 42 is the method of any one of embodiments 38 to 41, wherein the at least one first primer comprises at least one positive (+) sense primer and at least one negative (-) sense primer.

Embodiment 43 is the method of embodiment 42, wherein at least one sense primer hybridizes to at least one tag sequence and at least one negative primer hybridizes to a target polynucleotide or one or more fragments thereof, at least one negative primer hybridizes to at least one tag sequence and at least one positive primer hybridizes to a target polynucleotide or one or more fragments thereof, or at least one positive primer hybridizes to at least one tag sequence and at least one negative primer hybridizes to at least one different tag sequence.

Embodiment 44 is the method of any one of embodiments 35 to 43, wherein more than one first amplicon is generated.

Embodiment 45 is the method of any one of embodiments 38 to 44, wherein the at least one first primer comprises any primer or set or sets of reverse primers described herein.

Embodiment 46 is the method of any one of embodiments 35 to 45, wherein the amplification method is PCR.

Embodiment 47 is the method of any one of embodiments 35 to 46, wherein at least one first amplicon is purified.

Embodiment 48 is the method of any one of embodiments 35 to 47, further comprising amplifying at least a portion of the at least one first amplicon with at least one second primer to produce at least one second amplicon.

Embodiment 49 is the method of embodiment 48, wherein the at least one second primer hybridizes to at least one tag sequence introduced into the at least one first amplicon or one or more fragments thereof.

Embodiment 50 is the method of any one of embodiments 48 to 49, wherein the at least one second primer comprises at least one tag.

Embodiment 51 is the method of any one of embodiments 48 to 50, wherein at least one second primer is nested by at least one nucleotide within at least one first primer.

Embodiment 52 is the method of any one of embodiments 48 to 51, wherein the at least one second primer comprises at least one positive (+) sense primer and at least one negative (-) sense primer.

Embodiment 53 is the method of embodiment 52, wherein at least one sense primer hybridizes to at least one tag sequence and at least one negative sense primer hybridizes to at least one first amplicon or one or more fragments thereof, at least one negative sense primer hybridizes to at least one tag sequence and at least one positive sense primer hybridizes to at least one first amplicon or one or more fragments thereof, or at least one positive sense primer hybridizes to at least one tag sequence and at least one negative sense primer hybridizes to at least one different tag sequence.

Embodiment 54 is the method of any one of embodiments 48 to 53, wherein more than one second amplicon is generated.

Embodiment 55 is the method of embodiment 54, wherein at least one second primer is nested within at least one first primer, and at least one other second primer is not nested within at least one first primer.

Embodiment 56 is the method of any one of embodiments 48 to 55, wherein the at least one second primer comprises any primer or set or sets of opposing primers (opposed primers) described herein.

Embodiment 57 is the method of any one of embodiments 48-56, wherein the amplification method is PCR.

Embodiment 58 is the method of any one of embodiments 48 to 57, wherein at least one first amplicon is purified.

Embodiment 59 is the method of any one of embodiments 48-57, wherein at least one second amplicon is purified.

Embodiment 60 is the method of embodiment 58, wherein at least one second amplicon is also purified.

Embodiment 61 is a method of preparing a nucleic acid library comprising ligating a sequence tag to a target polynucleotide.

Embodiment 62 is the method of embodiment 61, wherein the tag comprises an adapter molecule.

Embodiment 63 is the method of embodiment 62, wherein the tag further comprises an additional sequence.

Embodiment 64 is the method of any one of embodiments 61 to 63, wherein the target polynucleotide is used directly from the sample.

Embodiment 65 is the method of any one of embodiments 61 to 63, wherein the target polynucleotide is the product of the method of any one of embodiments 1 to 33.

Embodiment 66 is a capture oligomer comprising in the 5 'to 3' direction: a capture sequence, an internal extension blocking sequence, a complement of the capture sequence, and a target hybridization sequence, wherein the complement of the capture sequence is configured to anneal to the capture sequence in the absence of an extended target sequence that anneals to the complement of the target hybridization sequence and the capture sequence.

Embodiment 67 is the capture oligomer of embodiment 66, wherein the capture oligomer has the formula 5' -A1-C-L-B-A2-C ' -A3-RB-A4-THS-X-3', wherein A1 is an optionally present first additional sequence; c is the capture sequence, L is the optional linker, B is the internal extension blocking sequence, A2 is the optional second additional sequence, C' is the complement of the capture sequence, A3 is the optional third additional sequence, RB is the optional reversible extension blocking sequence, A4 is the optional fourth additional sequence, and THS is the target hybridization sequence; and X is an optionally present blocking moiety.

Embodiment 68 is a combination comprising a capture oligomer and a complementary oligomer, wherein: (a) the capture oligomer comprises in the 5 'to 3' direction: a capture sequence comprising a first portion and a second portion, an internal extension blocking sequence, a spacer sequence comprising a first portion and a second portion, and a target hybridization sequence; and (b) the complementary oligomer comprises in the 3 'to 5' direction: a complement of the second portion of the capture sequence, and a complement of at least a first portion of the spacer sequence, wherein the complement of the second portion of the capture sequence and the complement of at least a first portion of the spacer sequence are configured to anneal to the capture oligomer simultaneously in the absence of the complement of the spacer sequence.

Embodiment 69 is a combination of embodiment 68, wherein the capture oligomer has the formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3', wherein A1 is an optionally present first additional sequence, C1 is a first portion of a capture sequence, C2 is a second portion of a capture sequence, B is an internal extension blocking sequence, A2 is an optionally present second additional sequence, S1 is a first portion of a spacer sequence, S2 is a second portion of a spacer sequence, A3 is an optionally present third additional sequence, RB is an optionally present reversible extension blocking sequence, A4 is an optionally present fourth additional sequence, THS is a target hybridization sequence, and X is an optionally present blocking portion.

Embodiment 70 is the combination of any of embodiments 68 or 69, wherein the complementary oligomer has the formula: 5' -S1' -A2' -L-C2' -X-3', wherein S1' is the complement of at least a first portion of the spacer sequence and A2' is the optionally present complement of a second additional sequence optionally present in the capture oligomer; l is an optionally present linker, C2' is the complement of the second part of the capture sequence, and X is a selectively present blocking moiety.

Embodiment 71 is a method of capturing a target polynucleotide from a composition, the method comprising: contacting the target polynucleotide with the capture oligomer of any one of embodiments 66 or 67, wherein the target hybridization sequence of the capture oligomer anneals to the target polynucleotide at a site comprising the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the capture sequence that anneals to the capture oligomer such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a second capture reagent comprising a complementary sequence of the capture sequence and either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the second capture reagent; and separating the complex from the composition, thereby capturing the target polynucleotide.

Embodiment 72 is a method of capturing a target polynucleotide from a composition, the method comprising: contacting the composition with the combination of any of embodiments 68 to 70, wherein the target hybridization sequence of the capture oligomer anneals to the target polynucleotide at a site comprising the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the spacer sequence that anneals to the capture oligomer such that the complement oligomer is displaced to a degree sufficient to make the capture sequence of the capture oligomer available for binding; contacting the capture sequence of the capture oligomer with a second capture reagent comprising a complementary sequence of the capture sequence and either (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the second capture reagent; and separating the complex from the composition, thereby capturing the target polynucleotide.

Embodiment 73 is a method of capturing a target polynucleotide from a composition, the method comprising: contacting a target polynucleotide with a capture oligomer comprising in the 5 'to 3' direction: a capture sequence, an optional internal extension blocking sequence, an optional spacer sequence, and a target hybridization sequence configured to anneal to a target polynucleotide; contacting the capture oligomer with a first capture reagent comprising a complement of the capture sequence (either before, simultaneously with, or after contacting the target polynucleotide with the capture oligomer); providing a second capture reagent comprising a complement of a sequence in the capture oligomer other than the capture sequence, wherein if some or all of the capture oligomer is not annealed to the target polynucleotide, the second capture reagent contacts the capture oligomer that is not annealed to the target polynucleotide; separating the first complex and the second complex from the composition, wherein the first complex comprises the target polynucleotide and the second complex comprises the capture oligomer that is not annealed to the target polynucleotide; and selectively eluting the target polynucleotide or a subcomplex comprising the target polynucleotide from the first complex; optionally, wherein (a) the first capture reagent comprises (i) a binding partner and (e.g., biotin) or (ii) a solid support (e.g., bead or surface) and/or (b) the second capture reagent comprises (i) a binding partner and (e.g., biotin) or (ii) a solid support (e.g., bead or surface).

Embodiment 74 is the method of any one of embodiments 71 to 73, wherein the input material is the output material of any one of claims 1 to 34.

Embodiment 75 is the method of any one of embodiments 71 to 73, wherein the input material is the output material of any one of claims 35 to 65.

Embodiment 76 is a method of producing a nucleic acid cluster immobilized on a solid support, the method comprising amplifying a target polynucleotide in a reaction mixture comprising a solid support immobilized first primer and a non-immobilized second primer to produce a solid support immobilized target nucleic acid amplicon and a free target nucleic acid amplicon.

Embodiment 77 is the method of embodiment 76, wherein the reaction mixture does not contain a non-immobilized first primer.

Embodiment 78 is the method of embodiment 76, wherein the reaction mixture further comprises a non-immobilized first primer.

Embodiment 79 is the method of embodiment 78, wherein the relative ratio of the concentrations of the non-immobilized second primer and the first primer is about 1:1.

Embodiment 80 is the method of embodiment 78, wherein the relative ratio of the concentrations of the non-immobilized second primer and the first primer is between about 2:1 and 1000:1 or higher.

Embodiment 81 is the method of embodiment 78, wherein the relative ratio of the concentrations of the non-immobilized first primer and the second primer is between about 2:1 and 1000:1 or higher.

Embodiment 82 is the method of any one of embodiments 76 to 81, wherein the target polynucleotide is added to the reaction mixture at a concentration that approximates the poisson distribution level on the solid support.

Embodiment 83 is the method of any one of embodiments 76 to 82, wherein the method comprises hybridizing the target polynucleotide to a first primer immobilized on a solid support prior to initiating amplification.

Embodiment 84 is the method of embodiment 83, wherein the solid support is washed after hybridization of the target polynucleotide to the immobilized first primer of the solid support and before amplification begins.

Embodiment 85 is the method of any one of embodiments 76 to 84, wherein an unfixed target nucleic acid amplicon is produced in addition to a surface-immobilized target nucleic acid amplicon.

Embodiment 86 is the method of embodiment 85, wherein one or more strands of the non-immobilized target nucleic acid amplicon are hybridized to the solid support immobilized first primer and amplified to produce additional solid support immobilized target nucleic acid.

Embodiment 87 is the method of embodiment 86, wherein said hybridization of said target nucleic acid amplicon not immobilized to said solid support immobilized first primer occurs in the immediate vicinity of the site of solid support-immobilized target nucleic acid amplicon production in the preceding amplification stage.

Embodiment 88 is the method of any one of embodiments 76 to 87, wherein the cluster is monoclonal.

Embodiment 89 is the method of any one of embodiments 76 to 88, wherein the target polynucleotide comprises at least 2 different target polynucleotides, and amplifying comprises:

Hybridizing different target polynucleotides to first primers immobilized to the solid support at spatially separated locations on the solid support;

Solid support-immobilized target nucleic acid amplicon clusters of each hybridized target polynucleotide are produced at and around each spatially-spaced apart location, wherein at least a portion of each cluster does not overlap with at least a portion of an adjacent cluster.

Embodiment 90 is the method of any one of embodiments 76 to 89, wherein the solid support immobilized first primer and/or the non-immobilized second primer or first primer comprises any primer or set or sets of opposing primers described herein.

Embodiment 91 is the method of any one of embodiments 76 to 90, wherein the amplification method is recombinase polymerase amplification.

Embodiment 92 is the method of any one of embodiments 76 to 91, wherein the target polynucleotide comprises a target nucleic acid output as claimed in any one of embodiments 1 to 75.

Embodiment 93 is the method of any one of embodiments 76 to 91, wherein the target polynucleotide is used directly from the sample.

Embodiment 94 is the method of any one of embodiments 76 to 91, wherein the target polynucleotide comprises the first primer binding sequence and/or the second primer binding sequence added as the tag sequence of embodiment 22 or the tag sequence described in embodiments 41 or 50.

Embodiment 95 is the method of any one of embodiments 76 to 93, wherein the solid support comprises a surface of a semiconductor chip.

Embodiment 96 is the method of embodiment 94, wherein the surface of the semiconductor chip further comprises an array of three-dimensional features.

Embodiment 97 is the method of embodiment 95, wherein the three-dimensional feature comprises a hole.

Embodiment 98 is the method of any one of embodiments 94 to 96, wherein the semiconductor chip further comprises an ISFET sensor array.

Embodiment 99 is a method of producing a nucleic acid cluster immobilized on a solid support, the method comprising amplifying a target polynucleotide, the method further comprising:

Providing a linear target nucleic acid comprising a first portion of a first primer binding sequence on a first portion of the linear target nucleic acid, a second portion of the first primer binding sequence on a second portion of the linear target nucleic acid, and a second primer sequence between the first portion and the second portion of the linear target nucleic acid;

providing a solid support comprising a first primer and a second primer immobilized on a surface of the solid support, wherein the first primer comprises a first primer sequence and the second primer comprises a second primer sequence;

hybridizing a first portion and a second portion of a first primer binding sequence of a target nucleic acid to one of the immobilized first primers;

ligating the first portion and the second portion of the first primer binding sequence of the target nucleic acid to produce a circular target nucleic acid comprising the first primer binding sequence and the second primer sequence;

Extending one of the immobilized first primers along the circular target nucleic acid as a template to produce a first extended target nucleic acid strand comprising at least one copy of the first primer sequence and at least one copy of the second primer binding sequence;

Hybridizing at least one copy of a second primer binding sequence on the first extended target nucleic acid strand to at least one immobilized second primer;

extending at least one copy of one of the immobilized second primers along the first extended target nucleic acid strand to produce a second extended target nucleic acid strand comprising at least one copy of the second primer sequence and at least one copy of the first primer binding sequence; and

Hybridizing at least one of the first primer binding sequences on the second extended target nucleic acid strand to the other of the immobilized first primers; and

The other of the immobilized first primers is extended along the second extended target nucleic acid strand as a template to produce a third extended target nucleic acid strand comprising at least one copy of the first primer sequence and at least one copy of the second primer binding sequence.

Embodiment 100 is the method of embodiment 99, wherein the amplification method is rolling circle amplification.

Embodiment 101 is the method of any one of embodiments 99 to 100, wherein the target polynucleotide comprises a target nucleic acid output as claimed in any one of embodiments 1 to 75.

Embodiment 102 is the method of any one of embodiments 99 to 100, wherein the target polynucleotide is used directly from the sample.

Embodiment 103 is the method of any one of embodiments 99 to 102, wherein the target polynucleotide comprises the first primer binding sequence and/or the second primer sequence added as the tag sequence of embodiment 22 or the tag sequence described in embodiments 41 or 50.

Embodiment 104 is the method of embodiment 103, wherein the one or more additional sequences added to the target polynucleotide comprise a first portion of a first primer binding sequence on a first end of the target polynucleotide, a portion or all of a second primer sequence in an interior region of the target polynucleotide, and a second portion of the first primer binding sequence on a second end of the target polynucleotide.

Embodiment 105 is the method of any one of embodiments 99 to 104, wherein the solid support comprises a surface of a semiconductor chip.

Embodiment 106 is the method of embodiment 105, wherein the surface of the semiconductor chip further comprises an array of three-dimensional features.

Embodiment 107 is the method of embodiment 106, wherein the three-dimensional feature comprises a hole.

Embodiment 108 is the method of any one of embodiments 105-107, wherein the semiconductor chip further comprises an ISFET sensor array.

Embodiment 109 is a method of producing a nucleic acid cluster immobilized on a solid support, the method comprising hybridizing a target polynucleotide in a reaction mixture comprising a first oligonucleotide immobilized on a solid support to produce a solid support immobilized target polynucleotide.

Embodiment 110 is the method of embodiment 109, wherein the solid support immobilized first oligonucleotide comprises a capture oligonucleotide, a primer, or a tethered (tethering) oligonucleotide.

Embodiment 111 is the method of any one of embodiments 109 to 110, wherein the solid support immobilized first oligonucleotides are immobilized in discrete regions of the solid support surface.

Embodiment 112 is the method of embodiment 111, wherein immobilizing comprises spotting (spotting) or directly synthesizing the oligonucleotides on the surface of the solid support.

Embodiment 113 is the method of any one of embodiments 109 to 112, wherein the immobilized nucleic acid cluster is monoclonal.

Embodiment 114 is the method of any one of embodiments 109 to 112, wherein the method further comprises at least first and second solid support immobilized oligonucleotides, and further wherein at least first and second nucleic acid clusters are generated.

Embodiment 115 is the method of embodiment 114, wherein at least the first and second nucleic acid clusters are monoclonal.

Embodiment 116 is the method of any one of embodiments 109 to 115, wherein the solid support comprises a surface of a semiconductor chip.

Embodiment 117 is the method of embodiment 116, wherein the surface of the semiconductor chip further comprises an array of three-dimensional features.

Embodiment 118 is the method of embodiment 117, wherein the three-dimensional feature comprises a hole.

Embodiment 119 is the method of any one of embodiments 116-118, wherein the semiconductor chip further comprises an ISFET sensor array.

Embodiment 120 is a method of producing a population of nucleic acid clusters immobilized on a solid support, the method comprising amplifying a population of target polynucleotides by recombinase polymerase amplification in a reaction mixture comprising a solid support immobilized first primer and a non-immobilized second primer to produce solid support immobilized target nucleic acid amplicons and free target nucleic acid amplicons, wherein at least a portion of each cluster does not overlap with at least a portion of an adjacent cluster.

Embodiment 121 is the method of embodiment 120, wherein one or more strands of the free target nucleic acid amplicon that are not immobilized are hybridized to a first primer immobilized on a solid support and amplified to produce additional solid support immobilized target nucleic acid.

Embodiment 122 is the method of embodiment 120 or 121, wherein the target polynucleotide has a universal region at the 3 'and/or 5' end.

Embodiment 123 is the method of any one of embodiments 120-122, wherein the solid support comprises wells, and the majority of the wells contain cloned amplicons.

Embodiment 124 is the method of embodiment 123, wherein the amplicon in the well is sequenced.

Embodiment 125 is the method of embodiment 124, wherein sequencing is performed using a semiconductor chip comprising an ISFET sensor array.

Embodiment 126 is a method of producing a population of nucleic acid clusters immobilized on a solid support, the method comprising amplifying a population of target polynucleotides by recombinase polymerase amplification in a reaction mixture comprising a solid support immobilized first primer and a non-immobilized second primer to produce solid support immobilized target nucleic acid amplicons and free target nucleic acid amplicons, wherein clusters are randomly distributed on the solid support and at least a portion of each cluster does not overlap at least a portion of an adjacent cluster.

Embodiment 127 is the method of embodiment 126, wherein the solid support comprises three-dimensional features.

Embodiment 128 is the method of embodiment 127, wherein the three-dimensional feature is a hole.

Embodiment 129 is the method of any one of embodiments 126 to 128, wherein the solid support comprises a semiconductor chip.

Embodiment 130 is the method of embodiment 129, wherein the chip comprises an ISFET sensor array.

Embodiment 131 is a method of determining a nucleotide sequence of a target polynucleotide, the method comprising:

immobilizing a target polynucleotide or derivative thereof to a solid support;

optionally amplifying the immobilized target polynucleotide or derivative thereof to produce clusters;

annealing the sequencing primer to the immobilized polynucleotide, derivative thereof, or amplicon product thereof to produce a target nucleic acid/sequencing primer complex;

Binding a sequencing enzyme to the target nucleic acid/sequencing primer complex;

Nucleotides are added sequentially and the signal measured after each addition to determine the nucleotide sequence of the target polynucleotide.

Embodiment 132 is the method of any one of embodiment 131, wherein the target polynucleotide is used directly from the sample.

Embodiment 133 is the method of any one of embodiments 131 or 132, wherein the target polynucleotide is prepared from a sample.

Embodiment 134 is the method of embodiment 133, wherein preparing the sample comprises the method of any one of embodiments 1 to 33, embodiments 35 to 65, embodiments 66 to 75, embodiments 76 to 130.

Embodiment 135 is the method of any one of embodiments 131 to 134, wherein the immobilized oligonucleotides are sequencing primers.

Embodiment 136 is the method of embodiment 135, wherein the step of immobilizing and the step of sequencing primer hybridizing are the same step.

Embodiment 137 is the method of any one of embodiments 131 to 134, wherein immobilizing and optionally amplifying a target polynucleotide or derivative thereof comprises the method of any one of embodiments 76 to 108.

Embodiment 138 is the method of any one of embodiments 131 to 134 or 137, wherein annealing the sequencing primer to the immobilized polynucleotide, derivative thereof, or amplicon product thereof to produce the target nucleic acid/sequencing primer complex and binding the sequencing enzyme to the target nucleic acid/sequencing primer complex occurs in the same step.

Embodiment 139 is the method of embodiments 131-138, wherein the sequencing enzyme is thermostable.

Embodiment 140 is the method of any one of embodiments 131 to 139, wherein the method further comprises a flow cell that facilitates the delivery of fluid to and removal of fluid from the solid support.

Embodiment 141 is the method of any one of embodiments 131 to 140, wherein the solid support comprises a surface of a semiconductor chip.

Embodiment 142 is the method of embodiment 141, wherein the surface of the semiconductor chip further comprises an array of three-dimensional features.

Embodiment 143 is the method of embodiment 142, wherein the three-dimensional feature comprises a hole.

Embodiment 144 is the method of any one of embodiments 141 to 143, wherein the semiconductor chip further comprises an ISFET sensor array.

Embodiment 145 is the method of embodiment 144, wherein measuring the signal after each addition to determine the nucleotide sequence of the target polynucleotide comprises detection by an ISFET sensor.

Embodiment 146 is the method of any one of embodiments 131 to 145, wherein determining the sequence comprises using a computerized analysis algorithm.

Embodiment 147 is a system comprising an instrument and a cartridge removably insertable into the instrument.

Embodiment 148 is the system of embodiment 147, wherein the assay cartridge comprises any one or any combination of the following:

A sample input unit;

An incubation unit;

A lysing unit;

a Magnetic Separation Unit (MSU),

A library preparation unit;

A Copy Control (CC) unit; and

Cluster generation/sequencing unit.

Embodiment 149 is the system of embodiment 148, wherein the sample input unit comprises a sample inlet, an input/output valve, and a fluid channel between and in fluid connection with the sample inlet and the input/output valve.

Embodiment 150 is the system of any one of embodiments 148-149, further comprising a fluid channel, and fluidly connected between the sample inlet and at least one chamber in the cartridge.

Embodiment 151 is the system of embodiment 150, wherein the at least one chamber in the cartridge comprises at least one lysing chamber.

Embodiment 152 is the system of any one of embodiments 148-151, wherein the sample input is a blood collection tube (e.g., a vacuum blood collection tube (Becton Dickinson)).

Embodiment 153 is the system of any one of embodiments 148-152, wherein the incubation unit comprises at least one incubation chamber, at least one input/output valve, a fluid channel between and fluidly connected to the at least one incubation chamber and the at least one input/output valve, and when two or more incubation chambers are present, a fluid channel between and fluidly connected to the at least one incubation chamber and at least one additional incubation chamber.

Embodiment 154 is the system of embodiment 153, wherein the at least one incubation chamber interfaces with a heating element when the cartridge is inserted into the instrument.

Embodiment 155 is the system of any one of embodiments 153-154, wherein the at least one incubation chamber comprises at least one lysis chamber.

Embodiment 156 is the system of any one of embodiments 153-155, wherein the system comprises more than one incubation unit.

Embodiment 157 is the system of any of embodiments 147-156, wherein the lysing unit comprises at least one lysing chamber, an input/output valve, a fluid channel between and in fluid connection with the at least one lysing chamber and the input/output valve, and when two or more lysing chambers are present, a fluid channel between and in fluid connection with the at least one lysing chamber and at least one additional lysing chamber.

Embodiment 158 is the system of embodiment 157, wherein the at least one lysing chamber comprises a rotating paddle or impeller.

Embodiment 159 is the system of embodiment 158, wherein the rotating paddle or impeller is capable of interfacing with an actuator on the instrument when the cartridge is inserted into the instrument.

Embodiment 160 is the system of any one of embodiments 147-159, wherein the MS unit comprises one or more MS chambers.

Embodiment 161 is the system of embodiment 160, wherein the one or more MS chambers interface with a heating element on the instrument when the cartridge is inserted into the instrument.

Embodiment 162 is the system of any one of embodiments 160-161, wherein one or more MS chambers interface with a magnetic element on the instrument when the cartridge is inserted into the instrument.

Embodiment 163 is the system of any of embodiments 160-162, wherein the MS unit further comprises an input/output valve and a fluid channel between and fluidly connected to the one or more MS chambers and the input/output valve.

Embodiment 164 is the system of any one of embodiments 160-163, wherein the MS unit further comprises one or more pneumatic ports in fluid connection with the one or more MS chambers, wherein the one or more pneumatic ports are capable of interfacing with a pneumatic manifold on the instrument when the cartridge is inserted into the instrument.

Embodiment 165 is the system of embodiment 164, wherein the MS unit further comprises one or more condensate trap chambers positioned between and fluidly connected to the one or more MS chambers and the one or more pneumatic ports.

Embodiment 166 is the system of any one of embodiments 164 to 165, wherein:

The one or more MS chambers include two or more MS chambers fluidly connected to the one or more input/output valves via one or more fluid channels located between the two or more MS chambers and the one or more input/output valves;

each of the two or more MS chambers is fluidly connected to a separate pneumatic port; and

Optionally, each of the two or more MS chambers is fluidly connected to a separate condensate trap chamber disposed between the MS chamber and the pneumatic port.

Embodiment 167 is the system of any of embodiments 147-166, wherein the library preparation unit comprises one or more amplification reaction chambers.

Embodiment 168 is the system of embodiment 167, wherein the one or more amplification reaction chambers interface with a heating element on the instrument when the cartridge is inserted into the instrument.

Embodiment 169 is the system of any one of embodiments 167 to 168, wherein the library preparation unit further comprises one or more input/output valves and one or more fluid channels positioned between and fluidly connected to the one or more amplification reaction chambers and the one or more input/output valves.

Embodiment 170 is the system of any one of embodiments 167-169, wherein the library preparation unit further comprises lyophilized amplification reagents in one or more amplification reaction chambers or in one or more fluidic channels.

Embodiment 171 is the system of any one of embodiments 167 to 170, wherein the library preparation unit further comprises one or more pneumatic ports in fluid connection with the one or more amplification reaction chambers, wherein the one or more pneumatic ports are capable of interfacing with a pneumatic manifold on the instrument when the cartridge is inserted into the instrument.

Embodiment 172 is the system of embodiment 171, wherein the library preparation unit further comprises one or more amplification aliquot chambers, the amplification aliquot chambers being located between and fluidly connected to the one or more amplification reaction chambers and the one or more input/output valves.

Embodiment 173 is the system of any of embodiments 171-172, wherein:

The one or more amplification reaction chambers include two or more amplification reaction chambers fluidly connected to a single output/input valve via a single fluid channel, the single fluid channel being located between the two or more amplification reaction chambers and the single input/output valve;

each of the two or more amplification reaction chambers is in fluid connection with a separate pneumatic port; and

Optionally, each of the two or more amplification reaction chambers is in fluid connection with a separate amplification aliquot chamber disposed between the amplification reaction chamber and a single input/output valve.

174 Embodiment 174 is the system of embodiment 173, wherein:

The one or more amplification reaction chambers include additional amplification reaction chambers separate from the two or more amplification reaction chambers;

The further amplification reaction chamber is in fluid connection with the further input/output valve via a further fluid channel between the further amplification reaction chamber and the further input/output valve, wherein the further input/output valve and the further fluid channel are separate from the input/output valve and the fluid channel in fluid connection with two or more chambers; and

Optionally in fluid connection with a further amplification aliquoting chamber arranged between the further amplification reaction chamber and the further input/output valve, wherein the further amplification aliquoting chamber is separated from the amplification aliquoting chamber in fluid connection with two or more amplification reaction chambers.

Embodiment 175 is the system of any one of embodiments 147-174, wherein the CC unit comprises one or more CC chambers.

Embodiment 176 is the system of embodiment 175, wherein the one or more CC chambers interface with a heating element on the instrument when the cartridge is inserted into the instrument.

Embodiment 177 is the system of any one of embodiments 175-176, wherein the one or more CC chambers interface with a magnetic element on the instrument when the cartridge is inserted into the instrument.

Embodiment 178 is the system of any one of embodiments 175-177, wherein the CC unit further comprises an input/output valve and a fluid channel between and fluidly connected to the one or more CC chambers and the input/output valve.

Embodiment 179 is the system of any one of embodiments 175-178, wherein the CC unit further comprises one or more pneumatic ports in fluid connection with the one or more CC chambers, wherein the one or more pneumatic ports are capable of interfacing with a pneumatic manifold on the instrument when the cartridge is inserted into the instrument.

Embodiment 180 is the system of embodiment 179, wherein the CC unit further comprises one or more condensate trap chambers positioned between and fluidly connected to the one or more CC chambers and the one or more pneumatic ports.

Embodiment 181 is the system of any one of embodiments 179 to 180, wherein:

the one or more CC chambers include two or more sub-CC chambers fluidly connected to a single input/output valve via a single fluid channel located between the two or more CC chambers and the input/output valve;

Each of the two or more CC chambers is fluidly connected to a separate pneumatic port; and

Optionally, each of the two or more CC chambers is fluidly connected to a separate condensate trap chamber disposed between the CC chamber and the pneumatic port.

Embodiment 182 is the system of any one of embodiments 147-181, wherein the cluster generation/sequencing unit comprises a flow cell capable of delivering fluid to and removing fluid from the solid support, further wherein a surface of the solid support is within a boundary of the flow cell and in contact with the fluid within the flow cell.

Embodiment 183 is the system of embodiment 182, wherein the flow cell is fluidly connected to one or more input/output valves.

Embodiment 184 is the system of any one of embodiments 182-183, wherein the flow cell is in fluid connection with one or more sequencing manifold ports, wherein the one or more sequencing manifold ports interface with a sequencing manifold on the instrument when the cassette is inserted into the instrument.

Embodiment 185 is the system of any one of embodiments 182 to 184, wherein the flow cell or solid support interfaces with a heating element on the instrument when the cartridge is inserted into the instrument.

Embodiment 186 is the system of any of embodiments 185, wherein the solid support further comprises an integrated heating element.

Embodiment 187 is the system of any one of embodiments 182 to 184, wherein the solid support further comprises an integrated heating element.

Embodiment 188 is the system of any one of embodiments 182-187, wherein the flow cell is capable of being connected to a semiconductor chip, and further wherein at least a portion of a surface of the chip is in fluid contact with at least a portion within the flow cell.

Embodiment 189 is the method of embodiment 188, wherein the surface of the semiconductor chip further comprises an array of three-dimensional features.

Embodiment 190 is the method of embodiment 189, wherein the three-dimensional feature comprises a hole.

Embodiment 191 is the method of any of embodiments 188 to 190, wherein the semiconductor chip further comprises an ISFET sensor array.

Embodiment 192 is the system of any one of embodiments 147-191, further comprising a fluid channel fluidly connected between at least one chamber in the cartridge and at least another chamber in the cartridge.

Embodiment 193 is the system of any of embodiments 147-192, further comprising a fluid channel fluidly connected between at least one chamber other than the waste chamber in the cartridge and at least one waste chamber in the cartridge.

Embodiment 194 is the system of embodiment 193, further comprising an input/output valve, a fluid passageway between and fluidly connected to at least one chamber other than the waste chamber and the input/output valve, and a fluid passageway between and fluidly connected to at least one waste chamber and the input/output valve.

Embodiment 195 is the system of any one of embodiments 147 to 191, further comprising a kit removably insertable into an instrument.

Embodiment 196 is the system of embodiment 192, wherein the kit comprises one or more reagent chambers fluidly connected to one or more input/output valves.

Embodiment 197 is the system of any one of embodiments 147 to 193, wherein one, some, or each of the input/output valves is a sealed pneumatic/pipette interface (SPI) valve.

Embodiment 198 is the system of any one of embodiments 147-194, wherein one, some, or each chamber is fluidly connected to an SPI valve.

Embodiment 199 is the system of any one of embodiments 147-195, wherein the sample input is in fluid connection with an SPI valve.

Embodiment 200 is the system of any one of claims 194-196, wherein the SPI valve comprises a flexible, solid valve body comprising a separable portion that separates and seals around the pipette tip to maintain a pressure differential on opposite sides of the valve when the pipette tip is inserted therein, and seals on itself to maintain a pressure differential on opposite sides of the valve when the pipette tip is removed therefrom.

Embodiment 201 is the system of any one of embodiments 147 to 200, wherein the instrument comprises one or more of any combination of:

A cartridge interface;

a kit interface;

thermal device and interface

Magnet arrangement and interface

Mechanical cracker and interface

Ultrasonic processing apparatus and interface

A cartridge fluid manifold;

A kit fluid manifold;

Pneumatic device and interface

A solid support interface;

One or more sensors;

One or more CPUs and associated devices and electronic equipment; and

A user interface.

Embodiment 202 is the system of any one of embodiments 147-201, wherein the instrument comprises a liquid handler comprising a pipette configured to transfer material from each of the input/output valves.

Embodiment 203 is the system of embodiment 202, wherein the instrument comprises at least one gantry robot capable of moving in x, y, and z dimensions.

Embodiment 204 is a method of performing the analysis of a target polynucleotide in a sample as claimed in any one of embodiments 131 to 146 in a system as claimed in any one of embodiments 147 to 203, the method comprising any one or more of the following in any combination:

a. Processing the sample in any one or any combination (including all) of sample input, incubation, lysis, copy control, and/or MS units to obtain a prepared target polynucleotide;

b. Amplifying a portion of the prepared target polynucleotide in a library preparation unit to obtain an amplified target nucleic acid;

c. controlling the number of output copies in the copy control unit;

d. generating one or more clusters in a cluster generation/sequencing unit;

e. Sequencing the target nucleic acid prepared in any one of steps a) to d) or any combination thereof in a cluster generation/sequencing unit.

Embodiment 204 is a method of analyzing a target polynucleotide in a sample as claimed in any one of embodiments 131 to 146 in a system as claimed in any one of embodiments 147 to 203, the method comprising any one or more of the following in any combination:

a) Processing the sample in any one or any combination (including all) of sample input, incubation, lysis, copy control, and/or MS units to obtain a prepared target polynucleotide;

b) Amplifying a portion of the prepared target polynucleotide in a library preparation unit to obtain an amplified target nucleic acid;

c) Controlling the number of output copies in the copy control unit;

d) Generating one or more clusters in a cluster generation/sequencing unit;

e) Sequencing the target nucleic acid prepared in any one or any combination of steps a) to d) in a cluster generation/sequencing unit.

Embodiment 205 is the method of embodiment 204, wherein preparing the target polynucleotide in the sample comprises the method of any one of embodiments 1 to 34.

Embodiment 206 is the method of any one of embodiments 204 to 205, wherein preparing the target polynucleotide in the sample comprises:

Inputting a sample in a sample input unit;

Transferring the sample to at least one incubation chamber in an incubation unit, wherein the at least one incubation chamber is preloaded with a lysis reagent;

mixing the sample with a lysis reagent to produce a sample lysis reagent mixture;

Heating the sample lysis reagent mixture in at least one incubation chamber;

Transferring the sample-lysis reagent mixture from the at least one incubation chamber to at least one lysis chamber in a lysis unit, wherein the at least one lysis chamber is preloaded with additional lysis reagent;

Lysing cells present in a sample lysis reagent mixture to produce a lysate;

Passing the lysate from the at least one lysis chamber through one or more first MS chambers having at least one docked heating element and into one or more second MS chambers, wherein the one or more second MS chambers are preloaded with a target capture reagent comprising at least one capture oligonucleotide;

Mixing the lysate with a target capture reagent to produce a lysate-target capture reagent mixture;

transferring the lysate-target capture reagent mixture to at least one incubation chamber;

Heating the lysate-target capture reagent mixture in at least one incubation chamber to enable annealing of the capture oligonucleotides to the target polynucleotide to produce capture oligomer-target complexes;

Transferring the capture oligomer-target complex into one or more third MS chambers, wherein the one or more third MS chambers are preloaded with capture beads (e.g., magnetic capture beads)

Mixing the capture oligomer-target complexes with the capture beads to produce capture oligomer-target-capture bead complexes;

The method comprises immobilizing capture beads by passing capture oligomer-target capture bead complexes through one or more first MS chambers that are docked with at least one magnetic element, removing lysate from the immobilized capture beads, adding a wash buffer to the immobilized capture beads and then removing the wash buffer from the immobilized capture beads to wash the immobilized capture beads, and adding an elution buffer to the immobilized capture beads to elute target polynucleotides from the immobilized capture beads, thereby producing prepared target polynucleotides.

Embodiment 207 is the method of embodiment 206, wherein transferring comprises passing through an input/output valve.

Embodiment 208 is the method of any one of embodiments 206-207, wherein mixing comprises selectively pressurizing and depressurizing the two or more chambers through two or more pneumatic ports forming independent fluid connections therewith.

Embodiment 209 is the method of any one of embodiments 206 to 208, wherein the pre-loaded reagent comprises a lyophilized reagent.

Embodiment 210 is the method of any one of embodiments 206 to 209, wherein the cleavage reagent comprises proteinase K.

Embodiment 211 is the method of any one of embodiments 206 to 210, wherein heating the sample lysis reagent mixture in the at least one incubation chamber comprises heating at about 60 ℃.

Embodiment 212 is the method of any one of embodiments 206 to 211, wherein the additional lysing agent comprises zirconium beads.

Embodiment 213 is the method of any one of embodiments 206 to 212, wherein lysing cells present in the sample-lysing reagent mixture comprises mixing using a rotating paddle or impeller.

Embodiment 214 is the method of any one of embodiments 206 to 213, wherein the one or more first MS chambers comprise a serpentine channel.

Embodiment 215 is the method of any one of embodiments 206 to 214, wherein passing the lysate from the one or more lysis chambers through the one or more MS chambers comprises heating at about 95 ℃.

Embodiment 216 is the method of embodiment 215, wherein heating comprises denaturing the double-stranded nucleic acid (if present).

Embodiment 217 is the method of any one of embodiments 206 to 216, wherein heating the lysate-target capture reagent mixture in at least one incubation chamber comprises heating at about 60 ℃.

Embodiment 218 is the method of any one of embodiments 204 to 217, wherein amplifying comprises the method of any one of claims 2.1 to 2.26.

Embodiment 219 is the method of any one of embodiments 204 to 218, wherein amplifying comprises:

Transferring the isolated target nucleic acid from the one or more STC chambers to a first PCR chamber of the one or more PCR chambers;

generating a first target nucleic acid amplicon in a first PCR chamber of the one or more PCR chambers;

Transferring the first target nucleic acid amplicon from a first PCR chamber of the one or more PCR chambers to a second PCR chamber of the one or more PCR chambers;

optionally diluting the first target nucleic acid amplicon; and

A second target nucleic acid amplicon is generated in a second PCR chamber of the one or more PCR chambers.

Embodiment 220 is the method of any one of embodiments 204 to 219, further comprising:

Transferring the amplified target nucleic acid from the PCR unit to the CC unit; and

The copy number of the amplified target nucleic acid is controlled in the CC unit to obtain a target nucleic acid therein at near a predetermined copy level.

Embodiment 221 is the method of embodiment 220, wherein controlling copy number comprises the method of any one of embodiments 66-75.

Embodiment 222 is the method of any one of embodiments 204 to 221, further comprising transferring the target nucleic acid from any other unit in the assay cartridge to a cluster generation/sequencing unit, and generating target nucleic acid clusters immobilized on a surface within the flow cell boundary in the cluster generation/sequencing unit.

Embodiment 223 is the method of embodiment 222, wherein generating the cluster comprises the method of any one of embodiments 76 to 130.

Embodiment 224 is the method of any one of embodiments 204 to 223, wherein after the sample is initially introduced and the run is started, all processes are performed in an automated manner in a continuous workflow within the system without human intervention.

Embodiment 225 is a method of analyzing a target polynucleotide in a sample as claimed in any one of embodiments 131 to 146 in a system as claimed in any one of embodiments 147 to 203, wherein the target polynucleotide is analyzed directly from the sample.

Embodiment 226 is an apparatus comprising any one or more of any combination of the following:

A cartridge interface;

A thermal device and an interface;

a magnet device and an interface;

a mechanical cracking device and an interface;

An ultrasonic processing device and an interface;

A cartridge fluid manifold;

A pneumatic device and an interface;

A solid support interface;

One or more sensors;

one or more CPUs and associated equipment and electronic devices; and

A user interface.

Embodiment 227 is one or more cassettes comprising any one or more of any combination of:

A sample input unit;

An incubation unit;

A lysing unit;

a Magnetic Separation Unit (MSU);

A library preparation unit;

a Copy Control (CC) unit;

a cluster generation/sequencing unit;

a waste liquid unit;

A dry reagent storage unit;

a liquid reagent storage unit for measuring a specific reagent;

a liquid reagent storage unit for batch and sequencing specific reagents; and

And a waste liquid unit.

Claims (105)

1. A method for analyzing a target in a sample, the method comprising:

introducing a sample into the cartridge;

introducing the cartridge into an instrument capable of automatically manipulating the sample within the cartridge:

isolating a target nucleic acid from the sample;

Amplifying the isolated target nucleic acid; and

Amplified target nucleic acids are sequenced using second generation sequencing,

Wherein the sample is maintained within the cassette throughout the isolation step, amplification step and sequencing step.

2. The method of claim 1, wherein the isolating step, amplifying step, and sequencing step are performed within 8 hours or less after the sample is introduced into the cassette.

3. The method of claim 1, wherein the target nucleic acid comprises fungal nucleic acid present in the sample at a level as low as 3 copies.

4. The method of claim 1, wherein the target nucleic acid comprises bacterial nucleic acid present in the sample at a level as low as 3 copies.

5. The method of claim 1, wherein the target nucleic acid comprises viral nucleic acid present in the sample at a level as low as a single copy.

6. The method of claim 1, wherein the cartridge has an external volume of about 3 liters or less.

7. The method of claim 6, wherein the cartridge has an external volume of about 2.5 liters or less.

8. The method of claim 7, wherein the cartridge has an external volume of about 2.1 liters or less.

9. The method of claim 6, wherein the cassette has a longest linear dimension of about 200mm or less.

10. The method of claim 9, wherein the cassette has a longest linear dimension of about 160mm or less.

11. The method of claim 1, wherein the sample is selected from the group consisting of a biological sample, a clinical sample, an environmental sample, and a food sample.

12. The method of claim 11, wherein the sample is a biological sample obtained from a subject and untreated prior to introduction into the cassette.

13. The method of claim 1, wherein the separating step comprises digesting proteins in the sample.

14. The method of claim 13, comprising digesting the protein with proteinase K.

15. The method of claim 1, wherein the isolating step comprises lysing organisms to release the target nucleic acid.

16. The method of claim 15, wherein lysing comprises mechanical lysing.

17. The method of claim 16, wherein the mechanically lysing comprises flowing the sample into a lysing chamber within the cartridge and rotating a paddle within the lysing chamber.

18. The method of claim 17, wherein the mechanically lysing further comprises adding zirconium beads to the lysing chamber prior to rotating the paddle within the lysing chamber.

19. The method of claim 1, wherein the isolating step comprises denaturing the target nucleic acid.

20. The method of claim 19, wherein denaturing comprises thermal denaturation.

21. The method of claim 1, wherein the isolating step comprises capturing the target nucleic acid by:

annealing a target capture oligonucleotide to the target nucleic acid to form a complex;

binding the complex to a solid support; and

Unbound material is removed from the solid support.

22. The method of claim 21, wherein removing unbound material comprises washing the solid support-bound complex with a wash reagent.

23. The method of claim 21, wherein the amplifying step is performed on a solid support-bound target nucleic acid.

24. The method of claim 21, further comprising eluting the target nucleic acid from the washed solid support to produce an isolated target nucleic acid.

25. The method of claim 23, wherein the amplifying step is performed directly on the eluted target nucleic acid without an intervening step.

26. The method of claim 20, wherein the separating step automatically separates the target nucleic acid from a sample having a volume between about 1mL and about 25 mL.

27. The method of claim 1, wherein the separation step comprises only one purification step.

28. The method of claim 1, wherein the isolated nucleic acid is amplified in the absence of a quantification.

29. The method of claim 1, wherein the amplifying step comprises:

performing a first amplification on the isolated target nucleic acid using a first primer set to produce a first amplification product;

diluting the first amplification product and aliquoting into a plurality of aliquots;

performing a second amplification of the target nucleic acid in the plurality of aliquots using a plurality of second primer sets to produce a plurality of second amplification products; and

Pooling the second amplification product.

30. The method of claim 29, wherein one or more primers in the first primer set are identical to one or more primers in the plurality of second primer sets.

31. The method of claim 30, wherein the amplifying step further comprises purifying the pooled second amplification products to produce the amplified target nucleic acids.

32. The method of claim 30, wherein one or more of the first amplification and the second amplification comprises PCR amplification.

33. The method of claim 30, wherein the plurality of aliquots comprises at least 10 separate aliquots.

34. The method of claim 30, wherein the first PCR amplification and the second PCR amplification are performed without quantification.

35. The method of claim 30, wherein one or more of the plurality of second primer sets are nested relative to the first primer set.

36. The method of claim 1, wherein the amplifying step comprises copy control of the amplified target nucleic acid prior to the sequencing step.

37. The method of claim 1, wherein the amplification step comprises only one purification step.

38. The method of claim 1, wherein the amplified target nucleic acid is sequenced in the absence of a quantification.

39. The method of claim 1, wherein the step of sequencing comprises:

The amplified target nucleic acid is immobilized above a semiconductor surface comprising an Ion Sensitive Field Effect Transistor (ISFET) sensor within a cassette.

40. The method of claim 39, wherein all products of the amplifying step flow over the semiconductor surface without intervening steps.

41. The method of claim 39, wherein the amplified target nucleic acid is immobilized by a capture oligomer bound over the ISFET sensor, wherein the capture oligomer hybridizes to a portion of the target nucleic acid.

42. The method of claim 41, wherein the surface comprises an array of ISFET sensors, each ISFET sensor having an aperture located thereabove.

43. The method of claim 41, wherein at least one aperture is positioned over a plurality of ISFET sensors in the ISFET sensor array.

44. The method of claim 42, wherein one or more of the wells comprises a surface-bound forward primer that hybridizes to a portion of the target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, and wherein the sequencing step comprises double-ended sequencing.

45. The method of claim 42, wherein one or more of the pores or an inter-pore gap between one or more of the pores comprises a plurality of bound inert oligomers that do not hybridize to the target nucleic acid.

46. The method of claim 39, wherein the amplified target nucleic acid is immobilized by a universal capture oligomer that binds over the ISFET sensor, wherein the universal capture oligomer hybridizes to a universal binding site.

47. The method of claim 46, wherein the amplifying step comprises amplifying the isolated target nucleic acid using a primer comprising the universal binding site.

48. The method of claim 46, wherein the amplifying step comprises ligating an adapter to the isolated target nucleic acid, the adapter comprising the universal binding site.

49. The method of claim 46, wherein the step of sequencing comprises clonal amplification of an immobilized target nucleic acid.

50. The method of claim 49, wherein the clonal amplification comprises recombinase polymerase amplification.

51. The method of claim 49, wherein the clonal amplification comprises rolling circle amplification.

52. The method of claim 49, wherein the clonal amplification comprises bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification.

53. A system, comprising:

A sample cartridge, the sample cartridge comprising:

Sample input;

a sample preparation unit capable of receiving a sample from the sample input and separating a target nucleic acid from the sample;

A library preparation unit capable of receiving isolated target nucleic acids from the sample preparation unit and amplifying the isolated target nucleic acids; and

A sequencing unit capable of receiving amplified target nucleic acids from the library preparation unit and sequencing the amplified target nucleic acids; and

An apparatus, comprising:

A cartridge interface comprising a physical connection and an electronic connection through which the instrument is capable of driving the sample and reagents within the cartridge to move and communicate with the sequencing unit.

54. The system of claim 53, wherein the one or more reagents required to isolate the target nucleic acid, amplify the isolated target nucleic acid, and sequence the amplified nucleic acid are dry reagents, the instrument being capable of reconstructing the one or more reagents.

55. The system of claim 53, further comprising one or more kits comprising one or more reagents required to isolate the target nucleic acid, amplify the isolated target nucleic acid, and sequence the amplified nucleic acid;

The instrument is capable of transferring reagents from the one or more kits to the sample cartridge.

56. The system of claim 55, wherein the sample cartridge and the one or more kits comprise a Sealed Pneumatic Interface (SPI) port, and

Wherein the instrument is capable of transferring the one or more reagents from the one or more kits to the sample cartridge via the SPI port using one or more pipettes.

57. The system of claim 56, wherein the instrument comprises a 3-degree-of-freedom pipette stage capable of transferring the one or more reagents.

58. The system of claim 53, which is capable of isolating, amplifying and sequencing target fungal nucleic acid at as low as 3 copy levels in the sample.

59. The system of claim 53, capable of isolating, amplifying and sequencing target bacterial nucleic acids present in the sample at as low as 3 copy levels.

60. The system of claim 53, capable of isolating, amplifying and sequencing target viral nucleic acids present in the sample down to a single copy level.

61. The system of claim 53, wherein the cartridge has an external volume of about 3 liters or less.

62. The system of claim 61, wherein the cartridge has an external volume of about 2.5 liters or less.

63. The system of claim 62, wherein the cartridge has an external volume of about 2.1 liters or less.

64. The system of claim 61, wherein the cassette has a longest linear dimension of about 200mm or less.

65. The system of claim 64, wherein the cassette has a longest linear dimension of about 160mm or less.

66. The system of claim 53, wherein the instrument has a volume of about 150 liters or less.

67. The system of claim 66, wherein the instrument has a volume of about 135 liters or less.

68. The system of claim 53, wherein the instrument has a longest linear dimension of about 700mm or less.

69. The system of claim 68, wherein the instrument has a longest linear dimension of about 650mm or less.

70. The system of claim 53, wherein the sample cartridge is capable of receiving biological samples, clinical samples, environmental samples, and food samples.

71. The system of claim 53, wherein the sample cartridge is capable of receiving an untreated biological sample.

72. The system of claim 53, wherein isolating the target nucleic acid comprises digesting proteins in the sample.

73. The system of claim 72, wherein the instrument is capable of exposing the sample to proteinase K in the sample preparation unit.

74. The system of claim 53, wherein the sample preparation unit is capable of lysing organisms to release the target nucleic acid.

75. The system of claim 74, wherein the sample preparation unit comprises a lysis chamber comprising a rotating paddle, the instrument being capable of flowing the sample into the lysis chamber and interfacing with the sample cartridge to rotate the rotating paddle to mechanically lyse organisms in the sample.

76. The system of claim 75, further comprising zirconium beads in the lysis chamber.

77. The system of claim 53, wherein isolating the target nucleic acid comprises denaturing the target nucleic acid.

78. The system of claim 77, wherein the instrument is capable of providing thermal energy to the sample preparation unit to denature nucleic acids in the sample preparation unit.

79. The system of claim 53, wherein the instrument is operable to:

exposing the sample to a target capture oligonucleotide and a solid support in the sample preparation unit to anneal the target capture oligonucleotide to the target nucleic acid, thereby forming a complex and binding the complex to the solid support.

80. The system of claim 79, wherein the instrument is further capable of introducing a wash buffer into the solid support bound complex and separating the solid support bound complex from unbound sample.

81. The system of claim 80, wherein the instrument is capable of transferring the isolated solid support-bound complexes to the library preparation unit and amplifying solid support-bound target nucleic acids.

82. The system of claim 79, wherein the instrument is capable of introducing an elution buffer into the isolated solid support-bound complex to elute the target nucleic acid from the solid support and transferring the eluted target nucleic acid to the library preparation unit for amplification.

83. The system of claim 79, wherein the instrument is capable of introducing amplification reagents into the solid support-bound complex and amplifying the target nucleic acid within the sample preparation unit.

84. The system of claim 53, capable of automatically containing a sample received by the sample input in a volume between about 1mL and about 25 mL.

85. The system of claim 53, wherein the instrument is capable of interfacing with the library preparation unit of the sample cartridge to introduce a desired reagent and provide thermal energy to:

performing a first amplification on the isolated target nucleic acid using a first primer set to produce a first amplification product;

diluting the first amplification product and aliquoting it into a plurality of aliquots;

Performing a second amplification of the target nucleic acid in the plurality of aliquots using a plurality of second primer sets to produce a plurality of second amplification products; and

Pooling the second amplification product.

86. The system of claim 85, wherein one or more primers in the first set of primers are identical to one or more primers in the plurality of second sets of primers.

87. The system of claim 85, wherein the instrument is further capable of purifying pooled second amplification products to produce the amplified target nucleic acids.

88. The system of claim 85, wherein one or more of the first amplification and the second amplification comprises PCR amplification.

89. The system of claim 85, wherein the plurality of aliquots comprises at least 10 separate aliquots.

90. The system of claim 53, further capable of copy control of one or more of the isolated target nucleic acid and the amplified target nucleic acid and controlling the number of output copies transferred to the library preparation unit or the sequencing unit, respectively.

91. The system of claim 53, wherein the sequencing unit comprises a semiconductor surface comprising an array of Ion Sensitive Field Effect Transistor (ISFET) sensors, each ISFET sensor having an aperture located thereabove, the instrument being capable of immobilizing amplified target nucleic acid thereabove, the ISFET sensor array in electronic communication with the instrument via electronic connection of the cartridge interface when a sample cartridge is positioned therein.

92. The system of claim 91, wherein the instrument is capable of flowing all output from the library preparation unit into the well on the semiconductor surface.

93. The system of claim 91, comprising a capture oligomer bound over the ISFET sensor array, wherein the capture oligomer is configured to hybridize to a portion of a target nucleic acid.

94. The system of claim 91, wherein at least one of the apertures is positioned over a plurality of ISFET sensors in the ISFET sensor array.

95. The system of claim 91, wherein one or more of the wells comprises a surface-bound forward primer that hybridizes to a portion of the target nucleic acid and a surface-bound reverse primer that hybridizes to a portion of the target nucleic acid, the instrument being capable of double-ended sequencing.

96. The system of claim 91, wherein one or more of the pores and the inter-pore gap between the pores comprises a plurality of bound inert oligomers that do not hybridize to the target nucleic acid.

97. The system of claim 91, comprising a universal capture oligomer conjugated over the ISFET sensor array, wherein the universal capture oligomer is configured to hybridize to a universal binding site.

98. The system of claim 97, wherein the instrument is capable of interfacing with the library preparation unit to amplify the isolated target nucleic acid using primers comprising the universal binding sites.

99. The system of claim 97, wherein the instrument is capable of ligating an adapter to the isolated target nucleic acid in the sample preparation unit or the library preparation unit, the adapter comprising the universal binding site.

100. The system of claim 97, wherein the instrument is capable of interfacing with the sequencing unit for clonal amplification of an immobilized target nucleic acid.

101. The system of claim 100, wherein the clonal amplification comprises a recombinase polymerase amplification.

102. The system of claim 100, wherein the clonal amplification comprises rolling circle amplification.

103. The system of claim 100, wherein the clonal amplification comprises bridge PCR, strand displacement amplification, or loop-mediated isothermal amplification.

104. The system of claim 53, wherein the physical connection and the electronic connection comprise a pneumatic system for driving movement of fluid within the sample cartridge.

105. The system of claim 53, wherein the cartridge interface further comprises a physical connection and an electronic connection through which the instrument is capable of communicating with one or more of the sample preparation unit and the library preparation unit.