Methods for spatial profiling using RNA-templated ligation

Abstract

Provided herein are methods for detecting analytes of interest using RNA-templated ligation to interrogate spatial gene expression in a sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 952,736, filed December 23, 2019; U.S. Provisional Patent Application No. 62 / 969,458, filed February 3, 2020; U.S. Provisional Patent Application No. 63 / 087,061, filed October 2, 2020; and U.S. Provisional Patent Application No. 63 / 108,088, filed October 30, 2020. The contents of each of these applications are incorporated herein by reference in their entirety. Background Art

[0003] Cells within a tissue differ in cell morphology and / or function due to different analyte levels (e.g., gene and / or protein expression) within the different cells. The specific location of a cell within a tissue (e.g., the position of the cell relative to neighboring cells or the position of the cell relative to the tissue microenvironment) can affect, for example, the cell's morphology, differentiation, fate, viability, proliferation, behavior, signal transduction, and crosstalk with other cells in the tissue.

[0004] Spatial heterogeneity has been previously investigated using techniques that typically provide data on a few analytes in the context of intact tissue or tissue sections (e.g., tissue sections), or provide data on important analytes from individual, single cells, but cannot provide information on the location of single cells from the original biological sample (e.g., tissue).

[0005] Typically, targeting of specific analytes in a biological sample utilizes capture probes that target common transcript sequences, such as poly(A) mRNA-like tails. However, this approach can detect a large number of off-target analytes. Methods (e.g., RNA-templated ligation) provide an alternative to indiscriminate capture of common transcript sequences. See, e.g., Yeakley, PLoS One, 25;12(5):e0178302 (2017), which is incorporated herein by reference in its entirety. However, there remains a need to develop an alternative approach to capture common transcript sequences (e.g., poly(A) mRNA-like tails) of target analytes that can detect analytes across the entire transcriptome while providing information about the spatial location and abundance of the target analyte. Summary of the Invention

[0006] Targeted RNA capture is an attractive alternative to poly (A) mRNA capture for interrogate spatial gene expression in samples (e.g., FFPE tissues). Compared to poly (A) mRNA capture, the targeted RNA capture described herein is less affected by RNA degradation associated with FFPE fixation, compared to methods that rely on oligo dT capture and mRNA reverse transcription. Further targeted RNA capture as described herein allows sensitive measurement of specific genes of interest that may otherwise be missed by full transcriptomics methods. Targeted RNA capture can be used to capture a defined set of RNA molecules of interest, or it can also be used for full transcriptome levels, or any level in between. When combined with spatial methods disclosed herein, the position and abundance of RNA targets can be determined.

[0007] In one aspect, the present disclosure features a method for determining the position of an analyte in a biological sample, comprising: (a) providing a biological sample on an array comprising a plurality of capture probes, wherein the capture probes in the plurality comprise: (i) a spatial barcode and (ii) a capture domain; (b) contacting a first probe and a second probe with the biological sample, wherein the first probe and the second probe each comprise one or more sequences substantially complementary to the sequence of the analyte, and wherein the second probe comprises a capture probe capture domain; (c) hybridizing the first probe and the second probe to the analyte; (d) generating a ligation product by connecting the first probe and the second probe; (e) releasing the ligation product from the analyte; (f) hybridizing the ligation product with the capture domain; and (g) determining (i) all or part of the sequence of the ligation product bound to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the position of the analyte in the biological sample. In some cases, the method comprises determining the abundance and position of the analyte in the biological sample. In some cases, the method comprises determining the abundance of the analyte at a certain position in the biological sample.

[0008] In some embodiments, the first probe and the second probe are substantially complementary to adjacent sequences of the analyte. In some embodiments, the first probe and the second probe hybridize to multiple sequences on the analyte that are not adjacent to each other.

[0009] In some embodiments, the method further comprises hybridizing a third probe to the first probe and the second probe, wherein the third probe comprises a first sequence that is substantially complementary to a portion of the first probe and a second sequence that is substantially complementary to a portion of the second probe. In some embodiments, hybridization of the first probe and the second probe to the analyte is performed at a first temperature. In some embodiments, hybridization of the third probe to the first probe and the second probe is performed at a second temperature. In some embodiments, the first temperature is a temperature higher than the second temperature. In some embodiments, the first temperature is from about 50°C to about 75°C, from about 55°C to about 70°C, or from about 60°C to about 65°C. In some embodiments, the second temperature is from about 15°C to about 35°C, from about 20°C to about 30°C, or from about 25°C to about 30°C. In some embodiments, the first probe is extended with a DNA polymerase, thereby filling the gap between the first probe and the second probe and generating an extended first probe.

[0010] In some embodiments, the first probe comprises a sequence that is substantially complementary to a first target sequence of the analyte. In some embodiments, the second probe further comprises: (i) a first sequence that is substantially complementary to a second target sequence of the analyte; (ii) a linker sequence; (iii) a second sequence that is substantially complementary to a third target sequence of the analyte; and (iv) a capture domain of the capture probe that is capable of binding to the capture domain of the capture probe. In some embodiments, the first target sequence of the analyte is directly adjacent to the second target sequence of the analyte. In some embodiments, the second target sequence is not directly adjacent to the third target sequence on the analyte. In some embodiments, the second target sequence and the third target sequence are: (i) on different exons of the analyte, or (ii) located in the same exon of the analyte but are not adjacent on the analyte.

[0011] In some embodiments, the second probe comprises a sequence that is substantially complementary to a third target sequence of the analyte. In some embodiments, the first probe comprises: (i) a first sequence that is substantially complementary to the first target sequence of the analyte; (ii) a linker sequence; and (iii) a second sequence that is substantially complementary to the second target sequence of the analyte. In some embodiments, the second target sequence is directly adjacent to the third target sequence. In some embodiments, the first target sequence is not directly adjacent to the second target sequence on the analyte. In some embodiments, the first target sequence and the second target sequence: (i) are on different exons of the analyte, or (ii) are located in the same exon but are not directly adjacent on the analyte.

[0012] In some embodiments, the adapter sequence comprises a total of about 1 nucleotide to about 100 nucleotides.In some embodiments, the adapter further comprises a barcode sequence that serves as a proxy for identifying the analyte.

[0013] In some embodiments, the first probe comprises at least two ribonucleobases at the 3' end, and wherein the second probe comprises a phosphorylated nucleotide at the 5' end.

[0014] In some embodiments, generating a ligation product comprises ligating (i) the first probe and the second probe or (ii) the extended first probe and the second probe using enzymatic ligation or chemical ligation, wherein the enzymatic ligation utilizes a ligase. In some embodiments, the ligase is one or more of T4 RNA ligase (Rn12), splintR ligase, single-stranded DNA ligase, or T4 DNA ligase.

[0015] In some embodiments, the second probe comprises a pre-adenylated phosphate group at its 5′ end, and wherein the first probe comprises at least two ribonucleobases at its 3′ end. In some embodiments, the step of generating a ligation product comprises ligating the 3′ end of the first probe to the 5′ end of the second probe using a ligase that does not require adenosine triphosphate for ligase activity. In some embodiments, the ligase is selected from the group consisting of a thermostable 5′ App DNA / RNA ligase, truncated T4 RNA ligase 2, truncated T4 RNA ligase 2K227Q, truncated T4 RNA ligase 2KQ, Chlorella virus PBCV-1 DNA ligase, or any combination thereof.

[0016] In some embodiments, the first probe further comprises a functional sequence, wherein the functional sequence is a primer sequence.

[0017] In some embodiments, the method further comprises providing a capture probe capture domain blocking portion that interacts with the capture probe capture domain. In some embodiments, the method further comprises releasing the capture probe capture domain blocking portion from the capture probe capture domain before step (f). In some embodiments, the capture probe capture domain comprises a polyadenylation (poly (A)) sequence or its complement. In some embodiments, the capture probe capture domain blocking portion comprises a polyuridine sequence, a polythymidine sequence, or both. In some embodiments, releasing the polyuridine sequence from the poly (A) sequence comprises denaturing the ligation product or contacting the ligation product with a nuclease, exonuclease, or ribonuclease. In some embodiments, the capture probe capture domain comprises a sequence complementary to all or part of the capture domain of the capture probe. In some embodiments, the capture probe capture domain comprises a degenerate sequence.

[0018] In some embodiments, the first probe and / or the second probe is a DNA probe.

[0019] In some embodiments, the third probe is a DNA probe.

[0020] In some embodiments, the capture domain blocking moiety of the capture probe is a DNA probe.

[0021] In some embodiments, the releasing step (f) comprises removing the attached probe from the analyte.

[0022] In some embodiments, (i) releasing the ligated product from the analyte or (ii) releasing the capture probe from the capture domain binding domain, wherein the release of the capture domain blocking portion comprises contacting the ligated probe with an endoribonuclease. In some embodiments, the endoribonuclease is one or more of RNase H, RNase A, RNase C, or RNase I. In some embodiments, RNase H comprises RNase HI, RNase H2, or RNase HI and RNase H2.

[0023] 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 fresh or frozen tissue sample. In some embodiments, the tissue sample is a FFPE tissue sample, and the tissue sample is de-crosslinked. In some embodiments, the biological sample is pre-stained. In some embodiments, the biological sample is pre-stained with immunofluorescence or immunohistochemistry. In some embodiments, the biological sample is pre-stained with hematoxylin and eosin.

[0024] In some embodiments, the method further comprises contacting the biological sample with a permeabilizing agent, wherein the permeabilizing agent is selected from an organic solvent, a detergent and an enzyme, or a combination thereof. In some embodiments, the permeabilizing agent is selected from the group consisting of endopeptidases, proteases, sodium dodecyl sulfate (SDS), polyethylene glycol tert-octylphenyl ether, polysorbate 80 and polysorbate 20, N-lauroyl sarcosine sodium salt solution, saponin, Triton X-100 TM and Tween-20 TM In some embodiments, the endopeptidase is pepsin or proteinase K.

[0025] In some embodiments, the method further comprises fixing the biological sample before step (a). In some embodiments, the step of fixing the biological sample is performed using one or both of methanol and acetone.

[0026] In some embodiments, the analyte comprises RNA. In some embodiments, the RNA is mRNA.

[0027] In some embodiments, the determining step includes amplifying all or part of the ligation product that specifically binds to the capture domain. In some embodiments, the amplified product includes (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement. In some embodiments, the determining step includes sequencing. In some embodiments, the sequencing step includes in situ sequencing, Sanger sequencing, next generation sequencing, and nanopore sequencing.

[0028] In another aspect, the present disclosure features a kit comprising: (a) a substrate comprising a plurality of capture probes comprising a spatial barcode and a capture domain; (b) a system comprising: a plurality of first probes and a second probe, wherein the first probe and the second probe each comprise a sequence substantially complementary to the analyte, and wherein the second probe comprises a capture binding domain; and (c) instructions for performing the method of any of the preceding claims.

[0029] On the other hand, the present disclosure features a kit comprising: (a) an array comprising a plurality of capture probes; (b) a plurality of probes comprising a first probe and a second probe, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte, wherein the second probe comprises: (i) a capture domain of the capture probe capable of binding to the capture domain of the capture probe, and (ii) a linker sequence; (c) a plurality of enzymes comprising ribonucleases and ligases; and (d) instructions for performing the method of any of the preceding claims.

[0030] On the other hand, the present disclosure features a kit comprising: (a) an array comprising a plurality of capture probes; (b) a plurality of probes comprising a first probe and a second probe, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte, wherein the first probe comprises a linker sequence, and wherein the second probe comprises a capture domain of the capture probe that is capable of binding to the capture domain of the capture probe; (c) a plurality of enzymes comprising ribonucleases and ligases; and (d) instructions for performing the method of any of the preceding claims.

[0031] In some embodiments, the kit includes a second probe comprising a pre-adenylated phosphate group at its 5' end, and a ligase that does not require ATP for ligase activity.

[0032] On the other hand, the present disclosure features a composition comprising: a spatial array comprising capture probes, wherein the capture probes comprise a spatial barcode and a capture domain; a biological sample on the spatial array, wherein the biological sample comprises a plurality of analytes of interest; a first probe oligonucleotide and a second probe oligonucleotide that hybridize and link together with the analytes, wherein the first probe oligonucleotide and the second probe oligonucleotide each comprise a sequence that is substantially complementary to an adjacent sequence of the analyte, and wherein one of the first probe or the second probe comprises a capture probe capture domain.

[0033] In some embodiments, the composition further comprises an RNase H enzyme. In some embodiments, the composition further comprises a ligase. In some embodiments, the probe oligonucleotide that does not comprise a capture domain of the capture probe comprises a functional domain.

[0034] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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 herein by reference. To the extent that publications, patents, patent applications, and items of information incorporated by reference conflict with the disclosure contained in this specification, the present specification is intended to control and / or take precedence over any conflicting material.

[0035] Where values ​​are described in a range, it should be understood that the description includes disclosure of all possible sub-ranges within that range as well as disclosure of specific values ​​within that range, regardless of whether a specific value or specific sub-range is explicitly stated.

[0036] The term "each" when referring to a group of items is intended to identify an individual item in the group, but not necessarily every item in the group unless expressly stated otherwise or unless the context of usage clearly indicates otherwise.

[0037] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following alternatives: "A," "B," "A or B," and "A and B."

[0038] Various embodiments of the features of the present invention are described herein. However, it should be understood that these embodiments are provided as examples only, and that many variations, changes, and substitutions may be made by those skilled in the art without departing from the scope of the present disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of the present disclosure.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following drawings illustrate certain embodiments of the features and advantages of the present invention. These embodiments are not intended to limit the scope of the appended claims in any way. The same reference numerals in the drawings represent the same elements.

[0041] Figure 1 An exemplary spatial analysis workflow is shown.

[0042] Figure 2 is a schematic diagram showing an example of a barcoded capture probe as described herein.

[0043] Figure 3 is a schematic diagram illustrating a cleavable capture probe, wherein the cleaved capture probe can enter a non-permeabilized cell and bind to a target analyte within a sample.

[0044] Figure 4 is a schematic diagram of an exemplary multiple spatial barcoding feature.

[0045] Figure 5 is a schematic diagram showing an exemplary arrangement of barcoding features within an array.

[0046] Figure 6 is a schematic diagram showing an exemplary workflow for RNA-templated ligation.

[0047] Figure 7 is a schematic diagram showing an exemplary workflow for capturing ligation products on a substrate comprising capture probes.

[0048] Figure 8 is a schematic diagram showing an example of a first probe and a second probe containing a linker sequence.

[0049] Figure 9 is a schematic diagram showing an example of a second probe and a first probe including an adapter sequence.

[0050] Figure 10 is a schematic diagram showing examples of a first probe, a second probe, and a spanning probe.

[0051] Figure 11 is a schematic diagram showing an example of RNA-templated ligation using a first probe oligonucleotide, a second probe oligonucleotide, and a third probe oligonucleotide.

[0052] Figure 12 A-12E show various methods of chemically mediated nucleic acid ligation. Figure 12 A illustrates the formation of the triazole bond. Figure 12 B illustrates the formation of a phosphorothioate bond. Figure 12 C illustrates the formation of an amide bond. Figure 12 D illustrates the formation of a phosphoramidate bond. Figure 12E illustrates the coupling reaction.

[0053] Figure 13 An exemplary RNA-templated ligation workflow is shown.

[0054] Figure 14 The results of enrichment PCR for detecting various probes are shown. R1 = Run 1; R2 = Run 2. (1) represents the band of ligation product with capture probe. (2), (3), and (4) represent non-ligation products.

[0055] Figure 15 Read count fractions for various combinations of conditions and probes are shown.

[0056] Figures 16A-16B The matrix for specific probe combinations is shown.

[0057] Figures 17A-17C Displays the total probe oligonucleotide counts ( Figure 17A ), non-specific probe oligonucleotide counts ( Figure 17B ) and gene-specific probe oligonucleotide counts ( Figure 17C ) in mouse brain tissue.

[0058] Figures 18A-18E Target genes with spatial expression in mouse brain tissue are shown.

[0059] Figures 19A-19F Displays the total probe oligonucleotide counts ( Figure 19A ) and gene-specific probe oligonucleotides ( Figures 19B-19F ) in mouse brain tissue.

[0060] Figure 20A Shown are the median unique molecular identifiers (UMIs) / cell versus the mean reads / cell under different hybridization conditions.

[0061] Figure 20B Shown are the median genes / cell versus the mean reads / cell for different hybridization conditions.

[0062] Figure 21A Hematoxylin staining of sections of triple-positive breast cancer tissue samples is shown.

[0063] Figure 21B Shown are the median UMI / cell count versus the mean reads / cell for different conditions.

[0064] Figure 21C t-SNE projections of spots in eight different clusters are shown.

[0065] Figure 21D Display and Figure 21AThe same tissue, with different clusters (n=8 clusters) expressed in different areas of the tissue.

[0066] Figure 21E The expression of estrogen receptor (ESR1) is shown.

[0067] Figure 21F Shows the expression of estrogen receptor and progesterone receptor (PGR).

[0068] Figure 21G Shows expression of ERBB2 (also known as HER2).

[0069] Figure 22A Hematoxylin staining of sections of ovarian cancer samples is shown.

[0070] Figure 22B Shown are the median UMI / cell versus the average reads / cell for different conditions.

[0071] Figure 22C Display and Figure 25 A. Same tissue, with different clusters (n=8 clusters) expressed in different areas of the tissue.

[0072] Figure 22D t-SNE projections of spots in eight different clusters are shown.

[0073] Figure 23 Median UMI / cell versus reads / cell for different de-crosslinking conditions are shown.

[0074] Figure 24 Shown are the median UMI / cell versus reads / cell (top panel) and the median genes / cell versus reads / cell (bottom panel) for different treatment conditions (RNase).

[0075] Figure 25 Shown are the median UMI / cell versus reads / cell (top panel) and the median genes / cell versus reads / cell (bottom panel) for different treatment conditions (time and temperature). Detailed Description of the Invention

[0077] Targeted RNA capture is an attractive alternative to poly(A) mRNA capture for interrogate spatial gene expression in FFPE tissues. Compared to poly(A) mRNA capture, targeted RNA capture is less affected by RNA degradation associated with FFPE fixation than methods that rely on oligo dT capture and mRNA reverse transcription; allows sensitive measurement of specific genes of interest (which might otherwise be missed by whole transcriptomics approaches); and is scalable, with probes demonstrated to target a large portion of the transcriptome.

[0078] Spatial analysis methods and compositions as described herein can provide a large amount of analytes and / or expression data of various analytes in biological samples with high spatial resolution while retaining natural spatial background information. Spatial analysis methods and compositions can include, for example, using capture probes including spatial barcodes (for example, providing nucleic acid sequences of information about the position or orientation of analytes in cells or tissue samples (such as mammalian cells or mammalian tissue samples)), and capture domains that can be combined with analytes (such as proteins and / or nucleic acids) produced by cells and / or present therein. Spatial analysis methods and compositions can also include using capture probes with capture domains that capture intermediates (intermediate agents) for indirect detection of analytes. For example, intermediates can include nucleic acid sequences (such as barcodes) associated with intermediates. Therefore, the detection of intermediates indicates the analyte in cells or tissue samples.

[0079] Non-limiting aspects of methods and compositions for spatial analysis are described in U.S. Patent Nos. 10,774,374, 10,724,078, 10,480,022, 10,059,990, 10,041,949, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, 7,709,198, U.S. Patent Application Publication Nos. 2020 / 239946, 2020 / 080136, 2020 / 0277663, 2020 / 024641, 2019 / 3 30617, 2019 / 264268, 2020 / 256867, 2020 / 224244, 2019 / 194709, 2019 / 161796, 2019 / 085383, 2019 / 055594, 2018 / 216161, 2018 / 051322 , 2018 / 0245142, 2017 / 241911, 2017 / 089811, 2017 / 067096, 2017 / 029875, 2017 / 0016053, 2016 / 108458, 2015 / 000854, 2013 / 171621, WO 2018 / 091676, WO2020 / 176788, 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; Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev. C, dated June 2020), and / or the Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020), both available on the 10x Genomics support documents website, can be used in any combination. Other non-limiting aspects of spatial analysis methods and compositions are described herein.

[0080] Some common terms that may be used in the present disclosure may be found in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Typically, a "barcode" is a label or identifier that conveys or is capable of conveying information (e.g., information about an analyte, beads, and / or capture probe in a sample). A barcode may be part of an analyte or may be independent of the analyte. A barcode may be attached to an analyte. A specific barcode may be unique relative to other barcodes. For purposes of the present invention, an "analyte" may include any biological substance, structure, part, or component to be analyzed. The term "target" may similarly refer to an analyte of interest.

[0081] Analytes can be roughly divided into two categories: 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-connected or O-connected), lipoproteins, phosphoproteins, specific phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, protein ubiquitination variants, protein sulfate variants, viral proteins (e.g., viral capsids, viral envelopes, viral shells, viral appendages, viral glycoproteins, viral spikes, etc.), extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, analytes can be located in subcellular locations, including, for example, organelles, such as mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, excretion vesicles, vacuoles, lysosomes, etc. In some embodiments, analytes can be peptides or proteins, including but not limited to antibodies and enzymes. Other examples of analytes can be found in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. In some embodiments, the analyte can be detected indirectly, such as through detection of an intermediate, such as a ligation product or an analyte capture agent (eg, an oligonucleotide-conjugated antibody), such as those described herein.

[0082] "Biological samples" are typically obtained from an object to be analyzed using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and typically include cells and / or other biological materials from the object. In some embodiments, the biological sample can be a tissue section. In some embodiments, the biological sample can be a fixed and / or stained biological sample (e.g., a fixed and / or stained tissue section). Non-limiting examples of staining agents include tissue stains (e.g., hematoxylin and / or eosin) and immunostains (e.g., fluorescent stains). In some embodiments, biological samples (e.g., fixed and / or stained biological samples) can be imaged. Biological samples are also described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0083] In some embodiments, the biological sample is permeabilized with one or more permeabilization agents. For example, permeabilization of the biological sample can facilitate the capture of the analyte. Exemplary permeabilization agents and conditions are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0084] Array-based spatial analysis methods relate to transferring one or more analytes from a biological sample to a feature array on a substrate, wherein each feature is associated with a unique spatial position on the array. Subsequent analysis of the transferred analytes includes determining the homogeneity of the analytes and the spatial position of the analytes in the biological sample. The spatial position of the analyte in the biological sample is determined based on the features that the analyte in the array is combined (e.g., directly or indirectly) and the relative spatial position of the features on the array.

[0085] "Capture probe" refers to any molecule capable of capturing (directly or indirectly) and / or labeling 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 embodiments, the capture probe may include a cleavage domain and / or a functional domain (e.g., a primer binding site, such as for next generation sequencing (NGS)). See, for example, WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. The generation of capture probes can be achieved by any suitable method, including those described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0086] In some embodiments, any suitable multiplexing technology (e.g., as described in WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663) can be used to detect (e.g., simultaneously or sequentially) more than one analyte type (e.g., nucleic acids and proteins) from a biological sample.

[0087] In some embodiments, one or more analyte capture agents can be used to detect one or more analytes (e.g., protein analytes). As used herein, an "analyte capture agent" refers to a substance 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 feature) to identify the analyte. In some embodiments, the analyte capture agent comprises: (i) an analyte binding portion (e.g., which can bind to an analyte), such as an antibody or an antigen-binding fragment thereof; (ii) an analyte binding portion barcode; and (iii) an analyte capture sequence. As used herein, the term "analyte binding portion barcode" refers to a barcode that is associated with an analyte binding portion or otherwise identifies an analyte binding portion. As used herein, the term "analyte capture sequence" refers to a region or portion that is configured to hybridize to a capture domain of a capture probe, bind to a capture domain of a capture probe, couple to a capture domain of a capture probe, or otherwise interact with a capture domain of a capture probe. In some cases, the analyte binding portion barcode (or portion thereof) may be capable of being removed (e.g., cleaved) from the analyte capture agent. Additional descriptions of analyte capture agents can be found in WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0088] There are at least two methods of associating a spatial barcode with one or more adjacent cells so that the spatial barcode identifies the content of one or more cells and / or one or more cells as being associated with a specific spatial position. One method is to promote analyte or analyte surrogate (proxy) (e.g., intermediate) to move out of the cell and toward a spatial barcoding array (e.g., including spatial barcoding capture probes). In some cases, a spatial barcoding array having capture probes (as further described herein) is brought into contact with a biological sample, and the biological sample is permeabilized so that the analyte migrates from the sample to the array. The analyte interacts with the capture probes on the spatial barcoding array. Another method is to crack the spatial barcoding capture probes from the array and promote the spatial barcoding capture probes toward the biological sample and / or into or onto the biological sample. In some cases, a spatial barcoding array having capture probes (as further described herein) can be brought into contact with the sample. The spatial barcoding capture probes are cracked and then interact with the cells in the biological sample provided. This interaction can be a covalent or non-covalent cell surface interaction. The interaction can be an intracellular interaction promoted by a delivery system or a cell penetrating peptide. Once the spatially barcoded capture probe is associated with a specific cell, the sample can be optionally removed for analysis. The sample can be selectively separated before analysis. Once the labeled cell is associated with the spatially barcoded capture probe, the capture probe can be analyzed to obtain spatially resolved information about the labeled cell.

[0089] In some cases, sample preparation may include placing the sample on a slide, fixing the sample and / or staining the biological sample for imaging. Bright field (for imaging of sample hematoxylin and eosin staining) and / or fluorescence (for imaging of features) modes can then be used to image the stained sample on the array. Alternatively, the sample can be decolorized before permeabilization. In some embodiments, the analyte is subsequently released from the sample, and the capture probe hybridization or binding of the released analyte is formed by forming a spatial barcoded array. The sample is then removed from the array, and the capture probe is cracked from the array. The biological sample and array are then optionally imaged for a second time in one or both modes, while the analyte is reverse transcribed into cDNA, and an amplicon library is prepared and sequenced. The images are then spatially superimposed to associate the biological sample information identified by the spatial method. When the sample and array do not have a second imaging, a point coordinate file is provided instead. The point coordinate file replaces the second imaging step. In addition, amplicon library preparation and sequencing can be performed using unique PCR adapters.

[0090] In some cases, another exemplary workflow is disclosed that utilizes a spatial barcoding array on a substrate, wherein the spatial barcoding capture probes are gathered in areas called features. The spatial barcoding capture probe can include a cleavage domain, one or more functional domains, a spatial barcode, a unique molecular identifier, and a capture domain. The spatial barcoding capture probe can also include a 5' end modification for reversibly attaching to a substrate. The spatial barcoding array is contacted with a biological sample and the sample is permeabilized by applying a permeabilization reagent. The permeabilization reagent can be administered by placing the array / sample assembly in a bulk solution. Alternatively, a permeabilization reagent can be administered to the sample via an anti-diffusion medium and / or a physical barrier (e.g., a cover), wherein the sample is sandwiched between the anti-diffusion medium and / or barrier and the substrate comprising the array. Analytes are migrated to the spatial barcoding capture array using any number of techniques disclosed herein. For example, analyte migration can be performed using an anti-diffusion medium cover and passive migration. As another example, for example, using an electrophoretic transfer system, analyte migration can be active migration. Once the analyte is in proximity to the spatially barcoded capture probes, the capture probes can hybridize or otherwise bind to the target analyte.The biological sample can optionally be removed from the array.

[0091] The capture probe can be optionally cleaved from the array, and the captured analyte can be spatially barcoded by performing a reverse transcriptase first-chain cDNA reaction. The first-chain cDNA reaction can optionally be performed using a template conversion oligonucleotide. For example, the template conversion oligonucleotide can hybridize in a template-independent manner with a poly (C) tail added to the 3' end of the cDNA by reverse transcriptase. The original mRNA template and template conversion oligonucleotide can be denatured from the cDNA, and then the spatial barcoding capture probe can hybridize with the cDNA and can generate a complement of the cDNA. The first-chain cDNA can then be purified and collected for downstream amplification steps. PCR can be used to amplify the first-chain cDNA, in which the forward and reverse primers flank the spatial barcode and analyte regions of interest to produce a library associated with a specific spatial barcode. In some embodiments, the library preparation can be quantitatively and / or quality controlled to verify the success of the library preparation step. In some embodiments, the cDNA comprises a synthetic sequencing (SBS) primer sequence. The library amplicons are sequenced and analyzed to decode spatial information.

[0092] In some cases, the capture probe can be used to initiate, replicate, and thereby produce, from a template (e.g., a DNA or RNA template, such as an analyte or intermediate (e.g., a ligation product or analyte capture agent), or a portion thereof), or a derivative thereof, an optionally barcoded extension product (see, e.g., WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, regarding extended capture probes). In some cases, the capture probe can be used to form a ligation product with a template (e.g., a DNA or RNA template, such as an analyte or intermediate, or a portion thereof), thereby producing a ligation product that serves as a template surrogate.

[0093] "Capture probe" refers to any molecule capable of capturing (directly or indirectly) and / or labeling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or 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 cases, the capture probe can include a functional sequence useful for subsequent processing. In some cases, the capture probe can be reversibly attached to a substrate via a connector. The capture probe may include one or more functional sequences, which may include a sequencer-specific flow cell attachment sequence, such as a P5 or P7 sequence, and a functional sequence, which may include a sequencing primer sequence, such as an R1 primer binding site, an R2 primer binding site. In some embodiments, the sequence is a P7 sequence, and the sequence is an R2 primer binding site. The capture probe may also include a spatial barcode and / or a unique molecular identifier and a capture domain. The different sequences of the capture probe do not need to be in a sequential manner as shown in this embodiment, but the capture domain should be placed in a position on the barcode where analyte capture and extension of the capture domain may occur to create a copy of the analyte.

[0094] Figure 2 It is a schematic diagram illustrating an example of a capture probe as described herein. As shown in the figure, capture probe 202 is optionally coupled to feature 203 by a cleavage domain 201 such as a disulfide bond linker. Capture probe may include a functional sequence useful to subsequent processing, such as a functional sequence 204, which may include a sequencer-specific flow cell attachment sequence, such as a P5 or P7 sequence, and a functional sequence 205, which may include a sequencing primer sequence, such as an R1 primer binding site. In some embodiments, sequence 204 is a P7 sequence, and sequence 205 is an R1 primer binding site. Spatial barcode 206 may be included in the capture probe for barcoding target analytes. Typically, functional sequences may be selected to be compatible with any of a variety of sequencing systems, such as ion torrent protons (Ion Torrent Proton) or PGM, Illumina sequencers, PacBio, Oxford nanopores (Oxford Nanopore) and their requirements. In some embodiments, functional sequences may be selected to be compatible with non-commercial sequencing systems. Examples of such sequencing systems and techniques for which appropriate 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. Additionally, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems.

[0095] In some embodiments, the spatial barcode 206, functional sequence 204 (e.g., flow cell attachment sequence) and 205 (e.g., sequencing primer sequence) can be common to all probes attached to a given feature. The spatial barcode can also include a capture domain 207 to facilitate capture of target analytes.

[0096] In some cases, capture probes are introduced into cells using cell-penetrating peptides. Figure 3 Schematic diagram illustrating a cleavable capture probe comprising a cell-penetrating peptide, wherein the cleaved capture probe can enter non-permeabilized cells and bind to analytes within the sample. Capture probe 301 comprises a cleavage domain 302, a cell-penetrating peptide 303, a reporter molecule 304, and a disulfide bond (-SS-). 305 represents all other parts of the capture probe, such as the spatial barcode and capture domain.

[0097] In some cases, the present disclosure provides multiplexed spatial barcoding features. Figure 4 is a schematic diagram of an exemplary multiplexed spatial barcoding feature. Figure 4 , a feature 401 (e.g., a bead, a location on a slide or other substrate, a well on a slide or other substrate, a partition on a slide or other substrate, etc.) can be coupled to spatially barcoded capture probes, where the spatial barcoded probes for a particular feature can have the same spatial barcode, but different capture domains designed to associate the spatial barcode of the feature with more than one target analyte. For example, a feature can be coupled to four different types of spatially barcoded capture probes, each type of spatially barcoded capture probe having a spatial barcode 402. One type of capture probe associated with the feature comprises a combination of a spatial barcode 402 and a poly(T) capture domain 403 designed to capture mRNA target analytes. A second type of capture probe associated with the feature comprises a combination of a spatial barcode 402 and a random N-mer capture domain 404 for gDNA analysis. A third type of capture probe associated with the feature comprises a spatial barcode 402 combined with a capture domain complementary to an analyte capture agent 405 of interest. The fourth type of capture probe associated with this feature includes a spatial barcode 402 in combination with a capture probe that can specifically bind to a nucleic acid molecule 406 that can function in a CRISPR assay (e.g., CRISPR / Cas9). Figure 4 Only four different capture probe barcoding constructs are shown, but capture probe barcoding constructs can be customized for analysis of any given analyte associated with nucleic acids and can be combined with such constructs. For example, Figure 4The scheme shown in can also be used for the simultaneous analysis of other analytes disclosed herein, including but not limited to: (a) mRNA, lineage tracing constructs, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNA enzyme-seq, and / or MNA enzyme-seq) cell surface or intracellular proteins and metabolites, and perturbations (e.g., CRISPR-crRNA / sgRNA, TALEN, zinc finger nucleases and / or antisense oligonucleotides as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, barcoding markers (e.g., MHC multimers described herein) and V(D)J sequences of immune cell receptors (e.g., T cell receptors). In some embodiments, the perturbation agent can be a small molecule, an antibody, a drug, an aptamer, miRNA, a physical environment (e.g., temperature change), or any other known perturbation agent.

[0098] Other features of the capture probe are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, each of which is incorporated herein by reference in its entirety. The generation of the capture probe can be achieved by any suitable method, including those described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, each of which is incorporated herein by reference in its entirety.

[0099] As used herein, an "extended capture probe" refers to a capture probe having additional nucleotides added to the end (e.g., the 3' or 5' end) of the capture probe to extend the total length of the capture probe. For example, an "extended 3' end" means that additional nucleotides are added to the 3'-most nucleotide of the capture probe to extend the length of the capture probe, for example, by a polymerization reaction for extending 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 comprises adding a nucleic acid sequence complementary to the nucleic acid sequence of an analyte or intermediate that specifically binds to the capture domain of the capture probe to the 3' end of the capture probe. In some embodiments, the capture probe is extended using reverse transcription. In some embodiments, the capture probe is extended using one or more DNA polymerases. The extended capture probe includes the sequence of the capture probe and the spatial barcode sequence of the capture probe.

[0100] In some embodiments, the extended capture probes are amplified (e.g., in bulk solution or on an array) to generate quantities sufficient for downstream analysis (e.g., by DNA sequencing). In some embodiments, the extended capture probes (e.g., DNA molecules) serve as templates for an amplification reaction (e.g., polymerase chain reaction).

[0101] Other variations of the spatial analysis method are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, including an imaging step in some embodiments. Analysis of the captured analyte (and / or intermediate or portion thereof), for example, including sample removal, extension of the capture probe, sequencing (e.g., sequencing of a cleaved extended capture probe and / or a cDNA molecule complementary to the extended capture probe), sequencing on an array (e.g., using, for example, in situ hybridization or an in situ ligation method), time domain analysis and / or proximity capture, are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Some quality control measures are also described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0102] Spatial information can provide information of biological and / or medical importance. For example, the methods and compositions described herein can allow: identification of one or more biomarkers for a disease or condition (e.g., for diagnosis, prognosis, and / or for determining therapeutic efficacy); identification of candidate drug targets for treating a disease or condition; identification (e.g., diagnosis) of a subject as having a disease or condition; identification of the stage and / or prognosis of a subject's disease or condition; identification of a subject as having an increased likelihood of developing a disease or condition; monitoring the progression of a subject's disease or condition; determination of the efficacy of a treatment for a subject's disease or condition; identification of a subpopulation of patients for whom a treatment is effective for a disease or condition; modification of a treatment for a subject having a disease or condition; selection of subjects for participation in a clinical trial; and / or selection of a treatment for a subject having a disease or condition.

[0103] Spatial information can provide information of biological importance. For example, the methods and compositions described herein can allow: identification of transcriptome and / or proteome expression profiles (e.g., in healthy and / or diseased tissues); identification of multiple analyte types in close proximity (e.g., nearest neighbor analysis); determination of upregulated and / or downregulated genes and / or proteins in diseased tissues; characterization of the tumor microenvironment; characterization of tumor immune responses; characterization of cell types and their co-localization in tissues; identification of genetic variation within tissues (e.g., based on gene and / or protein expression profiles associated with specific disease or disorder biomarkers).

[0104] Typically, for methods based on spatial arrays, substrates serve to support capture probes that are directly or indirectly attached to array features. A "feature" is an entity that serves as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in the array are functionalized for analyte capture. Exemplary substrates are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Exemplary features and geometric properties of arrays can be found in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0105] Figure 5 Depicted are exemplary arrangements of barcoded features in an array. From left to right, Figure 5 Shown are (left) a slide comprising six spatially barcoded arrays, (center) a magnified schematic view of one of the six spatially barcoded arrays showing a grid of barcoded features associated with a biological sample, and (right) a magnified schematic view of a portion of the array showing the specific identities of multiple features in the array (labeled as ID578, ID579, ID560, etc.).

[0106] Typically, when a biological sample is contacted with a substrate comprising a capture probe (e.g., a substrate having a capture probe embedded, spotted, printed, or manufactured on a substrate, or a substrate having features comprising a capture probe (e.g., beads, holes, areas on a substrate)), the analyte and / or intermediate (or portion thereof) can be captured. As used herein, "contacting" a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that the capture probe can interact with the analyte from the biological sample (e.g., covalent or non-covalent binding (e.g., hybridization)). Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0107] In some cases, spatial analysis can be performed by attaching and / or introducing a molecule (e.g., a peptide, lipid, or nucleic acid molecule) with a barcode (e.g., a spatial barcode) to a biological sample (e.g., a cell in a biological sample). In some embodiments, multiple molecules (e.g., multiple nucleic acid molecules) with multiple barcodes (e.g., multiple spatial barcodes) are introduced into a biological sample (e.g., multiple cells in a biological sample) for spatial analysis. In some embodiments, after attaching and / or introducing the molecule with the barcode to the biological sample, the biological sample can be physically separated (e.g., dissociated) into a single cell or a cell population for analysis. Some such spatial analysis methods are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0108] In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to the analyte. In some cases, for example, spatial analysis can be performed using RNA template ligation (RTL). The method of RTL has been described previously. See, for example, Credle et al., Nucleic Acids Res. 2017 Aug 21;45(14):e128. Typically, RTL involves hybridization of two oligonucleotides to adjacent sequences on the analyte (e.g., an RNA molecule, such as an mRNA molecule). In some cases, the oligonucleotides are DNA molecules. In some cases, one of the oligonucleotides includes at least two ribonucleobases at the 3′ end and / or the other oligonucleotide includes a phosphorylated nucleotide at the 5′ end. In some cases, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., SplintR ligase) ligates the two oligonucleotides together to produce a ligation product. In some cases, the two oligonucleotides hybridize to sequences that are not adjacent to each other. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some cases, polymerase (for example, DNA polymerase) can extend one of oligonucleotide before connecting.After connecting, connection product is discharged from analyte.In some cases, utilize endonuclease (for example, RNase H) to discharge connection product.The connection product of release can then be caught by the capture probe (for example, replacing the direct capture of analyte) on array, optionally amplified and sequenced, thus determine the position and optionally abundance of analyte in biological sample.

[0109] During spatial information analysis, sequence information of the spatial barcode associated with the 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 spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific positions in the feature array on the substrate. For example, a specific spatial barcode can be associated with a specific array position before the array is manufactured, and the sequence of the spatial barcode can be stored together with the specific array position information (e.g., in a database) so that each spatial barcode is uniquely mapped to a specific array position.

[0110] Alternatively, specific spatial barcodes can be deposited at predetermined locations in the feature array during manufacturing, such that at each location, only one type of spatial barcode is present, thereby uniquely associating the spatial barcode with a single feature of the array. If necessary, the array can be decoded using any of the methods described herein so that the spatial barcode is uniquely associated with the array feature location, and this mapping can be stored as described above.

[0111] When the sequence information of the capture probe and / or analyte is obtained during the spatial information analysis, the position of the capture probe and / or analyte can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature position. In this way, a specific capture probe and a captured analyte are associated with a specific position in the feature array. Each array feature position represents the position of a coordinate reference point (e.g., array position, fiducial marker) relative to the array. Therefore, each feature position has an "address" or position in the coordinate space of the array.

[0112] Some exemplary spatial analysis workflows are described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. See, for example, WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, which begin with "In some non-limiting examples of the workflows described herein, the sample can be immersed in ...". See also, for example, Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev C, dated June 2020), and / or Visium Spatial Tissue Optimization Reagent Kits User Guide (e.g., Rev C, dated July 2020).

[0113] In some embodiments, spatial analysis can be performed using specialized hardware and / or software, such as any system described in WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, or any one or more of the devices or methods described in WO2020 / 123320.

[0114] Suitable systems for performing spatial analysis may include parts, for example, for holding the chamber (for example, flow cell or sealable fluid-tight chamber) of biological sample. Biological sample can be fixed in for example biological sample container. One or more fluid chambers can be connected to chamber and / or sample container by fluid conduit, and fluid can be transported to chamber and / or sample container by fluid pump, vacuum source or other devices (generating pressure gradient to drive fluid flow) that are connected to fluid conduit. One or more valves also can be connected to fluid conduit to regulate the flow of reagent from reservoir to chamber and / or sample container.

[0115] The system may optionally include a control unit comprising one or more electronic processors, an input interface, an output interface (e.g., 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, writable and / or rewritable storage medium). The control unit may optionally be connected to one or more remote devices via a network. The control unit (and its components) may generally perform any steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) may perform any steps or features described herein. The system may optionally include one or more detectors (e.g., CCD, CMOS) for capturing an image. The system may also optionally include one or more light sources (e.g., LED-based, diode-based, laser-based) for irradiating a sample, a substrate with features, an analyte from a biological sample captured on the substrate, and various control and calibration media.

[0116] The system may optionally include software instructions encoded and / or implemented in one or more tangible storage media and hardware components (e.g., application-specific integrated circuits). When the software instructions are executed by a control unit (particularly an electronic processor) or an integrated circuit, the control unit, the integrated circuit, or other components executing the software instructions may cause the control unit, the integrated circuit, or other components executing the software instructions to perform any of the method steps or functions described herein.

[0117] In some cases, the systems described herein can detect (e.g., register images) biological samples on an array. Exemplary methods for detecting biological samples on an array are described in PCT application number 2020 / 061064 and / or U.S. patent application serial number 16 / 951,854.

[0118] Before the analyte is transferred from the biological sample to the feature array on the substrate, the biological sample can be aligned with the array. The alignment of the biological sample and the feature array including the capture probe can promote spatial analysis, which can be used to detect the difference in the presence and / or level of analytes in different positions in the biological sample, for example, to generate a three-dimensional graph of the presence and / or level of analytes. Exemplary methods for generating two-dimensional and / or three-dimensional graphs of the presence and / or level of analytes are described in PCT application number 2020 / 053655, and spatial analysis methods are generally described in WO2020 / 061108 and / or U.S. patent application serial number 16 / 951,864.

[0119] In some cases, one or more fiducial markers can be used to align a map of analyte presence and / or levels with an image of a biological sample, e.g., an object placed in the field of view of an imaging system appears in the resulting image, as described in WO 2020 / 123320, PCT Application No. 2020 / 061066, and / or U.S. Patent Application Serial No. 16 / 951,843. Fiducial markers can be used as reference points or measurement scales for alignment (e.g., to align a sample and an array, to align two substrates, to determine the position of a sample or array on a substrate relative to a fiducial marker) and / or for quantitative measurement of size and / or distance.

[0120] RNA capture using RNA-templated ligation

[0121] (a) General Background

[0122] Although technologies such as whole genome sequencing and whole exome sequencing can be used, the disadvantage of these technologies is that they provide a large amount of information and increase experimental costs. In the case where people prefer to study a limited number of analytes, this paper provides a method for being captured by target RNA. The derivative (for example, connection product) of the captured analyte provides the specificity of the enhancement about analyte detection. This is because it is necessary to hybridize at least two probes specific to the target to promote the connection and final capture of nucleic acid with the target.

[0123] refer to Figure 1 In an exemplary embodiment of the present disclosure, a method for identifying the location of an analyte in a biological sample is provided. In some cases, the method includes 101 contacting the biological sample with a spatially barcoded capture probe array. In some cases, the array is on a substrate and the array includes a plurality of capture probes, wherein the capture probes in the plurality include: (i) a spatial barcode and (ii) a capture domain. After the biological sample is placed on the array, the biological sample 102 is contacted with a first probe and a second probe, wherein the first probe and the second probe each contain one or more sequences that are substantially complementary to the sequence of the analyte, and wherein the second probe contains a capture probe capture domain; the first probe and the second probe 103 hybridize with the complementary sequences in the analyte. After hybridization, a ligation product comprising the first probe and the second probe 104 is produced, and the ligation product is released from the analyte. The released ligation product is then freed 105 to hybridize with the capture domain of the probe on the array. After capture, (i) all or part of the sequence of the ligation product specifically bound to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement 106, can be determined, and the determined sequences 107 of (i) and (ii) can then be used to identify the location of the analyte in the biological sample.

[0124] refer to Figure 13In another non-limiting example, a biological sample is deparaffinized, stained, and imaged 1301. After decolorization and cross-linking 1302, a probe is added to the sample and hybridized to the analyte 1303. In some cases, the probe is a DNA probe. In some cases, the probe is a diribose-containing probe. The probe is ligated 1304 and then released 1305 using an endonuclease such as RNase H. The ligated probe is captured on an array 1306 by a capture probe, extended 1307 using a polymerase, and denatured 1308. After quality control cleanup 1309, the abundance and location of the analyte is determined.

[0125] Non-limiting examples of the methods disclosed herein are shown in Figure 6 After a biological sample is contacted with a substrate comprising a plurality of capture probes and with (a) a first probe 601 having a target hybridization sequence 603 and a primer sequence 602 and (b) a second probe 604 having a target hybridization sequence 605 and a capture domain (e.g., a poly A sequence) 606, the first probe 601 and the second probe 604 hybridize 610 with an analyte 607. A ligase 621 ligates 620 the first probe to the second probe, thereby generating a ligation product 622. The ligation product is released 630 from the analyte 631 by digesting the analyte using an endoribonuclease 632. The sample is permeabilized 640 and the ligation product 641 is able to hybridize with the capture probes on the substrate.

[0126] Also provided herein are methods for identifying the location of an analyte in a biological sample comprising a second probe comprising a pre-adenylated phosphate group at its 5' end, which enables ligation using a ligase that does not require adenosine triphosphate for ligase activity.

[0127] Also provided herein are methods for identifying the location of an analyte in a biological sample that includes one or more spanning probes in addition to a first and a second probe. The use of spanning probes allows for greater flexibility in designing RTL probes, primarily by increasing the number of sequences in the analyte that can serve as alternative target sequences.

[0128] Also provided herein are methods for identifying the location of an analyte in a biological sample, comprising optimized hybridization, washing, and release steps.

[0129] In some embodiments, as Figure 7As shown, the ligation product 701 includes a capture probe capture domain 702, which can bind to a capture probe 703 (e.g., a capture probe directly or indirectly immobilized on a substrate 704). In some embodiments, the methods provided herein include contacting 705 a biological sample with a substrate 704, wherein the capture probe 703 is immobilized to the substrate (e.g., directly or indirectly immobilized to the substrate). In some embodiments, the capture probe capture domain 702 of the ligation product specifically binds to the capture domain 706. The capture probe may also include a unique molecular identifier (UMI) 707, a spatial barcode 708, a functional sequence 709, and a cleavage domain 710.

[0130] In some embodiments, the method provided herein includes the permeabilization of a biological sample so that the capture probe can be more easily combined with the connection probe captured (that is, compared to no permeabilization). In some embodiments, a reverse transcription (RT) reagent can be added to the permeabilized biological sample. The capture probe 711 can be extended by incubation with the RT reagent to produce spatial barcoded full-length cDNA 712 and 713 from the captured analyte (e.g., polyadenylated mRNA). A second chain reagent (e.g., a second chain primer, an enzyme) can be added to the biological sample on the slide to initiate second chain synthesis.

[0131] In some embodiments, the cDNA can be denatured 714 from the capture probe template and transferred (e.g., to a clean tube) for amplification and / or library construction. The spatially barcoded full-length cDNA can be amplified 715 by PCR prior to library construction. The cDNA can then be enzymatically fragmented and size-selected to optimize the size of the cDNA amplicons. P5716, i5717, i7718, and P7719 can serve as sample indexes, and TruSeq read 2 can be added by end repair, A-tailing, adapter ligation, PCR, or the like. The cDNA fragments can then be sequenced using paired-ended sequencing using TruSeq read 1 and TruSeq read 2 as sequencing primer sites.

[0132] (b) Probes for RNA-templated ligation

[0133] The methods provided herein utilize probe pairs (or sets; these terms are interchangeable). In some cases, the probe pairs are designed so that each probe hybridizes to an analyte sequence that is specific for the analyte (e.g., compared to the entire genome). That is, in some cases, a single probe pair can be specific for a single analyte.

[0134] In other embodiments, probe can be designed so that one of a pair of probes is a probe that hybridizes with a specific sequence. Then, another probe can be designed to detect interested mutation. Therefore, in some cases, a plurality of second probes can be designed and can be changed so that each is combined with a specific sequence. For example, the second probe can be designed to hybridize with the wild-type sequence, and another second probe can be designed to detect mutant sequences. Therefore, in some cases, probe groups can include a first probe and two second probes (or vice versa).

[0135] On the other hand, in some cases, the probe can be designed so that it covers a conserved region of the analyte. Thus, in some cases, the probe (or probe pair) can hybridize to similar analytes in a biological sample (e.g., to detect conserved or similar analytes) or to similar analytes in different biological samples (e.g., across different species).

[0136] In some embodiments, probe groups cover all or almost all genomes (for example, human genome). When probe groups are designed to cover the entire genome (for example, human genome), method disclosed herein can detect analyte in an unbiased manner. In some cases, a probe oligonucleotide is designed to cover a kind of analyte (for example, transcript). In some cases, design more than one probe oligonucleotide pair (for example, a probe pair comprising the first probe and the second probe) to cover a kind of analyte (for example transcript). For example, at least two, three, four, five, six, seven, eight, nine, ten or more probe groups can be used for hybridizing with a single analyte. The factor to be considered during designing probes is the presence of a variation (for example, SNP, mutation) or multiple isotypes of unigene expression. In some cases, probe oligonucleotide is not hybridized with the entire analyte (for example, transcript), but probe oligonucleotide is hybridized with the portion of the entire analyte (for example, transcript).

[0137] In some cases, about 5000, 10,000, 15,000, 20,000, or more probe oligonucleotide pairs (e.g., probe pairs comprising a first probe and a second probe) are used in the methods described herein. In some cases, about 20,000 probe oligonucleotide pairs are used in the methods described herein.

[0138] In some cases, RNA capture is targeted RNA capture (targeted RNA capture). Targeted RNA capture using the methods disclosed herein allows for the examination of a subset of RNA analytes from the entire transcriptome. In some embodiments, the subset of analytes includes individual target RNAs. In some embodiments, the analyte subset includes two or more targeted RNAs. In some embodiments, the analyte subset includes one or more mRNAs transcribed from one or more targeted genes. In some embodiments, the analyte subset includes one or more mRNA splice variants of one or more targeted genes. In some embodiments, the analyte subset includes non-polyadenylated RNA in a biological sample. In some embodiments, the analyte subset includes detecting mRNAs with one or more single nucleotide polymorphisms (SNPs) in a biological sample.

[0139] In some embodiments, the analyte subset includes mRNAs that mediate the expression of a group of genes of interest. In some embodiments, the analyte subset includes mRNAs that share identical or substantially similar sequences, which are translated into polypeptides having similar functional groups or protein domains. In some embodiments, the analyte subset includes mRNAs that do not have identical or substantially similar sequences, which are translated into proteins that do not share similar functional groups or protein domains. In some embodiments, the analyte subset includes mRNAs that are translated into proteins that play a role in the same or similar biological pathways. In some embodiments, biological pathways are associated with pathological diseases. For example, capture by target RNA can detect genes that are overexpressed or underexpressed in cancer.

[0140] In some embodiments, the subset of analytes includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, about 55, about 60, about 65, about 70, about 75, about 76, about 77, about 78, about 79, about 80, about 81. about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 600, about 700, about 800, about 900, or about 1000 analytes.

[0141] In some cases, the methods disclosed herein can detect the abundance and location of at least 5,000, 10,000, 15,000, 20,000, or more different analytes.

[0142] In some embodiments, the subset of analytes detected by the target RNA capture methods provided herein includes a majority of the transcriptome of one or more cells. For example, the subset of analytes detected by the target RNA capture methods provided herein can include at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more of the mRNA present in the transcriptome of one or more cells.

[0143] In some cases, the probe is a DNA probe. In some cases, the probe is a diribose-containing probe.

[0144] Other embodiments of probes and probe sets are described herein.

[0145] (i) First probe

[0146] In some embodiments, the methods described herein include a first probe. As used herein, a "first probe" can refer to a probe that hybridizes to all or part of an analyte and can be linked to one or more other probes (e.g., a second probe or a spanning probe). In some embodiments, a "first probe" can be used interchangeably with a "first probe oligonucleotide."

[0147] In some embodiments, the first probe includes ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the first probe includes deoxyribonucleotides. In some embodiments, the first probe includes deoxyribonucleotides and ribonucleotides. In some embodiments, the first probe includes a deoxyribonucleic acid that hybridizes with the analyte, and includes a part of an oligonucleotide that is not a deoxyribonucleic acid. For example, in some embodiments, the part that is not a deoxyribonucleic acid of the first oligonucleotide is a ribonucleic acid as described herein or any other non-deoxyribonucleic acid nucleic acid. In some embodiments, the first probe includes deoxyribonucleotides, and the hybridization of the first probe and the mRNA molecule causes DNA: RNA hybrid. In some embodiments, the first probe only includes deoxyribonucleotides and produces DNA: RNA hybrid after the first probe and the mRNA molecule hybridize.

[0148] In some embodiments, the method comprises a first probe comprising one or more sequences that are substantially complementary to one or more sequences of the analyte. In some embodiments, the first probe comprises a sequence that is substantially complementary to a first target sequence in the analyte. In some embodiments, the sequence of the first probe that is substantially complementary to the first target sequence in the analyte is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the first target sequence in the analyte.

[0149] In some embodiments, the first probe comprises a sequence having from about 10 nucleotides to about 100 nucleotides (e.g., from about 10 nucleotides to about 90 nucleotides, from about 10 nucleotides to about 80 nucleotides, from about 10 nucleotides to about 70 nucleotides, from about 10 nucleotides to about 60 nucleotides, from about 10 nucleotides to about 50 nucleotides, from about 10 nucleotides to about 40 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 10 nucleotides to about 20 nucleotides, from about 20 nucleotides to about 100 nucleotides, from about 2 0 nucleotides to about 90 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 30 nucleotides, about 30 nucleotides to about 100 nucleotides, about 30 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 70 nucleotides, about 30 nucleotides to about 60 nucleotides , about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 40 nucleotides, about 40 nucleotides to about 100 nucleotides, about 40 nucleotides to about 90 nucleotides, about 40 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 40 nucleotides to about 60 nucleotides, about 40 nucleotides to about 50 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 90 nucleotides, about 50 nucleotides to about 80 nucleotides, about 50 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 60 nucleotides to about 100 nucleotides, about 60 nucleotides to about 90 nucleotides, about 60 nucleotides to about 80 nucleotides, about 60 nucleotides to about 70 nucleotides, about 70 nucleotides to about 100 nucleotides, about 70 nucleotides to about 90 nucleotides, about 70 nucleotides to about 80 nucleotides, about 80 nucleotides to about 100 nucleotides, about 80 nucleotides to about 90 nucleotides, or a sequence of about 90 nucleotides to about 100 nucleotides).

[0150] In some embodiments, the sequence of the first probe that is substantially complementary to a sequence in the analyte comprises a sequence of about 5 nucleotides to about 50 nucleotides (e.g., about 5 nucleotides to about 45 nucleotides, about 5 nucleotides to about 40 nucleotides, about 5 nucleotides to about 35 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, about 10 nucleotides to about 50 nucleotides). , about 10 nucleotides to about 45 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 35 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 15 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 45 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 35 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 20 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 45 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 35 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 45 nucleotides, about 25 nucleotides to about 40 nucleotides, about 25 nucleosides nucleotides to about 35 nucleotides, about 25 nucleotides to about 30 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 45 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 35 nucleotides, about 35 nucleotides to about 50 nucleotides, about 35 nucleotides to about 45 nucleotides, about 35 nucleotides to about 40 nucleotides, about 40 nucleotides to about 50 nucleotides, about 40 nucleotides to about 45 nucleotides, or about 45 nucleotides to about 50 nucleotides).

[0151] In some embodiments, the first probe comprises a functional sequence. In some embodiments, the functional sequence comprises a primer sequence.

[0152] In some embodiments, the first probe comprises at least two ribonucleobases at its 3' end. In this case, the second probe oligonucleotide comprises a phosphorylated nucleotide at its 5' end. In some embodiments, the first probe comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten ribonucleobases at its 3' end.

[0153] like Figure 6 As shown, a non-limiting example of a first probe 601 , which may be referred to as an LHS probe, includes a functional sequence 602 , a sequence 603 that is substantially complementary to a first target sequence in an analyte 607 , and two ribonucleobases at the 3′ end.

[0154] In some embodiments, the first probe includes a helper sequence that does not hybridize to the analyte. In some embodiments, the helper sequence can be used to hybridize to other probes.

[0155] (ii) Second probe

[0156] In some embodiments, the methods described herein include a second probe. As used herein, a "second probe" can refer to a probe that hybridizes with all or part of an analyte and can be connected to one or more other probes (e.g., a first probe or a spanning probe). In some embodiments, a "second probe" can be used interchangeably with a "second probe oligonucleotide." It will be understood by those skilled in the art that the order of the probes is arbitrary, and therefore the contents of the first probe and / or second probe disclosed herein are interchangeable.

[0157] In some embodiments, the second probe includes ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the second probe includes deoxyribonucleotides. In some embodiments, the second probe includes deoxyribonucleotides and ribonucleotides. In some embodiments, the second probe includes a deoxyribonucleic acid that hybridizes with the analyte, and includes the part of an oligonucleotide that is not a deoxyribonucleic acid. For example, in some embodiments, the part that is not a deoxyribonucleic acid of the second probe is a ribonucleic acid as described herein or any other non-deoxyribonucleic acid nucleic acid. In some embodiments in which the second probe includes deoxyribonucleotides, the hybridization of the second probe and the mRNA molecule causes DNA: RNA hybrid. In some embodiments, the second probe only includes deoxyribonucleotides and produces DNA: RNA hybrid after the first probe hybridizes with the mRNA molecule.

[0158] In some embodiments, the method includes a second probe comprising one or more sequences that are substantially complementary to one or more sequences of the analyte. In some embodiments, the second probe comprises a sequence that is substantially complementary to a second target sequence in the analyte. In some embodiments, the sequence of the second probe that is substantially complementary to the second target sequence in the analyte is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the second target sequence in the analyte.

[0159] In some embodiments, the second probe comprises a sequence having from about 10 nucleotides to about 100 nucleotides (e.g., from about 10 nucleotides to about 90 nucleotides, from about 10 nucleotides to about 80 nucleotides, from about 10 nucleotides to about 70 nucleotides, from about 10 nucleotides to about 60 nucleotides, from about 10 nucleotides to about 50 nucleotides, from about 10 nucleotides to about 40 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 10 nucleotides to about 20 nucleotides, from about 20 nucleotides to about 100 nucleotides, from about 2 0 nucleotides to about 90 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 60 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 30 nucleotides, about 30 nucleotides to about 100 nucleotides, about 30 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 70 nucleotides, about 30 nucleotides to about 60 nucleotides , about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 40 nucleotides, about 40 nucleotides to about 100 nucleotides, about 40 nucleotides to about 90 nucleotides, about 40 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 40 nucleotides to about 60 nucleotides, about 40 nucleotides to about 50 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 90 nucleotides, about 50 nucleotides to about 80 nucleotides, about 50 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 60 nucleotides to about 100 nucleotides, about 60 nucleotides to about 90 nucleotides, about 60 nucleotides to about 80 nucleotides, about 60 nucleotides to about 70 nucleotides, about 70 nucleotides to about 100 nucleotides, about 70 nucleotides to about 90 nucleotides, about 70 nucleotides to about 80 nucleotides, about 80 nucleotides to about 100 nucleotides, about 80 nucleotides to about 90 nucleotides, or a sequence of about 90 nucleotides to about 100 nucleotides).

[0160] In some embodiments, the sequence of the second probe that is substantially complementary to a sequence in the analyte comprises a sequence of about 5 nucleotides to about 50 nucleotides (e.g., about 5 nucleotides to about 45 nucleotides, about 5 nucleotides to about 40 nucleotides, about 5 nucleotides to about 35 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 10 nucleotides, about 10 nucleotides to about 50 nucleotides). , about 10 nucleotides to about 45 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 35 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 15 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 45 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 35 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 20 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 45 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 35 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 45 nucleotides, about 25 nucleotides to about 40 nucleotides, about 25 nucleosides nucleotides to about 35 nucleotides, about 25 nucleotides to about 30 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 45 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 35 nucleotides, about 35 nucleotides to about 50 nucleotides, about 35 nucleotides to about 45 nucleotides, about 35 nucleotides to about 40 nucleotides, about 40 nucleotides to about 50 nucleotides, about 40 nucleotides to about 45 nucleotides, or about 45 nucleotides to about 50 nucleotides).

[0161] In some embodiments, the second probe includes a capture probe capture domain sequence. As used herein, a "capture probe capture domain" is a sequence, domain, or portion that can specifically bind to the capture domain of a capture probe. In some embodiments, "capture domain capture domain" can be used interchangeably with "capture probe binding domain." In some embodiments, the second probe includes a sequence from 5' to 3': a sequence that is substantially complementary to a sequence in the analyte and a capture probe capture domain.

[0162] In some embodiments, the capture probe capture domain includes a poly (A) sequence. In some embodiments, the capture probe capture domain includes a polyuridine sequence, a polythymidine sequence or both. In some embodiments, the capture probe capture domain includes a random sequence (e.g., a random hexamer or octamer). In some embodiments, the capture probe capture domain is complementary to the capture domain in the capture probe that detects a specific target of interest. In some embodiments, a capture probe capture domain blocking portion that interacts with the capture probe capture domain is provided. In some embodiments, the capture probe capture domain blocking portion includes a sequence that is complementary or substantially complementary to the capture probe capture domain. In some embodiments, the capture probe capture domain blocking portion prevents the capture probe capture domain from binding to the capture probe when present. In some embodiments, before the capture probe capture domain (e.g., present in a connected probe) is combined with the capture probe, the capture probe capture domain blocking portion is removed. In some embodiments, the capture probe capture domain blocking portion includes a polyuridine sequence, a polythymidine sequence or both. In some embodiments, the capture probe capture domain sequence includes ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the capture probe binding domain sequence comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capture probe binding domain sequence comprises at least 25, 30, or 35 nucleotides.

[0163] In some embodiments, the second probe includes a phosphorylated nucleotide at the 5' end. The phosphorylated nucleotide at the 5' end can be used in a ligation reaction to connect the second probe to the first probe.

[0164] like Figure 6 As shown, a non-limiting example of a second probe 604 , which may be referred to as an RHS probe, includes a sequence 605 that is substantially complementary to a second target sequence on an analyte 607 and a capture probe capture domain 606 .

[0165] In some embodiments, the second probe includes a helper sequence that does not hybridize to the analyte. In some embodiments, the helper sequence can be used to hybridize to other probes.

[0166] (iii) Multi-probe

[0167] In some embodiments, the target RNA capture method disclosed herein includes multiple probe oligonucleotides. In some embodiments, the method includes 2, 3, 4 or more probe oligonucleotides. In some embodiments, each probe oligonucleotide includes ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, each probe oligonucleotide includes deoxyribonucleotides. In some embodiments, each probe oligonucleotide includes deoxyribonucleotides. In some embodiments, each probe oligonucleotide includes deoxyribonucleotides and ribonucleotides.

[0168] In some cases, multiple probes span different target sequences, and multiple sequential ligation steps are performed to determine the location and abundance of the analyte.

[0169] In some cases, the method includes using a first probe and a plurality of second probes (or vice versa), wherein the plurality of second probes hybridize to different sequences (e.g., wild type and mutant sequences, different isoforms, splice variants) to determine the sequence of the analyte. It should be understood that the method can be used to detect single mutations (e.g., point mutations, SNPs, splice variants, etc.) or can be used to detect polynucleotide mutations (e.g., insertions, deletions, etc.).

[0170] Method provided herein can be applicable to single nucleic acid molecule or multiple nucleic acid molecules.The method for analyzing the sample comprising nucleic acid molecules can include: multiple nucleic acid molecules (for example, RNA molecules) are provided, wherein each nucleic acid molecule includes the first target region (for example, the first target sequence) and the second target region (for example, the second target sequence), multiple first probe oligonucleotides and multiple second probe oligonucleotides.In some cases, one or more target regions of the nucleic acid molecules of multiple nucleic acid molecules can include identical sequence.The first and second target regions (for example, the first and second target sequences) of the nucleic acid molecules in multiple nucleic acid molecules can be adjacent to each other.

[0171] (iv) A first probe having a linker sequence

[0172] Also provided herein is a method for identifying the location of an analyte in a biological sample, wherein the method comprises a first probe comprising a linker and a second probe. Using a pair of probes, wherein the first probe comprises a linker sequence, allows for greater flexibility in designing RTL probes, primarily by increasing the number of sequences in the analyte that can serve as optional target sequences.

[0173] As used herein, "linker sequence" can refer to one or more nucleic acid sequences on a probe (e.g., a sequence of the first probe, second probe, or spanning probe, analyte-specific sequence connecting the probes, located between the sequences that hybridize to the analyte). In some embodiments, the linker includes a sequence that is substantially non-complementary to the sequence of the target analyte or the first probe, second probe, or analyte-specific sequence spanning the probe. In some embodiments, the linker sequence includes ribonucleotides, deoxyribonucleotides, and / or synthetic nucleotides, wherein the sequence within the linker is substantially non-complementary to the target analyte or the first probe, second probe, or analyte-specific sequence spanning the probe.

[0174] In some embodiments where the first and / or second probe comprises a linker sequence, the linker sequence can comprise a total of about 10 nucleotides to about 100 nucleotides, or any subrange described herein.

[0175] In some embodiments, the linker sequence comprises a barcode sequence that serves as a surrogate for identifying the analyte. In some embodiments, the barcode sequence is a sequence that is at least 70% identical to the sequence in the analyte (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical). In some embodiments where the linker sequence comprises a barcode sequence, the barcode sequence is located 5′ to the linker sequence. In some embodiments where the linker sequence comprises a barcode sequence, the barcode sequence is located 3′ to the linker sequence. In some embodiments, the barcode sequence is located between two linker sequences. In this case, the two linker sequences flanking the barcode sequence can be considered to be part of the same linker sequence.

[0176] In some embodiments in which the first and / or second probe comprises a linker sequence, the linker sequence can comprise ribonucleotides, deoxyribonucleotides, and / or synthetic nucleotides.

[0177] A non-limiting example of a method for identifying the location of an analyte in a biological sample includes a first probe and a second probe comprising a linker sequence, comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein the capture probes in the plurality of capture probes comprise a capture domain and a spatial barcode; (b) contacting the biological sample with the first probe and the second probe, wherein a portion of the first probe and a portion of the second probe are substantially complementary to adjacent sequences of the analyte, wherein the first probe comprises: (i) a first sequence substantially complementary to a first target sequence of the analyte; (ii) a linker sequence; (iii) a second sequence substantially complementary to a second target sequence of the analyte; and wherein the second probe comprises a sequence substantially complementary to a third target sequence of the analyte and a capture probe capture domain capable of binding to the capture domain of the capture probe; (c) hybridizing the first probe and the second probe to the analyte; (d) connecting the first probe and the second probe to thereby produce a connection product; (e) releasing the connection product from the analyte; (f) hybridizing the capture probe binding domain to the capture domain; and (g) determining (i) all or part of the sequence of the connection product specifically bound to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the position of the analyte in the biological sample.

[0178] A non-limiting example of a method for identifying the location of an analyte in a biological sample, wherein the method comprises a first probe comprising a linker and a second probe comprising Figure 8 The components shown are as follows. The first probe 801 includes a functional sequence 802, a first sequence 803 that is substantially complementary to a first target sequence 804 of the analyte, a linker sequence 805, and a second sequence 806 that is substantially complementary to a second target sequence 807 of the analyte. The second probe 808 includes a sequence 809 that is substantially complementary to a third target sequence 810 of the analyte, and a capture probe capture domain 811 that is capable of binding to the capture domain of the capture probe.

[0179] 1) First probe

[0180] In some embodiments where the first probe comprises a linker sequence, the first probe comprises a first sequence substantially complementary to a first target sequence of the analyte, a linker sequence, and a second sequence substantially complementary to a second target sequence of the analyte. In some embodiments, the first probe comprises, from 5′ to 3′, a first sequence substantially complementary to the first target sequence of the analyte, a linker sequence, and a second sequence substantially complementary to the second target sequence of the analyte.

[0181] In some embodiments where the first probe comprises a linker sequence, the first probe comprises a functional sequence. In some embodiments, the first probe comprises a functional sequence, a first sequence substantially complementary to a first target sequence of the analyte, a linker sequence, and a second sequence substantially complementary to a second target sequence of the analyte. In some embodiments, the functional sequence comprises a primer sequence.

[0182] In some embodiments where the first probe comprises a linker sequence, the first probe comprises at least two ribonucleobases at the 3' end. In some embodiments, the first probe comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten ribonucleobases at the 3' end.

[0183] In some embodiments in which the first probe comprises a linker, the first probe comprises a sequence of about 10 nucleotides to about 300 nucleotides (e.g., about 10 nucleotides to about 300 nucleotides, about 10 nucleotides to about 250 nucleotides, about 10 nucleotides to about 200 nucleotides, about 10 nucleotides to about 150 nucleotides, about 10 nucleotides to about 100 nucleotides, about 10 nucleotides to about 50 nucleotides, about 50 nucleotides to about 300 nucleotides, about 50 nucleotides to about 250 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about about 150 nucleotides, about 50 nucleotides to about 100 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 250 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 150 nucleotides, about 150 nucleotides to about 300 nucleotides, about 150 nucleotides to about 250 nucleotides, about 150 nucleotides to about 200 nucleotides, about 200 nucleotides to about 300 nucleotides, about 200 nucleotides to about 250 nucleotides, or about 250 nucleotides to about 300 nucleotides).

[0184] In some embodiments where the first probe comprises a linker sequence, the first probe comprises a first sequence that is substantially complementary to a first target sequence of the analyte. In some embodiments, the first sequence of the first probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the first target sequence in the analyte. In some embodiments, the first sequence of the first probe that is substantially complementary to the first target sequence can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0185] In some embodiments where the first probe comprises a linker, the first probe comprises a second sequence that is substantially complementary to a second target sequence of the analyte. In some embodiments, the second sequence of the first probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the second target sequence in the analyte. In some embodiments, the second sequence of the first probe that is substantially complementary to the second target sequence can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0186] In some embodiments, the first probe comprising a linker sequence comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the first probe comprising a linker sequence comprises deoxyribonucleotides. In some embodiments, the first probe comprising a linker sequence comprises deoxyribonucleotides and ribonucleotides. In some embodiments in which the first probe comprising a linker sequence comprises deoxyribonucleotides, hybridization of the first probe with the mRNA molecule produces a DNA: RNA hybrid. In some embodiments, the first probe comprising a linker sequence comprises only deoxyribonucleotides, and produces a DNA: RNA hybrid after hybridization of the first probe with the mRNA molecule.

[0187] 2) Second probe

[0188] In some embodiments where the first probe comprises a linker sequence, the second probe comprises a sequence substantially complementary to a third target sequence of the analyte and a capture probe capture domain capable of binding to the capture domain of the capture probe. In some embodiments where the first probe comprises a linker, the second probe comprises a sequence of about 10 nucleotides to about 100 nucleotides, or any subrange described herein.

[0189] In some embodiments where the first probe comprises a linker, the sequence of the second probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a third target sequence in the analyte. In some embodiments, the sequence of the second probe that is substantially complementary to the third target sequence can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0190] In some embodiments where the first probe comprises a linker sequence, the first target sequence is not adjacent to the second target sequence. For example, the first target sequence and the second target sequence are located on different exons of the same mRNA molecule. In another example, the first target sequence and the second target sequence are located on the same exon of the same mRNA molecule but are not adjacent. In some cases, the first probe and the second probe hybridize to sequences that are at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides apart.

[0191] In some embodiments where the first probe comprises a linker sequence, the second target sequence is directly adjacent to the third target sequence.

[0192] In some embodiments, the first probe comprises a joint sequence, and the second probe comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the second probe comprises deoxyribonucleotides. In some embodiments, the second probe comprises deoxyribonucleotides and ribonucleotides. In some embodiments, the second probe comprises deoxyribonucleotides, and the hybridization of the second probe and mRNA molecule causes DNA: RNA hybrid. In some embodiments, the second probe only comprises deoxyribonucleotides and produces DNA: RNA hybrid after the second probe and mRNA molecule hybridize.

[0193] (v) Second probe with a linker

[0194] Also provided herein are methods for identifying the location of an analyte in a biological sample, wherein the method comprises a first probe and a second probe comprising a linker sequence. Using a pair of probes, wherein the second probe comprises a linker sequence, allows for greater flexibility in designing RTL probes, primarily by increasing the number of sequences in the analyte that can serve as alternative target sequences.

[0195] A non-limiting example of a method for identifying the location of an analyte in a biological sample, wherein the method includes a first probe and a second probe comprising a linker, comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein the capture probes in the plurality of capture probes include a capture domain and a spatial barcode; (b) contacting the biological sample with the first probe and the second probe, wherein a portion of the first probe and a portion of the second probe are substantially complementary to adjacent sequences of the analyte, wherein the first probe includes a sequence substantially complementary to a first target sequence of the analyte, and wherein the second probe includes: (i) a first sequence substantially complementary to a second target sequence of the analyte; (ii) a linker sequence; (iii) a second sequence substantially complementary to a second target sequence of the analyte; (iii) a second sequence that is substantially complementary to a third target sequence of the analyte; and (iv) a capture probe binding domain that is capable of binding to the capture domain of the capture probe; (c) hybridizing the first probe and the second probe to the analyte; (d) ligating the first probe and the second probe, thereby producing a ligation product; (e) releasing the ligation product from the analyte; (f) hybridizing the capture probe binding domain to the capture domain; and (g) determining (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0196] A non-limiting example of a method for identifying the location of an analyte in a biological sample, wherein the method comprises a first probe and a second probe comprising a linker, may comprise e.g. Figure 9 The components shown are as follows. The first probe 901 includes a functional sequence 902 and a sequence 903 that is substantially complementary to a first target sequence 904 of the analyte. The second probe 905 includes a first sequence 906 that is substantially complementary to a second target sequence 907 of the analyte, a linker sequence 908, a second sequence 909 that is substantially complementary to a second target sequence 910 of the analyte, and a capture probe capture domain 911 that is capable of binding to the capture domain of the capture probe.

[0197] 1) First probe

[0198] In some embodiments where the second probe comprises a linker sequence, the first probe comprises a sequence that is substantially complementary to the first target sequence of the analyte.

[0199] In some embodiments where the second probe comprises a linker sequence, the first probe comprises a functional sequence. In some embodiments, the first probe comprises a functional sequence and a sequence substantially complementary to a first target sequence of the analyte. In some embodiments, the functional sequence comprises a primer sequence. In some embodiments, the first probe oligonucleotide comprises, from 5′ to 3′, a functional sequence and a sequence substantially complementary to the first target sequence.

[0200] In some embodiments where the linker is on the second probe, the first probe comprises a sequence of about 10 nucleotides to about 100 nucleotides, or any subrange described herein.

[0201] In some embodiments where the second probe comprises an adapter sequence, the sequence of the first probe that is substantially complementary to the first target sequence of the analyte is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the first target sequence in the analyte. In some embodiments, the sequence that is substantially complementary to the first target sequence can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0202] In some embodiments where the second probe comprises a linker sequence, the first probe comprises at least two ribonucleobases at the 3' end. In some embodiments, the first probe comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten ribonucleobases at the 3' end.

[0203] In some embodiments, the second probe comprises a joint sequence, and the first probe comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the first probe comprises deoxyribonucleotides. In some embodiments, the first probe comprises deoxyribonucleotides and ribonucleotides. In some embodiments, the first probe comprises deoxyribonucleotides, and the hybridization of the first probe and mRNA molecule causes DNA: RNA hybrid. In some embodiments, the first probe only comprises deoxyribonucleotides and produces DNA: RNA hybrid after the first probe and mRNA molecule hybridize.

[0204] 2) Second probe

[0205] In some embodiments where the linker is located on the second probe, the second probe comprises (i) a first sequence that is substantially complementary to a second target sequence of the analyte; (ii) a linker sequence (e.g., any of the exemplary linker sequences described herein); (iii) a second sequence that is substantially complementary to a third target sequence of the analyte; and (iv) a capture probe capture domain that is capable of binding to a capture domain of the capture probe (e.g., any of the exemplary capture probe capture domains described herein).

[0206] In some embodiments where the second probe comprises a linker sequence, the second probe comprises a sequence of about 10 nucleotides to about 300 nucleotides, or any subrange described herein.

[0207] In some embodiments where the second probe comprises a linker sequence, the second probe comprises a first sequence that is substantially complementary to a second target sequence of the analyte. In some embodiments, the first sequence of the second probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the second target sequence in the analyte. In some embodiments, the first sequence of the second probe that is substantially complementary to the second target sequence can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0208] In some embodiments where the second probe comprises a linker sequence, the second probe comprises a second sequence that is substantially complementary to a third target sequence of the analyte. In some embodiments, the second sequence of the second probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the third target sequence in the analyte. In some embodiments, the second sequence of the second probe that is substantially complementary to the third target sequence can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0209] In some embodiments where the second probe includes a linker sequence, the second target sequence is not adjacent to the third target sequence in the mRNA molecule. For example, the second target sequence and the third target sequence are located in different exons of the same mRNA molecule. In another example, the second target sequence and the third target sequence are located in the same exon of the same mRNA molecule but are not adjacent.

[0210] In some embodiments where the second probe comprises a linker sequence, the first target sequence is directly adjacent to the second target sequence.

[0211] In some embodiments, the second probe comprising a linker sequence comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the second probe comprising a linker sequence comprises deoxyribonucleotides. In some embodiments, the second probe comprising a linker sequence comprises deoxyribonucleotides and ribonucleotides. In some embodiments in which the second probe comprising a linker sequence comprises deoxyribonucleotides, the hybridization of the second probe with the mRNA molecule produces a DNA: RNA hybrid. In some embodiments, the second probe comprising a linker sequence comprises only deoxyribonucleotides, and produces a DNA: RNA hybrid after the second probe hybridizes with the mRNA molecule.

[0212] (vii) Probe combination having a linker on the first probe and the second probe

[0213] Also provided herein is a method for identifying the location of an analyte in a biological sample, wherein the method comprises a first probe comprising a linker sequence and a second probe comprising a linker sequence. Using a pair of probes, wherein both the first and second probes comprise a linker sequence, allows for greater flexibility in designing RTL probes, primarily by increasing the number of sequences in the analyte that can serve as alternative target sequences.

[0214] (c) A probe combination comprising a first probe, a second probe, and a spanning probe

[0215] Also provided herein is a method for identifying the position of an analyte in a biological sample, wherein the method comprises a first probe, a spanning probe, and a second probe. Using a spanning probe can achieve greater flexibility in designing an RTL probe, primarily by increasing the sequence that can be used as an optional target sequence in the analyte. In some cases, using a spanning probe can also be used to interrogate variants (e.g., splice variants) that span a greater distance, which can be interrogated using a first or second probe with a linker sequence.

[0216] A non-limiting example of a method for identifying the location of an analyte in a biological sample, wherein the method includes a first probe, a spanning probe, and a second probe, comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises a capture domain and a spatial barcode; (b) contacting the biological sample with a first probe, a second probe, and one or more spanning probes, wherein the first probe is substantially complementary to a first portion of the analyte, wherein the second probe is substantially complementary to a second portion of the analyte and further comprises a capture probe binding domain, and wherein the spanning probe comprises: (i) a first sequence substantially complementary to a first target sequence of the analyte, and (ii) a second sequence that is substantially complementary to a second target sequence of the analyte; (c) hybridizing the first probe, the second probe, and the spanning probe to the analyte; (d) ligating the first probe, the one or more spanning probes, and the second probe, thereby producing a ligation product that is substantially complementary to the analyte; (e) releasing the ligation product from the analyte; (f) hybridizing the capture probe binding domain to the capture domain; and (g) determining (i) all or part of the sequence, or its complement, of the ligation product that specifically binds to the capture domain, and (ii) all or part of the sequence, or its complement, of the spatial barcode, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0217] A non-limiting example of a method for identifying the location of an analyte in a biological sample, wherein the method includes a first probe, a second probe, and a spanning probe, may include, for example Figure 10The components are shown. The first probe 1001 includes a functional sequence 1002 and a sequence 1003 that is substantially complementary to a first portion 1004 of the analyte. The spanning probe 1005 includes a first sequence 1006 that is substantially complementary to a first target sequence 1007 of the analyte, a linker sequence 1008, and a second sequence 1009 that is substantially complementary to a second target sequence 1010 of the analyte. The second probe 1011 includes a sequence 1012 that is substantially complementary to a second portion 1013 of the analyte and a capture probe capture domain 1014.

[0218] (i) First probe

[0219] In some embodiments where the method comprises spanning probes, the first probe comprises a sequence that is substantially complementary to the first portion of the analyte. In some embodiments, the sequence of the first probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the first portion of the analyte. In some embodiments, the sequence of the first probe that is substantially complementary to the first portion of the analyte may comprise a sequence of about 5 nucleotides to about 50 nucleotides or any sub-range as described herein. In some embodiments, the first probe comprises a functional sequence. In some embodiments, the functional sequence is a primer sequence.

[0220] In some embodiments where the method comprises spanning probes, the first probe comprises at least two ribonucleobases at the 3' end. In some embodiments, the first probe comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten ribonucleobases at the 3' end.

[0221] In some embodiments where the method comprises a spanning probe, the first probe comprises, from 5' to 3': a functional sequence, a sequence substantially complementary to a first portion of the analyte, and two or more ribonucleobases.

[0222] In some embodiments where the method comprises crossing probes, the first probe comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the first probe comprises deoxyribonucleotides. In some embodiments, the first probe comprises deoxyribonucleotides and ribonucleotides. In some embodiments where the first probe comprises deoxyribonucleotides, the hybridization of the first probe and the mRNA molecule causes DNA: RNA hybrid. In some embodiments, the first probe only comprises deoxyribonucleotides and produces DNA: RNA hybrid after the first probe and the mRNA molecule hybridize.

[0223] (ii) Spanning probes

[0224] In some embodiments of the method comprising a spanning probe, the spanning probe comprises a first sequence that is substantially complementary to a first target sequence of the analyte and a second sequence that is substantially complementary to a second target sequence of the analyte. In some embodiments, the spanning probe comprises a first sequence that is substantially complementary to the first target sequence of the analyte, a functional sequence, and a second sequence that is substantially complementary to the second target sequence of the analyte. In some embodiments, the spanning probe comprises, from 5′ to 3′, a first sequence, a functional sequence, and a second sequence. In some embodiments, the functional sequence is a linker sequence. The linker sequence may comprise a total of about 10 nucleotides to about 100 nucleotides, or any number within the subranges described herein.

[0225] In some embodiments, the functional sequence includes a barcode sequence. In some embodiments, the spanning probe can include a linker and a barcode sequence. In this case, the linker sequence can be located on both sides of the barcode, the barcode can be at the 5' end of the linker sequence, or the barcode can be at the 3' end of the linker sequence. In some embodiments, the barcode sequence is flanked by a 5' linker sequence (e.g., any exemplary linker sequence described herein) and a 3' linker sequence (e.g., any exemplary linker sequence described herein).

[0226] In some embodiments, a spanning probe from 5' to 3' includes: a first sequence, a 5' adapter sequence, a barcode, a 3' adapter sequence, and a second sequence.

[0227] In some embodiments, a spanning probe includes a sequence of about 10 nucleotides to about 300 nucleotides, or any subrange described herein.

[0228] In some embodiments, the first sequence of the spanning probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the first target sequence of the analyte. In some embodiments, the second sequence of the spanning probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the second target sequence of the analyte.

[0229] In some embodiments, the first sequence spanning the probe and the second sequence spanning the probe are substantially complementary to sequences within the same exon.

[0230] In some embodiments, the first target sequence of the analyte and the second target sequence of the analyte are located in the same exon. In this case, the first target sequence and the second target sequence are not directly adjacent.

[0231] In some embodiments, the first sequence spanning the probe and the second sequence spanning the probe are substantially complementary to sequences within different exons of the same gene. In some embodiments, the first target sequence of the analyte and the second target sequence of the analyte are located in different exons of the same gene.

[0232] In some embodiments, the spanning probe comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the spanning probe comprises deoxyribonucleotides. In some embodiments, the spanning probe comprises deoxyribonucleotides and ribonucleotides. In some embodiments where the spanning probe comprises deoxyribonucleotides, hybridization of the spanning probe with the mRNA molecule produces a DNA: RNA hybrid. In some embodiments, the spanning probe comprises only deoxyribonucleotides and produces a DNA: RNA hybrid after hybridization of the spanning probe with the mRNA molecule.

[0233] (iii) Second probe

[0234] In some embodiments where the method comprises a spanning probe, the second probe comprises a sequence substantially complementary to a second portion of the analyte and a capture probe capture domain (eg, any of the exemplary capture probe capture domains described herein).

[0235] In some embodiments where the method comprises a spanning probe, the sequence of the second probe is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the second portion of the analyte. In some embodiments, the sequence of the second probe that is substantially complementary to the second portion of the analyte can comprise a sequence of about 5 nucleotides to about 50 nucleotides, or any subrange described herein.

[0236] In some embodiments where the method comprises a spanning probe, a first portion of the analyte is directly adjacent to a first target sequence, and / or a second portion of the analyte is directly adjacent to a second target sequence. In this case, the sequence of the first probe is linked to the first sequence of the spanning probe, and the sequence of the second probe is linked to the second sequence of the spanning probe. In some embodiments, the spanning probe comprises at least two ribonucleic acids at its 3′ end, the first probe comprises at least two ribonucleic acids at its 3′ end, or both. In some embodiments, the spanning probe comprises a phosphorylated nucleotide at its 5′ end, the second probe comprises a phosphorylated nucleotide at its 5′ end, or both.

[0237] In some embodiments where the method comprises crossing probes, the second probe comprises ribonucleotides, deoxyribonucleotides and / or synthetic nucleotides that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the second probe comprises deoxyribonucleotides. In some embodiments, the second probe comprises deoxyribonucleotides and ribonucleotides. In some embodiments where the second probe comprises deoxyribonucleotides, hybridization of the second probe with the mRNA molecule causes DNA: RNA hybridization. In some embodiments, the second probe only comprises deoxyribonucleotides and produces DNA: RNA hybrid after the second probe hybridizes with the mRNA molecule.

[0238] (iv) A probe combination comprising a first probe, a second probe, and a multi-spanning probe

[0239] Also provided herein is a method for identifying the position of an analyte in a biological sample, wherein the method comprises a first probe, at least two spanning probes and a second probe. Using two or more spanning probes can achieve greater flexibility when designing an RTL probe, primarily by increasing the sequence that can be used as an optional target sequence in the analyte. In some cases, using two or more spanning probes can also be used to query variants (e.g., splice variants) that span a greater distance that can be queried using a spanning probe.

[0240] A non-limiting example of a method for identifying the location of an analyte in a biological sample, wherein the method includes a first probe, two or more spanning probes, and a second probe, comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein the capture probes in the plurality of capture probes include a capture domain and a spatial barcode; (b) contacting the biological sample with a first probe, a second probe, and two spanning probes, wherein the first probe is substantially complementary to a first portion of the analyte, wherein the second probe is substantially complementary to a second portion of the analyte and further includes a capture probe binding domain, and wherein the first spanning probe includes: (i) a first sequence substantially complementary to a first target sequence of the analyte, and (ii) a second sequence substantially complementary to a second target sequence of the analyte; and The two spanning probes include (i) a third sequence that is substantially complementary to a third target sequence of the analyte, and (ii) a fourth sequence that is substantially complementary to a fourth target sequence of the analyte; (c) hybridizing the first probe, the second probe and the spanning probe to the analyte; (d) connecting the first probe, one or more spanning probes and the second probe to produce a ligation product that is substantially complementary to the analyte; (e) releasing the ligation product from the analyte; (f) hybridizing the capture probe binding domain to the capture domain; and (g) determining (i) all or part of the sequence of the ligation product specifically bound to the capture domain or its complementary sequence, and (ii) all or part of the sequence of the spatial barcode or its complementary sequence, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0241] In some embodiments, the method comprising one or more spanning probes comprises at least two, at least three, at least four, at least five, or more spanning probes. In this case, the one or more spanning probes comprise (i) a third sequence that is substantially complementary to a third target sequence of the analyte, and (ii) a fourth sequence that is substantially complementary to a fourth target sequence of the analyte.

[0242] In some embodiments where the method comprises two (or more) spanning probes, the first target sequence is located in the first exon, the second target sequence is located in the second exon, and the third target sequence and the fourth target sequence are located in the third exon. In some embodiments where the method comprises two (or more) spanning probes, the first target sequence is located in the first exon, the second target sequence is located in the second exon, and the third target sequence is located in the third exon, and the fourth target sequence is located in the fourth exon. In some embodiments where the method comprises two (or more) spanning probes, the first target sequence and the second target sequence are located in the first exon, and the third target sequence and the fourth target sequence are located in the second exon. In some embodiments where the method comprises two (or more) spanning probes, the first target sequence and the second target sequence are located in the first exon, and the third target sequence is located in the second exon, and the fourth target sequence is located in the third exon.

[0243] In some embodiments, wherein the method comprises two (or more) spanning probes, the method comprises: ligating a first probe to a spanning probe, ligating the spanning probe to one or more other spanning probes, and ligating the one or more other spanning probe spanning oligonucleotides to a second probe, thereby generating a ligation product comprising one or more sequences substantially complementary to the analyte. In some embodiments, wherein the method comprises two (or more) spanning probes, the method comprises: ligating a first probe to one or more other spanning probes, ligating the one or more other spanning probes to the spanning probe, and ligating the spanning probe to a second probe, thereby generating a ligation product comprising one or more sequences substantially complementary to the analyte.

[0244] In some embodiments, each other spanning probe may include a functional sequence (e.g., any functional sequence described herein). For example, each other spanning probe may include a linker sequence (e.g., any exemplary linker sequence described herein). In another example, each other spanning probe may include a barcode sequence (e.g., any exemplary barcode sequence described herein) and a linker sequence (e.g., any linker sequence described herein). In some embodiments where the other spanning probe includes a barcode and a linker, the linker sequence may be flanked by the barcode, the barcode may be 5′ of the linker sequence, or the barcode may be 3′ of the linker sequence. In some embodiments, the barcode sequence is flanked by a 5′ linker sequence (e.g., any exemplary linker sequence described herein) and a 3′ linker sequence (e.g., any exemplary linker sequence described herein). In some embodiments, the other spanning probe may include, from 5′ to 3′, a first sequence, a 5′ linker sequence, a barcode, a 3′ linker sequence, and a second sequence.

[0245] (d) Prehybridization method

[0246] (i) Imaging and staining

[0247] Before adding probe, in some cases, multiple staining agents and staining techniques can be used to dye the biological sample. In some cases, the biological sample is a section (for example, 10 μm section) on a slide. In some cases, the biological sample is dried after being placed on a slide. In some cases, the biological sample is dried at 42 ° C. In some cases, drying occurs for about 1 hour, about 2 hours, about 3 hours, or until the section becomes transparent. In some cases, the biological sample can be dried overnight (for example, in a desiccant at room temperature).

[0248] In some embodiments, any number of biological stains can be used to stain the sample, including but not limited to acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hodgkin's stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some cases, the methods disclosed herein include imaging the biological sample. In some cases, sample imaging occurs before deamination of the biological sample. In some cases, the sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and / or PAS staining techniques. PAS staining is typically performed after formaldehyde or acetone fixation. In some cases, the stain is H&E stain.

[0249] In some embodiments, a biological sample can be stained using a detectable marker as described elsewhere herein (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes). In some embodiments, only one type of staining agent or a technique is used to stain the biological sample. In some embodiments, staining includes biological staining techniques, such as H&E staining. In some embodiments, staining includes using fluorescently coupled antibodies to identify analytes. In some embodiments, two or more different types of staining agents or two or more different staining techniques are used to stain the biological sample. For example, a biological sample can be prepared by staining and imaging using a technique (e.g., H&E staining and bright field imaging) and then staining and imaging the same biological sample using another technique (e.g., IHC / IF staining and fluorescence microscopy).

[0250] In some embodiments, biological sample can be decoloured.The decontamination or decolorization method of biological sample are known in the art, and usually depend on the character of the staining agent being applied to sample.For example, H&E staining can be by washing sample in HCl or any other acid and decolouring (for example, selenic acid, sulfuric acid, hydroiodic acid, benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid, succinic acid, salicylic acid, tartaric acid, sulfurous acid, trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid, orthophosphoric acid, arsenic acid, selenious acid, chromic acid, citric acid, hydrofluoric acid, nitrous acid, isocyanic acid, formic acid, hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonic acid, hydrogen sulfide or its combination).In some embodiments, decolouring can be included in 1,2,3,4,5 or more washings in acid (for example HCl).In some embodiments, decolouring can comprise HCl being added in downstream solution (for example permeabilization solution).In some embodiments, decolouring can comprise dissolving the enzyme (for example pepsin) used in the disclosed method in acid (for example HCl) solution. In some embodiments, after decolorizing hematoxylin with acid, other reagents can be added to the decolorizing solution to increase the pH for other applications. For example, SDS can be added to the acid decolorizing solution to increase the pH compared to the acid decolorizing solution alone. As another example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. These stains can be removed using techniques such as cleavage of disulfide bonds by washing with reducing agents and detergents, treatment with hygrotropic salts, treatment with antigen retrieval solutions, and treatment with acidic glycine buffer. For example, methods for multiple staining and destaining are described in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6: 8390, Pirici et al., J. Histochem. Cytochem. 2009; 57: 567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57: 899-905, the entire contents of which are incorporated herein by reference.

[0251] In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) can be performed as part or additional parts of the exemplary spatial workflow presented herein. For example, tissue sections can be fixed according to the methods described herein. Biological samples can be transferred to arrays (for example, capture probe arrays), wherein analytes (for example, proteins) are detected using immunofluorescence protocols. For example, samples can be rehydrated, sealed and permeabilized (3XSSC, 2% BSA, 0.1% Triton X, 1U / μl RNAse inhibitors, 4°C for 10 minutes), followed by staining with a fluorescent primary antibody (1: 100 in 3XSSC, 2% BSA, 0.1% Triton X, 1U / μl RNAse inhibitors, 4°C for 30 minutes). Biological samples can be washed, covered with a glass slide (in glycerol+1U / μl RNAse inhibitor), imaged (for example, using a confocal microscope or other device capable of fluorescence detection), washed and processed (according to analyte capture or spatial workflow as described herein).

[0252] In some cases, a glycerol solution and a coverslip can be added to the sample. In some cases, the glycerol solution can include a counterstain (e.g., DAPI).

[0253] As used herein, antigen retrieval buffer can improve antibody capture in IF / IHC protocols. An exemplary protocol for antigen retrieval can be preheating the antigen retrieval buffer (e.g., to 95° C.), immersing the biological sample in the heated antigen retrieval buffer for a predetermined time, then removing the biological sample from the antigen retrieval buffer and washing the biological sample.

[0254] In some embodiments, optimizing permeabilization may be useful for identifying intracellular analytes. Permeabilization optimization can include selecting a permeabilizing agent, the concentration of the permeabilizing agent, and the duration of permeabilization. Tissue permeabilization is discussed elsewhere herein.

[0255] In some embodiments, blocking arrays and / or biological samples during preparation of labeling biological samples can reduce the non-specific binding of antibodies to arrays and / or biological samples (reduce background). Some embodiments provide blocking buffer / blocking solution that can be applied before and / or during the application of a marker, wherein the blocking buffer can include a blocking agent and an optional surfactant and / or saline solution. In some embodiments, the blocking agent can be bovine serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk (e.g., skim milk powder), casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polyvinyl pyrrolidone (PVP), biotin blocking agent, peroxidase blocking agent, levamisole, Carnoy's solution, glycine, lysine, sodium borohydride, pontamine sky blue, Sudan black, trypan blue, FITC blocking agent and / or acetic acid. The blocking buffer / blocking solution can be applied to the array and / or the biological sample prior to and / or during labeling of the biological sample (eg, using an antibody conjugated to a fluorophore).

[0256] (ii) Sample preparation for probe application

[0257] In some cases, biological sample is dewaxed. Dewaxing can be achieved using any method known in the art. For example, in some cases, biological sample is processed with a series of washing solutions including xylene and ethanol of various concentrations. In some cases, dewaxing method includes processing xylene (for example, washing 3 times for 5 minutes each time). In some cases, the method also includes treating with ethanol (for example, 100% ethanol, washing twice for each time, 10 minutes each time; 95% ethanol, washing twice for each time, 10 minutes each time; 70% ethanol, washing twice for each time, 10 minutes each time; 50% ethanol, washing twice for each time, 10 minutes each time). In some cases, after the ethanol wash, the biological sample can be washed with deionized water (for example, washing twice for each time, 5 minutes each time). It will be appreciated that those skilled in the art can adjust these methods to optimize dewaxing.

[0258] In some cases, the biological sample is decrosslinked. In some cases, the biological sample is decrosslinked in a solution containing TE buffer (including Tris and EDTA). In some cases, the TE buffer is alkaline (e.g., at a pH of about 9). In some cases, decrosslinking occurs at about 50°C to about 80°C. In some cases, decrosslinking occurs at about 70°C. In some cases, decrosslinking occurs at 70°C for about 1 hour. Just before decrosslinking, the biological sample can be treated with acid (e.g., 0.1M HCl, about 1 minute). After the decrosslinking step, the biological sample can be washed (e.g., with 1x PBST).

[0259] In some cases, the method for preparing the biological sample for probe application includes permeabilization to the sample. In some cases, phosphate buffer is used to permeabilize the biological sample. In some cases, phosphate buffer is PBS (e.g., 1x PBS). In some cases, phosphate buffer is PBST (e.g., 1x PBST). In some cases, the permeabilization step is carried out repeatedly (e.g., 3 times for each 5 minutes).

[0260] In some cases, the method of preparing a biological sample for probe application includes the steps of equilibrating and blocking the biological sample. In some cases, equilibration is performed using a pre-hybridization (pre-Hyb) buffer. In some cases, the pre-Hyb buffer is RNase-free. In some cases, the pre-Hyb buffer is free of bovine serum albumin (BSA), Denhardt's solution, or other biological materials that may be contaminated by nucleases.

[0261] In some cases, the equilibration step is performed multiple times (e.g., 2 times, 5 minutes each; 3 times, 5 minutes each). In some cases, the biological sample is blocked with a blocking buffer. In some cases, the blocking buffer includes a carrier (e.g., tRNA), such as yeast tRNA from Saccharomyces cerevisiae (e.g., at a final concentration of 10-20 μg / mL). In some cases, the blocking can be performed for 5, 10, 15, 20, 25, or 30 minutes.

[0262] Any of the aforementioned steps can be optimized for performance. For example, the temperature can be changed. In some cases, the prehybridization method is carried out at room temperature. In some cases, the prehybridization method is carried out at 4°C (in some cases, changing the time range provided herein).

[0263] (e) Hybridization probe

[0264] In some embodiments, the method for being captured by target RNA provided herein includes hybridizing a first probe oligonucleotide and a second probe oligonucleotide (e.g., a probe pair). In some cases, each of the first and second probe oligonucleotides includes a sequence that is substantially complementary to one or more sequences (e.g., one or more target sequences) of an analyte of interest. In some embodiments, the first probe and the second probe are completely adjacent to each other (i.e., there is no nucleotide gap) or are combined with complementary sequences on the same transcript.

[0265] In some cases, the method includes the hybridization of probe groups, wherein the probe is about 1 to about 100nM to the concentration in culture medium. In some cases, the concentration of probe is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500nM. In some cases, the concentration of probe is 5nM. In some cases, the probe is diluted in hybridization (Hyb) buffer. In some cases, the concentration of probe is 5nM in Hyb buffer.

[0266] In some cases, probe hybridization occurs at about 50° C. In some cases, the temperature range for probe hybridization is about 30° C. to about 75° C., about 35° C. to about 70° C., or about 40° C. to about 65° C. In some embodiments, the temperature is about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., or about 70° C. In some cases, probe hybridization occurs for about 30 minutes, about 1 hour, about 2 hours, about 2.5 hours, about 3 hours, or more. In some cases, probe hybridization occurs at 50°C for about 2.5 hours.

[0267] In some cases, the hybridization buffer includes SSC (e.g., 1xSSC) or SSPE. In some cases, the hybridization buffer includes formamide or ethylene carbonate. In some cases, the hybridization buffer includes one or more salts, such as Mg salts such as MgCl2, Na salts such as NaCl, Mn salts such as MnCl2. In some cases, the hybridization buffer includes Denhardt's solution, dextran sulfate, polysucrose, PEG or other hybridization rate accelerators. In some cases, the hybridization buffer includes a carrier, such as yeast tRNA, salmon sperm DNA and / or lambda phage DNA. In some cases, the hybridization buffer includes one or more blockers. In some cases, the hybridization buffer includes an RNase inhibitor. In some cases, the hybridization buffer may include BSA, sequence-specific blockers, non-specific blockers, EDTA, RNase inhibitors, betaine, TMAC or DMSO. In some cases, the hybridization buffer may also include detergents, such as Tween, Triton-X100, Sarkosyl and SDS. In some cases, the hybridization buffer includes nuclease-free water, DEPC water.

[0268] In some embodiments, the first probe oligonucleotide and the second probe oligonucleotide bind to complementary sequences that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides away from each other. In some embodiments, when the first and second probe oligonucleotides are separated from one another by one or more nucleotides, nucleotides are connected between the first and second probe oligonucleotides. In some embodiments, when the first and second probe oligonucleotides are separated from one another by one or more nucleotides, deoxyribonucleotides are connected between the first and second probe oligonucleotides.

[0269] In some cases, after hybridization, the biological sample is washed with a post-hybridization wash buffer. In some cases, the post-hybridization wash buffer comprises one or more of SSC, yeast tRNA, formamide, ethylene carbonate, and nuclease-free water.

[0270] Additional embodiments regarding probe hybridization are further provided.

[0271] (i) Hybridization temperature

[0272] In some embodiments, the method utilizes oligonucleotides that include deoxyribonucleic acid (rather than strictly utilizing ribonucleotides) at the attachment site. Using deoxyribonucleic acid in the methods described herein produces more uniform efficiency, which can be easily controlled and flexible for various applications.

[0273] In one non-limiting example, the methods disclosed herein include contacting a biological sample with a plurality of oligonucleotides (e.g., probes) comprising a first oligonucleotide (e.g., a first probe) and a second oligonucleotide (e.g., a second probe), wherein the first oligonucleotide (e.g., the first probe) and the second oligonucleotide (e.g., the second probe) are complementary to a first sequence present in the analyte and a second sequence present in the analyte, respectively; hybridizing the first oligonucleotide (e.g., the first probe) and the second oligonucleotide (e.g., the second probe) to the analyte at a first temperature; hybridizing the first oligonucleotide (e.g., the first probe) and the second oligonucleotide (e.g., the second probe) to a third oligonucleotide (e.g., a splint oligonucleotide) at a second temperature, such that the first oligonucleotide (e.g., the first probe) and the second oligonucleotide (e.g., the second probe) are complementary to each other. , second probe) adjacent to each other; connecting a first oligonucleotide (e.g., the first probe) to a second oligonucleotide (e.g., the second probe) to produce a ligation product; contacting the biological sample with a substrate, wherein the capture probe is immobilized on the substrate, wherein the capture probe includes a spatial barcode and a capture domain; allowing the ligation product to specifically bind to the capture domain; and determining (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the position of the analyte in the biological sample; wherein the first oligonucleotide (e.g., the first probe), the second oligonucleotide (e.g., the second probe) and the third oligonucleotide are DNA oligonucleotides, and wherein the first temperature is a higher temperature than the second temperature.

[0274] A non-limiting example of the method is shown in Figure 11 A biological sample including an analyte 1101 is contacted with a first probe 1102 and a second probe 1103. The first probe 1102 and the second probe 1103 hybridize to the analyte at a first target sequence 1104 and a second target sequence 1105, respectively. The first probe and the second probe include free ends 1107-1110. Figure 11As shown, the first and second target sequences are not directly adjacent in the analyte. After hybridization, the unbound first and second probes are washed away. Then, the third oligonucleotide 1106 hybridizes with the first and second probes at 1108 and 1109, respectively. After hybridization, the first probe is extended 1112 and a connection product comprising the first probe sequence and the second probe sequence is produced. Alternatively, as an alternative to extending the first probe, the third oligonucleotide is used to "bind" the first probe and the second probe together. In this case, the first probe and the second probe bound together by the third oligonucleotide can be referred to as a connection product. The connection product is then contacted with a substrate 1111, and the connection product is bound to the capture probe 1113 of the substrate 1111 at different spatial positions on the array. In some embodiments, the biological sample is contacted with the substrate 1111 before contacting the first probe and the second probe.

[0275] In some embodiments, the first oligonucleotide (e.g., a first probe) and the second oligonucleotide (e.g., a second probe) hybridize to the analyte at a first temperature. In some embodiments, the first temperature ranges from about 50°C to about 75°C, about 55°C to about 70°C, or about 60°C to about 65°C. In some embodiments, the first temperature is about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C.

[0276] In some embodiments, after the step of hybridizing the first oligonucleotide (e.g., first probe) and the second oligonucleotide (e.g., second probe) to the analyte, a washing step is performed to remove unbound oligonucleotides (e.g., probes). The washing step can be performed using any washing method and solution described herein.

[0277] In some embodiments, after the step of hybridizing the first oligonucleotide (e.g., first probe) and the second oligonucleotide (e.g., second probe) to the analyte, a third oligonucleotide (e.g., splint oligonucleotide) is added to the analyte. In some embodiments, the third oligonucleotide is an oligonucleotide. In some embodiments, the third oligonucleotide is a DNA oligonucleotide.

[0278] In some embodiments, the third oligonucleotide comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a portion of the first probe oligonucleotide (e.g., the portion of the first probe that is not hybridized to the analyte (e.g., the helper sequence). In some embodiments, the third oligonucleotide comprises a sequence that is 100% complementary to a portion of the first oligonucleotide (e.g., the first probe). In some embodiments, the third oligonucleotide comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a portion of the second probe oligonucleotide (e.g., the portion of the second probe that is not hybridized to the analyte (e.g., the helper sequence). In some embodiments, the 3rd oligonucleotide comprises a sequence that is 100% complementary to a portion of the second oligonucleotide (e.g., the second probe). In some embodiments, the 3rd oligonucleotide hybridizes with the first oligonucleotide (e.g., the first probe) at the complementary portion. In some embodiments, the 3rd oligonucleotide hybridizes with the second oligonucleotide (e.g., the second probe) at the complementary portion.

[0279] In some embodiments, the 3rd oligonucleotide hybridizes with the first oligonucleotide (e.g., the first probe) and the second oligonucleotide (e.g., the second probe) at the second temperature. In some embodiments, the second temperature is lower than the first temperature of the first and second oligonucleotides (e.g., the first and second probes) in conjunction with the analyte. In some embodiments, the second temperature is in the range of about 15°C to about 35°C, about 20°C to about 30°C or about 25°C to about 30°C. In some embodiments, the first temperature is about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C or about 35°C. Including the third, or the method for splint oligonucleotides has been described in U.S. Patent No. 2019 / 0055594A1, the entire contents of which are incorporated herein by reference.

[0280] In some embodiments, after the step of the 3rd oligonucleotide and analyte hybridization, washing steps are carried out to remove unbound 3rd oligonucleotide.Washing steps can be carried out using any washing method and solution as described herein.In some embodiments, after washing steps, the first and second oligonucleotide (for example, the first and second probes) are bound to (for example, hybridize to) analyte, and the 3rd oligonucleotide is bound to (for example, hybridize to) the first and second oligonucleotide (for example, at the part that the first and second probes are not combined with analyte).

[0281] In some embodiments, the first oligonucleotide (e.g., the first probe), the second oligonucleotide (e.g., the second probe) and the third oligonucleotide are added to the biological sample simultaneously. Then, in some embodiments, the temperature is adjusted to a first temperature to allow the first oligonucleotide (e.g., the first probe) and the second oligonucleotide (e.g., the second probe) to hybridize with the analyte in the biological sample. Then, the temperature is adjusted to a second temperature to allow the third oligonucleotide to hybridize with the first oligonucleotide and the second oligonucleotide.

[0282] In some embodiments where the third oligonucleotide hybridizes to the first and second probes, wherein the first and second probes hybridize to target sequences that are not directly adjacent in the analyte, the third oligonucleotide is extended to fill the gap between the first and second probes. In some cases, a polymerase (e.g., a DNA polymerase) can extend one of the probes (e.g., the first probe) prior to ligation. For example, Figure 11 As shown, the first probe 1102 extends 1112 to fill a gap 1114 between the first probe 1102 and the second probe 1103 .

[0283] In some embodiments, carry out the connection step.Can use any method as described herein to connect.In some embodiments, described step comprises connecting the first oligonucleotide (for example, the first probe) and the second oligonucleotide (for example, the second probe), forms connection product.In some embodiments, the third oligonucleotide is used as oligonucleotide splint to promote the connection of the first oligonucleotide (for example, the first probe) and the second oligonucleotide (for example, the second probe).In some embodiments, connection is chemical connection.In some embodiments, connection is enzymatic connection.In some embodiments, ligase is T4RNA ligase (Rn12), splintR ligase, single-stranded DNA ligase or T4 DNA ligase.

[0284] (ii) Hybridization buffer

[0285] In some embodiments, the first probe and the second probe are hybridized with the analyte in a hybridization buffer. In some cases, the hybridization buffer contains formamide. In other cases, the hybridization buffer does not contain formamide. Formamide is unfriendly to humans and is known to have health hazards. Chemically, it oxidizes over time, thereby affecting the shelf life of reagents and, most importantly, the efficacy of reagents. Therefore, the methods described herein may include a buffer that does not contain formamide, including a hybridization buffer that does not contain formamide.

[0286] In some embodiments, the hybridization buffer that does not contain formamide is saline-sodium citrate (SSC) hybridization buffer.In some embodiments, SSC is present in SSC hybridization buffer with about 1xSSC to about 6xSSC (for example, about 1xSSC to about 5xSSC, about 1xSSC to about 4xSSC, about 1xSSC to about 3xSSC, about 1xSSC to about 2xSSC, about 2xSSC to about 6xSSC, about 2xSSC to about 5xSSC, about 2xSSC to about 4xSSC, about 2xSSC to about 3xSSC, about 3xSSC to about 5xSSC, about 3xSSC to about 4xSSC, about 4xSSC to about 6xSSC, about 4xSSC to about 5xSSC, or about 5xSSC to about 6xSSC).In some embodiments, SSC is present in SSC hybridization buffer with about 2xSSC to about 4xSSC.In some embodiments, SSPE hybridization buffer can be used.

[0287] In some embodiments, the SSC hybridization buffer comprises a solvent. In some embodiments, the solvent comprises ethylene carbonate instead of formamide (2020, Kalinka et al., Scientia Agricola 78(4): e20190315). In some embodiments, the ethylene carbonate is present in the SSC hybridization buffer at about 10% (w / v) to about 25% (w / v) (e.g., about 10% (w / v) to about 20% (w / v)), about 10% (w / v) to about 15% (w / v), about 15% (w / v) to about 25% (w / v), about 15% (w / v) to about 20% (w / v), or about 20% (w / v) to about 25% (w / v)). In some embodiments, the ethylene carbonate is present in the SSC hybridization buffer at about 15% (w / v) to about 20% (w / v). In some embodiments, the ethylene carbonate is present in the SSC hybridization buffer at about 10% (w / v), about 11% (w / v), about 12% (w / v), about 13% (w / v), about 13% (w / v), about 14% (w / v), about 15% (w / v), about 16% (w / v), about 17% (w / v), about 18% (w / v), about 19% (w / v), about 20% (w / v), about 21% (w / v), about 22% (w / v), about 23% (w / v), about 24% (w / v), or about 25% (w / v). In some embodiments, the ethylene carbonate is present in the SSC hybridization buffer at about 13% (w / v).

[0288] In some embodiments, the SSC hybridization buffer is at a temperature of about 40°C to about 60°C (e.g., about 40°C to about 55°C, about 40°C to about 50°C, about 40°C to about 40°C about 45°C, about 45°C to about 60°C, about 45°C to about 55°C, about 45°C to about 50°C, about 50°C to about 60°C, about 50°C to about 50°C about 55°C, or about 55°C to about 60°C). In some embodiments, the SSC hybridization buffer is at a temperature of about 45°C to about 55°C, or any subrange described herein. In some embodiments, the temperature of the SSC hybridization buffer is about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., or about 60° C. In some embodiments, the temperature of the SSC hybridization buffer is about 50° C.

[0289] In some embodiments, the SSC hybridization buffer further comprises one or more of a carrier, a crowder, or an additive. Non-limiting examples of carriers that may be included in the hybridization buffer include yeast tRNA, salmon sperm DNA, lambda phage DNA, glycogen, and cholesterol. Non-limiting examples of molecular aggregates that may be included in the hybridization buffer include Ficoll, dextran, Denhardt's solution, and PEG. Non-limiting examples of additives that may be included in the hybridization buffer include binding blockers, RNase inhibitors, Tm regulators, and adjuvants for relaxing secondary nucleic acid structures (e.g., betaine, TMAC, and DMSO). In addition, the hybridization buffer may include detergents such as SDS, Tween, Triton-X100, and sarkosyl (e.g., N-lauroylsarcosine sodium salt). Those skilled in the art will appreciate that the buffer for nucleic acid hybridization may include many different compounds that can enhance hybridization reactions.

[0290] (f) Washing

[0291] In some embodiments, the method disclosed herein further comprises a washing step. The washing step removes any unbound probe. The washing step can be performed between any steps in the method disclosed herein. For example, a washing step can be performed after adding the probe to the biological sample. Thus, the free / unbound probe is washed away, leaving only the probe hybridized with the analyte. In some cases, multiple (i.e., at least 2, 3, 4, 5 or more) washing steps occur between the methods disclosed herein. The washing step can be performed at various times (e.g., 1, 2, 3, 4 or 5 minutes) and temperatures (e.g., room temperature; 4°C as known in the art and determined by those skilled in the art).

[0292] In some cases, washing steps are performed using a wash buffer. In some cases, a wash buffer comprises SSC (e.g., 1xSSC). In some cases, a wash buffer comprises PBS (e.g., 1xPBS). In some cases, a wash buffer comprises PBST (e.g., 1xPBST). In some cases, a wash buffer may also comprise formamide or contain no formamide.

[0293] Additional embodiments regarding washing steps are provided herein.

[0294] (i) Wash buffer without formamide

[0295] In some embodiments, after connecting the first probe and the second probe, one or more unhybridized first probes, one or more unhybridized second probes or both are removed from the array. In some embodiments, after connecting the first probe, one or more spanning probes and the second probe, one or more unhybridized first, second and / or spanning probes are removed from the array. In some embodiments, after connecting the first probe, the second probe and the third oligonucleotide, one or more unhybridized first probes, one or more unhybridized second probes or one or more third oligonucleotides or all of the above are removed from the array.

[0296] In some embodiments, a prehybridization buffer is used to wash the sample. In some embodiments, a phosphate buffer is used. In some embodiments, multiple washing steps are performed to remove unbound oligonucleotides.

[0297] In some embodiments, removing comprises washing one or more unhybridized probes (eg, the first probe, the second probe, the spanning probe, the additional spanning probes, and the third oligonucleotide) from the array in a wash buffer that does not contain formamide.

[0298] In some embodiments, the formamide-free wash buffer is an SSC wash buffer. In some embodiments, the SSC is present in the SSC wash buffer at about 0.01xSSC to about 1xSSC (e.g., about 0.01x SSC to about 0.5x SSC, 0.01x SSC to about 0.1xSSC, about 0.01x SSC to about 0.05x SSC, about 0.05x SSC to about 1x SSC, about 0.05x SSC to about 0.5x SSC, about 0.05x SSC to about 0.1x SSC, about 0.1x SSC to about 1x SSC, about 0.1x SSC to about 0.5x SSC, or about 0.5xSSC to about 1x SSC). In some embodiments, SSC is present in the SSC wash buffer at about 0.01 x SSC, about 0.02 x SSC, about 0.03 x SSC, about 0.04 x SSC, about 0.05 x SSC, about 0.06 x SSC, about 0.07 x SSC, about 0.08 x SSC, about 0.09 x SSC, about 0.1 x SSC, about 0.2 x SSC, about 0.3 x SSC, about 0.4 x SSC, about 0.5 x SSC, about 0.6 x SSC, about 0.7 x SSC, about 0.8 x SSC, about 0.9 x SSC, or about 0.1 x SSC. In some embodiments, SSC is present in the SSC wash buffer at about 0.1 x SSC.

[0299] In some embodiments, the SSC wash buffer comprises a detergent. In some embodiments, the detergent comprises sodium dodecyl sulfate (SDS). In some embodiments, SDS is present at about 0.01% (v / v) to about 0.5% (v / v) (e.g., about 0.01% (v / v) to about 0.4% (v / v), about 0.01% (v / v) to about 0.3% (v / v), about 0.01% (v / v) to about 0.2% (v / v), about 0.01% (v / v) to about 0.1% (v / v), about 0.05% (v / v) to about 0.5% (v / v), about 0.05% (v / v) to about 0.4% (v / v), about 0.05% (v / v) to about 0.3% (v / v), about 0.05% (v / v) to about 0.2% (v / v), about 0.05% (v / v) to about 0.1% (v / v), about 0.1% (v / v) to about 0.5% (v / v), about 0.1% (v / v) to about 0.4% (v / v), about 0.1% (v / v) to about 0.3% (v / v), about 0.1% (v / v) to about 0.2% (v / v), about 0.2% (v / v) to about 0.5% (v / v), about 0.2% (v / v) to about 0.4% (v / v), about 0.2% (v / v) to about 0.3% (v / v), about 0.3% (v / v) to about 0.5% (v / v), about 0.3% (v / v) to about 0.4% (v / v), or about 0.4% (v / v) to about 0.5% (v / v)) in the SSC wash buffer. In some embodiments, SDS is present in the SSC wash buffer at about 0.01% (v / v), about 0.02% (v / v), about 0.03% (v / v), about 0.04% (v / v), about 0.05% (v / v), about 0.06% (v / v), about 0.07% (v / v), about 0.08% (v / v), about 0.09% (v / v), about 0.10% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), or about 0.5% (v / v). In some embodiments, SDS is present in the SSC hybridization buffer at about 0.1% (w / v). In some embodiments, sarkosyl may be present in the SSC wash buffer.

[0300] In some embodiments, the SSC wash buffer comprises a solvent. In some embodiments, the solvent comprises ethylene carbonate. In some embodiments, ethylene carbonate is present in the SSC wash buffer at about 10% (w / v) to about 25% (w / v) or any subrange described herein. In some embodiments, ethylene carbonate is present in the SSC wash buffer at about 15% (w / v) to about 20% (w / v). In some embodiments, ethylene carbonate is present in the SSC wash buffer at about 16% (w / v).

[0301] In some embodiments, the temperature of the SSC wash buffer is from about 50°C to about 70°C (e.g., from about 50°C to about 65°C, from about 50°C to about 60°C, from about 50°C to about 50°C about 55°C, from about 55°C to about 70°C, from about 55°C to about 65°C, from about 55°C to about 60°C, from about 60°C to about 70°C, from about 60°C to about 65°C, or from about 65°C to about 70°C). In some embodiments, the SSC wash buffer is at a temperature of from about 55°C to about 65°C. In some embodiments, the temperature of the SSC wash buffer is about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, or about 70°C. In some embodiments, the SSC wash buffer is at a temperature of about 60°C.

[0302] In some embodiments, the method includes releasing the ligation product, wherein releasing is performed after washing the array to remove one or more unhybridized first and second probes.

[0303] (g) Connection

[0304] In some embodiments, after the probe oligonucleotides (e.g., a first probe, a second probe, a spanning probe, additional spanning probes, and / or a third oligonucleotide) are hybridized to the analyte, the probes (e.g., a first probe, a second probe, a spanning probe, additional spanning probes, and / or a third oligonucleotide) can be ligated together to produce a single ligation product comprising one or more sequences complementary to the analyte. As described herein, ligation can be performed enzymatically or chemically.

[0305] In some cases, the ligation is an enzymatic ligation reaction using a ligase (e.g., T4 RNA ligase (Rn12), SplintR ligase, single-stranded DNA ligase, or T4 DNA ligase). For a description of KOD ligase, see, e.g., Zhang et al.; RNA Biol. 2017; 14(1): 36-44, which is incorporated herein by reference in its entirety. Following the enzymatic ligation reaction, the probes (e.g., the first probe, the second probe, the spanning probe, the additional spanning probes, and / or the third oligonucleotide) are considered to be ligated.

[0306] In some embodiments, the polymerase catalyzes the synthesis of the complementary strands of the ligation product to produce a double-stranded ligation product. In some cases, the polymerase is a DNA polymerase. In some embodiments, the polymerase has a 5' to 3' polymerase activity. In some embodiments, the polymerase has a 3' to 5' exonuclease activity for proofreading. In some embodiments, the polymerase has a 5' to 3' polymerase activity and a 3' to 5' exonuclease activity for proofreading.

[0307] In some embodiments, probe (for example, the first probe, the second probe, the spanning probe, other spanning probes and / or the third oligonucleotide) can each include a reaction portion so that when hybridized with the target and exposed to appropriate connection conditions, the probe oligonucleotides can be connected to each other. In some embodiments, the probe oligonucleotides comprising a reaction portion are chemically connected. For example, the first probe that can hybridize with the first target area (for example, the first target sequence or the first part) of nucleic acid molecules can include a first reaction portion, and the second probe oligonucleotide that can hybridize with the second target area (for example, the second target sequence or the second part) of nucleic acid molecules can include a second reaction portion. When the first and second probes hybridize with the first and second target areas (for example, the first and second target sequences) of nucleic acid molecules, the first and second reaction portions can be adjacent to each other. The reaction portion of the probe can be selected from the following non-restrictive group: azide, alkynes, nitrone (for example, 1,3-nitrone), stressed olefins (for example, trans-cyclic olefins, such as cyclooctene or oxybornadiene), tetrazine, tetrazole, iodide, thiosalts (for example phosphorothioate), acid, amine and phosphate. For example, the first reactive part of the first probe may include an azide moiety, and the second reactive part of the second probe may include an alkyne moiety. The first and second reactive parts can react to form a connecting portion. The reaction between the first and second reactive parts can be, for example, a cycloaddition reaction, such as a stress-promoted azide-alkyne cycloaddition, a copper-catalyzed azide-alkyne cycloaddition, a stress-promoted alkyne-nitrone cycloaddition, a Diels-Alder reaction, a [3+2] cycloaddition, a [4+2] cycloaddition, or a [4+1] cycloaddition; a thiol-ene reaction; a nucleophilic substitution (substation) reaction; or other reactions. In some cases, the reaction between the first and second reactive parts can produce a triazole moiety or an isoxazoline moiety. The reaction between the first and second reactive parts can include subjecting the reactive part to suitable conditions, such as suitable temperature, pH, or pressure, and providing one or more reagents or catalysts for the reaction. For example, the reaction between the first and second reactive parts can be catalyzed by a copper catalyst, a ruthenium catalyst, or a stressed substance (such as difluorooctyne, dibenzylcyclooctyne, or diarylazacyclooctyne). The reaction between the first reactive portion of the first probe that hybridizes to the first target region (e.g., the first target sequence or the first portion) of the nucleic acid molecule and the second reactive portion of the third probe oligonucleotide that hybridizes to the second target region (e.g., the first target sequence or the first portion) of the nucleic acid molecule can connect the first probe and the second probe to provide a connected probe. After connection, the first and second probes can be considered to be connected. Thus, the reaction of the first and second reactive portions can include a chemical connection reaction, such as a copper-catalyzed 5' azide to 3' alkyne "click" chemistry reaction to form a triazole bond between the two probe oligonucleotides.In other non-limiting examples, an iodide moiety can be chemically linked to a phosphorothioate moiety to form a phosphorothioate linkage, an acid can be linked to an amine to form an amide linkage, and / or a phosphate and an amine can be linked to form a phosphoramidate linkage.

[0308] Figure 12 A-12E illustrates examples of representative reactions. Figure 12 A shows the chemical ligation reaction where the alkyne moiety 1202 and the azide moiety 1204 react to form a triazole bond 1206 in a copper-mediated cycloaddition. Figure 12 B shows the chemical ligation reaction of phosphorothioate group 1208 and iodide group 1210 to form phosphorothioate bond 1212. Figure 12 C shows the chemical ligation reaction of acid 1214 and amine 1216 to form amide bond 1218. Figure 12 D shows the chemical ligation reaction of the phosphate moiety 1220 and the amine moiety 1222 to form the phosphoramidate bond 1224. Figure 12 E shows the coupling reaction of two species 1226 and 1228.

[0309] In some cases, ligation is performed in a ligation buffer. In the case of probe ligation on a probe containing a diribose, the ligation buffer can include T4 RNA ligase buffer 2, an enzyme (e.g., RNL2 ligase) and nuclease-free water. In the case of probe ligation on a DNA probe, the ligation buffer can include Tris-HCl pH 7.5, MnCl2, ATP, DTT, a substitute solution (e.g., glycerol), an enzyme (e.g., SplintR ligase) and nuclease-free water.

[0310] In some embodiments, the ligation buffer includes other reagents. In some cases, the ligation buffer includes adenosine triphosphate (ATP) added during the ligation reaction. The sealing catalyzed by the DNA ligase of the nicked DNA substrate is first activated by ATP hydrolysis, resulting in the covalent addition of the AMP group to the enzyme. After binding to the nick site in the DNA duplex, the ligase transfers AMP to the phosphorylated 5'-end of the nick, forming a 5'-5' pyrophosphate bond. Finally, the ligase catalyzes the attack of the OH group at the 3' end of the nick on the pyrophosphate bond, thereby sealing it, and then releasing the ligase and AMP. If the ligase separates from the substrate before the 3' attack, for example, due to premature AMP reloading of the enzyme, the 5'AMP remains at the 5' end, blocking further connection attempts. In some cases, ATP is added during the ligation reaction at a concentration of about 1 μM, about 10 μM, about 100 μM, about 1000 μM or about 10000 μM.

[0311] In some embodiments, a cofactor is added during the ligation process to aid in ligation of the probe oligonucleotides. In some cases, the cofactor includes magnesium ions (Mg 2+ In some cases, the cofactor includes a manganese ion (Mn 2+ ). In some cases, Mg 2+ In some cases, Mn 2+ In some cases, the concentration of MgCl2 is about 1 mM, about 10 mM, about 100 mM, or about 1000 mM. In some cases, the concentration of MnCl2 is about 1 mM, about 10 mM, about 100 mM, or about 1000 mM.

[0312] In some embodiments, the ligation product includes a capture probe capture domain that can bind to a capture probe (e.g., a capture probe directly or indirectly immobilized on a substrate). In some embodiments, the methods provided herein include contacting a biological sample with a substrate, wherein the capture probe is immobilized to the substrate (e.g., directly or indirectly immobilized to the substrate). In some embodiments, the capture probe capture domain of the ligation probe specifically binds to the capture domain.

[0313] In some cases, after ligation, the biological sample is washed with a post-ligation wash buffer. In some cases, the post-ligation wash buffer comprises one or more of SSC (e.g., 1xSSC), ethylene carbonate or formamide, and nuclease-free water. In some cases, the biological sample is washed at about 50°C to about 70°C at this stage. In some cases, the biological sample is washed at about 60°C.

[0314] (i) Ligation on a second probe comprising a pre-adenylated 5′ phosphate

[0315] Provided herein is a method for determining the position of a target nucleic acid in a biological sample, comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein the capture probes of the plurality of capture probes comprise a capture probe domain and a spatial barcode; (b) hybridizing the target nucleic acid in the biological sample with a first probe and a second probe, wherein the first probe comprises, from 3′ to 5′, a sequence substantially complementary to the capture domain, and a sequence substantially complementary to the first sequence in the target nucleic acid, and has a pre-adenylated phosphate group at its 5′ end; the second probe comprises a sequence substantially complementary to the second sequence in the target nucleic acid; (c) ligating the 3′ end of the second probe to the 5′ end of the first probe by using a ligase that does not require adenosine triphosphate to provide ligase activity to produce a ligation product; (d) releasing the ligation product from the target nucleic acid and allowing the capture domain of the ligation product to specifically bind to the capture domain of the capture probe; and (e) determining (i) all or part of the sequence corresponding to the ligation product, or its complement, and (ii) all or part of the sequence corresponding to the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the position of the target nucleic acid in the biological sample.

[0316] In some cases, a ligase that does not require adenosine triphosphate to provide ligase activity (e.g., thermostable 5′ App DNA / RNA ligase, truncated T4 RNA ligase 2 (trRnl2), truncated T4 RNA ligase 2 K227Q, truncated T4 RNA ligase 2KQ, Chlorella virus PBCV-1 DNA ligase, and combinations thereof). See, e.g., Nichols et al., "RNA Ligases," Curr. Protocol. Molec. Biol. 84(1):3.15.1-.4 (2008); Viollet et al., "T4 RNA Ligase 2 Truncated Active Site Mutants: Improved Tools for RNA Analysis," BMC Biotechnol. 11:72 (2011); and Ho et al., "Bacteriophage T4 RNA Ligase 2 (gp24.1) Exemplifies a Family of RNA Ligases Found in All Phylogenetic Domains," PNAS 99(20):12709-14 (2002), each of which is incorporated herein by reference in its entirety for illustrations of T4 RNA ligases. RNA ligase and truncated T4 RNA ligase. Thermostable 5′AppDNA / RNA ligase is an enzyme belonging to the ligase family that catalyzes the ligation of the 3′ end of ssRNA or ssDNA to 5′-adenylated ssDNA or 5′-adenylated ssRNA. Truncated T4 RNA ligase 2 is an enzyme belonging to the ligase family that catalyzes nicking of dsRNA and the ligation of ssRNA to ssRNA. When annealed to an RNA complement, it can also ligate the 3′ end of RNA or DNA to 5′-pDNA, and when annealed to a DNA complement, it can ligate the 3′ end of RNA to 5′-pRNA, but with lower efficiency. Truncated T4 RNA ligase 2K227Q is an enzyme belonging to the ligase family that catalyzes the ligation of the 3′ end of ssRNA to 5′-adenylated ssDNA and 5′-adenylated ssRNA. Compared to truncated T4 RNA ligase 2, it produces fewer byproducts. Truncated T4 RNA ligase 2KQ is an enzyme belonging to the ligase family that catalyzes the ligation of the 3′ end of ssRNA to 5′ adenylated ssDNA and 5′ adenylated ssRNA.It is preferred for ligating ssRNA to pre-adenylated adaptors and has reduced side products compared to the truncated T4 RNA ligase 2.

[0317] In some embodiments, the T4 RNA ligase comprises a K227Q mutation. See Viollet et al., "T4 RNA Ligase 2 Truncated ActiveSite Mutants: Improved Tools for RNA Analysis," BMC Biotechnol. 11, which is incorporated herein by reference in its entirety.

[0318] In some cases, a cofactor is added during the ligation process to facilitate ligation of the first and second probes. In some cases, the cofactor includes magnesium ions (Mg 2+ In some cases, the cofactor includes a manganese ion (Mn 2+ ). In some cases, Mg 2+ In some cases, Mn 2+ In some cases, the concentration of MgCl2 is about 1 mM to about 10 mM. In some cases, the concentration of MnCl2 is about 1 mM to about 10 mM.

[0319] In some cases, the ligation occurs at a pH in the range of about 6.5 to about 9.0, about 6.5 to about 8.0, or about 7.5 to about 8.0.

[0320] In some embodiments, the ligation buffer comprises an enzyme storage buffer. In some embodiments, the enzyme storage buffer comprises glycerol. In some embodiments, the ligation buffer is supplemented with glycerol. In some embodiments, the glycerol is present in the ligation buffer at a total volume of 15% v / v.

[0321] (h) Permeabilization and release of ligation products

[0322] In some embodiments, method provided herein includes a permeabilization step. In some embodiments, permeabilization is performed using a protease. In some embodiments, the protease is an endopeptidase. Operable endopeptidase includes but is not limited to trypsin, chymotrypsin, elastase, thermolysin, pepsin, clostripain, glutamyl endopeptidase (GluC), ArgC, peptidyl-aspartic endopeptidase (ApsN), endopeptidase LysC and endopeptidase LysN. In some embodiments, the endopeptidase is pepsin. In some embodiments, after (for example, by connecting the first probe and the second probe hybridized with adjacent sequences in the analyte) producing connection products, the biological sample is permeabilized. In some embodiments, the biological sample is permeabilized in the following steps simultaneously or before: the biological sample is contacted with the first probe and the second probe, the first probe and the second probe are hybridized with the analyte, the connection products are produced by connecting the first probe and the second probe, and the connection products are released from the analyte.

[0323] In some embodiments, method provided herein includes the permeabilization of biological samples, so that capture probe can be more easily combined with the connection probe of capture (that is, compared with no permeabilization). In some embodiments, reverse transcription (RT) reagent can be added to the permeabilization biological sample. Hatch together with RT reagent and can produce the full-length cDNA of spatial bar coding from the analyte (for example, polyadenylated mRNA) of capture. Second chain reagent (for example, second chain primer, enzyme) can be added to the biological sample on the slide to start the second chain synthesis.

[0324] In some cases, the permeabilization step includes applying a permeabilization buffer to the biological sample. In some cases, the permeabilization buffer includes a buffer (e.g., Tris pH 7.5), MgCl2, a sarkosyl detergent (e.g., sodium lauroyl sarcosinate), an enzyme (e.g., proteinase K and nuclease-free water. In some cases, the permeabilization step is carried out at 37°C. In some cases, the permeabilization step is carried out for about 20 minutes to 2 hours (e.g., about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours, or about 2 hours). In some cases, the release step is carried out for about 40 minutes.

[0325] In some embodiments, after the ligation product is produced, the ligation product is released from the analyte. In some embodiments, an endoribonuclease is used to release the ligation product from the analyte. In some embodiments, the endoribonuclease is RNase H, RNase A, RNase C, or RNase I. In some embodiments, the endoribonuclease is RNase H. RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA when hybridized with DNA. RNase H is part of a conserved family of ribonucleases found in many different organisms. There are two main classes of RNase H: RNase H1 and RNase H2. Retroviral RNase H enzymes are similar to prokaryotic RNase H1. All of these enzymes share a common characteristic: they are able to cleave the RNA component of RNA:DNA heteroduplexes. In some embodiments, the RNase H is RNase H1, RNase H2, or RNase H1 or RNase H2. In some embodiments, RNase H includes, but is not limited to, RNase HII from Pyrococcus furiosus, RNase HII from Pyrococcus horikoshi, RNase HI from Thermococcus litoralis, RNase HI from Thermus thermophilus, RNase HI from Escherichia coli, or RNase HII from Escherichia coli.

[0326] In some cases, release step is carried out using release buffer. In some cases, release buffer includes one or more of buffer (e.g., Tris pH 7.5), enzyme (e.g., RNase H) and nuclease-free water. In some cases, release step is carried out at 37 ° C. In some cases, release step is carried out for about 20 minutes to 2 hours (e.g., about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours or about 2 hours). In some cases, release step is carried out for about 30 minutes.

[0327] In some cases, the releasing step occurs before the permeabilization step. In some cases, the releasing step occurs after the permeabilization step. In some cases, the releasing step occurs simultaneously with the permeabilization step (e.g., in the same buffer).

[0328] (i) Blocking probe

[0329] In some embodiments, the capture probe capture domain is blocked before adding the second probe oligonucleotide to the biological sample. This can prevent the capture probe capture domain from prematurely hybridizing to the capture domain.

[0330] In some embodiments, the blocking probe is used to block or modify the free 3' end of the capture probe capture domain. In some embodiments, the blocking probe can hybridize with the capture probe capture domain of the second probe to shield the free 3' end of the capture probe capture domain. In some embodiments, the blocking probe can be a hairpin probe or a partially double-stranded probe. In some embodiments, the free 3' end of the capture probe capture domain of the second probe can be blocked by chemical modification, such as adding an azidomethyl group as a chemically reversible blocking portion so that the capture probe does not include a free 3' end. Before the second probe contacts the substrate, blocking or modifying the capture probe capture domain, particularly blocking or modifying the free 3' end of the capture probe capture domain, can prevent the second probe from hybridizing with the capture domain (for example, preventing the capture of the poly (A) of the capture probe capture domain by the poly (T) capture domain). In some embodiments, the blocking probe can be referred to as the capture probe capture domain blocking portion.

[0331] In some embodiments, the blocking probe can be reversibly removed. For example, a blocking probe can be applied to block the free 3' end of one or both of the capture probe capture domain and / or capture probe. Blocking the interaction between the capture probe capture domain and the capture probe on the substrate can reduce the non-specific capture of the capture probe. After the second probe is hybridized with the analyte and connected to the first probe, one or more spanning probes or the third oligonucleotide, the blocking probe can be removed from the 3' end of the capture probe capture domain and / or capture probe, and the connection product can migrate to the capture probe on the substrate and bind thereto. In some embodiments, removal includes denaturing the blocking probe from the capture probe capture domain and / or capture probe. In some embodiments, removal includes removing a chemically reversible capping portion. In some embodiments, removal includes digesting the blocking probe with an RNase (e.g., RNase H).

[0332] In some embodiments, the blocking probe is an oligo(dT) blocking probe. In some embodiments, the oligo(dT) blocking probe can have a length of 15-30 nucleotides. In some embodiments, an oligo(dT) blocking probe can have a length of 10-50 nucleotides, e.g., 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, 15-20, 20-50, 20-45, 20-40, 20-35, 20-30, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, 40-50, 40-45, or 45-50 nucleotides. In some embodiments, the analyte-capture agent can be blocked at different temperatures (eg, 4°C and 37°C).

[0333] (j) Biological samples

[0334] The methods disclosed herein can be performed on any type of sample. In some embodiments, the sample is fresh tissue. In some embodiments, the sample is a frozen sample. In some embodiments, the sample is pre-frozen. In some embodiments, the sample is a formalin-fixed paraffin-embedded (FFPE) sample.

[0335] The object from which a biological sample can be obtained can be a healthy or asymptomatic individual, an individual suffering from or suspected of having a disease (e.g., cancer) or an individual with a pre-treatment of the disease, and / or an individual needing treatment or suspecting the need for treatment. In some cases, a biological sample can include one or more pathological cells. Pathological cells can have metabolic characteristics, gene expression, protein expression and / or morphological characteristics that change. The example of disease includes inflammatory disorders, metabolic disorders, nervous system disorders and cancer. In some cases, a biological sample includes cancer or tumor cells. Cancer cells can derive from solid tumors, hematological malignancies, cell lines, or can be obtained as circulating tumor cells. In some cases, a biological sample is a heterogeneous sample. In some cases, a biological sample is a heterogeneous sample including tumor or cancer cells and / or stromal cells.

[0336] In some cases, the cancer is breast cancer. In some cases, the breast cancer is triple positive breast cancer (TPBC). In some cases, the breast cancer is triple negative breast cancer (TNBC).

[0337] In some cases, the cancer is colorectal cancer. In some cases, the cancer is ovarian cancer. In certain embodiments, the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin or non-Hodgkin lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, myeloma, salivary gland cancer, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or a type of head or neck cancer. In certain embodiments, the cancer treated is desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer, or an operable or inoperable brainstem glioma. In some embodiments, the subject is human.

[0338] FFPE samples are typically highly cross-linked and fragmented, so this type of sample allows limited RNA recovery using traditional detection techniques. In some embodiments, provided herein is a method for capturing target RNA that is less affected by RNA degradation associated with FFPE fixation than other methods (e.g., utilizing oligo-dT to capture and mRNA reverse transcription). In some embodiments, provided herein is a method for sensitively measuring specific genes of interest that would otherwise be missed by full transcriptomic methods.

[0339] In some cases, FFPE samples are stained (e.g., using H&E). The methods disclosed herein are compatible with H&E and will allow coverage of the morphological background of transcriptomic analysis. However, as needed, when the positioning of the nucleus is required, some samples may be stained only with nuclear stains, such as using only hematoxylin and not eosin staining samples.

[0340] In some embodiments, biological samples (such as tissue sections) can be fixed with methanol, stained with hematoxylin and eosin, and then imaged. In some embodiments, fixing, staining and imaging occur before one or more probes are hybridized with the sample. Some embodiments of any workflow described herein may also include a decolorization step (e.g., a hematoxylin and eosin decolorization step) after sample imaging and before the sample is permeabilized. For example, decolorization can be performed by performing one or more (e.g., one, two, three, four or five) washing steps (e.g., one or more (e.g., one, two, three, four or five) washing steps using a buffer solution comprising HCl). These images can be used for mapping spatial gene expression patterns back to biological samples. Permeabilization enzymes can be used for permeabilizing biological samples directly on a slide.

[0341] In some embodiments, the FFPE sample is dewaxed, permeabilized, equilibrated, and blocked prior to the addition of the target probe oligonucleotide. In some embodiments, dewax is performed using xylene. In some embodiments, dewax comprises multiple washes with xylene. In some embodiments, dewax comprises multiple washes with xylene, followed by multiple rounds of graded alcohol to remove the xylene, and then washing the sample with water. In some aspects, the water is deionized water. In some embodiments, equilibration and blocking comprise incubating the sample in pre-Hyb buffer. In some embodiments, the pre-Hyb buffer comprises yeast tRNA. In some embodiments, permeabilizing the sample comprises washing the sample with phosphate buffered saline. In some embodiments, the buffer is PBS. In some embodiments, the buffer is PBST.

[0342] (k) Determination of ligation product sequence

[0343] After ligation products from the sample are hybridized or otherwise associated with the capture probes, the barcoded constructs resulting from the hybridization / association are analyzed according to any of the methods described above in combination with general cell-based spatial analysis methods.

[0344] In some embodiments, after the biological sample is contacted with the substrate comprising the capture probe, a removal step may optionally be performed to remove all or part of the biological sample from the substrate. In some embodiments, the removal step comprises enzymatic and / or chemical degradation of the biological sample cells. For example, the removal step may comprise treating the biological sample with an enzyme (e.g., a protease, e.g., proteinase K) to remove at least a portion of the biological sample from the substrate. In some embodiments, the removal step may comprise ablation of the tissue (e.g., laser ablation).

[0345] In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting the location of an analyte, e.g., a biological analyte) from a biological sample (e.g., present in a biological sample), the method comprising: (a) optionally staining and / or imaging the biological sample on a substrate; (b) permeabilizing the biological sample on the substrate (e.g., providing a solution comprising a permeabilizing agent); (c) contacting the biological sample with an array comprising a plurality of capture probes, wherein the capture probes in the plurality capture the biological analyte; and (d) analyzing the captured biological analyte to spatially detect the biological analyte; wherein the biological sample is removed in whole or in part from the substrate.

[0346] In some embodiments, the biological sample is not removed from the substrate. For example, before releasing the capture probe (for example, the capture probe that is bound to the analyte) from the substrate, the biological sample is not removed from the substrate. In some embodiments, this release includes cracking the capture probe from the substrate (for example, by a cracking domain). In some embodiments, this release does not include releasing the capture probe from the substrate (for example, a copy of the capture probe bound to the analyte can be prepared, and the copy can be released from the substrate, for example, by denaturation). In some embodiments, after the biological sample is released from the substrate, before analyzing the analyte bound to the capture probe, the biological sample is not removed from the substrate. In some embodiments, during the period of removing the capture probe from the substrate and / or during the period of analyzing the analyte bound to the capture probe after being released from the substrate, the biological sample remains on the substrate. In some embodiments, during the removal (for example, by denaturation) of the copy of the capture probe (for example, complement), the biological sample remains on the substrate. In some embodiments, analysis of analytes bound to capture probes from a substrate can be performed without subjecting the biological sample to enzymatic and / or chemical degradation of cells (e.g., permeabilized cells) or ablation of tissue (e.g., laser ablation).

[0347] In some embodiments, at least a portion of the biological sample is not removed from the substrate. For example, before releasing a capture probe (e.g., a capture probe bound to an analyte) from the substrate and / or analyzing the analyte bound to the capture probe released from the substrate, a portion of the biological sample can be retained on the substrate. In some embodiments, before analyzing the analyte bound to the capture probe from the substrate, at least a portion of the biological sample is not subjected to enzymatic and / or chemical degradation of cells (e.g., permeabilized cells) or ablation (e.g., laser ablation) of the tissue.

[0348] In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting the location of an analyte, e.g., a biological analyte) from a biological sample (e.g., present in a biological sample), comprising: (a) optionally staining and / or imaging the biological sample on a substrate; (b) permeabilizing the biological sample on the substrate (e.g., providing a solution comprising a permeabilizing agent); (c) contacting the biological sample with an array comprising a plurality of capture probes, wherein the capture probes in the plurality capture the biological analyte; and (d) analyzing the captured biological analyte to spatially detect the biological analyte; wherein the biological sample is not removed from the substrate.

[0349] In some embodiments, provided herein are methods for spatially detecting a biological analyte of interest from a biological sample, comprising: (a) staining and imaging the biological sample on a substrate; (b) providing a solution comprising a permeabilizing agent to the biological sample on the substrate; (c) contacting the biological sample with an array on the substrate, wherein the array comprises one or more capture probes, thereby allowing the one or more capture probes to capture the biological analyte of interest; and (d) analyzing the captured biological analytes, thereby spatially detecting the biological analyte of interest; wherein the biological sample is not removed from the substrate.

[0350] In some embodiments, the method further comprises performing spatial transcriptomic analysis on a region of interest in a biological sample. In some embodiments, one or more capture probes comprise a capture domain. In some embodiments, one or more capture probes comprise a unique molecular identifier (UMI). In some embodiments, one or more capture probes comprise a cleavage domain. In some embodiments, the cleavage domain comprises a sequence recognized and cleaved by uracil DNA glycosylase, apurinic / apyrimidinic (AP) endonuclease (APE1), U uracil specific excision reagent (USER) and / or endonuclease VIII. In some embodiments, one or more capture probes do not comprise a cleavage domain and are not cleaved from the array.

[0351] In some embodiments, the capture probe can be extended ("extended capture probe", e.g., as described herein). For example, extending the capture probe can include generating cDNA from the captured (hybridized) RNA. The process involves synthesizing a complementary strand of the hybrid nucleic acid, e.g., generating cDNA based on the captured RNA template (RNA that hybridizes to the capture domain of the capture probe). Thus, in the initial step of extending the capture probe (e.g., cDNA generation), the captured (hybridized) nucleic acid (e.g., RNA) serves as a template for extension (e.g., a reverse transcription step).

[0352] In some embodiments, the capture probe is extended using reverse transcription. For example, reverse transcription includes using reverse transcriptase to synthesize cDNA (complementary or copy DNA) from RNA (such as messenger RNA). In some embodiments, reverse transcription is carried out when the tissue is still in situ, to produce an analyte library, wherein the analyte library includes a spatial barcode from a neighboring capture probe. In some embodiments, the capture probe is extended using one or more DNA polymerases.

[0353] In some embodiments, the capture domain of the capture probe includes a primer for generating a complementary strand of nucleic acid that hybridizes to the capture probe, for example, a primer for DNA polymerase and / or reverse transcription. The nucleic acid (e.g., DNA and / or cDNA) molecule produced by the extension reaction comprises the sequence of the capture probe. The extension of the capture probe, such as DNA polymerase and / or reverse transcription reaction, can be performed using various suitable enzymes and protocols.

[0354] In some embodiments, full-length DNA (e.g., cDNA) molecules are produced. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if the nucleic acid (e.g., RNA) is partially degraded in the tissue sample, the captured nucleic acid molecule will be different from the initial RNA length in the tissue sample. In some embodiments, the 3' end of the extended probe (e.g., the first-strand cDNA molecule) is modified. For example, a linker or adapter can be connected to the 3' end of the extended probe. This can be accomplished by using a single-stranded ligase such as T4 RNA ligase or CircleGase TM (available from Lucigen, Middleton, Wisconsin). In some embodiments, a template switching oligonucleotide is used to extend the cDNA to produce a full-length cDNA (or as close to a full-length cDNA as possible). In some embodiments, a second-strand synthesis auxiliary probe (a partially double-stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe) can be ligated to the 3′ end of the extended probe, e.g., a first-strand cDNA molecule, using a double-stranded ligase (e.g., T4 DNA ligase). Other enzymes suitable for the ligation step are known in the art and include, for example, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase (9°N) DNA ligase, and Pyrococcus sp. (strain 9°N) DNA ligase. TM DNA ligase, New England Biolabs), Ampligase TM (available from Lucigen, Middleton, Wisconsin) and SplintR (available from New England Biolabs, Ipswich, Massachusetts). In some embodiments, a polynucleotide tail (e.g., a poly(A) tail) is incorporated into the 3' end of the extended probe molecule. In some embodiments, the polynucleotide tail is enzymatically incorporated using terminal transferase activity.

[0355] In some embodiments, the double-stranded extended capture probe is treated to remove any unextended capture probe prior to amplification and / or analysis (e.g., sequence analysis). This can be achieved by a variety of methods, for example, using an enzyme to degrade the unextended probe, such as an exonuclease or a purification column.

[0356] In some embodiments, the extended capture probe is amplified to produce a quantity sufficient for analysis, such as by DNA sequencing. In some embodiments, the first strand (e.g., a DNA and / or cDNA molecule) of the extended capture probe is used as a template for an amplification reaction (e.g., a polymerase chain reaction).

[0357] In some embodiments, the amplification reaction uses a primer comprising an affinity group to incorporate the affinity group onto an extended capture probe (e.g., RNA-cDNA hybrid). In some embodiments, the primer comprises an affinity group and the extended capture probe comprises an affinity group. The affinity group can correspond to any affinity group described above.

[0358] In some embodiments, the extended capture probe comprising an affinity group can be coupled to a substrate that is specific for the affinity group. In some embodiments, the substrate can comprise an antibody or antibody fragment. In some embodiments, the substrate comprises avidin or streptavidin, and the affinity group comprises biotin. In some embodiments, the substrate comprises maltose and the affinity group comprises maltose binding protein. In some embodiments, the substrate comprises maltose binding protein and the affinity group comprises maltose. In some embodiments, the amplified extended capture probe can serve to release the extended probe from the substrate surface as long as a copy of the extended probe is not fixed to the substrate.

[0359] In some embodiments, the capture probe or its complement or amplicon of extension is released. The step of releasing the capture probe or its complement or amplicon of extension from the substrate surface can be achieved in a variety of ways. In some embodiments, the capture probe or its complement of extension is released from the array by nucleic acid cleavage and / or denaturation (e.g., denaturing double-stranded molecules by heating).

[0360] In some embodiments, the extended capture probe or its complement or amplicon is released from the surface of the substrate (e.g., an array) by physical means. For example, when the extended capture probe is indirectly immobilized on the array substrate, for example, by hybridization with the surface probe, it is sufficient to destroy the interaction between the extended capture probe and the surface probe. Methods for destroying the interaction between nucleic acid molecules include denaturing double-stranded nucleic acid molecules as known in the art. A direct method for releasing DNA molecules (i.e., stripping the extended probe array) is to use a solution that interferes with the hydrogen bonds of the double-stranded molecules. In some embodiments, the extended capture probe is released by applying a heated solution (e.g., water or a buffer of at least 85°C, such as water of at least 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, or 99°C). In some embodiments, a solution comprising salts, surfactants, etc. is added to further destabilize the interaction between nucleic acid molecules to release the extended capture probe from the substrate.

[0361] In some embodiments, the extended capture probe includes a cleavage domain, and the extended capture probe is released from the substrate surface by cleavage. For example, the cleavage domain of the extended capture probe can be cleaved by any of the methods described herein. In some embodiments, prior to the step of amplifying the extended capture probe, the extended capture probe is released from the substrate surface, for example by cleaving the cleavage domain in the extended capture probe.

[0362] In some embodiments, the probe complementary to the capture probe of extension can be contacted with substrate. In some embodiments, when the probe is in contact with substrate, the biological sample can be in contact with substrate. In some embodiments, the biological sample can be removed from substrate before contacting the substrate with the probe. In some embodiments, a detectable marker (e.g., any detectable marker described herein) can be used to label the probe. In some embodiments, the probe that is not specifically bound (e.g., hybridized) to the capture probe of extension can be washed off. In some embodiments, a probe complementary to the capture probe of extension can be detected on substrate (e.g., imaging, any detection method described herein).

[0363] In some embodiments, the probe complementary to the capture probe extended can be about 4 nucleotides to about 100 nucleotides long. In some embodiments, the probe complementary to the capture probe extended (e.g., detectable probe) can be about 10 nucleotides to about 90 nucleotides long. In some embodiments, the probe complementary to the capture probe extended (e.g., detectable probe) can be about 20 nucleotides to about 80 nucleotides long. In some embodiments, the probe complementary to the capture probe extended (e.g., detectable probe) can be about 30 nucleotides to about 60 nucleotides long. In some embodiments, the probe complementary to the capture probe extended (e.g., detectable probe) can be about 40 nucleotides to about 50 nucleotides long. In some embodiments, the number of probes complementary to the extended capture probes (e.g., detectable probes) can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75 about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, and about 99 nucleotides in length.

[0364] In some embodiments, about 1 to about 100 probes can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 1 to about 10 probes can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 10 to about 100 probes can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 20 to about 90 probes can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 30 to about 80 probes (e.g., detectable probes) can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 40 to about 70 probes can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 50 to about 60 probes can be contacted to a substrate and specifically bind (e.g., hybridize) to an extended capture probe. In some embodiments, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 93, about 94, about 95, about 96, about 97, about 98, and about 99 probes contact the substrate and specifically bind (e.g., hybridize) to the extended capture probes.

[0365] In some embodiments, a probe can be complementary to a single analyte (e.g., a single gene). In some embodiments, a probe can be complementary to one or more analytes (e.g., analytes in a gene family). In some embodiments, a probe (e.g., a detectable probe) can be used to detect a group of genes associated with a disease (e.g., cancer, Alzheimer's disease, Parkinson's disease).

[0366] In some cases, the ligated probes and capture probes can be amplified or replicated to produce multiple cDNA molecules. In some embodiments, the cDNA can be denatured from the capture probe template and transferred (e.g., to a clean tube) for amplification and / or library construction. Spatially barcoded cDNA can be amplified by PCR prior to library construction. The cDNA can then be enzymatically fragmented and size-selected to optimize the size of the cDNA amplicon. The P5 and P7 sequences used to capture the amplicon on the sequencing flow cell (Illumina sequencer) can be appended to the amplicon, i7 and i5 can be used as sample indexes, and TruSeq read 2 can be added by End Repair, A-tailing addition, adapter ligation, and PCR. The cDNA fragments can then be sequenced using paired-ended sequencing using TruSeq read 1 and TruSeq read 2 as sequencing primer sites. The additional sequences are specific to Illumina sequencers or sequencing instruments that utilize these sequences; however, the skilled artisan will appreciate that additional or alternative sequences used by other sequencing instruments or technologies are also applicable to the above methods.

[0367] In some embodiments, the entire sample can be sequenced by directly barcoding the sample by hybridizing to capture probes or analyte capture agents that are hybridized to, bound to, or associated with the cell surface or introduced into the cell, as described above.

[0368] A variety of different sequencing methods can be used to analyze barcoded analytes (e.g., ligation products). In general, the polynucleotides sequenced can be, for example, nucleic acid molecules, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA or DNA / RNA hybrids, and nucleic acid molecules with nucleotide analogs).

[0369] The order-checking of polynucleotide can be carried out by various systems.More generally, nucleic acid amplification, polymerase chain reaction (PCR) (for example, digital PCR and droplet digital PCR (ddPCR), quantitative PCR, real-time PCR, multiplex PCR, PCR-based single plex method, emulsion PCR) and / or isothermal amplification can be used to order-check.The non-limiting example of the method that genetic material is ordered-checking includes but is not limited to DNA hybridization method (for example, Southern blotting), restriction enzyme digestion method, Sanger sequencing, next generation sequencing (for example, single molecule real-time sequencing, nanopore sequencing and Polony sequencing), connection method and microarray method.

[0370] (1) Kit

[0371] In some embodiments, the present invention also provides a test kit including one or more reagents to detect one or more analytes described herein. In some cases, the test kit includes a substrate comprising multiple capture probes, and the capture probes include a spatial bar code and a capture domain. In some cases, the test kit includes multiple probes (for example, a first probe, a second probe, one or more spanning probes and / or a third oligonucleotide).

[0372] Non-limiting examples of kits for performing any of the methods described herein include: (a) a substrate comprising a plurality of capture probes comprising a spatial barcode and a capture domain; (b) a system comprising: a plurality of first probes and a second probe, wherein the first probe and the second probe each comprise a sequence substantially complementary to the analyte, and wherein the second probe comprises a capture binding domain; and (c) instructions for performing the method of any of the preceding claims.

[0373] Another non-limiting example of a kit for performing any of the methods described herein includes: (a) an array comprising a plurality of capture probes; (b) a plurality of probes comprising a first probe and a second probe, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte, wherein the second probe comprises (i) a capture probe binding domain that is capable of binding to the capture domain of the capture probe, and (ii) a linker sequence; (c) a plurality of enzymes comprising ribonucleases and ligases; and (d) instructions for performing the method of any of the preceding claims.

[0374] Another non-limiting example of a kit for performing any of the methods described herein includes: (a) an array comprising a plurality of capture probes; (b) a plurality of probes comprising a first probe and a second probe, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte, wherein the first probe comprises a linker sequence, and wherein the second probe comprises a capture probe binding domain that is capable of binding to the capture domain of the capture probe; (c) a plurality of enzymes comprising ribonucleases and ligases; and (d) instructions for performing the method of any of the preceding claims.

[0375] In some embodiments of any of the kits described herein, the kit comprises a second probe comprising a preadenylated phosphate group at its 5' end and a first probe comprising at least two ribonucleobases at its 3' end. Example

[0376] Example 1. Spatial gene expression analysis of FFPE fixed samples using RNA-templated ligation

[0377] Others have demonstrated in situ ligation. See Credle et al., Nucleic Acids Research, Vol. 45, No. 14, August 21, 2017, p. e128, https: / / doi.org / 10.1093 / nar / gkx471 (2017). However, previous methods utilized hybridization using poly(A) tails, which resulted in off-target binding. Here, the poly(A) tail on one probe oligonucleotide was switched to another sequence.

[0378] As an overview, non-limiting examples of RNA-templated ligation on FFPE-fixed samples are as follows: Figure 13 FFPE fixed samples are deparaffinized, stained (e.g., H&E stained), and imaged 1301. The sample is decolorized (e.g., using HCl) and decrosslinked 1302. After decrosslinking, the sample is treated with a prehybridization buffer (e.g., a hybridization buffer without the first and second probes), the probes are added to the sample, the probes are hybridized, and the sample is washed 1303. A ligase is added to the sample to ligate the hybridized probes to produce ligation products, and the sample is then washed 1304. The probes are released from the analyte by contacting the biological sample with RNase H 1305. The sample is then permeabilized to facilitate capture of the ligation products by capture probes on the substrate 1306. The ligation products hybridized to the capture probes are then extended 1307. The extended capture probes are denatured 1308. The denatured, extended capture probes are indexed, and the amplified library is quality controlled 1309 before sequencing.

[0379] FFPE mouse brain tissue sections were dewaxed, permeabilized with PBST, and equilibrated twice with pre-Hyb buffer for 5 minutes each. Samples were decrosslinked using either TE or PBS-Tween decrosslinking reagents. For TE decrosslinking, 100 μl of TE buffer (pH 9.0) (Genemed 10-0046) was added to each sample. Thermocycler protocols were performed on samples treated with TE according to Table 1.

[0380] Table 1. Decrosslinking Thermal Cycler Protocol

[0381]

[0382] Following the thermal cycler protocol, place the slides in the metal box and follow the method described in Table 2.

[0383] Table 2. Decrosslinking with HCl*

[0384]

[0385] *Note: Remove all liquid at each step.

[0386] RTL probes are designed to hybridize to adjacent sequences of each analyte of interest (e.g., mRNA sequence) in the genome, including estrogen receptor, progesterone receptor, and ERBB2, also known as HER2. Here, 20,056 probe pairs (e.g., RHS and LHS probes) were added to each tissue sample to capture 19,490 different genes. Two RTL probes (left (LHS) probe and right (RHS) probe (see, e.g., Figure 6 )) are added simultaneously and hybridize to adjacent sequences of the target mRNA, forming an RNA:DNA duplex. After decrosslinking, the PBS-Tween is removed and the DNA probes (1 nM each) are added to the tissue sample in hybridization buffer to allow the DNA probes to hybridize to their respective mRNA targets. The hybridization buffer contains SSC, formamide, or an equivalent, yeast tRNA as a carrier, and the RHS and LHS DNA probes.

[0387] One probe oligonucleotide (e.g., RHS probe or 3' probe) contains a non-target functional sequence at its 5' end, while the other probe oligonucleotide (e.g., LHS probe or 5' probe) contains a non-target poly A sequence at its 3' end. DNA probes were added to the tissue sample and incubated at 50°C for 2 hours and 30 minutes according to the thermal cycler protocol described in Table 3.

[0388] Table 3. Hybridization scheme

[0389]

[0390] After incubation at 50°C for 2.5 hours, the hybridization buffer was removed and the tissue was washed twice, each wash consisting of a 5-minute wash at 50°C in post-hybridization wash buffer preheated to 50°C. The post-hybridization wash buffer (post-hyb buffer) consisted of SSC, yeast tRNA, and nuclease-free water.

[0391] To ligate two adjacently hybridized diriboprobes on the target mRNA as described above, each sample was incubated with RNL2 T4 DNA ligase reaction mixture (ligation mix). The ligation reaction mixture included: T4 RNA ligase buffer (NEB B0293S), RNL2 ligase, and nuclease-free water.

[0392] To ligate two adjacent DNA probe oligonucleotides hybridized on the target mRNA as described above, SplintR (NEB) in ligation buffer was added to each sample. The ligase reaction mixture for each sample consisted of: Tris-HCl (pH 7.5), MnCl2, ATP, DTT, replacement buffer, SplintR ligase (NEB), and nuclease-free water. Ligation and wash steps were performed as described in Table 4.

[0393] After DNA probe ligation, tissue samples were washed twice in SSC / formamide post-ligation wash buffer at 60°C for 5 minutes each.

[0394] Table 4. Connection scheme

[0395]

[0396] *Post-ligation wash buffer (each sample) includes: SSC, formamide or equivalent, and nuclease-free water.

[0397] RNase H is added to digest the RNA strand of the hybridized RNA:DNA duplex. Briefly, the RNA of the DNA:RNA hybrid is digested by incubating the sample with an RNase H mixture at 37°C for 30 minutes, wherein the RNase H mixture includes: RNase H buffer and RNase H. After incubation, while maintained at 37°C, the biological sample is permeabilized to release the connected RTL probe using 1.25mg / mL Proteinase K. Specifically, the Proteinase K solution includes (each sample): Tris (pH 7.5), MgCl2, sarkosyl or SDS, Proteinase K (enzyme) and nuclease-free water. The sample is incubated in the Proteinase K solution at 37°C for at least 5 minutes. The sample is then washed 3 times with 2XSSC.

[0398] The released, ligated DNA probes, acting as surrogates for the target mRNA, are allowed to hybridize to the capture domain on the capture probes immobilized on the spatial array through the poly A tail at the 3′ end of the RHS probe. The captured ligated probes are replicated, using the capture probes as templates, and the extension products are released from the spatial array. Briefly, the tissue was incubated with a second-strand extension mixture containing Kapa Hifi DNA polymerase at 53°C for 25 minutes. After incubation, the second-strand extension mixture was removed from the tissue and the tissue was washed with 2X SSC. KOH solution was added to each tissue well, the tissue was incubated at room temperature for 10 minutes to release the extension products from the spatial array, and the supernatant from each tissue well was transferred for quantification and library preparation. Sample quantification was performed using qPCR and KAPA SYBR FAST qPCR master mix according to the manufacturer's instructions. Briefly, the KAPA SYBR master mix was prepared by adding qPCR primer cDNA_F and qPCR primer sRNA_R2. The thermal cycler protocol consisted of 3 minutes at 98°C followed by 30 cycles of 5 seconds at 98°C and 30 seconds at 63°C. For library preparation, samples were indexed using an Amp Mix containing dual-index primers and an Amp Mix. The nucleic acids were then sequenced and analyzed.

[0399] As controls, experiments were performed in parallel in which no ligase was added, pepsin was added before RNase H treatment, or pepsin was added after RNase H treatment.

[0400] like Figure 14-1 As shown in 9, the spatial expression of linked probe pairs highlights underlying mouse brain tissue physiology. Figure 14 PCR results are shown, demonstrating that ligation products are captured and amplifiable regardless of whether permeabilization occurs before or after RNase H treatment (positive control = standard, negative control = minus ligase). Arrow 1 points to the PCR band representing the desired ligation product, while arrows 2, 3, and 4 represent non-ligation products. Figure 15 The nonspecific probes detected as a fraction of the total reads for each condition are shown. Figure 16A -B shows that most probe combinations are specific. Arrows indicate RHS probes with increased background, thus including some nonspecific hybridization. Looking at the total counts in Figure 17, gene-specific LHS+RHS probes overlap with tissue footprints (see Figure 17A -C, where black circles indicate tissue footprints).

[0401] In addition, probes specific for various genes (e.g., Grp88, Penk, Plp1, Nptrx, and Mpb2) were first added to the sample in hybridization buffer, first at 60°C for 30 minutes and then at 45°C for 2 hours (see, e.g., Figures 18 and 19). When target-specific UMIs are counted, more detailed expression patterns can be observed. See, e.g., Figures 19B-19F This analysis showed that longer hybridization times resulted in higher sensitivity. Figures 18A-18E , white circles indicate spots reporting the expression of the indicated genes.

[0402] In contrast to the 2-hour hybridization discussed above, overnight hybridization was performed. Analysis of mouse brain samples using 21,833 probe pairs (e.g., RHS and LHS probes) added to each tissue sample to capture 21,604 different genes showed that overnight hybridization, and therefore longer hybridization, resulted in higher sensitivity compared to the positive control (see Figures 20A-20B Mouse probe design was derived from targets from Appris (see Rodriguez et al., Nucleic Acids Research, 46: D213-217, doi: 10.1093 / nar / gkx997 (2018), which is incorporated herein by reference in its entirety) and GENCODE. All probe pairs were non-overlapping and typically included approximately one probe pair per gene.

[0403] Example 2. Spatial gene expression analysis of triple-positive breast cancer (TPBC) using RNA-templated ligation (RTL)

[0404] This example demonstrates that RTL can be performed on samples to identify analyte abundance and spatial location in an unbiased manner.

[0405] The abundance and spatial location of analytes in two-year-old triple-positive ("TPBC", HER2, estrogen receptor, and progesterone receptor positive) breast cancer samples stored in FFPE were examined. The TPBC samples were interrogated with DNA probes by an RNA-templated ligation method. Prior to the ligation step, the TPBC tissue samples were dewaxed and stained according to established protocols. For example, FFPE TPBC tissue samples were preheated in a water bath (40°C), sectioned (10 μm), dried at 42°C for several hours, and then placed in a desiccator at room temperature overnight. The dried sectioned tissue was dewaxed by baking at 60°C, washed through a series of xylene and EtOH, and rinsed several times in water. After rinsing, the dewaxed tissue was stained with hematoxylin according to established protocols. The stained tissue was imaged to identify areas of tumor and stroma. See Figure 21A .

[0406] Tissues were decrosslinked to remove formaldehyde crosslinks from the sample, thereby releasing the analyte for RNA-templated ligation. Briefly, tissue samples were incubated with HCl solution for 1 minute, repeated twice, for a total of 3 minutes. Following the HCl incubation, tissue sections were incubated in TE pH 9.0 at 70°C for 1 hour. The TE was removed and the tissue was incubated in 1x PBS-Tween for 15 minutes.

[0407] RTL probes are designed to hybridize to adjacent sequences in the genome to respective analytes of interest (eg, mRNA sequences), including estrogen receptor, progesterone receptor, and ERBB2, also known as HER2.

[0408] Probe design was derived from targets from Appris (see Rodriguez et al., Nucleic Acids Research, 46: D213-217, doi: 10.1093 / nar / gkx997 (2018), which is incorporated herein by reference in its entirety) and GENCODE. All probe pairs were non-overlapping and typically included approximately one probe pair per gene.

[0409] Here, 20,056 probe pairs (e.g., RHS and LHS probes) were added to each tissue sample to capture 19,490 different genes in the human genome, including ESR1, PGR, and HER2. Two RTL probes (left (LHS) probe and right (RHS) probe) were added to each analyte (see, e.g., Figure 6 )) are added simultaneously and hybridize at adjacent sequences of the target mRNA, forming an RNA:DNA double-stranded structure.

[0410] After de-crosslinking, DNA probes (1 nm of each probe) are added to the tissue sample in hybridization buffer for hybridization of the DNA probes to their respective mRNA targets. One probe oligonucleotide (e.g., RHS probe or 3' probe) contains a non-target functional sequence at its 5' end, while the other probe oligonucleotide (e.g., LHS probe or 5' probe) contains a non-target poly A sequence at its 3' end. Briefly, hybridization buffer with DNA probes is added to the tissue sample, and the tissue is incubated at 50°C for approximately 2.5 hours. The hybridization / DNA probe buffer is removed, and the tissue is washed by adding post-hybridization buffer without DNA probes and incubating at 50°C for 5 minutes, for a total of 3 post-hybridization washes.

[0411] To ligate two DNA probe oligonucleotides that were adjacently hybridized on the target mRNA as described above, SplintR (NEB) in ligation buffer was added to each tissue sample, and the tissue was incubated for 60 minutes at 37° C. After DNA probe ligation, the tissue samples were washed twice for 5 minutes each in SSC / formamide post-ligation wash buffer at 60° C.

[0412] RNase H is then added to digest the RNA strand of the hybridized RNA:DNA duplex. Briefly, the RNA of the DNA:RNA hybrid is digested by incubating the tissue with RNase H at 37°C for 30 minutes. The biological sample is then permeabilized to release the attached RTL probes and contact them with the multiple capture probes attached to the slide. Specifically, after 30 minutes, the tissue is washed and permeabilized by adding 1.25 mg / ml proteinase K, incubating at 37°C for at least 5 minutes, and then washing to remove the protease.

[0413] The released, connected DNA probe serves as a surrogate for the target mRNA and is allowed to hybridize with the capture domain on the capture probe (capture probe fixed on the spatial array) through the poly A tail at the 3' end of the RHS probe. The captured connected probe is replicated, using the capture probe as a template, and the extension product is released from the spatial array. Briefly, the tissue is incubated at 53°C for 25 minutes with a second chain extension mixture containing KapaHifi DNA polymerase (Roche). After incubation, the extension mixture is removed from the tissue and the tissue is washed with SSC. KOH solution is added to each tissue well, the tissue is incubated at room temperature for 10 minutes to release the extension product from the spatial array, and then the supernatant in each tissue well is transferred for quantification, library preparation and sequencing on an Illumina NextSeq sequencing instrument.

[0414] Assessing the entire transcriptome can help better understand TPBC heterogeneity. For example, it was observed that using 20,056 probe pairs (e.g., RHS and LHS probes) to capture 19,490 different genes showed comparable results to the positive control in terms of the number of UMIs per cell and the number of genes per cell ( Figure 21B ). Analysis of the spatial location and abundance of each RTL ligation probe hybridized to the array revealed eight (8) distinct expression clusters, indicating that differential gene expression and the location of expressed genes can be determined using RTL probes. Figures 21C-21D . refer to Figure 21C Each point in the figure is a point on the array. Each point is assigned to a cluster, represented by a black circle. In some cases, the points assigned to the cluster are outside the circle indicated on the figure. Figure 21D Each point on the array is assigned to a cluster and each cluster is partially indicated by a circle. In some cases, a point is assigned to a cluster but is not within the designated circle on the array. Finally, the expression of individual analytes is determined. TPBC samples often show elevated levels of estrogen receptors, progesterone receptors, and ERRB2 (HER2). Figures 21E-21G As shown in A.

[0415] Embodiment A1. A method for determining the location of an analyte in a biological sample, comprising:

[0416] (a) contacting a biological sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain;

[0417] (b) contacting the biological sample with a first probe oligonucleotide and a second probe oligonucleotide, wherein the first probe oligonucleotide and the second probe oligonucleotide each comprise a sequence that is substantially complementary to an adjacent sequence of the analyte, and wherein the second probe oligonucleotide comprises a capture probe capture domain;

[0418] (c) hybridizing the first probe oligonucleotide and the second probe oligonucleotide to the analyte in a hybridization buffer without formamide;

[0419] (d) ligating the first probe oligonucleotide to the second probe oligonucleotide, thereby generating a ligation product;

[0420] (e) releasing the ligation product from the analyte and hybridizing the ligation product to the capture domain; and

[0421] (f) determining (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0422] Embodiment A2. The method of embodiment A1, wherein the capture probe further comprises one or more functional domains, unique molecular identifiers, cleavage domains, and combinations thereof.

[0423] Embodiment A3. The method of embodiment A1 or A2, wherein the array comprises one or more features on a substrate.

[0424] Embodiment A4. The method of embodiment A3, wherein the one or more features comprise beads.

[0425] Embodiment A5. The method of embodiment A3, wherein the substrate comprises a glass slide.

[0426] Embodiment A6. The method of any one of embodiments A1 to A5, wherein the formamide-free hybridization buffer is a saline-sodium citrate (SSC) hybridization buffer.

[0427] Embodiment A7. The method of embodiment A6, wherein the SSC is present in the SSC hybridization buffer at about 1 x SSC to about 6 x SSC.

[0428] Embodiment A8. The method of embodiment A6 or A7, wherein SSC is present in the SSC hybridization buffer at about 2x SSC to about 4x SSC.

[0429] Embodiment A9. The method of any one of embodiments A6-A8, wherein the SSC hybridization buffer comprises a solvent.

[0430] Embodiment A10. The method of embodiment A9, wherein the solvent comprises ethylene carbonate.

[0431] Embodiment A11. The method of any one of embodiments A6-A10, wherein the ethylene carbonate is present in the SSC hybridization buffer at about 10% (w / v) to about 25% (w / v).

[0432] Embodiment A12. The method of any one of embodiments A6-A11, wherein the ethylene carbonate is present in the SSC hybridization buffer at about 15% (w / v) to about 20% (w / v).

[0433] Embodiment A13. The method of any one of embodiments A6-A12, wherein the ethylene carbonate is present in the SSC hybridization buffer at about 13% (w / v).

[0434] Embodiment A14. The method of any one of embodiments A6-A13, wherein the SSC hybridization buffer is at a temperature of about 40°C to about 60°C.

[0435] Embodiment A15. The method of any one of embodiments A6 to A14, wherein the SSC hybridization buffer is at a temperature of about 45°C to about 55°C.

[0436] Embodiment A16. The method of any one of embodiments A6-A15, wherein the SSC hybridization buffer is at a temperature of about 50°C.

[0437] Embodiment A17. The method of any one of embodiments A6 to A16, wherein the SSC hybridization buffer further comprises one or more of yeast tRNA, a polymerization promoter, or an additive.

[0438] Embodiment A18. The method of any one of embodiments A1 to A17, wherein the ligating in step (d) comprises a ligase.

[0439] As in embodiment A19. The method of embodiment A18, wherein the ligase is one or more of T4 RNA ligase (Rnl2), SplintR ligase, single-stranded DNA ligase, or T4 DNA ligase.

[0440] Embodiment A20. The method of embodiment A19, wherein the ligase is T4 DNA ligase.

[0441] Embodiment A21. The method of any one of Embodiments A1-A20, further comprising removing one or more unhybridized first probe oligonucleotides, one or more unhybridized second probe oligonucleotides, or both from the array.

[0442] Embodiment A22. The method of embodiment A21, wherein removing comprises washing one or more unhybridized first probe oligonucleotides, one or more unhybridized second probe oligonucleotides, or both from the array in a wash buffer that does not contain formamide.

[0443] Embodiment A23. The method of embodiment A21 or A22, wherein the formamide-free wash buffer is SSC wash buffer.

[0444] Embodiment A24. The method of any one of embodiments A21 to A23, wherein SSC is present in the SSC wash buffer at about 0.01 x SSC to about 1 x SSC.

[0445] Embodiment A25. The method of any one of embodiments A21 to A24, wherein SSC is present in the SSC wash buffer at about 0.1 x SSC.

[0446] Embodiment A26. The method of any one of embodiments A21 to A25, wherein the SSC wash buffer comprises a detergent.

[0447] Embodiment A27. The method of embodiment A26, wherein the detergent comprises sodium dodecyl sulfate (SDS).

[0448] Embodiment A28. The method of any one of embodiments A21 to A26, wherein SDS is present in the SSC wash buffer at about 0.01% (v / v) to about 0.5% (v / v).

[0449] Embodiment A29. The method of any one of embodiments A21 to A28, wherein SDS is present in the SSC wash buffer at about 0.1% (v / v).

[0450] Embodiment A30. The method of any one of embodiments A21 to A29, wherein the SSC wash buffer comprises a solvent.

[0451] Embodiment A31. The method of any one of Embodiments A21-A30, wherein the solvent comprises ethylene carbonate.

[0452] Embodiment A32. The method of any one of embodiments A21-A31, wherein the ethylene carbonate is present in the SSC wash buffer at about 10% (w / v) to about 25% (w / v).

[0453] Embodiment A33. The method of any one of embodiments A21-A32, wherein the ethylene carbonate is present in the SSC wash buffer at about 15% (w / v) to about 20% (w / v).

[0454] Embodiment A34. The method of any one of embodiments A21-A33, wherein the ethylene carbonate is present in the SSC wash buffer at about 16% (w / v).

[0455] Embodiment A35. The method of any one of embodiments A21 to A34, wherein the SSC wash buffer is at a temperature of about 50°C to about 70°C.

[0456] Embodiment A36. The method of any one of embodiments A21 to A35, wherein the SSC wash buffer is at a temperature of about 55°C to about 65°C.

[0457] Embodiment A37. The method of any one of embodiments A21-A36, wherein the SSC wash buffer is at a temperature of about 60°C.

[0458] Embodiment A38. The method of any one of embodiments A1 to A37, wherein the releasing in step (e) comprises contacting the ligation product with an endoribonuclease.

[0459] Embodiment A39 is a method as described in embodiment A38, wherein the endoribonuclease is one or more of RNase H, RNase A, RNase C or RNase I.

[0460] Embodiment A40. The method of embodiment A38 or A39, wherein the endoribonuclease is RNase H.

[0461] Embodiment A41. The method of embodiment A40, wherein the RNase H comprises RNase H1, RNase H2, or both.

[0462] Embodiment A42. The method of any one of Embodiments A1-A41, further comprising extending the 3' end of the capture probe using the ligation product as a template for an extension reaction.

[0463] Embodiment A43. The method of embodiment A42, wherein extending the 3' end of the capture probe comprises reverse transcribing the analyte to generate a sequence complementary to the analyte.

[0464] Embodiment A44. The method of embodiment A43, wherein the reverse transcription analyte comprises reverse transcriptase.

[0465] Embodiment A45. The method of any one of embodiments A1 to A44, wherein the analyte is RNA.

[0466] Embodiment A46. The method of any one of embodiments A1 to A45, wherein the RNA is mRNA.

[0467] Embodiment A47. The method of any one of embodiments A1 to A46, wherein the biological sample is a tissue sample.

[0468] Embodiment A48. The method of embodiment A47, wherein the tissue sample is a tissue section.

[0469] Embodiment A49. The method of any one of embodiments A1 to A46, wherein the biological sample is a fresh-frozen biological sample.

[0470] Embodiment A50. The method of any one of embodiments A1 to A46, wherein the biological sample is a fixed biological sample.

[0471] Embodiment A51. The method of embodiment A41, wherein the fixed biological sample is a formalin-fixed paraffin-embedded sample.

[0472] Embodiment A52. The method of any one of embodiments A1 to A51, further comprising permeabilizing the biological sample.

[0473] Embodiment A53. The method of embodiment A52, wherein permeabilizing the biological sample occurs prior to releasing the ligation product from the analyte.

[0474] Embodiment A54. The method of embodiment A52 or A53, wherein permeabilizing the biological sample comprises an endopeptidase.

[0475] Embodiment A55. The method of any one of embodiments A1 to A54, further comprising amplifying the ligation products before contacting the biological sample with the array.

[0476] Embodiment A56. The method of any one of embodiments A1 to A55, wherein the determining in step (f) comprises sequencing.

[0477] Embodiment A57. The method of any one of Embodiments A1 to A56, further comprising a capture probe capture domain blocking moiety that specifically binds to the capture probe capture domain.

[0478] Embodiment A58. The method of any one of Embodiments A1-A57, further comprising releasing the capture probe capture domain blocking moiety from the capture probe capture domain prior to contacting the biological sample with the array.

[0479] Embodiment A59. The method of any one of embodiments A1 to A58, wherein the capture probe capture domain comprises a homopolymeric sequence.

[0480] Embodiment A60. The method of embodiment A59, wherein the capture domain of the capture probe comprises a poly(A) sequence.

[0481] Implementation Method B

[0482] Embodiment B1. A method for identifying the location of an analyte in a biological sample, the method comprising:

[0483] (a) contacting a biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises a capture domain and a spatial barcode;

[0484] (b) contacting the biological sample with a first probe and a second probe, wherein a portion of the first probe and a portion of the second probe are substantially complementary to adjacent sequences of the analyte,

[0485] wherein the first probe comprises a sequence substantially complementary to a first target sequence of the analyte,

[0486] The second probe comprises:

[0487] (i) a first sequence that is substantially complementary to a second target sequence of the analyte;

[0488] (ii) a linker sequence;

[0489] (iii) a second sequence that is substantially complementary to a third target sequence of the analyte; and

[0490] (iv) a capture domain of a capture probe capable of binding to the capture domain of a capture probe;

[0491] (c) hybridizing the first probe and the second probe to the analyte;

[0492] (d) ligating the first probe to the second probe, thereby generating a ligation product;

[0493] (e) releasing the ligation product from the analyte;

[0494] (f) hybridizing the capture domain of the capture probe to the capture domain; and

[0495] (g) determining (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0496] Embodiment B2. The method of embodiment B1, wherein the second probe comprises, from 5' to 3', a first sequence, a linker sequence, a second sequence, and a capture probe capture domain.

[0497] Embodiment B3. The method of any one of embodiments B1-B2, wherein the first target sequence for the analyte is directly adjacent to the second target sequence for the analyte.

[0498] Embodiment B4. The method of any one of embodiments B1-B3, wherein the second target sequence is not directly adjacent to the third target sequence on the analyte.

[0499] Embodiment B5. The method of any one of embodiments B1 to B4, wherein the second target sequence and the third target sequence are on different exons of the analyte.

[0500] Embodiment B6. The method of any one of embodiments B1 to B4, wherein the second target sequence and the third target sequence are within the same exon of the analyte but are not directly adjacent.

[0501] Embodiment B7. The method of any one of embodiments B1 to B6, wherein the linker sequence comprises a total of about 1 nucleotide to about 100 nucleotides.

[0502] Embodiment B8. The method of embodiment B7, wherein the linker further comprises a barcode sequence that serves as a surrogate for identifying the analyte.

[0503] Embodiment B9. A method for identifying the location of an analyte in a biological sample, the method comprising:

[0504] (a) contacting a biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises a capture domain and a spatial barcode;

[0505] (b) contacting the biological sample with a first probe and a second probe, wherein a portion of the first probe and a portion of the second probe are substantially complementary to adjacent sequences of the analyte,

[0506] The first probe comprises:

[0507] (i) a first sequence that is substantially complementary to a first target sequence of an analyte;

[0508] (ii) a linker sequence;

[0509] (iii) a second sequence that is substantially complementary to a second target sequence of the analyte; and

[0510] wherein the second probe comprises a sequence substantially complementary to a third target sequence of the analyte and a capture domain of the capture probe capable of binding to the capture domain of the capture probe;

[0511] (c) hybridizing the first probe and the second probe to the analyte;

[0512] (d) ligating the first probe to the second probe, thereby generating a ligation product;

[0513] (e) releasing the ligation product from the analyte;

[0514] (f) hybridizing the capture domain of the capture probe to the capture domain; and

[0515] (g) determining (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0516] Embodiment B10. The method of embodiment F9, wherein the second target sequence is directly adjacent to the third target sequence.

[0517] Embodiment B11. The method of any one of embodiments B9-B10, wherein the first probe comprises, from 5′ to 3′, a first sequence, a linker sequence, and a second sequence.

[0518] Embodiment B12. The method of any one of embodiments B9 to B11, wherein the first probe further comprises a functional sequence.

[0519] Embodiment B13. A method as described in embodiment B13, wherein the functional sequence is a primer sequence.

[0520] Embodiment B14. The method of embodiment B12 or B13, wherein the first probe comprises, from 5′ to 3′, a functional sequence, a first sequence, a linker sequence, and a second sequence.

[0521] Embodiment B15. The method of any one of embodiments B9 to B14, wherein the first target sequence is not directly adjacent to the second target sequence on the analyte.

[0522] Embodiment B16. The method of embodiment B15, wherein the first target sequence and the second target sequence are located on different exons.

[0523] Embodiment B17. The method of embodiment B15, wherein the first target sequence and the second target sequence are within the same exon but are not directly adjacent.

[0524] Embodiment B18. The method of any one of embodiments B9 to B17, wherein the linker sequence comprises a total of about 1 nucleotide to about 100 nucleotides.

[0525] Embodiment B19. The method of embodiment B18, wherein the linker further comprises a barcode sequence that serves as a surrogate for identifying the analyte.

[0526] Embodiment B20. A method for identifying the location of an analyte in a biological sample, the method comprising:

[0527] (a) contacting a biological sample with a substrate comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises a capture domain and a spatial barcode;

[0528] (b) contacting the biological sample with the first probe, the second probe, and the one or more spanning probes,

[0529] wherein the first probe is substantially complementary to a first portion of the analyte,

[0530] wherein the second probe is substantially complementary to the second portion of the analyte and further comprises a capture probe capture domain, and

[0531] The spanning probes include:

[0532] (i) a first sequence that is substantially complementary to a first target sequence of an analyte, and

[0533] (ii) a second sequence that is substantially complementary to a second target sequence of the analyte;

[0534] (c) hybridizing the first probe, the second probe, and the spanning probe to the analyte;

[0535] (d) ligating the first probe, the one or more spanning probes, and the second probe, thereby generating a ligation product that is substantially complementary to the analyte;

[0536] (e) releasing the ligation product from the analyte;

[0537] (f) hybridizing the capture domain of the capture probe to the capture domain; and

[0538] (g) determining (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

[0539] Embodiment B21. The method of embodiment B20, wherein the spanning oligonucleotide further comprises a functional sequence.

[0540] Embodiment B22. A method as described in embodiment B20 or B, wherein the spanning oligonucleotide comprises from 5' to 3': the first sequence, the functional sequence and the second sequence.

[0541] Embodiment B23. The method of any one of embodiments B20-B22, wherein the functional sequence comprises a linker sequence.

[0542] Embodiment B24. The method of embodiment B23, wherein the linker sequence comprises a total of about 1 nucleotide to about 100 nucleotides.

[0543] Embodiment B25. The method of any one of embodiments B20 to B24, wherein the functional sequence comprises a barcode sequence.

[0544] Embodiment B26. The method of embodiment B25, wherein the barcode sequence comprises a sequence that serves as a surrogate for identifying the analyte.

[0545] Embodiment B27. The method of any one of embodiments B20 to B26, wherein the functional sequence comprises one or more linker sequences and a barcode sequence.

[0546] Embodiment B28. The method of embodiment B27, wherein the functional sequence comprises a barcode sequence flanked by adapter sequences.

[0547] Embodiment B29. The method of any one of embodiments B20 to B28, wherein the linker sequence comprises a total of about 1 nucleotide to about 100 nucleotides.

[0548] Embodiment B30. The method of any one of embodiments B20 to B29, wherein the first sequence spanning the probe and the second sequence spanning the probe are substantially complementary to sequences within the same exon.

[0549] Embodiment B31. The method of embodiment B30, wherein the first target sequence of the analyte and the second target sequence of the analyte are located in the same exon.

[0550] Embodiment B32. The method of any one of embodiments B20 to B29, wherein the first sequence spanning the probe and the second sequence spanning the probe are substantially complementary to sequences within different exons of the same gene.

[0551] Embodiment B33. The method of embodiment B32, wherein the first target sequence of the analyte and the second target sequence of the analyte are located on different exons of the same gene.

[0552] Embodiment B34. The method of any one of embodiments B20-33, wherein the first portion of the analyte is directly adjacent to the first target sequence, and / or wherein the second portion of the analyte is directly adjacent to the second target sequence.

[0553] Embodiment B35. The method of any one of embodiments B20-B34, wherein the spanning probe comprises at least two ribonucleic acids based on the 3' end.

[0554] Embodiment B36. The method of any one of embodiments B20-B35, wherein the spanning probe comprises a phosphorylated nucleotide at the 5′ end.

[0555] Embodiment B37. The method of any one of embodiments B20 to B36, wherein the one or more spanning probes comprises one spanning probe.

[0556] Embodiment B38. The method of any one of embodiments B20 to B36, wherein the one or more spanning probes comprises at least two, at least three, at least four, at least five, or more spanning probes.

[0557] Embodiment B39. The method of embodiment B38, wherein the one or more spanning probes comprise:

[0558] (i) a third sequence that is substantially complementary to a third target sequence of the analyte, and

[0559] (ii) a fourth sequence that is substantially complementary to a fourth target sequence of the analyte.

[0560] Embodiment B40. The method of embodiment B39, wherein the first target sequence is located in a first exon, the second target sequence is located in a second exon, and the third and fourth target sequences are located in a third exon.

[0561] Embodiment B41. The method of embodiment B39, wherein the first target sequence is located in a first exon, the second target sequence is located in a second exon, the third target sequence is located in a third exon, and the fourth target sequence is located in a fourth exon.

[0562] Embodiment B42. The method of any one of embodiments B38 to B41, wherein the method comprises connecting:

[0563] First probe to spanning probe,

[0564] spanning probes to one or more other spanning probes, and

[0565] One or more additional spanning probes span the oligonucleotide to the second probe, thereby generating a ligation product that is substantially complementary to the analyte.

[0566] Embodiment B43. The method of any one of embodiments B38 to B42, wherein the one or more additional spanning probe oligonucleotides further comprise a functional sequence.

[0567] Embodiment B44. The method of embodiment B43, wherein the functional sequence comprises (i) a linker sequence, (ii) a barcode sequence, or (ii) one or more linkers and a barcode sequence.

[0568] Embodiment B45. The method of any one of embodiments B38 to B44, wherein the one or more additional spanning probes comprise at least two ribonucleic acids based on the 3′ terminus.

[0569] Embodiment B46. The method of any one of embodiments B38 to B45, wherein the one or more additional spanning probes comprise a phosphorylated nucleotide at the 5′ end.

[0570] Embodiment B47. The method of any one of embodiments B20 to B46, wherein the first probe further comprises a functional sequence.

[0571] Embodiment B48. A method as described in embodiment B47, wherein the functional sequence is a primer sequence.

[0572] Embodiment B49. The method of any preceding embodiment, wherein the first probe comprises at least two ribonucleobases at the 3' end.

[0573] Embodiment B50. The method of any preceding embodiment, wherein the second probe comprises a phosphorylated nucleotide at the 5' end.

[0574] Embodiment B51. The method of any preceding embodiment, further comprising providing a capture probe capture domain blocking portion that interacts with the capture probe capture domain.

[0575] Embodiment B52. The method of embodiment B51, further comprising releasing the capture probe capture domain blocking portion from the capture probe capture domain prior to step (f).

[0576] Embodiment B53. The method of any preceding embodiment, wherein the capture probe capture domain comprises a polyadenylation (poly(A)) sequence or its complement.

[0577] Embodiment B54. The method of embodiment B53, wherein the capture domain blocking portion of the capture probe comprises a polyuridine sequence, a polythymidine sequence, or both.

[0578] Embodiment B55. The method of embodiment B52, wherein releasing the polyuridine sequence from the poly(A) sequence comprises denaturing the ligation product or contacting the ligation product with an endonuclease or exonuclease.

[0579] Embodiment B56. The method of any preceding embodiment, wherein the capture domain of the capture probe comprises a sequence that is complementary to all or part of the capture domain of the capture probe.

[0580] Embodiment B57. The method of any preceding embodiment, wherein the capture domain of the capture probe comprises a degenerate sequence.

[0581] Embodiment B58. The method of any of the preceding embodiments, wherein the ligation step comprises using enzymatic ligation or chemical ligation.

[0582] Embodiment B59. The method of embodiment B58, wherein the enzymatic ligation uses a ligase.

[0583] Embodiment B60. The method of embodiment B59, wherein the ligase is one or more of T4 RNA ligase (Rnl2), splintR ligase, single-stranded DNA ligase, or T4 DNA ligase.

[0584] Embodiment B61. The method of embodiment B60, wherein the ligase is T4 RNA ligase 2 (Rnl2) ligase.

[0585] Embodiment B62. The method of any preceding embodiment, wherein the first probe, the second probe, and the one or more spanning probes are DNA probes.

[0586] Embodiment B63. The method of embodiment B62, wherein steps (b) and (c) each produce an RNA:DNA hybrid.

[0587] Embodiment B64. The method of any of the preceding embodiments, wherein steps (b) and (c) are performed substantially simultaneously.

[0588] Embodiment B65. The method of any of the preceding embodiments, wherein the biological sample comprises a FFPE sample.

[0589] Embodiment B66. The method of embodiment B65, wherein the tissue sample is an FFPE tissue sample and the tissue sample is de-crosslinked.

[0590] Embodiment B67. A method as described in any of the preceding embodiments, wherein the biological sample comprises a tissue section.

[0591] Embodiment B68. A method as described in any of the preceding embodiments, wherein the biological sample comprises a fresh frozen sample.

[0592] Embodiment B69. A method as described in any of the preceding embodiments, wherein the biological sample comprises living cells.

[0593] Embodiment B70. The method of any preceding embodiment, wherein the analyte comprises RNA and / or DNA.

[0594] Embodiment B71. A method as described in any of the preceding embodiments, wherein the analyte is RNA.

[0595] Embodiment B72. The method of embodiment B71, wherein the RNA is mRNA.

[0596] Embodiment B73. A method as described in any of the preceding embodiments, wherein the biological sample is pre-stained.

[0597] Embodiment B74. The method of embodiment B73, wherein the biological sample is pre-stained with hematoxylin and eosin (H&E).

[0598] Embodiment B75. The method of embodiment B73 or B74, wherein the biological sample is previously stained using immunofluorescence or immunohistochemistry.

[0599] Embodiment B76. The method of any preceding embodiment, further comprising contacting the biological sample with a permeabilizing agent.

[0600] Embodiment B77. The method of any preceding embodiment, wherein the releasing step comprises removing the attached probe from the analyte.

[0601] Embodiment B78. The method of embodiment B77, wherein the releasing step comprises contacting the attached probe with an endoribonuclease.

[0602] Embodiment B79. A method as described in embodiment B78, wherein the endoribonuclease is one or more of RNase H, RNase A, RNase C or RNase I.

[0603] Embodiment B80. The method of embodiment B79, wherein the RNase H comprises RNase H1, RNase H2, or RNase H1 and RNase H2.

[0604] Embodiment B81. The method of any of the preceding embodiments, wherein the determining step comprises amplifying all or a portion of the ligation product that specifically binds to the capture domain.

[0605] Embodiment B82. The method of embodiment B81, wherein the amplification is isothermal.

[0606] Embodiment B83. The method of embodiment B81, wherein the amplification is not isothermal.

[0607] Embodiment B84. A method as described in any of embodiments B81-B83, wherein the amplification product comprises (i) all or part of the sequence of the ligation product that specifically binds to the capture domain, or its complement, and (ii) all or part of the sequence of the spatial barcode, or its complement.

[0608] Embodiment B85. A method as described in any of the preceding embodiments, wherein the determining step comprises sequencing.

[0609] Embodiment B86. A method as described in embodiment B85, wherein the sequencing step includes in situ sequencing.

[0610] Embodiment B87. A kit comprising:

[0611] (a) An array comprising a plurality of capture probes;

[0612] (b) a plurality of probes comprising a first probe and a second oligonucleotide, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte, wherein the second probe comprises (i) a capture domain of a capture probe capable of binding to the capture domain of the capture probe and (ii) a linker sequence;

[0613] (c) a plurality of enzymes including ribonucleases and ligases; and

[0614] (d) Instructions for using the kit.

[0615] Embodiment B88. A kit comprising:

[0616] (a) An array comprising a plurality of capture probes;

[0617] (b) a plurality of probes comprising a first probe and a second oligonucleotide, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte, wherein the first probe comprises a linker sequence, and wherein the second probe comprises a capture probe capture domain capable of binding to a capture domain of a capture probe;

[0618] (c) a plurality of enzymes including ribonucleases and ligases; and

[0619] (d) Instructions for using the kit.

[0620] Embodiment B89. A kit comprising:

[0621] (a) An array comprising a plurality of capture probes;

[0622] (b) a plurality of probes comprising a first probe and a second oligonucleotide, wherein the second probe comprises a capture domain of a capture probe capable of binding to a capture domain of a capture probe;

[0623] (c) a plurality of spanning probes, wherein the spanning probes in the plurality of spanning probes comprise a first sequence, a linker sequence, and a second sequence, wherein the first probe and the first sequence of the spanning probe are substantially complementary to an adjacent sequence of the analyte, and wherein the second probe and the second sequence of the spanning probe are substantially complementary to an adjacent sequence of the analyte;

[0624] (d) a plurality of enzymes including ribonucleases and ligases; and

[0625] (e) Instructions for using the kit.

[0626] Embodiment B90. The kit of any one of embodiments B87-B89, wherein the ribonuclease is RNase H.

[0627] Embodiment B91. The kit of any one of embodiments B87 to B90, wherein the ligase is one or more of T4 RNA ligase (Rnl2), splintR ligase, single-stranded DNA ligase, or T4 DNA ligase.

[0628] Embodiment B92. The kit of embodiment B91, wherein the ligase is T4 RNA ligase 2 (Rnl2) ligase.

Claims

1. A method for determining the location of an analyte in a biological sample, comprising: (a) providing a biological sample on an array comprising a plurality of capture probes, wherein a capture probe in the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (b) contacting a first probe and a second probe with a biological sample, wherein the first probe and the second probe each comprise one or more sequences substantially complementary to a sequence of the analyte, and wherein the second probe comprises a capture probe capture domain; (c) hybridizing the first probe and the second probe to the analyte; (d) generating a ligation product by ligating the first probe and the second probe; (e) releasing the ligation product from the analyte; (f) hybridizing the ligation product to the capture domain; and (g) determining (i) all or part of the sequence of the ligation product bound to the capture domain, or its complement, and (ii) the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

2. A method for determining the location of an analyte in a biological sample, comprising: (a) providing a biological sample on a first substrate, wherein the biological sample comprises an analyte; (b) contacting a first probe and a second probe with a biological sample, wherein the first probe and the second probe each comprise one or more sequences substantially complementary to a sequence of the analyte, and wherein the second probe comprises a capture probe capture domain; (c) hybridizing the first probe and the second probe to the analyte; (d) generating a ligation product by ligating the first probe and the second probe; (e) contacting the biological sample on the first substrate with a second substrate, wherein the second substrate comprises a spatial array comprising a plurality of capture probes immobilized on the second substrate, wherein the capture probes of the plurality of capture probes comprise (i) a spatial barcode comprising a sequence that provides a location of the analyte and (ii) a capture domain, wherein 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 spatial array; (f) releasing the ligation product from the analyte; (g) hybridizing the ligation product to the capture domain; and (h) determining (i) all or part of the sequence of the ligation product bound to the capture domain, or its complement, and (ii) the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample.

3. The method of claim 1 or 2, wherein the first probe and the second probe are substantially complementary to adjacent sequences of the analyte.

4. A method for determining the location of an analyte in a biological sample, comprising: (a) providing a biological sample placed on a first substrate; (b) contacting a plurality of first and second probes with a biological sample, wherein the plurality of first and second probes target a plurality of nucleic acids in the biological sample, and wherein the first and second probes in the plurality comprise sequences that are substantially complementary to sequences that are not adjacent to each other on the analyte, and wherein the second probe comprises a capture domain of the capture probe that is complementary to all or a portion of a capture domain of the capture probe; (c) hybridizing the first probe and the second probe to the analyte; (d) generating a ligation product by ligating the first probe and the second probe; (e) releasing the ligation product from the analyte; (f) hybridizing the ligation product to a capture domain of a capture probe immobilized on the array, wherein the capture probe further comprises a spatial barcode; and (g) determining (i) all or part of the sequence of the ligation product bound to the capture domain, or its complement, and (ii) the sequence of the spatial barcode, or its complement, and using the determined sequences of (i) and (ii) to identify the location of the analyte in the biological sample. 5 . The method of claim 4 , wherein the first probe is extended, thereby (i) filling a gap between the first probe and the second probe and (ii) generating an extended first probe. The method of claim 5 , wherein the first probe is extended by DNA polymerase, RNA polymerase, or reverse transcriptase.

7. The method of claim 5, wherein the first probe is extended by Mu polymerase, VENT polymerase, or Taq polymerase.

8. The method of any one of claims 4-7, wherein determining further comprises identifying the presence or absence of: a splice variant, an insertion or deletion, or a single nucleotide polymorphism.

9. The method of any one of claims 4-7, wherein the array is on a first substrate.

10. The method of any one of claims 4-7, wherein the array is on a second substrate.

11. The method of claim 10, wherein 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 array.

12. The method of any one of claims 1, 2, and 4, further comprising hybridizing a third probe to the first probe and the second probe, wherein the third probe comprises a first sequence that is substantially complementary to a portion of the first probe, and a second sequence that is substantially complementary to a portion of the second probe.

13. The method of claim 12, wherein hybridizing the first probe and the second probe to the analyte is performed at a first temperature. The method of claim 13 , wherein hybridization of the third probe to the first probe and the second probe is performed at a second temperature. The method of claim 14 , wherein the first temperature is higher than the second temperature.

16. The method of any one of claims 13-15, wherein the first temperature is 50°C to 75°C.

17. The method of any one of claims 13-15, wherein the first temperature is 55°C to 70°C.

18. The method of any one of claims 13-15, wherein the first temperature is 60°C to 65°C.

19. The method of claim 14 or 15, wherein the second temperature is 15°C to 35°C.

20. The method of claim 14 or 15, wherein the second temperature is 20°C to 30°C.

21. The method of claim 14 or 15, wherein the second temperature is 25°C to 30°C.

22. The method of claim 5 or 6, wherein the first probe is extended with a DNA polymerase.

23. The method of any one of claims 1, 2, and 4, wherein the first probe comprises a sequence that is substantially complementary to a first target sequence of the analyte.

24. The method of claim 23, wherein the second probe further comprises: (i) a first sequence that is substantially complementary to a second target sequence of the analyte; (ii) a linker sequence; and (iii) a second sequence that is substantially complementary to a third target sequence of the analyte.

25. The method of claim 24, wherein the first target sequence for the analyte is directly adjacent to the second target sequence for the analyte.

26. The method of claim 24 or 25, wherein the second target sequence is not directly adjacent to the third target sequence on the analyte.

27. The method of claim 26, wherein the second target sequence and the third target sequence are (i) on different exons of the analyte or (ii) are located in the same exon of the analyte but are not adjacent on the analyte.

28. The method of claim 24, wherein the second probe comprises a sequence that is substantially complementary to a third target sequence of the analyte.

29. The method of claim 28, wherein the first probe comprises: (i) a first sequence that is substantially complementary to a first target sequence of an analyte; (ii) a linker sequence; and (iii) a second sequence that is substantially complementary to a second target sequence of the analyte.

30. The method of claim 28 or 29, wherein the second target sequence is directly adjacent to the third target sequence.

31. The method of claim 28 or 29, wherein the first target sequence is not directly adjacent to the second target sequence on the analyte.

32. The method of claim 31, wherein the first target sequence and the second target sequence are (i) on different exons of the analyte or (ii) are located within the same exon but are not directly adjacent on the analyte.

33. The method of claim 24 or 29, wherein the linker sequence comprises a total of 1 nucleotide to 100 nucleotides.

34. The method of claim 33, wherein the adapter sequence further comprises a barcode sequence that serves as a surrogate for identifying the analyte.

35. The method of any one of claims 1, 2, and 4, wherein the first probe comprises at least two ribonucleobases at the 3' end, and wherein the second probe comprises a phosphorylated nucleotide at the 5' end.

36. The method of any one of claims 1, 2, and 4, wherein generating a ligation product comprises ligating (i) the first probe to the second probe or (ii) the extended first probe to the second probe using enzymatic ligation or chemical ligation, wherein the enzymatic ligation uses a ligase.

37. The method of claim 36, wherein the ligase is T4 RNA ligase (Rn12), Chlorella DNA ligase, single-stranded DNA ligase, or T4 DNA ligase.

38. The method of claim 37, wherein the ligase is PBCV-1 ligase.

39. The method of any one of claims 1, 2, and 4, wherein the second probe comprises a pre-adenylated phosphate group at its 5' end, and wherein the first probe comprises at least two ribonucleobases at its 3' end.

40. The method of claim 39, wherein generating a ligation product comprises ligating the 3' end of the first probe to the 5' end of the second probe using a ligase that does not require adenosine triphosphate for ligase activity.

41. The method of claim 40, wherein the ligase is selected from the group consisting of thermostable 5' App DNA / RNA ligase, truncated T4 RNA ligase 2, truncated T4 RNA ligase 2K227Q, truncated T4 RNA ligase 2KQ, Chlorella virus PBCV-1 DNA ligase, or any combination thereof.

42. The method of any one of claims 1, 2, and 4, wherein the first probe further comprises a functional sequence, wherein the functional sequence is a primer sequence.

43. The method of any one of claims 1, 2, and 4, further comprising providing a capture probe capture domain blocking moiety that interacts with the capture probe capture domain.

44. The method of claim 43, further comprising releasing the capture probe capture domain blocking moiety from the capture probe capture domain prior to hybridizing the ligation product to the capture domain.

45. The method of claim 44, wherein the capture probe capture domain comprises a polyadenylation (poly(A)) sequence or its complement.

46. ​​The method of claim 45, wherein the capture probe capture domain blocking portion comprises a polyuridine sequence, a polythymidine sequence, or both.

47. The method of claim 46, wherein releasing the polyuridine sequence from the poly (A) sequence comprises denaturing the ligation product or contacting the ligation product with an endonuclease, exonuclease, or ribonuclease.

48. The method of any one of claims 1, 2, and 4, wherein the capture domain of the capture probe comprises a sequence that is complementary to all or part of the capture domain of the capture probe.

49. The method of any one of claims 1, 2, and 4, wherein the capture probe capture domain comprises a degenerate sequence.

50. The method of any one of claims 1, 2, and 4, wherein the first probe and / or the second probe is a DNA probe.

51. The method of claim 12, wherein the third probe is a DNA probe.

52. The method of claim 43, wherein the capture probe capture domain blocking moiety is a DNA probe.

53. The method of any one of claims 1, 2, and 4, wherein releasing the ligation product from the analyte comprises removing the ligation product from the analyte.

54. The method of claim 43, wherein (i) releasing the ligation product from the analyte or (ii) releasing the capture probe capture domain blocking portion from the capture domain binding domain comprises contacting the ligation product with an endoribonuclease.

55. The method of claim 54, wherein the endoribonuclease is RNase H, RNase A, RNase C, or RNase I.

56. The method of claim 55, wherein the RNase H comprises RNase H1, RNase H2, or RNase H1 and RNase H2.

57. The method of any one of claims 1, 2, and 4, wherein the biological sample is a tissue sample.

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

59. The method of claim 58, wherein the tissue sample is an FFPE tissue sample and the tissue sample is de-crosslinked.

60. The method of any one of claims 1, 2, and 4, wherein the biological sample is pre-stained.

61. The method of claim 60, wherein the biological sample is previously stained using immunofluorescence or immunohistochemistry.

62. The method of claim 60, wherein the biological sample is previously stained with hematoxylin and eosin.

63. The method of any one of claims 1, 2, and 4, wherein the method further comprises contacting the biological sample with a permeabilizing agent, wherein the permeabilizing agent is an organic solvent, a detergent, an enzyme, or a combination thereof.

64. The method of claim 63, wherein the permeabilizing agent is selected from the group consisting of protease, sodium dodecyl sulfate (SDS), polyethylene glycol tert-octylphenyl ether, polysorbate 80 and polysorbate 20, N-lauroyl sarcosine sodium salt solution, and saponin.

65. The method of claim 63, wherein the permeabilizing agent is selected from the group consisting of endopeptidase, Triton X-100 TM and Tween-20 TM .

66. The method of claim 65, wherein the endopeptidase is pepsin or proteinase K.

67. The method of any one of claims 1, 2, and 4, wherein the method further comprises fixing the biological sample prior to providing the biological sample.

68. The method of claim 67, wherein the biological sample is fixed using one or both of methanol and acetone.

69. The method of any one of claims 1, 2, and 4, wherein the analyte comprises RNA.

70. The method of claim 69, wherein the RNA is mRNA.

71. The method of any one of claims 1, 2, and 4, wherein said determining comprises amplifying all or a portion of said ligation product that is bound to said capture domain, thereby generating an amplified product.

72. The method of claim 71, wherein the amplification product comprises (i) all or part of the sequence of the ligation product bound to the capture domain, or its complement, and (ii) the sequence of the spatial barcode, or its complement.

73. The method of any one of claims 1, 2, and 4, wherein said determining comprises sequencing.

74. The method of claim 73, wherein the sequencing comprises in situ sequencing, Sanger sequencing, next generation sequencing, or nanopore sequencing.

75. The method of any one of claims 1, 2, and 4, wherein contacting the biological sample with a first probe and a second probe comprises contacting the biological sample with 5000 or more probe pairs comprising the first probe and the second probe.

76. The method of any one of claims 1, 2, and 4, wherein contacting the biological sample with the first probe and the second probe comprises contacting the biological sample with 100 or more probe pairs comprising the first probe and the second probe.

77. A composition comprising: a spatial array comprising capture probes, wherein the capture probes comprise a spatial barcode and a capture domain; A biological sample on the spatial array, wherein the biological sample comprises a plurality of analytes of interest and a first probe oligonucleotide and a second probe oligonucleotide that hybridize and are linked to the analytes of the plurality of analytes of interest, wherein the first probe oligonucleotide and the second probe oligonucleotide each comprise a sequence that is substantially complementary to an adjacent sequence of the analytes, and wherein one of the first probe or the second probe comprises a capture probe capture domain.

78. A composition comprising: (a) a biological sample disposed on a first substrate, wherein the biological sample comprises an analyte; and (b) a second substrate comprising a spatial array comprising a plurality of capture probes immobilized on the second substrate, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode comprising a sequence providing a location of an analyte and (ii) a capture domain, wherein 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 spatial array; (c) a ligation product comprising a first probe and a second probe, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to a sequence of an analyte, and wherein one of the first probe or the second probe comprises a capture domain of a capture probe that hybridizes to a capture domain of a capture probe on a second substrate.

79. A composition comprising: (a) a biological sample disposed on a first substrate, wherein the biological sample comprises a target nucleic acid; and (b) an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode comprising a sequence providing a location of a target nucleic acid and (ii) a capture domain; (c) a ligation product comprising a first probe and a second probe, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to a sequence that is not adjacent to each other on the target nucleic acid, and wherein one of the first probe or the second probe comprises a capture domain of the capture probe that hybridizes to the capture domain of the capture probe; and (d) Polymerase.

80. The composition of claim 79, wherein the polymerase is a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

81. The composition of claim 79, wherein the polymerase is Mu polymerase, VENT polymerase, or Taq polymerase.

82. The composition of claim 79 or 80, wherein the polymerase is a DNA polymerase.

83. The composition of claim 79 or 80, wherein the array is on a second substrate.

84. The composition of claim 83, wherein 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 array.

85. The composition of any one of claims 77-79, wherein the composition further comprises an RNase H enzyme.

86. The composition of any one of claims 77-79, wherein the composition further comprises a ligase.

87. The composition of any one of claims 77-79, wherein one of the first probe or second probe oligonucleotide that does not comprise a capture domain of a capture probe comprises a functional domain.

88. The composition of any one of claims 77-79, further comprising 100 or more ligation product pairs, wherein the ligation product pairs of the 100 or more ligation product pairs comprise a first probe and a second probe ligated together, and wherein the ligation product pairs hybridize to the capture domain of the capture probe.

89. The composition of any one of claims 77-79, wherein the biological sample is a tissue sample, and wherein the tissue sample is a formalin-fixed, paraffin-embedded tissue sample, a fresh tissue sample, or a frozen tissue sample.

90. The composition of any one of claims 77-79, wherein the biological sample is previously stained with hematoxylin and eosin, immunofluorescence, or immunohistochemistry.

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