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WO2024145441A1 - Procédés, compositions et kits pour déterminer un emplacement d'un acide nucléique cible dans un échantillon biologique fixe - Google Patents

Procédés, compositions et kits pour déterminer un emplacement d'un acide nucléique cible dans un échantillon biologique fixe Download PDF

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Publication number
WO2024145441A1
WO2024145441A1 PCT/US2023/086181 US2023086181W WO2024145441A1 WO 2024145441 A1 WO2024145441 A1 WO 2024145441A1 US 2023086181 W US2023086181 W US 2023086181W WO 2024145441 A1 WO2024145441 A1 WO 2024145441A1
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Prior art keywords
capture
rna
biological sample
sequence
nucleic acid
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PCT/US2023/086181
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English (en)
Inventor
Layla KATIRAEE
Ashley HAYES
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10X Genomics, Inc.
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Application filed by 10X Genomics, Inc. filed Critical 10X Genomics, Inc.
Publication of WO2024145441A1 publication Critical patent/WO2024145441A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the capture probe includes a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof.
  • the method includes permeabilizing the biological sample.
  • the permeabilizing includes use of a protease.
  • the protease includes pepsin or proteinase K.
  • the method includes generating a polynucleotide including: (i) the spatial barcode or a complement thereof; (ii) the analyte capture sequence or a complement thereof; and (iii) all or a portion of the sequence of the target nucleic acid or a complement thereof.
  • kits including: (a) an array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) a plurality of analyte capture sequences, where an analyte capture sequence of the plurality of analyte capture sequences is capable of hybridizing to the capture domain and includes a pre-adenylated 5’ end and a blocked 3’ end; and (c) a ligase.
  • the capture probe includes one or more functional domains, a cleavage domain, a unique molecular identifier, or a combination thereof.
  • the analyte capture sequence includes a homopolymeric nucleotide sequence.
  • the homopolymeric nucleotide sequence includes a poly(A) sequence.
  • the ligase includes an RNA ligase, preferably a T4 RNA ligase, more preferably T4 RNA ligase 2.
  • the kit includes one or more permeabilization reagents.
  • the one or more permeabilization reagents includes a protease, and optionally, where the protease includes proteinase K, pepsin, or collagenase.
  • the RNA is an mRNA, rRNA, tRNA, miRNA, lincRNA, antisense RNA, viral RNA, siRNA, snoRNA, or piRNA.
  • the substrate is the array. In some embodiments, the substrate is a glass slide. In some embodiments, the method includes aligning the substrate with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array, optionally where the array is included in a second substrate.
  • incorporating the analyte capture sequence includes ligating the analyte capture sequence to the 3’ end of the RNA.
  • the ligating includes the use of a ligase.
  • the ligase includes an RNA ligase, preferably a T4 RNA ligase, more preferably T4 RNA ligase 2.
  • the method includes imaging the biological sample. In some embodiments, the method includes staining the biological sample. In some embodiments, the staining includes hematoxylin and/or eosin staining. In some embodiments, the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof. In some embodiments, the capture probe includes a cleavage domain, one or more functional domains, a unique molecular identifier, and combinations thereof.
  • the method includes permeabilizing the biological sample.
  • the permeabilizing includes the use of a protease.
  • the protease includes pepsin, collagenase, or proteinase K.
  • the array includes one or more features selected from the group consisting of: a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead.
  • the method includes migrating the product from the biological sample to the array.
  • the migrating includes electrophoresis.
  • the biological sample is a tissue sample. In some embodiments, the tissue sample is a fresh-frozen tissue sample. In some embodiments, the tissue sample is a fixed tissue sample, and optionally, where the fixed tissue sample is a formalin-fixed paraffin-embedded tissue sample, an acetone-fixed tissue sample, a methanol- fixed tissue sample, or a paraformaldehyde-fixed tissue sample. In some embodiments, the biological sample is a tissue section. In some embodiments, the tissue section is a fresh- frozen tissue section. In some embodiments, the tissue section is a fixed tissue section. In some embodiments, the fixed tissue section is a formalin-fixed paraffin-embedded tissue section, an acetone- fixed tissue section, a methanol-fixed tissue section, or a paraformaldehyde-fixed tissue section.
  • the method includes contacting the biological sample with a DNase.
  • the 3’ blocked end of the analyte capture sequence includes one or more carbon atoms. In some embodiments, the 3’ blocked end of the analyte capture sequence includes a biotin moiety. In some embodiments, the 3’ blocked end of the analyte capture sequence includes one or more inverted nucleotides.
  • the analyte capture sequence includes a homopolymeric nucleotide sequence including DNA or RNA. In some embodiments, the homopolymeric nucleotide sequence includes from about 20 nucleotides to about 50 nucleotides. In some embodiments, the homopolymeric nucleotide sequence includes from about 25 nucleotides to about 35 nucleotides. In some embodiments, the homopolymeric nucleotide sequence includes a poly(A) sequence.
  • the method includes contacting the biological sample with one or more ribosomal RNA depletion probes.
  • the one or more ribosomal RNA depletion probes includes nucleic acid probes complementary to ribosomal RNA and a binding moiety.
  • the one or more ribosomal RNA depletion probes hybridize to the ribosomal RNA, thereby generating a ribosomal depletion probe/ribosomal RNA complex.
  • the binding moiety is biotin.
  • the method includes contacting the biological sample with one or more mitochondrial RNA depletion probes.
  • the one or more mitochondrial RNA depletion probes includes nucleic acid probes complementary to mitochondrial RNA and a binding moiety.
  • the one or more mitochondrial RNA depletion probes hybridize to the mitochondrial RNA, thereby generating a mitochondrial depletion probe/mitochondrial RNA complex.
  • the binding moiety is biotin.
  • the ribosomal depletion probe/ribosomal RNA complex and/or the mitochondrial depletion probe/mitochondrial RNA complex are removed.
  • the removal includes the use of an RNase.
  • the RNase is RNase Hl, RNase H2, or a thermostable RNase H.
  • the removal includes the use of streptavidin.
  • compositions including: (a) a target nucleic acid, where the target nucleic acid is a non-polyadenylated nucleic acid or a truncated polyadenylated nucleic acid; (b) an analyte capture sequence including a 5’ pre-adenylated end and a 3’ blocked end, where the analyte capture sequence is capable of hybridizing to a capture domain of a capture probe; and (c) a ligase.
  • the ligase includes RNA ligase 2.
  • the 3’ blocked end of the analyte capture sequence includes one or more carbon atoms. In some embodiments, the 3’ blocked end of the analyte capture sequence includes a biotin moiety. In some embodiments, the 3’ blocked end of the analyte capture sequence includes one or more inverted nucleotides.
  • the analyte capture sequence includes a homopolymeric nucleotide sequence including DNA or RNA. In some embodiments, the homopolymeric nucleotide sequence includes from about 20 nucleotides to about 50 nucleotides. In some embodiments, the homopolymeric nucleotide sequence includes about 30 nucleotides. In some embodiments, the homopolymeric nucleotide sequence includes a poly(A) sequence.
  • the target nucleic acid is an RNA.
  • the RNA is mRNA, rRNA, tRNA, miRNA, viral RNA, siRNA, snoRNA, or piRNA.
  • the target nucleic acid is a non-polyadenylated target nucleic acid or a truncated polyadenylated target nucleic acid.
  • FIG. 2A shows a perspective view of an exemplary sample handling apparatus in a closed position.
  • FIG. 3A shows the first substrate angled over (superior to) the second substrate.
  • FIG. 3B shows that as the first substrate lowers, and/or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium.
  • FIG. 7 shows exemplary 7 capture domains on capture probes.
  • Spatial analysis methodologies described herein can provide a vast amount of analyte and/or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context.
  • Spatial analysis methods can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or a nucleic acid) produced by and/or present in a cell.
  • a spatial barcode e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample
  • a capture domain that is capable of binding to an analyte (e.g.,
  • Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
  • non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments.
  • viral proteins e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.
  • the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • organelles e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc.
  • analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
  • an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • an intermediate agent for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.
  • a “biological sample” is ty pically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery', and laser capture microscopy’ (LCM), and generally includes cells and/or other biological material from the subject.
  • the biological sample is a tissue sample.
  • the biological sample e.g., tissue sample
  • TMA tissue microarray
  • a tissue microarray contains multiple representative tissue samples - which can be from different tissues or organisms - assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time.
  • Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates.
  • the biological sample e.g.. the tissue
  • a matrix e.g., optimal cutting temperature (OCT) compound to facilitate sectioning.
  • OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens.
  • the sectioning is performed by cryosectioning, for example using a microtome .
  • the methods further comprise a thawing step, after the cryosectioning.
  • the biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat.
  • a biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX).
  • the biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy.
  • Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and/or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities.
  • an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid.
  • the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetonemethanol mixture). In some instances when methanol, acetone, or an acetone-methanol mixture is used to fix the biological sample, the sample is not decrosslinked at a later step. In instances when the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone-methanol) fixation or acetone fixation, the biological sample is referred to as “fresh frozen”.
  • the biological sample can be fixed using PAXgene.
  • the biological sample can be fixed using PAXgene in addition, or alternatively to, a fixative disclosed herein or known in the art (e.g.. alcohol, acetone, acetone-alcohol. formalin, paraformaldehyde).
  • PAXgene is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and bio-molecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B. et al.. J Proteome Res.
  • RNA integrity of fixed (e.g., FFPE) samples can be low er than a fresh sample, thereby making it more difficult to capture RNA directly, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule.
  • RTL probes that hybridize to RNA target sequences in the transcriptome, one can avoid a requirement for RNA analytes to have both a poly(A) tail and target sequences intact. Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples.
  • the tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject.
  • the sample is a mouse sample.
  • the sample is a human sample.
  • the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen.
  • the sample is a human or mouse breast tissue sample.
  • the sample is a human or mouse brain tissue sample.
  • the sample is a human or mouse lung tissue sample.
  • the sample is a human or mouse tonsil tissue sample.
  • a capture probe can include a cleavage domain and/or a functional domain (e.g., a primer-binding site, such as for nextgeneration sequencing (NGS)).
  • NGS nextgeneration sequencing
  • FIG. 1A shows an exemplary' sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102 (e g., a parasitic organism), and a second substrate (e.g., array slide 104 including an array having spatially barcoded capture probes 106) are brought into proximity with one another.
  • a liquid reagent drop e.g., permeabilization solution 105
  • the permeabilization solution 105 may release analytes or analyte derivatives (e g., intermediate agents; e.g., ligation products) that can be captured by the capture probes of the array 106.
  • one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including spatially barcoded capture probes 106).
  • the one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.
  • the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and 500 microns, between about 2 microns and 400 microns, between about 2 microns and 300 microns, between about 2 microns and 200 microns, between about 2 microns and 100 microns, between about 2 microns and 25 microns, or between about 2 microns and 10 microns), measured in a direction orthogonal to the surface of first substrate that supports the biological sample.
  • a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and
  • the liquid reagent e.g., the permeabilization solution 105 fills the volume of the chamber 150 and may create a permeabilization buffer that allows analytes (e.g., mRNA transcripts and/or other molecules) or analyte derivatives (e.g., intermediate agents; e.g., ligation products) to diffuse from the biological sample 102 toward the capture probes of the second substrate (e.g., slide 104).
  • flow of the permeabilization buffer may deflect transcripts and/or molecules from the biological sample 102 and may affect diffusive transfer of analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) for spatial analysis.
  • a partially or fully sealed chamber 150 resulting from the one or more spacers 110, the first substrate, and the second substrate may reduce or prevent flow from undesirable convective movement of transcripts and/or molecules over the diffusive transfer from the biological sample 102 to the capture probes.
  • the sandwiching process methods described above can be implemented using a variety of hardware components.
  • the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sample handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in, e.g., US. Patent Application Pub. No. 2021/0189475, and PCT Publ. No. WO 2022/061152 A2. each of which are incorporated by reference in their entirely.
  • an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and/or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration).
  • the adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration.
  • spacers may be applied to the first substrate 206 and/or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching.
  • the permeabilization solution e.g., permeabilization solution 305
  • the first member 204 may then close over the second member 210 and form the sandwich configuration.
  • Analytes or analyte derivatives e.g., intermediate agents; e.g., ligation products
  • the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and/or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas.
  • FIGs. 3A-3C depict a side view and a top view of an exemplary angled closure workflow 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary implementations.
  • a first substrate e.g., slide 303
  • a second substrate e.g., slide 304 having capture probes 306
  • FIG. 3A depicts the first substrate (e.g., the slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304).
  • reagent medium e.g., permeabilization solution
  • FIG. 3A depicts the reagent medium on the right hand side of side view, it should be understood that such depiction is not meant to be limiting as to the location of the reagent medium on the spacer.
  • the first substrate and/or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate.
  • FIG. 3C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the second substrate and maintaining a separation distance and optionally the approximately parallel arrangement between the two substrates.
  • the spacer 310 fully encloses and surrounds the biological sample 302 and the capture probes 306, and the spacer 310 form the sides of chamber 350 which holds a volume of the reagent medium 305
  • FIG. 3C depicts the first substrate (e.g., the slide 303 including biological sample 302) angled over (superior to) the second substrate (e.g., slide 304) and the second substrate comprising the spacer 310.
  • an exemplary angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate comprising the spacer 310.
  • the reagent medium be free from air bubbles betw een the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present betw een the substrates may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and/or elimination of air bubbles betw een the two substrates (e.g., slide 303 and slide 304) during a permeabilization step (e.g., step 104). In some aspects, it may be possible to reduce or eliminate bubble formation between the substrates using a variety of filling methods and/or closing methods. In some instances, the first substrate and the second substrate are arranged in an angled sandwich assembly as described herein.
  • the substrate 406 is further lowered toward the substrate 402 (or the substrate 402 is raised up toward the substrate 406) and the dropped side of the substrate 406 may contact and may urge the reagent medium toward the side opposite the dropped side and creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the substrates.
  • the reagent medium 401 fills the gap between the substrate 406 and the substrate 402.
  • the linear flow front of the liquid reagent may form by squeezing the 401 volume along the contact side of the substrate 402 and/or the substrate 406. Additionally, capillary flow may also contribute to filling the gap area.
  • the reagent medium (e.g., 105 in FIG. 1A) comprises a permeabilization agent.
  • the permeabilization agent can be removed from contact with the biological sample (e.g., by opening sample holder).
  • Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X- 100TM, Tween-20TM, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K).
  • the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution).
  • a dried permeabilization reagent is applied or formed as a layer on the first substrate or the second substrate or both prior to contacting the biological sample and the array.
  • a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.
  • the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1 minute, about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45 minutes, or about an hour. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1-60 minutes.
  • the device is configured to control a temperature of the first and second substrates. In some embodiments, the temperature of the first and second members is lowered to a first temperature that is below room temperature.
  • a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and/or contents of the one or more cells, as associated with a particular spatial location.
  • One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes).
  • Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and/or into or onto the biological sample.
  • capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see. e.g., Section (II)(b)(vii) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663 regarding extended capture probes).
  • a template e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see. e.g., Section (II)(b)(vii) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020
  • capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that sen e as proxies for the template.
  • a template e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof
  • extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g.. sequencing.
  • extended capture probes e.g., DNA molecules
  • can act as templates for an amplification reaction e.g., a polymerase chain reaction.
  • the methods described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and/or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and/or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and/or selection of a treatment for a subject with a disease or disorder.
  • Exemplary methods for identifying spatial information of biological and/or medical importance can be found in U.
  • Analyte capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of PCT Publication No. WO2020/176788 and/or U.S. Patent Application Publication No. 2020/0277663.
  • FIG. 5 is a schematic diagram showing an exemplary capture probe, as described herein.
  • the capture probe 502 is optionally coupled to a feature 501 by a cleavage domain 503, such as a disulfide linker.
  • the capture probe can include a functional sequence 504 that are useful for subsequent processing.
  • the functional sequence 504 can include all or a part of sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence), all or a part of a sequencing primer sequence, (e.g., a R1 primer binding site, a R2 primer binding site), or combinations thereof.
  • the capture probe can also include a spatial barcode 505.
  • the capture probe can also include a unique molecular identifier (UMI) sequence 506. While FIG.
  • UMI unique molecular identifier
  • Such splint oligonucleotide in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence complementary' to a sequence of a nucleic acid analyte, a portion of a connected probe described herein, a capture handle sequence described herein, and/or a methylated adaptor described herein.
  • the feature 701 can be coupled to spatially -barcoded capture probes, wherein the spatially-barcoded probes of a particular feature can possess the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with more than one target analyte.
  • a feature may be coupled to four different types of spatially -barcoded capture probes, each type of spatially-barcoded capture probe possessing the spatial barcode 702.
  • One type of capture probe associated with the feature includes the spatial barcode 702 in combination with a poly(T) capture domain 703, designed to capture mRNA target analytes.
  • a second type of capture probe associated with the feature includes the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis.
  • the spatial barcode 505 and functional sequences 504 is common to all of the probes attached to a given feature.
  • the UMI sequence 506 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.
  • a polymerase e.g., a DNA polymerase
  • the ligation product is released from the analyte.
  • the ligation product is released using an endonuclease (e.g.. RNase H).
  • the ligation product is removed using heat.
  • the ligation product is removed using KOH.
  • the released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.
  • the extended ligation products can be denatured 9014 from the capture probe and transferred (e.g., to a clean tube) for amplification, and/or library construction.
  • the spatially-barcoded ligation products can be amplified 9015 via PCR prior to library construction.
  • P5 9016 and P7 9019 can be used as sequences that are complementary to sequencing probes for immobilization of the library on the sequencing flow cell and i5 9017 and i7 9018 can be used as sample indexes.
  • the amplicons can then be sequenced using paired-end sequencing using TruSeq Read I and TruSeq Read 2 as sequencing primer sites.
  • an analyte binding moiety’ barcode (or portion thereof) may be able to be removed (e g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of PCT Publication No. WO2020/176788 and/or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020/0277663.
  • FIG. 10 is a schematic diagram of an exemplary analyte capture agent 1002 comprised of an analyte-binding moiety 1004 and an analyte-binding moiety barcode domain 1008.
  • the exemplary’ analyte-binding moiety 1004 is a molecule capable of binding to an analyte 1006 and the analyte capture agent is capable of interacting with a spatially-barcoded capture probe.
  • the analyte-binding moiety can bind to the analyte 1006 with high affinity and/or with high specificity.
  • FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 1124 and an analyte capture agent 1126.
  • the feature-immobilized capture probe 1124 can include a spatial barcode 1108 as well as functional sequences 1106 and a UMI 1110, as described elsewhere herein.
  • the capture probe can be affixed 1104 to a feature such as a bead 1102.
  • the capture probe can also include a capture domain 1112 that is capable of binding to an analyte capture agent 1126.
  • the analyte-binding moiety barcode domain of the analyte capture agent 1126 can include a functional sequence 1118, analyte binding moiety’ barcode 1116, and an analyte capture sequence 1114 that is capable of binding (e.g., hybridizing) to the capture domain 1112 of the capture probe 1124.
  • the analyte capture agent can also include a linker 1120 that allows the analyte-binding moiety barcode domain (e.g.. including the functional sequence 1118. analyte binding barcode 1116. and analyte capture sequence 1114) to couple to the analyte binding moiety 1122.
  • the linker is a cleavable linker.
  • the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker.
  • the cleavable linker is a disulfide linker.
  • a disulfide linker can be cleaved by use of a reducing agent, such as dithiothreitol (DTT), Beta-mercaptoethanol (BME), or Tris (2- carboxyethyl) phosphine (TCEP).
  • sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample.
  • Various methods can be used to obtain the spatial information.
  • specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate.
  • specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.
  • specific spatial barcodes can be deposited at predetermined locations in an array of features during fabncation such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array.
  • the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.
  • Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample.
  • the biological sample can be mounted for example, in a biological sample holder.
  • One or more fluid chambers can be connected to the chamber and/or the sample holder via fluid conduits, and fluids can be delivered into the chamber and/or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow.
  • One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and/or sample holder.
  • the biological sample Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and/or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and/or level. Exemplary methods to generate a two- and/or three-dimensional map of the analyte presence and/or level are described in PCT Publication No. W02020/053655 and spatial analysis methods are generally described in PCT Publication No. W02021/102039 and/or U.S. Patent Application Publication No. 2021/0155982. each of which is incorporated herein by reference in their entireties.
  • Also provided herein are methods for processing a target nucleic acid in a biological sample including: (a) providing an array including a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a spatial barcode and (ii) a capture domain; (b) incorporating an analyte capture sequence to a 3 ’ end of the target nucleic acid in the biological sample, where the biological sample is disposed on a substrate, and where the analyte capture sequence includes a pre-adenylated 5’ end and a blocked 3’ end, thereby generating a product; (c) hybridizing the analyte capture sequence of the product to the capture domain of the capture probe; and (d) extending the capture probe using the product as an extension template, thereby generating an extended capture probe.
  • staining the fresh-frozen biological sample includes the use of a biological stain including, but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, safranin. or any combination thereof.
  • significant time e.g., days, months, or years
  • the ligase is T4 RNA ligase 2. In some embodiments, the ligase is thermostable 5’ App DNA/RNA ligase. In some embodiments, the analyte capture sequence is covalently attached to a 3 ' end of the target nucleic acid.
  • the target nucleic acid is an RNA.
  • the RNA is mRNA, rRNA, tRNA, miRNA, viral RNA, siRNA, snoRNA, or piRNA.
  • RNA molecules in fixed biological samples generally have degraded or missing poly(A) tails.
  • Typical capture on a spatial array relies on the presence of a poly(A) sequence present in the RNA molecule to hybridize with a poly(T) capture domain.
  • the T4 Rnl2 enzyme does not require ATP for ligation, however, it does require a pre-adenylated substrate for successful ligation which also confers specificity during the ligation reaction. Additionally, the pre-adenylated oligonucleotide included a block at its 3’ end to prevent ligation of the pre-adenylated oligonucleotide to another pre-adenylated oligonucleotide. In this experiment, a three carbon atom spacer was used as a 3‘ blocker, however, other 3 ’ blocking mechanisms are known in the art and described herein.

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Abstract

La présente invention concerne des procédés, des compositions et des kits pour déterminer l'emplacement d'acides nucléiques cibles et/ou traiter des acides nucléiques cibles à partir d'échantillons biologiques. Les procédés comprennent généralement l'incorporation d'une séquence de capture d'analyte à une extrémité d'un analyte d'acide nucléique dans un échantillon biologique in situ pour faciliter la capture ultérieure de l'analyte sur un substrat comprenant un réseau de sondes de capture. Dans certains modes de réalisation, la séquence de capture d'analyte comprend une séquence nucléotidique homopolymère, telle qu'une séquence poly(A).
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