Methods, compositions, and kits for spatial detection of genetic variants

Abstract

The present disclosure features methods, compositions, and kits for the spatial detection of genetic variants in a biological sample by using branched ligation.

Description

[0001] METHODS, COMPOSITIONS, AND KITS FOR SPATIAL DETECTION OF GENETIC VARIANTS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 516,651, filed July 31. 2023, which is herein incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] Cells within a tissue of a subject have differences in cell morphology and / or function due to varied analyte levels (e.g., gene and / or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment) can affect, e.g., the cell’s morphology, differentiation, fate, viability7, proliferation, behavior, signaling and cross-talk with other cells in the tissue.

[0006] Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provides substantial analyte data for dissociated tissue (i.e., single cells), but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).

[0007] High-throughput methods are available for spatial analysis to determine the identity, abundance, and distribution of nucleic acid genetic variants within cells of a biological sample, for example, a tissue sample or tissue section. Such methods include array-based spatial transcriptomics assays. The biological sample can be placed on an array or on a substrate and aligned with an array7to improve specificity and efficiency for the identification and / or characterization of a nucleic acid (e.g., DNA or RNA) within the biological sample. Increased sensitivity7for differentiating between wild-ty pe nucleic acids and genetic variant nucleic acids is important for accurately identifying genetic variants. For example, during genetic variant analysis, probes specific to either wild-type nucleic acids or nucleic acids with genetic variants can compete against each other resulting in inaccurate determination of the presence, location, and / or frequency of a genetic variant. Therefore, there exists a need for improved compositions, methods, and kits with increased accuracy and sensitivity7for identifying genetic variants (e.g., single nucleotide variants or polymorphisms, insertions, deletions, etc.) in spatial analysis assays.

[0008] SUMMARY The present disclosure features methods, compositions, and kits that can be used to analyze the presence, location, and / or frequency of genetic variants in a target nucleic acid in a biological sample using a target specific nucleic acid probe (e.g., a first probe), a blocking oligonucleotide, and a second probe in combination with capture probes in an array format. A probe (e.g., a first probe) hybridizes to a target nucleic acid at a region that is downstream of the location of a possible genetic variant. When the first probe is extended, for example, with a DNA polymerase or a reverse transcnptase (RT), the extended probe will incorporate a complementary sequence of a genetic variant or w ild-type sequence. However, accuracy of the extended probe incorporating the complementary sequence of the genetic variant depends on several factors. Mistakes in extension by DNA polymerase or RT or competition between probes in traditional templated ligation reactions can produce inaccurate readouts of the presence, location, and / or frequency of genetic variants in the biological sample. Thus, there is a need for improved methods, compositions, and kits with increased accuracy and / or sensitivity7when determining the presence, location, and / or frequency of genetic variants.

[0009] Thus, included herein are methods, compositions, and kits, for determining the presence, location, and / or frequency of genetic variants with an increased accuracy and / or sensitivity in spatial analysis assays. More specifically, the methods, compositions, and kits disclosed herein include a blocking oligonucleotide including: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3' blocking moiety. The blocking oligonucleotide prevents the first probe from being extended beyond the blocking oligonucleotide such that the extended probe can be ligated to a second probe via branch ligation. By designing a known stopping point for the extension, extended probes, including either wild-type sequences or genetic variant sequences, the resulting ligation products can be about the same length and more conducive for subsequent analysis (e.g., sequencing), leading to increased sensitivity and accuracy.

[0010] Thus provided herein are methods for determining a location of a genetic variant in a target nucleic acid in a biological sample, the method including: (a) providing an arrayincluding 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) contacting a plurality of first probes and a plurality of blocking oligonucleotides w ith the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary7to the target nucleic acid at a region that is downstream of the genetic variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary' to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety ; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid; (d) extending the first probe using the target nucleic acid as a template, thereby generating an extended first probe, where the extended first probe includes a sequence complementary to the genetic variant; (e) contacting a plurality of second probes with the biological sample, where a second probe of the plurality7of second probes includes a partially double-stranded nucleic acid molecule including a capture probe capture domain; (f) ligating the extended first probe and the second probe, thereby generating a ligation product; (g) hybridizing the ligation product to the capture probe capture domain; and (h) determining (i) all or a portion of the sequence of the ligation product, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the genetic variant in the target nucleic acid in the biological sample.

[0011] In some embodiments, the 3' blocking moiety is selected from the group consisting of: a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

[0012] In some embodiments, the genetic variant includes a single nucleotide variant or polymorphism. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more single nucleotide variants or polymorphisms.

[0013] In some embodiments, second probe includes a 5' phosphate group.

[0014] In some embodiments, the capture probe capture domain is substantially complementary7to the capture probe capture domain on the array.

[0015] In some embodiments, the ligating includes chemical ligation or enzy matic ligation. In some embodiments, the enzymatic ligation includes use of a ligase. In some embodiments, the ligase includes a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some embodiments, the ligase includes a pre-activated T4 DNA ligase.

[0016] In some embodiments, the extending the first probe using the target nucleic acid as a template in (d) includes the use of a polymerase or a reverse transcriptase. In some embodiments, the reverse transcriptase is a non-displacing reverse transcriptase. In some embodiments, the non-displacing reverse transcriptase includes a Thermococcus gorgonarius mutant reverse transcription xenopolymerase.

[0017] In some embodiments, the method includes releasing the ligation product from the target nucleic acid. In some embodiments, the releasing includes the use of an endoribonuclease. In some embodiments, the endoribonuclease includes RNase H, RNase A, RNase C, and / or RNase I, and optionally, where the RNase H includes RNase Hl, RNase H2, or both.

[0018] In some embodiments, the capture probe includes one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.

[0019] In some embodiments, the method includes contacting the biological sample with a permeabilization reagent. In some embodiments, the permeabilization reagent includes pepsin, proteinase K. or collagenase.

[0020] In some embodiments, the method includes extending the capture probe using the ligation product as a template, thereby generating an extended capture probe. In some embodiments, the method includes extending the ligation product using the capture probe as a template, thereby generating an extended ligation product. In some embodiments, the method includes amplifying the extended capture probe or the extended ligation product.

[0021] In some embodiments, the determining in (h) includes sequencing.

[0022] In some embodiments, the biological sample is disposed on the array. In some embodiments, the biological sample is disposed on a substrate. In some embodiments, the method includes aligning the substrate including the biological sample with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array.

[0023] In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample is a 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, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone-fixed tissue section. In some embodiments, the tissue section is a fresh frozen tissue section. In some embodiments, the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.

[0024] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, optionally, mRNA.

[0025] In some embodiments, the first probe includes a functional domain, where the functional domain is a primer binding site or a sequence specific site.

[0026] In some embodiments, a 3' end of the first probe includes one or more mismatched nucleotides. In some embodiments, a 3' end of the first probe includes a dideoxynucleotide. In some embodiments, the dideoxynucleotide includes ddATP, ddCTP, ddGTP, or ddTTP.

[0027] Also provided herein are methods for determining a presence or absence of a single nucleotide variant in a biological sample, the method 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) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is downstream of the single nucleotide variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic at a region that is upstream of the single nucleotide variant and (ii) a 3' blocking moiety; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid; (d) extending the first probe using the target nucleic acid as a template, thereby generating an extended first probe; (e) contacting a plurality of second probes with the biological sample, where a second probe of the plurality of second probes includes a partially double-stranded nucleic acid molecule including a capture probe capture domain; (f) ligating the extended first probe and the second probe, thereby generating a ligation product; (g) hybridizing the ligation product to the capture probe capture domain; and (h) determining (i) all or part of the sequence of the ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the presence or absence of the single nucleotide variant in the biological sample.

[0028] In some embodiments, the determining in (h) includes correlating a location of the single nucleotide variant on the array to a location of the single nucleotide variant in the biological sample.

[0029] In some embodiments, the 3' blocking moiety is selected from the group consisting of: a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

[0030] In some embodiments, the target nucleic acid includes at least one single nucleotide variant. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more single nucleotide variants.

[0031] In some embodiments, the second probe includes a 5' phosphate group. In some embodiments, the second probe includes a capture probe capture domain that is substantially complementary to the capture probe capture domain on the array.

[0032] In some embodiments, the ligating includes chemical ligation or enzymatic ligation. In some embodiments, the enzymatic ligation includes use of a ligase. In some embodiments, the ligase includes a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some embodiments, the ligase includes a pre-activated T4 DNA ligase. In some embodiments, the extending the first probe using the target nucleic acid as a template in (d) includes the use of a polymerase or a reverse transcriptase. In some embodiments, the reverse transcriptase enzyme is a non-displacing reverse transcriptase.

[0033] In some embodiments, the method includes releasing the ligation product from the target nucleic acid. In some embodiments, the releasing includes the use of an endoribonuclease. In some embodiments, the endoribonuclease includes RNase H, RNase A, RNase C, and / or RNase I, and optionally, where the RNase H includes RNase Hl, RNase H2, or both.

[0034] In some embodiments, the capture probe includes one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.

[0035] In some embodiments, the method includes contacting the biological sample with a permeabilization reagent. In some embodiments, the permeabilization reagent includes pepsin, proteinase K, or collagenase.

[0036] In some embodiments, the method includes extending the capture probe using the ligation product as a template thereby generating an extended capture probe. In some embodiments, the method includes extending the ligation product using the capture probe as a template, thereby generating an extended ligation product.

[0037] In some embodiments, the method includes amplifying the extended capture probe and / or the extended ligation product.

[0038] In some embodiments, the determining in (h) includes sequencing.

[0039] In some embodiments, the biological sample is disposed on the array. In some embodiments, the biological sample is disposed on a substrate. In some embodiments, the method includes aligning the substrate including the biological sample with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array.

[0040] In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample is a 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, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone-fixed tissue section. In some embodiments, the tissue section is a fresh frozen tissue section. In some embodiments, the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.

[0041] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, optionally, mRNA. In some embodiments, the first probe includes a functional domain, where the functional domain is a primer binding site or a sequence specific site.

[0042] In some embodiments, a 3' end of the first probe includes one or more mismatched nucleotides. In some embodiments, a 3' end of the first probe includes a dideoxynucleotide. In some embodiments, the dideoxynucleotide includes ddATP, ddCTP, ddGTP, or ddTTP.

[0043] Also provided herein are compositions including: (a) a spatial 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 first probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the first probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe including a partially double-stranded nucleic acid molecule, where the second probe includes a capture probe capture domain.

[0044] In some embodiments, the 3' blocking moiety is selected from the group consisting of a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

[0045] In some embodiments, the target nucleic acid includes a genetic variant. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more genetic variants. In some embodiments, the genetic variant includes an insertion or a deletion. In some embodiments, the genetic variant includes a single nucleotide variant or polymorphism.

[0046] In some embodiments, the second probe includes a 5' phosphate group. In some embodiments, the second probe includes a capture probe capture domain that is substantially complementary to the capture probe capture domain on the array.

[0047] In some embodiments, the composition includes a polymerase or a reverse transcriptase. In some embodiments, the reverse transcriptase is a non-displacing reverse transcriptase.

[0048] In some embodiments, the composition includes a biological sample. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a 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, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone- fixed tissue section. In some embodiments, the tissue section is a fresh frozen tissue section. In some embodiments, the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.

[0049] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is RNA, optionally, mRNA.

[0050] In some embodiments, a 3' end of the first probe includes one or more mismatched nucleotides. In some embodiments, a 3' end of the first probe includes a dideoxynucleotide. In some embodiments, the dideoxynucleotide includes ddATP, ddCTP. ddGTP, or ddTTP.

[0051] Also provided herein are kits including: (a) a spatial 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) (i) a plurality of first probes, where a first probe of the plurality of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is dow nstream of a genetic variant, and (ii) a plurality of second probes, where a second probe of the plurality of second probes includes a partially double-stranded nucleic acid molecule and where the second probe includes a capture probe capture domain; (c) a plurality of blocking oligonucleotides, where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3' blocking moiety7; (d) a ligase; and (e) instructions for performing any of the methods described herein.

[0052] In some embodiments, the capture probe includes one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.

[0053] In some embodiments, the ligase includes a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some embodiments, the ligase is a pre-activated T4 DNA ligase.

[0054] In some embodiments, the kit includes a polymerase. In some embodiments, the kit includes a reverse transcriptase. In some embodiments, the reverse transcriptase enzyme is a non-displacing reverse transcriptase.

[0055] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0056] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0057] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

[0060] FIG. 1A shows an exemplary sandwiching process where a first substrate (e.g., a slide), including a biological sample, and a second substrate (e g., array slide) are brought into proximity with one another.

[0061] FIG. IB shows a fully formed sandwich configuration creating a chamber formed from the one or more spacers, the first substrate, and the second substrate.

[0062] FIG. 2A shows a perspective view' of an exemplary sample handling apparatus in a closed position.

[0063] FIG. 2B shows a perspective view of an exemplary sample handling apparatus in an open position.

[0064] FIG. 3A shows the first substrate angled over (superior to) the second substrate.

[0065] 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.

[0066] FIG. 3C shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate. FIG. 4A shows a side view of the angled closure workflow.

[0067] FIG. 4B shows a top view of the angled closure workflow.

[0068] FIG. 5 is a schematic diagram showing an example of a barcoded capture probe, as described herein.

[0069] FIG. 6 shows a schematic illustrating a cleavable capture probe.

[0070] FIG. 7 shows exemplary capture domains on capture probes.

[0071] FIG. 8 shows an exemplary arrangement of barcoded features within an array.

[0072] FIG. 9A shows an exemplary workflow for performing templated capture and producing a ligation product, and FIG. 9B shows an exemplary' workflow for capturing a ligation product from FIG. 9A on a substrate.

[0073] FIG. 10 is a schematic diagram of an exemplary analyte capture agent.

[0074] FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 1124 and an analyte capture agent 1126.

[0075] FIG. 12 from top to bottom is a schematic diagram depicting a method of detecting a genetic variant (e.g., SNPs) using a first probe and a blocking oligonucleotide hybridized to a target nucleic acid. The first probe can be extended to incorporate complementary sequences of genetic variants until the extended probe reaches or nearly reaches the blocking oligonucleotide. After extension, partially double-stranded second probe including a capture probe capture domain can then be ligated to the extended probe.

[0076] FIG. 13 is a schematic diagram depicting specificity improvements of the method shown in FIG. 12 by incorporation of either a mismatched base (top) or a dideoxynucleotide (bottom) to limit extension of the first probe and non-specific ligation. Unextended first probes are shown on the far right.

[0077] DETAILED DESCRIPTION

[0078] A. Spatial Analysis Methods

[0079] 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. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.

[0080] Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos. 11,447,807, 11,352.667, 11,168,350, 11,104,936, 11,008,608, 10.995.361, 10,913,975. 10,774.374, 10,724,078, 10,640.816, 10.494,662, 10,480.022. 10,364,457, 10,317,321, 10,059,990, 10,041,949, 10,030,261, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, and 7,709,198; U.S. Patent Application Publication Nos. 2020 / 0239946, 2020 / 0080136, 2020 / 0277663, 2019 / 0330617, 2020 / 0256867. 2020 / 0224244, 2019 / 0085383, and 2013 / 0171621; PCT Publication Nos. WO2018 / 091676, W02020 / 176788, WO2017 / 144338, and WO2016 / 057552; Non-patent literature references Rodriques et al., Science 363(6434): 1463-1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2) :e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36: 1197-1202. 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e g., Rev F, dated January 2022); and / or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), both of which are available at the lOx Genomics Support Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in its entirety. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.

[0081] Some general terminology that may be used in this disclosure can be found in Section (I)(b) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. Typically, a '‘barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and / or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.

[0082] Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of 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. In some embodiments, 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. In some embodiments, 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, which is herein incorporated by reference. In some embodiments, 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.

[0083] A “biological sample” is typically 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. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample (e.g.. tissue sample) is a tissue microarray (TMA). 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 can be paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these tissue cores into a single recipient (microarray) block at defined array coordinates.

[0084] The biological sample as used herein can be any suitable biological sample described herein or known in the art. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a solid tissue sample. In some embodiments, the biological sample is a tissue section (e.g., a fixed tissue section). In some embodiments, the tissue is flash-frozen and sectioned. Any suitable method described herein or known in the art can be used to flash-freeze and section the tissue sample. In some embodiments, the biological sample, e.g., the tissue, is flash-frozen using liquid nitrogen before sectioning. In some embodiments, the biological sample, e.g., a tissue sample, is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane.

[0085] In some embodiments, the biological sample, e.g., the tissue, is embedded in 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. In some embodiments, the sectioning is performed by cryosectioning, for example, using a microtome. In some embodiments, the methods further comprise a thawing step, after the cryosectioning.

[0086] The biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat. In addition to the subjects described above, the biological sample can be obtained from non-mammalian organisms (e.g.. a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungus, an amphibian, or a fish (e.g., zebrafish)). A biological sample can be obtained from a prok ar ote such as a bacterium, e.g., Escherichia coll. Staphylococci or Mycoplasma pneumoniae,' an archaeon; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. 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. In some embodiments, 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. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy.

[0087] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0088] Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.

[0089] In some embodiments, the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, for example methanol. In some embodiments, instead of methanol, acetone or an acetone-methanol mixture can be used. In some embodiments, the fixation is performed after sectioning. In some instances, when the biological sample is fixed using a fixative including an alcohol (e.g.. methanol or acetone-methanol mixture), the biological sample is not decrosslinked afterward. In some preferred embodiments, the biological sample is fixed using a fixative including an alcohol (e.g., methanol or an acetone-methanol mixture) after freezing and / or sectioning. In some instances, the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone-methanol 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 frozen7’. In some embodiments, fixation of the biological sample e.g., using acetone and / or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g., glass slide, such as a positively charged glass slide).

[0090] In some embodiments, the biological sample, e.g.. the tissue sample, is fixed e.g., immediately after being harvested from a subject. In such embodiments, the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PF A) or formalin. In some embodiments, the fixative induces crosslinks within the biological sample. In some embodiments, after fixing e.g., by formalin or PF A, the biological sample is dehydrated via sucrose gradient. In some instances, the fixed biological sample is treated with a sucrose gradient and then embedded in a matrix e.g., OCT compound. In some instances, the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix e.g., OCT compound after fixation. In some embodiments, when a fixed frozen tissue sample is treated with a sucrose gradient, the sample can be rehydrated with an ethanol gradient. In some embodiments, the PFA or formalin fixed biological sample, which can be optionally dehydrated via sucrose gradient and / or embedded in OCT compound, is then frozen e.g., for storage or shipment. In such instances, the biological sample is referred to as “fixed frozen”. In preferred embodiments, a fixed frozen biological sample is not treated with methanol. In preferred embodiments, a fixed frozen biological sample is not paraffin embedded. Thus, in preferred embodiments, a fixed frozen biological sample is not deparaffinized. In some embodiments, a fixed frozen biological sample is rehydrated using an ethanol gradient.

[0091] In some instances, the biological sample (e.g., a fixed frozen tissue sample) is treated with a citrate buffer. Citrate buffer can be used for antigen retrieval to decrosslink antigens and fixation medium in the biological sample for antigen retrieval. Thus, any suitable decrosslinking agent can be used in addition to or alternatively to citrate buffer. In some embodiments, for example, the biological sample (e.g., a fixed frozen tissue sample) is decrosslinked using TE buffer.

[0092] In any of the foregoing, the biological sample can further be stained, imaged, and / or destained. For example, in some embodiments, a fresh frozen tissue sample or fixed frozen tissue sample is stained (e.g., via eosin and / or hematoxylin), imaged, destained (e.g.. via HC1), or a combination thereof. In some embodiments, when a fresh frozen tissue sample is fixed in methanol, the sample is treated with isopropanol prior to being stained (e.g., via eosin and / or hematoxylin), imaged, destained (e.g., via HC1), or a combination thereof. In some embodiments when a fixed frozen tissue sample is treated with a sucrose gradient, the sample can be rehydrated using an ethanol gradient before being stained, (e.g.. via eosin and / or hematoxylin), imaged, destained (e.g., via HC1), decrosslinked (e.g., via TE buffer or citrate buffer), or a combination thereof. In some embodiments, the biological sample can undergo further fixation (e.g., while mounted on a substrate), stained, imaged, and / or destained. For example, a fixed frozen biological sample may be subject to an additional fixing step (e.g., using PF A) before optional ethanol rehydration, staining, imaging, and / or destaining.

[0093] In any of the foregoing, the biological sample can be fixed using PAXgene. For example, 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, an acid, and a soluble organic compound that preserves morphology and bio-molecules. PAXgene provides 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. 2010 Oct 1 ;9(10):5188-96; Kap M. et al., PLoS One.; 6(ll):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol.; 146(l):25-40 (2016), each of which is hereby incorporated by reference in its entirety, for a description and evaluation of PAXgene for tissue fixation. Thus, in some embodiments, when the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, the fixative is PAXgene. In some embodiments, a fresh frozen tissue sample is fixed with PAXgene. In some embodiments, a fixed frozen tissue sample is fixed with PAXgene.

[0094] In some embodiments, the biological sample, e.g., the tissue sample is fixed, for example in methanol, acetone, acetone-methanol, PF A, PAXgene or is formalin-fixed and paraffin-embedded (FFPE). In some embodiments, the biological sample comprises intact cells. In some embodiments, the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet. FFPE samples are used in some instances in the RNA-templated ligation (RTL) methods disclosed herein. A limitation of direct RNA capture for fixed samples is that the RNA integrity of fixed (e.g., FFPE) samples can be lower than a fresh sample, thereby capturing RNA directly from fixed samples, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule, can be more difficult. However, by utilizing RTL probes that hybridize to RNA target sequences in the trans criptome, RNA analytes can be captured without requiring that both a poly(A) tail and target sequences remain intact. Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples. The biological sample, e.g., tissue sample, can be stained, and imaged prior, during, and / or after each step of the methods described herein. Any of the methods described herein or known in the art can be used to stain and / or image the biological sample. In some embodiments, the imaging occurs prior to destaining the sample. In some embodiments, the biological sample is stained using an H&E staining method. In some embodiments, the tissue sample is stained and imaged for about 10 minutes to about 2 hours (or any of the subranges of this range described herein). Additional time may be needed for staining and imaging of different types of biological samples.

[0095] The tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject. In some instances, the sample is a mouse sample. In some instances, the sample is a human sample. In some embodiments, the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. In some instances, the sample is a human or mouse breast tissue sample. In some instances, the sample is a human or mouse brain tissue sample. In some instances, the sample is a human or mouse lung tissue sample. In some instances, the sample is a human or mouse tonsil tissue sample. In some instances, the sample is a human or mouse liver tissue sample. In some instances, the sample is a human or mouse bone, skin, kidney, thymus, testes, or prostate tissue sample. In some embodiments, the tissue sample is derived from normal or diseased tissue. In some embodiments, the sample is an embryo sample. The embry o sample can be a non-human embry o sample. In some instances, the sample is a mouse embryo sample.

[0096] Biological samples are also described in Section (I)(d) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference.

[0097] The following embodiments can be used with any of the methods described herein. In some embodiments, the biological sample (e.g.. a fixed and / or stained biological sample) is imaged. In some embodiments, the biological sample is visualized or imaged using bright field microscopy. In some embodiments, the biological sample is visualized or imaged using fluorescence microscopy. The biological sample can be visualized or imaged using additional methods of visualization and imaging known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. In some embodiments, the sample is stained and imaged prior to adding reagents for analyzing captured analytes as disclosed herein to the biological sample.

[0098] In some embodiments, the methods include staining the biological sample. In some embodiments, the staining includes the use of hematoxylin and / or eosin. Non-limiting examples of stains include histological stains (e g., hematoxylin and / or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI (4',6-diamidino-2- phenylindole), eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red. Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunw ald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky7, silver, Sudan, Wright’s, and / or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation.

[0099] In some embodiments, the staining includes the use of a detectable label, such as a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

[0100] In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)( 13) or the Exemplary Embodiments Section of PCT Publication No.

[0101] WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. Briefly, in any of the methods described herein, the method includes permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of the extension products to the capture probes on the array. In some embodiments, the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, and methanol), a detergent (e g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), an enzyme (an endopeptidase, an exopeptidase, a protease), or a combination thereof. In some embodiments, the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X-100™, Tween-20™, or a combination thereof. In some embodiments, the endopeptidase is pepsin. In some embodiments, the endopeptidase is Proteinase K. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, which is herein incorporated by reference.

[0102] Array-based spatial analysis methods can involve the transfer of one or more analytes or derivatives thereof from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array.

[0103] A 'capture probe” refers to any molecule capable of capturing (directly or indirectly) and / or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI) and a capture domain). In some instances, the capture probe includes a homopolymer sequence, such as a poly(T) sequence. In some embodiments, 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)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. which is herein incorporated by reference. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. which is herein incorporated by reference.

[0104] In some instances, a capture probe and a nucleic acid analyte interaction (or any other nucleic acid to nucleic acid interaction) occurs because the sequences of the two nucleic acids are substantially complementary to one another. By “substantial,” “substantially” and the like, two nucleic acid sequences can be complementary when at least 60% of the nucleotide residues of one nucleic acid sequence are complementary to nucleotide residues of the other nucleic acid sequence. The complementary residues within a particular complementary nucleic acid sequence need not always be contiguous with each other, but can be interrupted by one or more non-complementary residues within the complementary nucleic acid sequence. In some embodiments, at least 60%, but less than 100%, of the residues of one of the two complementary nucleic acid sequences are complementary to residues of the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95% or 99% of the residues of one nucleic acid sequence are complementary to residues of the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. In some embodiments, the biological sample is mounted on a first substrate and the substrate comprising the array of capture probes is a second substrate. In this configuration, one or more analytes or analyte derivatives (e.g.. intermediate agents; e.g.. ligation products) can then be released from the biological sample and migrate to the second substrate comprising an array of capture probes. In some embodiments, the release and migration of the analytes or analyte derivatives to the second substrate comprising the array of capture probes occurs in a manner that preserves the original spatial context of the analytes in the biological sample. This method can be referred to as a sandwiching process, which is described e.g., in U.S. Patent Application Pub. No. 2021 / 0189475 and PCT Pub. Nos. WO 2021 / 252747 Al, WO 2022 / 061152 A2, and WO 2022 / 140028 Al, each of which is herein incorporated by reference.

[0105] FIG. 1A shows an exemplary sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102. 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. As shown in FIG. 1 , a liquid reagent drop (e.g., permeabilization solution 105) is introduced on the second substrate in proximity to the capture probes 106 and in between the biological sample 102 and the second substrate (e.g.. slide 104 including an array having spatially barcoded capture probes 106). 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.

[0106] During the exemplary sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the capture probes (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., array slide 104) is in an inferior position to the first substrate (e.g., slide 103). In some embodiments, the first substrate (e.g., slide 103) may be positioned superior to the second substrate (e.g., slide 104). A reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates. The reagent medium may be a permeabilization solution, which permeabilizes and / or digests the biological sample 102. In some embodiments, where the biological sample 102 has been pre-permeabilized, the reagent medium is not a permeabilization solution. Herein, the reagent medium may also comprise one or more of a monovalent salt, a divalent salt, ethylene carbonate, and / or glycerol. In some embodiments, analytes (e.g.. mRNA transcripts) and / or analyte derivatives (e.g., intermediate agents; e.g., ligation products) of the biological sample 102 may release from the biological sample, and actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106. Alternatively, in certain embodiments, migration of the analyte or analyte derivative (e.g., intermediate agent; e.g., ligation product) from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration). Exemplary methods of electrophoretic migration are described in WO 2020 / 176788, and U.S. Patent Application Pub. No. 2021 / 0189475, each of which is hereby incorporated by reference in its entirety.

[0107] As further shown, 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.

[0108] In some embodiments, 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. In some instances, the separation distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In some embodiments, the separation distance is less than 50 microns. In some embodiments, the separation distance is less than 25 microns. In some embodiments, the separation distance is less than 20 microns. The separation distance may include a distance of at least 2 pm.

[0109] FIG. IB shows a fully formed sandwich configuration 125 creating a chamber 150 formed from the one or more spacers 110, the first substrate (e.g.. the slide 103), and the second substrate (e.g., the slide 104 including an array 106 having spatially barcoded capture probes) in accordance with some example implementations. In the example of FIG. IB, 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). In some aspects, 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 (e.g., slide 103), and the second substrate (e.g., slide 104) may reduce or prevent flow from undesirable movement (e.g., convective movement) of transcripts and / or molecules during the diffusive transfer from the biological sample 102 to the capture probes.

[0110] The sandwiching process methods described above can be implemented using a variety of hardware components. For example, 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 is incorporated by reference in its entirety.

[0111] In some embodiments of a sample holder, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a biological sample. The first retaining mechanism can be configured to retain the first substrate disposed in a first plane. The sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane. The sample holder can further include an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism can be configured to align the first and second members along the first plane and / or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane. The adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and / or move the first member along an axis orthogonal to the first plane.

[0112] In some embodiments, the adjustment mechanism includes a linear actuator. In some embodiments, the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and / or the second member. In some embodiments, the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and / or the second member. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0. 1 mm / sec. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member with an amount of force of at least 0. 1 lbs.

[0113] FIG. 2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes a first member 204, a second member 210, optionally an image capture device 220, a first substrate 206, optionally a hinge 215, and optionally a mirror 216. The hinge 215 may be configured to allow the first member 204 to be positioned in an open or closed configuration by opening and / or closing the first member 204 in a clamshell manner along the hinge 215.

[0114] FIG. 2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206. In the example of FIG. 2B, the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206.

[0115] In some aspects, when the sample handling apparatus 200 is in an open position (e.g., in FIG. 2B), the first substrate 206 and / or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200, such as within the first member 204 and the second member 210, respectively. As noted, the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration.

[0116] In some aspects, after the first member 204 closes over the second member 210, 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 permeabilization of the sample (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.

[0117] In some embodiments, the biological sample (e.g., sample 102 from FIG. 1A) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample is aligned with the barcoded array of the second substrate (e.g., the slide 104 from FIG. 1A), e.g., when the first and second substrates are aligned in the sandwich configuration. Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g.. via an automated alignment mechanism). After or before alignment, 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. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 206 and / or the second substrate 212. 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) may be captured by the capture probes of the array and may be processed for spatial analysis.

[0118] In some embodiments, during permeabilization, 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.

[0119] Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. FIGs. 3A-3C depict a side view' and a top view' of an exemplary angled closure w orkflow' 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. FIG. 3A depicts the first substrate (e.g., slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304). As shown, reagent medium (e.g., permeabilization solution) 305 is located on the spacer 310 toward the right-hand side of the side view in FIG. 3A. While 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.

[0120] FIG. 3B shows that as the first substrate lowers, and / or as the second substrate rises, the dropped side of the first substrate (e.g., a side of the slide 303 angled toward the slide 304) may contact the reagent medium 305. The dropped side of the slide 303 may urge the reagent medium 305 toward the opposite direction (e.g., towards an opposite side of the spacer 310. towards an opposite side of the slide 303 relative to the dropped side). For example, in the side view of FIG. 3B the reagent medium 305 may be urged from right to left as the sandwich is formed.

[0121] In some embodiments, 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.

[0122] 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. As shown in the top view of FIG. 3C, 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.

[0123] While 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, it should be understood that an exemplary7angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate comprising the spacer 310.

[0124] It may be desirable that the reagent medium be free from air bubbles between the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present between 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 between the two substrates (e.g., slide 303 and slide 304) during permeabilization (e.g., step 104). In some aspects, bubble formation between the substrates may be reduced or eliminated 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. For example, during the sandwiching of the two substrates (e.g., the slide 303 and the slide 304), an angled closure workflow may be used to suppress or eliminate bubble formation.

[0125] FIG. 4A is a side view of the angled closure workflow 400 in accordance with some exemplary implementations. FIG. 4B is a top view of the angled closure workflow 400 in accordance with some exemplary implementations. As shown at step 405, reagent medium 401 is positioned to the side of the substrate 402 contacting the spring.

[0126] At step 410, the dropped side of the angled substrate 406 contacts the reagent medium 401 first. The contact of the substrate 406 with the reagent medium 401 may form a linear or low curvature flow front that fills the gap between the two substrates 406 and 402 uniformly with the slides closed.

[0127] At step 415, the substrate 406 is further lowered tow ard 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, thereby creating a linear or low curvature flow front that may prevent or reduce bubble trapping betw een the substrates.

[0128] At step 420, the reagent medium 401 fills the gap between the substrate 406 and the substrate 402. The linear flow front of the liquid reagent may be formed by squeezing the reagent medium 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.

[0129] In some embodiments, the reagent medium (e.g., 105 in FIG 1A) comprises a permeabilization agent. In some embodiments, following initial contact between the biological sample and a permeabilization agent, the permeabilization agent can be removed from contact with the biological sample (e.g., by opening the sample holder). Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, or methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin. Triton X- 100™, Tween-20™, or sodium dodecyl sulfate (SDS)). and enzymes (e.g.. trypsin or other proteases (e g., proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS orN-lauroylsarcosine sodium salt solution).

[0130] In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants, such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. In some embodiments, the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, elastase, and proteinase K. In some embodiments, the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises an RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, and RNase I. In some embodiments, the reagent medium comprises one or more of sodium dodecyl sulfate (SDS) or a sodium salt thereof, proteinase K, pepsin, N-lauroylsarcosine. and RNase.

[0131] In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG is from about 2K to about 16K. In some embodiments, the PEG is about 2K, about 3K, about 4K, about 5K, about 6K, about 7K, about 8K, about 9K, about 10K, about UK, about 12K. about 13K. about 14K. about 15K. or about 16K. In some embodiments, the PEG is present at a concentration from about 2% to about 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v / v).

[0132] In certain embodiments a dried permeabilization reagent is applied or formed as a layer on the first substrate, the second substrate, or both prior to contacting the biological sample with the array. For example, a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.

[0133] In some instances, 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.

[0134] In some instances, 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.

[0135] There are at least two methods to associate 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 release or cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and / or into or onto the biological sample. In some cases, 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, which is herein incorporated by reference). In some cases, 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 ligation products that sen e as proxies for the template.

[0136] As used herein, an “extended capture probe’7refers to a capture probe having additional nucleotides added to a terminus (e.g., 3' or 5' end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3' end” indicates additional nucleotides were added to the most 3' nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3’ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture probe capture domain. In some embodiments, the capture probe is extended using a reverse transcriptase. In some embodiments, the capture probe is extended using one or more DNA polymerases. In some embodiments, the extended capture probes include the sequence of the capture domain, the sequence of the spatial barcode of the capture probe, and the complementary sequence of the template used for extension of the capture probe.

[0137] In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for subsequent analysis, e.g., sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction).

[0138] As used herein an “extended probe” refers to a first probe hybridized to a target nucleic acid and is extended by one or more nucleotides using the target nucleic acid (e.g., RNA or DNA) as a template. First probes are substantially complementary to a target nucleic acid at a region of the target nucleic acid that is downstream (i.e.. to the 3' direction) of one or more genetic variant(s). These hybridized first probes can be extended with a polymerase or a reverse transcriptase, and the resulting extended probes comprising the complementary sequence of one or more genetic variant(s) can be ligated to a second probe (e.g.. second probes described herein) via branch ligation.

[0139] As used herein a "‘blocking oligonucleotide” refers to an oligonucleotide hybridized to a target nucleic acid at a region of the target nucleic acid that is upstream (i.e., to the 5' direction) of one or more genetic variant(s). The blocking oligonucleotide can be substantially complementary (as defined herein) to the target nucleic acid. In some embodiments, the blocking oligonucleotide includes a 3' blocking moiety. Various blocking moieties are known in the art (e.g., moieties sufficient to block extension). Non-limiting examples of blocking moieties include a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

[0140] Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of PCT Publication No. W02020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. Analysis of captured analytes (and / or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes using the capture analyte as a template, sequencing (e g., of a cleaved extended capture probe and / or a cDNA molecule complementary' to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and / or proximity capture, is described in Section (II)(g) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. Some quality' control measures are described in Section (II)(h) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. which is herein incorporated by reference.

[0141] Spatial information can provide information of medical importance. For example, 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.S. Patent Application Publication Nos. 2021 / 0140982, 2021 / 0198741, and 2021 / 0199660, each of which is herein incorporated by reference.

[0142] Spatial information can provide information of biological importance. For example, the methods described herein can allow for: identification of transcriptome and / or proteome expression profiles (e g., in healthy and / or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor or proximity based analysis); determination of up-regulated and / or down-regulated genes and / or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in healthy and diseased tissue; and identification of genetic variants within tissues (e.g., based on gene and / or protein expression profiles associated with specific disease or disorder biomarkers).

[0143] Typically, for spatial array-based methods, a substrate can function as a support for direct or indirect attachment of capture probes to features of the array. A "feature” is an entity7that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (IT)(d)(iii), and (II)(d)(iv) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference.

[0144] Generally, analytes and / or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and / or “contacting,” a biological sample with a substrate refers to any contact (e.g.. direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e g., hybridize)) with analytes from the biological sample. 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. which is herein incorporated by reference. FIG. 5 is a schematic diagram showing an exemplary capture probe, as described herein. As shown, 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 is 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 a combination 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. 5 shows the spatial barcode 505 as being located upstream (5') of UMI sequence 506, it is to be understood that capture probes wherein UMI sequence 506 is located upstream (5') of the spatial barcode 505 is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain 507 to facilitate capture of a target analyte. The capture domain can have a sequence complementary to a sequence of a nucleic acid analyte. The capture domain can have a sequence complementary to a connected probe described herein. The capture domain can have a sequence complementary to an analyte capture sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. A 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.

[0145] FIG. 6 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to analytes within the sample. The capture probe 601 can contain a cleavage domain 602, a cell penetrating peptide 603, a reporter molecule 604, and a disulfide bond (-S-S-). 605 represents all other parts of a capture probe, for example a spatial barcode and a capture domain.

[0146] FIG. 7 is a schematic diagram of an exemplary7multiplexed spatially-barcoded feature. In FIG. 7, 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. For example, a feature may include four different ty pes of spatially -barcoded capture probes, each ty pe of spatially-barcoded capture probe possessing the spatial barcode 702. One type of capture probe associated with the feature can include 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 can include the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis. A third type of capture probe associated with the feature can include the spatial barcode 702 in combination with a capture domain complementary to the analyte capture agent of interest 705. A fourth type of capture probe associated with the feature can include the spatial barcode 702 in combination with a capture probe that can specifically bind a nucleic acid molecule 706 that can function in a CRISPR assay (e.g., CRISPR / Cas9). While only four different capture probe-barcoded constructs are shown in FIG. 7, capture-probe barcoded constructs can be tailored for analyses of any given analyte associated with a nucleic acid and capable of binding with such a construct. For example, the schemes shown in FIG. 7 can also be used for concurrent analysis of other analytes disclosed herein, including, but not limited to: (a) mRNA, a lineage tracing construct, cell surface or intracellular proteins and / or metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or MNase-seq), cell surface or intracellular proteins and metabolites, and a perturbation agent (e.g., a CRISPR crRNA / sgRNA, TALEN, zinc finger nuclease, and / or antisense oligonucleotide as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, a barcoded labelling agent (e.g., the MHC multimers described herein), and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). In some embodiments, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature) change, or any other known perturbation agents.

[0147] The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio. Oxford Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with noncommercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.

[0148] In some embodiments, the spatial barcode 505 and functional sequence 504 are common to all of the probes attached to a given feature. In some embodiments, 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.

[0149] FIG. 8 depicts an exemplary arrangement of barcoded features within an array. From left to right, FIG. 8 shows (left) a slide including six spatially-barcoded arrays, (center) an enlarged schematic of one of the six spatially -barcoded arrays, showing a grid of barcoded features in relation to a biological sample, and (right) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (e.g., labelled as ID578, ID579, ID580, etc ).

[0150] In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference.

[0151] In some cases, spatial analysis can be performed by attaching and / or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and / or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. which is herein incorporated by reference.

[0152] In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug 21; 45(14):el28, which is herein incorporated by reference. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3' end and / or the other oligonucleotide includes a phosphorylated nucleotide at the 5' end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence or a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g.. a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNase H). In some instances, the ligation product is removed using heat. In some instances, the ligation product is removed using potassium hydroxide (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.

[0153] In some instances, one or both of the oligonucleotides may hybridize to genomic DNA (gDNA) which can lead to false positive sequencing data from ligation events on gDNA (off target) in addition to the desired (on target) ligation events on target nucleic acids, (e.g., mRNA). Thus, in some embodiments, the disclosed methods can include contacting the biological sample with a deoxyribonuclease (DNase). The DNase can be an endonuclease or exonuclease. In some embodiments, the DNase digests single-stranded and / or doublestranded DNA. Suitable DNases include, without limitation, a DNase I and a DNase II. Use of a DNase as described can mitigate false positive sequencing data from off target gDNA ligation events.

[0154] A non-limiting example of templated ligation methods disclosed herein is depicted in FIG. 9A. After a biological sample is contacted with a substrate including a plurality of capture probes and contacted with (a) a first probe 901 having a target-hybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a targethybridization sequence 905 and a capture domain (e.g., a poly(A) sequence) 906, the first probe 901 and the second probe 904 hybridize 910 to an analyte 907. A ligase 921 ligates 920 the first probe to the second probe 904, thereby generating a ligation product 922. The ligation product 922 is then released 930 from the analyte 931 by digesting the analyte 907 using an endoribonuclease 932. The sample is permeabilized 940 and the ligation product 941 can hybridize to a capture probe on the substrate. Methods and composition for spatial detection using templated ligation have been described in PCT Publication No. WO 2021 / 133849 Al, U.S. Pat. Nos. 11,332,790 and 11,505,828, each of which is incorporated by reference in its entirety.

[0155] In some embodiments, as shown in FIG. 9B, the ligation product 9001 includes a capture probe capture domain 9002, which can bind to a capture probe 9003 (e.g., a capture probe immobilized, directly or indirectly, on a substrate 9004). In some embodiments, methods provided herein include contacting 9005 a biological sample with a substrate 9004, wherein the capture probe 9003 is affixed to the substrate (e.g.. immobilized to the substrate, directly or indirectly). In some embodiments, the capture probe capture domain 9002 of the ligated product 9001 specifically binds to the capture domain 9006. The capture probe can also include a unique molecular identifier (UMI) 9007, a spatial barcode 9008, a functional sequence 9009, and a cleavage domain 9010.

[0156] In some embodiments, methods provided herein include permeabilization of the biological sample such that the capture probe can more easily capture the ligation products (i.e., compared to no permeabilization). In some embodiments, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can be used to extend the capture probes 9011 to produce spatially-barcoded full-length cDNA 9012 and 9013 from the captured ligation products.

[0157] In some embodiments, the extended ligation products can be denatured 9014, released 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 sequences can be used as flow cell capture probes 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 1 and TruSeq Read 2 as sequencing primer sites.

[0158] In some embodiments, detection of one or more analytes (e.g., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, 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, which is herein incorporated by reference.

[0159] 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 1002 is capable of interacting with a spatially- barcoded capture probe. The analyte binding moiety 1004 can bind to the analyte 1006 with high affinity and / or with high specificity. The analyte capture agent 1002 can include: (i) an analyte binding moiety barcode domain 1008, which serves to identify the analyte binding moiety, and (ii) an analyte capture sequence, which can hybridize to at least a portion or an entirety of a capture domain of a capture probe. The analyte-binding moiety 1004 can include a polypeptide and / or an aptamer. The analyte-binding moiety’ 1004 can include an antibody or antibody fragment (e.g.. an antigen-binding fragment).

[0160] 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 sequence 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 1124 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 1126 can also include a linker 1120 that allows the analytebinding moiety barcode domain (e.g., including the functional sequence 1118, analyte binding moiety barcode 1116, and analyte capture sequence 1114) to couple to the analyte binding moiety 1122. In some embodiments, the linker 1120 is a cleavable linker. In some embodiments, the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, chemical-cleavable linker, thermal-cleavable linker, or an enzyme cleavable linker. In some instances, 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). During analysis of spatial information, 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. In some embodiments, specific capture probes and their captured analytes are associated with specific locations in an array of features on a substrate. For example, 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.

[0161] Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication 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. Where necessary, 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.

[0162] When sequence information is obtained for capture probes and / or analytes during analysis of spatial information, the locations of the capture probes and / or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.

[0163] Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of PCT Publication No. W02020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. See, for example, the Exemplary7embodiment starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed. . . ” of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. which is herein incorporated by reference. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and / or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), each of which is herein incorporated by reference in its entirety. In some embodiments, spatial analysis can be performed using dedicated hardware and / or software, such as any of the systems described in Sections (II)(e)(ii) and / or (V) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and / or Sample and Array Alignment Devices and Methods. Informational labels of PCT Publication No. W02020 / 123320. which is herein incorporated by reference.

[0164] Suitable systems for performing spatial analysis can include components, such as a chamber (e.g., a flow cell or a 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.

[0165] The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid-state storage medium such as, but not limited to. a magnetic, optical, or other solid state, persistent, writeable and / or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the methods and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the methods or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.

[0166] The systems can optionally include software instructions encoded and / or implemented in one or more of tangible storage media and hardware components, such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the methods or functions described herein. In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Publication No. W0202I / 102003 and / or U.S. Patent Application Publication No. 2021 / 0150707, each of which is incorporated herein by reference in its entirety.

[0167] 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-dimensional 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 its entirety.

[0168] In some cases, a map of analyte presence and / or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. W02020 / 123320, WO 2021 / 102005, and / or U.S. Patent Application Publication No. 2021 / 0158522, each of which is incorporated herein by reference in its entirety. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, or to determine a location of a sample or array on a substrate relative to a fiducial marker) and / or for quantitative measurements of sizes and / or distances.

[0169] B. Determining a Location of a Genetic Variant in a Biological Sample

[0170] The present disclosure features methods, compositions, and kits that can be used to analyze the presence, location, and / or frequency of genetic variants in a target nucleic acid in a biological sample using a target specific nucleic acid probe (e.g., a first probe), a blocking oligonucleotide, and a second probe in combination with capture probes in an arrayed format. A probe (e.g., a first probe) hybridizes to a target nucleic acid at a region that is downstream of the location of a possible genetic variant in the target nucleic acid. When the first probe is extended, for example, with a DNA polymerase or a reverse transcriptase, the extended probe will incorporate a complementary sequence of a genetic variant or wild-tj pe sequence. However, accuracy of the extended probe incorporating the complementary sequence of the genetic variant depends on several factors. Mistakes in extension by a DNA polymerase or reverse transcriptase or competition between probes in traditional templated ligation reactions can produce inaccurate readouts of the presence, location, and / or frequency of genetic variants in the biological sample. Thus, there is a need for improved methods, compositions, and kits with increased accuracy and / or sensitivity’ when determining the presence, location, and / or frequency of genetic variants in a biological sample.

[0171] More specifically, the methods, compositions, and kits disclosed herein include a blocking oligonucleotide including: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3' blocking moiety. The blocking oligonucleotide prevents the first probe from being extended beyond the blocking oligonucleotide such that the extended probe can be ligated to a second probe via branch ligation. The second probe is a partially double-stranded molecule that can be ligated to the extended probe. The second probe also includes a capture probe capture domain that is substantially complementary to a capture domain of a capture probe on a spatial array.

[0172] Branch ligation (e g., 3' branch ligation) is described in Wang, L., 3' Branch ligation: a novel method to ligate non-compl ementary DNA to recessed or internal 3' OH ends in DNA or RNA, DNA Res., 26(1): 45-53 (2019), which is incorporated herein by reference in its entirety. Briefly, 3' branch ligation can be performed using T4 DNA ligase at non- conventional DNA ends formed by nicks, gaps, and overhangs. For example, blunt-end DNA donors (e.g., second probes) are partially double-stranded DNA molecules that can be ligated to extended first probes. In some embodiments, the partially double-stranded DNA molecule includes a dideoxy 3' terminus which inhibits self-ligation. In some embodiments, the longer strand of the partially double-stranded DNA molecule (e.g., the second probe) is phosphorylated at its 5' end. DNA acceptor oligonucleotide complexes can be assembled with a first probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the first probe is extended to or near the blocking oligonucleotide. The partially doublestranded DNA molecule (e.g.. second probe) can be ligated to the extended probe, as shown in FIG. 12. Branch ligation can be effective for ligation of nucleic acid molecules having nicks (e.g., lacking phosphates), gaps (e.g., 1 nucleotide to 8 nucleotides), 3' recessive ends, or blunt ends.

[0173] Thus provided herein are methods for determining a location of a genetic variant in a target nucleic acid in a biological sample, the method 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) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of the genetic variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid; (d) extending the first probe using the target nucleic acid as a template, thereby generating an extended first probe, where the extended first probe includes a sequence complementary to the genetic variant; (e) contacting a plurality of second probes with the biological sample, where a second probe of the plurality of second probes includes a partially double-stranded nucleic acid molecule including a capture probe capture domain; (f ligating the extended first probe and the second probe, thereby generating a ligation product; (g) hybridizing the ligation product to the capture probe capture domain; and (h) determining (i) all or a portion of the sequence of the ligation product, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the genetic variant in the target nucleic acid in the biological sample.

[0174] Also provided herein are methods for determining a presence or absence of a single nucleotide variant in a biological sample, the method 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) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, where a first probe of the plurality of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is downstream of the single nucleotide variant and where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially complementary to the target nucleic at a region that is upstream of the single nucleotide variant and (ii) a 3' blocking moiety; (c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid; (d) extending the first probe using the target nucleic acid as a template, thereby generating an extended first probe; (e) contacting a plurality of second probes with the biological sample, w here a second probe of the plurality' of second probes includes a partially double-stranded nucleic acid molecule including a capture probe capture domain; (f) ligating the extended first probe and the second probe, thereby generating a ligation product; (g) hybridizing the ligation product to the capture probe capture domain; and (h) determining (i) all or part of the sequence of the ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the presence or absence of the single nucleotide variant in the biological sample.

[0175] As described above, array-based spatial transcriptomics methods can measure analyte and / or gene expression data for a variety of analytes (e.g.. target nucleic acids), or proxies thereof (e.g., ligation products), within a biological sample at high spatial resolution, while retaining native spatial context.

[0176] In some embodiments, a genetic variant includes any genetic variation or modification compared to a canonical nucleic acid. For example, non-limiting examples of genetic variants include one or more single nucleotide variants or polymorphisms, one or more nucleotide deletions, one or more nucleotide insertions, one or more nucleic acid rearrangements, and one or more nucleotide duplications. In some embodiments, a genetic variant is at least one single nucleotide variant (SNV) or single nucleotide polymorphism (SNP). In some embodiments, a genetic variant includes multiple SNVs or SNPs (e.g., 1. 2. 3, 4. 5 or more SNVs or SNPs ). In some embodiments, the target nucleic acid comprises numerous genetic variants at a particular location, for example a “hot spot” of variants clustered in a particular location on the target nucleic acid.

[0177] In some embodiments, the target nucleic acid is RNA. Non-limiting examples of RNA include various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. The RNA can be a transcript (e.g., present in a tissue section). The RNA can be small (e.g.. less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. The RNA can be circular RNA. The RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA). The RNA can be from an RNA virus, for example RNA viruses from Group III, IV or V of the Baltimore classification system. The RNA can be from a retrovirus, such as a virus from Group VI of the Baltimore classification system. In some embodiments, the target nucleic acid is DNA. Non-limiting examples of DNA analytes include genomic DNA. methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and viral DNA.

[0178] In the methods described herein, the first probe is extended after hybridizing to the target nucleic acid (e.g., hybridized at a region that is upstream of a genetic variant), thereby generating an extended probe. The first probe can be extended by a polymerase or a reverse transcriptase. Extension is performed with an enzyme (e.g., a polymerase, a reverse transcriptase) that is non-strand displacing. Non-limiting examples of non-strand displacing polymerases are described herein. For example, a Thermococcus gorgonarius mutant reverse transcription xenopolymerase as found in U.S. Provisional Patent Application No. 63 / 467,541 lacks strand displacement activity. Thermococcus kodakaraensis K0D1 DNA polymerase, or KOD polymerase, Takagi et al., Characterization of DNA Polymerase from Pyrococcus sp. Strain K0D1 and Its Application to PCR, App Env Micro, 63(11):4504-4510) (1997)), also lacks strand displacement activity. Also, TgoRTX polymerase described in U.S. Patent Serial Number 18 / 319,864 lacks strand displacement activity. Generally, the first probe is extended to or near a blocking oligonucleotide that is also hybridized to the nucleic acid. In some embodiments, the first probe includes a functional domain (e.g., a primer binding site or a sequencing specific site). In some embodiments, the blocking oligonucleotide includes a 3' blocking moiety. Non-limiting examples of 3’ blocking moieties include a carbon spacer (e.g., one, two, three, or more carbon molecules), an inverted nucleotide, dideoxynucleotides, and nucleotides with a substituted 3' OH group. After extension of the first probe, the extended probe and the second probe (e.g., a partially double-stranded DNA molecule) can be ligated via branch ligation as described herein. In some embodiments, the second probe includes a capture probe capture domain (e.g., a sequence capable of hybridizing to the capture domain of a capture probe).

[0179] In some embodiments, the first probe includes one or more mismatched nucleotides at its 3' end. In some embodiments, the first probe includes a dideoxynucleotide at its 3' end. In some embodiments, the dideoxynucleotide includes one of ddATP, ddCTP, ddGTP. or ddTTP. Modifications at the 3' end of the first probe as described above can increase specificity of the extension reaction and reduce the likelihood of ligation of first probes (i.e., unextended first probes or first probes that have not undergone nucleic acid extension) to second probes. In some embodiments, the second probe includes a 5' phosphate group. The 5' phosphate group permits ligation to a 3' hydroxyl group (i.e., a 3' hydroxyl group of an extended first probe).

[0180] In some embodiments, the extended probe and the second probe are ligated via chemical ligation. In some embodiments, the extended probe and the second probe are ligated via enzymatic ligation. In some embodiments, enzymatic ligation includes the use of a ligase. In some embodiments, the ligase is one or more of a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlorella virus DNA ligase, a single-stranded DNA ligase, a T4 DNA ligase, and a combination thereof. In some embodiments, the ligase is a pre-activated T4 DNA ligase.

[0181] In some embodiments, after generating a ligation product, the ligation product is released or removed from the target nucleic acid. In some embodiments, the ligation product is removed from the target nucleic acid using an endoribonuclease. In some embodiments, the endoribonuclease is RNase H, RNase A, RNase C, and / or RNase I. In some embodiments, the endoribonuclease is RNase H. RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA, when hybridized to DNA. RNase H is part of a conserved family of ribonucleases which are present in many different organisms. There are two primary classes of RNase H: RNase Hl and RNase H2. Retroviral RNase H enzymes are similar to the prokaryotic RNase Hl. All of these enzymes share the characteristic that they are able to cleave the RNA component of an RNA: DNA heteroduplex. In some embodiments, the RNase H is RNase Hl , RNase H2, or RNase Hl , or RNase H2. In some embodiments, the RNase H includes but is not limited to RNase HII from Pyrococcus furiosus, RNase HII from Pyrococcus horikoshi, RNase HI from Thermococcus litoralis. RNase HI from Thermus thermophilus, RNAse HI from E. coll, or RNase HII from E. coll.

[0182] In some embodiments, the target nucleic acid is released from the ligation product via denaturation. In some embodiments, denaturation includes the use of heat. In some embodiments, the denaturation includes the use of potassium hydroxide. In some embodiments, denaturation includes the use of both heat and potassium hydroxide.

[0183] After or before the target nucleic acid is released from the ligation product, the ligation product can hybridize to a capture domain of a capture probe via the capture probe capture domain of the second probe (e.g., incorporated into the ligation product). In some embodiments, the capture probe is extended using the hybridized ligation product as a template, thereby generating an extended capture probe (as defined herein). In some embodiments, the ligation product is extended (e.g., extended towards the array) using the capture probe as a template, thereby generating an extended ligation product. In some embodiments, both the capture probe and the ligation product are extended using each other, respectively, as a template. In some embodiments, the extended capture probe and / or the extended ligation product are removed (e.g., cleaved, denatured, etc.) from the array. In some embodiments, the extended capture probe and / or the extended ligation product are amplified, thereby producing amplicons of the extended capture probe and / or the extended ligation product.

[0184] In some embodiments, the determining step includes sequencing. In some embodiments, the sequencing includes high-throughput sequencing. In some embodiments, the determining includes correlating a location of the genetic variant(s) on the array to a location of the genetic variant(s) in the biological sample.

[0185] In some embodiments, the method includes washing the array to remove unhybridized first probes and / or unhybridized blocking probes.

[0186] In some embodiments, the array includes one or more features. In some embodiments, features are directly or indirectly attached or fixed to a substrate. In some embodiments, the features are not directly or indirectly attached or fixed to a substrate, but instead, for example, are disposed within an enclosed or partially enclosed three-dimensional space (e.g., wells or divots). For example, the plurality7of capture probes can be located on features on a substrate. In some embodiments, features include, but are not limited to, a spot, an inkjet spot, a masked spot, a pit, a post, a well, a ridge, a divot, a hydrogel pad, and a bead (e.g., a hydrogel bead).

[0187] In some embodiments, the biological sample can be stained. In some embodiments, the biological sample is stained after fixation. In some embodiments, the biological sample is stained before fixation. In some embodiments, the staining includes optical labels as described herein, including, but not limited to, fluorescent (e.g., fluorophore), radioactive (e.g., radioisotope), chemiluminescent (e.g., a chemiluminescent compound), a bioluminescent compound, calorimetric, or colorimetric detectable labels. In some embodiments, the staining includes a fluorescent antibody directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample. In some embodiments, the staining includes an immunohistochemistry stain directed to a target analyte (e.g., cell surface or intracellular proteins) in the biological sample. In some embodiments, the staining includes a chemical stain, such as hematoxylin and eosin (H&E) or periodic acid-schiff (PAS). In some embodiments, staining the 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. In some embodiments, significant time (e.g., days, months, or years) can elapse between staining and / or imaging the biological sample.

[0188] In some embodiments, the biological sample is imaged. In some embodiments, the biological sample is imaged after fixation. In some embodiments, the biological sample is imaged before fixation. In some embodiments, imaging includes one or more of expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy.

[0189] In some embodiments, the biological sample is permeabilized. Permeabilization of a biological sample can occur on a substrate where the substrate is aligned with the array such that at least a portion of the biological sample is aligned with at least a portion of the array or directly on an array including a plurality of capture probes. In some embodiments, the biological sample is permeabilized with a protease. In some embodiments, the protease is one or more of pepsin, Proteinase K, and collagenase.

[0190] The biological sample can be applied to any of the variety' of arrays described herein. In some embodiments, the plurality of capture probes includes, e.g., in a 5' to a 3' direction, a spatial barcode and a capture domain. In some embodiments, the capture domain hybridizes to a capture sequence. In some embodiments, the capture domain is a poly(T) capture domain. In some embodiments, the capture domain is not a poly(T) sequence. In some embodiments, the capture domain is a fixed sequence. As used herein, a “fixed sequence” is anon-random sequence. For example, the capture domain and the capture probe capture domain of the second probe each can be any sequence as long as the respective sequences are substantially complementary' to one another to facilitate hybridization.

[0191] In some embodiments, a capture probe can include one or more functional domains, and / or a cleavage domain. A functional domain typically includes a functional nucleotide sequence for a subsequent analytical step in the overall analysis procedure. In some embodiments, the functional domain can include a sequencing specific site or a primer binding site. In some embodiments, the functional domain can include an amplification (e.g., PCR) sequence. In some embodiments, a capture probe includes a unique molecular identifier as described herein. In some embodiments, the unique molecular identifier is located 5' to the capture domain in the capture probe. The methods disclosed herein can be performed on any type of biological sample. In some embodiments, the biological sample is a fresh tissue sample. In some embodiments, the biological sample is a frozen tissue sample. In some embodiments, the biological sample was previously frozen. In some embodiments, the biological sample is a fixed tissue sample. In some embodiments, the fixed tissue sample is a formalin-fixed, paraffin embedded (FFPE) sample.

[0192] In some embodiments, the biological sample is a tissue section. In some embodiments, the sample is a fresh tissue section. In some embodiments, the biological sample is a frozen tissue section. In some embodiments, the biological sample was previously frozen. In some embodiments, the biological sample is a fixed tissue section. In some embodiments, the fixed tissue section is a formalin-fixed, paraffin embedded (FFPE) section, an acetone-fixed tissue section, a methanol-fixed tissue section, or a paraformaldehyde-fixed tissue section.

[0193] Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy. In some instances, the biological sample can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, cancer, and / or diseases associated with a genetic variant (e.g., any of the genetic variants described herein). In some instances, the biological sample comprises nucleic acids with one or more genetic variants. In some instances, the one or more genetic variants is associated with a disease or disease state. In some instances, the biological sample includes cancer or tumor cells. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. In some instances, the biological sample is a heterogeneous sample. In some instances, the biological sample is a heterogeneous sample that includes tumor or cancer cells and / or stromal cells.

[0194] FFPE samples generally are heavily cross-linked and fragmented, and therefore this ty pe of sample allows for limited RNA recovery' using conventional detection techniques. In certain embodiments, methods of targeted RNA capture provided herein are less affected by RNA degradation associated with FFPE fixation than other methods (e.g., methods that take advantage of oligo-dT capture and reverse transcription of mRNA). In certain embodiments. methods provided herein enable sensitive measurement of specific genes of interest that otherwise might be missed with a whole transcriptomic approach.

[0195] In some embodiments, the FFPE sample or section is deparaffmized, permeabilized, equilibrated, and blocked before target probe oligonucleotides are added. In some embodiments, deparaffmization includes using xylenes. In some embodiments, deparaffinization includes multiple washes with xylenes. In some embodiments, deparaffmization includes multiple washes with xylenes followed by removal of xylenes using multiple rounds of graded alcohol followed by washing the sample with water. In some aspects, the water is deionized water.

[0196] Library Preparation

[0197] In some embodiments, the target nucleic acids or complements thereof and other proxies of target nucleic acids (e.g., ligation products), and / or amplicons of such products, can be prepared for subsequent applications, such as generation of a sequencing library and next-generation sequencing. Generating sequencing libraries are known in the art. For example, the target nucleic acids, proxies of target nucleic acids and / or their complements thereof can be purified and collected for subsequent amplification steps. The amplification products can be amplified using PCR, where primer binding sites flank the spatial barcode and target nucleic acid, or a complement thereof, generating a library associated with a particular spatial barcode. In some embodiments, the library preparation can be quantitated and / or quality controlled to verify the success of the library preparation steps. The library amplicons are sequenced and analyzed to decode spatial information of the target nucleic acid or proxy thereof (e.g., a ligation product).

[0198] Alternatively or additionally, the amplicons can then be enzymatically fragmented and / or size-selected in order to provide for desired amplicon size. In some embodiments, when utilizing an Illumina® library preparation methodology, for example, P5 and P7, sequences can be added to the amplicons thereby allowing for capture of the library preparation on a sequencing flow cell (e.g., on Illumina sequencing instruments). Additionally. i7 and i5 can index sequences be added as sample indexes if multiple libraries are to be pooled and sequenced together. Further, Read 1 and Read 2 sequences can be added to the library preparation for sequencing purposes. The aforementioned sequences can be added to a library preparation sample, for example, via End Repair, A-tailing, Adaptor Ligation, and / or PCR. The cDNA fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites, although other methods are known in the art.

[0199] Compositions

[0200] The present disclosure also features compositions for the spatial detection of genetic variants via branched ligation. Thus, provided herein are compositions including: (a) a spatial 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 first probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the first probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe including a partially double-stranded nucleic acid molecule, where the second probe includes a capture probe capture domain.

[0201] Also provided herein are compositions including: (a) a spatial 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) an extended probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the extended probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe including a partially double-stranded nucleic acid molecule, and where the second probe includes a capture probe capture domain.

[0202] Also provided herein are compositions including: (a) a spatial 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) an extended probe and a blocking oligonucleotide hybridized to a target nucleic acid, where the extended probe includes a sequence substantially complementary to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide includes: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and (c) a second probe ligated to the extended probe, wherein the second probe includes a partially double-stranded nucleic acid molecule, and where the second probe includes a capture probe capture domain.

[0203] Also provided herein are compositions including: (a) a spatial 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; and (b) a partially double-stranded ligation product including a capture probe capture domain.

[0204] Also provided herein are compositions including: (a) a spatial 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; and (b) a partially double-stranded ligation product hybridized to the capture probe capture domain.

[0205] In some embodiments, the 3' blocking moiety is selected from the group consisting of a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

[0206] In some embodiments, the target nucleic acid includes at least one genetic variant. In some embodiments, the target nucleic acid includes at least two, at least three, at least four, at least five or more genetic variants. In some embodiments, at least one genetic variant includes one or more indels. In some embodiments, at least one genetic variant includes one or more single nucleotide variants or polymorphisms.

[0207] In some embodiments, the second probe includes a 5' phosphate group.

[0208] In some embodiments, the extended probe is immediately adjacent to the blocking oligonucleotide. In some embodiments, the extended probe is at least one nucleotide away from the blocking oligonucleotide. In some embodiments, the extended probe is at least 2, at least 3, at least 4. at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least, 16, at least 17, at least 18, at least 19. at least 20 or more nucleotides away from the blocking oligonucleotide.

[0209] In some embodiments, the second probe includes a capture probe capture domain that is capable of hybridizing to the capture probe capture domain on the array. For example, the capture probe capture domain sequence is substantially complementary to a sequence of the capture probe capture domain on the array.

[0210] In some embodiments, the composition includes a polymerase enzyme or a reverse transcriptase enzyme. In some embodiments, the reverse transcriptase enzyme is a nondisplacing reverse transcriptase enzyme.

[0211] In some embodiments, the composition includes a biological sample. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a 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, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone- fixed tissue section. In some embodiments, the tissue section is a fresh frozen tissue section. In some embodiments, the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.

[0212] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acid is mRNA.

[0213] In some embodiments, the first probe includes one or more mismatched nucleotides at its 3' end. The one or more mismatched nucleotides are not complementary to the target nucleic acid to which the first probe hybridizes. In some embodiments, the first probe includes a dideoxynucleotide at its 3' end. In some embodiments, the dideoxy nucleotide includes one of ddATP, ddCTP, ddGTP, or ddTTP.

[0214] Kits

[0215] In addition to the methods and compositions described herein, the present disclosure also features kits for the spatial detection of genetic variants via branched ligation. Thus provided herein are kits including: (a) a spatial 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) (i) a plurality of first probes, where a first probe of the plurality’ of first probes includes a sequence substantially complementary to a target nucleic acid at a region that is dow nstream of a genetic variant, and (ii) a plurality of second probes, where a second probe of the plurality of second probes includes a partially double-stranded nucleic acid molecule and where the second probe includes a capture probe capture domain; (c) a plurality of blocking oligonucleotides, where a blocking oligonucleotide of the plurality of blocking oligonucleotides includes: (i) a sequence substantially’ complementary’ to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3’ blocking moiety; (d) a ligase; and (e) instructions for performing any of the methods described herein.

[0216] The capture probes (e.g.. any of the capture probes descnbed herein) can further include capture probe include one or more functional domains (e.g., a sequencing specific site, a primer binding site, etc.), a unique molecular identifier, a cleavage domain, and any combination thereof.

[0217] In some embodiments, the first probe includes a functional domain.

[0218] In some embodiments, the second probe includes a 5' phosphate group. In some embodiments, the ligase is one or more of a T4 RNA ligase (Rnl2), a PBCV- 1 DNA ligase, a Chlorella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase. In some embodiments, the ligase is a pre-activated T4 DNA ligase.

[0219] In some embodiments, the kit includes a polymerase enzyme. Any suitable polymerase enzyme can be used with the methods described herein, where the polymerase includes non-displacing activity. In some embodiments, the kit includes a reverse transcriptase enzyme. In some embodiments, the reverse transcriptase enzyme is a nondisplacing reverse transcriptase enzyme.

[0220] EXAMPLES

[0221] Example 1. Spatial Detection of Genetic Variants via Branched Ligation

[0222] FIG. 12 shows an exemplary method for spatially detecting genetic variant(s). More specifically, beginning at the top, a first probe and a blocking oligonucleotide can be contacted with a biological sample. The first probe includes a sequence that is substantially complementary to a target nucleic acid in the biological sample and hybridizes to the target nucleic acid at a region that is downstream (i.e. , 3') of the genetic variant(s). Optionally, the first probe further includes a functional domain (e.g., any of the functional domains described herein). The blocking oligonucleotide is also substantially complementary to the target nucleic acid and hybridizes to the target nucleic acid at a region that is upstream (i.e., 5') of the genetic variant(s). In some examples, the blocking oligonucleotide includes a 3' blocking moiety. Any suitable blocking moiety can be used. Non-limiting examples of 3' blocking moieties include a carbon spacer (e g., one, two, three, four or more carbon molecules), an inverted nucleotide, one or more dideoxynucleotides, and a nucleotide with a substituted 3' OH group.

[0223] As shown in FIG. 12 (middle), the first probe can be extended to generate an extended first probe that incorporates complementary sequence(s) of the genetic variant(s) in the target nucleic acid if present. In some examples, the extended first probe is extended with a polymerase (e.g., a DNA polymerase). In some examples, the extended first probe is extended with a reverse transcriptase (e.g., any suitable reverse transcriptase). In some examples, neither the polymerase nor the reverse transcriptase possesses strand displacing activity, i.e., the extended first probe is extended using a non-displacing extension enzyme. The blocking oligonucleotide stops extension of the non-displacing extension enzy me (e.g., the polymerase or the reverse transcriptase). In some examples, the extended probe is extended completely to the blocking oligonucleotide. In some examples, the extended probe is extended one or more nucleotides short of the blocking oligonucleotide (i.e., leaving a “gap’?) between the extended first probe and the blocking oligonucleotide. In some examples, this gap can be the result of steric hindrance based on the selected polymerase or reverse transcriptase.

[0224] As shown in FIG. 12 (bottom), a second probe can be contacted with the biological sample where the second probe is a partially double-stranded molecule that includes a capture probe capture domain. A branch-like structure can be formed between the extended first probe, the second probe, and the blocking oligonucleotide. The extended first probe and the second probe can be ligated to one another, thereby generating a ligation product. For example, the second probe comprises a partially double-stranded DNA molecule, which is phosphorylated on the 5' end of the double-stranded DNA molecule. The double-stranded DNA molecule can also include a capture probe capture domain sequence that can hybridize to a capture domain of a capture probe on a spatial array, i.e., the capture probe capture domain sequence is substantially complementary to a sequence of the capture probe capture domain. The phosphorylated, partially double-stranded DNA molecule, or the second probe, can be added to the biological sample and ligated to the first probe, for example, using a T4 DNA ligase, etc. As such, this method of branch ligation allows non-complementary ends to be ligated together, such that the extended first probe can be ligated to a double-stranded DNA molecule that includes a capture probe capture domain that, in turn, can be captured by a capture probe on a spatial array. The 3' branch ligation described herein is further described in Wang, L. et al., as discussed above.

[0225] In some examples, additional methods are used to further increase specificity of the extension reaction. For example, FIG. 13 is a schematic diagram depicting specificity improvements by incorporating either a mismatched base (top) or a dideoxynucleotide (bottom) to limit unintended extension of the first probe. Reverse transcriptases having exonucleolytic activity can be used to remove the one or more mismatched bases at the 3' end of the first probe. For example, the reverse transcriptase can be used to digest the mismatched base(s) prior to extension (i.e., reverse transcription) of the hybridized first probe. These modifications can increase the specificity of the extension reaction and reduce the likelihood of non-specific ligation of unextended first probes to second probes. Similarly, a dideoxynucleotide can be incorporated at the 3' end of the first probe to increase specificity of the extension reaction and reduce non-specific ligation. For example, a modification at the 3' end of the first probe using one or more ddNTPs, a 3' flap, etc. can be removed in a 3 '-5' direction by the reverse transcriptase, while the reverse transcriptase facilitates polymerization in the 5 '-3' direction of the first probe. While some unextended probes may remain hybridized to the target nucleic acid, the mismatched base or dideoxynucleotide modification can reduce the likelihood of the 3' branched ligation from occurring, i.e., if the 3' end modifications of the first probe are not removed, there will be no ligation event with the 3' branched oligonucleotide. FIG. 13 also shows unextended probes on the far right with either a mismatched base or a dideoxynucleotide. Ligation cannot occur between the first probe and the second probe if the first probe has not been extended.

[0226] After ligation of the first probe and the second probe, the target nucleic acid can be released from the ligation product. Any suitable method of releasing the target nucleic acid can be used, including, but not limited to RNase treatment (e.g., any of the RNases described herein) and / or denaturation. Denaturation can be performed with potassium hydroxide and / or heat. The released ligation product can include the capture probe capture domain from the second probe which interacts with (e.g., hybridizes to) a capture domain of a capture probe on a spatial array (e.g., any of the arrays described herein). The captured ligation product and / or the capture probe can be extended using each other, respectively, as a template. The resulting products (e.g., extended capture probes, extended ligation products) can be removed from the array, optionally amplified, and analyzed (e.g., sequenced) to determine the location and / or abundance of a genetic variant in a biological sample.

Claims

WHAT IS CLAIMED IS:

1. A method for determining a location of a genetic variant in a target nucleic acid in a biological sample, the method comprising:(a) providing an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;(b) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, wherein a first probe of the plurality of first probes comprises a sequence substantially complementary to the target nucleic acid at a region that is downstream of the genetic variant and wherein a blocking oligonucleotide of the plurality of blocking oligonucleotides comprises: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety;(c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid;(d) extending the first probe using the target nucleic acid as a template, thereby- generating an extended first probe, wherein the extended first probe comprises a sequence complementary to the genetic variant;(e) contacting a plurality of second probes with the biological sample, wherein a second probe of the plurality- of second probes comprises a partially double-stranded nucleic acid molecule comprising a capture probe capture domain;(f) ligating the extended first probe and the second probe, thereby generating a ligation product;(g) hybridizing the ligation product to the capture probe capture domain; and(h) determining (i) all or a portion of the sequence of the ligation product, or a complement thereof, and (ii) a sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the location of the genetic variant in the target nucleic acid in the biological sample.

2. The method of claim 1, wherein the 3' blocking moiety is selected from the group consisting of: a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

3. The method of claim 1 or 2, wherein the genetic variant comprises a single nucleotide variant or polymorphism.

4. The method of claim 3, wherein the target nucleic acid comprises at least two, at least three, at least four, at least five or more single nucleotide variants or polymorphisms.

5. The method of any one of claims 1-4, wherein the second probe comprises a 5' phosphate group.

6. The method of any one of claims 1-5, wherein the capture probe capture domain is substantially complementary to the capture probe capture domain on the array.

7. The method of any one of claims 1-6, wherein the ligating comprises chemical ligation or enzymatic ligation.

8. The method of claim 7, wherein the enzymatic ligation comprises use of a ligase.

9. The method of claim 8, wherein the ligase comprises a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlor ella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.

10. The method of claim 9, wherein the ligase comprises a pre-activated T4 DNA ligase.

11. The method of any one of claims 1-10, wherein the extending the first probe using the target nucleic acid as a template in (d) comprises the use of a polymerase or a reverse transcriptase.

12. The method of claim 11. wherein the reverse transcriptase is a non-displacing reverse transcriptase.

13. The method of claim 12, wherein the non-displacing reverse transcriptase comprises a Thermococcus gorgonarius mutant reverse transcription xenopolymerase.

14. The method of any one of claims 1-13, further comprising releasing the ligation product from the target nucleic acid.

15. The method of claim 14, wherein the releasing comprises the use of an endoribonuclease.

16. The method of claim 15. wherein the endoribonuclease comprises RNase H, RNase A, RNase C, or RNase I, and optionally, wherein the RNase H comprises RNase Hl, RNase H2, or both.

17. The method of any one of claims 1-16, wherein the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.

18. The method of any one of claims 1-17, further comprising contacting the biological sample with a permeabilization reagent.

19. The method of claim 18, wherein the permeabilization reagent comprises pepsin, proteinase K, or collagenase.

20. The method of any one of claims 1 -19, further comprising extending the capture probe using the ligation product as a template, thereby generating an extended capture probe.

21. The method of any one of claims 1-20, further comprising extending the ligation product using the capture probe as a template, thereby generating an extended ligation product.

22. The method of claim 20 or 21, further comprising amplifying the extended capture probe or the extended ligation product.

23. The method of any one of claims 1-22, wherein the determining in (h) comprises sequencing.

24. The method of any one of claims 1-23, wherein the biological sample is disposed on the array.

25. The method of any one of claims 1-23, wherein the biological sample is disposed on a substrate.

26. The method of claim 25. wherein the method further comprises aligning the substrate comprising the biological sample with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array.

27. The method of any one of claims 1-26, wherein the biological sample is a tissue sample.

28. The method of claim 27, wherein the biological sample is a tissue section.

29. The method of claim 28. wherein the tissue section is a fixed tissue section.

30. The method of claim 29, wherein the fixed tissue section is a formalin-fixed, paraffin- embedded tissue section, a methanol-fixed tissue section, a paraformaldehy de tissue section, or an acetone-fixed tissue section.

31. The method of claim 28, wherein the tissue section is a fresh frozen tissue section.

32. The method of claim 27, wherein the tissue sample is a fixed tissue sample or a fresh- frozen tissue sample.

33. The method of any one of claims 1-32, wherein the target nucleic acid is DNA.

34. The method of any one of claims 1-32, wherein the target nucleic acid is RNA. optionally, mRNA.

35. The method of any one of claims 1-34, wherein the first probe further comprises a functional domain, wherein the functional domain is a primer binding site or a sequence specific site.

36. The method of any one of claims 1-35, wherein a 3' end of the first probe comprises one or more mismatched nucleotides.

37. The method of any one of claims 1-35, wherein a 3' end of the first probe comprises a dideoxynucleotide.

38. The method of claim 37, wherein the dideoxynucleotide comprises ddATP, ddCTP, ddGTP, or ddTTP.

39. A method for determining a presence or absence of a single nucleotide variant in a biological sample, the method comprising:(a) providing an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;(b) contacting a plurality of first probes and a plurality of blocking oligonucleotides with the biological sample, wherein a first probe of the plurality7of first probes comprises a sequence substantially complementary to a target nucleic acid at a region that is downstream of the single nucleotide variant and wherein a blocking oligonucleotide of the plurality of blocking oligonucleotides comprises: (i) a sequence substantially complementary to the target nucleic at a region that is upstream of the single nucleotide variant and (ii) a 3' blocking moiety;(c) hybridizing the first probe and the blocking oligonucleotide to the target nucleic acid;(d) extending the first probe using the target nucleic acid as a template, thereby generating an extended first probe;(e) contacting a plurality of second probes with the biological sample, wherein a second probe of the plurality of second probes comprises a partially double-stranded nucleic acid molecule comprising a capture probe capture domain;(f) ligating the extended first probe and the second probe, thereby generating a ligation product;(g) hybridizing the ligation product to the capture probe capture domain; and(h) determining (i) all or part of the sequence of the ligation product, or a complement thereof, and (ii) the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to determine the presence or absence of the single nucleotide variant in the biological sample.

40. The method of claim 39, wherein the determining in (h) further comprises correlating a location of the single nucleotide variant on the array to a location of the single nucleotide variant in the biological sample.

41. The method of claim 39 or 40, wherein the 3' blocking moiety is selected from the group consisting of: a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

42. The method of any one of claims 39-41, wherein the target nucleic acid comprises at least one single nucleotide variant.

43. The method of claim 42, wherein the target nucleic acid further comprises at least two, at least three, at least four, at least five or more single nucleotide variants.

44. The method of any one of claims 39-43. wherein the second probe comprises a 5' phosphate group.

45. The method of any one of claims 39-44, wherein the second probe comprises a capture probe capture domain that is substantially complementary’ to the capture probe capture domain on the array.

46. The method of claim 39, wherein the ligating comprises chemical ligation or enzymatic ligation.

47. The method of claim 46, wherein the enzymatic ligation comprises use of a ligase.

48. The method of claim 47, wherein the ligase comprises a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlor ella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.

49. The method of claim 48, wherein the ligase comprises a pre-activated T4 DNA ligase.

50. The method of any one of claims 39-49, wherein the extending the first probe using the target nucleic acid as a template in (d) comprises the use of a polymerase or a reverse transcriptase.

51. The method of claim 50, wherein the reverse transcriptase enzyme is a non-displacing reverse transcriptase.

52. The method of any one of claims 39-51, further comprising releasing the ligation product from the target nucleic acid.

53. The method of claim 52, wherein the releasing comprises the use of an endoribonuclease.

54. The method of claim 53, wherein the endoribonuclease comprises RNase H, RNase A, RNase C, or RNase I, and optionally, wherein the RNase H comprises RNase Hl, RNase H2, or both.

55. The method of any one of claims 39-54, wherein the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.

56. The method of any one of claims 39-55, further comprising contacting the biological sample with a permeabilization reagent.

57. The method of claim 56, wherein the permeabilization reagent comprises pepsin, proteinase K. or collagenase.

58. The method of any one of claims 39-57, further comprising extending the capture probe using the ligation product as a template thereby generating an extended capture probe.

59. The method of any one of claims 39-58, further comprising extending the ligation product using the capture probe as a template, thereby generating an extended ligation product.

60. The method of claim 58 or 59, further comprising amplifying the extended capture probe and / or the extended ligation product.

61. The method of any one of claims 39-60, wherein the determining in (h) comprises sequencing.

62. The method of any one of claims 39-61, wherein the biological sample is disposed on the array.

63. The method of any one of claims 39-61, wherein the biological sample is disposed on a substrate.

64. The method of claim 63, wherein the method further comprises aligning the substrate comprising the biological sample with the array, such that at least a portion of the biological sample is aligned with at least a portion of the array.

65. The method of any one of claims 39-65, wherein the biological sample is a tissue sample.

66. The method of claim 65, wherein the biological sample is a tissue section.

67. The method of claim 66, wherein the tissue section is a fixed tissue section.

68. The method of claim 67, wherein the fixed tissue section is a formalin-fixed, paraffin- embedded tissue section, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone-fixed tissue section.

69. The method of claim 66, wherein the tissue section is a fresh frozen tissue section.

70. The method of claim 65, wherein the tissue sample is a fixed tissue sample or a fresh- frozen tissue sample.

71. The method of any one of claims 39-70, wherein the target nucleic acid is DNA.

72. The method of any one of claims 39-70, wherein the target nucleic acid is RNA, optionally. mRNA.

73. The method of any one of claims 39-72, wherein the first probe further comprises a functional domain, wherein the functional domain is a primer binding site or a sequence specific site.

74. The method of any one of claims 39-73, wherein a 3' end of the first probe comprises one or more mismatched nucleotides.

75. The method of any one of claims 39-73. wherein a 3' end of the first probe comprises a dideoxynucleotide.

76. The method of claim 75, wherein the dideoxynucleotide comprises ddATP, ddCTP, ddGTP, or ddTTP.

77. A composition comprising:(a) a spatial array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;(b) a first probe and a blocking oligonucleotide hybridized to a target nucleic acid, wherein the first probe comprises a sequence substantially complementary7to the target nucleic acid at a region that is downstream of a genetic variant and the blocking oligonucleotide comprises: (i) a sequence substantially complementary to the target nucleic acid at a region that is upstream of the genetic variant, and (ii) a 3' blocking moiety; and(c) a second probe comprising a partially double-stranded nucleic acid molecule, wherein the second probe comprises a capture probe capture domain.

78. The composition of claim 77, wherein the 3' blocking moiety is selected from the group consisting of a carbon spacer, an inverted nucleotide, a dideoxynucleotide, and a nucleotide with a substituted 3' OH group.

79. The composition of claim 77 or 78, wherein the target nucleic acid comprises a genetic variant.

80. The composition of claim 79, wherein the target nucleic acid comprises at least two, at least three, at least four, at least five or more genetic variants.

81. The composition of claim 79 or 80, wherein the genetic variant comprises an insertion or a deletion.

82. The composition of claim 79 or 80, wherein the genetic variant comprises a single nucleotide variant or polymorphism.

83. The composition of any one of claims 77-82, wherein the second probe comprises a 5' phosphate group.

84. The composition of any one of claims 77-83, wherein the second probe comprises a capture probe capture domain that is substantially complementary to the capture probe capture domain on the array.

85. The composition of any one of claims 77-84, further comprising a polymerase or a reverse transcriptase.

86. The composition of claim 85, wherein the reverse transcriptase is a non-displacing reverse transcriptase.

87. The composition of any one of claims 77-86, further comprising a biological sample.

88. The composition of claim 87, wherein the biological sample is a tissue sample.

89. The composition of claim 88, wherein the tissue sample is a tissue section.

90. The composition of claim 89, wherein the tissue section is a fixed tissue section.

91. The composition of claim 90, wherein the fixed tissue section is a formalin-fixed, paraffin-embedded tissue section, a methanol-fixed tissue section, a paraformaldehyde tissue section, or an acetone-fixed tissue section.

92. The composition of claim 89, wherein the tissue section is a fresh frozen tissue section.

93. The composition of claim 88, wherein the tissue sample is a fixed tissue sample or a fresh-frozen tissue sample.

94. The composition of any one of claims 77-93, wherein the target nucleic acid is DNA.

95. The composition of any one of claims 77-93, wherein the target nucleic acid is RNA. optionally, mRNA.

96. The composition of any one of claims 77-95, wherein a 3' end of the first probe comprises one or more mismatched nucleotides.

97. The composition of any one of claims 77-95, wherein a 3' end of the first probe comprises a dideoxynucleotide.

98. The composition of claim 97, wherein the dideoxynucleotide comprises ddATP, ddCTP, ddGTP, or ddTTP.

99. A kit comprising:(a) a spatial array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode, and (ii) a capture domain;(b) (i) a plurality of first probes, wherein a first probe of the plurality of first probes comprises a sequence substantially complementary to a target nucleic acid at a region that is downstream of a genetic variant, and(ii) a plurality of second probes, wherein a second probe of the plurality of second probes comprises a partially double-stranded nucleic acid molecule and wherein the second probe comprises a capture probe capture domain;(c) a plurality of blocking oligonucleotides, wherein a blocking oligonucleotide of the plurality7of blocking oligonucleotides comprises: (i) a sequence substantially complementary7to the target nucleic acid at a region that is upstream of the genetic variant and (ii) a 3' blocking moiety;(d) a ligase; and(e) instructions for performing the method of any one of claims 1-74.

100. The kit of claim 99, wherein the capture probe further comprises one or more functional domains, a unique molecular identifier, a cleavage domain, and a combination thereof.

101. The kit of claim 99 or 100, wherein the ligase comprises a T4 RNA ligase (Rnl2), a PBCV-1 DNA ligase, a Chlor ella virus DNA ligase, a single stranded DNA ligase, or a T4 DNA ligase.

102. The kit of claim 101, wherein the ligase is a pre-activated T4 DNA ligase.

103. The kit of any one of claims 99-102, further comprising a polymerase.

104. The kit of any one of claims 99-103, further comprising a reverse transcriptase.

105. The kit of claim 104, wherein the reverse transcriptase enzyme is a non-displacing reverse transcriptase.

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