HK1196858A - Systems and methods for genetic and biological analysis - Google Patents

Abstract

The invention relate to systems and methods for sequencing polynucleotides, as well as detecting reactions and binding events involving other biological molecules. The systems and methods may employ chamber-free devices and nanosensors to detect or characterize such reactions in high-throughput. Because the system in many embodiments is reusable, the system can be subject to more sophisticated and improved engineering, as compared to single use devices.

Description

System and method for genetic and biological analysis

Cross Reference to Related Applications

Priority and benefit of U.S. provisional application No.61/491,081 filed on 27/5/2011, U.S. provisional application No.61/565,651 filed on 1/12/2011, U.S. provisional application No.61/620,381 filed on 4/2012, and U.S. application serial No. 13/397,581 filed on 15/2/2012, each of which is incorporated herein by reference in its entirety, are claimed herein. The PCT application was filed on day 29, 5/2012 with the us acceptance office, since day 28, 5/month was federal holiday.

The present application further relates to PCT/US2011/054769, which is incorporated herein by reference in its entirety.

Statement regarding federally sponsored research and development

Aspects of the invention are accomplished with government support in conjunction with the U.S. Department of Health and Human Services with the help of qualified Therapeutic Discovery Grant awarded by the IRS. The united states government has certain rights in this invention.

Background

Rapid and cost-effective genetic and biological analysis methods, including high-throughput DNA sequencing, remain an important aspect in driving personalized medicine and diagnostic testing. Current high throughput or miniaturized systems have limitations. For example, current systems for DNA sequencing, including those employing optical detection, are cumbersome and expensive, and have limited throughput. While some systems use sensors and sequencing flow cells to address these limitations, these are typically disposable items that add significant cost to the user and limit the complexity of the sensor, as the sensor must be cost effectively manufactured for single use. Emulsion PCR offers some advantages, however sequencing of clonal DNA populations may exhibit limited accuracy when sequencing is not performed "in phase" throughout the clonal population, which in turn may actually result in short read lengths.

There is a need for systems and methods for genetic and biological analysis, particularly for sensitive and cost-effective highly parallel or clonal sequencing reactions.

Disclosure of Invention

Aspects and embodiments described herein relate to systems and methods for polynucleotide sequencing and detection of reactions and binding events involving other biomolecules. These systems and methods may employ chamber-less devices and/or nanosensors to detect and/or characterize such reactions at high throughput. Because the system is reusable in many embodiments, the system can be subjected to more complex and improved engineering than a single-use device.

In some embodiments, the invention provides methods and systems for sequencing polynucleotides, which may be single double-stranded or single-stranded polynucleotides alone, or in other embodiments, clonal populations of polynucleotides. For example, one aspect of the invention provides a method for parallel or clonal polynucleotide sequencing, the method comprising: a first portion of the population of target polynucleotides is sequenced, phase error (phase error) corrected, and then a second downstream portion of the population of target polynucleotides is sequenced. In various embodiments, the polynucleotide sequencing can include one or more of: sequencing clones on a bead array, electronic detection of nucleotide incorporation, and an electron well for separating or concentrating reaction components.

For example, phase error can be corrected by adding a combination of three nucleotide bases to stop the polynucleotide population at the first occurrence of the excluded base. Phase errors may also be corrected by combinations and/or sequences of incorporation reactions as detailed herein. Alternatively or in addition, phase errors can be corrected by reversibly incorporating chain terminating nucleotides into the in-phase polynucleotide chain. Alternatively or in addition, phase errors may be corrected by adding one or more polynucleotide clips (clamps) that hybridize to the target polynucleotide to stop the sequencing reaction. In some embodiments, the clip is denatured, destabilized, or degraded to continue the sequencing reaction. In other embodiments, at least one clip has a 3 'terminator nucleotide that cannot be extended, so upon removal of the 3' terminator nucleotide, the clip becomes a primer for subsequent downstream sequencing.

Rephasing can be performed periodically or, alternatively, the signal loss of the reaction can be monitored and rephasing performed to restore the sequencing signal.

In another aspect, the invention provides a method for reducing lead phase error in parallel or clonal polynucleotide sequencing. The method according to this aspect comprises sequencing a population of target polynucleotides in the presence of a competition reaction, wherein the competition reaction comprises nucleotide bases or nucleotide derivatives of all four nucleotide bases. Typically, three of the four nucleotide bases will not be incorporated into the growing polynucleotide strand, thereby reducing the propensity of the polymerase to incorporate incorrect nucleotides. According to this aspect, polynucleotide sequencing may optionally include one or more of: sequencing clones on a bead array, electronic detection of nucleotide incorporation, and an electron well for separating or concentrating reaction components. Various nucleotide derivatives are known and described herein that can be bound by a polymerase, but do not incorporate into the growing polynucleotide strand.

In yet other aspects, the invention provides a method for reducing lag phase errors in parallel or clonal polynucleotide sequencing reactions. According to this aspect, the method comprises stacking a polymerase on or near the population of target polynucleotides during the sequencing reaction such that the polymerase is substantially available for each active polymerization site. Alternatively or in addition, the method comprises binding a repair protein or a single-stranded DNA binding protein to the population of target polynucleotides. Optionally, the polynucleotide sequencing reaction comprises one or more of: sequencing clones on a bead array, electronic detection of nucleotide incorporation, and an electron well for separating or concentrating reaction components. Stacking can be the result of natural binding of a polymerase to a primer (including a non-extendable primer that hybridizes to a template polynucleotide).

In still other aspects, the invention provides methods for repeated nucleotide sequencing such that sequence data for several sequencing runs (run) can be analyzed. According to this aspect, the method comprises providing a circularised DNA sequencing template and sequencing the template by determining the order in which nucleotides are incorporated by a DNA polymerase having 5 'to 3' exonuclease activity. This aspect may also optionally include one or more of the following: sequencing clones on a bead array, electronic detection of nucleotide incorporation, and an electron well for separating or concentrating reaction components. The DNA polymerase according to this aspect may be highly persistent and have reduced exonuclease activity. Highly persistent polymerases can bind on or near biosensors suitable for determining nucleotide incorporation.

In some embodiments, the method sequences a single DNA molecule by attaching a polymerase to a biosensor in a volume, and allowing the DNA template with the associated primer to enter the volume and hybridize and be retained by the polymerase (or a proximity polymerase). The sequence can then be determined after the primer is extended by the polymerase.

In yet other aspects, the present invention provides a chamber-less apparatus comprising: an array of electromagnetic sensors, a magnetic carrier for carrying or holding a molecule of interest on or near the electromagnetic sensors, and a mechanism for removing the magnetic carrier by liquid flow and/or electromagnetic removal. The electromagnetic sensor may be one of a nanoneedle or a nanobridge, and the apparatus may further comprise a local amplifier (local amplifier). In some embodiments, the electromagnetic sensor has a narrow structure and is etched under the structure so that both sides of the sensor surface are accessible for changes in pH or conductivity. The devices and methods described herein can include one or more improvements, including the introduction of materials with reduced zeta potentials, the use of reagents that allow polynucleotide incorporation and sensitive pH measurement, and the design and fabrication of nanosensors that are at optimal distances and configurations relative to the bead or matrix that holds the polynucleotide template for the sequencing reaction.

Other aspects and embodiments of the invention will be apparent to those skilled in the art based on the following detailed description.

Drawings

Fig. 1A shows a schematic detail of a complete integrated system and some subsystems. FIG. 1B shows schematic details of the sample and library preparation subsystem. FIG. 1C shows schematic details of the DNA fragmentation and purification subsystem. Fig. 1D shows the PDMS library preparation subsystem.

Fig. 2 shows a magnetic and virtual confinement array.

FIG. 3 shows a Comsol simulation of the electric field for a virtual aperture.

Fig. 4A-4D show schematic diagrams, pictures and manufactured products of PDMS valving subsystems.

Fig. 5 shows an embodiment of a combined NanoNeedle (NanoNeedle) sensor and magnetic array element.

Fig. 6 shows two forms of magnetic arrays in which magnetic beads can be positioned in fixed positions.

Fig. 7 shows an embodiment of a magnetic array that can position beads in fixed positions.

Fig. 8 schematically depicts elements of an array that utilize "leak valves" to position beads.

Fig. 9 depicts simulated voltage and current curves associated with redox reactions for detection.

FIG. 10 is a graph showing the charge distribution of DNA binding proteins.

Fig. 11 shows a schematic diagram of a magnetic array for planar magnetic particles.

Fig. 12 schematically illustrates a combined magnet, virtual well and nanoneedle array element with a bead.

Figures 13A-13E show illustrations, pictures, and photomicrographs of various enrichment module embodiments.

Figure 14 illustrates the bead loading density of a prior flow cell.

Fig. 15 schematically illustrates a valving system and interface to a multi-channel flow cell with adjacent valve control.

Fig. 16 schematically illustrates a simple model of impedance in nanoneedle and bead array elements.

Fig. 17 schematically depicts an undercut (under-etched) stacked nanoneedle.

Fig. 18 is a photomicrograph of an undercut stacked nanoneedle array.

Fig. 19 schematically depicts a 2D array of underetched stacked nanoneedles.

Fig. 20 is a photomicrograph of a 2D array of undercut stacked nanoneedles.

Fig. 21A-C schematically depict elements of a single-sided contact nanoneedle array.

Fig. 22A-D schematically and diagrammatically show elements of a double-sided contact nanoneedle array.

Fig. 23 schematically depicts the elements of a NanoBridge (NanoBridge).

Fig. 24A-C schematically depict views of an annular nanobridge.

Fig. 25 schematically depicts a nanoneedle array for single molecule sequencing.

Fig. 26 illustrates sequencing data from a nanoneedle array element and the linearity of the sequencing data.

Detailed Description

The present invention provides methods and systems for DNA sequencing and other types of biological or genetic analysis. The present invention provides methods and systems for sequencing a clonal population of DNA or an array of single molecule DNA, including by electronic sequencing, thereby providing a low cost and convenient sequencing platform. In some aspects, the invention provides methods for monitoring and/or correcting phase errors during sequencing of a population of DNA clones to improve accuracy and read length. Furthermore, the present invention provides methods and sensors for sequencing single molecules of DNA, thereby avoiding such phase errors. In other aspects, the invention provides arrays, including magnetic arrays, and virtual reactors for highly parallel reactions. In some embodiments, these systems include nanosensors for detecting biological reactions or interactions (including incorporation of nucleotides) in a DNA sequencing process. Furthermore, the present invention provides an integrated system for amplifying and sequencing a DNA sample.

Monitoring and correction of sequencing phase errors

As used herein, "phase error" is defined as an event in which some template polynucleotides in a clonal population are extended more or less than in the consensus state (consensus state). This type of phase error is considered to be "leading (leading)" in a fragment having a base added to a position where a base should not be added to the consensus fragment. For other template molecules where no base should be added relative to the consensus fragment, the polynucleotide is considered "lagging" (stretching). Since the polymerase is imperfect, some phase errors are inevitable in colonies with long extension reactions as part of the colony-based sequencing process. Phase error limits the read length of commercial clonal sequencing systems.

"lead sequencing incorporation error" refers to a sequence that leads a dominant sequence by incorrect nucleotide addition. This incorrect addition may be caused by polymerase error, especially when high concentrations of dntps are used in non-competitive reactions. Alternatively, lead sequencing incorporation errors may be caused by inadequate washing or non-specific binding of dntps, which can then be released and incorporated. "late sequencing incorporation error" refers to a sequence that lags behind the dominant sequence due to missing the addition of the correct nucleotide; this may occur due to non-optimal reaction conditions, steric hindrance, secondary structure or other sources of polymerase inhibition. Longer cycle times may give the polymerase more opportunity to incorporate the wrong nucleotide. Also, inaccessible DNA can lead to insufficient opportunity to incorporate the correct nucleotide. It is contemplated that the temperature, step time, polymerase choice, nucleotide concentration, salt concentration, and buffer choice can be optimized to minimize incorporation errors.

For example, the DNA sample may have a TGTTC sequence in the first region following the region complementary to the primer. The fluid circulation may introduce dCTP first, dTTP second then dATP third then dATP and dGTP fourth then with intervening wash steps. In the first part of the fluid cycle, dCTP molecules that are part of the first cycle may not be washed out of the pore structure correctly. In a second part of the fluid cycle, dTTP molecules that are in-flowing as part of the second cycle may not be properly washed out of the pore structure. No dntps should be incorporated during the first and second portions of the first fluidic cycle. During the third part of the fluidic cycle, dATPs can be introduced and incorporated because dATP is complementary to the first base, T, of the sample. Any non-specifically bound dCTP molecules that stop non-specific binding may also be incorporated during this third portion of the fluid cycle. These unbound dCTP molecules can be incorporated after incorporation of the dATP molecules. After incorporation of the dCTP molecule, two dATP molecules can be subsequently incorporated, which can result in some molecules of the clonal beads having lead sequencing phase errors. Some molecules of the monoclonal beads may thus become "out of phase".

When a polymerase is provided with a single nucleotide or nucleotide analogue at a time, the error rate is typically significantly higher than when all four nucleotides or nucleotide analogues are provided. This may occur, although according to k_pol/K_d,appThere are large differences in measured catalytic efficiency, and mismatched nucleotides may be 4 logs or more lower than matched nucleotides. Most of which are due to K_d,appThe difference in (a). For example, Klenow polymerase has every 10⁶-10⁸The rate of erroneous incorporation of one base out of the individual bases. In contrast, polymerase extension reactions performed by current commercial systems utilizing single native dNTP incorporation can be limited to 100-1000 bases. The polymerases in these systems spend almost all their time trying to misincorporate bases, resulting in significant "leading" phase errors. In addition, polymerases that are unable to incorporate bases in an incorporation fluid flow cycle can cause dephasing (phasing) due to either a lack of the polymerase followed by the presence of the polymerase in a subsequent incorporation fluid flow cycle, or due to a sufficiently low combination of dNTP concentration and incorporation time that bases are not incorporated, resulting in a "lagging" phase error. Even when the nucleotide added to the system is the nucleotide to be added next, the reaction time must be long enough to complete the reaction of a homopolymer, which may be 8 or more nucleotides, or a DNA strand, which may be less sterically hindered.

In one aspect, the invention provides methods for parallel or clonal polynucleotide sequencing. In certain embodiments, the method comprises sequencing a first portion of the population of template polynucleotides and correcting for phase error. Sequencing of a second downstream portion of the population of target polynucleotides is then continued. In various embodiments, sequencing can include one or more of: sequencing clones of a polynucleotide population array (e.g., a bead array), electronic detection of nucleotide incorporation, and electronic wells for separating or concentrating components of a sequencing reaction. In various embodiments, the present invention provides methods for monitoring and correcting leading and lagging phases, and the various methods described herein may be used alone or in any combination.

"clone" as used herein means that substantially all of the beads or population of particles can have the same template nucleic acid sequence. In some embodiments, there may be two populations associated with a single sample DNA fragment, as desired by "mate pair", "paired end" or other similar methods; these populations may be present in substantially similar numbers on the beads or particles, and may be randomly distributed on the beads or particles.

In some embodiments, the colonies are rephased by incorporating nucleotides in a different order to provide sequencing than would otherwise be normally performed. For example, if the system has predominantly lagging phase errors (unlike leading phase errors), e.g. only 1% lagging error per base (and all four different bases have similar lagging error rates), then after 20 bases have been incorporated, only more than 75% of the colony members may be in phase, while more than 20% may lag by one base. By the time 70 bases have been sequenced, less than half of the colony members will be in phase, 35% will lag one base, 13% will lag two bases, and 3% will lag three bases. Thus, for the following exemplary incorporation sequence example (… CGATCGATCGA) where the ideal position is shown in bold, 50% of the colonies will be in phase at the fourth base (T), 35% will lag by 1 base (A), 13% will lag by two bases (G), and 3% will lag by 3 bases (C). If the previous base incorporation order was CGAT and C was provided, the lag error will continue and will rise slightly. Conversely, if C is excluded and the next base provided is G, the leading base will not be extended, and the portion of the colony that lags two bases at the first G shown will be extended; if A is next provided, then most of the colonies will now be in phase. If a combination of three bases without C, such as GAT, is provided one or more times, any phase error will be concentrated in the C bases. Thus statistically some sequences may become more out of phase, but most sequences may be made more in phase. Colonies can also be rephased using a combination of two bases in a very similar manner, and a mixture of two bases and three base sets can be used. After rephasing, the system can be restored to a combination of four bases and rephasing can be repeated as often as necessary.

In other embodiments, a combination of four bases may not be used at all, but only alternating sets of three and two bases may be used. In yet another embodiment, four bases are added in any combination of three and two base sets of nucleotides, in some embodiments alternating combinations of two or three base sets are used. In some embodiments, the base set can also include the use of unincorporable nucleotides (incorporable nucleotides). In other embodiments, the concentration of nucleotides and/or unincorporable nucleotides used in combinations of two, three, or four bases may vary from cycle to cycle or from group to group.

In certain embodiments, the phase error is corrected by excluding at least one nucleotide base from the sequencing reaction. For example, the phase error can be corrected by adding a combination of three nucleotide bases, stopping each nascent polynucleotide in the clonal population at the first occurrence of the excluded nucleotide base.

In certain other embodiments, phase error is corrected by reversibly incorporating a chain terminating nucleotide into the in-phase polynucleotide chain. Once the lagging phase strand has caught up to the in-phase strand, the terminating nucleotide is removed. This approach may be most advantageous when the sequence being sequenced includes a homopolymer region. For example, in the sequence fragment …. AGCTCCC, where the in-phase portion of the colony has incorporated the T base, where most of the lagging sequence has incorporated C, G and the a base as the last base of the colony member, if a C ' stop nucleotide is provided followed by the base combinations AGT, a significant bimodal population may exist, where the sequences …. AGC ' and …. AGCTC ' predominate. The terminator can then be removed from the C 'nucleotide and another C' terminator nucleotide can be provided, yielding two major sequences: …, AGC and AGCTCC'. The C' terminated nucleotide can be followed by base combinations AGT, resulting in two populations: … AGCT and … AGCTCC'. The terminator can then be removed and the non-terminating C nucleotide can then be provided, resulting in predominantly a single sequence: … AGCTCCC.

In some embodiments, phase errors are expected at certain locations (e.g., homopolymeric regions) based on the reference sequence, thus allowing phase correction to be effectively performed where appropriate in sequencing.

These methods may be most effective for those systems that have a major source of error, such as hysteresis error. Combinations of bases useful for rephasing can be added separately so that the complete order of incorporation can be determined, or can be added together so that rephasing can be accomplished with a small portion of missing data. In some embodiments, the reversible terminator may be reused during a sequencing process or method, and may be combined with incorporatable or non-incorporatable nucleotides.

In yet other embodiments, phase error is corrected by adding one or more oligonucleotide clips that hybridize to the target polynucleotide to stop the sequencing reaction, thereby removing the phase error. Such clips may be PNA fragments, DNA fragments or other natural or non-natural molecules that specifically bind to DNA sequences. In a system for targeted resequencing, a particular oligonucleotide may be used as a "clip". The "clip" may be provided while the primer sequence may be provided, before the primer sequence may be provided, after the primer sequence may be provided, before any sequencing reaction has been completed, or after some sequencing reaction has been completed. A plurality of different targeting or non-targeting clips may be provided for each template.

The clips may be random or targeted to specific regions of the DNA template. The DNA fragments or other clips may be further stabilized by the use of histones, cationic protamines, recombinases, and other molecules known to stabilize duplex DNA. The sequencing reactions (sequences interactions) may then continue until the point where the clips are located. Additional incorporation reactions can be performed using a single base, a combination of two bases, a combination of three bases, or using four bases simultaneously. The clips may be positioned such that there is (on average) 10 to 50 bases between the 3 'end of the primer and the clip, or may be positioned such that there is (on average) 10 to 100 bases between the 3' end of the primer and the clip, or may be positioned such that there is (on average) 100 to 500 bases between the 3 'end of the primer and the clip, or may be positioned such that there is (on average) 300 to 500 bases between the 3' end of the primer and the clip, or may be positioned such that there is (on average) 1000 to 5000 bases or more between the 3 'end of the primer and the clip, or may be positioned such that there is (on average) 2000 to 5000 bases between the 3' end of the primer and the clip.

In some embodiments, a clip can have a particular number of bases that can specifically hybridize, and can have additional bases that can be used to stabilize the clip. If the sequence of the clip is not targeted to a particular region, but is a non-targeted clip, several criteria can be used to select the sequence of the clip, including the stability of the clip, the frequency of the selected clip sequence in the genome of interest or a similarly characterized genome or chromosome of interest or transcriptome of interest. The hybridization stability of an intact clip, including any non-specific base such as deoxyinosine, 5-nitroindole, or abasic nucleotide, or may include any universal base described in US7,575,902 (which is incorporated herein by reference in its entirety), and may include the stability of the base selected to be the particular base of the clip.

In some embodiments, the clip can comprise 5, 6, 7, 8, 9, 10, or more specific bases. The clip can be used with a variety of DNA colonies, wherein substantially all of the colonies can have a different DNA sequence from the other DNA colonies. In some embodiments, a single-clamp type comprising a single set of specific hybridizing dNTPs may be used. In other embodiments, a multi-clamp type is used, wherein the number, order, or spacing of specifically hybridizing bases may be different. For example, two different hexamer clips may be used to reduce the average spacing, measured in DNA base units, from primer to clip or between one clip and the next, that would occur if only one of the two hexamer clips were used, but may be greater than that which would occur if one pentamer clip were used. In some embodiments, the spacing from primer to clip or clip to clip, measured in units of DNA bases, may vary due to the selection of clip sequences, as there is a significant difference (greater than 20-fold) in the performance of different hexamers in the transcriptome (Anderson et al RNA V14 (5)).

In some embodiments, the clips are subsequently removed (after phasing) by raising the temperature, changing the pH or ion concentration, resulting in denaturation of the clips, but leaving longer extension primers that can then be further extended after clip removal. In other embodiments, the clip may comprise a nicking site (nicksite) that can be subsequently nicked by a suitable nickase or endonuclease, which can destabilize the clip sufficiently to denature it. In some embodiments, a clip may comprise a cleavable linker site, wherein the cleavable linker site may be chemically cleavable or optically cleavable. In some embodiments, a base terminating at the 3' position can be provided as part of a clip. The terminator can then be removed after the nucleotides have been added in order to achieve rephasing. The terminator may be removed using a chemical or photochemical process. In some embodiments, a combination of different types of cleavable linker sites (e.g., unique cleavable linker sites) are used for different clips, such that clips with different linker types can be provided before any sequencing has begun or after sequencing has begun, and the clips can be denatured using different cleavable mechanisms in an order that allows the template DNA to be rephased multiple times.

In some embodiments, the method comprises adding clips after the sequencing and rephasing process has occurred. In other embodiments, the process of adding additional clips may be repeated multiple times, such as 2-5 times, 4-10 times, or more than 10 times.

In some embodiments, data can be collected for all bases prior to the position adjacent to the clip as incorporation can occur; in other embodiments, data collection may not be performed for all bases prior to the position adjacent to the clip.

In some embodiments, the clip can be used in combination with a non-strand displacing polymerase such that when the polymerase reaches the clip through a polymerization process, the polymerase cannot displace the clip. In other embodiments, the 5 ' bases of the clip can be ligated using a non-natural linker that is not cleaved by the 5 ' to 3 ' exonuclease activity that the polymerase may have. In alternative embodiments, the polymerase may be a non-strand displacing polymerase and may further lack 5 'to 3' exonuclease activity.

In further embodiments, a strand displacing polymerase can be used in combination with a clamp that is resistant to strand displacement by the strand displacing polymerase. The clip, particularly at the 5' end of the clip, may be comprised of non-natural bases that are resistant to strand displacement activity of a strand displacing polymerase. Such bases may comprise abasic bases such as depurination bases, or synthetic bases such as PNA, arabino derivatives of nucleobases, ribonucleotides, 2 '-O-alkylribonucleotides, 2' -O-methylribonucleotides, or bases with methylphosphonate linkages.

In some embodiments, the clip serves as a primer after phasing. The clip may include a reversible terminator at its 3' end, wherein a primer extension reaction is performed until the clip substantially prevents further extension. Further extension may be followed by removal of the terminator from the 3' end of the clip, which allows the polymerase to prime a primer extension reaction starting from the clip/primer.

In some embodiments, a single clip/primer is used for a colony or group of colonies, wherein the distance between the primer and the clip can be significantly greater than the average sequencing length before phase shifting would normally occur, e.g., when the clip is used in order to determine the structure of DNA, e.g., to create a scaffold and remove sequence uncertainty due to repetitive sequences. In some embodiments, the distance between the primer and the clip/primer may be twice as long as the average sequence "read length" prior to phase shifting, or may be two to five times as long as the average sequence "read length" prior to phase shifting, or may be five to twenty times as long as the average sequence "read length" prior to phase shifting, or may be twenty to fifty times as long as the average sequence "read length" prior to phase shifting. Additional clips/primers may optionally be used in this embodiment to extend the read length, and/or to further elucidate the structure of the DNA colonies. In some embodiments, the average read length is about 50 nucleotides, or about 100 nucleotides, or about 200 nucleotides, or about 300 nucleotides, or about 400 nucleotides, or about 500 nucleotides. In other embodiments, clips/primers are used for one colony or group of colonies, wherein the distance between the primer and the clip/primer or from a clip/primer to the next clip/primer may be similar to the average sequencing length before phase shifting would normally occur, e.g., as desired to expand the read length of the average sequencing length.

If there is any variation in the incorporation stop due to interaction between the clip (including any stabilizing moiety) and the polymerase (or ligase), the clip rephasing method may be combined with one of the methods described previously, which may be advantageous as the sequence of the clip is known, thereby allowing the addition of a base other than the first base of the clip sequence, possibly followed by the addition of a base other than the second base of the clip sequence, or any stop at any other known part of the clip sequence.

To allow short hybridization probes as rephasing agents, stabilizing compounds such as hydralazine or antitumor antibiotic cc-1065 can be used. Also, the probe may be a PNA or LNA probe, which may provide dual functions, i.e. providing a stronger binding and by using a terminator at the 3' end of the probe for example without the need to prevent the probe from being extended by a polymerase. In addition, the probe may be a single strand, a duplex that can hybridize to the target DNA to produce a more stable triplex, or a triplex that can hybridize to the target DNA to form a quadruplex. In some embodiments, the probe may be provided in two or more portions, where one portion may be hybridized single stranded and the second portion may be hybridized to produce triplexes. In some embodiments, additional portions of the probe complex may be provided, allowing quadruplexes to be formed, or duplexes to be formed with more than two portions in addition to the prototemplate.

In other embodiments, the lagging phase shift can be reduced by "stacking" polymerases on or near the DNA to be extended and sequenced such that many polymerases are available for each active polymerization site. The stacking may result from the natural binding of a polymerase to DNA. Such binding can occur normally, for example, when Klenow polymerase is used, where the Klenow polymerase has intrinsic ssDNA and dsDNA binding.

Further, in some embodiments, the DNA is provided with a 3 ' terminated random primer in addition to a universal or targeted primer, wherein the universal or targeted primer may not terminate, and wherein the polymerase can bind at the 3 ' terminating end of the random primer, as well as the 3 ' end of the universal or targeted primer. Since the random primer may be terminated, the random primer may not be extended and thus may not contribute to the signal associated with extending the strand from the universal or targeted primer. In this embodiment, the polymerase may lack 3 'to 5' exonuclease activity so that the random primer cannot be degraded, resulting in loss of stacking capacity.

In an alternative embodiment, a random primer using nucleotide analogs at least at the 3 'end can be used in place of the 3' terminated random primer, wherein the polymerase will bind to the random primer but will not bind to it It is extended. In this embodiment, the polymerase may have 3 'to 5' exonuclease activity, as long as the 3 'to 5' exonuclease activity is virtually inactive in removing nucleotide analogs. In some embodiments, it may be desirable for the polymerase to bind to the 3 'terminated random primer, or at least the random primer having one or more nucleotide analogs at the 3' terminal position, to have K_dIs smaller. The random primer containing a nucleotide analog may be chimeric, wherein the chimera comprises natural nucleotides and nucleotide analogs, multiple types of nucleotide analogs, or natural nucleotides and multiple types of nucleotide analogs.

In a further embodiment, the random primer may be a 3 ' terminated random primer, wherein the 3 ' end of the random primer further comprises a phosphorothioate nucleotide at the 3 ' (terminated) position, such that the random primer is further resistant to 3 ' to 5 ' exonuclease activity. 3' phosphorothioate primers are commercially available from, for example, IDT (Integrated DNA technologies). In this embodiment, a native polymerase having 3 'to 5' exonuclease activity, such as phi29, may be used without the need to mutate the polymerase to inactivate the exonuclease activity and thereby prevent degradation of the random primers. Such phosphorothioates may be alpha-S or alpha-R stereoisomers. The random primer may also contain a 5 ' phosphorothioate so that 5 ' to 3 ' exonuclease activity may be inhibited. Alternatively, the random primer comprises a 3 'inverted dT, the 3' position of which relative to the random primer acts to prevent polymerization and exonuclease activity. Dideoxynucleotides can be used as terminators. The terminator may be a reversible terminator, a virtual terminator, a terminator attached to a nucleotide base, or a terminator attached to any position of a sugar of a nucleotide. The nucleotides in the random primer may be natural bases or may be synthetic bases. The random primer may comprise dntps, or may comprise a chimera with PNA, RNA, LNA, 5-nitroindole, deoxyinosine, or other non-natural dntps in combination.

The stacked polymerase can also bind to the surface of the bead, the surface of the sensor, the gap regions between the sensors, groups, linkers or polymers attached to the bead, sensor or gap regions. Such binding may be to additional strands of non-extendible exonuclease resistant DNA, synthetic DNA or other linear polymers, or may be other binding groups such as antibodies, wherein the binding groups may be directly bound to the polymer, or may be bound to an intermediate protein that may be complexed with the polymerase.

To ensure that the stacked polymerase is able to replace a polymerase that has dissociated from the active incorporation site of the DNA strand in order to incorporate bases and prevent lagging phase shifts within a desired error rate, the K from the polymerase of the active incorporation site_offAnd K of stacked polymerases_offThe relative kinetic relationship between stacking sites and numbers and extension phase must be appropriate. For example, if K_offFor active incorporation site and stacking site K_offAnd K_onAre identical and K_offCorresponding to 20 cycles of incorporation fluid, if there are 20 stacked polymerases, there is less than 50% chance that another polymerase will become available for binding to the active incorporation site. This can be achieved by lowering the K at the stacking site _offAnd increasing stacking site K_onTo improve, it is reminded that loss of polymerase due to fluid flow may be a problem. K can be reduced by using non-natural bases, terminators, or the association of proteins with binding sites that normally reduce the persistence of the polymerase_off。

Alternatively or in addition, the polymerase may be a single type of polymerase or may be a combination of different types of polymerases. Generally, commercial, more persistent polymerases have lower incorporation error rates, and thus it may be desirable to primarily use highly persistent polymerases. One type of polymerase has a significantly longer K than the other type of polymerase_offMay be desirable. Make shorter K_offThe polymerase of (3) can be used to substitute for any higher persistence of dissociation (longer K)_off) The polymerase of (2) may also be suitable, so that the polymerase isWill be useful for incorporating any base as appropriate. Thus, the K of less persistent polymerases_offA length of time below that allotted for the incorporation fluid cycle may be appropriate to ensure that the polymerase will be available for incorporation when the more persistent polymerase dissociates from the binding site of the extended primer.

In some embodiments, where it is preferred to use two or more types of polymerase, it may be desirable to preferentially bind the more persistent polymerase to the primer to be extended. It may therefore be desirable to have a more processive polymerase bind to the primer to be extended before adding a less processive polymerase, which may have ssDNA and/or dsDNA binding moieties associated with it. In other embodiments, it may be desirable to modify or mutate the polymerase so that binding moieties can be added to the polymerase so that the polymerase can bind directly to ssDNA or portions of dsDNA.

The accumulation of polymerase can be replenished periodically. The replenishment can occur at each incorporation cycle or after an appropriate number of incorporation cycles have occurred. The number of cycles between replenishments may vary depending on the stacking method. For example, if the stacking method is storage on random primers, the number of stacking positions decreases with extension of the universal or targeting primer, as the random primers can be replaced by polymerase using strand displacement or 5 'to 3' exonuclease digestion of the random primers. For example, if a second binding mechanism exists for binding to double-stranded DNA, the amount of polymerase stacked can be maintained.

In certain embodiments, the method comprises monitoring the reaction for signal loss, and rephasing to restore sequencing signal. For example, data from a sequencing reaction can be monitored and rephasing can be performed when it is observed to be necessary, e.g., to see a signal level that may be lower than expected for a single base but would be desirable if there was a lagging phase error or to see a reduction in the signal level observed for a single base. The observations can take into account the nominal sequence in determining whether the signal is statistically caused by a lagging phase error. The observations may comprise a histogram of signal levels for one sequencing fluid cycle or a set of sequencing fluid cycles.

In some embodiments, rephasing can be performed on any clonal sequencing system (including those that utilize four incorporatable nucleotides, as well as all those systems described above for dephasing minimization). In some embodiments, rephasing is performed with emulsion PCR, or alternatively, with a magnetic array or bead array as described herein, optionally with electronic sequencing.

In one embodiment of the invention, compensation may be made to reduce phase error by determining the expected background level per cycle per location using earlier and/or later data. The phase error expected for each base, for each base in the sequence context, and the amount of lag and lead errors previously determined can be used to help determine the actual base. This error correction may also take into account phase errors from adjacent reactions on the array, as well as the effect of the adjacent reactions on the signal received from each sensor.

In some embodiments, the distribution between leading and lagging phase errors is affected such that one type of phase error may occur at a higher rate than another type of phase error. In one embodiment, the concentration of dntps can be limited so that lagging phase errors are more likely than leading phase errors. In further embodiments, rephasing may then be performed to correct for more likely types of phase errors. In other embodiments, the method is used to correct two types of phase errors, where the method corrects for one type of phase error and then corrects for the other type of phase error. The method of phase correction may be repeated periodically during the sequencing process. In some embodiments, the fluid mode used for rephasing may be fixed. For example, a fixed pattern may have a fixed number of fluid sequencing cycles between performing rephasing methods, or the number of fluid sequencing cycles may vary during the sequencing process, e.g., by reducing the number of fluid sequencing cycles between performing rephasing methods.

In another aspect, the invention provides a method for reducing lead phase error in parallel or clonal polynucleotide sequencing. The method according to this aspect comprises sequencing the population of polynucleotides in the presence of a competition reaction. The competition reaction includes nucleotide bases or nucleotide derivatives of all four nucleotide bases, three of which are not incorporated into the growing polynucleotide strand. Sequencing may include one or more of: sequencing clones of a polynucleotide population array (e.g., a bead array), electronic detection of nucleotide incorporation, and electronic wells for separating or concentrating components of a sequencing reaction. In various embodiments, the non-incorporatable nucleotide may be selected from (without limitation) PNA nucleotides, LNA nucleotides, ribonucleotides, adenine monophosphate, adenine diphosphate, adenosine, deoxyadenosine, guanine monophosphate, guanine diphosphate guanosine, deoxyguanosine, thymine monophosphate, thymine diphosphate thymine, 5-methyluridine, thymidine, cytosine monophosphate, cytosine diphosphate, cytidine, deoxycytidine, uracil monophosphate, uracil diphosphate, uridine, and deoxyuridine. Typically, unincorporable nucleotides are bound by the polymerase, but cannot be incorporated into the growing polynucleotide strand by the polymerase. In some embodiments, the concentration of non-incorporable nucleotides is related to the activity of the polymerase on each non-incorporable nucleotide. The non-incorporable nucleotides or nucleotide analogs may be unlabeled, photo-labeled or charge-labeled.

In some embodiments, the concentration of dntps that can be incorporated can be increased relative to the concentration of dntps that can be incorporated, allowing for a reduction in lead and lag error rates.

In another aspect, the invention provides a method for reducing lag phase error in a population of polynucleotide templates. The method comprises stacking a polymerase on or near the target polynucleotide during the sequencing reaction such that the polymerase is available for each or substantially each active polymerization site.

Alternatively or in addition, the method comprises binding a repair protein or single-stranded DNA binding protein to the target polynucleotide, in order to, inter alia, remove secondary structure or aid in persistence. In some embodiments, it may be desirable to reduce the secondary structure of single-stranded DNA that may otherwise interfere with polymerase activity, resulting in a lagging phase error. In one embodiment, a protein that binds to single-stranded DNA is used. Such proteins may include repair proteins such as bacterial RecA DNA repair protein, HIV nucleocapsid protein, T4 phage gene product 32, calf thymus UP1, EB virus BALF2, or commercial single-stranded binding proteins such as Epicentre e coli (e.coli) single-stranded binding protein, as well as many other proteins. In some embodiments, the single-strand binding protein may also contribute to the persistence of the polymerase. In other embodiments, other moieties may also contribute to the persistence of the polymerase, such as epstein barr virus BMRF1, triple-stranded saccharomyces cerevisiae (s. cerevisiae) proliferating cell nuclear antigen, T4 phage gene product 45, thioredoxin (thioredoxin), escherichia coli PolIII holoenzyme, eukaryotic clamp protein PCNA, or other DNA sliding clamp proteins, or other double-stranded or single-stranded DNA binding moieties.

In some embodiments, polymerase persistence may be enhanced by polymerase mutations, such as the addition of a helix-hairpin-helix domain to Phi29 polymerase, or the addition of a thioredoxin binding domain, the addition of an archaeal slide clamp, DNA binding protein Sso7d, zinc finger domain, leucine zipper, and other possibilities using a chimeric (chimeric) polymerase as described by Salas et al in PNAS 10716506. In some embodiments, the polymerase may be further modified to use more than one persistence-enhancing mutation, such as mutations that add double-stranded and single-stranded binding moieties at the respective ends of the polymerase. In some embodiments, such binding moieties also bind indirectly to the polymerase, such that the streptavidin moiety is added, e.g., by mutating the polymerase, and the biotin moiety is added to the single-stranded DNA binding moiety and/or the double-stranded DNA binding moiety, such as those described above, by mutation. The polymerase will thus bind to the single-or double-stranded DNA binding moiety by streptavidin biotin binding and further bind to the DNA by said single-or double-stranded DNA binding moiety. In other embodiments, other binding moieties are employed to bind the polymerase to the single-stranded or double-stranded DNA binding moiety, wherein additional moieties may be added to each of the polymerase and the single-stranded or double-stranded binding moiety by mutation, wherein the additional moieties added to the polymerase and the single-stranded or double-stranded binding moiety have a mutual binding affinity. In a further embodiment, one portion may be added to one of the polymerase, the single-stranded DNA binding portion and the double-stranded DNA binding portion by mutation, wherein the portion added by mutation may then bind to another of the polymerase, the single-stranded DNA binding portion and the double-stranded DNA binding portion, such that the polymerase binds to either the single-stranded or double-stranded DNA binding portion.

In some embodiments, stacking is achieved by using polymerase to bind to a non-extendable primer. In certain embodiments, the non-extendable primer is not affected by the 3' exonuclease activity of the polymerase. In some embodiments, the non-extendable primer is a 3' terminated random primer and the extension primer is a universal or targeted primer. The polymerase binds at the 3 'terminated end of the random primer and also to the 3' end of the universal or targeting primer. For example, a 3 ' terminated primer can comprise a phosphorothioate nucleotide at the 3 ' termination position such that the 3 ' terminated primer is resistant to 3 ' to 5 ' exonuclease activity.

Sequencing according to this aspect may comprise one or more of: sequencing clones of a population array of nucleotides (e.g., a bead array), electronic detection of nucleotide incorporation, and electronic wells for separating or concentrating components of a sequencing reaction.

With respect to electronic sequencing, it may be desirable in some embodiments to use a lower ion concentration than is optimal for synthesis in order to have a sufficiently low ion concentration for improved detector operation. This can lead to phasing errors, and shorter sequence lengths than desired. It may be desirable to have a longer sequence length than is possible with the low ion concentration. Thus, in one embodiment, the effective read length is increased by alternating the optimal detection conditions with the optimal synthesis conditions. For example, the method may comprise performing a sequencing reaction on the full length possible while using the low ion concentration required for optimal reading of the DNA extension reaction, melting the extended primer strands, introducing new primers and dntps, and continuing the synthesis reaction while using the ion concentration optimal for synthesis. The process of melting the extended primer strands, introducing new primers and dntps, and continuing the synthesis reaction while using the optimal ion concentration for synthesis, and subsequently changing the conditions to conditions suitable for detection can be repeated many times until the process no longer yields useful data. Since the determination of how many synthesis steps to use may be statistical, the process can be reversed, synthesized using optimal synthesis conditions, and then synthesized using conditions suitable for detection; the extended primer strand may then be subsequently melted, new primers introduced, and ion concentrations suitable for detection used.

To optimize detection sensitivity, concentrations of ions and/or dntps that are lower than may be required for proper enzyme kinetics may be desirable. This may result in longer incorporation times and/or more lagging phase errors than desired. In some embodiments, it may be desirable to use more than one dNTP concentration during a single incorporation cycle. For example, it may be desirable to perform a single incorporation cycle with a low concentration of dntps and/or ions in order to optimize the sensitivity and signal-to-noise ratio of the sensor. Thus, a solution with a low concentration of dntps and/or ions can flow into the flow cell so that the assay can be performed. This may be followed by a solution with a concentration of dntps and/or ions that is optimal for incorporation into the extended primer so that minimal dephasing can occur. In alternative embodiments, a high concentration of dntps can be flowed into the flow cell so that a rapid, optimal incorporation reaction can occur, thereby providing minimal dephasing. This may be followed immediately by a reagent solution with low or no dntps and a suitably low ion concentration so that an optimal sensor reading can be made.

In another embodiment, a reagent solution having an optimal concentration of dntps and/or ions for rapid incorporation of nucleotides can be flowed into the flow cell to rapidly incorporate a significant portion of dntps into the various colonies as desired, followed very rapidly by flowing a reagent solution optimal for optimal sensor reading into the flow cell, followed by flowing a reagent solution having a concentration of dntps and/or ions into the flow cell at a concentration suitable for incorporation of nucleotides into the extension primers of the various primers. The first reagent solution may have a nucleotide concentration that may be high enough for the reagent solution to be used over an extended period of time, and polymerase incorporation errors may occur at significant levels.

Because the efficiency of the polymerase may be different for each base, it is desirable to use different dntps at different concentrations, use different buffers, different cation concentrations, different polymerase concentrations, different types of polymerases, or any combination thereof to optimize the incorporation efficiency, minimize the amount of phase error, minimize the amount of incorporation error, and maximize the read length.

In certain embodiments, the concentration levels of different nucleotides or non-incorporable nucleotide analogs are matched to the relative activity of the polymerase for each nucleotide or nucleotide analog. For example, dTTP binding rates have been measured to differ more than two-fold relative to other nucleotides. The other three nucleotides may be very close in their polymerase binding rates, but still differ by more than 10% relative to each other. The difference may be even greater when comparing the polymerase binding rates of different non-incorporable nucleotide analogs relative to the native nucleotide.

In further embodiments, the concentration of non-incorporatable nucleotides required for equivalent polymerase extension is higher, equal to, or lower than the concentration of optimal primer extension with the lowest dephasing for one or more incorporatable nucleotides or nucleotide analogs that can be provided for a sequencing reaction that does not utilize non-incorporatable nucleotides or nucleotide analogs, such that the probability of misincorporation of a nucleotide or nucleotide analog is lower than if the concentration of the non-incorporatable nucleotide analog were provided such that the polymerase extension efficiencies matched, or if the non-incorporatable nucleotide analog was provided at a concentration that has a lower polymerase extension efficiency relative to the incorporatable nucleotide or nucleotide analog. Alternatively, the non-incorporatable nucleotide analogs can be provided at a concentration that has a lower polymerase binding rate relative to the incorporatable nucleotides or nucleotide analogs, such that the reaction can proceed at a higher rate than would occur if the polymerase binding rate of the non-incorporatable nucleotides were equal to or higher than the incorporatable nucleotide analog binding rate.

In other embodiments, the incorporatable nucleotide or concentration of nucleotides provided can be varied such that the incorporation rates of the different dntps can be more equal. The concentration can be varied as desired for different buffer conditions, pH, polymerase and interaction with any polymerase clamp complex or other moiety that can be used to stabilize the polymerase.

In some embodiments, non-incorporable nucleotides are used as part of a set of reagents that are not intended to result in nucleobase incorporation. Such a set of reagents may, for example, be intended to wash out a previous set of incorporatable nucleobases prior to introducing a new set of incorporatable nucleobases. This washing step may also include a phosphatase that degrades the nucleobase triphosphate into a diphosphate or a monophosphate, so that any residual triphosphate will degrade and will therefore become unincorporable. Non-incorporatable nucleobases may occupy a position in the polymerase pocket that is complementary to the next base, and may help effectively increase the persistence of the polymerase, as the polymerase's finger will remain closed, thereby attempting to incorporate non-incorporatable nucleobases and reducing dissociation of the polymerase from DNA.

The above process can be used for the following reaction conditions: wherein there may be three non-incorporable nucleotide analogs and one incorporable nucleotide or nucleotide analog, or wherein there may be two non-incorporable nucleotide analogs and two incorporable nucleotides or nucleotide analogs, or wherein there may be one non-incorporable nucleotide analog and three incorporable nucleotides or nucleotide analogs.

Detection methods that may be used with the above reaction conditions and non-incorporatable nucleotide analogs may include any form of electronic sensing of the incorporation or incorporation event, including ISFET, CHEMFET, nanoneedle, nanobridge, chemiluminescent detection, fluorescent detection, including detection of quantum dots or other non-standard fluorophores, and detection of intercalating fluorophores, using fluorescent moieties.

In further embodiments of the invention employing bead arrays and electronic sequencing (as described herein), empty beads or empty sensor regions (sensor regions without associated colonies) may be used as references; such a reference may compensate for changes in temperature, changes in conductivity or pH of the bulk reagent, or local changes in conductivity or pH. Control of the system will help limit and characterize the phase error, thereby extending the read length.

The common practice for FET pH sensors is to use a reference electrode; some designs of FET pH sensor arrays use a reference channel for each detection channel; others have reference channels for a set of detection channels. However, the local pH of the detector is affected by the presence of DNA colonies and varies as the length of the second strand of DNA is extended by the polymerization reaction. When using a chemical method that introduces a single type of nucleotide into the flow cell at once, many detector channels will not react on the detector; in practice most detector channels will not react. Thus, in one embodiment, using an adjacent detector as a reference channel provides the data analysis algorithm with the opportunity to measure pH or ion concentration as it changes in the detector adjacent to the detector with the polymerization reaction occurring. This allows for detection of pH or ion concentration levels or other sources of local noise of the detector of interest, and may also allow for detection of crosstalk, allowing for monitoring and changing of the crosstalk deconvolution function.

In certain embodiments, the use of empty beads (with no DNA colonies, or with colonies that do not contain the same primers as will be used for the sequencing-by-synthesis reaction) ensures that some detectors will not have a polymerization reaction occurring on them. Beads with DNA colonies containing appropriate primers can be introduced into the flow cell using a set of random positions on the detector. Subsequently, a set of empty beads as described above may be introduced into the flow cell, whereby the empty beads may occupy random positions that have not been occupied by beads already present in the flow cell. As reagents are introduced into the flow cell, the pH and/or ion concentration levels are then monitored using empty beads, thereby enabling the analysis algorithm to better determine background levels and/or crosstalk deconvolution functions.

In another embodiment, empty beads are introduced in pairs with sample beads and a differential amplifier is used to determine the signal, thereby avoiding the need for an analysis algorithm that directly deconvolves background and crosstalk variations.

In still other embodiments, adjacent beads or sensor regions that do not have an incorporation reaction in the current fluidic cycle may be used as a reference. Since typically most beads or sensor areas will not have an incorporation reaction when sequencing large populations of different sequences, these sites without incorporation reaction can be used as additional references. As a reference, beads or sensor regions without an incorporation reaction may also provide a better reference relative to empty sensors or empty beads, since DNA polymerase and beads will be present in the volume of interest, and any changes in surface chemistry and resulting background counter ion concentration will likely better match. It is likely that different colonies on a bead or sensor region may have colony DNA and/or extension primers of a different length than colony DNA and/or extension primers associated with other beads and/or sensor regions and therefore may present different amounts of charge that can interact with the sensor.

Thus, in some embodiments of the invention, software may be required to compensate for the relative lengths and resulting different charge and signal levels of the DNA and/or extension primers associated with the sensor, as well as the position of the beads or colonies relative to the sensor, which may have an effect. The software may keep a record of the signal level: associating a bead with each sensor prior to introducing the bead into the sensor; after the beads are introduced but before the primers and/or polymerase are introduced; after introducing the primer and/or polymerase but before introducing the first nucleotide in the sequencing-by-synthesis reaction; (ii) associated with colonies without primers; (ii) associated with colonies that may have DNA of different lengths; and signal levels associated with colonies with hybridized primers, which may have DNA of different lengths. The software can track how many bases have been added to each primer. The signal level may be an absolute level, or a relative level between different sensors. The software may use many other adjacent or closest sensors as references to determine the signal level of the individual sensors, thereby compensating for the DNA length of each group of colonies, the length of the extended primers and the signal level that may be due to the positioning of the beads or colonies relative to the sensors. The software can also compensate for the relative position of the sensor with respect to the transition between the reagent volume without dNTPs and the reagent volume with dNTPs. The software can further compensate for variations in dNTP concentration with diffusion and/or dNTP concentration depletion due to incorporation into the extension primer. The amount of diffusion can be characterized by the following data: early data from the same chip, which may be in the same sequencing run or from a previous sequencing run, or sequencing data from a previous chip used on the same instrument, or data from other chips used on other instruments. The expected consumption level can be determined based on data generated as the transition between the reagent volume without dNTPs and the reagent volume with dNTPs passes through the flow cell.

In some embodiments, control beads with known sequences or with different known sequences are used. The known sequences may have homopolymer runs of different known lengths and may be used to calibrate the response of the system to better determine the length of a homopolymer run of unknown length. Known sequences can also be useful in determining signal or background signal levels, as the length of the extended primer and whether an incorporation event has occurred will be known in advance. Control beads can be used to distinguish instrument problems from sample preparation problems. Control beads with known sequences can be generated outside the system and introduced with the DNA colonies attached thereto, or the control DNA can be mixed with or introduced before or after the sample DNA to generate DNA colonies on the beads or otherwise associated with the sensor. In some embodiments, signal levels can be monitored and stored in a manner similar to that described herein for normalization by adjacent beads.

In a similar manner to optical aberrations, diffusion of species (species) detected by the detectors will cause cross-talk between different detectors. In one embodiment of the invention, the deconvolution of data obtained from different sensors on the array may be performed in a manner similar to that used to deconvolute the point spread function from the optical system. The deconvolution function used may depend in part on the temperature of the flow cell at the time of detection, as well as the flow rate through the flow cell, which often may lead to more crosstalk "downstream" of a particular colony. The deconvolution function used for the deconvolution may be a fixed deconvolution function, or may be derived as part of a best-fit algorithm.

The sensor array may be self-calibrating, allowing for calibration of variables such as amplification efficiency, bead size and load, bead location on the sensor, etc. Typically, when a DNA sample is amplified to produce a monoclonal population of DNA on a bead, a first primer can be ligated to the sample DNA prior to the amplification reaction. During sequencing, a second primer is provided that is complementary to the first primer that has been ligated to the DNA sample. The second primer may be several bases shorter than the first primer. Thus, each monoclonal bead has a density of known initial sequences independent of amplification efficiency that will be the portion of the first primer that is not matched by the complementary second primer. This may allow for prior knowledge of the base sequence, and may include calibration sequences such as homopolymer runs of known length.

In alternative embodiments, such calibration may occur after the sequencing reaction is complete, or alternatively after many bases have been sequenced in the sequencing reaction. Statistically, most of the fluidic cycles in a sequencing reaction will not result in base incorporation on individual beads. The next most common result of fluid circulation will be the incorporation of a single base. Thus, the data set can be analyzed and an appropriate signal level can be set for each bead.

In further embodiments, ongoing compensation/calibration may be performed when the signal level of base incorporation in the fluidic cycle decreases during sequencing, and the background without base incorporation in the fluidic cycle is the result of factors such as: loss of some clonal population on the bead, sequencing phase lead or sequencing phase lag of some clonal population on the bead, or other factors. Thus, the signal level can be calculated as to how much signal can be expected to be generated at each point in the sequencing process for a fluid cycle with single base incorporation, no base incorporation, or multiple base incorporation due to homopolymer runs.

In some embodiments, reverse phase alignment can be performed in which a polymerase having 3 'to 5' exonuclease activity is used with a dNTP pool that lacks at least one dNTP. A polymerase with 3 'to 5' exonuclease activity will remove the base, back to the next dNTP in the pool of dntps provided, at which point equilibrium will be reached and no further nucleotides will be removed. This may be done to remove any bases that have been incorporated due to the leading phase error. In other embodiments, one or more base types that do not allow exonuclease activity, such as phosphorothioate nucleotides, may be incorporated. Exonuclease activity can then be used by removing unincorporable nucleotides, thereby improving the kinetics of exonuclease activity. In other embodiments, an exo-polymerase may be used for initial incorporation, followed by the use of an exo + polymerase or another nuclease to remove any bases, back to phosphorothioate nucleotides or other nucleotides resistant to nuclease activity.

Sequencing of single and/or repeated polynucleotides

In another aspect, the invention provides a method for sequencing repeated and/or single polynucleotides. The method of this aspect comprises providing a circularised DNA sequencing template and sequencing the template by determining the order in which nucleotides are incorporated by a DNA polymerase having 5 'to 3' exonuclease activity. Sequencing according to this aspect may comprise one or more of: sequencing clones of a polynucleotide population array (e.g., a bead array), electronic detection of nucleotide incorporation, and electronic wells for separating or concentrating components of a sequencing reaction. In various embodiments, the DNA polymerase is highly processive and has reduced exonuclease activity. In addition, the DNA polymerase can be bound on or near a biosensor suitable for determining incorporation of nucleotides. In some embodiments, the method comprises pre-binding a polymerase to the polynucleotide prior to sequencing. According to this aspect, the present invention avoids the need for corrective phasing or rephasing.

Single DNA molecules can be sequenced by a nanoneedle biosensor (described in detail herein). The polymerase is attached to the sensor. The DNA sample with the associated primers can then be brought into the volume with the sensor with the polymerase attached using, for example, pressure induced flow, electroosmotic induced flow and/or migration or similar means. Single molecules from the DNA sample can then be bound by a polymerase attached to the sensors in the sensor array. Additional single DNA molecules may also be bound by other polymerases bound to other sensors in the sensor array.

To allow for repeated assays of the same DNA sample, the DNA sample may be circularized, and the polymerase may be a strand displacing polymerase. Thus, the DNA sample can be repeatedly sequenced by continuing the primer extension reaction through multiple cycles completely around the circular DNA sample. The data from this chain can then be converted to a more accurate consensus sequence with reduced data processing. In a clear advantage over systems employing fluorophore detection, the system of this aspect takes advantage of the full capacity of the polymerase to read length, without being hindered by the shortening of the read length by phototoxicity.

A single molecule is one instance of a monoclonal population, where the population is 1. Therefore, the idea regarding monoclonal DNA is generally applicable also to the case of a single-molecule state.

Fig. 25 depicts and illustrates an apparatus and method whereby a single DNA molecule 2507 can be sequenced through a nanoneedle biosensor array 2500. Polymerase 2506 can be attached to sensor 2501. The DNA sample with the associated primers can then be brought into the volume with the sensor with the polymerase attached using, for example, pressure induced flow, electroosmotic induced flow and/or migration or the like. Single molecules 2507 from the DNA sample are then bound by the polymerase attached to the sensors 2501 in the sensor array 2500. Additional single DNA molecules 2507 may also be bound by other polymerases 2506 bound to sensors 2501 in sensor array 2500.

In one embodiment, one of the four native dntps 2502 then flows into channel volume 2504 with a sensor. A dNTP can be bound and incorporated if it is complementary to the next base in the sample DNA 2507. The resulting change in local charge of the extended primer DNA can then be detected by the nanoneedle sensor 2501, allowing the incorporation event to be detected at each appropriate location of the sensor array 2500. If the sample has more than one contiguous base complementary to a type of dNTP2502 that has been introduced into the channel volume 2504 with the sensor 2501, then a second or subsequent binding and incorporation of dNTP2502 can be detected by the nanoneedle sensor 2501. The dntps 2502 can then be washed away from the channel volume 2504 containing the sensor 2501.

In certain embodiments, one of the four native dntps is then flowed into the volume with the sensor. If the dNTP is complementary to the next base in the sample DNA, it is bound and incorporated. The resulting local charge change can then be detected by the nanoneedle sensors, which may be the result of a charge change in the extended primer DNA, or may be the result of other charge changes, allowing the incorporation event to be detected at each appropriate location of the sensor array. If the sample has more than one contiguous base complementary to a dNTP type that has been introduced into the volume with the sensor, a second or subsequent binding and incorporation of dntps can be detected by the nanoneedle sensor. The dntps can then be washed out of the volume containing the sensor.

Different dntps can then flow into the sensor array volume, allowing the detection of the incorporation event. Subsequent cycles of washing, introduction of one of the four dntps at a time, and detection of incorporation events allow the determination of different sample DNA sequences.

In yet another embodiment, up to four different nucleotides can be delivered simultaneously, and which nucleotide is incorporated can be determined by observing the kinetics associated with the incorporation reaction.

In an alternative embodiment, the sample DNA may bind to one of the polymerase, the sensor or the region between the sensors in close proximity (in mediated proximity to) the sensor such that the bound polymerase can bind to the sample DNA after the primers have been introduced into the system and allowed to hybridize to the sample DNA. Subsequently, after primer extension and the related determination of the sample DNA sequence are completed, the extended primers can be melted by changing the temperature or pH of the solution in which the sample DNA is solvated or both the temperature and pH of the solution. The sample can then be resequenced by reintroducing the primers and restoring the temperature or pH of the solution in which the sample DNA is solvated to conditions suitable for primer extension, including appropriate nucleotide and cation concentrations.

In some embodiments, the nucleotide may be a native dNTP. In other embodiments, dntps can be modified with charge-altering structures. The charge-altering structures can be associated, bound, and coupled to polyphosphoric acid and subsequently cleaved as part of the incorporation process, thereby avoiding the need for a separate process for cleaving, separating, or removing the charge-altering structures.

In an alternative embodiment, the charge-altering structure is a terminator and is therefore associated, bound or coupled to the 3' position of the sugar of the dNTP and may therefore act as a terminator. Detection may occur as a result of the incorporation process or may result from cleavage of the charge-altering structure.

In other embodiments, the charge altering structure may be associated, bound or coupled to the 2 'or 4' position of the dNTP saccharide. In still further embodiments, the charge-altering structure may be associated with, bound to, or coupled to the base of the nucleotide. The charge-altering structure can act as a terminator, preventing the incorporation of additional dntps.

The linkage, association or coupling may be disrupted by physical processes such as temperature changes, or may be disrupted by chemical processes, or may be disrupted by photochemical reactions. The linkage, association or coupling may be disrupted after each nucleotide incorporation, or several nucleotides may be incorporated, and the number of incorporated nucleotides may be determined by measuring the amount of charge added as a result of the incorporation.

In further embodiments, two or three nucleotides are used at a time, allowing for the addition of multiple bases and corresponding large signals at a time. After completion of primer extension and relevant data collection, the extended primers are melted, new primers are added, and the extension process can be performed again using a different order of dNTP combinations. This process determines which dntps do not follow the completion of the previous set of dntps, and information about the incorporation length, where the length determination need not be accurate.

To allow for repeated assays of the same DNA sample, the DNA sample may be circularized while the polymerase may be a strand displacement polymerase, or may be a polymerase with 5 'to 3' exonuclease activity. The DNA sample can thus be repeatedly sequenced by continuing the primer extension reaction for a number of cycles around the circular DNA sample. Among the obvious advantages over systems employing fluorophore detection, the systems in certain embodiments of the invention can employ the full capacity of the read length of the polymerase without being hindered by the reduction of the read length by phototoxicity. In some embodiments, a strand displacing enzyme may be used, thus resulting in an increase in charge and associated counter ions. In other embodiments, a polymerase enzyme having 5 'to 3' exonuclease activity may be used, thereby keeping the electrostatic charge constant while generating protons and/or hydroxide ions, which may be measured as an increase in conductivity, or as a result of the interaction of ions with the surface of an ISFET, ChemFET, or nanobridge sensor.

The polymerase bound or associated with the sensor may be a highly processive polymerase that allows the incorporation of more bases than less processive polymerases. The polymerase may be phi29, RepliPHI,T4 (E.coli T4), F-530, B104 or other highly processive polymerases. The polymerase may be modified to have reduced or no 3 'to 5' exonuclease activity, or the native form of the polymerase may have no or little 3 'to 5' exonuclease activity. Likewise, any 5 'to 3' exonuclease activity may be modified to reduce or nearly eliminate it. Thermostable polymerases or other types of DNA or RNA polymerases may be used, such as: vent (Thermococcus littoralis), Vent exo-, Deep Vent exo-, Taq (Thermus aquaticus), Hot Start Taq, Hot Ex Taq, Hot Start LA Taq, DreamTaq^TM、TopTaq、RedTaq、Taqurate、NovaTaq^TM、SuperTaq^TMStoffel fragment, discovery enzyme^TMdHPLC、9°Nm、LongAmp Taq, LongAmp Hot Start Taq, OneTaq,Crimson Taq, Hemo KlenaTaq, haire Hot Start II, DyNAzyme I, DyNAzyme II, M-MulV Reverse Transcript,Tth (Thermus thermophilus) HB-8), Tfl, Amlitherm ^TMBacillus DNA, DisplaceAce^TMPfu (Pyrococcus furiosus), PfuPfunds、ReproFast、PyroBest^TMVeraSeq, Mako, Manta, Pwo (Pyrococcus woseri), ExactRun, KOD (Pyrococcus kodakkaraensis), Pfx, Rerohot, Sac (Sulfolobus acidocaldarius), Sso (Sulfolobus solfataricus), Tru (Thermus rubrus), Pfx50^TM(Thermococcus zilligi), AccuPrime, and combinations thereof^TMGC-Rich (Pyrolobus fumarius), Pyrococcus GB-D, Tfi (Thermus filiformis), Tfi exo-, ThermalAce^TMTac (thermoacidophilum (Thermoplasma acidophilum)), (Mth Methanobacterium thermoautotrophicum (M. thermoautotrophicum)), Pab (Pyrococcus abyssi), pho (Pyrococcus horikoshii), B103(Picovirinae phage B103), Bst (Bacillus stearothermophilus), Bst large fragment, Bst2.0, Bst 2.Wa0RMStart, Bsu, Therminator^TM、Therminator^TMII、Therminator^TMIII、Therminator^TMGamma, T7DNA, Escherichia coli polymerase I, Kenow (Escherichia coli) fragment, Klenow fragment exo-, T4DNA, sulfolobus DNA polymerase IV, AMV reverse transcriptase, human polymerase mu-h6, DNA polymerase I (Escherichia coli), T7RNA (Escherichia coli T7), SP6 (Escherichia coli SP6) RNA, Escherichia coli Poly (A), Poly (U), T3 RNA.

The polymerase and/or DNA may be bound directly to or near the sensor, or may be bound via a linker.

In some embodiments, the recombinase polymerase amplified variants as described in US7,270,981, incorporated herein by reference, are used for sequencing. In some embodiments, the amplified DNA template may be double-stranded, and the input primer may be complexed with a recombinase such as RecA or RAD 51. The composite primer can bind to double-stranded DNA and displace a portion of both strands of the double-stranded DNA with the aid of a recombinase. The polymerase can then bind to the appropriate end of the primer, enabling the polymerase to incorporate the nucleobase and extend the primer. A single-stranded binding protein can be added that binds to a strand of double-stranded DNA that does not hybridize to a primer. Thus, a large number of counter ions may be present for sensing, whereby the counter ions may be associated with newly synthesized strands of DNA, and additional counter ions may be associated with the single-stranded binding protein. An additional advantage is the reduction of problems due to secondary structure created by the use of single stranded DNA templates.

Chamber-less reactor and virtual reactor

In other aspects, the invention provides a chamber-less apparatus for sequencing a polynucleotide. The device comprises an electromagnetic sensor, a magnetic carrier for carrying or holding the template polynucleotide on or near the electromagnetic sensor, and a mechanism for removing the magnetic carrier by liquid flow and/or electromagnetic removal. In certain embodiments, the electromagnetic sensor is one of a nanoneedle or a nanobridge, and the device further comprises a local amplifier. The electromagnetic sensor may have a narrow structure and may be etched under the structure so that both sides of the sensor surface are accessible for a change in pH or a change in conductivity.

In some embodiments, the system employs a Magnetic array as described in U.S. provisional application 61/389,484 entitled "Magnetic Arrays for emission-Free Polynucleotide Amplification and Sequencing," which is incorporated by reference herein in its entirety. This system is shown schematically in fig. 5.

Fig. 5 depicts a single element 500 and a magnet 516 in a nanoneedle array, where a substrate 504 may have an electrode 506 on the substrate 504 and under a bead 502, or an electrode 506 on a spacer or adhesion layer (not shown) between the electrode 506 and the substrate. The electrodes 502 may thus be located within the debye layer of the bead. A dielectric layer 508 may be located over the substrate 504 and may also cover a portion of the electrodes 506, and may further have recesses or openings (cutouts) that may be larger than the spacing required for the beads 502 when holding the beads 502 in place. The dielectric may serve a variety of functions, including providing a surface against which a magnetic force may pull the surface of the bead 502. The magnetic force created by the interaction of magnet 516 and the bead 502 is used to hold and position the bead 502, applying a force down to the electrode 502 and to dielectric 508. A second layer of dielectric 512 may be applied to the dielectric 508, providing important access of reagents to the beads 502, while further extending the height of the total thickness of dielectric material, so that an upper electrode 514 may be fabricated on the second layer of dielectric 512. The upper electrode 514, dielectric, and second dielectric 512 may be positioned such that the upper electrode may be within the debye length of the bead and may be further proximate to the midpoint of the bead 502. The upper electrode 514 may be above the centerline of the bead 502, particularly if the upper electrode 514 is within the debye length of the bead 502, or may be below the centerline of the bead 502, such that the top of the upper electrode 514 makes contact with the bead at an angle of 10-90 degrees from perpendicular to the point of contact between the bead 502 and the electrode 506. The angle to the perpendicular to the point of contact between the bead 502 and the top of the upper electrode may be 30-85 degrees, 45-80 degrees, or 60-75 degrees. The magnet 516 may be embedded in the matrix 504, above the matrix 504 or above the dielectric 508, but should be below the centerline of the bead 502 in order to apply a downward force to the bead 502, pulling the bead 502 downward toward the electrode 506. When the beads are in place in the array, the magnets 516 should be further offset from the center of the beads 502 in order to pull the beads 502 toward the upper electrode, thereby allowing the beads 502 to enter the bead 502 to within the debye length of the upper electrode 514. Each element of a single element 500 in the nanoneedle array and the magnet 516 may have an additional spacer layer or adhesion layer between the elements. The debye length may include a debye length that may be caused by a high concentration of salt, a low concentration of salt, deionized water, or an aqueous solution co-mixed with a non-aqueous fluid that is miscible in water.

Fig. 2 is a photomicrograph of a combined virtual well and magnetic array according to various embodiments described herein. Most locations in the array have a single bead located between the magnets at the point where the virtual well structure is located. Some locations have more than one bead. Most of the ends of the magnet also have beads located thereon.

The magnetic array may be used in a manner similar to that described in US7,682,837, which is incorporated herein by reference in its entirety.

As used herein, "bead" means a bead, moiety or particle that is spherical or non-spherical, wherein the bead, moiety or particle is porous or solid or a mixture of solid and porous, and may include magnetic beads that may be paramagnetic, superparamagnetic, diamagnetic or ferromagnetic.

As used herein, "bead capture features" means features that can temporarily hold a single bead in a fixed position relative to a sensor, and can include local magnetic structures on a substrate, which can include external magnets, local magnetic structures, van der waals forces, or gravity as a depression that fixes the bead position. The beads may be bound in place by covalent or non-covalent binding.

As used herein, "restricted" refers to the situation when a molecule (e.g., DNA) produced at one bead or particle remains associated with the same bead or particle, thereby substantially maintaining the clonal nature of these beads or particles.

As used herein, "separating" means preventing migration, diffusion, flow, or otherwise movement from one virtual well to another virtual well, if necessary, to maintain the clonal nature of these beads or particles.

As used herein, "localized magnetic features" means magnetic features produced on a substantially planar substrate to retain individual beads on the substantially planar substrate.

As used herein, "localized magnetic field" means a magnetic field that is present substantially in the volume between the north pole of a first magnetic region and the south pole of a second magnetic region or substantially in the volume between the north pole and the south pole of a single magnetic region.

As used herein, "particle" means a non-bead moiety, such as a molecule, an aggregate of molecules, a molecule bound to a solid particle, or a particle, among other forms known in the art.

As used herein, a "single-phase liquid" is a liquid having relatively uniform physical properties throughout (including properties such as density, refractive index, specific gravity, and the like), and may include aqueous solutions, miscible aqueous and organic mixtures, but excludes immiscible liquids such as oils and water. Among the physical properties that are believed not to potentially cause a liquid to be considered a non-single phase liquid are local changes in pH, charge density, and ion concentration or temperature.

As used herein, "substantially planar" should allow for small pedestals, raised segments, holes, depressions, or irregularities of no more than 40 μm relative to the local plane of the device. Variations due to warping, twisting, deep drawing or other planar deformations are not generally considered to constitute a part of the allowed deviations. Protrusions or recesses in excess of 40 μm may not be necessary for the purposes described herein, but do not prevent the device from being considered substantially planar. Fluid channels having dimensions greater than 40 μm and/or structures used to create the fluid channels also do not prevent the device from being viewed as substantially planar.

As used herein, a "virtual well" refers to a localized electric or magnetic field confinement region in which a species or group of species of interest, typically DNA or beads, does not normally migrate into an adjacent "virtual well" during a period of time necessary for a desired reaction or interaction.

As used herein, "electrode" is defined as any structure used to generate or apply an electric or magnetic pair in such an array. Such structures can be used for separations or operations in the delivery of biomolecules to specific regions in an array (e.g., the middle region of a confinement array or elements in a separation array) at a time of interest, resulting from the opening and closing of fields or forces.

In embodiments of the devices disclosed herein, the device comprises a sensing surface for sensing nucleotide incorporation, the sensing surface comprising a silicon nitride layer.

In embodiments of the devices disclosed herein, a plurality of magnetic beads are configured for carrying a template polynucleotide, wherein the magnetic beads have a low zeta potential at a pH level effective for nucleotide incorporation.

A virtual nanoreactor or "chamber-less array" can detect or manipulate particles (e.g., beads, cells, DNA, RNA, proteins, ligands, biomolecules, other particulate moieties, or combinations thereof) in an array, wherein the array captures, holds, confines, separates, or moves the particles by an electrical, magnetic, or electromagnetic force, and can be used for reactions and/or detection of and/or reactions involving the particles. The "virtual nanoreactors" provide a powerful tool for capturing/holding/manipulating beads, cells, other biomolecules or their carriers, and can then concentrate, confine or separate portions of pixels or regions of the array that are different from other pixels or regions of the array using electrical, magnetic or electromagnetic forces. In one embodiment, the array is in a fluid environment. Sensing may be by measuring charge, pH, current, voltage, heat, optical or other methods.

In certain embodiments, the chamber-less apparatus described herein allows for better washing of nucleotides during a sequencing reaction, leading sequencing phase errors can be reduced by reducing the number of remaining nucleotides, which can include unbound and non-specifically bound nucleotides that can subsequently be improperly incorporated into incorrect cycles.

In addition to DNA sequencing, a variety of different molecular biology applications using "chamberless" arrays or "virtual" nanoreactors are also contemplated. The array may be used as a tool, e.g., as a cell sorter, and the array may then also perform molecular biology on the cells, which may include sorting, determination or manipulation of one or more biochemical events or reactions of interest (e.g., drug screening or biomolecule detection). The virtual nanoreactor array can be used for cell monitoring and analysis, for example, the system can measure electrical characteristics of cells that are captured in the array and/or adjacent to sensing elements associated with the virtual nanoreactor. The array can be used to screen rare cells, for example, to detect responses in drug screening for drug development, or to select and detect cancer cells. In many embodiments, the assay or detection target includes cell biology, drug screening and monitoring of specific cell types, detection of DNA, RNA (nucleic acids), proteins, charged small molecules, ligands, or other biomolecules.

In other embodiments, the further electrode (or virtual wall/fence) element may comprise two further electrodes in the array associated with each electrical confinement or separation element.

In some embodiments, the system is used to capture multiple beads or cells in each pixel, and the size, shape or period of the electromagnetic field can be turned on, off or varied at desired times or in response to changes at the sensors associated with the elements in a "virtual wall" array that may be required for different applications. For example, the system may capture a set of beads or cells, and then increase the field strength to capture another portion that is less affected by the field. The electric field may be DC or AC or a combination of different combinations of said fields.

FIG. 3 is a graphical representation obtained from Comsol simulation depicting a 3D equipotential field strength curve 306 for one-quarter of a cylindrical structure 300 resulting from a field applied to electrodes 304A and 304B. Since the field is substantially radially asymmetric, the volume 308 where the field gradient is most concentrated is close to the central electrode 304B, at which point the beads may be held by a magnetic array as shown in fig. 2.

In other embodiments, the virtual nanoreactors are used in and/or in combination with multiple steps in biomolecular processes, e.g., bead enrichment of particles moving in an electric field, microfluidic sample preparation and library preparation, such as on-chip extraction of DNA, shearing of DNA, on-chip normalization of DNA concentration, emulsion-free amplification, sensing, which may include SensePlus sensing using dual-or multi-sensing sequencing detectors, which may utilize transient and/or steady-state electronic sequencing and rephasing methods to extend the read length of the sequencing process, wherein the multiple steps may result in a fully integrated electronic genome analyzer system.

In some embodiments, multiple arrays of virtual nanoreactors are used. Different arrays of virtual nanoreactors may be used for different biological reactions, processes or methods. In some embodiments, one array or set of arrays is used for extraction of DNA, wherein different arrays in the set of arrays or different members of a set may retain cells from different samples, or may retain different types of cells from a single sample, or a combination thereof. For example, different samples may be held in different arrays, and different types of cancer cells from a single sample tumor may be held in different regions of a single array. Different cell types may be sorted or isolated, or cell types may be determined from data derived from individual cells after the cells are captured and retained in individual locations on the array of virtual nanoreactors.

In some embodiments, several biological reactions, processes or methods may be performed on a single sample or portion while it is held in place within the virtual nanoreactor. In other embodiments, one or more biological reactions, processes, or methods may be performed on a sample or portion in a location of a virtual nanoreactor array prior to transferring or moving the sample or portion to another location in another virtual nanoreactor array.

In some embodiments, an array of electrical concentration and confinement virtual nanoreactors is used for purposes other than nucleic acid capture/separation as described elsewhere herein for nucleic acid sequencing and amplification. In some embodiments, electrical concentration and confinement is used with any charged moiety, wherein the time period, concentration of the moiety, mobility, concentration, local viscosity, percentage of crosslinking of the local polymer, or concentration of the charged moiety affects the spacing of the electrical confinement structures and/or the field strength and/or field gradient magnitude used, so as to maintain sufficient confinement within the individual virtual nanoreactors. The tolerable level of cross-contamination between different virtual nanoreactors can be different for different applications and different steps in a biological reaction, process or method. For example, if a nucleic acid amplification reaction process is performed on different samples in adjacent virtual nanoreactors, cross-contamination may be a problem during early thermal cycling of the PCR reaction, while cross-contamination during later cycling of the PCR reaction may not be of particular concern because the primers may be consumed in large amounts, thereby preventing significant amplification of cross-contaminated nucleotides from other virtual nanoreactors.

In some embodiments, the mobility of the nucleotide or other moiety is reduced using a polymer. For example, the polymer may be a partially entangled polymer such as POP-7^TMOr agarose, polyacrylamide or starch gels. The concentration and/or percentage of cross-linking may be varied as needed for different mobilities of different portions that are desired to be confined in the virtual nanoreactor. Sample portions or methods that can be used in introducing the biological reactions, processes or methodsThe polymer is introduced into the volume of the virtual nanoreactor before, simultaneously with or after the other part.

In some embodiments, the electric concentration and confinement is used to capture moieties to which other biomolecules or biological moieties have been bound, e.g., beads having a charge that can bind to an antibody, and wherein the beads can then be used to capture proteins, and wherein the beads can then be further captured using an electric concentration and confinement array.

In some embodiments, the virtual nanoreactors can be used for several different applications other than sequencing, for example, the virtual nanoreactors can be used as hybridization arrays (similar to Affy or Agilent DNA microarrays) where DNA is on beads and the beads are held in place by the virtual nanoreactors. In other embodiments, virtual nanoreactors are used for digital PCR, wherein the beads are introduced into an array. Detection may be by an array of electronic sensors associated with each element of the array, or detection may be by optical means to detect the presence or amount of a particular nucleic acid. Digital PCR can be used to quantify the concentration of a target within a sample in a relative manner.

In some embodiments, concentrating the sample in a virtual nanoreactor as described herein allows for higher sensitivity quantitation. In some embodiments, the use of concentration in some regions of the array and not in other regions may allow for higher concentration quantitative dynamic range. In further embodiments, the DC concentration field may be reversed in order to partially "push" the sample portion away from the virtual nanoreactor. In some embodiments, the combination of focusing, non-focusing, and pushing sample portions into different regions may help further expand the dynamic range of digital PCR or any other desired biological reaction, process, or method. Beads with different primers can be introduced into different regions of a virtual nanoreactor array as described herein, which allows for simultaneous digital PCR reactions on several targets.

In some embodiments, the virtual nanopore is used for a full extension reaction of DNA bound to a primer coupled to the bead. In further embodiments, the virtual nanoreactors can be used for ligation reaction detection.

In some embodiments, the system may use electromagnets, permanent magnets or electrodes or other different subsystems to generate electromagnetic fields for brief or partial time separation, or to hold or concentrate biomolecules or other moieties of interest (e.g., DNA, cells, proteins) or carriers of biomolecules or other moieties of interest (e.g., beads, particles or other moieties). The electromagnetic field may be a magnetic field, an electric field, or a combination of both. The electric field may be a DC field, an AC field, a pulsed DC field, a non-sinusoidal AC field, a pulsed AC field, or a combination thereof. Nanoreactors with virtual walls (or fences) can be used or created with electric or magnetic fields to hold, capture, concentrate, separate, or manipulate biomolecules or their carriers.

In some embodiments employing two or more sets of electrode structures, a virtual wall or separation wall or fence may be opened or closed, or the magnitude, shape or period of the electromagnetic field modified at a desired time, which may be a fixed time, or may be responsive to a change in a sensor associated with a particular member of the array of electrode structures. Turning on or off or modifying the magnitude, shape or period of the electromagnetic field may be used to control the movement of particles, beads, cells, biomolecules or other moieties of interest that are concentrated, confined or isolated in an array of electrode structures, or to control biomolecules or other moieties of interest that may be used in a reaction, such as an antigen or a second antibody in protein detection, or nucleotides in DNA or RNA sequencing, or secondary cells in cell interaction, or drugs or cells in drug screening and monitoring. This feature may be used to provide easy access and flexible operation and/or mixing.

In some embodiments, the virtual nanoreactor array normalizes the amount and/or concentration of DNA input into the system and can generate feedback and/or control of the entry, which can then be used to control the amount and/or concentration of the DNA or other biomolecules or other moieties input into the system described herein. The system may further be used for the purpose of real-time normalization of detection or sequencing arrays.

An array for capturing (separating, confining or concentrating) beads or cells or other biomolecules, particles or other moieties of interest by magnetic or electromagnetic or electrical capture and/or retention may be structured to contain two sets of "capture elements" for each array element (e.g., GENIUS rods or elements of a magnetic array as described elsewhere herein) to allow one set of capture elements to capture (separate, confine or concentrate) one moiety and then a second moiety of interest to be captured (separate, confine or concentrate). The capture may be performed in a different order and with a different structure, and each array element may comprise more than two sets of capture elements, and accordingly, additional capture steps may be performed at each array element.

In some embodiments, the electrical concentration and confinement array can be used to capture charged beads to which B cells have been bound, such as magnetic beads carrying proteins, polysaccharides, or other immunogens, and wherein the beads can then be used to capture B cells. The electric concentration and confinement array can then be used to capture charged beads. In some embodiments, an electrical concentration and confinement array is used to capture B cells that can bind to proteins, polysaccharides, or other immunogens.

In some embodiments, the electrical concentration and confinement array is used to capture charged beads that have been bound to other carbohydrates or glycolipids, e.g., charged beads that can bind proteins or peptides comprising carbohydrate-binding moieties. The charged beads may then be used to capture carbohydrates or glycolipids, and wherein the electrically concentrated and confined array may then be used to further capture the charged beads. Such carbohydrate or glycolipid binding moieties may comprise carbohydrate active enzymes such as glycoside hydrolases, lectins, galectins, endolysins, penetratins, selectins, adhesins (adherenes) or hyaluronic acid.

In some embodiments, the array is used for chemical screening applications, and the system can be used to test or monitor or assay non-biological molecules. For example, in applications where drug screening is desired to determine drug effects, the system may employ, for example, about 100, 1000, 10,000, or 1,000,000 different drug candidates, each on its own bead, wherein the beads may be captured and held in a "virtual nanoreactor" array. The system may then allow for the interaction or determination of an interaction between a bead and a cell or group of cells of interest, with electrical confinement for the separation of pixels in the array, providing a high throughput and rapid drug screening system.

In some embodiments, an electrical concentration and confinement array is used to capture a set of combined charged beads to which a plurality of different types of biomolecules have been bound. The set of combined charged beads may comprise a plurality of sets of charged beads having different types of biomolecules or biological moieties bound thereto. This may be one type of binding moiety that can bind one biomolecule or biological moiety on a set of charged beads, and a different type of binding moiety that can bind a different biomolecule or biological moiety on a set of different beads. The combined set of charged beads can further comprise a plurality of sets of charged beads, wherein the set of charged beads can comprise charged beads having a plurality of binding moieties bound thereto.

In further embodiments, the combined set of beads can further comprise a label, wherein the label distinguishes between different sets of beads. The label may be an optical label such as a fluorescent dye, a biochemical label such as DNA, a metal particle label, any other type of label, or a combination of different types of labels. The label can be used to determine which type of bead can be on each element of the electrical concentration and confinement feature of the array.

In some embodiments, different detection methods of the beads and interactions resulting from various biomolecular reactions can be observed as a result of the detection of the beads. The detection may be achieved as a result of the following sensors: a nanoneedle sensor, a nanobridge sensor, a ChemFET sensor, an ISFET detector, an optical sensor such as a fluorescence detector, a SERS detector, an absorption detector, a PH detector, a conductivity detector, a mass resonance detector, a calorimeter detector, or any other type of detector suitable for detecting a biomolecular or other type of reaction. In some embodiments, the sensors may be combined at each location in an array having electrical concentration and confinement features.

In some embodiments, electrical concentration and confinement is used to directly capture charged moieties, wherein the charged moieties bind to other biomolecules or biological moieties. For example, antibodies or antibodies that bind to proteins can be concentrated or limited, where the antibodies or antibodies that bind to proteins can then be used to capture proteins or other antibodies.

In some embodiments, a "virtual nanoreactor" array increases the reaction rate by concentrating biomolecules of interest, such as DNA, RNA, or other reactants or reagents for more efficient synthesis.

In some embodiments, it is desirable to integrate a valving system as part of the flow cell. The valving system enables a sample to flow to portions of the flow cell such that different samples are available for different portions of the flow cell. In other embodiments, the valving system is integrated adjacent to the flow cell, whereby the valving system and the flow cell can form a sealed interface with each other. In other embodiments, the valving system and the flow cell may be adjacent to each other on a single base (mount) to which the valving system and the flow cell may be mounted. The valving system may also include a blowdown valve such that fluid may be removed from the valving system prior to flowing into the portion of the flow cell. For example, if there is a significant dead volume in the valving system, it may be desirable to remove fluids that may have unacceptable levels of cross-contamination with previous fluids.

In some embodiments, it may be desirable to integrate a valving device and a flow cell. Such a valving configuration can include a variety of inputs, which can include inputs for four dntps (e.g., for sequencing reactions), which can also include buffers, salts, enzymes, and any other moieties required for nucleotide incorporation. The following reagent inputs may also be used: a variety of buffers and wash reagents, buffers containing polymerases (which may also contain salts and any other moieties required for polymerization), reagents required to strip any coatings from the flow cell, reagents that may be required to recoat the flow cell, buffers further including phosphatases, or other reagents.

In one embodiment, the valving device is fabricated from PDMS. In another embodiment, the valving device is made of glass, with magnetically or pneumatically activated elastomeric valves.

In some embodiments, it may be desirable to bond the valving and fluidic PDMS manifolds to the silicon device. It may be desirable to increase the bond strength between the PDMS and the silicon device.

In one embodiment of the present invention, it may be desirable to use plasma activated PDMS to improve the bond strength. Since plasma treatment with too much power or too much pressure can actually reduce the bond strength of PDMS to silicon, it may be important to use lower power levels and pressures. Suitable power and pressure levels are described by Tang et al in 2006j.phys.: conf.ser.34155. In one embodiment, it is suitable to use a pressure of 500 millitorr (mil Torr) to 30 milliTorr and a power level of 10-60 Watts, while using, for example, a 790 series Plasma-Therm.

For devices made of PDMS or other similar materials, a pressure valve may be used to control the flow of reagents. With this type of valve, it is possible to have several valves very close to each other and these valves may be very close to the central channel, reducing dead volume, as shown in fig. 4A, which shows a reagent valve system 400 with three reagent input lines 402 and valves 406, each of which may be configured to flow to an input of a flow cell 408 under the control of a pressure control line 404.

For more complex systems where more reagent input is required, the simple valve system 400 of fig. 4A is not sufficient because it has only three reagent input lines 402. In an alternative embodiment as shown in fig. 4B and 4C, more inputs can be used. This method also allows for the clearance of dead volume from the channel. In fig. 4B, the inputs include input ports for dATP, dTTP, dCTP, dGTP, buffer 1, buffer 2, and sample, and output ports for waste 1, waste 2, and waste 3. Control lines are located at appropriate locations for each of the input and output ports, with additional control lines to control the direction of flow between activated ports. The waste port is shown immediately before the flow cell so that any remaining reagent from the previous flow can be removed, allowing a very clean transition from one reagent to another without diffusion from any dead volume in the valving system. Fig. 4C depicts a valving system with an elliptical flow path such that all input valve port positions have paths from the input valve port position to the outlet (waste) port in both directions. The valves shown in fig. 4A may be used for each of the valves shown in fig. 4B, 4C, or in the physical embodiment of the reagent valving system 430 shown in fig. 4D, where a photograph of the PDMS valve system 440 is shown.

One embodiment allows for purging of each input line up to each input port control valve of air or other contamination so that when each input port is activated, the appropriate reagent can be introduced into the system. For example, to purge the dATP line, the dATP control line and the waste 1 overhead control line can be activated, allowing air and any undesired reagents in the dATP line to flow through the dATP valve and out of waste 1. In other embodiments, contaminants in the dCTP, dTTP and dGTP lines may be cleaned or cleared by activating the dCTP control line and the waste 1 above control line, the dTTP and waste 1 above control line, and the dGTP control line and the waste 1 below control line, respectively. To clear or remove contaminants from the sample line, the sample control line and the waste 2 control line may be activated.

After replacing the sequencing assembly, contaminants may need to be purged or removed from all lines. Also, it may be desirable to clean or remove contaminants from the reagent line after replacement or refilling of the reagent bottle or container. Further cleaning or removal of contaminants may be required over time without instrumentation, which may subject any reagent lines containing reagents that need to be cooled below ambient temperatures to degradation; for example, a polymerase in a reagent containing the polymerase may be subjected to prolonged exposure to ambient temperature.

In some embodiments, it is desirable to fill the manifold leading to the input of the flow cell so that any reagents remaining from previous use of the manifold can be removed. For example, the dATP control line, the upper liquid control line, and the waste 2 control line can be activated prior to introduction of the dATP into the flow cell. Then dATP reagent will flow from the dATP input line, around both sides of the upper liquid loop, through the channel between the upper liquid zone and the lower liquid zone, and out through the waste 2 valve into the waste 2 line. Alternatively, the dCTP control line, the overhead liquid control line, and the waste 2 control line can be activated prior to introducing dCTP into the flow cell. The dCTP reagent will then flow from the dCTP input line, around both sides of the upper liquid loop, through the channel between the upper and lower liquid zones, and out through the waste 2 valve into the waste 2 line. Likewise, the dTTP control line, the lower liquid control line, and the waste 2 control line may be activated prior to introduction of the dTTP into the flow cell. The dTTP reagent will then flow from the dTTP input line, around both sides of the lower liquid circuit, through the channel between the lower liquid zone and the lower liquid zone, and out through the waste 2 valve into the waste 2 line. Also, the dGTP control line, the lower liquid control line, and the waste 2 control line may be activated prior to introducing dGTP into the flow cell. The dGTP reagent will then flow from the dGTP input line, around both sides of the lower fluid circuit, through the channel between the lower fluid zone and the lower fluid zone, and out through the waste 2 valve into the waste 2 line.

Buffer 1 can be flowed through the main flow cell (dark blue) and out the waste 3 port by activating the B1C control line and the W3C control line. Alternatively, buffer 1 can be flowed out of the waste 2 port by activating the B1C control line and the W2C control line. In another alternative, the buffer 1 can be flowed out of the waste 1 line through the upper portion of the fluid manifold by activating the buffer 1 control line, the upper fluid control line, and the waste 1 upper control line. Likewise, the lower fluid manifold can be flushed with buffer 1 by activating the buffer 1 control line, the lower fluid control line, and the waste 1 lower control line. Activation of the flow in these combinations of zones in a time sequential manner or together enables decontamination of the entire liquid manifold as a wash or purge of air bubbles, other contaminants, or any other liquid that may have been introduced into the system via dATP, dTTP, dCTP, dGTP, buffer 2, or sample input ports.

In some embodiments, it is desirable to use a passivation layer on a silicon device having a higher bond strength than thermally grown silicon dioxide. Tang et al describe several passivation layers that provide improved bond strength, including PSG (PECVD phosphosilicate glass), USG (PECVD undoped silicate glass), Si ₃N₄(LPCVD silicon nitride).

In some systems using PDMS valving manifolds, a pin or needle inserted into the PDMS is used to connect the reagent lines to the valving manifold. While this provides a secure connection, connecting many reagent lines to the PDMS valved manifold is time consuming and error prone. Thus, in some embodiments, it may be desirable to use an interface manifold, wherein the reagent lines are connected to the interface manifold, rather than to the valving manifold, and the interface manifold may be connected to the valving manifold. The reagent line may be attached to a pin or needle that may be attached to the interface manifold. The pins or needles may be permanently attached to the interface manifold, secured in place with an adhesive, by welding or brazing, by using a press fit, or by some other means. Alternatively, the lines may be connected directly to the interface manifold, they may be retained on the interface manifold by fittings or O-rings, or by some other means known in the art.

In some embodiments, the interface manifold may be sealingly engaged to the valving manifold such that reagent may flow from the interface block into the valving manifold. The interface between the interface manifold and the valving manifold may be an interface used by a user to enable replacement of the chip/flow cell and/or the valving manifold.

In some embodiments, the interface manifold may have internal channels formed by bonding, which may include melt bonding, solvent bonding, or adhesive bonding.

In some embodiments, minimizing the path length to the active portion of the flow cell may be important for several reasons, including minimizing the amount of mixing of reagents, which occurs due to wall interactions and diffusion induced differences in flow rates at the center of the channel and at the edges of the channel. In some embodiments, it may also be desirable to minimize the temperature-independent volume in order to prevent degradation of reagents such as polymerases in the temperature-independent volume. In some embodiments, it may be desirable to minimize the tubing volume to simultaneously minimize cross-contamination of reagents that may also occur in a flow region common to multiple reagents due to non-specific binding to materials contacting the reagents.

FIG. 1 illustrates a schematic of one embodiment of the present invention in which magnetic or paramagnetic beads are held in place on a sensing region by a magnetic array. The magnetic array is described in U.S. provisional application 61/389,484 entitled "magnetic arrays for Emulsion-Free Polynucleotide Amplification and Sequencing," which is incorporated by reference herein in its entirety. The retained magnetic or paramagnetic beads may have a monoclonal population of DNA. The beads may be sized so that there is sufficient space for only one bead on each sensor, thus providing a one-to-one correspondence between sensors and beads. While there may be space for only one bead per sensor, there may be space between beads when the beads are aligned on the beads, resulting in reduced cross talk between sensors. For example, a set of beads may be about 10 microns in diameter, positioned on a sensor spanning about8microns (over sensors white area about8 microns), and the sensors may be spaced about 15 microns apart, resulting in a spacing of about 5 microns between the beads. The sensor may be larger in size than the beads if there is insufficient space to hold both beads on the sensor. The size of the beads, sensors and spacing may vary. In other embodiments, the beads can be greater than 10 microns in size, such as about 15 microns, about 20 microns, about 25 microns, or greater. In further embodiments, the beads may be less than 10 microns, such as about 5 microns, about 3 microns, about 2 microns, about 1 micron, or less than 1 micron. The size of the sensor may be set to align with the size of the beads and thus the size may span greater than or less than 8microns, possibly from less than 1 micron to about 1, 2, 3, 5, 10, 15, 20 or more microns. The spacing between sensors may also be greater than 15 microns, or may be less than 15 microns; the sensor spacing may range from less than 1 micron between sensors to about 1, 2, 3, 5, 10, 15, 20, 25 or more microns.

The chamberless magnetic retention structure as shown in fig. 1 may allow improved flow of nucleotides, polymerases, and other components as their flow is not impeded by the pore structure, as shown in this fig. 2, allowing for better washing, more complete incorporation of bases, and possibly faster cycle times if beads are located in the pores. In the pore structure, the beads and associated DNA as shown in fig. 2 hinder accessibility and flow, and thus higher concentrations of polymerase and nucleotides may be required to allow sufficient diffusion to all parts of the beads as shown in fig. 2. Due to misincorporation by the polymerase, the higher concentrations of dntps and polymerase can increase the error rate, resulting in a higher level of lead sequencing phase error than would occur using the chamber-less configuration shown in figure 1.

In some embodiments, the arrays described herein are reusable (e.g., not disposable). The sequencing cost has multiple components; for sequencing using electronic sensors, one of the major costs is the cost of the processed silicon itself, i.e. the sensor. This may be particularly true if the sensor is not reusable, but must be discarded after a single use. The magnetic array described above makes reuse rather simple, since the DNA does not bind to the sensor, and the beads can be easily removed by reducing or removing the magnetic field that holds the beads in place. Removal may be more difficult if a structure is used to hold the beads in place.

In one embodiment, the beads are held in place by a structure or array of wells and removed by applying a magnetic field such that the beads (which may be magnetic or paramagnetic beads) are drawn out of the wells and subsequently removed from the flow cell by flowing the reagents through the flow cell.

In some embodiments, the array of magnetic features is used for uses other than nucleic acid capture/isolation as otherwise described herein for sequencing and amplification of said nucleic acids. These uses include the capture of cells (e.g., cancer cells, B-cells), proteins, glycoproteins, glycolipids, antibodies, sugars or polysaccharides, and other moieties as already described.

In some embodiments, the relevant cells bound to the retained beads are lysed while the beads are held in place in the magnetic array. The retained beads may further comprise additional primers or primer sets for amplifying and/or sequencing the target nucleotide sequence. The primers may be universal primers, or primers that target specific sequences associated with the cell type bound to each bead, or may be universal primers or universal primer sets with barcodes. For example, the barcode is associated with the cell type bound to each bead, or is a combination of different primer types. In some embodiments, after cell lysis, reverse transcription and/or amplification reactions may be performed. In some embodiments, the amplification is a real-time PCR reaction, wherein the amount of a particular RNA can be determined for each bead, and thus for each cell type. In other embodiments, the amplification reaction is a PCR reaction or an isothermal reaction, and a clonal population can be generated on the beads, or a polyclonal population can be generated, wherein different primers can be used for each clonal type. In other embodiments, a sequencing-by-synthesis reaction is subsequently performed to determine the sequence of the amplified sequences associated with each bead type, and thus each cell type.

In further embodiments, after cell lysis, DNA, RNA, or other molecules having a particular charge may be retained, and beads may be removed. In some embodiments, additional beads may be introduced into the magnetic array. As described herein, the newly introduced beads can have a different primer type associated with the newly introduced beads.

In some embodiments, the array of magnetic features may be configured such that a preferred position is maintained by magnetic or paramagnetic beads or particles. Such a preferred position may be required in order to properly position the particle with respect to the one or more sensors and/or to keep the particle in a fixed position so that the particle and the charge attached to the particle do not move with respect to the one or more sensors. In some embodiments, as shown in FIG. 6, the preferred position may be caused, at least in part, by the configuration of the shape of the magnetic array elements. Fig. 6 illustrates two different configurations 600 in which at least some of the shapes in the array may be configured such that the density of the magnetic flux is more concentrated at one end of a member of the magnetic array (relative to the other end of some other member of the magnetic array). In the top two pairs of magnetic elements, the south pole of the left trapezoidal magnetic array element 604 is narrower than the north pole of the trapezoidal magnetic array element. Since the total flux level emanating from the magnetic array elements must be the same at both ends, the flux density at the narrower end of trapezoidal magnetic array element 604 will be higher than the wider end of the trapezoidal magnetic array element. Since a higher concentration of flux corresponds to a higher force applied to the magnetic or paramagnetic elements, the force exerted by the narrower end of the trapezoidal magnetic array element 604 on the magnetic or paramagnetic particles or beads 602 will be higher than the force exerted by the similarly sized magnetic array element 606 on the right side, where the elements on the magnetic array element 606 on the right side are similarly sized, but rectangular, and thus have a lower flux density and force. Likewise, the force exerted by the narrower end of the trapezoidal magnetic array element 604 on the magnetic or paramagnetic particle or bead 602 will be higher than the force exerted by the similarly sized trapezoidal magnetic array element 608 on the right side, with the element on the magnetic array element 608 on the right side facing the magnetic or paramagnetic particle or bead 602 with its wider end.

In another embodiment of the invention, additional features may be introduced as part of the fabrication of the magnetic array, thereby inhibiting the movement of magnetic or paramagnetic particles or beads 702 as shown in fig. 7. In the embodiment shown, the magnetic or paramagnetic particles or beads 702 are preferentially pulled toward the narrow end of the left trapezoidal magnetic array element 704 and into contact with two posts 710, and downward into contact with the array surface, thereby providing three contact points to sufficiently stabilize the magnetic or paramagnetic particles or beads 702. The pillars and magnetic array elements may provide a minimum surface contact area to allow maximum access of ions, dNTP enzymes and other moieties. The pillars and magnetic array members can be positioned with tight tolerances with respect to the sensor elements to provide reproducible signal levels between different members of the sensor array.

In a further embodiment, small holes are used so that magnetic or paramagnetic particles can rest in the upper corners of the holes. The well may be circular or, if the shape of the magnetic or paramagnetic structure is generally spherical, some shape other than circular, in order to allow better access of enzymes, dntps, ions and other moieties to the bottom of the magnetic or paramagnetic particle.

In some embodiments, magnetic arrays are used to generate clonal populations for hybridization detection, hybridization pullout, or sequencing. The assay may be performed with beads on the magnetic array at the location where amplification occurs, or the beads may be moved from the region or volume where the amplification reaction occurs to another location. The second location may also be magnetically immobilized to perform the assay, or may be immobilized differently, such as biotin streptavidin binding. In some embodiments, the sensor is positioned directly under the magnetic or paramagnetic particles to maximize the interaction between the charges associated with the beads and the DNA on the sensor. In other embodiments, the beads may be bound or associated in such a way that they are immobilized and not free to rotate. It may be desirable to locate the sensor off-center of the paramagnet, allowing access to a particle region that allows free access to the aqueous environment and to polymerase, dntps and other moieties, and may have an optimal enzymatic reaction that can then be read by the sensor, as compared to a region in direct contact with the surface where reactions may be inhibited due to the inability to access the aqueous environment.

In some embodiments, it is desirable to use the differential flow that occurs in the channel to rotate the magnetic or paramagnetic particles, thereby providing optimal access of the enzyme, dntps, and ions to all surfaces of the magnetic or paramagnetic particles. The differential flow is caused by a parabolic flow characteristic of small channels, where the volume flow rate (bulk flow rate) at the surface of the channel is zero and the maximum flow rate in the channel is usually highest in the center of the channel. This may result in a significant flow velocity difference between the bottom of the channel and the top of the magnetic or paramagnetic particle. The difference in flow rate between the top and bottom of the magnetic or paramagnetic particles will be a function of the particle size, the height and width of the channel, and the average flow velocity in the channel. DNA and/or other moieties that may be attached to magnetic or paramagnetic particles may provide drag or pull on top of the magnetic or paramagnetic particles due to the relatively high flow rate on top of the magnetic or paramagnetic particles. Instead, the flow rate at the bottom will remain substantially zero, thereby generating significant rotational power. Magnetic array elements, potentially in combination with other physical features, may maintain the position of the magnetic or paramagnetic particles.

In some embodiments, the flow rate is maintained at a constant flow rate while dNTPs and/or other reagents are introduced and flowed and while the sensor is read, thereby maintaining a constant average rotational speed. In other embodiments, it is desirable to reduce the flow rate while reading the sensor to prevent significant vibration and movement of the magnetic or paramagnetic particles. In still other embodiments, it may be desirable to increase the flow rate while flowing the reagent through the flow cell when reading the sensor in order to allow a larger surface area of the magnetic or paramagnetic particles to interact with the sensor. This may result in an enhanced averaging effect, which may reduce variations in sensor readings due to variations in DNA attachment density on the surface of magnetic or paramagnetic particles, or due to irregularities in particle shape.

In other embodiments, forces other than those resulting from changes in flow rate may be used to alter the rate of rotation or movement of the magnetic or paramagnetic particles. In some embodiments, the flux level may change due to movement of an external magnet that may be coupled through the magnetic array elements due to the higher permeability of the magnetic array elements. In an alternative embodiment, electromagnets are utilized to affect the amount of flux that interacts with the particles. These changes in the amount of magnetic flux can reduce the friction forces acting on the magnetic or paramagnetic particles, allowing more or less rotation.

In yet other embodiments, a magnetic or electric field is used to rotate the magnetic or paramagnetic particles, in part due to the polarizability of the beads and associated DNA.

In some embodiments, in addition to the force caused by the flow of reagents through the flow cell, a force is required to position the magnetic or paramagnetic particles in the appropriate locations in one-to-one correspondence with the sensors. In some embodiments, magnetic or paramagnetic particles may be flowed in a reagent stream into one or more flow cells and moved into a position relative to a sensor using magnets or electromagnets such that a higher proportion of the magnetic or paramagnetic particles are associated with the sensor in a one-to-one correspondence than would occur without the use of these additional magnets or electromagnets.

The magnetic array also allows for nearly complete distribution of beads to the array location. The low velocity flow is sufficient to allow the beads to be retained locally in the array in a one-to-one correspondence between beads and array positions without centrifugation. In one embodiment, if a higher level of packed sites than unfilled sites on the array is needed or desired, the reagent stream can be circulated so that the beads can be reintroduced into the flow cell. In another embodiment, the reagent flow may be stopped or slowed as the beads are introduced into the flow cell. In another embodiment, the direction of reagent flow may be reversed (possibly several times) to provide the beads with more opportunity to fill the array. In yet another embodiment, the beads are retained after flowing the beads into the flow cell by flowing the beads through an inlet or outlet of a storage location, thereby making them available for subsequent sequencing processes. To prevent the beads from sticking to locations other than the target bead locations, the flow may be increased to remove any weakly retained beads while still retaining properly retained beads. After completion of a chemical process such as sequencing or amplification, the beads can be removed by decreasing the retention field flux, by increasing the new field that pulls the beads away from the array, by increasing the flow rate of the fluid, by using an air-water interface associated with the bubbles, which can include surface tension, or any combination of the above steps.

In certain embodiments, the charged beads are retained with an array of electrodes using a DC field or a dielectrophoretic field, or both, as shown in fig. 3. Due to the use of a magnetic array, no pore structure is required to retain the beads, allowing free flow of the components in solution. To ensure that charged components in solution, such as DNA samples, nucleotides, enzymes and other charged moieties can flow easily through the volume on the array, a vibration frequency sufficient to retain the charged beads is used, but slow enough to allow these moieties in solution to flow or diffuse away from the retained beads. The addition of recesses associated with the sensors in a one-to-one correspondence may result in better alignment between the beads and the sensors, allowing for better detection. In alternative embodiments, pedestals (pedestals) or alignment posts (registration posts) or other three-dimensional structures are used, for example, in order that better fluid flow may be introduced.

In alternative embodiments where the beads may be positioned in one-to-one correspondence with the array, the beads may be brought into position by a magnetic or electric field and may subsequently be held in place by alternative methods such as DNA hybridization, biotin streptavidin binding, thiol binding, light activated binding, covalent binding, and the like. Binding can be initiated by a change in temperature, application of light, or by washing in a binding reagent or catalyst while the beads are held in a one-to-one correspondence by the magnetic or electric field. After binding occurs, the magnetic or electric field strength may be changed in intensity or frequency, possibly being turned off. This binding may be reversible, allowing the beads to wash out of the volume on the sensor array.

In some embodiments, the magnetic or paramagnetic particles may have a surface coating thereon having sufficient porosity to provide access therethrough for a polymerase or other enzyme as well as sample DNA, dNTP ions and other moieties. The coating may be configured such that the primers may be attached at appropriate intervals and may thereby provide a greater density of sample DNA and hence a greater charge density for interaction with the sensor located there. The coating may be a coating of agarose, polyacrylamide or other cross-linked polymer, or may be made of porous glass.

In other embodiments, the beads may be provided with a coating to minimize or reduce non-specific binding of DNA, protein, or other charged moieties relative to the amount of non-specific binding that may result when the DNA, protein, or other charged moieties interact with the beads without the surface coating. The coating may be similar to the coatings described herein for the surfaces of the flow cell, sensor, enrichment module, or magnetic array, and any of the coatings described herein for one surface may be used on the other surface.

In some embodiments, the beads have a magnetic core, which may have an impermeable coating thereon. The coating may bind, link or associate with multiple strands of DNA. For example, each DNA strand may be substantially the same rolling circle amplicon, thereby providing multiple strands of DNA, each having multiple consecutive copies of a DNA target.

Fig. 8 illustrates one embodiment of an alternative method and system for retaining beads in a one-to-one correspondence with sensors in an array. Fig. 8 illustrates a system 800 whereby a separate control line 810 is activated and thus a layer of structure 812 continuously expands into the flow cell volume 806 between the matrix 804 and the fluidic structure 802, forcing the beads 808 to disengage from more than a level one-to-one corresponding to the sensors by moving excess beads 808 upon activation of the control line 810. The beads 808 can then be fixed in place during the sequencing cycle. It can be seen in fig. 8 that there is sufficient space for the liquid to flow under the control line, but not enough space for the beads to move. When a set of sequencing cycles has been completed, the control line can be deactivated, and the beads can then be removed by fluid flow through the sensor array region 808.

In alternative embodiments, the number of beads may be lower than the number of sensors. The number of beads may be close to the number of sensors, with the control line activated to position the beads with the sensors. The positioning may be assisted by alternating the flow direction, introducing vibrations or oscillations or the like in the sensor array region, such that the beads undergo frequent movement until movement is prevented by a sufficiently activated control line which is too low to enable the beads to move from one sensor region to another. Further movement of the beads in combination with further activation of the control line will help to concentrate the beads more completely on the sensor.

In a further alternative embodiment, a structure having a shape similar to that shown in fig. 8 may be molded, machined, or otherwise formed such that the shape is similar to that which would occur when the control wire is fully activated. The structure can be slowly lowered relative to the bead covered sensor array.

In yet another embodiment, in order for a higher percentage of sensors to have associated beads, the beads may be attached to the sensors by biotin streptavidin binding, thiol binding, or the like, after a set of beads is positioned by one of the structures described above. Additional beads can then be introduced into the sensor array region and the process repeated. If the binding agent is located in the area above the sensor, as done previously, any excess beads captured or squeezed by the structure will not bind and can be washed away before starting the sequencing cycle.

In yet another embodiment, a significant excess of beads can be introduced into the sensor array region. A single control line at the exit of the sensor array region can be activated to capture the set of beads. The aqueous conditions and/or temperature may be varied to allow the beads to bind to the sensor. Excess beads can then be removed by releasing the control line, allowing the beads to flow out of the sensor array area with the aqueous reagent.

Some embodiments combine pH sensing with electrochemical detection due to the introduction of a reversibly reducing layer that can be fabricated based on previous sensor designs. Such sensors are available from SenovaSystems. During the sequencing cycle, if a base has been incorporated into the bead associated with the sensor, a reduction reaction will occur. The reduced level can be measured and after the sequencing cycle is completed, a voltage can be applied to the sensor, causing the surface to oxidize, returning it to its original state, at which time it can be used for the next sequencing cycle.

In some embodiments, magnetic beads are used without a magnetic array. The beads self-assemble into monolayers at uniform spacing, which spacing can be affected by using an external magnet to change the local field strength. The magnetic beads may be spaced at a pitch that matches the pitch of the sensor array and may then be bound to the sensor array by changing aqueous conditions, temperature, etc., as previously described. In order to enable alignment of the beads with the sensor, a slow translation or movement of the beads after binding may be appropriate. Such translation or movement may need to occur in multiple dimensions (which may include X, Y, θ) and spacing. The addition of recesses associated with the sensors in a one-to-one correspondence may result in better alignment between the beads and the sensors, allowing better detection.

Fig. 11 illustrates different embodiments in which magnetic, paramagnetic, non-magnetic particles may have shapes other than spherical for use in sensor arrays with magnetic retention (1102), sensor arrays with electrical confinement, or sensor arrays with self-assembled particles. The particles may be planar, circular, rectangular (1104), star-shaped, hexagonal (1106), or in another shape. In other embodiments, the particles may be dendritic, thereby enlarging the surface area of the particles. The dendritic particles may be generally spherical, planar, elliptical, or any other shape. In yet other embodiments, the particles may be porous; if the particle is porous, the pore size may be of sufficient size to allow DNA, polymerase, dntps and other moieties necessary for primer extension sequencing or other applications to move freely as appropriate.

In another embodiment, a magnetic rod array is not required and the beads can be replaced with DNA spheres produced by using rolling circle amplification. The DNA spheres can then be digested by DNA nucleases. In another embodiment, the spheres may be made of monomers or polymers such as polystyrene, which can be dissolved using an organic solvent such as acetone after sequencing, thereby releasing the attached DNA by the same process, both of which can then be removed from the flow cell by flowing reagents through the flow cell.

In yet another embodiment, the bead may be fixed in place by a bond between the moiety attached to the well and the moiety attached to the bead. The well may be shorter than the radius of the bead and may have a shape other than circular so that the reagent may flow around the bead, and the bead may be bound at several points at the entrance of the well. The binding may be streptavidin biotin binding, DNA binding, DNA PNA binding, PNA binding, thiol Au binding, photoactivated binding, covalent binding, or the like. The binding may be released by raising the temperature or by introducing an agent that reduces the affinity between the moieties on the wells and the moieties on the beads, thereby allowing the beads to be removed from the flow cell by flowing the agent through the flow cell. The beads can be further induced to move out of the wells to which they have been bound by sonication of the beads and the well structure. To remove the beads from the flow cell, sonication can be performed while the reagent is flowing through the flow cell.

In amplifying DNA in a chamberless system as described in provisional application 61/491081, various factors may be optimized. This includes, among other things, frequency, voltage, and the size and shape of the restriction "pool" used to restrict the polymerase, target DNA, and amplicons produced. If only the restriction is considered, it will be possible to limit almost any size of amplicon, regardless of the small size of the amplicon. However, in order to have a field strong enough to ensure proper confinement, the field may prevent proper activity of the polymerase to incorporate bases during PCR or isothermal amplification, or the polymerase and/or extended primer may be withdrawn from the complex of the primer extended from the target DNA and the polymerase. In one embodiment, it is desirable to optimize the combination of frequency, voltage, and size of the limiting pool according to the size of the amplicon.

In some embodiments, the amplification reaction is reverse PCR amplification, hot start PCR amplification, methylation specific PCR amplification (MSP), nested PCR amplification, reverse transcription reaction, reverse transcription PCR amplification (RT-PCR), descending PCR amplification, inter-sequence specific PCR amplification (ISSR-PCR), co-amplification at lower denaturation temperatures (COLD-PCR), solid phase amplification, bridge PCR amplification, or single primer bridge amplification.

In some embodiments, the amplification reaction is Helicase Dependent Amplification (HDA), nickase amplification (NEAR), recombinase polymerase Reaction (RPA), Transcription Mediated Amplification (TMA), self-sustained sequence replication (3SR), nucleic acid based amplification (NASBA), Signal Mediated Amplification of RNA Technology (SMART), loop mediated isothermal amplification of DNA (LAMP), Isothermal Multiple Displacement Amplification (IMDA), solid phase isothermal, bridge isothermal, Single Primer Isothermal Amplification (SPIA), circular helicase dependent amplification (cHDA), or rolling circle amplification.

In generating a DC field for electrophoretic concentration or confinement, electrolysis products can be constructed. These include hydronium ions and hydroxide ions. To minimize the effect of these ions, the DC field can be pulsed (pulsed) so that the net DC is fairly low. In some embodiments, the pulse duty cycle (pulse duty cycle) may be reduced after the DNA migrates closer to the center electrode. In other embodiments, the process may use DC to concentrate DNA, and then AC to maintain the concentration or confinement of the electrolysis products. In other embodiments, both DC and AC may be used for concentration and limiting.

In some embodiments, pulsed field gel electrophoresis is used. For example, a non-sinusoidal AC waveform may be used, where a higher positive voltage may be balanced by a longer but shorter (voltage but short) negative voltage to make the average voltage substantially zero. A higher positive voltage can be used for polymer concentration so that replication of DNA occurs in the polymer solution. The polymer solution may effectively cause a lower migration of DNA to the direction of the lower field than in the higher strength electric field, which may thereby increase the mobility of DNA. In this way, due to the balanced nature of the AC waveform, DNA can migrate more in the desired direction, while other molecules, such as Mg, move freely back and forth. The difference in migration may also be frequency dependent, thus allowing capture of DNA of different sizes. Pulsed field gel electrophoresis may be 1D or 2D and may use clamped electric field, transverse alternating field electrophoresis or spin gel electrophoresis, either of which may use a gel, an entangled polymer or another sieving matrix.

In generating the dielectrophoretic field, a sinusoidal waveform is typically used. While this may be ideal for applications strictly for confining or separating different species, it may lead to problems with systems where biochemical reactions can be performed within a confined volume. For example, high fields may cause localized heating. In one embodiment, a modified sinusoidal waveform may be used instead. For example, the modified sinusoidal waveform may remove voltage at the top of the sinusoidal curve or at any other point of the sinusoidal waveform, thereby allowing local diffusion, allowing hybridization of amplicons to primers, binding of polymerases to double stranded DNA, and binding and incorporation of nucleotides or nucleotide analogs. The field may then be restored after an appropriate period of time. The same process may occur at opposite sign peaks according to the modified sinusoidal waveform. In other embodiments, any other alternating current waveform may be used to focus or limit. Alternatively, the interruption of the sinusoidal waveform may occur only once per cycle, or may occur once every few cycles, or once over multiple cycles, so that any "scattered" amplicons may be captured to the region of lowest field strength and returned to the main volume of the confined volume. Alternatively, other waveforms such as square, trapezoidal, asymmetric, etc. may be used.

In some embodiments, monoclonal beads can be produced in a small microfluidic device. In one embodiment, the electrodes and magnets may be fabricated on a sheet, wherein the upper surface may be bonded or fused in place, thereby creating an integrated microfluidic device. The microfluidic device may have electrical and fluidic connections thereon. The microfluidic device can then be placed in good thermal contact with the first heated plate, for example by vacuum or air pressure. The second plate may be located above at a different temperature. Two temperatures may be selected to facilitate PCR amplification. After one temperature point is completed, the card may be transferred or brought into contact with an upper heated plate, for example by vacuum or air pressure. Because only the thin card and the reagents in the card need to change temperature, the system can have a fast temperature transition and consume minimal power.

In other embodiments that provide minimal thermal mass, electrodes built into the amplification microfluidic device may be used as resistive heaters to locally heat a liquid. In some embodiments, the change in resistance of the electrodes is used to measure temperature for better thermal control. In other embodiments, sensors such as nanobridge or nanoneedle (described herein) are used as temperature sensors to better control the region of interest.

In some embodiments, it may be desirable to perform the process from a single copy of DNAAnd (3) DNA amplification. If a polymerase error is generated during an early amplification of the amplification process, such as during the first cycle of a PCR amplification, the error will proliferate, making it possible that the correct and incorrect sequences cannot be distinguished. Thermostable polymerases generally have a much higher error rate than mesophilic polymerases or thermophilic polymerases, which may not be suitable for PCR because they are inactivated during the denaturation step of PCR. Thus, in some embodiments, the initial portion of the PCR reaction requires the use of a highly precise polymerase, where the highly precise polymerase does not have sufficient thermostability to prevent inactivation during the PCR process, but can provide better accuracy than a more thermostable polymerase. Highly accurate polymerases may have a low K_offSuch that the highly accurate polymerase substantially binds to the active extension site in the presence of other polymerases that may be sufficiently stable to prevent significant inactivation during the denaturation step of the PCR amplification.

In some embodiments, a highly accurate polymerase is introduced into the volume with the primer and template prior to introducing the more thermostable polymerase. In other embodiments, the heating activates the thermostable polymerase such that any heat-activated polymerase will be inactive for the first cycle of PCR.

In other embodiments, a combination of isothermal PCR amplification reactions is used. Initial amplification may be performed by a highly accurate non-thermostable polymerase, while subsequent amplification may be performed by a less accurate thermostable polymerase that is not substantially inactivated by the denaturation step of PCR.

In an alternative embodiment, clonal populations are generated in the area of individual sensors in a sensor array. The sensor may be a nanoneedle or a nanobridge or other sensor for detecting a polymerization event. In one embodiment, the primer is preferentially attached to the surface of the sensor. Due to the difference in materials, the primers may preferentially attach, wherein the material of the sensors is more favorable for attachment than the regions between the sensors of the sensor array. In an alternative embodiment, a mask may be applied to the area between the sensors of the sensor array, and then surface modification may be performed. Subsequently, the mask may be removed; leaving areas between the sensors of the sensor array that have not been surface modified. Surface modification may include attachment of biotin, application of a gold layer, and various other methods known in the art.

Primers can then be preferentially applied to regions on the surface of the sensors in the sensor array. In one embodiment, primer ligation results from biotin streptavidin binding, where streptavidin is attached to the 5' end of the primer. In another embodiment, a thiol group may be attached to the 5 'end of the primer, and then the 5' end of the primer may be bonded to a gold layer previously applied on the sensor, thereby forming an Au — S bond. If a PCR reaction is desired, the primer may be modified with DTPA such that 2 thiol-gold bonds are formed, thereby preventing decomposition that might otherwise occur at the temperatures of 60-95 ℃ commonly used in PCR.

After completion of one set of sequencing cycles, the primers were removed and replaced. Buffer conditions can be varied to weaken the biotin streptavidin bond, such as high concentrations of GuHCl at low pH; alternatively, the temperature may be increased above 70 ℃ to break the biotin streptavidin bonds. It is likewise possible to cleave thiol bonds at elevated temperatures. Aggressive means can be used as damage to the polymerase and DNA is no longer important. In one embodiment, an organic reagent is used to break the association, such as covalent binding, between the extended primer and the surface. After removal of the extended primers, new primers can be flowed into the volume above the sensor, thereby enabling the device to be reused for another set of sequencing cycles on another set of DNA samples.

In some embodiments as schematically depicted in fig. 12, the sensor array may have elements 1200 provided with an additional array of electrodes 1206, which may be used for dielectrophoretic concentration. Dielectrophoretic concentration may be performed initially to attract sample DNA, dntps and primers to each sensor region. Amplification can then begin at the region of each sensor where the DNA sample is located. Dielectrophoretic forces may also help to prevent cross-contamination between different sensor regions undergoing amplification due to retention of amplicons during amplification. To ensure that no polyclonal regions are generated, the concentration of input DNA needs to be low enough that most sensor regions have one or zero sample DNA molecules. The DNA sample may be single-stranded or double-stranded, depending on the amplification method. The amplification reaction may be a PCR reaction or an isothermal reaction. In some embodiments, the additional electrode 1206 is shown to have the same voltage relative to the voltage level of the sensor. In an alternative embodiment, the electrodes on either side of the sensor may have voltages of opposite sign relative to each other.

The sensor array element 1200 may be fabricated on a substrate 1212 and may have a magnet 1216 for retaining magnetic or paramagnetic particles or beads 1202, which may be held down against the electrode 1204, against the dielectric 1210 and/or upper electrode 1208. Detection may utilize the electrode 1204 and the upper electrode 1208, while dielectrophoretic concentration/confinement may utilize the electrode 1204 and the outer electrode 1206, wherein the outer electrode may comprise a single electrode, or may comprise multiple electrodes.

The amplification may be solid phase amplification with one primer on the surface of the bead and a second primer in solution, or the amplification may be solid phase amplification with all primers on the bead. Alternatively, amplification can be performed in which both primers are present in solution and one primer or both primers are also present on the bead.

The electrode configuration may take a variety of different forms, including planar electrodes on both major planes of the flow cell, or there may be one electrode on the opposite surface of the bead, with a set of smaller electrodes associated with each detector location.

Figure 12 illustrates the use of an amplified region above a sensor in a sensor array in a sequencing reaction. After the amplification reaction is complete, the volume above the sensor array may be washed to remove amplicons, polymerases, and dntps. Polymerase and individual dntps can then be flowed into the volume above the sensor array, allowing detection of binding, incorporation, and incorporation events, thereby determining the sequence of the different amplified sample DNA molecules.

In some embodiments, the sensor is used for a variety of purposes, such as, for example, detecting the presence of a bead when introduced to the bead, detecting amplification associated with the bead (e.g., real-time PCR amplification or end-point PCR amplification), and detecting a sequencing reaction.

When producing clonal beads, a high percentage of beads will have no DNA template. In addition, others may have poor amplification. These beads do not provide useful sequencing, so it is desirable to remove these beads to improve instrument throughput and reagent utilization. In some embodiments, an electric field is used to separate beads with no or minimal template. Beads on which amplification has occurred have more fixed negative charges from the amplified DNA, and they can be separated from beads on which no amplification has occurred by using electrophoretic separation. This allows for a situation as shown in fig. 3, where most of the positions in the magnetic array are described as occupied by beads where amplification reactions have taken place, and are therefore suitable for use in sequencing reactions.

Beads that are fully loaded with template have a higher charge and will therefore move further in the electric field than beads with only primer or a small amount of template. In one embodiment as shown in fig. 13A, 13B and 13C, this separation is performed in a flow-through module. The first fluid input 1311A allows for injection of mixed beads. A second inlet 1312A allows injection of a bead-free buffer solution. The first outlet 1311B is located downstream of the first inlet 1311A. The second outlet 1312B is located downstream of the second inlet 1312A.

The flow rate of the fluid may be set according to the fluid resistance or pumping speed so that more liquid flows into the second inlet. In the embodiment shown in fig. 13B, the width of the inlet and outlet can be varied to create different fluidic resistances, but other methods of varying fluidic resistances (e.g., different lengths or heights) or the use of flow restrictors in portions of the system external to the enrichment module 300 are contemplated. Similarly, the fluidic resistance of the first outlet 1311B and the second outlet may be varied to cause more liquid to flow out of the first outlet 1311B. In such an arrangement, beads that do not have a small velocity perpendicular to the flow will exit the first outlet port 1311B. Additional output channels may be added to aid in the separation of beads with intermediate levels of template. In some embodiments, the flow rate in each output channel may be directly controlled by providing a separate pump for each outlet channel.

A pair of electrodes 1313 may be provided which are capable of generating an electric field perpendicular to the fluid flow in the separation section 1310, such that beads loaded with template (which may be brought into the enrichment module 1300 through the inlet 1311A, while additional reagents may be introduced into the module through the second input 1312A) may migrate out of the flow path towards the second outlet 1312B. Fluid port 1309 allows connection to system piping.

The electrodes 1313 may be made of any electrode material compatible with electrophoresis. In some embodiments, discrete metal lines may be used, although metal traces are also contemplated. It is contemplated that a variety of metals may be used, such as platinum, platinum/iridium, gold, and other precious metals or alloys, as well as corrosion resistant materials, such as stainless steel. The use of non-metallic electrodes is also contemplated.

In another embodiment, the flow rate of the fluid can be set by the fluid resistance or pumping speed such that more or less fluid flows out of the first outlet than the second outlet, thereby allowing manipulation of the bead flow stream. In yet another embodiment, the bead flow may be regulated by inlet and outlet fluid flow.

The flow-through enrichment module 1300 can be constructed from a non-conductive material, such as molded plastic, glass, ceramic, or moldable polymer (e.g., PDMS), or from a conductive material that can be coated with a non-conductive coating, or a combination of these materials, or with other materials. The fluidic components can be fused, bonded, or held together with a clamping mechanism to create an enrichment module that includes a separation portion 1308. In one embodiment, the enrichment module can include a molded upper piece 1308, and a flat substrate 1302. In other embodiments, the enrichment module may consist of more than two pieces, e.g., three, four, five or more components. If two assemblies are used, both sides may have non-planar surfaces so that fluid or control channels may be formed in either assembly. If multiple components are used, any of them may be planar or shaped such that they include channels, recesses, or protrusions, or may be planar and shaped such that they include a combination of channels, recesses, or protrusions.

In some embodiments, a surface or a portion of a surface of one or more components of an enrichment module has a zeta potential sufficient to cause significant electroosmotic flow. It may be desirable to minimize any mixing or turbulence that may be caused by the electroosmotic flow. In some embodiments, a material such as TiO is selected₂、ZrO₂Or BaTiO₃Such that the zeta potential and the resulting electroosmotic flow are significantly reduced. In some embodiments, the zeta potential and the relationship between zeta potential and pH change may vary depending on the surface coating. In some embodiments, the zeta potential can vary significantly with changes in pH, as is the pH dependence of silica. In other cases, the zeta potential changes are very small with respect to the pH value, in particular in the pH range from pH7.5 to pH9, BaTiO₃This is the case.

In other embodiments, surface coatings such as PEG (polyethylene glycol), methylcellulose, n-dodecyl-B-D-maltoside, acrylamide, fluorinated alkane chains, PTFE, acrylates, or other crosslinked or partially crosslinked polymers are used to alter the zeta potential, or combinations of surface coatings are used to similarly minimize electroosmotic flow. In some embodiments, a polymer is used to fill the aqueous volume of the electroosmotic flow restriction.

In other embodiments, a physical structure as shown in fig. 13C is used to reduce or eliminate undesirable mixing and turbulence caused by electroosmotic flow. Such a structure may have a flow restricting portion 1320, and the flow restricting portion 1320 may be used on both sides of the separation portion 1308. The electric field may be distributed from an electrode (not shown) through the buffer reservoir 1322. The electrodes may be positioned at an auxiliary input port 1324 that may be used to pass buffer into and through the buffer reservoir 1322 and flow restrictor 1320. In an alternative embodiment, the electrodes are positioned in the buffer reservoir 1322, electrically connected to a voltage source, which may be located external to the enrichment module 1300. Input beads and reagents can be brought into the separation section 1308 through input ports 1311A and 1312A, or reagents can be brought in through input ports 1311A and 1312A while beads are brought in through central input port 1326. The beads may be separated between output ports 1311B and 1312B.

In an alternative embodiment, the electrodes are positioned in a buffer reservoir (reservoir) separate from the structure shown in fig. 13C, with a fluid connection to allow current to flow into the enrichment module. In some embodiments, the buffer reservoir allows electroosmotic flow more in one direction across the enrichment flow cell than with a circular flow that would be turbulent with a sealed flow cell, where any flow in one direction must match the flow in the opposite direction. In some embodiments, the voltage may be periodically stopped to allow the fluid reservoirs to return to their equilibrium state, where the liquid level in each reservoir is at the same level after electroosmotic pumping. In other embodiments, the volume or cross-section of the reservoir is significant relative to the electroosmotic pumping, such that a bead set or bead sets may be separated or enriched prior to allowing the fluid reservoir to return to its equilibrium state.

In other embodiments, the magnitude of the zeta potential is reduced by protecting the silanol groups with a compound that reduces the number of ionizable silanol groups, such as trimethylchlorosilane. Fig. 13D and 13E show the use of the structure as shown in fig. 13C in photomicrographs, where fig. 13D shows the input bead stream 1314 with no field applied, so all of the input beads 1314 are carried to the output 1311B as if they were lower charged beads 1314A, and no beads appear to be higher charged beads 1314B that are pulled to the outlet port 1312B. The flow restriction 1320 shown in fig. 13C is shown in greater detail, showing the fluid channel 1316, and the support column 1318. In fig. 13E, a field is applied to the separation portion 1308 and the input bead stream 1314 is separated into low-charged beads 1314A that are carried to output port 1311A, and higher-charged beads 1314B are pulled and carried to output port 1312B.

In some embodiments, the thicknesses or depths of separation portion 1308, fluid inputs 1311A, 1312A, fluid outputs 1311B, 1312B, separation portion 1308, electroosmotic flow restriction portion 1320, and buffer reservoir 1322 may be the same. In other embodiments, the thickness or depth of the different portions may be different, for example, the thickness at the electroosmotic flow restriction portion 1320 may be less than the thickness of the separation portion 1308 or the buffer reservoir 1322.

In some embodiments, the thickness or depth of the separation portion 1308 and other fluidic portions of the enrichment module is 10-1000 μm; in other embodiments, the thickness or depth of the enrichment module is 20-200 μm, 50-150 μm, 200-500 μm, or 70-130 μm.

In some embodiments, the width of the flow restrictor 1313 in the electroosmotic flow restriction section is 10-1000 μm, and in other embodiments the width of the flow restriction is 20-200 μm, 50-150 μm, 200-500 μm, or 70-130 μm.

In some embodiments, the length of the enrichment zone can be up to 2mm, 2mm to 10mm, 10mm to 100 mm. In some embodiments, the width of the enrichment zone can be up to 1mm, 1mm to 4mm, 4mm to 10mm, and 10mm to 100 mm.

In some embodiments, the enrichment module 1300 has a feedback system (not shown) to compensate for different charge levels that may exist between different batches of beads. Such a feedback system may then allow the electrophoresis voltage to be automatically adjusted for a particular batch of beads, and automatically readjusted for a subsequent batch of beads. The feedback system may use reflected light, absorbed light, refracted light, fluorescence, capacitive coupling with the beads, direct conductivity detection of the debye layer associated with the beads, ISFET/ChemFET detection of the beads, or any other suitable detection means may be used. The detection means may be configured such that detection of the beads is accomplished in or associated with one or more of the fluid outputs 1311B and 1312B, or may be configured such that detection of the beads is accomplished in or associated with the separation portion 1308.

In some embodiments, the feedback system is used during separation of a batch of beads, adjusting the flow rate and electroosmotic pressure so that the beads are optimally separated and flow into the nominal desired location in each output 1311B and 1312B. The flow rate control may be accomplished by using a variable pressure applied to the flow, a variable vacuum applied to the flow, a variable restriction in the flow, or any combination thereof.

In some embodiments, it may be desirable to concentrate the bead slurry. In one embodiment, the bead solution is passed over a magnet to retain the beads. By removing the magnetic field or using a higher flow rate, the beads can be released in a more concentrated form.

In some embodiments, negatively charged DNA is separated from proteins using enrichment module 300, including cell membrane fragments that can be mixed with the DNA after lysing cells that may be the origin of the DNA and the proteins and the cell membrane fragments. In some embodiments, it may be desirable to separate the DNA from most positively chargeable proteins, and it may also be desirable to separate the DNA from proteins that can be negatively charged at neutral pH, such as human serum apotransferrin, thyroglobulin, or BSA. Such proteins, which may be negatively charged at neutral pH, typically have a pKa value above 4.0, whereas the pKa value of DNA is 1.0. The electrophoretic mobility of proteins is typically much lower than that of highly negatively charged DNA, allowing for easy separation of DNA in the enrichment module 300. This separation can be performed at low pH values, such as pH values below 7, pH6-7, pH5-6, pH4-5 or pH3-4, allowing the enrichment module to run below the pKa of the protein and above the pKa of DNA.

In some embodiments, DNA can be captured by dielectrophoresis after separation from substantially any proteins and cell membranes. The dielectrophoretic capture may be implemented in the fluid outlets 311B, 312B, or may be implemented in a separate module. After the dielectrophoretic capture, the buffer may be changed, for example, from a low pH as previously described for effecting separation from the protein or cell membrane to a buffer suitable for PCR, isothermal amplification or sequencing, for example, where the pH is approximately optimal or otherwise suitable for polymerase activity, for example.

If beads stick to the electrode, the voltage applied to the electrode 1313 can be periodically reduced or even reversed if necessary. The voltage used may be greater than the voltage required for electrolysis (1.23V at 25C, pH 7) or may be less than the voltage required for electrolysis. Higher voltages and narrower spacing provide higher field strength and greater force on these beads. The voltage across the system can be calibrated by: beads with no or limited template are flowed and the voltage and/or flow rate is set so that the beads do not move far enough to enter the second outlet while the beads with template can be directed into the second outlet.

Non-flow-through enrichment modules are also contemplated. In one embodiment, the beads are introduced into a chamber and a magnetic field or gravity will pull the beads downward. An electric field is established that pulls the beads with the template upwards. In some embodiments, a capture membrane or filter may be added in front of the positive electrode to facilitate the concentration of the beads.

In one embodiment, the beads are removed from the flow cell by actions and methods performed in the same instrument, wherein the flow cell is used for detection reactions, such as sequencing reactions.

In another embodiment, the flow cell assembly is removed from the instrument in which the flow cell is used to detect a reaction, such as a sequencing reaction, and moved to another instrument or device, where the beads are removed from the flow cell.

In another embodiment, the flow cell assembly is removed from the instrument in which the flow cell is used to detect a reaction, such as a sequencing reaction, and transported to a central refurbishment site (central refurbishment site), where the beads are removed from the flow cell.

In another embodiment, the flow cell assembly is removed from the instrument in which the flow cell is used to detect a reaction, such as a sequencing reaction, and moved to another instrument or device, where the coating may be applied, removed, and/or reapplied to the flow cell and/or fluidic manifold.

In another embodiment, the flow cell assembly is removed from the instrument in which the flow cell is used to detect a reaction, such as a sequencing reaction, and transported to a central refurbishment site, where the coating can be applied, removed, and/or reapplied to the flow cell and/or the fluidic manifold.

In some embodiments, the flow cell and/or the fluid manifold have different surface coatings. Such surface coatings serve to reduce non-specific binding of moieties in various reagents to the surface of the flow cell or fluid manifold. In some embodiments, coatings intended to reduce non-specific binding may include PEG (polyethylene glycol), BSA (bovine serum albumin), PEI (polyethyleneimine), PSI (polysuccinimide), DDM (n-dodecyl-b-D-maltoside), fluorinated coatings, teflon coatings, silanized coatings, or other suitable coatings.

In one embodiment, the coating is applied, removed and/or reapplied to a flow cell and/or fluidic manifold by actions and methods performed in the same instrument, wherein the flow cell is used for detection reactions, such as sequencing reactions.

In some embodiments, the sensor combines pH sensing with electrochemical detection due to the introduction of a reversibly reducing layer that can be fabricated based on previous sensor designs. Such sensors are available from Senova Systems. During the sequencing cycle, if a base has been incorporated into the bead associated with the sensor, a reduction reaction will occur. The reduced level can be measured and after the sequencing cycle is completed, a voltage can be applied to the sensor, causing the surface to oxidize, returning it to its original state, at which time it can be used for the next sequencing cycle.

As shown in fig. 9, in some embodiments, a redox reaction can be performed, wherein the redox potential can include a combination of an AC potential 904 and a nominal DC potential 902, wherein the nominal DC potential 902 is half a sine wave that starts at zero volts, rises to a maximum, and returns to zero volts, wherein the AC potential 904 can be superimposed on the nominal DC. The waveform of the AC potential 904 may have a frequency 10 times the nominal DC potential 902, or the waveform of the AC potential 904 may have a frequency 10-100 times the nominal DC potential 902, or the waveform of the AC potential 904 may have a frequency 100-. The waveform of AC potential 904 may be a sinusoidal waveform, a triangular waveform, a square waveform, or any other kind of symmetrical or asymmetrical waveform. The waveform of the DC potential may be a half sine wave, an isosceles triangle waveform, a sawtooth waveform, or any other waveform that starts at zero volts, rises to a maximum value, and returns from there back to zero volts. The amplitude of the superimposed waveform of AC potential 904 may be constant or may vary during the DC potential 902 waveform, e.g., the AC waveform may be smaller when the DC waveform is close to zero and may increase when the DC waveform reaches its maximum potential. The current 906 produced by the combination of the AC potential 904 and the nominal DC potential 902 may be a non-linear function of the applied potential.

In one embodiment where the amount of data collected is minimized, it may be desirable to align active areas (active areas) that have the straight line nature of a semiconductor electronic device that normally exists, in addition to increasing the speed of the reaction. In previous systems, the location of the active reaction may not be well aligned with the detector array. Preferably, the detector electronics are arranged in a strictly linear fashion, as opposed to the convention of having reagent ingress and egress from the corners 1404A and 1404B of the chip 1400 as shown in fig. 14. This approach both prevents alignment with the reagent flow (reagent slug) and wastes a significant area of the chip because the reagent flow does not flow well (or at all) to the other corners of the chip 1402 and causes flow (and loading efficiency 1406) non-uniformity due to the large difference in cross-sectional area from the center of the chip to the inlet and outlet ports at the chip corners 1404A and 1404B.

In one embodiment, a plurality of fluid inlets 1512A as shown in fig. 15 may be used in a multi-flow cell sensor device 1500, allowing for greater utilization of chip area, and more uniform and aligned reagent flow relative to the chip and the chip reading structure. In some embodiments, samples can be introduced into different channels of a flow cell at different times, allowing different samples to be used without the need for barcode encoding or other means for sample identification. The multi-flow cell sensor apparatus can have a plurality of flow cells 1510, each flow cell having an input port 1512A, an output port 1512B, and a waste line 1516, and wherein each input and output port can be configured to optimize the uniformity of flow through the sensor array within the flow cell 1510, potentially having angled sidewalls and/or surface roughness to optimize the flow uniformity. The flow cell may further be configured with a valve 1504 and a control 1506 for the valve for controlling the flow of sample and/or other reagents in close proximity to the flow cell 1510. Input port 1502 may be used to introduce samples and other reagents.

In some embodiments, the sample is introduced at different times in a process or set of processes. For example, a second set of beads that have undergone an amplification reaction and thus have extended primers is introduced into a channel of a flow cell associated with a chip, where another channel of the chip may already have the first set of beads. The first set of beads may have undergone an amplification reaction and thus contain extended primers therein, and the channel with the first set of beads may have been exposed to one sequencing cycle, or may have been exposed to multiple sequencing cycles. In another embodiment, the second set of beads is introduced into the same channel in which the first set of beads is contained. In another embodiment, where amplification and sequencing can be performed in a single region of an array as described elsewhere herein, one set of beads in one channel can undergo isothermal amplification while a second set of beads in another channel can undergo a sequencing reaction.

In some embodiments, where a first process may require a temperature change and a second process does not, the second process may be temporarily stopped or suspended when the temperature change occurs, and then may be started after the temperature is restored to the previous temperature. For example, after the primer associated with the bead is extended, the amplification reaction process may require that the second strand be melted and removed so that the complementary unextended primer can hybridize to the primer associated with the bead so that the sequencing-by-synthesis reaction can begin. Prior to raising the temperature of a chip with multiple channels, a channel with a set of beads undergoing a sequencing reaction may halt the sequencing reaction and may actuate a co-located virtual nanoreaction well electrode. In this manner, partially hybridized extension primers remain in position with each virtual nanoreaction well, while extension primers that are not associated with a bead in a channel can be detached from the primer associated with the bead and then removed from the channel.

The system can split the sample onto multiple fluidic channels or chips if it is too large, or combine it if it is combinable (e.g., a sample with a barcode). In some embodiments, a sample provided to the instrument will be ready for sequencing. In other embodiments, the sample may be processed by the instrument to produce a sample ready for sequencing.

In another embodiment, multiple input ports to a single flow cell or electrowetting are used to introduce samples into portions of the flow cell, allowing more samples to be used at a time without the risk of cross-contamination.

In another embodiment, an electrowetting system may be provided to move the reagent within a portion of the flow cell without creating a reagent interface, thereby completely preventing any mixing of the reagent before it reaches the desired portion of the flow cell, and may thus provide an extremely fast reagent change over.

In some embodiments, for example, when flowing dNTP-containing reagents through a flow cell in which the sensor is located, it is desirable to have a faster transition between one reagent and another. This may help to provide a faster switch between a concentration of dntps present therein of substantially zero and reagents having dNTP concentrations desired for extension or further extension of the primer by polymerase incorporation. This may allow a short time between the start time of base incorporation and the time at which a significant percentage of the primer or extension primer already has newly introduced nucleotides incorporated into the appropriate position in the different colonies in the flow cell. This shorter time may allow for a greater change in the signal level of one or more sensors per unit time, which may improve the signal-to-noise ratio. Electronic sensors typically have noise, which may include thermal noise and flicker noise, both of which may be minimized by shortening the time interval for sensing the integrated signal with the sensor.

This fast switch is obtained in contrast to systems where a slower switch occurs between the concentration of dNTP present therein being essentially zero and the reagent having the dNTP concentration desired for extension or further extension of the primer by polymerase incorporation. This slower transition may occur due to diffusion of the dNTP from the dNTP-containing reagent solution to the reagent solution that originally had substantially zero dNTP. This may occur while switching through the long channel through which the reagents flow, prior to introduction to a flow cell in which the sensor is positioned for detection of a sequencing reaction or another reaction. Further mixing may result from changes in channel width, corners through which reagent flows, irregularities in the surface of the channel through which reagent flows, slugging of the channel through which reagent is introduced into the flow cell, or slugging of the flow through the flow cell.

In one embodiment, the channel length can be significantly shortened between: in one spot, a switch is made between a concentration of dNTP present that is substantially zero and a reagent having a dNTP concentration that is desired for extension or further extension of the primer by polymerase incorporation; in another point, a reagent is introduced into the flow cell. The channel length between the generation of the transition between the reagent and the flow cell may be 1 micron or less, 1-5 microns, 5-20 microns, 20-100 microns, 100 microns to 300 microns, 300 microns to 1 mm, 1 mm to 3 mm, 3 mm to 10 mm, or 10 mm to 30 mm. Typically, the shorter the distance in the fluidic system between the location where the transition between the reagents is made and the location where the agent is introduced into the flow cell, and the fewer the number of transitions in the fluidic system or the flow cross-sectional size of corners in the distance, the lower will be the diffusion of dntps or other moieties, and thus the shorter will be the time range between a reagent with a dNTP concentration of substantially zero and a reagent with a desired dNTP concentration at each sensor in the flow cell.

Minimizing the time of the polymerization reaction may also improve the signal-to-noise ratio associated with the detection of the polymerization reaction. The noise associated with the detector may be fairly consistent with time and the total integrated amount of the generated signal may be the same regardless of the length of time it is generated. Thus, the amount of noise that must be handled in the analysis can be minimized by speeding up the time associated with polymerase binding and/or by reducing the time taken to collect data by providing a fast conversion of dNTP concentration when dntps are introduced into the reagent stream. Thus, the noise bandwidth that must be handled by the analysis software can be reduced.

In some cases where the sensor is near the exit port, the number of dntps that need to be incorporated may be large enough such that depletion of the dntps can result in an increase in the time to generate the desired dNTP concentration for extension of the primer by polymerase incorporation. Lower dNTP concentrations, longer distances with more colonies in the flow cell, larger colonies with more primers for extension can all result in an increase in the time to generate sufficient dNTP concentration. In some embodiments, multiple input ports are utilized to provide input to a single flow cell to ensure availability of dntps at each sensor and/or colony. In some embodiments, a reagent channel is provided in an additional layer of the PDMS liquid manifold, above the flow cell. A repeating set of control lines similar to those shown in fig. 42 may be provided which can control a repeating set of valves similar to those shown in fig. 42 and flow through a repeating set of manifolds similar to those shown in fig. 42, which can then provide an alternative flow cell input with a short distance between the point in the fluid where the interface between the two reagents is created and the second or subsequent flow cell input port.

One important issue associated with the new generation sequencing is the enormous amount of data that is generated. Some systems may generate an average of 3000 or more data points for each useful base of sequencing data. Storage and analysis of the data adds significantly to the overall cost of new generation sequencing. In some embodiments, data reduction is performed in the simplest manner by obtaining less data. Polymerase activity can be significantly faster than the time required to pass a reagent with dntps completely through the flow cell; thus the DNA colonies near the flow cell inlet may have completely completed the next synthesis before dntps reach even colonies near the flow cell outlet. If data is acquired for the entire flow cell within the time required to detect a reaction occurring anywhere in the flow cell, much of the data will come from areas of the flow cell where no reaction has occurred. Depending on the time required for the dNTP reagent stream to pass through the flow cell, and the speed of polymerization, most of the colonies in the flow cell will either be waiting for dntps or will have completed their synthesis reactions, rather than incorporating dntps and thereby generating useful data.

In some embodiments, the reading of the detector electronics is synchronized with the movement of the reagent stream through the flow cell. The reagent stream containing dntps may enter the flow cell first but has not moved far enough in the flow cell for dntps to bind and incorporate into any colonies. At this point in time, data may not be collected at all. At a later point in time, the reagent flow will have entered the flow cell sufficiently to interact with the colony set in the first zone. At this point in time, data may be taken from the detector associated with the colony in the first region, but may not be taken from other regions of the flow cell. At a second later point in time, the reagent flow may have entered the flow cell sufficiently to begin interacting with the colony group in the second zone. At this point in time, depending on the speed of the reagent flow and the speed of the polymerase, data may be taken from the detector associated with the colonies in the second region, and may still need to be taken from the first region, but may not need to be taken from other regions of the flow cell. At a third later point in time, the reagent flow has sufficiently passed through the flow cell to begin interacting with the last group of colonies in the flow cell. At this point in time, data may be taken from the detectors associated with colonies in the last region. Depending on the speed of reagent flow, the speed of the polymerase, the length of the flow cell and the size of the colonies, some data may still need to be taken from previous regions, such as the region immediately preceding the last group of colonies, but may not need to be taken from other regions of the flow cell.

In other embodiments, time division multiplexing with phase delay may be performed to distinguish different samples from each other.

Since the speed of the valves used in the system can vary, and the size of the tubes, channels and ports can vary between the system and the consumable, the flow rate can also vary. To accommodate for the changes, a first set of data is taken from the system or consumable, which previous data may provide appropriate guidance regarding flow rates in the system, and more data may need to be collected so that capture of data relating to the sequencing reaction can be assured.

In one embodiment, the acquisition time may be adaptively determined from a previous column or from data from a previous cycle of the same column. For example, if a typical detection event occurs near the end of the acquisition time, additional delay may be added before the beginning of the next acquisition period for the downstream sample. Likewise, if a typical detection event occurs near the beginning of the collection time, the next collection session for the downstream sample may begin earlier.

The size of the region is shown in the figure as one colony width, but the width of the region may be greater than one colony width. For example, if the flow cell is 1000 colonies wide from the inlet to the outlet, the area may be one colony wide, 10 colonies wide, 100 colonies wide, 500 colonies wide, or any number in between. Thus, the area from which data is acquired as the reagent stream moves through the flow cell may vary in width from one sensor to tens of sensors to hundreds of sensors, moving with the reagent stream as it passes through the flow cell and as the polymerization reaction is completed.

Reading of the width of the area can be done when reading the chip, thereby preventing the need to generate and discard data. This may be done in a similar manner as may be used to read a partition of a CMOS image sensor, so that a subset of the total rows or columns may be read out at once. Depending on the structure of the device, it may be possible to select individual sensors, since it is possible to select individual pixels in some CMOS sensors. Alternatively, if the chip architecture is designed to read out a complete row at a time using a separate analog-to-digital converter for each column of the sensor array, the chip can be read out, thereby selecting which subset of rows are desired. The subset of rows will vary as the reagent flow progresses through the flow cell and as the region of the flow cell completes the polymerization reaction at the colonies in that region. In some embodiments, the separate analog-to-digital converters of the comparators are associated with each column, wherein a counter may also be associated with each column, thereby allowing simultaneous conversion of analog signals to digital signals while allowing more time, potentially many orders of magnitude more time, for analog-to-digital conversion with concomitant improvement in signal-to-noise ratio. In other embodiments, the analog-to-digital converter may be a successive approximation device. In other embodiments, a pixel parallel readout approach may be used.

The width of the collected data area must be large enough to account for a number of factors to ensure that all valid data is collected. These factors include variations in the flow rate of the reagent stream, which may flow slower as it approaches the edge of the flow cell due to interaction with the surface. Other factors may include changes in the rate of polymerization due to the concentration of polymerase, changes in the concentration of dntps, changes in temperature, changes in colony density, the number of repeats of the base incorporated for one or more colonies, and the like. Any of these may require the acquired data to be of a longer width.

As previously described, the dNTP used to extend the primer may be a native dNTP that can be incorporated by a native or modified polymerase, a modified dNTP, or both a native dNTP and a modified dNTP. If modified dNTPs are used, the modification may serve as a reversible terminator, a virtual terminator, or the charge of the incorporated nucleotide may be altered for easier detection. Thus, in some embodiments, the sequencing reaction incorporates all bases in a homopolymer run, or may incorporate one base at a time in a homopolymer run, thereby reducing the difficulty of determining the number of bases in a homopolymer run when the number of bases in a homopolymer run is large.

The kinetics associated with diffusion or binding of polymerase to the colony DNA may be significantly longer than the kinetics associated with diffusion, binding and incorporation of dntps. Thus, if a polymerase is added to the same reagent stream with dntps, the length of time for which polymerization occurs may be longer than if the polymerase is added to the reagent stream with the polymerase, followed by the reagent stream with dntps. Thus, it may be advantageous to achieve a minimization of the amount of data to get the polymerase into the flow cell, allowing the polymerase to bind to the DNA colony before dntps are introduced. If the polymerase is a continuous polymerase, such that the polymerase is well retained between cycles, the polymerase can be combined with dntps to eliminate the need for separate delivery. In another embodiment, delivery of a wash solution comprising a phosphatase and a polymerase can allow for efficient elimination of residual nucleotides and replenishment of the polymerase in one fluid delivery. In other embodiments, the reagent includes a phosphatase without a polymerase to effectively eliminate dntps by removing phosphate groups via hydrolysis. The phosphatase may be shrimp alkaline phosphatase, calf intestinal phosphatase, or another phosphatase.

In general, for most clonal DNA sequencing systems, it is desirable to have as much DNA on the surface as possible in order to maximize the amount of data signal. However, since the DNA is randomly located on the surface, the spacing of the DNA may cause steric hindrance in the subsequent polymerization reaction.

In many different sequencing applications, target DNA or primers can be bound to the surface of a substrate. As a result of the ligation process, the target DNA and primers may be randomly located on the surface and may be close enough to sterically hinder during polymerase extension. In some embodiments, a primer is attached, bound, or associated with a substrate while hybridizing to DNA that overlaps with the primer, which can provide a priming site for a polymerase. The primer and overlapping DNA may also contain a polymerase which may act as a spacer to prevent binding of the primers so that steric hindrance will occur, for example, when there is not enough space on each strand of DNA for the polymerase. The polymerase and overlapping DNA can then be removed so that the target DNA can hybridize to the substrate-attached, bound or associated primer for primer extension, which can be used for amplification purposes or for sequencing purposes. Alternatively, the primer may be in the form of a hairpin with an extended end to which the polymerase can bind, thereby eliminating the need for longer DNA. Even with biotin streptavidin binding of the template, the size of streptavidin (3nm) may not be sufficient to space the DNA molecules properly apart so that there is room for the polymerase (7 to 10 nm). In one embodiment, the target DNA may be spaced appropriately so that steric hindrance does not occur. This can be achieved by: for example, a complex having double stranded DNA and a polymerase is used that is smaller, similar or larger in size than the polymerase to be used for the sequencing reaction. Alternatively, other proteins that bind to double-stranded or single-stranded DNA and that may be of appropriate size may be used, including polymerases intended for subsequent sequencing reactions. The polymerase may be continuous, such that it remains bound during ligation, and may further have additional binding moieties associated with it to further enhance the ability of the polymerase to remain in place during DNA binding. Alternatively, a portion other than a protein may be used to space apart DNA, which is then removed, thereby creating a spaced apart DNA to avoid steric hindrance. In some embodiments, it may be desirable that the surface of the substrate may not be saturated with DNA. The diameter of the dsDNA strand is 20 angstroms (2nm), unlike the diameter of polymerases that may be greater than 100 angstroms. For example, E.coli 22S RNA polymerase has a diameter of 135 angstroms (Kitano et al, J.biochemistry 1969651-16); thus, DNA spaced apart using the polymerase can be spaced apart at a saturation level of 2% (2^2/13.5^ 2^ 100) relative to dsDNA bound to the substrate in a saturated configuration. The size of the polymerase can vary significantly, for example, it is reported that Bacillus stearothermophilus DNA polymerase I has a diameter of 9nm (Kiefer et al Nature Vol.39115304), as compared to the previously cited E.coli 22S RNAP of 13.5 nm. In some embodiments, it may be desirable to match the template copy number to the size of the sensor; thus, in some embodiments, it may be desirable to use a relatively small number of template copies. In other embodiments, a large number of copies of the template may need to be used. Thus, in some embodiments, it may be desirable to use from 1,000 to 1,00,000 template copies, or 10,000 and 100,000 template copies, or 100,000 and 1,000,000 template copies. In other embodiments, it may be desirable to use 1,000,000-100,000,000 template copies, such as 1,000,000-5,000,000 template copies, 5,000,000-20,000,000 template copies, or 20,000,000-100,000,000 template copies.

Steric hindrance may still occur when the spacing is equal to the size of the polymerase, when the desired length of the DNA template is long, such that much, if not most, of the DNA is not perpendicular to the substrate. In some embodiments, the DNA template is long enough to "clump," possibly taking up more space on the substrate than the polymerase. In certain instances, ssDNA and/or dsDNA binding moieties may be used to space apart DNA, wherein the DNA may be a DNA primer, and wherein the ssDNA and/or dsDNA binding moieties may bind to other spacer moieties, thereby minimizing steric hindrance by spacing and subsequent removal of the ssDNA and/or dsDNA binding moieties and associated additional spacer moieties. The spacer moiety may be another protein or may be a DNA of similar length to that desired for the DNA template. In some embodiments, the saturation level of ligated DNA relative to saturated ligated DNA may be 0.001-40%, or may be 0.01-15%, or may be 0.1-5%, or may be 0.5-2%, depending on the size of the polymerase required and the desired length of the template.

In some embodiments, steric hindrance will be significantly reduced by using a polymerase or other spacer while attaching, binding or associating DNA to the substrate, particularly a polymerase that is larger than the polymerase intended to extend the substrate-attached primer. In further embodiments, the polymerase may also have any other helper proteins or other moieties that may be used to increase the effective size of the complex.

In some embodiments, the debye length of the reading reagent solution is similar to the debye length of deionized water, which by definition has a value of 10^-7Molar H⁺And OH-concentration, and the resulting Debye length of 680 nm. In other embodiments, the ion concentration of the reading solution is about 1 micromolar, which results in a debye length of about 300 nm. This may allow reading during the reaction. In other embodiments, the reading solution comprises an ionic solvent that may not be a completely aqueous solvent, allowing for lower charge levels in the solution, thus enabling longer debye lengths, and allowing more beads to be sensed with a nanobridge (as described herein). For example, reading the solution may allow for sensing beads having a diameter greater than 1 micron, e.g., 2, 3, 5 microns or more. The reading solution comprising the non-aqueous solvent may have a conductivity lower than that of distilled water, allowing a higher proportion of current to pass through the counter ions associated with the DNA than through the bulk solution. The solvent may be miscible with water and may have the desired partial solubility; some representative solutionsAgents include DMSO, alcohols, and ethers. The miscible solvent may have a lower intrinsic ionic concentration, with a lower H than water ⁺And OH-concentration. The solvent may be used with water, in part to provide a low concentration of hydrogen ions. In some embodiments, the ionic concentration may be less than or equal to 1 micromolar, such as 1 micromolar to 0.5 micromolar, 0.1 micromolar to 0.5 micromolar, 0.01 micromolar to 0.1 micromolar, 0.001 micromolar to 0.01 micromolar, or less than 0.001 micromolar. In other embodiments, the ion concentration of the read reagent may be greater than 1 micromolar, e.g., an ion concentration of 1 millimolar, 2 millimolar, 5 millimolar or greater. In some embodiments, the sensor may be capable of detecting a change in local charge, local conductivity, or local hydrogen ion concentration.

Many commercial buffers used for polymerization contain large amounts of sodium chloride or potassium chloride that are not needed for polymerization and can be further heavily buffered. For example, NEB isothermal amplification buffer (1X), generally described as suitable for Bst polymerase, contains 20mM Tris-HCl, 10mM (NH)₄)₂SO₄、50mM KCl、2mM MgSO₄And 0.1% tween-20; the NEB phi29DNA polymerase reaction buffer (1X) contained 50mM Tris-HCl, 10mM (NH)₄)₂SO₄、10mM MgCl₂And 4mM dithiothreitol. When using a nanobridge or ISFET as a sensor, the buffer reagent interferes with the pH measurement, and the high ion concentration creates a high background level that can interfere with the measurement when using a nanoneedle sensor.

Thus, in some embodiments, it is desirable to use a buffer reagent with a lower pH buffer concentration and/or a lower total ion concentration.

In some embodiments, it is desirable to use reagents with very low ionic strength in order to maximize debye length. For such embodiments, it may be desirable to use reagents that do not have more salt than is required for the enzymatic reaction. For such agents, it is desirable to minimize the amount of salt, e.g., reduce or minimize the amount of NaCl or KCl, and use sufficient Mg. Sufficient Mg may include a concentration equal to the concentration of nucleotides used in the reagent, additional Mg acting as a counter ion to the DNA, and additional Mg used in the flow cell for DNA-associated polymerases. Thus, the concentration required will be a function of the nucleotide concentration, the amount and length of DNA in the flow cell, the number of polymerase molecules, and the volume of reagents used.

In some embodiments where the ionic concentration is very low, the pH may be affected by the surrounding air. In the formation of CO of carbonic acid₂Of any residual CO that may remain after any effort to minimize the presence of₂The pH will be lowered. The buffering agent contributes to the ion concentration; it is therefore also desirable to minimize the amount of buffering. The need to mitigate this conflict between having sufficient buffering and having sufficiently low ionic strength can be achieved by various embodiments. One embodiment uses two buffers simultaneously, e.g., Tris and HEPES combined, as opposed to Tris HCl, so that both Tris and HEPES can contribute to buffering. Ideally, both buffer solutions will have a high molecular weight/charge to reduce mobility. In another embodiment, a water-miscible organic reagent, such as an alcohol (e.g., ethanol), may be used.

In some embodiments, it is desirable to eliminate any monovalent cations, such as Na, from the buffer⁺Or K⁺Thereby avoiding changes in the distribution of counter ions on the DNA or beads relative to divalent Mg⁺⁺The competitive reaction of (1).

In some embodiments, the charge associated with the beads can narrow the charge/conductivity sensor, or reduce the signal-to-noise ratio of the sensor. Thus, in some embodiments, it is desirable to minimize the amount of charge present on the bead surface, for example, by varying the amount of sulfate or other negatively charged moieties on the surface. In some embodiments, it may be desirable to have a small amount of negative charge so that the DNA or nucleotides do not bind on the surface of the beads, but not enough charge so that the dynamic range of the sensor is not significantly reduced. In other embodiments, the beads may have a small positive charge, such that when the DNA primers are ligated, the beads become negatively charged. A solution containing a solvent such as ethanol can be used to solvate the beads to allow ligation of the DNA.

In yet another embodiment, linking may be used instead of polymerization. Four pools of probe oligonucleotides can be used, wherein the first base of each probe in a single pool of probes is the same. These probes may use a reversibly terminated tail, or may have a natural tail, such that multiple ligations may occur, with an accompanying increase in signal level. In a similar manner to the use of multiple dntps and polymerases, more than one pool of oligonucleotides (all probes starting from a single base) can be combined, also with an increase in ligation number and signal level. The second strand may be removed and a new primer introduced, wherein the length of the primer may be shorter or longer than the length of the previous primer.

In yet another embodiment, the ligated DNA molecule may have a hairpin primer, wherein a portion of the hairpin primer has a restriction site. Subsequently, after completion of primer extension and the relevant determination of the sample DNA sequence, the restriction site can be cleaved by an appropriate endonuclease or nickase, and the extended primer can be melted by changing one of the temperature or pH of the solution in which the sample DNA is solvated. The sample can then be resequenced after the temperature or pH of the solution in which the sample DNA is solvated is returned to conditions suitable for primer extension, including appropriate nucleotide and cation concentrations. In alternative embodiments, a strand displacing enzyme or an enzyme having 5 'to 3' exonuclease activity may be used, thereby avoiding the need to remove a second strand.

In a further embodiment, a linkage may be provided that can be chemically cleaved, thereby avoiding the need for enzymatic cleavage.

In some embodiments, it is desirable to minimize the number of counter ions associated with the polymerase and/or any other accessory proteins. Thus, it may be desirable to use a more divergent BLOSUM45 alignment to replace charged amino acids in the polymerase such as Glu, Asp, Lys, His and Arg with very conservative substitutions such as Glu to gin, Glu to His, Asp to Asn, Arg to gin, His to Tyr, Lys to Arg, Lys to gin, Lys to Glu, respectively, or with conservative substitutions such as Glu to Arg, Glu to Asn, Glu to His, Glu to Ser, Asp to gin, Asp to Ser, Arg to Asn, Arg to Glu, Arg to His, His to Arg, His to gin, His to Glu, Lys to Asn, Lys to Ser. The substitutions may be from charged to uncharged amino acids, or may be from uncharged to charged amino acids, wherein the amino acid that is changed from an uncharged amino acid to a charged amino acid may be adjacent to a charged amino acid of opposite charge, thereby sharing charge between the charged amino acids, thereby eliminating or reducing the need for counter ions.

In some embodiments, it may be desirable to perform the displacement on a portion of the protein that interacts directly with the fluid environment rather than with ssDNA to which the protein may bind. In some embodiments, it may be desirable to add additional positively charged amino acids in the positions that interact and bind with the ssDNA to provide tighter binding.

For example, as seen in fig. 10 (Hollis et al, PNAS98179557), the positively charged portion of a single SSB (normally ternary in vivo) is shown in darker grey as part of 1002, thereby showing the portion of the SSB that binds ssDNA. In contrast, fig. 10 shows the portion of the SSB that can be positively charged in darker grey as part of 1000, and which can therefore cause additional counter ions to accumulate when the SSB binds to ssDNA, resulting in an increase in the background current change due to the effect of the charge on the sensitive region of the nanobridge, or by locally increasing the conductivity of the volume near the ssDNA. In some embodiments, a DNA binding protein, which may be a polymerase or a protein with ssDNA or dsDNA binding affinity, may be mutated such that binding of the protein to a DNA strand of interest results in a lower background current change than would occur if the native DNA binding protein bound to the DNA strand of interest. In some embodiments, a change in background current due to binding of a mutated protein to the DNA strand of interest may not result in an observable change in background current relative to background current when no protein binds to the DNA strand of interest due to a decrease in charge of the mutated protein interacting with a fluid, or due to a decrease in the number of counter ions that may be associated with the DNA strand of interest due to an increase in charge interaction between the mutated protein and the DNA strand of interest. In other embodiments, a change in background current due to binding of a mutated protein to the DNA strand of interest may result in a decrease in background current relative to background current when no protein binds to the DNA strand of interest, either due to a decrease in charge of the mutated protein interacting with the fluid, or due to the mutated protein decreasing the number of counter ions that may be associated with the DNA strand of interest, as the charge interaction between the mutated protein and the DNA strand of interest increases.

In other embodiments, primers with nicking sites may be provided. In still further embodiments, multiple adjacent primers can be provided, thereby avoiding the need for an nicking endonuclease. The primer may be complementary to the attached primer, or may be complementary to a targeting portion of the DNA. The sequencing primer may comprise all or a portion of the primer used for clonal generation by the amplification reaction, or may comprise a region that may not be used as part of an amplification primer, or may comprise a region that is used as a primer in an amplification reaction and a region that is not used in an amplification reaction.

In some embodiments, the quality of the signal measurement may depend on the sharpness of the front impact of the reagent on the sensor, and the size of the collection time window may depend on when the reagent contacts the sensor, particularly for detecting transient pH changes due to base incorporation. The sharpness of the wavefront may be reduced by, for example, diffusion of the reagent as it moves through the fluid lines, valves, and the flow cell itself. To better control timing and minimize diffusion, and in particular to minimize the time required to achieve reagent delivery, the charge retention active reagent can be repelled by an electric field, e.g., dntps can be held away from the sensor on an axis perpendicular to the plane of the sensor array during initial delivery along the length of the flow cell from the input port to the output port. The electric field can then be turned off or reversed to pull the reagent toward the sensor. In some embodiments, the electric field may be activated on different portions of the sensor array at different times to allow for better control of the readout time window.

In some embodiments, the flow of reagents through the flow cell may be laminar flow and the reagents delivered to/at the top of the reaction chamber will stay there, mixing occurring only due to diffusion on an axis perpendicular to the flow of reagents. This can be used to delay incorporation until needed. In some embodiments, laminar flow and electric field can be combined to control delivery to the sensor.

In other embodiments, the temperature may be kept low to minimize polymerase activity while delivering reagents, and then increased to start the reaction.

To minimize the diffusion effect, all nucleotides required for sequence analysis may be present in the system, and the only reaction trigger may be magnesium ions. Magnesium ions have a higher diffusion rate than other reaction components such as polymerase and dntps. In these or other embodiments, the temperature of the reservoir and reaction may be maintained below the activation temperature of the polymerase. The reaction can be triggered by precise temperature control, overcoming diffusion limitations. For example, in these embodiments, the parallel reactions of the array may be nearly simultaneous. Activation temperatures are known in the art and/or may be determined experimentally for any particular embodiment. The activation temperature of Klenow polymerase is about 4 ℃ and the activation temperature of Taq is about 60 ℃. In yet other embodiments, the reaction may be triggered by the introduction of a desired reaction cofactor, which may be sequestered in the nanoparticle or vesicle prior to the reaction and released using a suitable external stimulus (e.g., laser or temperature).

In some embodiments, a "barcode" may be associated with each sample as part of the sample preparation process. In this process, short oligonucleotides are added to the primers, wherein each different sample uses a different oligonucleotide in addition to the primers. Primers and barcodes were attached to each sample as part of the library generation process. Thus, during the amplification process associated with the generation of each colony, primers and short oligonucleotides are also amplified. Because the association of barcodes is performed as part of the library preparation process, it is possible to use more than one library, and thus more than one sample, in generating a clonal population. Synthetic DNA barcodes may be included as part of the primers, where a different synthetic DNA barcode may be used for each library. In some embodiments, different libraries may be mixed as they are introduced into the flow cell, and the identity of each sample may be determined as part of the sequencing process.

The sample separation method may be used with a sample identifier. For example, a chip may have 4 separate channels and use 4 different barcodes to allow 16 different samples to run simultaneously. This allows the use of shorter barcodes, while still providing unambiguous sample identification.

Nano-sensor and detection method and system

In some embodiments, the sequence of the DNA colonies is determined using a charge or pH sensitive detector. Colonies can be generated on the beads, which can be transferred to a sensor site provided with a primer, polymerase and dntps, while observing changes in charge or pH due to incorporation of the dntps. There may be a one-to-one correspondence between sensor locations and colonies.

In embodiments of the devices disclosed herein, a plurality of nanoneedle sensors are employed having at least one electrode formed in the shape of a circular arc conforming to the edge of a recess in which one of the plurality of magnetic beads is located.

A nanosensor is a sensor designed to detect beads or particles that are less than one of 0.1, 1, 5, 10, or 20 microns as measured on a diameter or long axis for non-spherical beads or particles. Alternatively, the sensor may be sensitive to a moiety associated with the bead or particle or to a moiety associated with a reaction product or byproduct, wherein the reaction includes the moiety associated with the bead or particle. The moiety may comprise a DNA fragment, a hydrogen ion or other ion, which may be a counter ion, and thus associated with the bead or particle or a moiety bound or associated with the bead or particle. The nanosensor may include a nanobridge, a nanoneedle, or an ISFET sensor. The nanoneedle may be an impedance measuring sensor comprising two electrodes positioned for measuring the electrical conductivity of the local environment between the active areas of the electrodes. "NanoBridges" refers to resistive devices that may include sensors that may be responsive to electrical charge proximate to the active region of the resistive device, and wherein the resistive device may also be a semiconductor device.

In some embodiments, the nanoneedle functions as a pH sensor as described in U.S. provisional application 61/389,490 entitled "Integrated systems and methods for nuclear amplification and sequencing," which is incorporated herein by reference in its entirety.

These sensors may be used to detect transient characteristics associated with the incorporation event described in US7,932,034 (which is incorporated herein by reference in its entirety).

In an embodiment of the device disclosed herein, a plurality of nanobridge sensors are employed, having an active region partially surrounding and in close proximity to one of a plurality of magnetic beads. For example, the radius of the active region may be smaller than the radius of the magnetic bead.

In an embodiment of the device, the plurality of nanobridge sensors is adapted to measure the incorporation of nucleotides into the polynucleotide, and each nanobridge sensor has an active region and a conductive element. The conductive element has a work function that matches the work function of the active region.

In some embodiments of the invention, the sensor may be a nanoneedle sensor, a nanobridge sensor, an ISFET sensor, a chemFET, a nanowire FET, a carbon nanotube FET, other types of charge, conductivity, or pH detection sensor, or a combination of different types of sensors at each sensor location, where beads or colonies may be positioned for a sequencing reaction. A single sensor may detect using only a single mode, e.g., charge, conductivity, or pH, or a single sensor may detect using more than one mode, e.g., in response to charge and pH. These sensors may provide similar information, or they may provide supplemental information. For example, one sensor at a sensor location may respond to a change in pH, while another sensor at a sensor location may respond to a change in conductivity. In some embodiments, one sensor may detect a local change in pH, conductivity, or charge, while the other sensor serves as a reference. In one embodiment, the reference sensor may be placed so that it does not contact any reagents and may be used to compensate for changes in temperature, supply voltage, etc.

Changes in charge concentration can result from other sources, including binding of DNA to DNA directly attached to a sensor, which can be a nanobridge, nanoneedle, or FET, or can result from binding of charged cDNA, RNA, proteins, lipids, carbohydrates; the change in charge may also result from an enzymatic reaction, or any other chemical reaction that may be sufficiently localized to occur primarily within the sensing region of one sensor and within the sensing region of another sensor.

In some embodiments, a combination of nanoneedle sensors, nanobridge sensors, and magnetic retention structures are used. The nanoneedle structures may be located below the magnetic structures that make up the magnetic array elements, or the nanoneedle structures may be located on top of the magnetic structures that make up the magnetic array elements. In other embodiments, the nanoneedles may be positioned orthogonally or at some other angle to the structure making up the magnetic array elements.

In some embodiments, the nanoneedle sensor can measure impedance changes due to ions generated by a DNA polymerization event.

In other embodiments, the nanoneedles measure changes in the impedance surface due to incorporation of DNA. Each base of DNA has a negative charge. The charge becomes more negative due to the addition of the base. This additional charge attracts positive counter ions that can alter the conductivity of the DNA-coated bead surface. This impedance change may also result from molecules binding to DNA. Because the charge is associated with the immobilized molecules (DNA bound to the beads), the local fluid environment changes compared to the polymerization conditions. For example, one buffer may be used for base incorporation, while a second buffer may be used to measure the change in conductivity compared to the previous measurement.

In some embodiments, the nanoneedle sensor is configured to measure a change in impedance of a bead upon addition of a base to a template DNA attached to the bead. In other embodiments, the DNA template may be attached or associated with a substrate or a coating on the substrate, or may be attached or associated with an electrode of a device or a coating on the electrode. To improve performance, it is desirable to reduce other impedances.

The impedance of the sensor may be dominated by other impedances. For example, if the physical alignment between the nanoneedle electrodes and the beads to which the DNA is bound is not good, the impedance of the bulk reagent between the electrodes and the debye layer associated with the beads may be large relative to the impedance through the debye layer associated with the beads. For example, if the impedance of the bulk reagent constitutes 90% of the total impedance between the electrodes, and the impedance of the DNA and its associated counter ions on the bead constitutes 10% of the total impedance between the electrodes, a 1% change in the impedance of the DNA and associated counter ions will result in a 0.1% change in the total impedance between the electrodes. If the ionic concentration of the bulk solution is high, the impedance of the bulk reagent between the electrodes may be small relative to the impedance through the debye layer associated with the beads.

Fig. 16 schematically illustrates a simplified circuit 1600 of a nanoneedle sensor with beads. The sensor may have a parasitic capacitance 1614 and a parasitic resistance (not shown). The sensor may also have a double layer capacitance 1610 associated with each electrode 1612. Resistance 1606, which is generated by counter ions associated with the DNA or other sample bound, attached or associated with the beads, can be in parallel with resistance 1602, which is generated by bulk fluid, resistance 1606, which is generated by the conductivity of counter ions associated with the charge on the surface of the beads, and resistance 1608, which is generated by the conductivity of counter ions associated with the charge on the surface of the sensor. Additional portions of this circuit that may add complexity include the resistance between the double layer capacitance 1610 (not shown) and the resistance 1606 resulting from the conductivity through the counter ions associated with the charge on the bead surface, and the resistance 1608 resulting from the conductivity through the counter ions associated with the charge on the sensor surface.

Thus, in some embodiments, it is desirable to minimize the distance between the two electrodes and the beads. In other embodiments, it is desirable to measure the counter ions from as many beads as possible, thereby allowing averaging from as many intact surfaces of the beads as possible. In some embodiments, it may be desirable to position the electrodes on both sides of the beads, allowing current to flow across the entire surface of the beads in as smooth a manner as possible. In other embodiments, it is desirable to have electrodes that are as small as possible so that the current density of the current path emanating from the electrodes is not significantly higher than the current path at the point in the current path where the current density is the smallest. For example, if an electrode can be made infinitely small, the current density emitted by the infinitely small electrode will be infinitely large. In another example, the electrode comprises spherical caps at opposite ends of the bead, and the circumference of the circle formed by the spherical caps is half the length of the great circle of the bead. The maximum current density will be twice that at the spherical caps because it is in the middle of the great circle between the spherical caps. In some embodiments, the ratio of maximum current to minimum current on the bead surface may be 2: 1; in other embodiments, the ratio of maximum current to minimum current on the bead surface can be from 2:1 to 3:1, from 3:1 to 4:1, from 4:1 to 6:1, from 6:1 to 9:1, from 9:1 to 15:1, from 15:1 to 30:1, or from 30:1 to 100: 1.

In some embodiments, the electrode is fabricated using semiconductor technology, and the region of the electrode adjacent to the bead has a height equal to the thickness of the electrode. It may be desirable to keep the electrodes a small distance from the beads, such as from 0.1 to 0.3 debye length, from 0.3 to 1.0 debye length, from 1.0 to 3.0 debye length, from 3.0 to 10.0 debye length, or from 10.0 to 100 debye length. The debye length is considered to be the additive combination of the debye length of the bead and the electrode. Alternatively, it may be desirable for the electrode to have a length of a portion of half the circumference of a great circle of beads. The portion may be 0.01 to 0.03 of the half circumference of the great circle of the bead, 0.03 to 0.1 of the half circumference of the great circle of the bead, 0.1 to 0.3 of the half circumference of the great circle of the bead, 0.3 to 0.75 of the half circumference of the great circle of the bead. In some embodiments, the system maintains the distance between the electrode and the bead and makes the electrode a portion of one-half of the circumference of the great circle of beads.

In some embodiments, it may be desirable to reduce the current through the bulk reagent in order to partially maximize the measured current through the counter ions associated with the DNA bound, linked or associated with the beads. Thus, in some embodiments, it is desirable to physically reduce the volume of bulk reagent in proximity to the beads to maximize the impedance contribution of the DNA counter ions. In other embodiments, it is desirable to minimize the surface area of the structure that holds the beads close to the electrodes used to measure DNA counter ions. In another embodiment, it is desirable to minimize the zeta potential of the bead and/or to keep the bead at the surface of the structure near the electrode used to measure the DNA counter ions.

The nanoneedle structures can be fabricated in nanoneedle arrays, allowing a large number of single DNA molecules or colonies to be sequenced simultaneously.

As shown in fig. 17, a nanoneedle sensor structure 1700 may be fabricated using a silicon substrate 1701 and may have 800nm deep channels 1702 etched into the substrate. A 200nm thick silicon oxide layer 1703 may be fabricated on the substrate, followed by an 80nm thick conductive p + silicon layer 1704, followed by a 30nm thick silicon oxide layer 1705, followed by an 80nm thick conductive p + silicon layer 1706, followed by a 20nm thick silicon oxide layer 1707. The channels may be created after the structure is manufactured. The structure may be produced such that the oxide layer or resist layer covers all portions that may remain in the final structure. A chemical wet etch, plasma etch, or vapor phase etch may be used to remove silicon or other similar substrate from beneath the structure. An ion milling step may then be used to expose the conductive tips of the structure.

All thicknesses can vary, as can the material. The channels in the matrix may alternatively be fabricated in the channel volume using an oxide layer, with a resist layer. An oxide layer and a conductor layer can then be fabricated on top of the oxide and the resist, avoiding the need to undercut the structure. Fig. 18 illustrates a single-ended nanoneedle array fabricated in a similar manner as schematically depicted in fig. 17.

Such a structure may have sensors 1901 on both sides of a channel 1902 formed in a substrate 1903, as shown in fig. 19. The polymerase and/or target DNA1904 may be attached to the active region of the sensor. The sensor itself may be used to electrophoretically and/or dielectrophoretically localize the polymerase and/or target DNA to the active region of the sensor. The target DNA may be a single double stranded, single stranded or circularised DNA target or may be locally amplified at appropriate locations in the active region of the sensor as described in PCT/US 2011/054769. Fig. 20 illustrates an array of crossed nanoneedles fabricated in a similar manner to that schematically depicted in fig. 19.

Nucleotides or probes may then be provided 1905, and the sequencing-by-synthesis process or the sequencing-by-ligation process may begin.

To improve the sensitivity of the nanoneedle or nanobridge, a local amplifier may be provided. The amplifier may be a BJT or a FET. In some embodiments, the amplifier uses one amplifier circuit for each sensor, or multiple sensors sharing the same amplifier. In other embodiments, some amplification may be associated with each sensor, and additional amplification and/or other associated circuitry is shared or multiplexed between different sensors. The sensor can be manufactured in a narrow structure and can be etched under the structure so that both sides can obtain a change in pH or a change in conductivity. The surface of the device may be roughened, allowing more surface area for binding of sample molecules. The surface associated with the electrode of the nanoneedle may be gold or platinum, or may be platinum black, iridium oxide or Ppy/PSS to increase the surface area and associated double layer capacitance.

Electrical concentration of ions can be achieved to concentrate DNA, polymerase, primers, nucleotides and other reagents required for the active region of the nanoneedle or nanobridge sensor. The concentration allows more sample to be attached or associated with each sensor, thereby reducing the need for whole genome amplification.

Another factor that may prevent optimal measurement of DNA impedance on the bead includes counter ions caused by the debye layer associated with the zeta potential of the bead surface and/or counter ions caused by the debye layer associated with the zeta potential of the sensor surface. These counter ions may result in a desired current associated with the counter ions of the DNA on the beads, which may be a current in series and/or in parallel. In addition, as the zeta potential changes, the debye length and the associated number of counter ions may change consistently. The zeta potential may vary with changes in buffer conditions, including changes in pH, salt concentration, and various other factors. The consistent changes can therefore confound the determination of DNA. Therefore, it is desirable to minimize the zeta potential and minimize the change in zeta potential with changes in buffer conditions.

In some implementations, the sensor can be fabricated using silicon. Silica has a significant zeta potential magnitude at a pH typically useful for polymerization activity (e.g., pH 7-9); but the magnitude of the zeta potential of silicon nitride is significantly less than that of silicon dioxide. Thus, in some embodiments, silicon nitride is used at the interface between the silicon sensor device and any components that may come into contact with the silicon sensor device, thereby minimizing the zeta potential and accompanying current flow through counter ions that may be located in the associated debye layer.

In some embodiments, a coating is applied on the surface of the sensor. The sensor may be made of silicon, silicon dioxide, PDMS, Topaz^TMOr other various polymers or combinations thereof, wherein the electrodes and/or coatings may include, for example, TiO₂、ZrO₂Or indium tin oxide, BaTiO₃Such that the zeta potential and the resulting debye layer are significantly reduced. In other embodiments, a surface coating such as PEG (polyethylene glycol), PTFE, poly L-lysine, acrylates, methylcellulose, n-dodecyl-B-D-maltoside, acrylamide, fluorinated alkane chains, or other cross-linked or partially cross-linked polymers is incorporated to alter the zeta potential, or a combination of surface coatings is utilized to similarly minimize the zeta potential and accompanying Debye length. In other embodiments, the magnitude of the zeta potential is reduced by protecting the silanol groups with a compound that reduces the number of ionizable silanol groups, such as trimethylchlorosilane.

In some embodiments, it is desirable to reduce the zeta potential of the bead to which the DNA to be sensed is attached, thereby reducing the accompanying current due to the zeta potential generated by the counter ions associated with the bead surface. Thus, in some embodiments, it is desirable to make the beads from a material having a low zeta potential at the pH levels expected to be effective for polymerization, or the beads can be coated with a material having a low zeta potential at the pH levels expected to be effective for polymerization, such as PEG (polyethylene glycol), PTFE, poly L-lysine, acrylates, methylcellulose, n-dodecyl-B-D-maltoside, acrylamide, fluorinated alkane chains, or other crosslinked or partially crosslinked polymers for altering the zeta potential, or combinations of surface coatings can be utilized to similarly minimize the zeta potential and accompanying debye length.

In some embodiments, it may be desirable to minimize changes in pH that may be caused by buffering agents, while it may be desirable to minimize ion concentration. Therefore, the temperature of the molten metal is controlled,it is desirable to use agents with low buffering capacity while maintaining a fixed pH. In some cases, the buffer may be degassed as part of the assay or method; then when CO is present₂After dissolution into the buffer reagent, the buffer may undergo a change in pH. In some embodiments, it may be desirable to limit CO₂Interaction with a buffering agent. Thus, it may be desirable to vent atmospheric gases and provide a gas stream that does not include CO₂Other gases such as nitrogen, argon or other purified gases, or CO-free₂Or other gas mixture that may otherwise dissolve in the reagent buffer, and thereby change the ion concentration and/or pH.

In some embodiments, the system includes an external gas source such as an industrial gas cylinder. In some embodiments, the gas cylinder is located outside of the instrument in which the fluid is located. In other embodiments, the industrial cylinder is placed within a compartment within the instrument. In other embodiments, CO is used₂Scrubbers/deaerators, e.g. regenerable metal oxide systems, Kraft process systems, activated carbon systems, using systems such as Systec Or Poridex^TMThe membrane system of (1). The CO is₂The scrubber/degasser/debubbler may be built into the instrument or may be external to the instrument.

In some embodiments, it may be desirable to bring the sensor electrodes of a sensor, such as a nanoneedle sensor, in close proximity to the beads in order to minimize the amount of bulk reagent volume between the nanoneedle electrodes and the beads. One embodiment may retain beads in the depressions as shown in fig. 2A, 2B, and 2C. The depression may be formed of a material deposited on the substrate, and the material forming the depression may form a pair of nanoneedle electrodes on the material. The electrode may be formed with a circular arc which conforms to the edge of the recess and thus to the edge of the bead. In some embodiments, the depression may be substantially accessible to fluid on one or both sides, or the depression may have a width to depth ratio of less than 1.0, or may have a width to depth ratio of 1.0 to 0.9, 0.9 to 0.8, 0.8 to 0.7, 0.7 to 0.6, 0.6 to 0.5, 0.5 to 0.4, 0.4 to 0.3, 0.3 to 0.2, 0.2 to 0.1, or 0.1 to 0.01.

In some embodiments, the recess minimizes the volume of reagent proximate to the bead. The recess may be shaped to conform to the shape of the bead, whereby the bottom of the recess is narrower than the cross-section of the recess at the height of the electrode. In other embodiments, the electrodes are covered by an additional layer of material such that the effective depth of the recess is greater than half the diameter of the bead, thereby further reducing the volume of bulk reagent in proximity to the bead.

The electrode may thus contact the surface of the bead or may be within the debye length of the bead or particle surface and the DNA attached or bound thereto. In some embodiments, the electrodes are curved such that the electrodes conform to a curve having a radius similar to the radius of the bead, thereby allowing for better coupling between the electrodes and the bead. Such a device allows for the bulk reagent solution to have a minimal effect on the total impedance between the nanoneedle electrodes and for the DNA attached or bound to the bead or particle surface or counter ions near the DNA to have a maximal effect.

In some embodiments, the nanoneedles have an active region of the sensor shaped to fit a bead or other sample holding mechanism. For example, it may be shaped as an arc having an arc curve oriented to align with the curve of the bead. It may also have one nanoneedle of a pair of nanoneedles configured such that it is offset or "shorter" than the other nanoneedle of the pair, such that the inner radius of the arc has a larger diameter and the same centroid. The offset may allow for an increase in the volume of the sensing region associated with the nanoneedle and may also change the orientation of the field associated with the sensing region and thus the orientation of the sensing region, thereby bringing the sensing region more towards the center of the bead than parallel to the matrix.

In an alternative embodiment shown in schematic side view fig. 21A and schematic top view fig. 21B, one electrode 2105 may be attached directly to the substrate 2102 or another layer of the substrate, allowing for separation from the substrate. The second electrode 2105 of the nanoneedle may be connected to a dielectric 2113 part of the sensor for positioning the bead or particle 2101 in a fixed position. Thus, the bead or particle 2101 is in contact with both electrodes 2101, 2105, thereby minimizing the effect of the bulk reagent solution on the overall impedance between the nanoneedle electrodes, which is different from the impedance caused by counter-ions within the debye length associated with the bead or particle or DNA connected or bound to the bead or particle.

In some embodiments, one or both electrodes may be fabricated such that the electrodes conform to the curve of the bead so as to provide a lower and more regular impedance between the electrode and the bead. The bend may abut the edge of the recess or may be slightly distant from the edge of the recess to allow a larger interface area and lower current density.

In another embodiment as shown in fig. 21C, the bead or particle 2101 may be held in place on a substrate 2102. The first electrode 2105A of the nanoneedle 2100 may be attached directly to the substrate 2102 or to an adhesive layer (not shown) adhered to the substrate 2102. A dielectric layer 2114 may then be fabricated to cover the first electrode 2105A. A second electrode 2105B of nanoneedle 2100 may then be fabricated on dielectric 2114 and the first electrode 2105A of nanoneedle 2100. The second electrode 2105B may be short in order to follow the curve of the bead or particle. The difference in length will be a function of the diameter of the bead or particle 2101 and the thickness of the two electrodes 2105 and dielectric 2114 between the electrodes 2105. In this manner, the electrode 2105 can be in contact with the bead or particle 2101, or can be in close proximity to the bead or particle 2101, such that the impedance caused by counter ions within the debye length associated with the bead or particle 2101 and DNA connected or bound to the bead or particle 2101 is greater than the impedance of the bulk reagent.

In another embodiment, the electrodes are fabricated such that they do not abut the edges of the recess, but rather can be fabricated a short distance from the edges such that the current density in close proximity to the electrodes can be reduced.

In fig. 22A, a nanoneedle 2200 is schematically illustrated in a side view, wherein the nanoneedle 2200 has an electrode 2205 on each side of a recess in a dielectric 2203, wherein a bead 2201 may be held on a substrate 2202, and a metallization 2204 may be used for the electrode 2205. As shown in fig. 22A, the thickness of the dielectric material 2203 may be similar to half the diameter of the bead 2201, and the width of the recess may be slightly larger than the diameter of the bead 2201, while still allowing the bead 2201 to be within the debye length of the bead 2201 relative to the two electrodes 2205. The thickness of the dielectric 2203 can be a thickness that allows for holding the bead 2201, and holding the bead 2201 within the debye length of the bead 2201 of both electrodes 2205.

In fig. 22B, a nanoneedle 2200 is schematically illustrated in a side view, wherein the nanoneedle 2200 has an electrode 2205 on each side of a recess in a dielectric 2203, wherein a bead 2201 is held on a substrate 2202, and a metallization 2204 is used for the electrode 2205. As shown in fig. 22A, the dielectric material 2203 may be less than half the diameter of the bead 2201, the dielectric material 2203 may be one-fourth to one-third the thickness of the bead diameter, and the width of the recess may be less than the diameter of the bead 2201, such that the bead 2201 hovers over the substrate 2202. The close proximity of the bead 2202 and the electrodes 2205 maintains spatial proximity between the bead 2202 and the electrodes 2205 such that the bead 2201 is located within the debye length of the bead 2201 of both electrodes 2205.

In fig. 22C, a nanoneedle 2200 is schematically illustrated in a top view, wherein the nanoneedle 2200 has an electrode 2205, due to the fact that the diameter of the recess in the dielectric material 2203 is smaller than the diameter of the bead 2201, causing the electrode 2205 to bend so as to remain in close proximity to the bead, such that the bead remains in close proximity on an arc of a circle corresponding to the point of contact or close proximity between the bead 2201 and the electrode 2205.

Fig. 22D is a transparent three-dimensional perspective view of a nanoneedle structure 2200 similar to fig. 22B and 22C, but with a large number of fluid inlets to the bead 2201. The bead 2201 remains suspended over the substrate 2202 and held against an electrode 2205A (and is insulating head against insulating electrodes2205A) fabricated on the dielectric material 2203, wherein the electrode 2205A and the dielectric material 2203 are shaped to be within the debye length of said bead 2201, but are not bent to match the curvature of the bead 2201 such that there is not a line contact between the bead and the electrode 2205 and/or the dielectric material 2203, but rather a three or four point contact between the bead 2201 and the electrode or dielectric material 2203. Fig. 23D also shows a magnet 2208 applying a force to hold the bead 2201 in place in the nanoneedle structure 2200.

The nanoneedles may be configured in a double helix or serpentine pattern to increase the length of the nanobridge channel while decreasing its width. A sensing region that is too wide will have a relatively low impedance and may have a sensing region area with less local charge density variation than other regions (e.g., at the edges of the bead compared to the center of the bead). A sensing region that is "too wide" may therefore also have a smaller change in impedance, since only a portion of the sensing region may be significantly affected by the binding or reaction that results in a local change in charge. Conversely, a nanoneedle that is too long and thin may have so large an impedance that any current change may be too small to be sensed with a good signal-to-noise ratio. Thus, the width and length of the channel associated with the nanobridge sensor will need to be adjusted for the particular application for which the nanobridge sensor is intended.

In some embodiments, the nanoneedle is configured to have several active regions as part of a single nanoneedle. The active regions are located at different positions relative to a single sample, providing an average from several different areas of the sample area, such that variations in the position of the sample area, e.g. slight misalignment of the beads relative to the sensor or variations in packing density on the surface, will have less of an impact on the signal-to-noise ratio of the sensor.

Streaming potential was initially observed by Quinke in 1859 and is a well-known phenomenon in capillaries; it is a function of factors such as the flow rate, zeta potential and conductivity of the fluid. Thus, a voltage may be applied across the nanoneedle, and a change in flow rate or distance between the electrodes may result in a spatial or temporal change in the bias voltage applied across the nanobridge.

In other embodiments, it is desirable to orient the nanoneedle electrodes parallel to the flow of fluid so that there is no potentially variable flow potential applied between the electrodes of the nanoneedles, as would be the case if the electrodes were orthogonal to the flow of the fluid.

In some embodiments, the nanoneedle is coupled with a local capacitor or capacitor associated with one or both electrodes to prevent the effects of DC bias levels from the drive circuit or leakage from within the chip sensor from affecting the output signal.

In a further alternative embodiment, a nanobridge sensor structure 2300 as shown in fig. 23 is used in place of the nanoneedle sensor described above. The nanobridge sensor may be used in the same manner as a nanoneedle, including the use of circularized or linear DNA, linear or hairpin primers, polymerase enzymes as described for nanobridges, and may be fabricated into an array.

The nanobridge sensor structure 2300 may comprise a silicon-on-insulator device comprising a substrate 2360, a dielectric insulator 2310, two higher doped semiconductor regions 2304A and 2304B, a lower doped semiconductor active area region (active area region)2305, a further metallization layer 2340, which metallization layer 2340 may cover said semiconductor active area region 2305, and may have a further dielectric coating 2350 thereon.

In some embodiments, the nanobridge senses a local change in charge. The change in surface charge of the nanobridge surface adjacent to the flow cell may be caused by a change in charge in the second layer of the flow cell. These charge changes on the surface of the nanobridge may subsequently alter the charge distribution in the nanobridge and thus the conductivity of the nanobridge. The region of the nanobridge surface having a change in charge may thus have a change in conductivity in the relevant volume of the nanobridge, while other surface regions of the nanobridge may not have a change in surface charge and thus may not have a change in conductivity in its relevant volume. The conductivity may be increased or decreased depending on the type of semiconductor material (n-or p-type) from which the nanobridge is constructed, the amount and uniformity of doping in the nanobridge semiconductor material, and the sign (positive or negative) of the charge on the surface of the nanobridge, and whether the change in charge is an increase or decrease in the amount or density of surface charge.

In some embodiments, a change in charge on a bead located within the debye length causes a corresponding change in the amount of charge or concentration locally present in both layers of the bilayer. Said change in the amount of charge is directly related to the local ion concentration and thus also to the capacitance of the surface layer and the conductivity of the agent within the debye length. The charge change may be an increase or decrease depending on the relative charge of the surface layer and the charge change on the beads.

In some embodiments for sequencing clonal beads, a nanobridge sensor is used. The nanobridge sensor may be used in a manner similar to a nanoneedle.

In an alternative embodiment, the nanobridge detects local temperature changes and thus acts in part as a temperature sensor.

In an alternative embodiment, the nanobridge may be configured to operate as a temperature sensor and/or a pH sensor to detect incorporation of a nucleotide. This method is further described in U.S. patent application 20080166727 entitled "Heat and pHmeasurement for sequencing of DNA," which is incorporated herein in its entirety.

The present invention provides methods and systems for polynucleotide sequencing based on pH and/or temperature detection. In some embodiments, the systems and methods may further employ (or alternatively employ) dyes or quantum dots that allow for visual or optical detection of changes in pH and/or temperature. Such monitoring may allow for monitoring of the bulk solution, or may allow for local monitoring of the volume associated with each colony, or may allow for monitoring of both the bulk solution and the volume associated with each colony.

In other embodiments, the nanobridge sensor array is underetched, thereby further minimizing the size of the channel and maximizing the surface area that interacts with the charges generated by the DNA sequencing reaction. In other embodiments, the nanobridge sensor array may not be under-etched, or may be partially etched to provide a more robust structure. In still further embodiments, the nanobridge sensor array is configured to be arranged in a comb configuration, with the sensors interleaved between each other from both sides, with potential features, such as potential amplifiers, alternately arranged on one side and then the other. In another embodiment, the nanobridge sensor array is arranged such that potential features, such as amplifiers, are all arranged on one side of the sensor array.

In some embodiments, the nanobridge sensor is configured such that the width and length of the sensing channel are aligned for optimal sensitivity for the sensing application. The variation may be related to the following factors: the spacing and size of the sample region, the charge associated with sensing the desired portion, and the impedance of the nanobridge in the non-sensing region, such as the conductive portion of the nanobridge between the sensing region and the local amplifier, or other associated impedance.

In some embodiments, as shown in fig. 24A, 24B, and 24C, the sensor is a nanobridge sensor 2400 in which the active region is fabricated such that it partially surrounds the bead or particle 2401 and is in close proximity to the bead or particle 2401. The sensor may comprise a substrate 2402, on which substrate 2402 a layer 2403 of dielectric and/or semiconductor material may be applied. The active region 2405 of the nanobridge may be fabricated such that it mainly surrounds the bead or particle 2401. Metallization lines 2404 may be connected to more highly doped regions 2404A of semiconductor material, which are then joined with active regions 2405 of the nanobridge. Fig. 24A is a side view of a "loop" nanobridge where the inner portion of the active region 2405 is within the debye length of the bead or particle and DNA that can bind thereto. The active region may be partially or completely within the debye length of the bead or particle, thereby causing the impedance of the entire active region to change in response to a change in charge associated with or associated with the bead or particle and/or an incorporation event of a nucleotide or nucleotide analog. The diameter of the ring and associated support structure 2403 may be sized so that the beads fit tightly within the ring.

Alternatively, as shown in fig. 24B, the loops and support structure 2400 may be sized smaller than the diameter of the bead or particle 2401 so that the bead may rest on the loops of the active region 2405 of the nanobridge, especially when held by a magnetic field, ensuring that the loops are within the debye length of the bead or particle 2401 and the DNA bound thereto. Fig. 24C is a top view of a nanobridge 2400 implemented with a ring structure, showing the overlap of the beads 2401 on the active region 2405 of the nanoneedle, and the electrical conductor 2404 providing a means for measuring the impedance of the active region 2405.

In some embodiments, the shape of the nanobridge sensor is optimized to provide greater interaction with magnetic or paramagnetic particles. The nanobridge sensor may be shaped as a spiral, serpentine or other non-linear shape or a shape with a variable cross-section to provide a greater surface area while preserving a narrow channel for the flow of electrical current through the nanobridge channel.

The electrical conductor 2404 may be connected to a heavily doped region of the nanobridge (not shown), which then provides an electrical connection to the active region 2405 of the nanobridge. Alternatively, the electrical conductor 2404 of the nanobridge may be directly connected to the active region 105 of the nanobridge, which has an ohmic connection by fabricating the nanobridge electrical conductor 2404 such that the work function matches the work function of the active region 2405 of the nanobridge. The value of the work function of aluminum is close to that of lightly doped silicon, but not a perfect match. To produce a more perfect match, an aluminum alloy may alternatively be used.

Streaming potential was initially observed by Quinke in 1859 and is a well-known phenomenon in capillaries; it is a function of factors such as the flow rate, zeta potential and conductivity of the fluid. Thus, a voltage may be applied across the nanobridge ISFET or other chemFET sensor, and a change in flow rate or distance between electrodes may result in a spatial or temporal change in the bias voltage applied across the nanobridge.

In some embodiments, it may be desirable to use a reference electrode that can be fabricated between different nanobridge or ISFET sensors in an array to reduce variations in the bias voltage applied across the sensitive area of the sensor. In some embodiments, a reference electrode is fabricated on the nanobridge or ISFET array substrate between each of the arrayed nanobridges or ISFETs. The electrodes may be interconnected by metallization as part of the fabrication of the nanobridge or ISFET array. In other embodiments, the electrode sets are interconnected using metallization as part of the fabrication of the nanobridge or ISFET array. In other embodiments, the reference electrode is fabricated such that there is a fixed or variable number of nanobridge or ISFET sensors between each nanobridge or ISFET in the array of nanobridges or ISFETs. In further embodiments, the bias voltage difference may be compensated by software or firmware, wherein the effect of the bias voltage may be measured and plotted, and the plot used to compensate for changes in signal levels from the nanobridge or ISFET array.

In some embodiments, a reference electrode is used to bias a reagent fluid, which may be a nanobridge and/or nanoneedle array or chemFET, with respect to a sensor electrode or active region of a sensor device. In some embodiments, the reference electrode is configured as part of a sensor device. In further embodiments, there are a plurality of reference electrodes, wherein one or more reference electrodes are part of or associated with a flow cell associated with the sensor device. In other embodiments, two or more reference electrodes are associated with the sensor device. In some embodiments, multiple reference electrodes maintain substantially similar reagent voltages at all members of the array, which can be difficult in flow cells where the fluid thickness is thin enough to allow significant impedance at the sensor array surface.

In some embodiments, at least one additional electrode may be provided to bias the bulk reagent solution in the flow cell. The electrode may be the same electrode that is used to concentrate the sample and/or other reagents at other times. In some embodiments, the voltage applied to the electrodes may be used to bias the detector at the optimum point of its response curve, e.g., to provide an appropriate offset to optimize the amount of gain that can provide the maximum signal over the available dynamic range of the analog-to-digital converter, which can minimize a/D quantization error.

In some embodiments, the bias level can be modified as the sequencing reaction proceeds and the amount of charge in proximity to the sensor changes. In some implementations, a sensor may be used for the read, after which a change in bias level may occur, followed by a read of the sensor again, so that any non-linearity or undesirable shift caused by changing the bias voltage may be observed and may be compensated for by software. In some embodiments, a positive charge is provided on or near the bead or colony so that the sensor can be biased to an appropriate level.

In some embodiments, multiple reference electrodes may be used with a nanoneedle sensor, a nanobridge sensor, an ISFET sensor, or a chemFET sensor.

Electronic sensors such as chemfets can be designed with a wide dynamic range, as is the case with some pH sensors. They may alternatively be designed with a smaller dynamic range, but with a higher sensitivity. In one embodiment, the dynamic range of the sensor and the sensitivity of the sensor are optimized by including additional elements in the system that bias the active region. The element may be one or more reference electrodes, wherein a variable voltage may be applied between the reference electrode and the active region of the sensor (e.g., chemFET or nanobridge). The adjustment of the voltage may allow for a highly sensitive detection despite wide variations in the amount of charge interacting with the sensor. For example, the sensor can be optimized for use in sequencing reactions in which the target DNA is 100 base pairs long. Alternatively, if the target DNA is 1000 base pairs long, the sensor may no longer work within the dynamic range of the sensor. The voltage between the reference electrode and the active region can then be adjusted to allow the sensor to operate within its dynamic range. If the extended primer has been extended to 500 base pairs during the sequencing reaction, the sensor may again no longer be within its dynamic range. The reference voltage can again be changed to bring the sensor within its dynamic range. Additionally or alternatively, the back gate may be used in almost the same way. In a further refinement, the back gate may be segmented such that there may be different portions of the back gate for different regions of the sensor array. There may be many sections, so that there may be a separate back gate for each sensor, allowing compensation for different sequence dependent primer extension rates.

In some embodiments, the reference voltage is changed when a nanoneedle sensor, a nanobridge sensor, an ISFET sensor, or a chemFET sensor is employed.

In some embodiments, the incorporation of the polymerase is assayed to determine the sequence of the DNA target. Multiple assays may often be required to ensure that the incorporation profile is correctly captured and measured, for example, to determine the number of bases that have been incorporated in a homopolymer run. Such an assay can measure the byproducts of the incorporation reaction, such as PPi or hydronium ion. For large arrays of sensors, such assays may require very high data acquisition rates, which may challenge the sensitivity of the sensors, preventing insufficient signal-to-noise ratio, to provide the desired error rate associated with sequencing data. There may be difficulties associated with the error associated with the phase error and hence read length, and the error associated with accurately determining which and how many bases have been incorporated. This may be a result of the low ion concentration required for accuracy of the sensor and the higher concentration required for the polymerase to run accurately without phase error. Thus, in some embodiments, two or more different reagent conditions are used in the sequencing process, where at least one set of reagent conditions is optimized for the minimization of accuracy and dephasing of the polymerase, and the second reagent is optimized for detection, for example by having very low ionic strength. Reading the sensor separately from the incorporation event can improve the accuracy and read length of the sequencing data. In some embodiments, less data is needed because the sensor may no longer be forced to read at a high data rate to capture polymerase incorporation events, but may be read a few times, possibly as little as a single time. The electronics may also have a time constant that may be long enough to allow for significant reduction in sensor noise. Furthermore, in some embodiments, the reduced data requirements may simplify data processing hardware, data transmission requirements, and data storage requirements.

In some embodiments, the read buffer may have a lower ion concentration than the optimal concentration for the polymerase. In some embodiments, the ion concentration of the reading buffer may be one third of the ion concentration of the incorporation buffer; or in other embodiments, the ion concentration of the read buffer can be one third to one tenth, one tenth to one thirtieth, one thirtieth to one hundredth, or one hundredth to one thousandth of the ion concentration of the incorporation buffer.

In some embodiments, the pH of the incorporation buffer and the read buffer may be substantially the same pH. In other embodiments, the pH of the incorporation buffer and the read buffer can be significantly different, e.g., where the pH of the incorporation buffer is optimized for optimal activity and/or accuracy of the polymerase, e.g., pH8.5, and the read buffer has a pH that minimizes the conductivity of the read buffer, e.g., pH7.0 (e.g., when OH is present)^-And H⁺Is the same and is 10-⁷Moles). In some embodiments, the optimal pH for the lowest read buffer conductivity is slightly higher than pH7.0 because the mobility of OH-is lower than that of H +. Thus, in some embodiments, the pH of the read buffer is between pH6.5 and pH8.0, between pH6.8 and pH7.5, or between pH7.0 and pH7.2, and the pH of the incorporation buffer is between pH7.5 and pH9.0, between pH8.0 and pH8.8, or between pH7.0 and pH7.2 pH8.3-pH8.5。

In some embodiments, the nanoneedle sensor, nanobridge sensor, ISFET sensor, or chemFET sensor uses different reagent buffers.

In some embodiments, an integrator (integrator) is combined with the sensors to maximize the amount of time given to each sensor to reduce the read noise of each sensor. The integrator may include a capacitor associated with each sensor in the array. In other embodiments, the sensor is configured as a capacitive sensor, where no current flows, but rather charge accumulates during the chemical cycle. In some embodiments of integrated devices or capacitive devices, the sensor may have local amplification electronics for each pixel. In other embodiments, the charge moves to the readout port in a manner similar to a CCD.

In some embodiments, the integrator is used as part of a sensor, wherein the sensor comprises a nanoneedle sensor, a nanobridge sensor, an ISFET sensor, or a chemFET sensor.

There may be one or more read ports associated with each device. In some embodiments, each corner of the device may have a readout port; in other embodiments, there may be multiple ports along opposite sides of the device, allowing for reduced readout rates, and associated signal-to-noise improvement. In other embodiments, the readout circuitry may divide the array into columns or rows. In other embodiments, the readout circuitry may be placed under a channel support or channel separation feature. In further embodiments, there may be multiple sets of readout circuits, where the sensor array is divided into multiple sub-arrays, and the multiple sets of readout circuits are positioned such that the readout circuits are synchronized with the channel support or channel separation features. In some embodiments, it may be desirable to use a flow cell with a minimum reagent volume; it is therefore desirable to have a flow cell height that is as short as possible. For example, it may be desirable for the flow cell to be 300 microns high or lower, 100 microns high or lower, or 50 microns high or lower. In some embodiments, it may be desirable to use a semiconductor device, which may be 1 square centimeter or greater, possibly up to 10 square centimeters. A flow cell that is wide enough to cover a large portion of the width of the sensor chip can have significant difficulties in mechanical tolerances due to the flatness of one or both major surfaces of the flow cell relative to the other, especially if one surface is a molded plastic part or PDMS or similar polymer part. Thus, it may be desirable to use support posts, channels, or other support shapes to prevent the flatness tolerances from shrinking or expanding beyond desired tolerances.

In some embodiments, the system may use sensors, such as bridge sensors, arranged in a manner similar to Fin FETs, whereby two or three sides of the channel may readily interact with the surrounding environment, e.g., DNA bound to the surface of the channel. The sensor channel may have a vertical dimension perpendicular to the substrate that is greater than the horizontal cross-section of the channel. Such a device may have a higher sensitivity than a device having only one surface accessible to the sample.

In other embodiments, it may be desirable to use a material that provides a greater surface area than is possible with a planar electrode or a polished planar electrode. In some embodiments, it may be desirable to use platinum black, platinum metal sponge, or platinized metal, which may be platinized platinum, platinized titanium, platinized iridium, platinized niobium, platinized tantalum, platinized zirconium, or other platinized metal, as the electrode material. The electrode may be a reference electrode or may be an electrode that is part of a nanoneedle. In other embodiments, the electrode surface is fabricated from other members of the platinum group metal group: palladium, rhodium, ruthenium, iridium, or osmium, which can be used in the same manner as platinum to form electrode surfaces having a higher surface area than would be formed by a planar electrode or a polished electrode.

In some embodiments, the platinizing process may include cleaning the support material, possibly with aqua regia, HCl, and HNO₃Cleaning, followed by an electroplating process that can utilize chloroplatinic acid and lead acetate.

In other embodiments, the electrode surface may comprise iridium oxide, titanium nitrate, or polypyrrole/poly (styrene sulfonate) conductive polymers. The iridium oxide may be fabricated by sputtering using standard photolithographic processes. Malleo et al (Review of scientific instruments81,016104) describe the increase in effective interfacial capacitance of different materials relative to a bright platinum electrode in the range: 240 times higher for platinum black, 75 times higher for iridium oxide, and 790 times higher for polypyrrole/poly (styrene sulfonate) conducting polymer.

In some embodiments, a nanoneedle sensor, a nanobridge sensor, an ISFET sensor, or a chemFET sensor uses a sensor with a large surface area.

In some embodiments, nanoneedles, nanobridge, chemFET, or ISFET are fabricated such that sensors are produced on the surface of a substrate such as silicon, fused silica, glass, or other similar material. In other embodiments, the sensor is fabricated such that it protrudes vertically or horizontally above the substrate, so that the sensor has easier access to fluids and reagents. Easier access to fluids and reagents can reduce the time required for a sequencing reaction to occur, allowing for lower concentrations of reagents to be used, and increasing the sensitivity of the sensor by increasing the surface area associated with the active region of the sensor.

In some embodiments, the electrodes may be fabricated using an angular rotation deposition method, may employ a glancing angle deposition as described by Zhao et al (p59-73SPIE Vol5219Nanotubes and nanorowires), or may be fabricated using a PVD method as described in US6,046,097 (which is incorporated herein by reference in its entirety).

Figure 26 shows data from one run of a nanoneedle sequencing reaction, where the run data is scaled and displayed against a linear plot of base incorporation. Dntps that should not be incorporated are shown to overlap mostly with previous data, while multiple incorporation base data are shown to have a fairly good linear relationship (R2= 0.9974).

In alternative embodiments, the system or method detects the kinetics of a single molecule reaction, such as an enzymatic reaction. In some embodiments, the reaction may be a hybridization reaction, which may result in the beads or particles with attached hybridization probes being held in place on the sensors, and the change in charge proximate to one or more sensors resulting from the hybridization reaction may be measured. In alternative embodiments, the hybridization probe may be attached to or in proximity to a sensor, whereby the change in charge resulting from the progress of the hybridization reaction may be measured. In some embodiments, an electric field may be used to concentrate DNA from a reagent solution into a volume to which hybridization probes are attached, which may be on a bead or particle, or may be on or near the sensor. The electric field may be a DC field, an AC field, or a combination thereof.

In some embodiments, the real-time PCR reaction is monitored by monitoring the change in conductivity or the change in charge present due to incorporation of dntps into amplicons and/or release of pyrophosphate and hydronium ions with higher mobilities using one or more sensors. In an alternative embodiment, the isothermal reaction that amplifies the target DNA is detected by a change in conductivity resulting from incorporation of dntps into the amplicon. In other embodiments, the sensor monitors the progress of the immuno-PCR reaction, wherein a sandwich assay captures the antigen and a probe DNA oligomer is attached to the detector antibody, whereby the real-time PCR assay can detect and quantify the presence and quantity of the antigen by detection of conductivity or charge changes as previously described. In another embodiment, isothermal reactions detect and quantify the antigen of interest.

In some embodiments, detection of a protein can be achieved by direct measurement of a reaction, by measurement of a sandwich assay, or by measurement using an aptamer, or by other suitable methods in which a change in counter ion or change in charge associated with a target that can bind, link, or associate with a sensor.

In another embodiment, the presence and quantity of the target is detected using nucleic acid aptamers that bind to or are in proximity to one or more sensors. Aptamers can bind to the target, changing the charge, which can be detected by the sensor as previously described. In an alternative embodiment, the aptamer is linked to or in proximity to one or more of the sensors and the increase in conductivity due to binding of the target thereto is detected.

In another embodiment, blunt end attachment may be performed using ligands having different binding reagents at the 3 'and 5' ends of the ligands. The electrodes of the nanoneedle may be coupled with complementary reagents for binding, e.g. the 3 'end of the ligand may have a thiol group and one electrode may be made of gold, while the 5' end of the ligand may have a PNA sequence and the second electrode may have the complement of said PNA sequence. The strands of DNA may then be electrophoretically and/or dielectrophoretically concentrated to the nanoneedle region, where the DNA strands may then bind to one end associated with one electrode and the other end associated with a second electrode of the nanoneedle. The polymerase and primer may bind to the DNA strand, or may be introduced subsequently. The determination of the incorporation event can then be obtained by direct determination of the impedance of the DNA in combination with a direct determination of the greater conductivity of the counter-ions associated with the DNA.

In some embodiments, the sensor device, which may be a nanobridge or a nanoneedle, produces digital output data. The digital output may comprise any of a number of output physical/data link/protocol formats, including USB2, USB3, Firewire (Firewire), Gige, single-link or double-link DVI, HDMI, S/PIF, ADAT lightpipe, AES3, MADI-X, I²S, AC '97, MC' 97, McASP, super Video (S-Video), ATM, SONET, SDH, UTP, STP, AUI, HDLC, 802.1, ARP, VLAN, HDLC, ATM, frame Relay, Qin Q, PPP, BSC, DDCMP, Banyan, CDMA2000, DECnet, CDPD, FUNI, CDMA, X.25, GPRS, GR-303, H.323, NFS, ISDNSS7, TCIP, UMTS, WAP, XNS, MDLP, wireless broadband, etc.

The output may be in a compressed format such as MPEG1, MPEG2, MPEG4, DVA, AVI, MOV, MPG, video CD, RM, WMA, WMV, WAV, FLC, FLI, BMP, PCX, TGA, TIF, JPG, PCT, GIF, Flash, QuickTime, MP3, or a sequence thereof.

The sensor device may be configured to have more than one digital I/O connection and may have more than one output format; for example, one digital connection may be used to control the operation of the sensor, while one or more digital connections transmit data from the sensor to an additional device, which may be part of the instrument of which the sensor is a part. The additional device may be a data storage, a device or may be a computing device. The additional device may be a GPU, or a group of GPUs, such as a GPU array.

The data may be transferred from the sensor directly to a hard disk, from the sensor directly to a solid state drive, or from the sensor directly to a GPU cluster, GPU blade, or GPU server, CPU, or memory associated with a GPU or CPU. In some embodiments, an instrument or system may have more than one sensor. In such an instrument or system, data may be accumulated from one or more sensors and transferred from there directly to a hard disk, solid state drive, GPU blade, GPU server, GPU cluster, CPU, or memory associated with a GPU or CPU.

In some embodiments, a single sensor may have more than one digital output. In other embodiments, the digital output may be configured to be directly connected to another part of the system, such as a solid state drive or memory associated with a GPU or CPU, where two or more parts of the system may be part of a MCM (multi-chip module) or SIP (system in package). The MCM may be a layered MCM, a deposited MCM, a ceramic matrix MCM, or a chip stack MCM. The sensor may be part of the MCM or may be separate from said MCM. The sensor may be configured such that the sensor may be removed and a second sensor may be utilized. The sensors may be interconnected using sockets; the socket may be a zero insertion resistance socket for PGA (pin grid array), LGA (land grid array) socket or a commutator (slotket).

This data may be compared to data in a CAM (content addressable memory) or CAM memory that allows a selectable number of errors in DNA mapping, such as a ternary CAM. The CAM memory may have multiple levels in a manner similar to a TLB (translation lookaside buffer), where one level of the CAM or TLB may be faster and less than the other level of the CAM or TLB.

To improve the sensitivity of the nanoneedle or nanobridge, a local amplifier may be provided. The amplifier may be a BJT or a FET. The sensor can be fabricated in a narrow structure and etching can be performed under the structure to make it easy for both sides to obtain changes in pH, conductivity or local charge. The surface of the device may be roughened, allowing more surface area for binding sample molecules. Electrical confinement of ions may be achieved as described further below.

In some embodiments of the invention, the image sensor array may use amplifier designs similar to those in a CMOS active pixel image array; depending on the desired signal-to-noise ratio and whether global shutter equivalence (global shutter equivalence) is required if the integrated circuit is used, a three-transistor, four-transistor, five-transistor, or six-transistor circuit may be included. The amplifier structures may be arranged in a one-to-one correspondence with the image sensor array, possibly providing a significantly better signal-to-noise ratio than would otherwise be possible with a common amplifier of multiple sensors.

Integrated system

The present invention further provides methods and systems for localizing samples and reagents in volumes where desired reactions or binding can occur. This aspect of the invention may eliminate or reduce the need for whole genome amplification and thus reduce the required coverage.

In some embodiments, the DNA sequencer may be part of a larger system in which portions of the workflow are automated. These parts of the workflow that can be automated can include cell lysis, DNA purification, DNA amplification, DNA library preparation, colony generation, sequencing, preliminary analysis and base calling, localization of sequences relative to a reference (mapping), and determining the presence or absence of a genetic disease or other genetic characteristic. In some embodiments, the system may have further functionality, including devices that sort cells, such as cancer cells from blood, using flow cytometry or affinity extraction (affinity pullout) of desired or undesired cells.

It may be desirable to process multiple samples on a single chip because many items do not require the full capacity of the chip. Other items may have a single sample that would exceed the chip capacity. In some embodiments, one or more samples can be introduced into the instrument in a single tube, a tube row (tube strips), a 96-well plate, a 384-well plate, or the like. In some embodiments, the sample wells may be sealed to extend the life on the instrument. In other embodiments, the plate may be cooled to extend the life of the sample. In other embodiments, the sample can be accessed by an automated pipettor in a software selectable manner.

Prior to amplification, the beads need to be loaded with a single DNA fragment in order to generate monoclonal beads. Typically, the concentration of DNA is determined and then introduced to the beads in diluted form, such that on average less than 1 fragment will bind to each bead. Many beads have zero DNA fragments, fewer beads have a single fragment, and a minority has 2 or more fragments. The steps required for quantification often require separate instrumentation and separate processing.

In one embodiment, the target concentration may be generated by hybridization-based extraction. A controlled number of binding sites can be used to functionalize the solid support (e.g., the extraction bead). In some embodiments, these are DNA primer complements. The unamplified sample may have a known primer attached to each end. In some embodiments, the primer can hybridize to DNA on the draw bead. After the hybridization sites are fully occupied, the residual DNA can be washed away, and the DNA bound to these beads can then be denatured, thereby releasing a known amount of DNA.

In another embodiment, a primer attached to each DNA fragment binds to the primer complement and is detected using fluorescent detection of an intercalating dye. Since these primers are of known length, the signal level will be proportional to the number of fragments. In another embodiment, a polymerase and associated dntps can be introduced, thereby generating a full-length double-stranded DNA. When combined with information from the primer signal, the signal level of the full-length intercalating dye will allow determination of the average fragment length.

In another embodiment, dielectrophoresis is used to concentrate the DNA. The current is measured during or after concentration to determine the DNA concentration. In another embodiment, the pooled DNA is quantified by using an intercalating dye as described above. In another embodiment, the concentration of DNA is determined directly by light absorption. Light absorption determination may, for example, use a light source that generates light at 260 nm.

In one embodiment, the sample is made into a very dilute and/or small volume of sample reagent and loaded onto the beads. The DNA will bind to some beads, which are then amplified in a virtual reactor to produce beads with DNA. The sequencing primer can be made shorter than the complement attached to the sample DNA. Since the sequence is known, the correct dNTP can be added and detected. In one embodiment, a plurality of dnpts is added simultaneously. For example, if all the dNTPs are added, the polymerase will extend to the end of the fragment, thereby generating a large signal. The large signal may be generated as part of the amplification process. This may allow the number of beads with DNA to be detected and counted, even if the beads have minimal amplification. Knowing how many beads have a signal allows calculation of the appropriate dilution to produce the desired number of monoclonal beads.

Also, measurements made using electrical current, optical signals, or other signals indicative of the concentration of DNA in the sample can be used to determine the level of dilution, if any, that is required to utilize the DNA in the system in an optimal manner.

In some embodiments, dilution is required to properly generate colonies. Also, dilution may be required for nanopore systems in order to prevent pore clogging, but instead, to optimize the duty cycle that a pore can occupy with a DNA strand. Dilution can be accomplished as part of an emulsion PCR system, a bridge PCR system, a nanopore sequencing system, or a single molecule optical system.

In other embodiments, concentration may be achieved as part of the system, and may be accomplished by dielectrophoresis, hybridization, ethanol precipitation, or other methods, and may be used to increase the concentration of DNA to improve emulsion PCR systems, bridge PCR systems, nanopore sequencing systems, or single molecule optical systems.

The host system may perform the assay using software or hardware to determine the concentration of template DNA, which may then be concentrated or diluted as necessary before using the template DNA in the appropriate next step of the system, which may be amplification or sequencing. The determination step may also utilize an existing calibration step that may use a standard containing a known concentration of DNA, or may use a DNA concentration that is initially unknown, wherein the concentration is determined by a separate system. The determined concentration may be input, transferred or otherwise communicated to the host system. The host system may store any values required for local calibration in the host system, or it may be stored in a portion of a larger system or in a separate computer or database. The calibration information may also include additional information such as calibration time, operator, sample or standard used for calibration, or other information that may be determined to be relevant.

Many existing systems use whole genome amplification in order to have sufficient DNA for their protocols. Typical amplification methods may use degenerate primers and PCR, random hexamer and isothermal amplification or other methods for genomic DNA amplification. The amplification can amplify genomic DNA by a thousand-fold or more. Such amplification can introduce bias and is an additional cost in time and resources. The ability to reduce or eliminate the need to amplify a sample is desirable. In one embodiment, the beads to be loaded are loaded into a packed bed and the sample is pumped across the packed bed. The sample may be pumped through the bead bed multiple times to provide additional opportunities for sample binding. A high surface area to volume ratio should allow the use of a minimum of sample. The beads can then be moved into a flow cell so that they can be held in place by a magnetic array and local colonies can be generated on the beads by PCR or isothermal amplification.

In another embodiment, the sample is concentrated in the amplification zone using the existing electrodes of the emulsion-free nanoreactor. In one embodiment, multiple electrodes may be constructed on a single plane. In another embodiment, the electrodes may be added to a second plane parallel to the plane of the virtual reactor. In other embodiments, it is contemplated to use a mixture of AC and DC voltage inputs.

In other embodiments, whole genome amplification or targeted amplification, such as amplification of targeted exomes, conserved regions of a genome, cancer panels (panels), or other targets of interest, may be performed as part of a subsystem in an integrated system. The targeted amplification may also incorporate barcodes for different samples as part of the amplification process. The concentration of amplified DNA can then be determined as described herein using a clonal sequencing subsystem and method (which can be part of an integrated system) prior to clonal amplification for subsequent sequencing. Alternatively or in addition, DNA is sequenced directly using a single molecule sequencing subsystem and method that may be part of the integrated system.

Since many projects may not require full utilization of the sequencing chip or flow cell, it may be desirable to load multiple samples into different portions or regions of a single chip or flow cell. In one embodiment, the sample is directed to separate regions on the chip or flow cell separated by walls using valves integrated into the chip or flow cell assembly. Such valves may be PDMS valves integrated into the fluid path. In another embodiment, there may be separate regions with separate inputs and outputs. In another embodiment, the sample may be directed to a separate region on the chip or flow cell using a local electric field. A positive electric field may be applied to attract DNA to desired regions, while a negative electric field may be applied to repel DNA from undesired regions. In another embodiment, the sample can be directed into separate regions using electromagnets to control the positioning of magnetic or paramagnetic beads. In another embodiment, a self-sealing port may be used to deliver the sample into a separate channel. The self-sealing port may include a rubber septum and a needle.

In another embodiment, the sample may be injected at different time points, and the sensor signal relative to the signal previously determined for the sensor may be used to distinguish between new beads and the location of beads, where the bead location was previously empty.

In further embodiments, electrowetting or electro-electrowetting is used to deliver samples to different and separate regions of a chip or flow cell.

In some embodiments, the container of reagents may be cooled as needed, for example, reagents containing samples, polymerases, phosphatases, or other enzymes may need to be cooled, for example, to about 4 degrees celsius.

In some embodiments, the amount of reagent contained in the line leading from the reagent container to the valve manifold may contain a volume that is significant relative to the volume required to perform a sequencing reaction. To avoid the need to discard the reagents, for example, at the start of a new sequencing run, it may be necessary to cool the lines from where they connect to the reagent containers up to or near where they enter the valve manifold. In some embodiments, the valve manifold is sufficiently separated from where the reagents enter the flow cell where sequencing or another reaction takes place to allow the flow cell and valve manifold to operate at different temperatures, e.g., about 4 degrees celsius and 20-40 degrees celsius, respectively, which may also require cooling of the valve manifold.

In some embodiments, it may be desirable to use a system capable of performing more than one method of sequencing, where one method, process, or subsystem for sequencing provides one type of information, while a second method, process, or subsystem for sequencing may provide a second type of information. For example, in some embodiments, it may be desirable to provide a method in which the type of information elucidates the structure of the DNA sample such that sequence reads can span the length of a repeat sequence, such as a simple sequence repeat, a short tandem repeat, a microsatellite, a variable number of tandem repeats, a interspersed repeat such as a LINE repeat, a SINE repeat such as an Alu repeat, a direct repeat, or an inverted repeat, or other types of repeat sequences, that can prevent proper complete assembly of other desired sequence or sequences of genomic or DNA. In some embodiments, it may be desirable to use methods, processes or subsystems for sequencing that may elucidate DNA structure, for example, where the sequence may not need to be determined with high accuracy. In some embodiments, it may be desirable to correlate reads that may be separated by multiple bases, as in some systems, for example by pair-wise sequencing or stroboscopic sequencing. In some embodiments, it is desirable to detect, for example, single nucleotide polymorphisms using methods, processes, or subsystems for sequencing that can provide sequencing reads with very high accuracy, but which do not require long sequencing read lengths. In some embodiments, it may further be desirable to use methods, processes, or subsystems for sequencing that can provide the ability to provide many short reads with low accuracy (as may be required for whole transcriptome analysis).

In some embodiments, it may be desirable to use different methods for a single sample, to enable, for example, the detection of single nucleotide polymorphisms and structural rearrangements from a single sample. In some embodiments, it is desirable to divide the purified nucleic acid into two or more aliquots in a single system, so there may be different corresponding library preparation methods, subsystems or processes that may include amplification, and different amplification methods, subsystems and processes may be utilized as appropriate for the different desired sequencing methods, subsystems or processes. The size, concentration, or volume of different fractions may be similar, the same, or different, and may be suitably different depending on the different sequencing and/or library preparation methods, subsystems, or processes that may be used to achieve different desired sequencing results. In some embodiments, the method may differ for different fractions, but the subsystems used may be the same, and/or the subsystems used for different methods may be the same subsystems, where one method is used first, and then a second or more subsequent methods may be performed using the same subsystems. For example, it may be desirable to use different fragment lengths for different sequencing methods, where, for example, long fragments may be desirable for determining sequence structure, and short (or shorter) fragments may be desirable for determining single nucleotide polymorphisms. Thus, it may be desirable to break different fractions of the sample into different average fragment lengths, where the average fragment length of one fraction may be longer, possibly significantly longer, than the other fraction. In some embodiments, the fragmentation process may be the same and, for example, a sonicator may be used, but the time the sonicator applies energy to the fraction and/or the power level the sonicator applies to the fraction may be different, such that the fragmentation levels of the sample in two or more fractions may be different, possibly significantly different, and the resulting fragment lengths may be different, possibly significantly different. In other embodiments, it may be desirable to use a single system to produce both long and short fragments; in another embodiment, it may be desirable to fragment the DNA to a size suitable for the long fragments, remove the aliquots, and further fragment the remaining DNA to a size suitable for the short fragments.

In other embodiments, one aliquot of a nucleic acid sample may be genomic DNA for which single nucleotide polymorphisms may be determined, and a second aliquot may be RNA for which a transcriptome may be desired. The amplification methods, subsystems or processes may be different for the genomic DNA and RNA, wherein a conversion from RNA to cDNA may be achieved, and wherein the amplification protocol may be different for different aliquots, since the accuracy required for single nucleotide polymorphism amplification may be much higher than the accuracy required for conversion of RNA to cDNA and subsequent amplification of the cDNA. In some embodiments, amplification of cDNA for transcriptome analysis may require the use of lower concentrations and/or less expensive reagents than amplification of genomic DNA for single nucleotide polymorphism analysis. In further embodiments, amplification of cDNA for transcriptome analysis may require the use of shorter cycle times than amplification of genomic DNA for single nucleotide polymorphism analysis, which may speed response times, allowing transcriptome analysis to begin using the same sequencing subsystem that may subsequently be used by the genomic DNA single nucleotide polymorphism analysis. Any other combination of using different sequencing methods, subsystems, or processes to isolate nucleic acid material for subsequent analysis is contemplated.

In some embodiments, different types of sequencing detection methods, subsystems, or processes are used. For example, one subsystem may use single molecule sequencing as described in Church et al in US5,795,782, Haneck et al in US8,137,569, Korlach et al in US7,361,466, and Clark et al in US2011/0177498 (all incorporated herein by reference in their entirety), which may have low accuracy and may have very long sequence reads, while the other subsystem may use optical or electrochemical detection of synthetic sequencing as described in McKernan et al in US2009/0181385, balaubabramanian in US6,833,246, Nyren et al in US6,210891, Bridgeham et al in US7,282,370, Williams et al in US7,645,596, Rothberg et al in US7,948,015, toumazu et al in US8,114,591, and Miyahara et al in US7,888,013 (all incorporated herein by reference in their entirety). Thus, in some embodiments, a single system may have at least two different detection subsystems, wherein the two different detection subsystems may use different sequencing methods, sequencing detection methods, or sequencing processes, and wherein the different sequencing methods, sequencing detection methods, or sequencing processes may be performed on the same sample or different samples at the same time, or may be performed on the same sample or different samples at different times.

An exemplary integrated system is shown in the drawings.

Fig. 1A depicts a complete sequencing system 100, which may include an external computing device 102 and an integrated system 104. The integrated system may include a rack module 110, which may further include a fluidic interface subsystem 116, a set of sequencing/sample preparation cards 112, and a separate sequencing subsystem 114 on each sequencing/sample preparation card 112. The schematic of sequencing/sample preparation 120 includes library preparation 122, a reusable magnetic array 124, the magnetic array 124 may further comprise a sequence detector 126, the sequence detector 126 generating sequencing data 128.

Fig. 1B depicts a complete library preparation subsystem 130 that includes a sample cell input 131, cell lysis and protein removal 132 that produces unfragmented genomic DNA133, which unfragmented genomic DNA133 can be input into a fragmentation and isolation subsystem 134, which can then output fragmented genomic DNA136, which fragmented genomic DNA136 can be transported with a set of beads 135 to a virtual well array 137 for amplification, which can then be separated into an amplified bead set and an unamplified bead set using a field 138 in a bead enrichment module 139.

FIG. 1C schematically illustrates a genomic DNA fragmentation and isolation system 140, the system 140 comprising input unfragmented genomic DNA and fragmentation beads 142, which are input into a fragmentation system 144, wherein the unfragmented genomic DNA can be fragmented. Fragmented DNA may be separated by size in the channel 146 using pumping or electrophoretic forces from a pump or electrode 147 and then may be moved to the output of the separation channel 146 via a fluid output 148 and the fragmented DNA149 is output.

FIG. 1D shows one embodiment of a PDMS library preparation module 150, which module 150 includes a cleavage section 152, a protein removal section 154, and an amplification section 156.

While various embodiments have been described above, it should be understood that they have been presented by way of example only, and not limitation. Where methods and/or diagrams described indicate certain events, and/or flow patterns, and/or chemical reactions occur in a certain order, the order of certain events, and/or flow patterns, and/or chemical reactions may be modified. While embodiments have been particularly shown and described, it will be understood that various changes in form and/or detail may be made.

Although embodiments have been described as having particular combinations of features and/or components, other embodiments having any combination of features and/or components as discussed above may also be reasonable.

Claims (36)

1. A method for parallel or clonal polynucleotide sequencing comprising:

sequencing a first portion of a population of target polynucleotides;

correcting the phase error; and

sequencing a second downstream portion of the population of target polynucleotides, wherein the polynucleotide sequencing optionally comprises one or more of:

sequencing clones of the bead array,

electronic detection of nucleotide incorporation, and

an electron aperture for separating or concentrating the reaction components.

2. The method of claim 1, wherein phase error is corrected by adding a combination of three nucleotide bases.

3. The method of claim 1, wherein phase errors are corrected by a combination and/or sequence of incorporation reactions.

4. The method of claim 1, wherein the phase error is corrected by reversibly incorporating a chain terminating nucleotide into the in-phase polynucleotide chain.

5. The method of claim 1, wherein phase error is corrected by adding an oligonucleotide clip that hybridizes to the target nucleotide to stop a sequencing reaction.

6. The method of claim 5, wherein a plurality of clips are added.

7. The method of claim 5 or 6, wherein the clips are denatured, destabilized or degraded to continue the sequencing reaction.

8. The method of claim 5 or 6, wherein at least one clip has a 3 'terminator nucleotide that cannot be extended, and wherein the 3' terminator nucleotide can optionally be removed, thereby becoming a primer for subsequent downstream sequencing.

9. The method of claim 7 or 8, wherein the clips have unique cleavable sites.

10. The method of any one of claims 5-9, wherein the polymerase does not have strand displacement activity or substantial 5 ' to 3 ' exonuclease activity, or the clamp has a 5 ' moiety or nucleotide derivative that is not a substrate for the 5 ' to 3 ' exonuclease activity or displacement activity.

11. The method of any one of claims 5-10, wherein the clip comprises one or more Locked Nucleic Acids (LNA) or Peptide Nucleic Acids (PNA).

12. The method of any one of claims 1-11, further comprising monitoring the reaction for signal loss and rephasing to restore sequencing signal.

13. A method for reducing lead phase error in parallel or clonal polynucleotide sequencing, the method comprising:

sequencing a population of target polynucleotides in the presence of a competition reaction comprising nucleotide bases or nucleotide derivatives for all four nucleotide bases, wherein three of the four nucleotide bases are not incorporable into a growing polynucleotide strand;

Wherein the polynucleotide sequencing optionally comprises one or more of:

sequencing clones of the bead array,

electronic detection of nucleotide incorporation, and

an electron aperture for separating or concentrating the reaction components.

14. The method of claim 13, wherein at least one unincorporable nucleotide is selected from PNA nucleotides, LNA nucleotides, ribonucleotides, adenine monophosphate, adenine diphosphate, adenosine, deoxyadenosine, guanine monophosphate, guanine diphosphate guanosine, deoxyguanosine, thymine monophosphate, thymine diphosphate thymine, 5-methyluridine, thymidine, cytosine monophosphate, cytosine diphosphate cytidine, deoxycytidine, uracil monophosphate, uracil diphosphate, uridine, and deoxyuridine.

15. The method of claim 14, wherein the unincorporable nucleotide is bound by a polymerase, but cannot be incorporated into the growing polynucleotide strand by the polymerase.

16. The method of claim 14, wherein the concentration of the non-incorporable nucleotides is related to the activity of a polymerase for each non-incorporable nucleotide.

17. A method for reducing lag phase error in parallel or clonal polynucleotide sequencing, the method comprising:

Stacking a polymerase on or near a target polynucleotide population during a sequencing reaction such that the polymerase is substantially available for each active polymerization site and/or binds a repair protein or single-stranded DNA binding protein to the target polynucleotide population;

wherein the polynucleotide sequencing optionally comprises one or more of:

sequencing clones of the bead array,

electronic detection of nucleotide incorporation, and

an electron aperture for separating or concentrating the reaction components.

18. The method of claim 17, wherein the stacking is achieved by natural binding of a polymerase to the bound non-extendable primer.

19. The method of claim 18, wherein the non-extendable primer is not affected by the 3' exonuclease activity of the polymerase.

20. The method of claim 19, wherein the non-extendable primer is a 3 ' terminated random primer and the extension primer is a universal primer or a targeting primer, and wherein a polymerase binds at the 3 ' terminating end of the random primer and at the 3 ' end of the universal primer or the targeting primer.

21. The method of claim 20, wherein the 3 ' terminated primer comprises a phosphorothioate nucleotide at the 3 ' termination position such that the 3 ' terminated primer is resistant to 3 ' to 5 ' exonuclease activity.

22. A method for repeated nucleotide sequencing, comprising:

providing a circularised DNA sequencing template and sequencing said template by determining the order in which nucleotides are incorporated by a DNA polymerase having 5 'to 3' exonuclease activity;

wherein the polynucleotide sequencing optionally comprises one or more of:

sequencing clones of the bead array,

electronic detection of nucleotide incorporation, and

an electron aperture for separating or concentrating the reaction components.

23. The method of claim 22, wherein the DNA polymerase is highly processive and has reduced exonuclease activity, and wherein the highly processive polymerase binds on or near a biosensor suitable for determining incorporation of nucleotides.

24. The method of claim 23, further comprising melting the extension primer by one of a temperature change and a pH change.

25. The method of any one of claims 1-24, further comprising pre-binding a polymerase to the polynucleotide prior to sequencing.

26. The method of any one of claims 1-25, further comprising providing the target polynucleotide or template polynucleotide on a bead and contacting the bead with a polymerase/DNA primer complex.

27. A chamber-less device, comprising:

an array of electromagnetic sensors is provided which is,

a magnetic carrier for carrying or retaining a molecule of interest on or in the vicinity of the electromagnetic sensor, and

a mechanism for removing the magnetic carrier by liquid flow and/or electromagnetic removal.

28. The apparatus of claim 27, wherein the electromagnetic sensor is one of a nanoneedle or a nanobridge, and wherein the apparatus further comprises a local amplifier.

29. The apparatus of claim 28, wherein the electromagnetic sensor has a narrow structure and is etched under the structure to make it easy for both sides of the sensor surface to obtain a change in pH or a change in conductivity.

30. A method for sequencing a single DNA molecule, the method comprising:

attaching a polymerase to a biosensor in a volume and allowing the DNA sample with the associated primer to enter the volume and be retained by or in proximity to the polymerase;

determining the order of nucleotide incorporation after the primer is extended by the polymerase.

31. An apparatus for sequencing a polynucleotide, the apparatus comprising: a sensing surface for sensing nucleotide incorporation, the sensing surface comprising a silicon nitride layer, and TiO ₂、ZrO₂And BaTiO₃Thereby lowering the zeta potential.

32. The apparatus of claim 31, further comprising: a plurality of magnetic beads configured to carry a template polynucleotide, wherein the magnetic beads have a material with a low zeta potential at a pH level effective for nucleotide incorporation.

33. The device of claim 32, comprising a plurality of nanoneedle sensors having at least one electrode formed in a circular arc shape conforming to an edge of a recess in which one of the plurality of magnetic beads is disposed.

34. The device of claim 32, comprising a plurality of nanobridge sensors having an active region partially surrounding and in close proximity to one of the plurality of magnetic beads.

35. The device of claim 34, wherein the radius of the active region is smaller than the radius of the magnetic bead.

36. The device of claim 34, wherein the plurality of nanobridge sensors are adapted to determine the incorporation of nucleotides onto a polynucleotide, the nanobridge sensors each having an active region and a conductive element having a work function matching the work function of the active region.