Methods, compositions, and kits for spatial analysis by tagmentation

Abstract

Provided herein are methods, compositions, and kits for detecting analytes of interest from biological samples using tagmentation. The method comprises: (a) hybridising a nucleic acid analyte to a capture probe on an array, wherein the capture probe comprises: (i) a spatial barcode and (ii) a capture domain; (b) extending the capture probe using the nucleic acid analyte as a template, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule comprises a complement of the sequence of the nucleic acid analyte; and (c) tagmenting the barcoded nucleic acid molecule or a derivative thereof using a transposome comprising a transposase to insert a transposon end sequence into the barcoded nucleic acid molecule or the derivative thereof, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment comprises: (i) the barcode, (ii) the transposon end sequence, and (iii) at least a portion of a sequence of the nucleic acid analyte or a complement thereof.

Description

[0001] METHODS, COMPOSITIONS, AND KITS FOR SPATIAL ANALYSIS BY TAGMENTATION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 675,324, filed July 25, 2024. The contents of this application are incorporated by reference in their entireties.

[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, viability, proliferation, behavior, signaling and crosstalk 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 provide 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] Spatial transcriptomic methods can enable precise genome-wide mRNA expression profiling of biological samples, e.g., at a single cell scale. Such techniques can provide a vast amount of analyte and / or expression data for a variety of analytes of a biological sample at high spatial resolution while retaining native spatial context. However, some existing workflows, including those that require a template switching or template switch oligonucleotides (TSO), have multiple complex steps, which can be tedious, resourceconsuming, and time-consuming to perform. Improved workflows with more streamlined assaying steps are therefore desirable and may be helpful in expanding the utility of spatial transcriptomic methodologies and applications thereof.

[0008] SUMMARY

[0009] The present disclosure features improved methods, compositions, and kits for the spatial detection of target analytes of a biological sample. The present methods, compositions, and kits for spatially capturing analytes, such as nucleic acids, proteins, and / or metabolites, from a biological sample can bypass template switching. cDNA amplification, fragmentation, end repair, A-tailing, adapter ligation, and / or other processes required in existing workflows, thereby streamlining library construction and reducing reagent usage. More specifically, the methods include using a transposase to insert a sequencing primer sequence into a spatially barcoded nucleic acid analyte, or a derivative or complement thereof, to thereby generate spatially barcoded, adapter-ligated nucleic acid fragments that are suitable for library preparation and sequence analysis.

[0010] Thus, provided herein are methods of spatial analysis of a nucleic acid analyte of a biological sample that include: (a) hybridizing the nucleic acid analyte to a capture probe on an array, wherein the capture probe includes: (i) a barcode sequence and (ii) a capture domain;

[0011] (b) extending the capture probe using the nucleic acid analyte as a template, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule includes a sequence of the nucleic acid analyte or a complement thereof; and (c) tagmenting the barcoded nucleic acid molecule or a derivative thereof to insert a transposon end sequence into the barcoded nucleic acid molecule or the derivative thereof, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment includes: (i) the barcode sequence and (ii) the transposon end sequence.

[0012] In some instances, the methods further include, after the tagmenting in (c), extending the barcoded nucleic acid fragment or the derivative thereof, thereby generating an extended barcoded fragment comprising: (i) the barcode, (ii) the transposon end sequence, and (iii) at least the portion of the sequence of the nucleic acid analyte or the complement thereof.

[0013] In some instances, the methods further include, after extending the barcoded nucleic acid fragment or the derivative thereof, releasing the nucleic acid analyte from the capture probe on the array. In some instances, releasing is by treating the extended barcoded fragment with a base. In some instances, releasing is by treating the extended barcoded fragment with an RNase. In some instances the method further include, after releasing the nucleic acid analyte from the capture probe on the array: (i) hybridizing a primer to the extended barcoded fragment or a derivative thereof, and (ii) extending the primer, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

[0014] In some instances, the barcoded extension product is a single-stranded DNA molecule. In some instances, primer comprises a sequencing primer sequence. In some instances, primer comprises a sequence complementary to the transposon end sequence. In some instances, the methods ds also include releasing the barcoded extension product from the capture probe on the array. In some instances, the methods also include determining a sequence of the barcoded extension product or a derivative thereof.

[0015] In some instances, the methods also include sequencing the barcoded extension product or a derivative thereof. In some instances, the methods also include amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

[0016] In some instances, the methods also include, after the tagmenting in (c), releasing a sequence at a 3' end of the nucleic acid analyte hybridized to the capture probe, wherein the sequence is not hybridized to the capture probe in (a). In some instances, the releasing is by treating with a nuclease. In some instances, the nuclease is exonuclease T or RNase T.

[0017] In some instances, the methods also include, after the releasing, extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof. In some instances, the barcoded extension product is a single-stranded RNA / DNA hybrid molecule. In some instances, the methods include releasing the barcoded extension product from the capture probe on the array. In some instances, the methods include determining a sequence of the barcoded extension product or a derivative thereof. In some instances, the methods include sequencing the barcoded extension product or the derivative thereof.

[0018] In some instances, the methods include reverse transcribing the barcoded extension product or a derivative thereof prior to the sequencing. In some instances, the methods include amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

[0019] In some instances, the methods include, prior to the tagmenting in (c), releasing the nucleic acid analyte from the capture probe on the array, and wherein the barcoded nucleic acid molecule comprises the complement of the sequence of the nucleic acid analyte. In some instances, releasing is by treating with a base. In some instances, releasing is by treating with an RNase.

[0020] In some instances, the methods include, after the tagmenting in (c), extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof. In some instances, the methods include, after extending the barcoded nucleic acid fragment or the derivative thereof, releasing the barcoded extension product from the capture probe on the array. In some instances, releasing is by denaturing the barcoded extension product or a derivative thereof. In some instances, the denaturing is by heating. In some instances, the barcoded extension product is a single-stranded DNA molecule. In some instances, the methods also include determining a sequence of the barcoded extension product or a derivative thereof. In some instances, the methods also include sequencing the barcoded extension product or the derivative thereof. In some instances, the methods also include amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

[0021] In some instances, the methods also include determining: (i) the sequence of the barcode or a complement thereof, and (ii) all or a portion of a sequence of the barcoded nucleic acid fragment or a complement thereof. In some instances, the extending in (b) is using a reverse transcriptase. In some instances, the extending in (b) is using a DNA polymerase. In some instances, the tagmenting in (c) comprises contacting the barcoded nucleic acid molecule with a transposome comprising the transposon end sequence. In some instances, the transposome comprises a transposase. In some instances, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof.

[0022] In some instances, the transposon end sequence is inserted at a 3' end of the barcoded nucleic acid fragment. In some instances, the insertion occurs at a region that is at least 100 nucleotides from a 5' end of the barcoded nucleic acid molecule. In some instances, the transposon end sequence comprises a sequencing primer sequence. In some instances, the sequencing primer sequence is a P7 sequence. In some instances, the tagmenting in (c) is performed on the array.

[0023] In some instances, the methods include, after (b), releasing the barcoded nucleic acid molecule from the capture probe on the array, and wherein the tagmenting in (c) is performed off of the array. In some instances, the tagmenting in (c) is performed in a presence of a permeabilization agent. In some instances, the permeabilization agent comprises a detergent and / or enzyme. In some instances, the detergent is sodium dodecyl sulfate (SDS), and wherein the enzy me is selected from pepsin or proteinase K. In some instances, the methods include, prior to (a), contacting a biological sample comprising the nucleic acid analyte with the capture probes on the array. In some instances, the biological sample is mounted on a first substrate, the array is on a second substrate, and the method further comprises aligning the first substrate with the array on a second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array. In some instances, after the aligning, releasing the capture probe from the array, such that the released capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte.

[0024] In some instances, the methods include imaging the biological sample. In some instances, the methods include staining the biological sample. In some instances, the staining comprises immunofluorescence, immunohistochemistry, hematoxylin, and / or eosin staining. In some instances, the biological sample is a tissue sample. In some instances, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

[0025] In some instances, the nucleic acid analyte is an RNA molecule. In some instances, the RNA molecule is a messenger RNA (mRNA) molecule. In some instances, the nucleic acid analyte is a DNA molecule. In some instances, the DNA molecule is a genomic DNA molecule.

[0026] In some instances, the DNA molecule is coupled to an analyte binding moiety. In some instances, the analyte binding moiety is an antibody or a functional fragment thereof. In some instances, the DNA molecule comprises: (i) a capture handle sequence that is complementary to the capture domain of the capture probe, and (ii) an analyte binding moiety barcode that is associated with or identifies the analyte binding moiety. In some instances, the DNA molecule is coupled to the analyte binding moiety' by a linker. In some instances, the linker is a cleavable linker. In some instances, the cleavable linker is a disulfide linker, a photo-cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker.

[0027] In some instances, the barcoded nucleic acid molecule is a partially double-stranded RNA / DNA molecule. In some instances, the partially double-stranded RNA / DNA molecule comprises an RNA sequence of the nucleic acid analyte and a DNA sequence complementary' to the sequence of the nucleic acid analyte. In some instances, the barcoded nucleic acid molecule is a single-stranded DNA molecule. In some instances, the single-stranded DNA molecule comprises a sequence complementary to a sequence of the nucleic acid analyte. In some instances, the barcode identifies a spatial location on the array.

[0028] In some instances, the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, and wherein the common barcode is specific to each subset of the subsets of capture probes. In some instances, the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes. In some instances, the capture probe further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof. In some instances, the one or more functional domains comprises a sequencing primer sequence. In some instances, the array comprises one or more features. In some instances, the one or more features comprises a bead and the capture probe is attached to the bead.

[0029] Also disclosed herein is a method for processing a nucleic acid analyte, the method comprising: (a) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein each of the first probe and the second probe comprises a sequence that is substantially complementary to sequences of the nucleic acid analyte; (b) coupling the first probe and the second probe, thereby generating a connected probe, wherein the connected probe comprises a capture probe binding domain; (c) hybridizing the capture probe binding domain of the connected probe to a capture probe of a plurality of capture probes on an array, wherein the capture probe comprises a barcode and a capture domain; (d) extending the capture probe using the nucleic acid analyte as a template, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule comprises a sequence of the connected probe or a complement thereof; and (e) tagmenting the barcoded nucleic acid molecule to insert a transposon end sequence, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment comprises: (i) the barcode, (ii) the transposon end sequence, (iii) at least a portion of a sequence of the first probe or a complement thereof, and (iv) at least portion of a sequence of the second probe or a complement thereof.

[0030] In some instances, prior to the tagmenting in (e), releasing the connected probe from the array, and wherein the barcoded nucleic acid molecule comprises the complement of the sequence of the connected probe. In some instances, the releasing is by treating with a base.

[0031] In some instances, the releasing is by heating. In some instances, after the tagmenting in (e), extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

[0032] In some instances, the methods include releasing the barcoded extension product from the array. In some instances, the releasing is by denaturing the barcoded extension product or a derivative thereof. In some instances, the denaturing is by heating. In some instances, the barcoded extension product is a single-stranded DNA molecule. In some instances, the methods include determining a sequence of the barcoded extension product or a derivative thereof.

[0033] In some instances, the methods include sequencing the barcoded extension product or a derivative thereof. In some instances, the methods include amplifying the barcoded extension product or the derivative thereof prior to the sequencing. In some instances, the first probe and the second probe hybridize to adjacent sequences of the nucleic acid analyte. In some instances, the first probe and the second probe hybridize to sequences that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides apart. In some instances, the coupling the first probe and the second probe comprises ligating the first probe and the second probe. In some instances, after the hybridizing in (a), generating: (i) an extended first probe or (ii) an extended second probe by contacting with a polymerase, wherein the extended first probe comprises a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe, and the extended second probe comprises a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe.

[0034] In some instances, the coupling the first probe and the second probe comprises ligating: (i) the first probe and the extended second probe, (ii) the extended first probe and the second probe, or (iii) the extended first probe and the extended second probe. In some instances, the ligating is performed using a ligase. In some instances, the ligase is a Chlorella virus DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase. In some instances, the first probe or the second probe comprises the capture probe binding domain. In some instances, the first probe, the second probe, or the connected probe is a single-stranded DNA molecule.

[0035] In some instances, the methods include determining: (i) a sequence of the barcode or a complement thereof, and (ii) all or a portion of a sequence of the barcoded nucleic acid fragment or a complement thereof. In some instances, the extending in (d) is using a DNA polymerase. In some instances, the tagmenting in (e) comprises contacting the barcoded nucleic acid molecule with a transposome comprising the transposon end sequence. In some instances, the transposome comprises a transposase. In some instances, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof. In some instances, the transposon end sequence is inserted at a 3' end of the barcoded nucleic acid fragment. In some instances, the insertion occurs at a region that is at least 100 nucleotides from a 5' end of the barcoded nucleic acid molecule. In some instances, the transposon end sequence comprises a sequencing primer sequence. In some instances, the sequencing primer sequence is a P7 sequence. In some instances, the tagmenting in (e) is performed on the array. In some instances, after (d), releasing the barcoded nucleic acid molecule from the array, and wherein the tagmenting in (e) is performed off of the array. In some instances, the tagmenting in (e) is performed in a presence of a permeabilization agent. In some instances, the permeabilization agent comprises a detergent and / or enzyme. In some instances, the detergent is sodium dodec l sulfate (SDS), and wherein the enzyme is selected from pepsin or proteinase K. In some instances, prior to (a), contacting a biological sample comprising the nucleic acid analyte with the array.

[0036] In some instances, the biological sample is mounted on a first substrate, the array is on a second substrate, and the method further comprises aligning the first substrate with the array on a second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array. In some instances, after the aligning, releasing the capture probe from the array, such that the released capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte.

[0037] In some instances, the methods include imaging the biological sample. In some instances, the methods include staining the biological sample. In some instances, the staining comprises hematoxylin or eosin staining. In some instances, the biological sample is a tissue sample. In some instances, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

[0038] In some instances, the nucleic acid analyte is an RNA molecule. In some instances, the RNA molecule is a mRNA molecule. In some instances, the nucleic acid analyte is a DNA molecule. In some instances, the DNA molecule is a genomic DNA molecule.

[0039] In some instances, the DNA molecule is coupled to an analyte binding moiety. In some instances, the analyte binding moiety is an antibody or a functional fragment thereof. In some instances, the DNA molecule comprises: (i) a capture handle sequence that is complementary' to the capture domain of the capture probe, and (ii) an analyte binding moiety7barcode that is associated with or identifies the analyte binding moiety. In some instances, the DNA molecule is coupled to the analyte binding moiety by a linker. In some instances, the linker is a cleavable linker. In some instances, the cleavable linker is a disulfide linker, a photo-cleavable linker, a UV-cleavable linker, or an enzy me cleavable linker. In some instances, the barcoded nucleic acid molecule is a partially double-stranded DNA molecule.

[0040] In some instances, the partially double-stranded DNA molecule comprises a sequence of the first probe or a complement thereof and a sequence of the second probe or a complement thereof. In some instances, the barcode identifies a spatial location on the array. In some instances, the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, wherein the common barcode is specific to each subset of the subsets of capture probes. In some instances, the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes. In some instances, the capture probe further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof. In some instances, the one or more functional domains comprises a sequencing primer sequence. In some instances, the array comprises one or more features. In some instances, the one or more features comprises a bead and the capture probe is attached to the bead.

[0041] Also provided herein is a composition comprising: (a) a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a barcode and (ii) a capture domain; and (b) a transposome comprising a transposase and a transposon end sequence, wherein the transposon end sequence comprises a sequencing primer sequence.

[0042] In some instances, the composition comprises an analyte. In some instances, the analyte is a nucleic acid analyte.

[0043] In some instances, the substrate comprises an array, wherein the barcode identifies a spatial location on the array. In some instances, the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, wherein the common barcode is specific to each subset of the subsets of capture probes. In some instances, the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes. In some instances, the array comprises one or more features. In some instances, the one or more features comprises a bead and the capture probe is attached to the bead.

[0044] In some instances, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof. In some instances, the transposome comprises two transposon end sequences. In some instances, the composition further comprises a reverse transcriptase. In some instances, the composition further comprises a polymerase. In some instances, the polymerase is a DNA polymerase.

[0045] In some instances, the composition further comprises a plurality of dNTPs.

[0046] In some instances, the composition further comprises one or more permeabilization reagents. In some instances, the one or more permeabilization reagents comprises a protease. a surfactant, or a detergent. In some instances, the protease comprises Proteinase K, pepsin, or collagenase. In some instances, the composition further comprises an RNase. In some instances, the composition further comprises a DNase.

[0047] In some instances, the nucleic acid analyte comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe.

[0048] In some instances, the composition further comprises a first probe and a second probe, wherein each of the first probe and the second probe comprises a sequence that is substantially complementary to sequences of the nucleic acid analyte. In some instances, the first probe or the second probe comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe. In some instances, the composition includes a ligase. In some instances, the ligase is a Chlorella vims DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase. In some instances, the composition further includes a tissue sample, wherein the nucleic acid analyte is from the tissue sample.

[0049] In some instances, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

[0050] In some instances, the nucleic acid analyte is an RNA molecule. In some instances, the RNA molecule is a mRNA molecule. In some instances, the nucleic acid analyte is a DNA molecule. In some instances, the DNA molecule is a genomic DNA molecule. In some instances, the DNA molecule is coupled to an analyte binding moiety.

[0051] In some instances, the analyte binding moiety is an antibody or a functional fragment thereof.

[0052] Also provided herein are kits. In some instances, the kits include (a) a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a barcode and (ii) a capture domain: (b) a reverse transcriptase or a polymerase: (c) a transposome comprising a transposase and a transposon end sequence, wherein the transposon end sequence comprises a sequencing primer sequence; and (d) instructions for performing any one of the method described herein.

[0053] In some instances, the substrate comprises an array, wherein the barcode identifies a location on the array. In some instances, the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, wherein the common barcode is specific to each subset of the subsets of capture probes. In some instances, the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes. In some instances, the array comprises one or more features. In some instances, the one or more features comprises a bead and the capture probe is attached to the bead.

[0054] In some instances, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof. In some instances, the transposome comprises two transposon end sequences. In some instances, the kit comprises the reverse transcriptase. In some instances, the kit comprises the polymerase. In some instances, the polymerase is a DNA polymerase. In some instances, the kit also includes a plurality of dNTPs. In some instances, the kit also includes one or more permeabilization reagents. In some instances, the one or more permeabilization reagents comprises a protease, a surfactant, or a detergent. In some instances, the protease comprises Proteinase K, pepsin, or collagenase. In some instances, the kit also includes an RNase. In some instances, the kit also includes a DNase.

[0055] In some instances, the kit also includes a first probe and a second probe, wherein each of the first probe and the second probe comprises a sequence that is substantially complementary’ to sequences of a nucleic acid analyte. In some instances, the first probe or the second probe comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe. In some instances, the kit also includes a ligase. In some instances, the ligase is a Chlorella virus DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase.

[0056] 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 contradictory7material.

[0057] 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.

[0058] 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. 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.

[0059] BRIEF DESCRIPTION OF DRAWINGS

[0060] 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.

[0061] 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.

[0062] 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.

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

[0064] FIG. 2B shows a perspective view of an exemplary sample handling apparatus in an open position.

[0065] FIG. 3A shows the first substrate angled over (superior to) the second substrate.

[0066] 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.

[0067] 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.

[0068] FIG. 4A shows a side view of the angled closure workflow.

[0069] FIG. 4B shows a top view of the angled closure workflow.

[0070] FIG. 5 is a schematic diagram showing an example of a barcoded capture probe, as described herein.

[0071] FIG. 6 shows a schematic illustrating a cleavable capture probe.

[0072] FIG. 7 shows exemplar}' capture domains on capture probes.

[0073] FIG. 8 shows an exemplary arrangement of barcoded features within an array. FIG. 9A shows an exemplary' workflow for performing templated capture and producing a ligation product.

[0074] FIG. 9B shows an exemplary workflow for capturing a ligation product from FIG. 9A on a substrate.

[0075] FIG. 10 is a schematic diagram of an exemplary analyte capture agent.

[0076] FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature- immobilized capture probe 1124 and an analyte capture agent 1126.

[0077] FIG. 12 is a schematic diagram of a spatial analysis method that includes incorporation of a template switching oligonucleotide (TSO) prior to second strand synthesis, poly dT: poly -thymidine sequence; RT: reverse transcription; KOH: potassium hydroxide; qPCR: quantitative polymerase chain reaction; cDNA: complementary DNA; A-tail: addition of adenosines to the 3’ end of fragments; SI-PCR: sample index polymerase chain reaction.

[0078] FIG. 13 is a schematic diagram of an alternative spatial analysis method that includes tagmentation of the double-stranded nucleic acid analyte / cDNA molecule and subsequent analysis of a barcoded DNA molecule. P7: P7 adapter; SDS: sodium dodecyl sulfate.

[0079] FIG. 14 is a schematic diagram of an alternative spatial analysis method that includes tagmentation of the double-stranded nucleic acid analyte / cDNA molecule and subsequent analysis of a barcoded mRNA / DNA hybrid molecule. polyA: polyadenylated tail sequence.

[0080] FIG. 15 is a schematic diagram of an alternative spatial analysis method that includes tagmentation of the single-stranded cDNA molecule and subsequent analysis of a barcoded DNA molecule.

[0081] DETAILED DESCRIPTION

[0082] A. Spatial Analysis Methods

[0083] Spatial analysis methodologies described herein can provide a vast amount of analyte and / or expression data for a variety of analytes of 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 or a spatial barcode sequence (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.

[0084] 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; and 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 1 Ox 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.

[0085] 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.

[0086] 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.

[0087] 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 may 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.

[0088] 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. 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.

[0089] 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 plant, 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 prokaryote such as a bacterium, e.g., Escherichia cold Staphylococci ox 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 an organoid, 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 a 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.

[0090] 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.

[0091] 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. 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 the tissue sample. In some instances, when the biological sample is fixed using a fixative including an alcohol (e.g., methanol or acetonemethanol 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 stage. In instances when the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone-methanol) fixation or acetone fixation, the biological sample is referred to as "fresh frozen’; 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).

[0092] 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 PFA, 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 using 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.

[0093] In some instances, the biological sample (e.g., a fixed frozen tissue sample) is treated with a citrate buffer. Citrate buffer can be used to decrosslink antigens and fixation medium for antigen retrieval in the biological sample. Thus, any suitable decrosslinking agent can be used in addition, 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.

[0094] 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). decrosslmked (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.

[0095] 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 biomolecules. 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(1 l):e27704 (201 1); and Mathieson W. et al.. Am J Clin Pathol.; 146( l):25-40 (2016), each of w hich 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.

[0096] 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 integrity7of fixed (e.g., FFPE) samples can be lower than of 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. By utilizing RTL probes that hybridize to RNA target sequences in the transcriptome, 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.

[0097] 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 embryo sample can be a non-human embryo sample. In some instances, the sample is a mouse embryo sample.

[0098] 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.

[0099] 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.

[0100] 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, Romanowsky, silver, Sudan, Wright’s, and / or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation.

[0101] 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.

[0102] 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.

[0103] WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. Briefly, any of the methods described herein includes permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of 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, or methanol), a detergent (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), an enzyme (e.g., an endopeptidase, an exopeptidase, or 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, 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.

[0104] 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.

[0105] 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)). In some embodiments, a capture probe described herein is affixed to an array. 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.

[0106] 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 of 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) are then 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.

[0107] 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. 1A, 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. In some embodiments, the capture probes are affixed to the array.

[0108] 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 wherein 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'.

[0109] As further shoyvn, one or more spacers 110 may be positioned betw een 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 shoyvn as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.

[0110] 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 (pm) and about 1 mm (e.g., between about 2 pm and about 800 pm, betw een about 2 pm and about 700 pm, between about 2 pm and about 600 pm, between about 2 pm and about 500 pm, between about 2 pm and about 400 pm, between about 2 pm and about 300 pm, between about 2 pm and about 200 pm, between about 2 pm and about 100 jam, between about 2 jam and about 25 pm, or between about 2 |am and about 10 fam), 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 pm. In some embodiments, the separation distance is less than 50 pm. In some embodiments, the separation distance is less than 25 pm. In some embodiments, the separation distance is less than 20 pm. The separation distance may include a distance of at least 2 pm.

[0111] 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 undesirable movement (e.g., convective movement) of transcripts and / or molecules during the diffusive transfer from the biological sample 102 to the capture probes.

[0112] 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., U.S. Patent Application Pub. No. 2021 / 0189475 and PCT Publ. No. WO 2022 / 061152 A2, each of which is incorporated by reference in its entirety.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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. 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.

[0117] 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 sample permeabilization (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.

[0118] 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.

[0119] In some embodiments, during permeabilization, the image capture device 220 maycapture 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.

[0120] 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 workflow 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary- implementations.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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

[0125] 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 exemplary angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate comprising the spacer 310.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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 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 between the substrates.

[0130] 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.

[0131] 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™, SDS), and enzymes (e.g., try psin or other proteases (e.g., Proteinase K)). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N- lauroylsarcosine sodium salt solution). In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and 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. Exemplary7proteases include, e.g., pepsin, try psin, 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 comprises RNase A, RNase C, RNase H, or RNase I. In some embodiments, the reagent medium comprises one or more of SDS or a sodium salt thereof, Proteinase K, pepsin, N- lauroylsarcosine, and RNase.

[0132] In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG molecular weight 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).

[0133] 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, the second substrate, or both, and then dried.

[0134] 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.

[0135] 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.

[0136] There are at least tw o 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 cleave spatially barcoded capture probes from an array and promote the spatially barcoded capture probes towards and / or into or onto the biological sample.

[0137] 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 serve as proxies for the template.

[0138] As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to a terminus (e.g.. a 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 domain of the capture probe. 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.

[0139] In some embodiments, extended capture probes are amplified (e.g.. in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e g., sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction).

[0140] Additional variants of spatial analysis methods, including, in some embodiments, an imaging step, are described in Section (II)(a) of PCT Publication No. WO2020 / 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 captured 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 in its entirety.

[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 ty pes in close proximity (e.g., nearest neighbor or proximity based analysis); determination of up-regulated and / or down -reg dated 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] For spatial array-based methods, a substrate may function as a support for direct or indirect attachment of capture probes to features of the array. In some cases, capture probes are directly or indirectly affixed to the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all 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, which is herein incorporated by reference. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i). (II)(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 or wells) comprising capture probes). As used herein unless expressly stated otherw ise, “array” refers to any arrangement or collection of capture probes or features comprising capture probes on a substrate. In some embodiments of the disclosure, capture probes are provided on a substrate or array in an ordered manner. Alternatively, in some embodiments of the disclosure, capture probes are provided on a substrate or array in a random manner (i.e., a random array), such that a decoding step is required to determine the location of one or more features on the substrate. 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.

[0145] 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 combinations thereof. The capture probe can also include a spatial barcode 505. The capture probe can also include a unique molecular identifier (UMI) sequence 506. While FIG. 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 adapter described herein.

[0146] FIG. 6 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter a non-permeabilized cell and bind to analytes within the cell. 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.

[0147] FIG. 7 is a schematic diagram of an exemplary multiplexed 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 / or 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.

[0148] The functional sequences can generally be selected for compatibility with any of a variety7of 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.

[0149] 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.

[0150] FIG. 8 depicts an exemplary7arrangement 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.).

[0151] 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.

[0152] 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., a cell or cell nucleus 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., a plurality of cells or cell nuclei 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, single cell nuclei, 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.

[0153] 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 in its entirety. 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 probe binding domain (e.g., a poly(A) sequence or anon-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 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.

[0154] 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.

[0155] 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-hybridi ation 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 901 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 compositions for spatial detection using templated ligation have been described in PCT Publication No. WO 2021 / 133849 Al, U.S. Pat. Nos. 1 1,332,790 and 11,505,828, each of which is incorporated by reference in its entirety.

[0156] 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.

[0157] 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, polymerization (e.g., reverse transcription (RT)) reagents can be added to permeabilized biological samples. Incubation with the polymerization 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 (e.g., ligation products). The ligation products can be extended using the capture probe as a template to include a complement of the capture probe, thereby generating extended ligation products.

[0158] 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, i5 9017, i7 9018, and P7 9019 sequences 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.

[0159] 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.

[0160] 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 exemplary7analyte binding moiety 1004 is capable of binding to an analyte 1006 and the analyte capture agent 1002 is capable of interacting with a spatially barcoded capture probe. The analy te 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).

[0161] 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 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 analyte binding 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, thermal-cleavable, 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).

[0162] 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 the 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.

[0163] 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 each spatial barcode is 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.

[0164] 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 or a fiducial marker) of the array. Accordingly, each feature location has an ‘‘address’' or location in the coordinate space of the array.

[0165] Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which is herein incorporated by reference. See, for example, the Exemplary embodiment 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.

[0166] 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.

[0167] 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, on 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.

[0168] 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.

[0169] 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.

[0170] 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. W02021 / 102003 and / or U.S. Patent Application Publication No. 2021 / 0150707, each of which is incorporated herein by reference in its entirety.

[0171] 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.

[0172] 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.

[0173] 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 entirety7. 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, 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.

[0174] B. Methods of Spatial Analysis by Tagmentation-based Library Preparation

[0175] The present disclosure features improved methods, compositions, and kits for the spatial detection of target analytes from a biological sample. The present methods, compositions, and kits for spatially capturing analytes, such as nucleic acids, proteins, and / or metabolites, from a biological sample can bypass template switching, cDNA amplification, fragmentation, end repair, A-tailing, PCR adapter ligation, and / or other processes required in existing workflows, thereby streamlining library7construction and reducing reagent usage. More specifically, the present methods include using a transposase to insert a sequencing primer sequence into a spatially barcoded nucleic acid analyte, or a derivative or complement thereof, to generate a spatially barcoded, adapter-ligated nucleic acid fragments suitable for subsequent library preparation and sequence analysis. Thus, provided herein are methods of spatial analysis of a nucleic acid analyte from a biological sample that include: (a) hybridizing the nucleic acid analyte to a capture probe on an array, wherein the capture probe includes: (i) a barcode sequence and (ii) a capture domain;

[0176] (b) extending the capture probe, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule includes a sequence of the nucleic acid analyte or a complement thereof; and (c) tagmenting the barcoded nucleic acid molecule or a derivative thereof to insert a transposon end sequence into the barcoded nucleic acid molecule or the derivative thereof, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment includes: (i) the barcode sequence and (ii) the transposon end sequence.

[0177] In some cases, the methods further include, after the tagmenting in (c), extending the barcoded nucleic acid fragment or the derivative thereof, thereby generating an extended barcoded fragment comprising: (i) the barcode sequence and (ii) the transposon end sequence.

[0178] In some embodiments, the methods further include, after extending the barcoded nucleic acid fragment or the derivative thereof, releasing the nucleic acid analyte from the capture probe on the array. The releasing can be performed by treating the nucleic acid analyte hybridized to the capture probe on the array with a base or an RNase. In some embodiments, the methods further include, after releasing the nucleic acid analyte from the capture probe on the array: (i) hybridizing a primer to the extended barcoded fragment or a derivative thereof, and (ii) extending the primer, thereby generating a barcoded extension product comprising: (i) the barcode sequence or a complement thereof, and (ii) the transposon end sequence or a complement thereof. In some embodiments, the barcoded extension product is a single-stranded DNA molecule. In some embodiments, the primer includes a sequencing primer sequence. In some embodiments, the primer includes a sequence complementary to the transposon end sequence.

[0179] In some embodiments, the methods further include releasing the barcoded extension product from the array prior to sequence analysis and / or amplification. In some embodiments, the methods further include determining a sequence of the barcoded extension product or a derivative thereof. In some embodiments, the methods further include sequencing the barcoded extension product or a derivative thereof. In some embodiments, the methods further include amplifying the barcoded extension product or the derivative thereof prior to the sequencing. In some cases, the methods further include, after the tagmenting in (c), releasing a sequence at a 3' end of the nucleic acid analyte hybridized to the capture probe, wherein the sequence is not hybridized to the capture probe in (a). In some embodiments, the releasing is by treating with a nuclease, such as exonuclease T or RNase T.

[0180] In some embodiments, the methods further include, after the releasing, extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) the barcode sequence or a complement thereof, and (ii) the transposon end sequence or a complement thereof. In some embodiments, the barcoded extension product is a single-stranded RNA / DNA hybrid molecule.

[0181] In some embodiments, the methods further include releasing the barcoded extension product from the array. In some embodiments, the methods further include determining a sequence of the barcoded extension product or a derivative thereof. In some embodiments, the methods further include sequencing the barcoded extension product or a derivative thereof. In some embodiments, the methods further include reverse transcribing the barcoded extension product or the derivative thereof prior to the sequencing. In some embodiments, the methods further include amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

[0182] In some cases, the methods further include, prior to the tagmenting in (c), releasing the nucleic acid analyte from the capture probe on the array, and wherein the barcoded nucleic acid molecule includes the complement of the sequence of the nucleic acid analyte. In some embodiments, the releasing is by treating with a base or an RNase.

[0183] In some embodiments, the methods further include, after the tagmenting in (c), extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) the barcode sequence or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

[0184] In some embodiments, the methods further include, after extending the barcoded nucleic acid fragment or the derivative thereof, releasing the barcoded extension product from the capture probe on the array. In some embodiments, the releasing is performed by denaturing the barcoded extension product or a derivative thereof. In some embodiments, the denaturing is performed by heating. In some embodiments, the barcoded extension product is a single-stranded DNA molecule.

[0185] In some embodiments, the methods further include determining a sequence of the barcoded extension product or a derivative thereof. In some embodiments, the methods further include sequencing the barcoded extension product or a derivative thereof. In some embodiments, the methods further include amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

[0186] In some embodiments, the methods further include determining: (i) the barcode sequence or a complement thereof and (ii) all or a portion of a sequence of the barcoded nucleic acid fragment or a complement thereof. In some embodiments, the extending in (b) is performed using a reverse transcriptase. In some embodiments, the extending in (b) is performed using a DNA polymerase.

[0187] In some embodiments, the tagmenting in (c) includes contacting the barcoded nucleic acid molecule with a transposome comprising the transposon end sequence. In some embodiments, the transposome further includes a transposase. In some embodiments, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof. In some embodiments, the transposon end sequence is inserted at a 3' end of the barcoded nucleic acid fragment. In some embodiments, the insertion occurs at a region that is at least 100 nucleotides from a 5' end of the barcoded nucleic acid molecule. In some embodiments, the transposon end sequence includes a sequencing primer sequence. In some embodiments, the sequencing primer sequence is a P7 sequence.

[0188] In some embodiments, the tagmenting in (c) is performed on the array. Alternatively, the methods further include, after (b), releasing the barcoded nucleic acid molecule from the array, and wherein the tagmenting in (c) is performed off of the array. Tagmentation of target nucleic acids or derivatives thereof may be more efficient in solution as compared to on the array. Thus, in some embodiments, the methods further include releasing the barcoded nucleic acid molecule from the array, and wherein the tagmenting in (c) is performed off of the array.

[0189] In some embodiments, the tagmenting in (c) is performed in a presence of a permeabilization agent. In some embodiments, the permeabilization agent includes a detergent. In some embodiments, the detergent is sodium dodecyl sulfate (SDS).

[0190] In some embodiments, the methods further include, prior to (a), contacting a biological sample comprising the nucleic acid analyte with the array. In some embodiments, the biological sample is mounted on a first substrate, the array is on a second substrate, and the method further includes aligning the first substrate with the array on a second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array. In some embodiments, the methods further include, after the aligning, releasing the capture probe from the array, such that the released capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte.

[0191] In some embodiments, the methods further include imaging the biological sample.

[0192] In some embodiments, the methods further include staining the biological sample. In some embodiments, the staining includes hematoxylin or eosin staining.

[0193] In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

[0194] In some embodiments, the nucleic acid analyte is an RNA molecule, e.g., a messenger RNA (mRNA) molecule. In some embodiments, the nucleic acid analyte is a DNA molecule, e.g., a genomic DNA molecule. In some embodiments, the DNA molecule is coupled to an analyte binding moiety. In some embodiments, the analyte binding moiety is an antibody or a functional fragment thereof.

[0195] In some embodiments, the barcoded nucleic acid molecule is a partially doublestranded RNA / DNA molecule. In some embodiments, the partially double-stranded RNA / DNA molecule includes an RNA sequence of the nucleic acid analyte and a DNA sequence complementary to the sequence of the nucleic acid analyte.

[0196] In some embodiments, the barcoded nucleic acid molecule is a single-stranded DNA molecule. In some embodiments, the single-stranded DNA molecule includes a sequence complementary to a sequence of the nucleic acid analyte.

[0197] In some embodiments, the barcode sequence identifies a spatial location on the array. In some embodiments, the array includes subsets of capture probes, each subset of the subsets of capture probes includes a common barcode sequence, and wherein the common barcode is specific to each subset of the subsets of capture probes. As such, the common barcode sequence can be used to identify a capture probe as being from a given subset of capture probes and / or located at a given location on the array. In some embodiments, the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode sequence is common to the subset of capture probes.

[0198] In some embodiments, the capture probe further includes a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof. In some embodiments, the one or more functional domains includes a sequencing primer sequence.

[0199] In some embodiments, the array includes one or more features. In some embodiments, the one or more features includes a bead and the capture probe is attached to the bead. Also provided herein are methods of spatial analysis of a nucleic acid analyte from a biological sample that include: (a) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein each of the first probe and the second probe includes a sequence that is substantially complementary to sequences of the nucleic acid analyte; (b) coupling the first probe and the second probe, thereby generating a connected probe, wherein the connected probe includes a capture probe binding domain; (c) hybridizing the capture probe binding domain of the connected probe to a capture probe of a plurality of capture probes on an array, wherein the capture probe includes a barcode sequence and a capture domain; (d) extending the capture probe, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule includes a sequence of the connected probe or a complement thereof; and (e) tagmenting the barcoded nucleic acid molecule to insert a transposon end sequence, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment includes: (i) the barcode, (ii) the transposon end sequence, (iii) at least a portion of a sequence of the first probe or a complement thereof, and (iv) at least portion of a sequence of the second probe or a complement thereof.

[0200] In some embodiments, the methods further include, prior to the tagmenting in (e), releasing the connected probe from the capture probe on the array, and wherein the barcoded nucleic acid molecule includes the complement of the sequence of the connected probe. In some embodiments, the releasing is by treating with a base. In some embodiments, the releasing is by heating.

[0201] In some embodiments, the methods further include, after the tagmenting in (e), extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) the barcode sequence or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

[0202] In some embodiments, the methods further include, releasing the barcoded extension product from the capture probe on the array. In some embodiments, the releasing is by denaturing the barcoded extension product or a derivative thereof. In some embodiments, the denaturing is by heating. In some embodiments, the barcoded extension product is a singlestranded DNA molecule.

[0203] In some embodiments, the methods further include determining a sequence of the barcoded extension product or a derivative thereof. In some embodiments, the methods further include sequencing the barcoded extension product or a derivative thereof. In some embodiments, the methods further include amplifying the barcoded extension product or the derivative thereof prior to the sequencing. In some embodiments, the first probe and the second probe hybridize to adjacent sequences of the nucleic acid analyte. Alternatively, in some embodiments, the first probe and the second probe hybridize to sequences that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides apart.

[0204] In some embodiments, the coupling the first probe and the second probe includes ligating the first probe and the second probe.

[0205] In some embodiments, the methods further include, after the hybridizing in (a), generating: (i) an extended first probe or (ii) an extended second probe by contacting with a polymerase, wherein the extended first probe includes a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe, and the extended second probe includes a sequence complementary to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe.

[0206] In some embodiments, the coupling the first probe and the second probe includes ligating: (i) the first probe and the extended second probe, (ii) the extended first probe and the second probe, or (iii) the extended first probe and the extended second probe.

[0207] In some embodiments, the ligating is performed by using a ligase, such as a Chlorella virus DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase.

[0208] In some embodiments, the first probe or the second probe includes the capture probe binding domain.

[0209] In some embodiments, the first probe, the second probe, or the connected probe is a single-stranded DNA molecule.

[0210] In some embodiments, the methods further include determining: (i) the barcode sequence or a complement thereof, and (ii) all or a portion of a sequence of the barcoded nucleic acid fragment or a complement thereof.

[0211] In some embodiments, the extending in (d) is using a DNA polymerase.

[0212] In some embodiments, the tagmenting in (e) includes contacting the barcoded nucleic acid molecule with a transposome comprising the transposon end sequence. In some embodiments, the transposome includes a transposase. In some embodiments, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof.

[0213] In some embodiments, the transposon end sequence is inserted at a 3' end of the barcoded nucleic acid fragment. In some embodiments, the insertion occurs at a region that is at least 100 nucleotides from a 5' end of the barcoded nucleic acid molecule. In some embodiments, the transposon end sequence includes a sequencing primer sequence. In some embodiments, the sequencing primer sequence is a P7 sequence.

[0214] In some embodiments, the tagmenting in (c) is performed in a presence of a permeabilization agent. In some embodiments, the permeabilization agent includes a detergent. In some embodiments, the detergent is sodium dodecyl sulfate (SDS).

[0215] In some embodiments, the methods further include, prior to (a), contacting a biological sample comprising the nucleic acid analyte with the array. In some embodiments, the biological sample is mounted on a first substrate, the array is on a second substrate, and the method further includes aligning the first substrate with the array on a second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array. In some embodiments, the aligning comprises: (a) mounting the first substrate on a first member of a support device, the first member configured to retain the first substrate; (b) mounting the second substrate on a second member of the support device; (c) applying a reagent medium to the first substrate and / or the second substrate; and (d) operating an alignment mechanism of the support device to move the first member and / or the second member such that at least a portion of the biological sample is aligned with at least a portion of the array, and such that the portion of the biological sample and the portion of the array contact the reagent medium.

[0216] In some embodiments, the methods further include, after the aligning, releasing the capture probe from the array, such that the released capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte.

[0217] In some embodiments, the methods further include imaging the biological sample, e.g., by brightfield imaging and / or immunofluorescence.

[0218] In some embodiments, the methods further include staining the biological sample. In some embodiments, the staining includes hematoxylin or eosin staining.

[0219] In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample. In some cases, the fresh tissue or the frozen tissue sample is also fixed using a fixative agent.

[0220] In some embodiments, the nucleic acid analyte is an RNA molecule, e g., a mRNA molecule. In some embodiments, the nucleic acid analyte is a DNA molecule, e.g., a genomic DNA molecule. In some embodiments, the DNA molecule is coupled to an analy te binding moiety. In some embodiments, the analyte binding moiety is an antibody or a functional fragment thereof. In some embodiments, the antibody or a functional fragment thereof binds to a target protein or other analyte in the tissue sample.

[0221] In some embodiments, the barcoded nucleic acid molecule is a partially doublestranded RNA / DNA molecule. In some embodiments, the partially double-stranded RNA / DNA molecule includes an RNA sequence of the nucleic acid analyte and a DNA sequence complementary’ to the RNA sequence of the nucleic acid analyte.

[0222] In some embodiments, the barcode sequence identifies a spatial location on the array, such that when at least a portion of the biological sample is aligned with at least a portion of the array, the barcode sequence identifies a spatial location of the nucleic acid analyte in the biological sample, e.g., relative to a fiducial marker on the array. The array can include subsets of capture probes, where each subset of the subsets of capture probes includes a common barcode, and wherein the common barcode is specific to each subset of the subsets of capture probes. In some embodiments, the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode sequence is common to the subset of capture probes.

[0223] In some embodiments, the capture probe further includes a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof. In some embodiments, the one or more functional domains includes a sequencing primer sequence, e.g., a P5 sequence.

[0224] In some embodiments, the array includes one or more features. In some embodiments, the one or more features includes a bead and the capture probe is attached to the bead.

[0225] C. Compositions for Spatial Analysis by Tagmentaiion-based Library Preparation

[0226] In addition to the methods described herein, the present disclosure also features compositions for the spatial detection of target analytes from a biological sample. Thus, provided herein are compositions produced by any of the methods herein and illustrated in the figures herein. For example, provided herein are compositions including: (a) a substrate having a plurality of capture probes, where a capture probe of the plurality of capture probes includes: (i) a barcode sequence and (ii) a capture domain; (b) a nucleic acid analyte; and (c) a transposome having a transposase and a transposon end sequence, where the transposon end sequence includes a sequencing primer sequence.

[0227] In some embodiments, the substrate can include an array, where the barcode sequence of the capture probe identifies a location on the array. The array can include subsets of capture probes, such that each subset of the subsets of capture probes includes a common barcode that is specific (common) to a given subset of the subsets of capture probes. The capture probe can be from a subset of capture probes from the subsets of capture probes, such that the barcode sequence of the capture probe is common to the subset of capture probes. In some embodiments, the array can include one or more features. The one or more features can include a bead and the capture probe is attached to the bead.

[0228] In some embodiments, the transposase is a Tn5 transposase. a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof. For example, the transposase can be a recombinant transposase designed to append one or more sequencing adapters (e.g., one or more sequencing primer sequences) and fragment a target nucleic acid. The transposome can include two transposon end sequences. In some embodiments, the two transposon end sequences are the same. In other embodiments, the two transposon end sequences are different.

[0229] In some embodiments, the compositions include a reverse transcriptase. In some embodiments, the compositions include a polymerase. The polymerase can be a DNA polymerase. The compositions can further include a plurality of dNTPs for facilitating reverse transcription or nucleic acid polymerization.

[0230] In some embodiments, the compositions further include one or more permeabilization reagents. The one or more permeabilization reagents can include a protease, a surfactant, or a detergent. The protease can include Proteinase K, pepsin, or collagenase.

[0231] In some embodiments, the compositions further include an RNase. In some embodiments, the compositions further include a DNase.

[0232] In some embodiments, the nucleic acid analyte comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe.

[0233] In some embodiments, the compositions further include a first probe and a second probe, where each of the first probe and the second probe includes a sequence that is substantially complementary to sequences of the nucleic acid analyte. The first probe or the second probe can include a capture probe capture domain capable of hybridizing to the capture domain of the capture probe.

[0234] In some embodiments, the compositions further include a ligase. The ligase can be a Chlorella vims DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase.

[0235] In some embodiments, the compositions further include a tissue sample, such that the nucleic acid analyte is from the tissue sample. The tissue sample can be a formalin-fixed, paraffin-embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample. In some embodiments, the nucleic acid analyte can be an RNA molecule. The RNA molecule can be a mRNA molecule. In some embodiments, the nucleic acid analyte can be a DNA molecule. The DNA molecule can be a genomic DNA molecule. The DNA molecule can be coupled to an analyte binding moiety. The analyte binding moiety can be an antibody or a functional fragment thereof. The antibody or a functional fragment thereof can bind to a target protein or other analyte in the tissue sample.

[0236] D. Kits for Spatial Analysis by Tagmentation-based Library Preparation

[0237] In addition to the methods and compositions described herein, the present disclosure also features kits for the spatial detection of target analytes from a biological sample. Thus, provided herein are kits including: (a) a substrate having a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes: (i) a barcode sequence and (ii) a capture domain; (b) a reverse transcriptase or a polymerase: (c) a transposome comprising a transposase and a transposon end sequence, wherein the transposon end sequence includes a sequencing primer sequence; and (d) instructions for performing any method described herein.

[0238] In some embodiments, the substrate can include an array, where the barcode sequence of the capture probe identifies a location on the array. The array can include subsets of capture probes, such that each subset of the subsets of capture probes includes a common barcode that is specific to each subset of the subsets of capture probes. The capture probe can be from a subset of capture probes from the subsets of capture probes, such that the barcode sequence of the capture probe is common to the subset of capture probes. In some embodiments, the array can include one or more features. The one or more features can include a bead and the capture probe is attached to the bead.

[0239] In some embodiments, the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof. For example, the transposase can be a recombinant transposase designed to append one or more sequencing adapters (e.g., one or more sequencing primer sequences) and fragment a target nucleic acid. The transposome can include two transposon end or mosaic end (ME) sequences. In some embodiments, the two transposon end sequences are the same. In other embodiments, the two transposon end sequences are different.

[0240] In some embodiments, the kits include the reverse transcriptase. In some embodiments, the kits include the polymerase. The polymerase can be a DNA polymerase. The kits can further include a plurality of dNTPs for facilitating reverse transcription or nucleic acid polymerization.

[0241] In some embodiments, the kits further include one or more permeabilization reagents. The one or more permeabilization reagents can include a protease, a surfactant, or a detergent. The protease can include Proteinase K, pepsin, or collagenase.

[0242] In some embodiments, the kits further include an RNase. In some embodiments, the kits further include a DNase.

[0243] In some embodiments, the kits further include a first probe and a second probe, where each of the first probe and the second probe includes a sequence that is substantially complementary to sequences of a nucleic acid analyte. The first probe or the second probe can include a capture probe capture domain capable of hybridizing to the capture domain of the capture probe.

[0244] In some embodiments, the kits further include a ligase, e.g., a Chlorella virus DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase.

[0245] EXAMPLES

[0246] EXAMPLE 1 - Tagmentation of double-stranded RNA / cDNA hybrid extension product and analysis of synthesized second strand DNA product.

[0247] FIG. 12 is a schematic of an example workflow for spatial analysis of an mRNA analyte from a biological sample, e.g., a tissue sample. In this workflow, polyadenylated mRNA can be captured by hybridization to a capture domain of a capture probe on a substrate, e.g., a spatial array having a plurality of capture probes. The capture domain can include a poly(T) (e.g., poly dT) sequence that is configured to hybridized to the poly(A) sequence of the polyadenylated mRNA. The capture probe also includes a spatial barcode (gray box) that identifies the capture probe from the plurality of capture probes on the substrate or spatial array. From a spatial context, the spatial barcode of the capture probe can identify a location on the array. For example, the array can include subsets of capture probes associated with a location on the array, such that each subset of the subsets of capture probes includes a common spatial barcode that is specific to each subset of the subsets of capture probes. The capture probe can be from one subset of capture probes and the spatial barcode of the capture probe is common to that one subset of capture probes. In some cases, the array can include one or more features. The one or more features can include a bead and the capture probe is attached to the bead (not shown). The capture probe can also include a sequencing primer sequence, e.g., a P5 sequence. Following mRNA capture, the capture probe can be extended using a reverse transcriptase or a polymerase, and the mRNA as a template, thereby generating a complementary DNA (cDNA) molecule that includes both the spatial barcode and a sequence complementary to a sequence of the mRNA (e.g., a barcoded nucleic acid molecule; e.g., an extended capture probe). As shown in FIG. 12, the extension reaction also includes the addition of untemplated cytosine nucleotides (e.g., CCC) to the 3' end of the extended cDNA. The untemplated cytosine nucleotides provide a blunt end for hybridization to a template switch oligonucleotide (TSO) having a 3' riboguanosine sequence (e.g., rGrGrG). The TSO can be used to add a common 3' sequence to the cDNA that is used for cDNA amplification.

[0248] Following incorporation of the TSO sequence to the cDNA, the mRNA analyte can be washed away, e.g.. by treating with a base (KOH). A second strand primer can then hybridize to the barcoded cDNA via the TSO sequence to initiate second strand synthesis (extension) to generate a partially double-stranded cDNA molecule. The synthesized second strand, which includes the complement of the spatial barcode, can then be eluted from the array by denaturing the double-stranded cDNA molecule. The collected second strand cDNA can then be amplified and quantified by qPCR. The amplified cDNA can then be subjected to fragmentation, end repair, A-tailing, and adapter ligation (e g., using smRNA2 or T4 RNA ligase) prior to sample index-PCR (SI-PCR) library construction.

[0249] FIG. 13 is a schematic of a streamlined spatial analysis workflow herein that can bypass various processes in the workflow of FIG. 12. Similar to the workflow of FIG. 12. this alternative method involves capturing a poly adenylated mRNA analyte from a biological sample using a capture probe having a poly(T) sequence.

[0250] Following mRNA capture, the capture probe can be extended using a reverse transcriptase or a polymerase, and the mRNA as a template, thereby generating a cDNA molecule that includes both the spatial barcode and a sequence complementary to a sequence of the mRNA analyte. As shown in FIG. 13, the partially double-stranded RNA / cDNA can be subjected to tagmentation by contacting with a transposome complex that includes a transposase and one or more transposon end or mosaic end (ME) sequences. The one or more transposon end sequences can include one or more sequencing primer sequences, e.g., a P7 sequence, as shown. The transposase can facilitate fragmentation and insertion of one or more transposon end sequences to the partially double-stranded RNA / cDNA (i.e., tagmentation). Depending on the specificity of the transposase, insertion can occur at a region that is at least 100 nucleotides from an end (either 3' or 5') of the target molecule. In some cases, the insertion site is random. Alternatively, the insertion site can be non-random, e.g.. site-specific or sequence-specific. Optionally, the tagmentation can occur after releasing the target molecule from the array.

[0251] Following tagmentation, one or more detergents, e.g., SDS, can then be used to wash away the transposome complex and / or non-bound fragments from the array, including nucleic acid fragments that are not hybridized to the capture probe and thus, not affixed to the array. The remaining nucleic acid fragment (“the 3' element”) that includes the barcode sequence at its 5' end and a P7 sequence at its 3' end can be subsequently processed for library construction. The barcoded nucleic acid fragment can be extended to fill any gaps resulting from tagmentation. The top strand comprising a portion of the mRNA analyte can then be washed away, e.g., by treating with a base (e.g., KOH) or an RNase. Next, a second strand primer can then hybridize to the barcoded cDNA via the P7 sequence to initiate second strand synthesis (extension) to generate a partially double-stranded cDNA molecule. The synthesized second strand, which includes the complement of the barcode sequence, can then be eluted from the array by denaturing the double-stranded cDNA molecule. The collected second strand cDNA product can be amplified for SI-PCR library construction.

[0252] Using transposase-mediated tagmentation, the workflow of FIG. 13 thereby bypasses the fragmentation, end repair, A-tailing, and adapter ligation processes that precede library construction and sequencing.

[0253] EXAMPLE 2 - Tagmentation of double-stranded RNA / DNA extension product and analysis of single-stranded RNA / DNA hybrid product.

[0254] FIG. 14 shows another streamlined spatial analysis workflow herein that can bypass various processes in the workflow of FIG. 12. As previously described, a poly adenylated mRNA analyte from a biological sample can be captured using a capture probe having a poly(T) sequence.

[0255] Following mRNA capture, the capture probe can be extended using a reverse transcriptase or a polymerase, and the mRNA as a template, thereby generating a cDNA molecule that includes both the barcode sequence and a sequence complementary to a sequence of the mRNA analyte. As shown in FIG. 14, the partially double-stranded RNA / cDNA (i.e., one RNA strand hybridized to one DNA strand) can be subjected to tagmentation by contacting with a transposome complex that includes a transposase and one or more transposon end sequences, e.g., P7 sequences, as shown. The transposase can facilitate fragmentation and insertion of one or more transposon end sequences to the partially double-stranded RNA / cDNA (i.e., tagmentation). Optionally, the tagmentation can occur after releasing the target molecule from the array.

[0256] Similar to the workflow of FIG. 13, following tagmentation, one or more detergents, e.g., SDS, can then be used to wash away the transposome complex and / or non-bound fragments from the array. The remaining nucleic acid fragment (“the 3' element’") that includes the barcode sequence at its 5' end and a P7 sequence at its 3' end can subsequently be processed for library construction. Distinct from the workflow of FIG. 13, the barcoded nucleic acid fragment can then be treated using an exonuclease, e.g., exonuclease T, to cleave the 3' polyadenylated overhang of the mRNA analyte that is not hybridized (i.e., noncompl ementary) to the capture probe, thereby generating a blunt 3' end available for extension. The resulting barcoded nucleic acid fragment can then be extended to fill any gaps resulting from tagmentation, as well as toward the 5' end of the capture probe comprising the barcode sequence, such that the top strand includes a complement of the barcode sequence, a sequence of the mRNA analyte, and the transposon end sequence. Instead of washing away the top strand, the top strand can be eluted from the array by denaturing. The collected RNA / cDNA hybrid product can then be amplified for sample indexing-reverse transcription- PCR (SI-RT-PCR) library construction.

[0257] The workflow of FIG. 14 thereby bypasses the fragmentation, end repair, A-tailing, adapter ligation in the workflow of FIG. 12, as well as second strand synthesis in the workflow of FIG. 13.

[0258] EXAMPLE 3 - Tagmentation of single-stranded cDNA extension product and analysis of synthesized second strand DNA product.

[0259] FIG. 15 shows another streamlined spatial analysis workflow herein that bypasses various processes in the workflow of FIG. 12. As previously described, a poly adenylated mRNA analyte from a biological sample can be captured using a capture probe having a poly(T) sequence.

[0260] Following mRNA capture, the capture probe can be extended using a reverse transcriptase or a polymerase, and the mRNA as a template, thereby generating a cDNA molecule that includes both the barcode sequence and a sequence complementary to a sequence of the mRNA analyte. As shown in FIG. 15, departing from the workflows of FIGs. 13 and 14. the top strand mRNA can then be washed away, e.g., using a base (KOH) or an RNase. The remaining single-stranded cDNA molecule on the array can then be subjected to tagmentation by contacting with a transposome complex that includes a transposase and one or more transposon end sequences, e.g., P7 sequences, as shown. Optionally, the tagmentation can occur after releasing the target molecule from the array.

[0261] As previously described, following tagmentation, one or more detergents, e.g., SDS, can then be used to wash away the transposome complex and / or non-bound fragments from the array. The remaining nucleic acid fragment (“the 3' element”) that includes the barcode sequence at its 5' end and a P7 sequence at its 3' end can subsequently be processed for library construction. The resulting barcoded nucleic acid fragment can then be extended to fill any gaps resulting from tagmentation, as well as toward the 5' end of the capture probe comprising the barcode sequence, such that the bottom (“second”) strand shown includes a complement of the barcode sequence, a sequence of the mRNA analyte, and the transposon end sequence. The synthesized second strand can then be eluted from the array by denaturing the double-stranded cDNA molecule. The collected second strand cDNA product can be amplified for SI-PCR library' construction.

[0262] Using transposase-mediated tagmentation of the single-stranded cDNA, the w orkflow' of FIG. 15 thereby bypasses the fragmentation, end repair, A-tailing, adapter ligation in the w orkflow of FIG. 12, as w ell as second strand synthesis in the workflow of FIG. 13.

[0263] EXAMPLE 4 - Tagmentation by templated ligation.

[0264] While FIGs. 13-15 show example workflows for spatial analysis of target nucleic acids from a biological sample, such as mRNA. similar tagmentation methods can be used to analyze oligonucleotides that hybridize to target analytes. Such oligonucleotides serve as proxies for detecting target analytes to which the oligonucleotides bind. For instance, tagmentation-based spatial analysis can be performed using nucleic acid-templated ligation, e.g., RNA-templated ligation (RTL). In this method, two or more probes (e.g., a first probe and a second probe) can hybridize to adjacent or near-adjacent (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides from one another) a target mRNA analyte. The tw o or more probes can be DNA molecules. After hybridization, the two or more probes can be ligated together to generate a connected probe. The connected probe can include a capture probe capture domain configured to hybridize to a capture probe, e.g., the capture probe capture domain can include a poly(A) sequence. As previously described in EXAMPLES 1-3, the polyadenylated connected probe can then be captured by a barcoded capture probe having a poly(T) sequence, on a substrate or an array.

[0265] Following hybridization of the connected probe to the capture probe, the capture probe can be extended using a reverse transcriptase or a polymerase, and the connected probe as a template, thereby generating a complementary' DNA (cDNA) molecule that includes both the barcode sequence and a sequence complementary to a sequence of the connected probe.

[0266] Subsequent tagmentation of the extension product can be performed by various methods. In one aspect, the partially double-stranded DNA molecule can be subjected to tagmentation by contacting with a transposome complex to fragment the DNA molecule and insert one or more sequencing primer sequences, e.g., a P7 sequence. Since the tagmentation reaction depends on the specificity of the transposase. insertion can occur at a region that is at least 100 nucleotides from an end of the cDNA molecule. In some cases, the insertion site is random. Alternatively, the insertion site can be non-random, e.g., site-specific or sequencespecific. Optionally, the tagmentation can occur after releasing the cDNA from the array.

[0267] Following tagmentation of the cDNA, the transposome complex and / or non-bound fragments can be washed away from the array to enrich for the 3' element that remains affixed to the array and includes the barcode sequence at its 5' end and a P7 sequence at its 3' end. The barcoded nucleic acid fragment can be extended to fill any gaps resulting from tagmentation, as well as toward the 5' end of the capture probe comprising the barcode sequence, such that the second strand includes a complement of the barcode sequence, a sequence of the mRNA analyte (i.e., the DNA equivalent thereof), and the transposon end sequence. The synthesized second strand can then be eluted from the array by denaturing the double-stranded cDNA molecule. The collected second strand cDNA product can be amplified for SI-PCR library construction.

[0268] In another aspect, a single-stranded cDNA can be subjected to transposase-mediated tagmentation. The hybridized connected probe can be denatured and washed away, e.g., by treating with a base (KOH) or heating. The workflow may then proceed in a similar manner as the workflow of FIG. 15 in which the remaining single-stranded cDNA molecule on the array can then be subjected to tagmentation to fragment the cDNA and append one or more sequencing primer sequences, e.g., a P7 sequence. Since the tagmentation reaction depends on the specificity of the transposase, insertion can occur at a region that is at least 100 nucleotides from an end of the cDNA molecule, e.g., a 3' or 5' end. In some cases, the insertion site is random. Alternatively, the insertion site can be non-random, e.g.. site-specific or sequence-specific. Optionally, the tagmentation can occur after releasing the cDNA from the array.

[0269] Following tagmentation, the transposome complex and / or non-bound fragments from the array can be washed away from the array. The remaining nucleic acid fragment ("the 3' element”) that includes the barcode sequence at its 5' end and a P7 sequence at its 3' end can subsequently be processed for library' construction. For improved specificity to the original target analyte in the biological sample, the tagmentation reaction results in a barcoded nucleic acid fragment that includes a portion of the connected probe that comprises sequences that are complementary' to both the first probe and the second probe. Since the connected probe is complementary' to the original mRNA analyte, the cDNA generated from the connected probe template includes a sequence of the mRNA, i.e., the DNA equivalent thereof. The barcoded nucleic acid fragment can be extended to fill any gaps resulting from tagmentation, as well as toward the 5' end of the capture probe comprising the barcode sequence, such that the second strand includes a complement of the barcode sequence, a sequence of the mRNA analyte (i.e., the DNA equivalent thereof), and the transposon end sequence. The synthesized second strand can then be eluted from the array by denaturing the double-stranded cDNA molecule. The collected second strand cDNA product can be amplified for SI-PCR library construction.

[0270] As described, spatial analysis with transposase-mediated tagmentation can be used to identify a variety of different analytes (e.g., RNA and DNA) in a biological sample.

Claims

WHAT IS CLAIMED IS:

1. A method for processing a nucleic acid analyte, the method comprising:(a) hybridizing the nucleic acid analyte to a capture probe on an array, wherein the capture probe comprises: (i) a barcode and (ii) a capture domain;(b) extending the capture probe using the nucleic acid analyte as a template, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule comprises a sequence of the nucleic acid analyte or a complement thereof; and(c) tagmenting the barcoded nucleic acid molecule or a derivative thereof to insert a transposon end sequence into the barcoded nucleic acid molecule or the derivative thereof, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment comprises: (i) the barcode, (ii) the transposon end sequence, and (iii) at least a portion of a sequence of the nucleic acid analyte or a complement thereof.

2. The method of claim 1 , further comprising, after the tagmenting in (c), extending the barcoded nucleic acid fragment or the derivative thereof, thereby generating an extended barcoded fragment comprising: (i) the barcode, (ii) the transposon end sequence, and (iii) at least the portion of the sequence of the nucleic acid analyte or the complement thereof.

3. The method of claim 2, further comprising, after extending the barcoded nucleic acid fragment or the derivative thereof, releasing the nucleic acid analyte from the capture probe on the array.

4. The method of claim 3, wherein releasing is by treating the extended barcoded fragment with a base.

5. The method of claim 3, wherein releasing is by treating the extended barcoded fragment with an RNase.

6. The method of any one of claims 3-5, further comprising, after releasing the nucleic acid analyte from the capture probe on the array: (i) hybridizing a primer to the extended barcoded fragment or a derivative thereof, and (ii) extending the primer, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

7. The method of claim 6, wherein the barcoded extension product is a single-strandedDNA molecule.

8. The method of claim 6 or 7, wherein the primer comprises a sequencing primer sequence.

9. The method of any one of claims 6-8, wherein the primer comprises a sequence complementary to the transposon end sequence.

10. The method of any one of claims 6-9, further comprising releasing the barcoded extension product from the capture probe on the array.

11. The method of any one of claims 6-10, further comprising determining a sequence of the barcoded extension product or a derivative thereof.

12. The method of any one of claims 6-11, further comprising sequencing the barcoded extension product or a derivative thereof.

13. The method of claim 12, further comprising amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

14. The method of claim 1, further comprising, after the tagmenting in (c), releasing a sequence at a 3' end of the nucleic acid analyte hybridized to the capture probe, wherein the sequence is not hybridized to the capture probe in (a).

15. The method of claim 14, wherein the releasing is by treating with a nuclease.

16. The method of claim 15, wherein the nuclease is exonuclease T or RNase T.

17. The method of any one of claims 14-16, further comprising, after the releasing, extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

18. The method of claim 17, wherein the barcoded extension product is a single-stranded RNA / DNA hybrid molecule.

19. The method of claim 17 or 18, further comprising releasing the barcoded extension product from the capture probe on the array.

20. The method of any one of claims 17-19. further comprising determining a sequence of the barcoded extension product or a derivative thereof.

21. The method of any one of claims 17-19, further comprising sequencing the barcoded extension product or the derivative thereof.

22. The method of claim 21, further comprising reverse transcribing the barcoded extension product or a derivative thereof prior to the sequencing.

23. The method of claim 21, further comprising amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

24. The method of claim 1, further comprising, prior to the tagmenting in (c). releasing the nucleic acid analyte from the capture probe on the array, and wherein the barcoded nucleic acid molecule comprises the complement of the sequence of the nucleic acid analyte.

25. The method of claim 24, wherein releasing is by treating with a base.

26. The method of claim 24, wherein releasing is by treating with an RNase.

27. The method of any one of claims 24-26, further comprising, after the tagmenting in (c). extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

28. The method of claim 27, further comprising, after extending the barcoded nucleic acid fragment or the derivative thereof, releasing the barcoded extension product from the capture probe on the array.

29. The method of claim 28, wherein releasing is by denaturing the barcoded extension product or a derivative thereof.

30. The method of claim 29, wherein the denaturing is by heating.

31. The method of any one of claims 27-30. wherein the barcoded extension product is a single-stranded DNA molecule.

32. The method of any one of claims 27-31, further comprising determining a sequence of the barcoded extension product or a derivative thereof.

33. The method of any one of claims 27-31, further comprising sequencing the barcoded extension product or the derivative thereof.

34. The method of claim 33, further comprising amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

35. The method of any one of claims 1-34, further comprising determining: (i) the sequence of the barcode or a complement thereof, and (ii) all or a portion of a sequence of the barcoded nucleic acid fragment or a complement thereof.

36. The method of any one of claims 1-35, wherein the extending in (b) is using a reverse transcriptase.

37. The method of any one of claims 1-35, wherein the extending in (b) is using a DNA polymerase.

38. The method of any one of claims 1-37, wherein the tagmenting in (c) comprises contacting the barcoded nucleic acid molecule with a transposome comprising the transposon end sequence.

39. The method of claim 38, wherein the transposome comprises a transposase.

40. The method of claim 39, wherein the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof.

41. The method of any one of claims 1-40, wherein the transposon end sequence is inserted at a 3' end of the barcoded nucleic acid fragment.

42. The method of claim 41, wherein the insertion occurs at a region that is at least 100 nucleotides from a 5' end of the barcoded nucleic acid molecule.

43. The method of any one of claims 1-42, wherein the transposon end sequence comprises a sequencing primer sequence.

44. The method of claim 43, wherein the sequencing primer sequence is a P7 sequence.

45. The method of any one of claims 1-44, wherein the tagmenting in (c) is performed on the array.

46. The method of any one of claims 1-44, further comprising, after (b), releasing the barcoded nucleic acid molecule from the capture probe on the array, and wherein the tagmenting in (c) is performed off of the array.

47. The method of any one of claims 1-46, wherein the tagmenting in (c) is performed in a presence of a permeabilization agent.

48. The method of claim 47, wherein the permeabilization agent comprises a detergent and / or enzyme.

49. The method of claim 48. wherein the detergent is sodium dodecyl sulfate (SDS), and wherein the enzyme is selected from pepsin or proteinase K.

50. The method of any one of claims 1-49, further comprising, prior to (a), contacting a biological sample comprising the nucleic acid analyte with the capture probes on the array.

51. The method of claim 50, wherein the biological sample is mounted on a first substrate, the array is on a second substrate, and the method further comprises aligning the first substrate with the array on a second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array.

52. The method of claim 51, further comprising, after the aligning, releasing the capture probe from the array, such that the released capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte.

53. The method of any one of claims 50-52, further comprising imaging the biological sample.

54. The method of any one of claims 50-53, further comprising staining the biological sample.

55. The method of claim 54, wherein the staining comprises immunofluorescence, immunohistochemistry, hematoxylin, and / or eosin staining.

56. The method of any one of claims 50-55, wherein the biological sample is a tissue sample.

57. The method of claim 56, wherein the tissue sample is a formalin-fixed, paraffin- embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

58. The method of any one of claims 1-57, wherein the nucleic acid analyte is an RNA molecule.

59. The method of claim 58, wherein the RNA molecule is a messenger RNA (mRNA) molecule.

60. The method of any one of claims 1-57, wherein the nucleic acid analyte is a DNA molecule.

61. The method of claim 60, wherein the DNA molecule is a genomic DNA molecule.

62. The method of claim 60, wherein the DNA molecule is coupled to an analyte binding moiety.

63. The method of claim 62, wherein the analyte binding moiety' is an antibody or a functional fragment thereof.

64. The method of claim 62 or 63, wherein the DNA molecule comprises:(i) a capture handle sequence that is complementary to the capture domain of the capture probe, and(ii) an analyte binding moiety barcode that is associated with or identifies the analyte binding moiety.

65. The method of any one of claims 62-64, wherein the DNA molecule is coupled to the analyte binding moiety by a linker.

66. The method of claim 65, wherein the linker is a cleavable linker.

67. The method of claim 66, wherein the cleavable linker is a disulfide linker, a photocleav able linker, a UV-cleavable linker, or an enzyme cleavable linker.

68. The method of any one of claims 1-67, wherein the barcoded nucleic acid molecule is a partially double-stranded RNA / DNA molecule.

69. The method of claim 68, wherein the partially double-stranded RNA / DNA molecule comprises an RNA sequence of the nucleic acid analyte and a DNA sequence complementary to the sequence of the nucleic acid analyte.

70. The method of any one of claims 1-69, wherein the barcoded nucleic acid molecule is a single-stranded DNA molecule.

71. The method of claim 70, wherein the single-stranded DNA molecule comprises a sequence complementary to a sequence of the nucleic acid analyte.

72. The method of any one of claims 1-71, wherein the barcode identifies a spatial location on the array.

73. The method of any one of claims 1-72, wherein the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, and wherein the common barcode is specific to each subset of the subsets of capture probes.

74. The method of claim 73, wherein the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes.

75. The method of any one of claims 1-74, wherein the capture probe further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof.

76. The method of claim 75, wherein the one or more functional domains comprises a sequencing primer sequence.

77. The method of any one of claims 1-76, wherein the array comprises one or more features.

78. The method of claim 77, wherein the one or more features comprises a bead and the capture probe is attached to the bead.

79. A method for processing a nucleic acid analyte, the method comprising:(a) hybridizing a first probe and a second probe to the nucleic acid analyte, wherein each of the first probe and the second probe comprises a sequence that is substantially complementary’ to sequences of the nucleic acid analyte;(b) coupling the first probe and the second probe, thereby generating a connected probe, wherein the connected probe comprises a capture probe binding domain;(c) hybridizing the capture probe binding domain of the connected probe to a capture probe of a plurality of capture probes on an array, wherein the capture probe comprises a barcode and a capture domain;(d) extending the capture probe using the nucleic acid analyte as a template, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule comprises a sequence of the connected probe or a complement thereof; and(e) tagmenting the barcoded nucleic acid molecule to insert a transposon end sequence, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment comprises: (i) the barcode, (ii) the transposon end sequence, (iii) at least a portion of a sequence of the first probe or a complement thereof, and (iv) at least portion of a sequence of the second probe or a complement thereof80. The method of claim 79, further comprising, prior to the tagmenting in (e), releasing the connected probe from the array, and wherein the barcoded nucleic acid molecule comprises the complement of the sequence of the connected probe.

81. The method of claim 80, wherein the releasing is by treating with a base.

82. The method of claim 80, wherein the releasing is by heating.

83. The method of any one of claims 79-82, further comprising, after the tagmenting in (e), extending the barcoded nucleic acid fragment or a derivative thereof, thereby generating a barcoded extension product comprising: (i) a sequence of the barcode or a complement thereof, and (ii) the transposon end sequence or a complement thereof.

84. The method of claim 83, further comprising releasing the barcoded extension product from the array.

85. The method of claim 84, wherein releasing is by denaturing the barcoded extension product or a derivative thereof.

86. The method of claim 85. wherein the denaturing is by heating.

87. The method of any one of claims 83-86, wherein the barcoded extension product is a single-stranded DNA molecule.

88. The method of any one of claims 83-87, further comprising determining a sequence of the barcoded extension product or a derivative thereof.

89. The method of any one of claims 83-88, further comprising sequencing the barcoded extension product or a derivative thereof.

90. The method of claim 89. further comprising amplifying the barcoded extension product or the derivative thereof prior to the sequencing.

91. The method of any one of claims 79-90, wherein the first probe and the second probe hybridize to adjacent sequences of the nucleic acid analyte.

92. The method of any one of claims 79-90, wherein the first probe and the second probe hybridize to sequences that are at least 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, or more nucleotides apart.

93. The method of any one of claims 79-92, wherein the coupling the first probe and the second probe comprises ligating the first probe and the second probe.

94. The method of any one of claims 79-93, further comprising, after the hybridizing in (a), generating: (i) an extended first probe or (ii) an extended second probe by contacting with a polymerase, wherein the extended first probe comprises a sequence complementary' to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe, and the extended second probe comprises a sequence complementary' to a sequence between the sequence hybridized to the first probe and the sequence hybridized to the second probe.

95. The method of claim 94, wherein the coupling the first probe and the second probe comprises ligating: (i) the first probe and the extended second probe, (ii) the extended first probe and the second probe, or (iii) the extended first probe and the extended second probe.

96. The method of any one of claims 93-95, wherein the ligating is performed using a ligase.

97. The method of claim 96, wherein the ligase is a Chlorella virus DNA ligase, a singlestranded DNA ligase, or a T4 DNA ligase.

98. The method of any one of claims 79-97, wherein the first probe or the second probe comprises the capture probe binding domain.

99. The method of any one of claims 79-98. wherein the first probe, the second probe, or the connected probe is a single-stranded DNA molecule.

100. The method of any one of claims 79-99, further comprising determining: (i) a sequence of the barcode or a complement thereof, and (ii) all or a portion of a sequence of the barcoded nucleic acid fragment or a complement thereof.

101. The method of any one of claims 79-100, wherein the extending in (d) is using a DNA polymerase.

102. The method of any one of claims 79-101, wherein the tagmenting in (e) comprises contacting the barcoded nucleic acid molecule with a transposome comprising the transposon end sequence.

103. The method of claim 102, wherein the transposome comprises a transposase.

104. The method of claim 103, wherein the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof.

105. The method of any one of claims 79-104, wherein the transposon end sequence is inserted at a 3' end of the barcoded nucleic acid fragment.

106. The method of claim 105, wherein the insertion occurs at a region that is at least 100 nucleotides from a 5' end of the barcoded nucleic acid molecule.

107. The method of any one of claims 79-106, wherein the transposon end sequence comprises a sequencing primer sequence.

108. The method of claim 107, wherein the sequencing primer sequence is a P7 sequence.

109. The method of any one of claims 79-108, wherein the tagmenting in (e) is performed on the array.

110. The method of any one of claims 79-108, further comprising, after (d), releasing the barcoded nucleic acid molecule from the array, and wherein the tagmenting in (e) is performed off of the array.

111. The method of any one of claims 79-110, wherein the tagmenting in (e) is performed in a presence of a permeabilization agent.

112. The method of claim 111, wherein the permeabilization agent comprises a detergent and / or enzyme.

113. The method of claim 112, wherein the detergent is sodium dodecyl sulfate (SDS), and wherein the enzy me is selected from pepsin or proteinase K.

114. The method of any one of claims 79-113, further comprising, prior to the hybridizing in (a), contacting a biological sample comprising the nucleic acid analyte with the array.

115. The method of claim 114, wherein the biological sample is mounted on a first substrate, the array is on a second substrate, and the method further comprises aligning the first substrate with the array on a second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the array.

116. The method of claim 115, further comprising, after the aligning, releasing the capture probe from the array, such that the released capture probe migrates to the biological sample and the capture domain of the capture probe hybridizes to the nucleic acid analyte.

117. The method of any one of claims 114-116. further comprising imaging the biological sample.

118. The method of any one of claims 114-117. further comprising staining the biological sample.

119. The method of claim 118, wherein the staining comprises hematoxylin or eosin staining.

120. The method of any one of claims 114-119. wherein the biological sample is a tissue sample.

121. The method of claim 120, wherein the tissue sample is a formalin-fixed, paraffin- embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

122. The method of any one of claims 79-121, wherein the nucleic acid analyte is an RNA molecule.

123. The method of claim 122, wherein the RNA molecule is a messenger RNA (mRNA) molecule.

124. The method of any one of claims 79-121, wherein the nucleic acid analyte is a DNA molecule.

125. The method of claim 124, wherein the DNA molecule is a genomic DNA molecule.

126. The method of claim 124 or 125, wherein the DNA molecule is coupled to an analyte binding moiety.

127. The method of claim 126, wherein the analyte binding moiety is an antibody or a functional fragment thereof.

128. The method of claim 126 or 127, wherein the DNA molecule comprises:(i) a capture handle sequence that is complementary to the capture domain of the capture probe, and(ii) an analyte binding moiety barcode that is associated with or identifies the analyte binding moiety.

129. The method of any one of claims 126-128. wherein the DNA molecule is coupled to the analyte binding moiety by a linker.

130. The method of claim 129, wherein the linker is a cleavable linker.

131. The method of claim 130, wherein the cleavable linker is a disulfide linker, a photo- cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker.

132. The method of any one of claims 79-131, wherein the barcoded nucleic acid molecule is a partially double-stranded DNA molecule.

133. The method of claim 132, wherein the partially double-stranded DNA molecule comprises a sequence of the first probe or a complement thereof and a sequence of the second probe or a complement thereof.

134. The method of any one of claims 79-133, wherein the barcode identifies a spatial location on the array.

135. The method of any one of claims 79-134, wherein the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, wherein the common barcode is specific to each subset of the subsets of capture probes.

136. The method of claim 135, wherein the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes.

137. The method of any one of claims 79-136, wherein the capture probe further comprises a cleavage domain, one or more functional domains, a unique molecular identifier, or a combination thereof.

138. The method of claim 137, wherein the one or more functional domains comprises a sequencing primer sequence.

139. The method of any one of claims 79-138, wherein the array comprises one or more features.

140. The method of claim 139, wherein the one or more features comprises a bead and the capture probe is attached to the bead.

141. A composition comprising:(a) a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a barcode and (ii) a capture domain; and(b) a transposome comprising a transposase and a transposon end sequence, wherein the transposon end sequence comprises a sequencing primer sequence.

142. The composition of claim 141, further comprising an analyte.

143. The composition of claim 142. wherein the analyte is a nucleic acid analyte.

144. The composition of any one of claims 141-143, wherein the substrate comprises an array, wherein the barcode identifies a spatial location on the array.

145. The composition of any one of claims 141-144, wherein the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, wherein the common barcode is specific to each subset of the subsets of capture probes.

146. The composition of claim 145. wherein the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes.

147. The composition of any one of claims 141-146, wherein the array comprises one or more features.

148. The composition of claim 147, wherein the one or more features comprises a bead and the capture probe is attached to the bead.

149. The composition of any one of claims 141-148, wherein the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase. a Vibrio species transposase, or a functional derivative thereof.

150. The composition of any one of claims 141-149, wherein the transposome comprises two transposon end sequences.

151. The composition of any one of claims 141-150, further comprising a reverse transcriptase.

152. The composition of any one of claims 141-151, further comprising a polymerase.

153. The composition of claim 152, wherein the polymerase is a DNA polymerase.

154. The composition of any one of claims 141-153, further comprising a plurality of dNTPs.

155. The composition of any one of claims 141-154, further comprising one or more permeabilization reagents.

156. The composition of claim 155, wherein the one or more permeabilization reagents comprises a protease, a surfactant, or a detergent.

157. The composition of claim 156. wherein the protease comprises Proteinase K, pepsin, or collagenase.

158. The composition of any one of claims 141-157, further comprising an RNase.

159. The composition of any one of claims 141-158, further comprising a DNase.

160. The composition of any one of claims 143-159, wherein the nucleic acid analyte comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe.

161. The composition of any one of claims 141-160, further comprising a first probe and a second probe, wherein each of the first probe and the second probe comprises a sequence that is substantially complementary to sequences of the nucleic acid analyte.

162. The composition of claim 161, wherein the first probe or the second probe comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe.

163. The composition of claim 161 or 162, further comprising a ligase.

164. The composition of claim 163, wherein the ligase is a Chlorella virus DNA ligase, a single-stranded DNA ligase, or a T4 DNA ligase.

165. The composition of any one of claims 141-164, further comprising a tissue sample.

166. The composition of claim 165. wherein the tissue sample is a formalin-fixed, paraffin- embedded (FFPE) tissue sample, a frozen tissue sample, or a fresh tissue sample.

167. The composition of any one of claims 141-166, wherein the nucleic acid analyte is an RNA molecule.

168. The composition of claim 167, wherein the RNA molecule is a messenger RNA (mRNA) molecule.

169. The composition of any one of claims 141-166, wherein the nucleic acid analyte is a DNA molecule.

170. The composition of claim 169, wherein the DNA molecule is a genomic DNA molecule.

171. The composition of claim 169, wherein the DNA molecule is coupled to an analyte binding moiety.

172. The composition of claim 171. wherein the analyte binding moiety is an antibody or a functional fragment thereof.

173. A kit comprising:(a) a substrate comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a barcode and (ii) a capture domain;(b) a reverse transcriptase or a polymerase;(c) a transposome comprising a transposase and a transposon end sequence, wherein the transposon end sequence comprises a sequencing primer sequence; and(d) instructions for performing the method of any one of claims 1-140.

174. The kit of claim 173, wherein the substrate comprises an array, wherein the barcode identifies a location on the array.

175. The kit of claim 174, wherein the array comprises subsets of capture probes, each subset of the subsets of capture probes comprises a common barcode, wherein the common barcode is specific to each subset of the subsets of capture probes.

176. The kit of claim 175, wherein the capture probe is from a subset of capture probes from the subsets of capture probes, and wherein the barcode is common to the subset of capture probes.

177. The kit of any one of claims 173-176, wherein the array comprises one or more features.

178. The kit of claim 177. wherein the one or more features comprises a bead and the capture probe is attached to the bead.

179. The kit of any one of claims 173-178, wherein the transposase is a Tn5 transposase, a Mu transposase, a Tn7 transposase, a Vibrio species transposase, or a functional derivative thereof.

180. The kit of any one of claims 173-179, wherein the transposome comprises two transposon end sequences.

181. The kit of any one of claims 173-180, wherein the kit comprises the reverse transcriptase.

182. The kit of any one of claims 173-180, wherein the kit comprises the polymerase.

183. The kit of claim 182, wherein the polymerase is a DNA polymerase.

184. The kit of any one of claims 173-183, further comprising a plurality of dNTPs.

185. The kit of any one of claims 173-184, further comprising one or more permeabilization reagents.

186. The kit of claim 185, wherein the one or more permeabilization reagents comprises a protease, a surfactant, or a detergent.

187. The kit of claim 186, wherein the protease comprises Proteinase K, pepsin, or collagenase.

188. The kit of any one of claims 173-187, further comprising an RNase.

189. The kit of any one of claims 173-188, further comprising a DNase.

190. The kit of any one of claims 173-189, further comprising a first probe and a second probe, wherein each of the first probe and the second probe comprises a sequence that is substantially complementary to sequences of a nucleic acid analyte.

191. The kit of claim 190, wherein the first probe or the second probe comprises a capture probe capture domain that hybridizes to the capture domain of the capture probe.

192. The kit of any one of claims 173-191, further comprising a ligase.

193. The kit of claim 192, wherein the ligase is a Chlor ella virus DNA ligase, a singlestranded DNA ligase, or a T4 DNA ligase.

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