Compositions and methods for improved on-target spatial profiling

Abstract

Compositions and methods for enhanced resolution of spatial profiling of nucleic acids in biological samples including single-stranded (ss)DNA regions have been developed. The methods reduce or prevent off-target binding of analyte-binding probes with ssDNA within biological samples to enhance resolution, sensitivity, specificity and / or accuracy of spatial analysis of targeted analytes. The methods comprise extension using ssDNA as a template to provide a complementary second DNA strand, forming a "blocked sample" having less ssDNA than the biological sample prior to being treated in accordance with the disclosed methods. In some embodiments, the methods employ a multiplicity of block primers having designed or random sequences that hybridize with accessible regions of ssDNA within a biological sample. In some embodiments, the methods employ extension of one or more regions of ssDNA in a biological sample using a primer-free polymerase to provide a blocked sample.

Description

[0001] COMPOSITIONS AND METHODS FOR IMPROVED ON-TARGET SPATIAL PROFILING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 657,548 filed June 7, 2024, which is hereby incorporated by reference in its entirety.

[0004] REFERENCE TO THE SEQUENCE LISTING

[0005] The Sequence Listing XML submitted as a file named “GENOMX_2023_101_PCT_ST26.xml”, created on June 4, 2025, and having a size of 1,838 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0006] FIELD OF THE DISCLOSURE

[0007] The present disclosure is generally in the field of molecular profiling of analytes present in a biological sample, specifically enhanced systems for spatial profiling of nucleic acids.

[0008] BACKGROUND OF THE DISCLOSURE

[0009] Cells within a tissue of a subject have differences in cell morphology and / or function due to varied analyte levels (e.g., gene and / or protein expression) within the different cells.

[0010] The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell’s position relative to the tissue microenvironment) can affect, e.g., the cell’s morphology, differentiation, fate, viability, proliferation, behavior, and signaling and crosstalk with other cells in the tissue. Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a segment of a tissue or provide a lot of analyte data for single cells but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).

[0011] The spatial heterogeneity in developing systems has typically been studied via RNA hybridization, immunohistochemistry, fluorescent reporters, or purification or induction of pre-defined subpopulations and subsequent genomic profiling e.g., RNA- seq). Such approaches, however, rely on a small set of pre-defined markers, therefore introducing selection bias that limits discovery and increases the cost and labor required to localize RNA on transcriptome-wide basis. Methods for spatial profiling of analytes present in a biological sample include the use of spatially barcoded substrates and / or nucleic acid probes to detect analytes. The presence of single stranded nucleic acids in a sample can lead to off-target binding of analyte-specific probes, and reduced quality (e.g., resolution, specificity) of spatial analysis.

[0012] There is a need for enhanced methods for reducing off-target effects during spatial analysis of nucleic acids and / or proteins in biological samples.

[0013] SUMMARY

[0014] Compositions and methods for enhanced spatial profiling of analytes (e.g., nucleic acids) in a biological sample including single-stranded (ss)DNA have been developed. The methods reduce or prevent off-target binding of analyte-binding probes with ssDNA within the sample to enhance the resolution and accuracy of spatial analysis of the targeted analytes. In some embodiments, an extension reaction is performed using ssDNA as a template to provide a complementary second DNA strand. In some embodiments, the methods employ a multiplicity of block primers having targeted or random sequences that hybridize with accessible regions of ssDNA within a sample and extend the block primers using the ssDNA as a template to provide a complementary second DNA strand, forming a “blocked sample” in which there is less ssDNA than in the biological sample prior to the methods.

[0015] Methods for processing a biological sample typically include (a) performing an extension reaction in a biological sample, whereby the extension includes using one or more regions of ssDNA in the biological sample as template to generate double stranded DNA (dsDNA), thereby providing a blocked sample; (b) contacting the blocked sample with a probe or probe set to detect a target RNA analyte in the blocked sample; and (c) performing an amplification reaction using the probe or probe set and detecting a product of the amplification reaction in the blocked sample. In some forms the biological sample is contacted with a polymerase in (a) and an additional polymerase in (c). In some forms, the polymerase and the additional polymerase are different. In some forms, the additional polymerase is a Phi29 DNA polymerase. In some forms, the one or more region / s) of ssDNA includes genomic ssDNA. In some forms, the biological sample is de-crosslinked prior to performing the extension reaction. In some forms, a sequence of the probe or probe set, or a complement thereof, is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some forms, the product of the amplification reaction includes one or more barcode sequences corresponding to the target RNA analyte or complements thereof. In some forms, the amplification reaction includes rolling circle amplification (RCA). In some forms, the biological sample is a Formaldehyde-Fixed Paraffin-Embedded (FFPE) tissue sample or tissue section. In some forms, the de-crosslinking includes heating the biological sample. In some forms, the heating includes exposing the biological sample to a temperature of at least about 70 °C. In some forms, the probe or probe set includes a circularizable probe or probe set. In some forms, the circularizable probe is a padlock probe. In some forms, the methods further include ligating the circularizable probe or probe set to form a circular nucleic acid. In some forms, the ligating includes performing an enzymatic ligation to connect a 5’ end of the circularizable probe to a 3’ end of the circularizable probe. In some forms, the blocked sample is contacted with a plurality of probes or probe sets configured for detecting a plurality of target RNA analytes. In some forms, the one or more target analyte or the target RNA analyte includes mRNA.

[0016] In other forms, methods for processing a biological sample typically include the steps of

[0017] (a) contacting a biological sample including a nucleic acid molecule including one or more regions of ssDNA with a multiplicity of block primers configured to hybridize with the one or more regions of ssDNA present in the biological sample; (b) extending the block primers using the one or more regions of ssDNA to provide a blocked sample; and (c) optionally washing the blocked sample to remove any unhybridized block primers and / or block primer dimers.

[0018] In some embodiments the method further includes, prior to step (a), providing the biological sample disposed on a first substrate. In some embodiments the methods further include (d) contacting the biological sample with a first probe and optionally a second probe, wherein the first probe and optionally the second probe each include sequences that are substantially complementary to sequences of an RNA analyte present within the blocked sample, and wherein the first probe or optionally the second probe includes a capture probe binding domain. In some embodiments the method further includes (e) hybridizing the first probe and optionally the second probe to the RNA analyte, coupling the first probe and optionally the second probe, and ligating the probes to form a connected probe. In some embodiments the method further includes (f) aligning the biological sample with a second substrate including an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array includes a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes: (i) a spatial barcode; and (ii) a capture domain. In some embodiments the method further includes (g) releasing the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array to provide a released probe. In some embodiments the method further includes (h) hybridizing the released probe to the capture domain of the capture probe. In some embodiments, the capture probe binding domain is substantially complementary to the capture domain of the capture probe. In some embodiments the blocked sample includes less ssDNA than the biological sample prior to step (a).

[0019] In other forms, methods for analyzing an RNA analyte in a biological sample include the steps of (a) providing the biological sample disposed on a first substrate; (b) contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of single-stranded DNA (ssDNA) present in the biological sample; (c) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample, optionally wherein the blocked sample includes less ssDNA than the biological sample prior to step (b); (d) hybridizing a first probe and a second probe to the RNA analyte, wherein the first probe and the second probe each include a sequence that is substantially complementary to sequences of the RNA analyte, wherein the second probe includes a capture probe binding domain; (e) coupling the first probe and the second probe, thereby generating a connected probe; (f) aligning the first substrate with a second substrate including an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array includes a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes: i. a spatial barcode; and ii. a capture domain; (g) releasing the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array; and (h) hybridizing the connected probe via the capture probe binding domain to the capture domain of the capture probe. In some embodiments, the capture probe binding domain is substantially complementary to the capture domain of the capture probe.

[0020] In some embodiments the methods further include: (i) determining: I. all or a part of the sequence of the connected probe, or a complement thereof, and / or II. the sequence of the spatial barcode, or a complement thereof. In some embodiments the methods further include: (j) using the determined sequences of I and II to provide the sequence and / or location of the RNA analyte in the biological sample. In some embodiments extending the block primers across the length of the one or more regions of ssDNA includes contacting the biological sample with a polymerase enzyme. In some embodiments the polymerase enzyme is selected from the group including DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V. In some embodiments the RNA analyte includes mRNA. In some embodiments the one or more regions of ssDNA includes genomic ssDNA. In some embodiments, the biological sample includes a tissue sample. In some embodiments the method further includes permeabilizing the tissue sample, optionally permeabilizing the tissue sample prior to step (a). In some embodiments, the tissue sample includes a frozen and / or lyophilized tissue sample. For example, in some embodiments, the frozen tissue sample includes a fresh frozen tissue sample or a fresh frozen tissue section. In particular embodiments, the fresh frozen sample or fresh frozen tissue section is frozen following removal from a biological source, preferably without further processing. In some embodiments, the tissue sample includes a fixed tissue sample or a fixed tissue section. Exemplary fixatives are selected from the group including ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, and glutaraldehyde, or combinations thereof. In some embodiments, the fixed tissue sample includes a Paraffin-Embedded (PE) tissue sample, or the fixed tissue section includes an PE tissue section; and the method further includes deparaffinizing the PE tissue sample or PE tissue section. In some embodiments, the fixed tissue sample includes a Formaldehyde-Fixed Paraffin- Embedded ((FF)PE) tissue sample or the fixed tissue section includes an (FF)PE tissue section. In some embodiments, the deparaffinizing includes contacting the (FF)PE tissue sample or (FF)PE tissue section with a solvent, Exemplary solvents include: (i) a solvent including xylene; or (ii) a solvent including ethanol; or (iii) a solvent including xylene, followed by a solvent including ethanol. In some embodiments, the methods further include de-crosslinking the tissue sample, optionally de-crosslinking the tissue sample prior to step (a). In some embodiments, the de-crosslinking includes heating the biological sample and / or contacting the biological sample with an alkaline solvent and / or an acidic solvent. In some embodiments, the heating includes exposing the biological sample to a temperature of between about 70 °C and about 99 °C, inclusive. In some embodiments, the heating includes exposing the biological sample to a temperature of about 95 °C. In some embodiments, the de-crosslinking includes contacting the tissue sample with de-crosslinking buffer. In some embodiments, the tissue sample includes at least one dimension having a thickness of between about 1 pm and about 20 pm, inclusive; about 5 pm and about 15 m, inclusive; or about 10 pm and about 12 pm, inclusive.

[0021] In some embodiments, the tissue sample is disposed on a first substrate including a glass slide; in other embodiments, the method further includes staining and / or labelling the tissue sample, optionally before or after step (a). Exemplary stains include hematoxylin and / or eosin (H and E) stains. In some embodiments, the methods further include imaging the tissue sample. In some embodiments, the methods further include de-staining a stained tissue sample. In some embodiments, contacting the biological sample with a multiplicity of block primers occurs under conditions suitable for hybridizing the block primers to the one or more regions of ssDNA. In some embodiments, the multiplicity of block primers includes between one and one hundred million species of oligonucleotide sequences. In some embodiments, the oligonucleotide sequences include fixed sequence(s) and / or partially or completely randomly-generated sequences of between about 6 nucleotides and about 40 nucleotides, inclusive. In particular embodiments, the sequence of one or more of the oligonucleotide sequences includes GAGAATGTGAGTGAAGATGTATGGTGANNNNNNN (SEQ ID NO:1), wherein N is A, G, T, or C. In some embodiments, the multiplicity of block primers includes between one and one hundred thousand species of fixed oligonucleotide sequences of about 6 nucleotides and about 40 nucleotides, inclusive. In some embodiments, the multiplicity of block primers include a set of random hexamers including 4,096 species of oligonucleotide sequences. In some embodiments, a block primer of the multiplicity of block primers includes a guanine / cytosine (GC) content of from about 30% to about 70%, inclusive. In some embodiments, the 3’ terminus of the block primer includes guanine (G) or cytosine (C). In some embodiments, a block primer of the multiplicity of block primers includes one or more of DNA, LNA, PNA, UNA, TNA phosphorothioate and P5' phosphoramidate. In some embodiments, the step of contacting the biological sample with a multiplicity of block primers further includes contacting the biological sample with one or more reagents for block primer extension. In some embodiments, the one or more reagents for block primer extension includes a polymerase enzyme, deoxynucleotide triphosphates (dNTPs), and / or a suitable reaction buffer for block primer extension. In some embodiments, the polymerase enzyme includes a DNA polymerase that catalyzes synthesis of DNA from a DNA template. In some forms, the DNA polymerase enzyme does not catalyze the synthesis of DNA from an RNA template. In some embodiments, the polymerase enzyme is selected from DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V. In some embodiments, the polymerase enzyme is a thermostable DNA polymerase I. In some embodiments the thermostable DNA polymerase I is derived from Thermits aquaticus. In some embodiments, the method further includes, following the step of contacting the biological sample with a multiplicity of block primers, one or more steps for washing the biological sample to remove unbound / unhybridized block primers and / or primer extension reagents from the blocked sample. In exemplary forms, the washing includes: (i) contacting the blocked sample with a suitable solvent in an amount and time effective to solubilize the block primers and / or primer extension reagents; and (ii) removing the blocked sample from the solvent. In some embodiments, the solvent includes an aqueous buffer and optionally one or more additional reagents. An exemplary aqueous buffer includes PBS and a polysorbate. In some forms, the methods include repeating the washing steps (i) and (ii) one or more times. In some embodiments, the methods further include contacting the blocked sample with one or more additives in an amount and time effective to stabilize one or more regions of double strand DNA (dsDNA) formed by extension of one or more blocked primers in the blocked sample. An exemplary additive includes spermine and / or methylene blue. In some embodiments, the amount of ssDNA within the blocked sample is less than 50%, 40%, 30% 20%, 10%, 5%, 2%, 1%, or 0.1% of the amount of ssDNA within the biological sample prior to contacting the biological sample with the multiplicity of block primers. In some embodiments, the sequence of RNAs within the blocked sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of RNAs within the biological sample prior to contacting the biological sample with the multiplicity of block primers. In some embodiments, the abundance of RNAs within the blocked sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the abundance of RNAs within the tissue sample prior to contacting the biological sample with the multiplicity of block primers.

[0022] Methods including a first and second RNA-templated Ligation (RTL) probes are also provided In some embodiments, the first RTL probe hybridizes to a first sequence in the RNA analyte, and the second RTL probe hybridizes to a second sequence in the RNA analyte, wherein the first and second sequences are contiguous, and / or wherein one or more ends of the hybridized first and second RTL probes are adjacent to one another. In some embodiments, the first RTL probe hybridizes to a first sequence in the RNA analyte, and the second RTL probe hybridizes to a second sequence in the RNA analyte, wherein the first and second sequences are not contiguous, and / or wherein a 3’ end of the hybridized first RTL probe and a 5 ’end of the hybridized second RTL probes are separated by a gap or a 5’ end of the hybridized first RTL probe and a 3 ’end of the hybridized second RTL probes are separated by a gap. In some embodiments the methods further include filling the gap between the hybridized first and second RTL probes. In some embodiments the methods, further include contacting the biological sample with a polymerase enzyme to extend the first and / or second RTL probes using the RNA analyte as a template to fill in the gap between the hybridized first and second RTL probes. In some embodiments the methods, further include ligating the first and second RTL probes to form the connected probe. In some embodiments the ligating includes chemical ligation. In some embodiments, the ligating includes contacting the biological sample with a ligase enzyme. In some embodiments, the first RTL probe, or second RTL probe, or both first and second RTL probes have bound thereto one or more functional moieties. Exemplary functional moieties include one or more of a label, an imaging agent, a contrast agent or a dye. An exemplary label is a fluorophore.

[0023] In some embodiments, the polymerase enzyme is a DNA polymerase I derived from Thermits aquaticus. In some embodiments, the tissue sample includes tissue from a human cancer selected from the group including ovarian cancer, lung cancer, tonsil cancer, glioblastoma, and prostate cancer. In some embodiments, the methods further include: (d) contacting the blocked sample with a circularizable probe or probe set including sequences that are substantially complementary to sequences of an RNA analyte present within the blocked sample. In some embodiments, the methods further include: (e) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and (f) detecting the RCP in the biological sample. In some embodiments, detecting RCP in the sample includes detecting a barcode sequence or a complement thereof in the RCP. In some embodiments, detecting the barcode sequence or complement thereof includes: contacting the biological sample with a universal pool of detectably labeled probes and a first pool of intermediate probes, wherein the intermediate probes of the first pool of intermediate probes include hybridization regions complementary to the barcode sequence or complement thereof and reporter regions complementary to a detectably labeled probe of the universal pool of detectably labeled probes; detecting complexes formed between the barcode sequence or complement thereof, the intermediate probes of the first pool of intermediate probes, and the detectably labeled probes; and removing the intermediate probes of the first pool of intermediate probes and the detectably labeled probes. In some embodiments, detecting the barcode sequences or complements thereof further includes: contacting the biological sample with the universal pool of detectably labeled probes and a second pool of intermediate probes, wherein the intermediate probes of the second pool of intermediate probes include hybridization regions complementary to the barcode sequence or complement thereof and reporter regions complementary to a detectably labeled probe of the universal pool of detectably labeled probes; and detecting complexes formed between the barcode sequence or complement thereof, the intermediate probes of the second pool of intermediate probes, and the detectably labeled probes. In some embodiments, the barcode sequence or complement thereof is assigned a series of signal code that identifies the barcode sequence or complement thereof, and detecting the barcode sequence or a complement thereof includes decoding the barcode sequence or complement thereof by detecting the corresponding sequences of signal code detected from sequential hybridization, detection, and removal of sequential pools of intermediate probes and the universal pool of detectably labeled probes. In some embodiments, the series of signal code is a fluorophore sequence assigned to the corresponding barcode sequence or a complement thereof. In some embodiments, the detectably labeled probes are fluorescently labeled. In some embodiments, the method includes imaging the blocked sample to detect the RCP. In some embodiments, the imaging includes detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCP. In some embodiments, a sequence of the RCP is analyzed at a location in the biological sample or a matrix embedding the biological sample. In some embodiments, the sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

[0024] Kits of parts are also provided. An exemplary kit includes a multiplicity of block primers configured to hybridize with single- stranded DNA (ssDNA), and / or a polymerase enzyme, and instructions for performing the described methods. In some embodiments, the multiplicity of block primers includes between one and one hundred million species of fixed oligonucleotide sequence(s) and / or partially or completely randomly generated sequences each including about 6 nucleotides to about 40 nucleotides, inclusive. An exemplary sequence of one or more of the block primers includes GAGAATGTGAGTGAAGATGTATGGTGANNNNNNN (SEQ ID NO:1), wherein each “N” is, independently, A, G, T, or C. In some embodiments, the multiplicity of block primers includes between one and one hundred thousand species of oligonucleotide sequences including from about 6 nucleotides and about 40 nucleotides, inclusive. In an exemplary form, a multiplicity of block primers includes a set of random hexamers including 4,096 species of oligonucleotide sequences. In some embodiments, the described kits further include one or more of (i) a solvent suitable to solubilize the oligonucleotides and / or polymerase enzyme; (ii) deoxynucleotide triphosphates (dNTPs); (iii) a suitable reaction buffer for primer extension; and (iv) one or more inhibitors of the polymerase enzyme.

[0025] Methods for analyzing an RNA analyte in a biological sample are also provided. Typically, the methods include the steps of: (a) contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of singlestranded DNA (ssDNA) present in the biological sample; (b) extending the block primers across one or more regions of ssDNA to provide a blocked sample, optionally wherein the blocked sample includes less ssDNA than the biological sample prior to step (b); (c)hybridizing a first probe and a second probe to the RNA analyte, wherein the first probe and the second probe each include a sequence that is substantially complementary to sequences of the RNA analyte, wherein the second probe includes a capture probe binding domain; (d) coupling the first probe and the second probe, thereby generating a connected probe; (e) releasing the connected probe from the RNA analyte;(f) hybridizing the connected probe to a capture domain of a capture probe on an array, wherein the array includes a plurality of capture probes, wherein the capture probe is included in the plurality of capture probes, and wherein the capture probe includes: (i) a spatial barcode; and (ii) the capture domain. In some embodiments, the biological sample is disposed on a substrate including the array including the plurality of capture probes. Methods for processing a biological sample are also provided. Typically, the methods include: (a) contacting a biological sample including a nucleic acid with a polymerase, wherein the nucleic acid includes one or more regions of ssDNA; (b) performing an extension using the one or more regions of ssDNA as template to generate double stranded DNA (dsDNA), thereby providing a blocked sample; and (c) contacting the blocked sample with a probe or probe set to detect one or more target analyte in the blocked sample. In some embodiments, 87, further including: (d) detecting the probe or probe set in the biological sample. In some embodiments, the polymerase is a Phi29 DNA polymerase. In some embodiments, the polymerase is provided in a reaction mixture that includes deoxynucleotide triphosphates (dNTPs) and / or nucleotide triphosphates (NTPs). In some embodiments, wherein the reaction mixture includes a cofactor of the polymerase. In some embodiments, the cofactor of the polymerase is a di-cation selected from the group including Mg2+, Co2+, and Mn2+. In some embodiments, the blocked sample includes less ssDNA than the biological sample prior to step (a). In some embodiments, the method further includes, prior to step (a), providing the biological sample disposed on a first substrate. In some embodiments, the probe set includes a first probe and a second probe, and wherein the first probe and the second probe each include sequences that are substantially complementary to sequences of an RNA analyte present within the blocked sample. In some embodiments, the first probe or the second probe includes a capture probe binding domain.

[0026] In some embodiments, the method further includes coupling the first probe and the second probe and ligating the probes to form a connected probe. In some embodiments, a sequence of the probe or probe set, or a complement thereof, is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some embodiments, the probe or probe set includes a circular probe or a circularizable probe or probe set, wherein the circular probe or the circularizable probe or probe set includes a target recognition sequence complementary to a target sequence in a target RNA in the blocked sample and, wherein the circular probe or the circularizable probe or probe set hybridizes to the target RNA. In some embodiments, the method further includes performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP). In some embodiments, the method further includes detecting the RCP at a location in the blocked sample. In some embodiments, the circularizable probe or probe set includes one or more ribonucleotides. In some embodiments, a 3’ end and a 5’ end of the circularizable probe or probe set are ligated using the target RNA as a template. In some embodiments, the method includes imaging the blocked sample to detect the probe or probe set or a product thereof. In some embodiments, the imaging includes detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCP. In some embodiments, a sequence of the RCP is analyzed at a location in the biological sample or a matrix embedding the biological sample. In some embodiments, the sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof. In some embodiments, the sequence of the RCP product includes one or more barcode sequences or complements thereof. In some embodiments, the one or more barcode sequences or complements thereof correspond to the target RNA. In some embodiments, the RCP is immobilized in the biological sample and / or crosslinked to one or more other molecules in the biological sample. In some embodiments, wherein detecting the probe or probe set in the biological sample includes: contacting the blocked sample with a universal pool of detectably labeled probes and a first pool of intermediate probes, wherein the intermediate probes of the first pool of intermediate probes include hybridization regions complementary to the barcode sequence or complement thereof and reporter regions complementary to a detectably labeled probe of the universal pool of detectably labeled probes; detecting complexes formed between the barcode sequence or complement thereof, the intermediate probes of the first pool of intermediate probes, and the detectably labeled probes; and removing the intermediate probes of the first pool of intermediate probes and the detectably labeled probes. In some embodiments, the barcode sequence or complement thereof is assigned a series of signal codes that identifies the barcode sequence or complement thereof, wherein detecting the barcode sequences or complements thereof includes decoding the barcode sequence or complement thereof by detecting the corresponding sequence of signal codes detected from sequential hybridization, detection, and removal of sequential pools of intermediate probes and the universal pool of detectably labeled probes. In some embodiments, the series of signal codes are fluorophore sequences assigned to the corresponding barcode sequence or complement thereof. In some embodiments, the one or more regions of ssDNA includes genomic ssDNA. In some embodiments, the biological sample includes a tissue sample. In some embodiments, the methods further include permeabilizing the tissue sample, optionally permeabilizing the tissue sample prior to (a). In some embodiments, the tissue sample includes a frozen and / or lyophilized tissue sample. In some embodiments, the frozen tissue sample includes a fresh frozen tissue sample or a fresh frozen tissue section, optionally wherein the fresh frozen sample or fresh frozen tissue section is frozen following removal from a biological source, preferably without further processing. In some embodiments, the tissue sample includes a fixed tissue sample or a fixed tissue section. In some embodiments, the fixed tissue sample or fixed tissue section is fixed with a fixative selected from the group including ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, and glutaraldehyde, or combinations thereof. In some embodiments, the fixed tissue sample includes a Paraffin- Embedded (PE) tissue sample or the fixed tissue section includes an PE tissue section; and wherein the method further includes deparaffinizing the PE tissue sample or PE tissue section. In some embodiments, the fixed tissue sample includes a Formaldehyde- Fixed Paraffin-Embedded (FFPE) tissue sample or the fixed tissue section includes an FFPE tissue section. In some embodiments, the deparaffinizing includes contacting the (FF)PE tissue sample or (FF)PE tissue section with a solvent, optionally (i) a solvent including xylene; or (ii) a solvent including ethanol; or (iii) a solvent including xylene, followed by a solvent including ethanol. In some embodiments, the method further includes de-crosslinking the tissue sample, optionally de-crosslinking the tissue sample prior to step (a). In some embodiments, the de-crosslinking includes heating the biological sample and / or contacting the biological sample with an alkaline solvent and / or an acidic solvent. In some embodiments, the heating includes exposing the biological sample to a temperature of between about 70 °C and about 99 °C, inclusive. In some embodiments, the heating includes exposing the biological sample to a temperature of about 95 °C. In some embodiments, the de-crosslinking includes contacting the tissue sample with de-crosslinking buffer.

[0027] In some embodiments, the tissue sample is a tissue section between about 1 pm and about 50 pm in thickness, optionally wherein the tissue section is between about 5 pm and about 35 pm in thickness. In some embodiments, the tissue sample is a tissue section between about 10 pm and about 25 pm in thickness.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings illustrate certain forms of the features and advantages of this disclosure. These forms are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

[0030] FIG. 1A shows an exemplary sandwiching process where a first substrate (<?.g., a slide), including a biological sample, and a second substrate (e.g., array slide) are brought into proximity with one another.

[0031] FIG. IB shows a fully formed sandwich configuration creating a chamber formed from the one or more spacers, the first substrate, and the second substrate.

[0032] FIG. 2A shows a perspective view of an exemplary sample handling apparatus in a closed position.

[0033] FIG. 2B shows a perspective view of an exemplary sample handling apparatus in an open position. FIG. 3A shows the first substrate angled over (superior to) the second substrate.

[0034] FIG. 3B shows that as the first substrate lowers, and / or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium.

[0035] FIG. 3C shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate.

[0036] FIG. 4A shows a side view of the angled closure workflow.

[0037] FIG. 4B shows a top view of the angled closure workflow.

[0038] FIG. 5 is a schematic diagram showing an example of a barcoded capture probe, as described herein.

[0039] FIG. 6 shows a schematic illustrating a cleavable capture probe.

[0040] FIG. 7 shows exemplary capture domains on capture probes.

[0041] FIG. 8 shows an exemplary arrangement of barcoded features within an array.

[0042] FIG. 9A shows an exemplary workflow for performing a templated capture and producing a ligation product, and FIG. 9B shows an exemplary workflow for capturing a ligation product from FIG. 9A on a substrate.

[0043] FIG. 10 is a schematic diagram of an exemplary analyte capture agent.

[0044] FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1124 and an analyte capture agent 1126.

[0045] FIG. 12 is a schematic diagram depicting an example workflow of a method for preparing a blocked sample, starting from a dsDNA including multiple regions of singlestranded DNA. In some embodiments, the sample is contacted with block primers under conditions suitable for hybridizing the block primers to the ssDNA (1220); extending the block primers across the regions of ssDNA using a polymerase using the ssDNA as a template (1240); and subsequent removal of excess block primers and polymerase from the blocked sample (1260) are depicted.

[0046] FIG. 13 is a schematic diagram depicting an example workflow for preparing a blocked sample by extending ssDNA regions in the sample using a polymerase and the ssDNA as a template.

[0047] DETAILED DESCRIPTION

[0048] I. Spatial Analysis

[0049] Spatial analysis methodologies described herein can provide a vast amount of analyte and / or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte e.g., a protein and / or a nucleic acid) produced by and / or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with the intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.

[0050] Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos. 11,447,807, 11,352,667, 11,168,350, 11,104,936, 11,008,608, 10,995,361, 10,913,975, 10,774,374, 10,724,078, 10,640,816, 10,494,662, 10,480,022, 10,364,457, 10,317,321, 10,059,990, 10,041,949, 10,030,261, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, and 7,709,198; U.S. Patent Application Publication Nos. 2020 / 0239946, 2020 / 0080136, 2020 / 0277663, 2019 / 0330617, 2020 / 0256867, 2020 / 0224244, 2019 / 0085383, and 2013 / 0171621; PCT Publication Nos. WO2018 / 091676, WO2020 / 176788, WO2017 / 144338, and WO2016 / 057552; Non-patent literature references Rodriques et al., Science 363(6434): 1463- 1467, 2019; Lee et al., Nat. Protoc. 10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2) :e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and / or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), both of which are available at the lOx Genomics Support Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in their entireties. Further nonlimiting aspects of spatial analysis methodologies and compositions are described herein.

[0051] Some general terminology that may be used in this disclosure can be found in Section (I)(b) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and / or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.

[0052] Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some forms, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some forms, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Examples of nucleic acid analytes include, but are not limited to, DNA e.g., genomic DNA, cDNA) and RNA, including coding and non-coding RNA (e.g., mRNA, rRNA, tRNA, ncRNA). Additional examples of analytes can be found in Section (I)(c) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. In some forms, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.

[0053] A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In some forms, the biological sample is a tissue sample. In some forms, the biological sample (e.g., tissue sample) is a tissue microarray (TMA). A tissue microarray contains multiple representative tissue samples - which can be from different tissues or organisms - assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time. Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these tissue cores into a single recipient (microarray) block at defined array coordinates. In some embodiments, the biological sample is or comprises a cell pellet or a section of a cell pellet. In some embodiments, the biological sample is or comprises a cell block or a section of a cell block.

[0054] The biological sample as used herein can be any suitable biological sample described herein or known in the art. In some forms, the biological sample is a tissue. In some forms, the tissue sample is a solid tissue sample. In some forms, the biological sample is a tissue section (e.g., a fixed tissue section). In some forms, the tissue is flash- frozen and sectioned. Any suitable method described herein or known in the art can be used to flash-freeze and section the tissue sample. In some forms, the biological sample, e.g., the tissue, is flash-frozen using liquid nitrogen before sectioning. In some forms, the biological sample, e.g., a tissue sample, is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane.

[0055] In some forms, the biological sample, e.g., the tissue, is embedded in a matrix e.g., optimal cutting temperature (OCT) compound to facilitate sectioning. OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens. In some forms, the sectioning is performed using cryo-sectioning. In some forms, the methods further include a thawing step, after the cryo-sectioning.

[0056] The biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat. In addition to the subjects described above, the biological sample can be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegan.s), a fungus, an amphibian, or a fish (e.g., zebrafish)). A biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae', an archaeon; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). The biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and / or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. In some forms, an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre-disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy.

[0057] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0058] Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.

[0059] In some forms, the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, for example methanol. In some forms, instead of methanol, acetone, or an acetone-methanol mixture can be used. In some forms, the fixation is performed after sectioning. In some instances, the biological sample is not fixed with paraformaldehyde (PFA). In some instances, when the biological sample is fixed with a fixative including an alcohol (e.g., methanol or acetone-methanol mixture), it is not de-crosslinked afterward. In some preferred forms, the biological sample is fixed with a fixative including an alcohol (e.g., methanol or an acetone-methanol mixture) after freezing and / or sectioning. In some instances, the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone- methanol mixture). In some instances when methanol, acetone, or an acetone-methanol mixture is used to fix the biological sample, the sample is not de-crosslinked at a later step. In instances when the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone-methanol) fixation or acetone fixation, the biological sample is referred to as “fresh frozen”. In some forms, fixation of the biological sample e.g., using acetone and / or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g., glass slide, such as a positively charged glass slide).

[0060] In some forms, the biological sample, e.g., the tissue sample, is fixed e.g., immediately after being harvested from a subject. In such forms, the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PFA) or formalin. In some forms, the fixative induces crosslinks within the biological sample. In some forms, after fixing e.g., by formalin or PFA, the biological sample is dehydrated via sucrose gradient. In some instances, the fixed biological sample is treated with a sucrose gradient and then embedded in a matrix e.g., OCT compound. In some instances, the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix e.g., OCT compound after fixation. In some forms when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient. In some forms, the PFA or formalin fixed biological sample, which can be optionally dehydrated via sucrose gradient and / or embedded in OCT compound, is then frozen e.g., for storage or shipment. In such instances, the biological sample is referred to as “fixed frozen”. In preferred forms, a fixed frozen biological sample is not treated with methanol. In preferred forms, a fixed frozen biological sample is not paraffin embedded. Thus, in preferred forms, a fixed frozen biological sample is not deparaffmized. In some forms, a fixed frozen biological sample is rehydrated in an ethanol gradient.

[0061] In some instances, the biological sample {e.g., a fixed frozen tissue sample) is treated with a citrate buffer. Citrate buffer can be used for antigen retrieval to decrosslink antigens and fixation medium in the biological sample. Thus, any suitable decrosslinking agent can be used in addition to or alternatively to citrate buffer. In some forms, for example, the biological sample {e.g., a fixed frozen tissue sample) is decrosslinked with TE buffer.

[0062] In any of the foregoing, the biological sample can further be stained, imaged, and / or destained. For example, in some forms, a fresh frozen tissue sample or fixed frozen tissue sample is stained {e.g., via eosin and / or hematoxylin), imaged, destained {e.g., via HC1), or a combination thereof. In some forms, when a fresh frozen tissue sample is fixed in methanol, it is treated with isopropanol prior to being stained {e.g., via eosin and / or hematoxylin), imaged, destained {e.g., via HC1), or a combination thereof. In some forms when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient before being stained, {e.g., via eosin and / or hematoxylin), imaged, destained {e.g., via HC1), de-crosslinked {e.g., via TE buffer or citrate buffer), or a combination thereof. In some forms, the biological sample can undergo further fixation {e.g., while mounted on a substrate), stained, imaged, and / or destained. For example, a fixed frozen biological sample may be subject to an additional fixing step {e.g., using PFA) before optional ethanol rehydration, staining, imaging, and / or destaining. In any of the foregoing, the biological sample can be fixed using PAXgene. For example, the biological sample can be fixed using PAXgene in addition, or alternatively to, a fixative disclosed herein or known in the art e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde). PAXgene is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and biomolecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B. et al., J Proteome Res. 2010 Oct 1 ;9(10):5188-96; Kap M. et al., PLoS One:, 6(1 l):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol:, 146(1 ):25-40 (2016), each of which are hereby incorporated by reference in their entirety, for a description and evaluation of PAXgene for tissue fixation. Thus, in some forms, when the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, the fixative is PAXgene. In some forms, a fresh frozen tissue sample is fixed with PAXgene. In some forms, a fixed frozen tissue sample is fixed with PAXgene.

[0063] In some forms, the biological sample, e.g., the tissue sample is fixed, for example in methanol, acetone, acetone-methanol, PFA, PAXgene or is formalin-fixed and paraffin-embedded (FFPE). In some forms, the biological sample includes intact cells. In some forms, the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet. In some embodiments, the biological sample e.g., FFPE sample) is permeable after deparaffinization. In some embodiments, processing of the biological sample, such as de-waxing, allows the biological sample to become permeabilized. FFPE samples are used in some instances in the RNA-templated ligation (RTL) methods disclosed herein. A limitation of direct RNA capture for fixed samples is that the RNA integrity of fixed (e.g., FFPE) samples can be lower than a fresh sample, thereby making it more difficult to capture RNA directly, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule. However, by utilizing RTL probes that hybridize to RNA target sequences in the transcriptome, one can avoid a requirement for RNA analytes to have both a poly(A) tail and target sequences intact. Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples. The biological sample, e.g., tissue sample, can be stained, and imaged prior, during, and / or after each step of the methods described herein. Any of the methods described herein or known in the art can be used to stain and / or image the biological sample. In some forms, the imaging occurs prior to destaining the sample. In some forms, the biological sample is stained using an H&E staining method. In some forms, the tissue sample is stained and imaged for about 10 minutes to about 2 hours (or any of the subranges of this range described herein). Additional time may be needed for staining and imaging of different types of biological samples.

[0064] The tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject. In some instances, the sample is a mouse sample. In some instances, the sample is a human sample. In some forms, the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. In some instances, the sample is a human or mouse breast tissue sample. In some instances, the sample is a human or mouse brain tissue sample. In some instances, the sample is a human or mouse lung tissue sample. In some instances, the sample is a human or mouse tonsil tissue sample. In some instances, the sample is a human or mouse liver tissue sample. In some instances, the sample is a human or mouse bone, skin, kidney, thymus, testes, or prostate tissue sample. In some forms, the tissue sample is derived from normal or diseased tissue. In some forms, the sample is an embryo sample. The embryo sample can be a non-human embryo sample. In some instances, the sample is a mouse embryo sample.

[0065] Biological samples are also described in Section (I)(d) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0066] The following forms can be used with any of the methods described herein. In some forms, the biological sample (e.g., a fixed and / or stained biological sample) is imaged. In some forms, the biological sample is visualized or imaged using bright field microscopy. In some forms, the biological sample is visualized or imaged using fluorescence microscopy. Additional methods of visualization and imaging are known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. In some forms, the sample is stained and imaged prior to adding the primer to the biological sample.

[0067] In some forms, the methods include staining the biological sample. In some forms, the staining includes the use of hematoxylin and / or eosin. Non-limiting examples of stains include histological stains (e.g., hematoxylin and / or eosin) and immunological stains (e.g., fluorescent stains). In some forms, a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and / or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation.

[0068] In some forms, the staining includes the use of a detectable label selected from the group including a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

[0069] In some forms, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)( 13) or the Exemplary Forms Section of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Briefly, in any of the methods described herein, the method includes a step of permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of the extension products to the capture probes on the array. In some forms, the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, and methanol), a detergent (e.g., saponin, Triton X-100™, Tween- 20™, or sodium dodecyl sulfate (SDS)), an enzyme (an endopeptidase, an exopeptidase, a protease), or combinations thereof. In some forms, the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X-100™, Tween-20™, or combinations thereof. In some forms, the endopeptidase is pepsin. In some forms, the endopeptidase is Proteinase K. Additional methods for sample permeabilization are described, for example, in Jamur el al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference.

[0070] Array-based spatial analysis methods can involve the transfer of one or more analytes or derivatives thereof from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array.

[0071] A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and / or labelling an analyte (e.g., an analyte of interest) in a biological sample. In some forms, the capture probe is a nucleic acid or a polypeptide. In some forms, the capture probe includes a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI)) and a capture domain). In some instances, the capture probe includes a homopolymer sequence, such as a poly(T) sequence. In some forms, a capture probe can include a cleavage domain and / or a functional domain (e.g., a primer-binding site, such as for next-generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)- (vi)) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0072] In some instances, interaction between a capture probe and an analyte (or any other nucleic acid to nucleic acid interaction) occurs because the sequences of the two nucleic acids are substantially complementary to one another. By “substantial,” “substantially” and the like, two nucleic acid sequences can be complementary when at least 60% of the nucleotide residues of one nucleic acid sequence are complementary to nucleotide residues in the other nucleic acid sequence. The complementary residues within a particular complementary nucleic acid sequence need not always be contiguous with each other, and can be interrupted by one or more non-complementary residues within the complementary nucleic acid sequence. In some forms, at least 60%, but less than 100%, of the residues of one of the two complementary nucleic acid sequences are complementary to residues in the other nucleic acid sequence. In some forms, at least 70%, 80%, 90%, 95% or 99% of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence.

[0073] In some forms, the biological sample is mounted on a first substrate and the substrate including the array of capture probes is a second substrate. During this process, one or more analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) are released from the biological sample and migrate or transfer to the second substrate including an array of capture probes. In some forms, the release and migration / transfer of the analytes or analyte derivatives to the second substrate including the array of capture probes occurs in a manner that preserves the original spatial context of the analyte or analyte derivative in the biological sample. This method can be referred to as a sandwiching process, which is described e.g., in U.S. Patent Application Pub. No. 2021 / 0189475 and PCT Pub. Nos. WO 2021 / 252747 Al, WO 2022 / 061152 A2, and WO 2022 / 140028 Al.

[0074] Prior to transferring analytes from the biological sample to the array of features (e.g., containing capture probes) on the substrate, the biological sample can be aligned with the array. Alignment of a biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and / or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and / or level. Exemplary methods to generate a two- and / or three-dimensional map of the analyte presence and / or level are described in PCT Publication No. W02020 / 053655 and spatial analysis methods are generally described in PCT Publication No. W02021 / 102039 and / or U.S. Patent Application Publication No. 2021 / 0155982, each of which is incorporated herein by reference in their entireties.

[0075] FIG. 1A shows an exemplary sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102, and a second substrate (e.g., array slide 104 including an array having spatially barcoded capture probes 106) are brought into proximity with one another. As shown in FIG. 1A a liquid reagent drop (e.g., permeabilization solution 105) is introduced on the second substrate in proximity to the capture probes 106 and in between the biological sample 102 and the second substrate (e.g., slide 104 including an array having spatially barcoded capture probes 106). The permeabilization solution 105 may release analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) that can be captured by the capture probes of the array 106.

[0076] During the exemplary sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the capture probes (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., array slide 104) is in an inferior position to the first substrate (e.g., slide 103). In some forms, the first substrate (e.g., slide 103) may be positioned superior to the second substrate (e.g., slide 104). A reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates. The reagent medium may be a permeabilization solution which permeabilizes and / or digests the biological sample 102. In some forms wherein the biological sample 102 has been pre- permeabilized, the reagent medium is not a permeabilization solution. Herein, the reagent medium may also include one or more of a monovalent salt, a divalent salt, ethylene carbonate, and / or glycerol. In some forms, analytes (e.g., mRNA transcripts) and / or analyte derivatives (e.g., intermediate agents; e.g., ligation products) of the biological sample 102 may release from the biological sample, and actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106. Alternatively, in certain forms, migration of the analyte or analyte derivative (e.g., intermediate agent; e.g., ligation product) from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration).

[0077] Exemplary methods of electrophoretic migration are described in WO 2020 / 176788, and US. Patent Application Pub. No. 2021 / 0189475, each of which is hereby incorporated by reference.

[0078] As further shown, one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including spatially barcoded capture probes 106). The one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.

[0079] In some forms, the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and

[0080] 500 microns, between about 2 microns and 400 microns, between about 2 microns and

[0081] 300 microns, between about 2 microns and 200 microns, between about 2 microns and

[0082] 100 microns, between about 2 microns and 25 microns, or between about 2 microns and

[0083] 10 microns), measured in a direction orthogonal to the surface of first substrate that supports the biological sample. In some instances, the separation distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In some forms, the separation distance is less than 50 microns. In some forms, the separation distance is less than 25 microns. In some forms, the separation distance is less than 20 microns. The separation distance may include a distance of at least 2 pm.

[0084] FIG. IB shows a fully formed sandwich configuration 125 creating a chamber 150 formed from the one or more spacers 110, the first substrate (e.g., the slide 103), and the second substrate (e.g., the slide 104 including an array 106 having spatially barcoded capture probes) in accordance with some example implementations. In the example of FIG. IB, the liquid reagent e.g., the permeabilization solution 105) fills the volume of the chamber 150 and may create a permeabilization buffer that allows analytes (e.g., mRNA transcripts and / or other molecules) or analyte derivatives (e.g., intermediate agents; e.g., ligation products) to diffuse from the biological sample 102 toward the capture probes of the second substrate (e.g., slide 104). In some aspects, flow of the permeabilization buffer may deflect transcripts and / or molecules from the biological sample 102 and may affect diffusive transfer of analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) for spatial analysis. A partially or fully sealed chamber 150 resulting from the one or more spacers 110, the first substrate, and the second substrate may reduce or prevent flow from undesirable movement (e.g., convective movement) of transcripts and / or molecules during the diffusive transfer from the biological sample 102 to the capture probes.

[0085] The sandwiching process methods described above can be implemented using a variety of hardware components. For example, the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sample handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in, e.g., US. Patent Application Pub. No. 2021 / 0189475, and PCT Publ. No. WO 2022 / 061152 A2, each of which are incorporated by reference in their entirety.

[0086] In some forms of a sample holder, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate including a biological sample. The first retaining mechanism can be configured to retain the first substrate disposed in a first plane. The sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane. The sample holder can further include an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism can be configured to align the first and second members along the first plane and / or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane. The adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and / or move the first member along an axis orthogonal to the first plane.

[0087] In some forms, the adjustment mechanism includes a linear actuator. In some forms, the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and / or the second member. In some forms, the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and / or the second member. In some forms, the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0.1 mm / sec. In some forms, the linear actuator is configured to move the first member, the second member, or both the first member and the second member with an amount of force of at least 0.1 lbs.

[0088] FIG. 2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes a first member 204, a second member 210, optionally an image capture device 220, a first substrate 206, optionally a hinge 215, and optionally a mirror 216. The hinge 215 may be configured to allow the first member 204 to be positioned in an open or closed configuration by opening and / or closing the first member 204 in a clamshell manner along the hinge 215.

[0089] FIG. 2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206. In the example of FIG. 2B, the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206.

[0090] In some aspects, when the sample handling apparatus 200 is in an open position (e.g., in FIG. 2B), the first substrate 206 and / or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200 such as within the first member 204 and the second member 210, respectively. As noted, the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration. In some aspects, after the first member 204 closes over the second member 210, an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and / or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration). The adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration.

[0091] In some forms, the biological sample (e.g., sample 102 from FIG. 1A) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample is aligned with the barcoded array of the second substrate (e.g., the slide 104 from FIG. 1A), e.g., when the first and second substrates are aligned in the sandwich configuration. Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g., via an automated alignment mechanism). After or before alignment, spacers may be applied to the first substrate 206 and / or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 206 and / or the second substrate 212. The first member 204 may then close over the second member 210 and form the sandwich configuration. Analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) may be captured by the capture probes of the array and may be processed for spatial analysis.

[0092] In some forms, during the permeabilization step, the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and / or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas.

[0093] Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. FIGs. 3A-3C depict a side view and a top view of an exemplary angled closure workflow 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary implementations.

[0094] FIG. 3A depicts the first substrate (e.g., the slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304). As shown, reagent medium (e.g., permeabilization solution) 305 is located on the spacer 310 toward the right-hand side of the side view in FIG. 3A. While FIG. 3A depicts the reagent medium on the right hand side of side view, it should be understood that such depiction is not meant to be limiting as to the location of the reagent medium on the spacer.

[0095] FIG. 3B shows that as the first substrate lowers, and / or as the second substrate rises, the dropped side of the first substrate (e.g., a side of the slide 303 angled toward the second substrate) may contact the reagent medium 305. The dropped side of the first substrate may urge the reagent medium 305 toward the opposite direction (e.g., towards an opposite side of the spacer 310, towards an opposite side of the first substrate relative to the dropped side). For example, in the side view of FIG. 3B the reagent medium 305 may be urged from right to left as the sandwich is formed.

[0096] In some forms, the first substrate and / or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate.

[0097] FIG. 3C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the second substrate and maintaining a separation distance and optionally the approximately parallel arrangement between the two substrates. As shown in the top view of FIG. 3C, the spacer 310 fully encloses and surrounds the biological sample 302 and the capture probes 306, and the spacer 310 form the sides of chamber 350 which holds a volume of the reagent medium 305.

[0098] While FIG. 3C depicts the first substrate (e.g., the slide 303 including biological sample 302) angled over (superior to) the second substrate (e.g., slide 304) and the second substrate including the spacer 310, it should be understood that an exemplary angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate including the spacer 310.

[0099] It may be desirable that the reagent medium be free from air bubbles between the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present between the substrates may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and / or elimination of air bubbles between the two substrates (e.g., slide 303 and slide 304) during a permeabilization step (e.g., step 104). In some aspects, it may be possible to reduce or eliminate bubble formation between the substrates using a variety of filling methods and / or closing methods. In some instances, the first substrate and the second substrate are arranged in an angled sandwich assembly as described herein. For example, during the sandwiching of the two substrates (e.g., the slide 303 and the slide 304), an angled closure workflow may be used to suppress or eliminate bubble formation.

[0100] FIG. 4A is a side view of the angled closure workflow 400 in accordance with some exemplary implementations. FIG. 4B is a top view of the angled closure workflow

[0101] 400 in accordance with some exemplary implementations. As shown at 405, reagent medium 401 is positioned to the side of the substrate 402.

[0102] At step 410, the dropped side of the angled substrate 406 contacts the reagent medium 401 first. The contact of the substrate 406 with the reagent medium 401 may form a linear or low curvature flow front that fills the gap between the two substrates 406 and 402 uniformly with the slides closed.

[0103] At step 415, the substrate 406 is further lowered toward the substrate 402 (or the substrate 402 is raised up toward the substrate 406) and the dropped side of the substrate 406 may contact and may urge the reagent medium toward the side opposite the dropped side and creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the substrates.

[0104] At step 420, the reagent medium 401 fills the gap between the substrate 406 and the substrate 402. The linear flow front of the liquid reagent may form by squeezing the

[0105] 401 volume along the contact side of the substrate 402 and / or the substrate 406. Additionally, capillary flow may also contribute to filling the gap area.

[0106] In some forms, the reagent medium (e.g., 105 in FIG 1A) includes a permeabilization agent. In some forms, following initial contact between the biological sample and a permeabilization agent, the permeabilization agent can be removed from contact with the biological sample (e.g., by opening sample holder). Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some forms, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution).

[0107] In some forms, the reagent medium includes a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. In some forms, the reagent medium includes a protease. Exemplary proteases include, e.g., pepsin, trypsin, pepsin, elastase, and proteinase K. In some forms, the reagent medium includes a nuclease. In some forms, the nuclease includes an RNase. In some forms, the Rnase is selected from Rnase A, Rnase C, Rnase H, and Rnase I. In some forms, the reagent medium includes one or more of sodium dodecyl sulfate (SDS) or a sodium salt thereof, proteinase K, pepsin, N-lauroylsarcosine, and RNAse.

[0108] In some forms, the reagent medium includes polyethylene glycol (PEG). In some forms, the PEG is from about PEG 2K to about PEG 16K. In some forms, the PEG is PEG 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, UK, 12K, 13K, 14K, 15K, or 16K. In some forms, the PEG is present at a concentration from about 2% to 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v / v).

[0109] In certain forms a dried permeabilization reagent is applied or formed as a layer on the first substrate or the second substrate or both prior to contacting the biological sample and the array. For example, a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.

[0110] In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1 minute, about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45 minutes, or about an hour. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1-60 minutes.

[0111] In some instances, the device is configured to control a temperature of the first and second substrates. In some forms, the temperature of the first and second members is lowered to a first temperature that is below room temperature.

[0112] There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and / or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially -barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and / or into or onto the biological sample.

[0113] In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligations products that serve as proxies for the template.

[0114] As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3’ or 5’ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3’ end” indicates additional nucleotides were added to the most 3’ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some forms, extending the capture probe includes adding to a 3’ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some forms, the capture probe is extended by a reverse transcriptase. In some forms, the capture probe is extended using one or more DNA polymerases. In some forms, the extended capture probes include the sequence of the capture domain and the sequence of the spatial barcode of the capture probe.

[0115] In some forms, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., sequencing. In some forms, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction).

[0116] Additional variants of spatial analysis methods, including in some forms, an imaging step, are described in Section (II)(a) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Analysis of captured analytes (and / or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes, sequencing (e.g., of a cleaved extended capture probe and / or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and / or proximity capture, is described in Section (II)(g) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Some quality control measures are described in Section (II)(h) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0117] Spatial information can provide information of medical importance. For example, the methods described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and / or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stage and / or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and / or selection of a treatment for a subject with a disease or disorder. Exemplary methods for identifying spatial information of biological and / or medical importance can be found in U.S. Patent Application Publication Nos. 2021 / 0140982, 2021 / 0198741, and 2021 / 0199660.

[0118] Spatial information can provide information of biological importance. For example, the methods described herein can allow for: identification of transcriptome and / or proteome expression profiles (e.g., in healthy and / or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor or proximity based analysis); determination of up- and / or down-regulated genes and / or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their colocalization in healthy and diseased tissue; and identification of genetic variants within tissues (e.g., based on gene and / or protein expression profiles associated with specific disease or disorder biomarkers).

[0119] Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some forms, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0120] Generally, analytes and / or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or a substrate with features (e.g., beads, wells) including capture probes). As used herein, “contact,” “contacted,” and / or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0121] FIG. 5 is a schematic diagram showing an exemplary capture probe, as described herein. As shown, the capture probe 502 is optionally coupled to a feature 501 by a cleavage domain 503, such as a disulfide linker. The capture probe can include a functional sequence 504 that are useful for subsequent processing. The functional sequence 504 can include all or a part of sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence), all or a part of a sequencing primer sequence, (e.g., a R1 primer binding site, a R2 primer binding site), or combinations thereof. The capture probe can also include a spatial barcode 505. The capture probe can also include a unique molecular identifier (UMI) sequence 506. While FIG. 5 shows the spatial barcode 505 as being located upstream (5’) of UMI sequence 506, it is to be understood that capture probes wherein UMI sequence 506 is located upstream (5’) of the spatial barcode 505 is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain 507 to facilitate capture of a target analyte. The capture domain can have a sequence complementary to a sequence of a nucleic acid analyte. The capture domain can have a sequence complementary to a connected probe described herein. The capture domain can have a sequence complementary to a capture handle sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. Such splint oligonucleotide, in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence complementary to a sequence of a nucleic acid analyte, a portion of a connected probe described herein, a capture handle sequence described herein, and / or a methylated adaptor described herein.

[0122] FIG. 6 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to analytes within the sample. The capture probe 601 can contain a cleavage domain 602, a cell penetrating peptide 603, a reporter molecule 604, and a disulfide bond (-S-S-). 605 represents all other parts of a capture probe, for example a spatial barcode and a capture domain.

[0123] FIG. 7 is a schematic diagram of an exemplary multiplexed spatially-barcoded feature. In FIG. 7, the feature 701 can be coupled to spatially-barcoded capture probes, wherein the spatially-barcoded probes of a particular feature can possess the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with more than one target analyte. For example, a feature may be coupled to four different types of spatially-barcoded capture probes, each type of spatially-barcoded capture probe possessing the spatial barcode 702. One type of capture probe associated with the feature includes the spatial barcode 702 in combination with a poly(T) capture domain 703, designed to capture mRNA target analytes. A second type of capture probe associated with the feature includes the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis. A third type of capture probe associated with the feature includes the spatial barcode 702 in combination with a capture domain complementary to the analyte capture agent of interest 705. A fourth type of capture probe associated with the feature includes the spatial barcode 702 in combination with a capture probe that can specifically bind a nucleic acid molecule 706 that can function in a CRISPR assay (e.g., CRISPR / Cas9). While only four different capture probe-barcoded constructs are shown in FIG. 7, capture-probe barcoded constructs can be tailored for analyses of any given analyte associated with a nucleic acid and capable of binding with such a construct. For example, the schemes shown in FIG. 7 can also be used for concurrent analysis of other analytes disclosed herein, including, but not limited to: (a) mRNA, a lineage tracing construct, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or MNase-seq) cell surface or intracellular proteins and metabolites, and a perturbation agent (e.g., a CRISPR crRNA / sgRNA, TALEN, zinc finger nuclease, and / or antisense oligonucleotide as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, a barcoded labelling agent (e.g., the MHC multimers described herein), and a V(D)J sequence of an immune cell receptor (e.g., T- cell receptor). In some forms, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature change), or any other known perturbation agents.

[0124] The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore, etc., and the requirements thereof. In some forms, functional sequences can be selected for compatibility with noncommercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some forms, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.

[0125] In some forms, the spatial barcode 505 and functional sequences 504 is common to all of the probes attached to a given feature. In some forms, the UMI sequence 506 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.

[0126] FIG. 8 depicts an exemplary arrangement of barcoded features within an array. From left to right, FIG. 8 shows (L) a slide including six spatially-barcoded arrays, (C) an enlarged schematic of one of the six spatially-barcoded arrays, showing a grid of barcoded features in relation to a biological sample, and (R) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (labelled as ID578, ID579, ID580, etc.).

[0127] In some forms, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0128] In some cases, spatial analysis can be performed by attaching and / or introducing a molecule e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some forms, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some forms, after attaching and / or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells or cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0129] In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug 21; 45(14) :el 28. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). The terms “first analyte binding probe” and “first probe”, and “second analyte binding probe” and “second probe” as used herein are interchangeable. In some forms, a first and / or second probe or first and / or second analyte binding probes are formed entirely or include oligonucleotides. Therefore, in some forms, a first and second probes are oligonucleotide probes, e.g., RTL probes. In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3’ end and / or the other oligonucleotide includes a phosphorylated nucleotide at the 5’ end. In some instances, one of the two oligonucleotides includes a capture binding capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chorella virus DNA Ligase, a single-stranded DNA ligase, or a T4 DNA ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNAse H). In some instances, the ligation product is removed using heat. In some instances, the ligation product is removed using KOH. The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample. A non-limiting example of templated ligation methods disclosed herein is depicted in FIG. 9A. After a biological sample is contacted with a substrate including a plurality of capture probes and contacted with (a) a first probe 901 having a targethybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a target-hybridization sequence 905 and a capture domain (e.g., a poly- A sequence) 906, the first probe 901 and a second probe 904 hybridize 910 to an analyte 907. A ligase 921 ligates 920 the first probe to the second probe thereby generating a ligation product 922. The ligation product is released 930 from the analyte 931 by digesting the analyte using an endoribonuclease 932. The sample is permeabilized 940 and the ligation product 941 is able to hybridize to a capture probe on the substrate. Methods and composition for spatial detection using templated ligation have been described in PCT Publ. No. WO 2021 / 133849 Al, U.S. Pat. Nos. 11,332,790 and 11,505,828, each of which is incorporated by reference in its entirety.

[0130] In some forms, as shown in FIG. 9B, the ligation product 9001 includes a capture probe capture domain 9002, which can bind to a capture probe 9003 (e.g., a capture probe immobilized, directly or indirectly, on a substrate 9004). In some forms, methods provided herein include contacting 9005 a biological sample with a substrate 9004, wherein the capture probe 9003 is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). In some forms, the capture probe capture domain 9002 of the ligated product specifically binds to the capture domain 9006. The capture probe can also include a unique molecular identifier (UMI) 9007, a spatial barcode 9008, a functional sequence 9009, and a cleavage domain 9010.

[0131] In some forms, methods provided herein include permeabilization of the biological sample such that the capture probe can more easily bind to the captured ligated probe (z.e., compared to no permeabilization). In some forms, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can extend the capture probes 9011 to produce spatially-barcoded full-length cDNA 9012 and 9013 from the captured analytes (e.g., polyadenylated mRNA). Second strand reagents e.g., second strand primers, template switch oligonucleotides, enzymes) can be added to the biological sample on the slide to initiate second strand synthesis.

[0132] In some forms, cDNA can be denatured 9014 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 9015 via PCR prior to library construction. The cDNA can then be enzymatically fragmented and size-selected in order to optimize the cDNA amplicon size. P5 9016, i5 9017, i7 9018, and P7 9019, and can be used as sample indexes, and TruSeq Read 2 can be added via End Repair, A-tailing, Adaptor Ligation, and PCR. The cDNA fragments can then be sequenced using paired- end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites.

[0133] In some forms, detection of one or more analytes (e. ., protein analytes) can be performed using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some forms, the analyte capture agent includes: (i) an analyte binding moiety (e.g., that binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) analyte binding moiety barcode; and (iii) an analyte capture sequence. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of PCT Publication No. WO2020 / 176788 and / or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020 / 0277663.

[0134] FIG. 10 is a schematic diagram of an exemplary analyte capture agent 1002 included of an analyte-binding moiety 1004 and an analyte-binding moiety barcode domain 1008. The exemplary analyte -binding moiety 1004 is a molecule capable of binding to an analyte 1006 and the analyte capture agent is capable of interacting with a spatially -barcoded capture probe. The analyte -binding moiety can bind to the analyte 1006 with high affinity and / or with high specificity. The analyte capture agent can include an analyte-binding moiety barcode domain 1008, a nucleotide sequence (e.g., an oligonucleotide), which can hybridize to at least a portion or an entirety of a capture domain of a capture probe. The analyte-binding moiety barcode domain 1008 can include an analyte binding moiety barcode and a capture handle sequence described herein. The analyte-binding moiety 1004 can include a polypeptide and / or an aptamer. The analyte-binding moiety 1004 can include an antibody or antibody fragment (e.g., an antigen-binding fragment). FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1124 and an analyte capture agent 1126. The feature- immobilized capture probe 1124 can include a spatial barcode 1108 as well as functional sequences 1106 and UMI 1110, as described elsewhere herein. The capture probe can be affixed 1104 to a feature (e.g., bead) or array 1102. The capture probe can also include a capture domain 1112 that is capable of binding to an analyte capture agent 1126. The analyte capture agent 1126 can include a functional sequence 1118, analyte binding moiety barcode 1116, and a capture handle sequence 1114 that is capable of binding to the capture domain 1112 of the capture probe 1124. The analyte capture agent can also include a linker 1120 that allows the capture agent barcode domain 1116 to couple to the analyte binding moiety 1122.

[0135] During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some forms, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.

[0136] Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.

[0137] When sequence information is obtained for capture probes and / or analytes during analysis of spatial information, the locations of the capture probes and / or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array. Some exemplary spatial analysis workflows are described in the Exemplary Forms section of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. See, for example, the Exemplary form starting with “In some non-limiting examples of the workflows described herein, the sample can be immersed. . of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and / or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022).

[0138] In some forms, spatial analysis can be performed using dedicated hardware and / or software, such as any of the systems described in Sections (II)(e)(ii) and / or (V) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and / or Sample and Array Alignment Devices and Methods, Informational labels of PCT Publication No. W02020 / 123320.

[0139] Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biological sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and / or the sample holder via fluid conduits, and fluids can be delivered into the chamber and / or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and / or sample holder.

[0140] The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and / or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.

[0141] The systems can optionally include software instructions encoded and / or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.

[0142] In some cases, the systems described herein can detect e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Publication No. W02021 / 102003 and / or U.S. Patent Application Publication No. 2021 / 0150707, each of which is incorporated herein by reference in their entireties.

[0143] In some cases, a map of analyte presence and / or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. W02020 / 123320, WO 2021 / 102005, and / or U.S. Patent Application Publication No. 2021 / 0158522, each of which is incorporated herein by reference in their entireties. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and / or for quantitative measurements of sizes and / or distances.

[0144] IL Methods of Reducing or Preventing Non-specific Probe Interactions

[0145] Enhanced methods for spatial analysis of an analyte (e.g., a target nucleic acid analyte) in a biological sample containing single-stranded DNA (ssDNA), e.g., partially single-stranded genomic DNA or single- stranded genomic DNA, have been developed. In some embodiments, the methods enhance the resolution, sensitivity, specificity and / or accuracy of spatial analysis by reducing off-target binding of probes to non-target nucleic acids, such as genomic ssDNA, within the same biological sample. In some forms, the ssDNA is present in the biological sample due to a prior manipulation of the biological sample, such as, but not limited to, de-crosslinking of the biological sample. In some forms, the ssDNA is present in the biological sample due to DNA degradation in the biological sample. In some instances, the degradation is due to harsh treatment conditions on the biological sample (e.g., treatment at high temperature for decrosslinking). In some embodiments, the spatial analysis is performed using capture probes as described in Section I or using microscopy as a readout as described in Section III.

[0146] In some forms, the methods enhance the resolution, sensitivity, specificity and / or accuracy of spatial analysis of a target nucleic acid analyte within a biological sample by reducing the amount of single-stranded DNA (ssDNA) in the biological sample. In some examples, the ssDNA is a partially single- stranded DNA molecule. Thus, the disclosed methods reduce ssDNA regions available for hybridization to oligonucleotide probes intended for target analyte detection (e.g., RNA hybridization), thereby reducing false positives and other off-target effects during spatial analysis. In some embodiments, the method comprises performing extension using the ssDNA region as template to generate a blocked sample, wherein the blocked sample comprises an extended DNA strand complementary to the ssDNA template, thereby forming double stranded DNA. An exemplary method includes contacting a tissue sample containing ssDNA with a multiplicity of block primers configured to hybridize with the ssDNA, hybridizing the primers to ssDNA in the tissue sample, and extending the block primers using the ssDNA as template (e.g., across the length of the ssDNA) to provide a blocked sample having less ssDNA than the tissue sample prior to contacting with the multiplicity of block primers according to the methods. In some forms, the methods further include one or more steps of washing the sample to remove extension reagents from the blocked sample. In some forms, the methods further include one or more steps of washing the sample to remove any primers (e.g., excess or unhybridized block primers) and / or PCR reagents from the blocked sample.

[0147] The methods for reducing the amount of ssDNA within a sample are designed to be implemented within any spatial analysis protocol, for example, to reduce the amount of ssDNA within the sample prior to contacting the sample with probes specific for one or more target nucleic acid analytes within the sample. Therefore, methods including spatial analysis of a blocked sample are also provided. In some forms, one or more steps for blocking the sample are implemented within one or more steps of a spatial analysis protocol. In some forms, one or more steps for blocking the sample are performed prior to a spatial analysis assay. Typically, the amount of, and / or variety of different species of target analytes captured by capture probes according to a spatial analysis method performed using a sample contacted with block primers according to the described methods for reducing ssDNA is the same or greater than the amount of target analytes that are captured by the capture probes according to a spatial analysis methodology performed using an equivalent sample not contacted with block primers according to the described methods. In some forms, the amount of, and / or variety of different species of target analytes captured by capture probes according to a spatial analysis methodology performed using a sample contacted with block primers according to the described methods for reducing ssDNA is at least 0.1% greater, up to 100% greater than the amount of target analytes that are captured by the capture probes according to a spatial analysis methodology performed using an equivalent sample not contacted with block primers according to the described methods. For example in some forms, the amount of, and / or variety of different species of target analytes captured by capture probes according to a spatial analysis methodology performed using a sample contacted with block primers according to the described methods for reducing ssDNA is up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% or more that 100% greater than the amount of target analytes that are captured by the capture probes according to a spatial analysis methodology performed using an equivalent sample not contacted with block primers according to the described methods. In other forms, the described methods for reducing ssDNA in a sample reduce the overall number of and / or variety of analytes that are that are captured by the capture probes according to a spatial analysis methodology performed using an equivalent sample not contacted with block primers according to the described methods for reducing ssDNA. For example, in some forms, the methods reduce the amount of non-target analytes that are captured by capture probes. For example, in some forms, the methods reduce the amount of off-target analytes and / or increase the proportion of target analytes that are captured relative to the total amount captured as compared with an equivalent sample not contacted with block primers according to the described methods.

[0148] Therefore, in some forms, the methods decrease or preclude off-target recognition of ssDNA in a sample by target analyte-binding oligonucleotide (e.g., probes) and thereby enhance the sensitivity, specificity and / or signal over background of spatial analyses of one or more target analytes in a sample. In some embodiments, a method disclosed herein relates to the detection of target nucleic acids sequences (e.g., target RNAs) in situ at one or more locations in a blocked sample wherein background signal is reduced and / or sensitivity is increased. In some embodiments, a method disclosed herein relates to the detection of target nucleic acids sequences (e.g., target RNAs) in situ using probe hybridization. In some aspects, the spatial analysis of the blocked sample results in improved sensitivity (number of detected signals), specificity, signal intensity, and / or improved signal to noise, compared to an equivalent sample with ssDNA wherein extension has not been performed to generate dsDNA using the ssDNA as template, e.g., according to the described methods. For example, the amount of target analytes detected according to a spatial analysis method performed using a blocked sample according to the described methods for reducing ssDNA is the same or greater than the amount of target analytes detected in an equivalent sample wherein the ssDNA has not been used as template to generate dsDNA according to the described methods. In some forms, the amount of, and / or variety of different species of target analytes detected according to a spatial analysis method performed using a blocked sample according to the described methods for reducing ssDNA is at least 0.1% greater, up to 100% greater than the amount of target analytes detected in an equivalent sample wherein the ssDNA has not been used as template to generate dsDNA. For example in some forms, the amount of, and / or variety of different species of target analytes detected according to a spatial analysis method performed using a blocked sample according to the described methods for reducing ssDNA is up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% or more that 100% greater than the amount of target analytes detected in an equivalent sample wherein the ssDNA has not been used as template to generate dsDNA. In other forms, the described methods for reducing ssDNA in a sample reduce the overall number of and / or variety of analytes that are detected in a sample wherein the ssDNA has not been used as template to generate dsDNA for reducing ssDNA in the sample. For example, in some forms, the methods reduce the amount of non-target analytes that are detected. For example, in some forms, the methods reduce the amount of off- target analytes and / or increase the proportion of target analytes that are detected relative to the total amount detected as compared with an equivalent sample wherein the ssDNA has not been used as template to generate dsDNA according to the described methods.

[0149] An overview of the described methods for reducing or preventing non-specific probe interactions with ssDNA is depicted in Figure 12. A section of DNA within a nonblocked sample, including double stranded DNA having one or more single- stranded regions of DNA (1210) is illustrated, with three distinct regions of single stranded DNA depicted (1201, 1202 and 1203). Each region of single-stranded DNA includes a distinct ssDNA sequence and represents a potential site for non-specific binding of one or more target analyte-specific probes. In some embodiments, the methods include a step of contacting (1220) the dsDNA with a plurality of species of block primers (1204) under conditions permissive for hybridization of the primers to each of the three regions of single stranded DNA (1201, 1202 and 1203), to form an intermediate block primerbound dsDNA (1230). In some embodiments, block primers (1204) are not required for forming a complementary second DNA strand using the regions of single stranded DNA (1201, 1202 and 1203) as template. The methods include a step of extending (1240) the block primer-bounds across the dsDNA with a polymerase (e.g., a DNA-dependent polymerase) (1205) under conditions suitable for extension of the block primers across the regions of single-stranded DNA, using the ssDNA as a template, to form an intermediate block primer-extended dsDNA (1250) having dNTPs incorporated therein. In some embodiments, the method comprises a step of extending a free 3’ end of DNA using a single stranded region of ssDNA hybridized thereto as template with a polymerase (e.g., a DNA-dependent polymerase) to form an extended dsDNA having dNTPs incorporated therein. In some embodiments, external block primers are added to the biological sample for extension. In some embodiments, external primers are not used for the extension, e.g., as shown in Figure 13. The methods then optionally include one or more steps to wash the sample (1260), to remove unbound block primers and / or polymerase, to provide a blocked sample, including double-stranded DNA (1270) in which regions of single- stranded DNA breaks are removed. Each of the method steps is described in more detail, below.

[0150] Figure 13 depicts DNA within a non-blocked sample, including double stranded DNA having one or more single-stranded regions of DNA (1310), with three distinct regions of single stranded DNA depicted (1301, 1302 and 1303). Each region of singlestranded DNA includes a distinct ssDNA sequence and represents a potential site for non-specific binding of one or more target analyte- specific probes. In some embodiments, the methods include a step of contacting (1320) the dsDNA having one or more single- stranded regions of DNA with a polymerase (e.g., a DNA-dependent polymerase) (1304) and dNTPs under conditions suitable for extension (1330) across the regions of single-stranded DNA to form a blocked sample comprising dsDNA having the dNTPs incorporated therein. In some embodiments, the method comprises extending a free 3’ end of DNA (e.g., at a region where there is a break in the strand) using a single stranded region of ssDNA as template with a polymerase e.g., a DNA-dependent polymerase) to form an extended dsDNA (1340) having dNTPs incorporated therein.

[0151] In some embodiments, a generated blocked sample is used for spatial analysis e.g., using capture probes as described in Section I or an in situ assay using microscopy as a readout as described in Section III).

[0152] A. Sample Preparation Methods for Reducing ssDNA

[0153] Methods for reducing the amount of single-stranded DNA (ssDNA) in a sample, such as a biological sample, are provided. In some instances, the methods provide block primers that hybridize to and extend throughout one or more regions of ssDNA within the sample, such as genomic ssDNA. In some instances, the methods provide a workflow for performing extension at one or more regions of ssDNA within the sample, such as genomic ssDNA.

[0154] In some forms, the methods comprise contacting a biological sample including a ssDNA region with a DNA polymerase to extend a free 3’ end of DNA using the one or more regions of ssDNA present in the biological sample as template to provide a blocked sample. In some forms, provided herein is a method for performing an extension in a biological sample using one or more regions of ssDNA as template to generate double stranded DNA (dsDNA), thereby providing a blocked sample.

[0155] In some forms, the methods include one or more steps of:

[0156] (a) contacting a biological sample including ssDNA with a multiplicity of block primers configured to hybridize with one or more regions of ssDNA present in the biological sample; and

[0157] (b) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample.

[0158] Typically, the blocked sample includes less ssDNA than the tissue sample. In some forms, the blocked sample contains dsDNA in regions corresponding to the one or more regions of ssDNA in the biological sample.

[0159] It is contemplated that, in some forms, the presence of excess unbound or dimerized block primers may interfere with subsequent spatial analysis methods. Therefore, in some forms, the methods include one or more steps of:

[0160] (c) optionally washing the blocked sample to remove any unbound block primers and / or block primer dimers. (a) Contacting a biological sample comprising ssDNA with a multiplicity of block primers

[0161] In some embodiments, the method uses one or more blocked primers to perform extension. For example, the methods include one or more steps for contacting a biological sample including or potentially including one or more regions of ssDNA with one or more species of block primers. The block primers are configured to hybridize with the ssDNA present in the sample. Typically, the methods contact the sample with an amount of block primers sufficient to hybridize to all of the ssDNA in the sample. In some forms, the multiplicity of block primers collectively hybridize to a multiplicity of sites within the same ssDNA, such that a single region of ssDNA is hybridized to more than one block primer. In some forms, the contacting is carried out under conditions that allow the probes to hybridize to the ssDNA upon contact. In other forms, the contacting occurs under conditions that are not optimal for hybridization. Therefore, in some forms, the methods include one or more steps for providing conditions suitable for hybridizing the block probes to ssDNA within the sample.

[0162] Typically, the methods employ a DNA polymerase enzyme that is specific to DNA, e.g., a DNA polymerase that does not function with DNA- RNA hybrids. Therefore, if the block primers bind to RNA, they can be removed with wash steps and, after DNA polymerization the fragments attached to DNA will be longer so only RNA bound primers are removed.

[0163] (b) Extending DNA e.g., the block primers) using ssDNA as template

[0164] In some embodiments, the methods include performing extension of DNA using one or more regions of ssDNA as template to generate dsDNA. In some embodiments, the extension uses a single-stranded region of open or fragmented genomic DNA as template. In some instances, the extension reaction across the one or more regions of ssDNA creates double-stranded (ds)DNA in place of the ssDNA, thus “filling in” regions of ssDNA, for example, in genomic DNA to recreate dsDNA using the ssDNA as a template. The methods include one or more steps for extending the hybridized block primers across the one or more regions of ssDNA to create double- stranded (ds)DNA in place of the ssDNA, and to “fill in” regions of ssDNA, for example, in genomic DNA to recreate dsDNA using the ssDNA as a template. In some instances, block primers are not used in the extension reaction and extension is performed directly on a 3’ end of a DNA (e.g., at a break in the DNA strand at the region of ssDNA). In some instances, the ssDNA (e.g., gDNA) is endogenous to the biological sample. In some forms, the extending includes contacting the biological sample including ssDNA having block primers hybridized thereto with a polymerase enzyme, optionally together with reagents such as dNTPs required for extending a second strand of a nucleic acid using a first strand as template to form a blocked sample, in which a proportion of the ssDNA is reduced relative to the sample prior to contacting with the block primers. In preferred forms, the extending removes at least 50% of the ssDNA in the sample by converting the ssDNA to dsDNA. For example, in some forms, the blocked sample has only 40%, 30%, 20%, 10%, 5%, 1% or less than 1% of the amount of ssDNA relative to the sample prior to contacting with the block primers.

[0165] Typically, extending DNA (e.g., hybridized block primers) across ssDNA requires one or more reagents for primer extension, such as a polymerase enzyme, and / or deoxynucleotide triphosphates (dNTPs), and / or a suitable reaction buffer for primer extension. Therefore, in some forms, the step of extending the DNA e.g., the hybridized block primers) across the ssDNA includes providing to the sample one or more reagents for primer extension, such as a polymerase enzyme, and / or deoxynucleotide triphosphates (dNTPs), and / or a suitable reaction buffer for primer extension.

[0166] In some forms, to avoid undesired binding or extension of block primers with RNAs within a sample, the methods require a DNA-dependent DNA polymerase that does not extend the block primers on an RNA template.

[0167] Typically, the methods require a DNA-specific DNA polymerase enzyme. Typically, the methods do not include a polymerase enzyme that functions in the presence of RNA and / or DNA / RNA hybrids.

[0168] Exemplary polymerase enzymes are selected from DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V. In some cases, a preferred polymerase enzyme is a thermostable DNA polymerase I, for example, a thermostable DNA polymerase I derived from Thermus aquaticus. In some embodiments, a suitable polymerase enzyme includes a DNA polymerase such phi29 (tp29) polymerase, Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some aspects, DNA polymerases that have been engineered or mutated to have desirable characteristics can be employed. In some embodiments, the polymerase is a phi29 DNA polymerase. (c) Removing unbound block primers and / or block primer dimers

[0169] In some forms, the methods include one or more steps to remove unbound and / or excess block primers and / or block primer dimers from the sample following hybridizing the block primers and / or extending the block primers.

[0170] Wash steps that can be implemented include contacting the blocked sample with one or more suitable solvents in an amount and for a time effective to solubilize unbound block primers or primer dimers in the sample. Suitable solvents include an aqueous or non-aqueous wash buffer.

[0171] Typically, a wash step includes contacting the sample with the solvent in an amount and for a time effective for unbound block primers and / or primer dimers to solubilize within the solvent and removing the solvent, including solubilized block primers and / or block primer dimers from the sample.

[0172] In some forms, one or more steps to remove unbound excess block primers and / or block primer dimers from the sample are carried out after a step of contacting a biological sample including a nucleic acid molecule including one or more regions of ssDNA with a multiplicity of block primers configured to hybridize with the one or more regions of ssDNA present in the biological sample;. In some forms, one or more steps to remove unbound excess block primers and / or block primer dimers from the sample are carried out after a step of extending the block primers using the one or more regions of ssDNA to provide a blocked sample, wherein the blocked sample includes less ssDNA than the biological sample prior to step (a). In some forms, removal of the excess block primers and / or primer dimers also effectively removes reagents required for the extension step. In preferred forms, the methods include one or more steps to remove unbound excess block primers and / or block primer dimers from the sample are after contacting a biological sample with a multiplicity of block primers and again after a step of extending the block primers.

[0173] In some forms, a wash step is repeated once or more than once. For example, in some forms, a wash step is repeated 2, 3, 4, 5, 6, 7 ,8, 9, 10 or more than 10 times.

[0174] An exemplary wash step includes:

[0175] (i) contacting the blocked tissue sample with a suitable solvent in an amount and time effective to solubilize the block primers and / or primer extension reagents; and

[0176] (ii) removing the blocked tissue sample from the solvent. An exemplary solvent for use in a wash step includes an aqueous buffer and optionally one or more additional reagents. Typically, the one or more additional reagents are solubilized within the wash buffer solvent. An exemplary wash buffer includes PBS and a polysorbate. In some forms, the one or more additional reagents includes a DNA polymerase. For example, in some forms, the wash buffer includes a DNA polymerase prior to addition of the wash buffer to a biological sample.

[0177] B. Streamlined Integrated Spatial Analysis Methods

[0178] Methods for spatial analysis of biological samples, e.g., ssDNA-blocked biological samples are also provided. In some forms, the methods for reducing the amount of ssDNA within a sample are implemented within the workflow of a spatial analysis protocol, for example, prior to contacting the sample with one or more spatial analysis probes.

[0179] In some forms, methods for spatial analysis require that the sample be disposed within or on a substrate, such as a slide or other matrix. Therefore, in some forms, methods including one or more of steps (a)-(c) (described above) further include, prior to step (a), providing the biological sample disposed on a first substrate.

[0180] 1. Supplemental Spatial Analysis Workflows

[0181] In some forms, the methods for spatial analysis implement the same or modified protocol for analysis of the ssDNA-blocked sample as typically applied to any other sample that is subjected to spatial analysis methodologies, such that the methods are supplemental to existing spatial analysis workflows. For example, in some forms, when the target analyte is an RNA, and when the methods include use of a first oligonucleotide probe and optionally a second oligonucleotide probe (e.g., first and second RTL probes), the methods include one or more steps of:

[0182] (d) hybridizing a first probe and optionally a second probe to one or more RNA analytes present within the sample. In some forms, according to spatial analysis workflows, the first probe and optionally the second probe each include a sequence that is substantially complementary to a sequence present in a target RNA analyte. In some forms, according to spatial analysis workflows, one or more of a first probe or second probe includes a capture probe binding domain. Therefore, in some forms, a first probe and optionally a second probe each include a sequence that is substantially complementary to a target RNA analyte, and whereby the first probe and / or second probe include a capture probe binding domain. In some forms, when the methods include a first RTL probe and a second RTL probe, the methods further include one or more steps of:

[0183] (e) optionally coupling the first probe and the second probe, thereby generating a connected probe.

[0184] Typically, a connected probe includes a single capture probe binding domain (e.g., derived from the first probe or second probe). The “capture probe binding domain” is a domain including a sequence that is complementary to a particular capture domain present in a capture probe on an array. In some forms, the capture probe binding domain includes a poly(A) sequence. In some forms, the capture probe binding domain includes a poly-uridine sequence, a poly-thymidine sequence, or a combination thereof. In some forms, the capture probe binding domain includes a random sequence (e.g., a random hexamer or octamer). In some forms, the capture probe binding domain is complementary to a capture domain in a capture probe. In some instances, the capture probe binding domain or a complement thereof is on the 5’ end of the first probe or second probe. In some instances, the capture probe binding domain or a complement thereof is on the 3’ end of one of the first probe or second probe.

[0185] In some forms, after hybridization of probe oligonucleotides (e.g., first and the second probes) to the target analyte, the probe oligonucleotides (e.g., the first probe and the second probe) are coupled (e.g., ligated) together, creating a single connected probe (e.g., a ligation product) that is complementary to the target analyte. Ligation can be performed enzymatically or chemically. For example, the first and second probes are hybridized to the first and second target regions of the analyte, and the first and second probes are subjected to a nucleic acid reaction to ligate them together. For example, the probes may be subjected to an enzymatic ligation reaction using a ligase (e.g., T4 RNA ligase (Rnl2), a SplintR ligase, or a T4 DNA ligase). See, e.g., Zhang L., et al:, Archaeal RNA ligase from Thermoccocus kodakarensis for template dependent ligation RNA Biol. 2017; 14(1): 36-44 for a description of KOD ligase. A skilled artisan will understand that various reagents, buffers, cofactors, etc. may be included in a ligation reaction depending on the ligase being used.

[0186] The connected probe (e.g., ligation product) that results from the coupling (e.g., ligation) of the first probe and second probe can serve as a proxy for the target analyte, as such an mRNA. In some forms, the methods include providing a plurality of first probes and a plurality of second probes, wherein a pair of probes for a target analyte includes both a first and second probe. Further, it is appreciated that probe pairs can be designed to cover any gene of interest. For example, a pair of probe oligonucleotides can be designed so that each analyte, e.g., a whole exome, a transcriptome, a genome, can conceivably be detected using a probe oligonucleotide pair. In some instances, probe pairs are designed to cover an entire transcriptome of a species (e.g., a mouse or a human). In some instances, probes are designed to cover a subset of a transcriptome e.g., a mouse or a human). In some instances, the methods disclosed herein utilize about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 15,000, about 20,000, or more probe pairs.

[0187] In some forms, when the methods further include providing the biological sample disposed on a first substrate, the methods include one or more steps of:

[0188] (f) aligning the first substrate with a second substrate including an array, such that at least a portion of the biological sample is aligned with at least a portion of the array. Typically, an array for use with methods for spatial analyses includes a plurality of capture probes. In some forms, a capture probe of the plurality of capture probes includes a spatial barcode and a capture domain, e.g., for capture of a capture domain binding domain of a target analyte-binding probe, a target nucleic acid analyte (e.g., mRNA) or a connected probe or ligation product.

[0189] In some forms, the methods further include one or more steps of:

[0190] (g) releasing the first probe or the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array to provide a released probe. Methods for releasing the nucleic acid component, including probe(s), from a sample typically include contacting the sample with one or more reagents for digesting or otherwise removing protein, lipid, carbohydrate and / or and other non-nucleic acid components from the sample. In some forms, to release the connected probe (e.g., a ligation product), an endoribonuclease (e.g., RNase A, RNase C, RNase H, or RNase I) is used. An endoribonuclease such as RNase H specifically cleaves RNA in RNA:DNA hybrids. In some forms, the connected probe (e.g., a ligation product) is released enzymatically. In some forms, the endoribonuclease is an RNase H. In some embodiments, the RNase H is RNase Hl or RNase H2. In some forms, releasing the first probe or the connected probe from the RNA analyte includes contacting the sample with an RNase, e.g., RNase H.

[0191] In some forms, according to a spatial analysis methodology, the methods further include one or more steps of: (h) hybridizing the released probe to the capture domain of the capture probe. The hybridizing typically occurs in the absence of any block primers or block primer dimers.

[0192] In some forms, step (h) further includes one or more steps for isolating, quantifying and / or characterizing spatial information for one or more captured analytes.

[0193] 2. Integrated Workflow Methodologies

[0194] In some forms, the methods for spatial analysis implement a modified protocol for analysis of the ssDNA-blocked sample. In some forms, the sample is mounted and / or prepared for spatial analysis according to existing methodologies and then additional steps are carried out to reduce ssDNA within the sample prior to and / or within the spatial analysis workflow. In some forms, implementing the described methods for reducing ssDNA within an existing spatial analysis workflow includes one or more changes to the workflow. For example, in some forms, when the target analyte is an RNA, and when the methods include a first probe and optionally a second probe, the methods include one or more steps of:

[0195] (a) providing the biological sample disposed on a first substrate;

[0196] (b) contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of single-stranded DNA (ssDNA) present in the biological sample;

[0197] (c) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample,

[0198] (d) hybridizing a first probe and a second probe to the RNA analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to sequences of the RNA analyte, and wherein the second probe comprises a capture probe binding domain;

[0199] (e) coupling the first probe and the second probe, thereby generating a connected probe;

[0200] (f) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises:

[0201] (i) a spatial barcode; and

[0202] (ii) a capture domain; (g) releasing the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array; and

[0203] (h) hybridizing the connected probe to the capture domain of the capture probe.

[0204] In some forms, the steps of contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of single- stranded DNA (ssDNA) and extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample are carried out as a single step, for example, including contacting the biological sample with a multiplicity of block primers together with one or more enzymes and reagents for extending the block primers on ssDNA. In some forms, the contacting occurs under conditions that facilitate hybridization of the block primers to ssDNA. In some forms, the step of contacting the biological sample with a multiplicity of block primers configured to hybridize with single- stranded DNA (ssDNA) and extending the block primers across the length of the ssDNA to provide a blocked sample are carried out as a single step together with the step of contacting the sample with a first, second or further probe. In some forms, the contacting occurs under conditions that facilitate hybridization of the block primers to one or more regions of ssDNA.

[0205] Therefore, in some forms, the methods include contacting the biological sample with a single composition including both block primers and analyte-binding probes (e.g., RTL probes or circularizable probes such as padlock probes). In other forms, the methods include simultaneously contacting the sample with two or more compositions collectively including block primers and analyte-binding probes. When block primers are added at the same time as a first and optionally a second or further analyte-binding probe, or within the same composition as a first and optionally a second or further analyte-binding probe, it is contemplated that the design of the block primers must be such that they do not hybridize with the analyte-binding probes. Therefore, in some forms, the block primers are designed to include one or more specific oligonucleotide sequence.

[0206] For example, in some forms, the methods include one or more steps of:

[0207] (a) providing the biological sample disposed on a first substrate;

[0208] (b) contacting the biological sample with a probe mixture, whereby the probe mixture includes (i) a multiplicity of block primers configured to hybridize with one or more regions of single-stranded DNA (ssDNA) present in the biological sample;

[0209] (ii) one or more reagents for extending the block primers across the length of the one or more regions of ssDNA; and

[0210] (iii) a first probe and optionally a second or further probe or further probe having one or more regions complementary to a target analyte, whereby the first probe and optionally second or further probe includes a capture probe binding domain;

[0211] (c) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample,

[0212] (d) hybridizing the first probe and optionally second or further probe to a target analyte;

[0213] (e) optionally coupling the first probe and second or further probe, thereby generating a connected probe;

[0214] (f) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises a plurality of capture probes, whereby a capture probe of the plurality of capture probes includes:

[0215] (i) a spatial barcode; and

[0216] (ii) a capture domain;

[0217] (g) releasing the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array;

[0218] (h) hybridizing the connected probe to the capture domain of the capture probe;

[0219] (i) determining

[0220] I. all or a part of the sequence of the connected probe, or a complement thereof, and / or

[0221] II. the sequence of the spatial barcode, or a complement thereof; and

[0222] (j) using the determined sequences of I and / or II to provide the sequence and / or location of the RNA analyte in the biological sample.

[0223] C. Block Primers

[0224] The design and quantity of block primers can be selected according to the requirements of the spatial analysis methodology that is to be implemented, and / or the characteristics of the biological sample and / or analyte that are the subject of the spatial analysis. In some forms, the block primers are designed to include sequences that are tagged or otherwise selectively appended to ssDNA within the sample. In some forms, the step of contacting the biological sample with a multiplicity of block primers occurs under conditions that facilitate hybridizing of the block primers to one or more regions of ssDNA. Therefore, in some forms, the design of the sequence of the block primers includes calculating a melting temperature, for example, by including a specific quantity of one or more nucleic acids within each block primer.

[0225] 1. Block Primer Design

[0226] In some forms, the block primer sequences include specifically designed sequence(s) and / or partially or completely randomly-generated sequences.

[0227] Since the block primers are designed to provide blocked samples that preserve the quantity and variety of RNAs within the original biological sample, the block primers can be designed to reduce or prevent blocking the RNA from binding to analyte-binding probes, such as RNA Templated Ligation (RTL) probes. Therefore, when the block primers are or include designed sequences, they can be configured such that they do not bind to non-ssDNA nucleic acids in a biological sample, such as RNA. When the block primers are or include designed sequences, they are typically, configured such that they do not form a DNA / RNA hybrid. Design considerations for block primers typically include a sequence having a prescribed annealing temperature that can be controlled to prevent or reduce binding to non-ssDNA, such as RNAs, etc.

[0228] In some forms, the block primers are only non-coding regions of DNA. Therefore, typically the block primers only bind to DNA, and will not hybridize with any RNA in the biological sample.

[0229] In some forms the block primers are composed of between about 6nucleotides and about 300 nucleotides, inclusive. For example, in some forms, block primers include between about 6 nucleotides and about 100 nucleotides, inclusive, between about 6 nucleotides and about 60 nucleotides, inclusive, between about 6 nucleotides and about 50 nucleotides, inclusive, between about 6 nucleotides and about 40nucleo tides, inclusive, between about 6 nucleotides and about 30 nucleotides, inclusive, between about 6 nucleotides and about 20 nucleotides, inclusive, between about 6 nucleotides and about 10 nucleotides, inclusive, or between about 6 nucleotides and about 8 nucleotides, inclusive. In some forms, the multiplicity of block primers includes between one and one hundred million species of oligonucleotide sequences. For example, in some forms, the multiplicity of block primers includes between one and one million species of oligonucleotide sequences, between about one and one hundred thousand species of oligonucleotide sequences, between about one and ten thousand species of oligonucleotide sequences, between about one and one thousand species of oligonucleotide sequences, between about one and one hundred species of oligonucleotide sequences, or between about one and twenty species of oligonucleotide sequences. In an exemplary method, the multiplicity of block primers includes between one and one hundred thousand species of designed, or “fixed” and / or randomly generated “random” oligonucleotide sequences having a size of about 6 nucleotides and about 40 nucleotides, inclusive. a. Fixed Block Primer Sequences

[0230] In some forms, one or more species of a multiplicity of species of block primers include one or more known, or “fixed” sequences.

[0231] Typically, fixed oligonucleotide sequences are user-defined sequences having a user-defined number of bases, melting temperature, configuration, etc. In some forms, fixed sequences include one more variations of one or more “core” or template sequence. In some forms, every species of a multiplicity of species of fixed sequences include one, two, three, four, five, six, seven, eight, nine, ten or more than ten common nucleosides, and include one, two, three, four, five, six, seven, eight, nine, ten or more than ten variant nucleosides. In some forms, the common nucleosides of a multiplicity of species of oligonucleotide sequences are contiguous. For example, in some forms, a multiplicity of species of oligonucleotide sequences of block primers include the same 3’ and / or same 5’ sequence(s). For example in some forms, a multiplicity of species of oligonucleotide sequences of block primers include the same “tail” or “tag” sequence. In some forms, the common nucleosides of a multiplicity of species of oligonucleotide sequences are noncontiguous.

[0232] In an exemplary form, the nucleic acid sequence of one or more of the block primers includes GAGAATGTGAGTGAAGATGTATGGTGANNNNNNN (SEQ ID NO:1), whereby each “N” is, independently, A, G, T, or C.

[0233] In some forms, a block primer of the multiplicity of block primers includes a guanine / cytosine (GC) content of from about 30% to about 70%, inclusive, such as 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. For example, in some forms, every block primer of a multiplicity of block primers includes a guanine / cytosine (GC) content of from about 30% to about 70%, inclusive, such as 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0234] In some forms, the 3’ terminus of one or more of the block primers includes guanine (G) or cytosine (C). For example, in some forms, every block primer of a multiplicity of block primers includes guanine (G) or cytosine (C) at the 3’ terminus. In some forms, the 3’, or 5’, or 3’ and 5’ terminus of one or more of the block primers includes a sequence of two or more than two thymine (T) residues. Therefore, in some forms, a block primer includes a poly d(T).

[0235] In some forms, a block primer of the multiplicity of block primers includes one or more of DNA, LNA, PNA, UNA, TNA phosphorothioate and P5' phosphoramidate. b. Random Block Primer Sequences

[0236] In some forms, one or more species of a multiplicity of species of block primers include one or more unknown, or “random” sequences. Methods for generating and synthesizing oligonucleotide primers having a random number and / or sequence of bases are known in the art. In some forms, a multiplicity of species of random block primers have the same number of bases. In other forms, a multiplicity of species of random block primers have varied numbers of bases.

[0237] In an exemplary form, the multiplicity of block primers includes a set of random hexamers including 4,096 species of oligonucleotide sequences. c. Functional Adducts of Block Primers

[0238] In some forms, one or more species of a multiplicity of species of fixed or random block primers include one or more functional moieties or adducts.

[0239] In some forms, the methods include one or more steps to modify the ssDNA within a sample prior to the step of contacting the biological sample with a multiplicity of block primers. For example, in some forms, the methods include adding a primer- specific affinity sequence to the ssDNA prior to contacting the biological sample with a multiplicity of block primers. In an exemplary form, the methods include one or more steps for adding a poly(A) sequence to the ssDNA in a sample and designing the multiplicity of block primers to include poly (dT). In another exemplary form, the methods include one or more steps for adding a poly(T) sequence to the ssDNA in a sample and designing the multiplicity of block primers to include poly (dA). In some forms, one or more of the block primers include, or have bound thereto one or more functional moieties. In some forms, a specific functional moiety is associated with / bound to a specific species of block primer. In some forms, two or more different species of block primers of a plurality of block primers include two or more different functional moieties. Therefore, in some forms, a plurality of block primers includes two or more species of block primers including two or more species of different functional moieties.

[0240] Exemplary functional moieties include one or more of a label, an imaging agent, a contrast agent or a dye. An exemplary label is a fluorophore. In some forms, one or more block primers of a plurality of block primers include one or more of a label or dye. In some forms, a specific label or dye is associated with / bound to a specific species of block primer. Therefore, in some forms, a plurality of block primers includes two or more species of different block primers including two or more species of different labels or dyes. In some forms, when one or more block primers of a plurality of block primers include one or more of a label or dye, the methods for blocking a sample include one or more steps of imaging a sample including block primers. The imaging can be carried out at a point before, during or after contacting and / or hybridizing and / or extending the block primers with the sample.

[0241] D. “Blocked” Double-Stranded DNA

[0242] The methods typically convert one or more regions of ssDNA within a sample into dsDNA, thereby creating a modified, “blocked” sample having a smaller proportion of ssDNA than in the same sample prior to the described methods.

[0243] Typically, the amount of ssDNA within the blocked sample is less than 50%, 40%, 30% 20%, 10%, 5%, 2%, 1%, or less than 0.1% of the amount of ssDNA within the biological sample prior to contacting with the block primers. Typically, the steps to block the sample have little or no effect upon the amount of other nucleic acid analytes within the sample. For example, in some forms, the amount and sequence of RNAs in the blocked sample is the same or almost the same as the amount of RNA within the sample prior to the steps to block the sample. In exemplary forms, the sequence of RNAs within the blocked sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of RNAs within the sample prior to the steps to block the sample. In exemplary forms, the amount of RNAs within the blocked sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the abundance of RNAs within the sample prior to the steps to block the sample.

[0244] In some forms, following steps for annealing and extending blocking probes across the one or more regions of ssDNA in a biological sample to create a blocked sample including newly formed second strands of DNA, the methods include one or more steps to stabilize the double- stranded DNA. For example, in some forms, the methods contact the blocked sample with one or more additives in an amount and time effective to stabilize the dsDNA in the blocked sample. Exemplary stabilizing agents include spermine and / or methylene blue.

[0245] In some forms, the DNA in a blocked sample is or includes genomic DNA. In some forms, the genomic DNA in the biological sample includes double stranded and some single stranded regions. In some forms, the genomic DNA in the biological includes fragmented genomic DNA. In some embodiments, the DNA in the biological sample includes open genomic DNA (e.g., at least partially open gDNA in some regions as ssDNA). In some forms, the blocked sample is a tissue sample.

[0246] In some forms, the methods include one or more steps of permeabilizing a blocked sample. In some forms, the methods include de-crosslinking the blocked sample. In some embodiments, a de-crosslinked tissue sample comprises at least partially open genomic DNA (e.g., at least partially open gDNA in some regions as ssDNA). In some embodiments, a blocked sample is formed by processing a de-crosslinked tissue sample comprising one or more regions of ssDNA. In some forms, the methods include decrosslinking the biological sample prior to contacting the biological sample with the multiplicity of blocked primers to generate the blocked sample. In some forms, the methods include de-crosslinking the biological sample prior to performing extension in the biological sample to generate the blocked sample. In exemplary forms, the decrosslinking includes heating the sample and / or contacting the sample with an alkaline solvent and / or an acidic solvent. In some forms, heating includes exposing the sample to a temperature of between about 70 °C and about 99 °C, inclusive. In some forms, heating includes exposing the sample to a temperature of at least about 70 °C. In some forms, heating includes exposing the sample to a temperature of at least about 75 °C. In some forms, heating includes exposing the sample to a temperature of at least about 80 °C. In some forms, heating includes exposing the sample to a temperature of at least about 85 °C. In some forms, heating includes exposing the sample to a temperature of at least about 90 °C. In preferred forms, the heating includes exposing the sample to a temperature of about 95 °C. In some forms, the de-crosslinking includes contacting the sample (e.g., tissue sample or tissue section) with a de-crosslinking buffer.

[0247] 1. Archival Blocked Samples

[0248] In some forms, a blocked sample prepared according to the described methods is not immediately used for an assay. For example, in some forms, a biological sample is blocked according to the disclosed methods, and then modified, for example, by freezing, drying, or other processes for preserving or storing a sample. In some forms, the modification includes modifying the temperature of the blocked sample, for example, by modifying the temperature to room temperature, or to a temperature below room temperature, such as 25°C, or 4°C, or 0°C, or a temperature below 0°C, such as -KFC, - 20°C, -80°C or below -80°C. Therefore, in some forms, the blocked sample is stored for a period of time in excess of one day, week, month, year or decade.

[0249] In some forms, the blocked sample is fixed following the described methods for blocking the sample. For example, in some forms, the blocked sample is fixed in formalin. In some forms, the blocked sample is embedded within a suitable matrix for storage and preservation of the blocked sample. In some forms, a blocked sample is fixed with a fixative selected from ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. Thus, in some forms, the blocked sample is a formalin-fixed paraffin embedded tissue sample, a paraformaldehyde fixed tissue sample, a methanol fixed tissue sample, or an acetone fixed tissue sample. In some instances, the blocked sample is fixed using PAXgene. PAXgene is a formalin- free, non-cross-linking fixative that preserves morphology and biomolecules. In some forms, the blocked sample is a Formalin-Fixed Paraffin- Embedded (FFPE) sample. Therefore, in some forms, the methods include de-waxing and / or de-paraffinizing the blocked sample prior to spatial analysis. In other forms, the blocked sample includes a frozen and / or lyophilized sample. In some forms, deparaffinizing a blocked sample includes contacting the blocked sample with a solvent. In some forms, a solvent for deparaffinizing a blocked sample includes xylene. In some forms, a solvent for deparaffinizing a blocked sample includes a solvent including ethanol. In some forms, a solvent for deparaffinizing a blocked sample includes a solvent including xylene, followed by a solvent including ethanol. a. Providing a Blocked Sample

[0250] Typically, the blocked sample that has been modified or processed for storage / archiving is ultimately used in one or more methods for spatial analysis. Therefore, in some forms, methods for spatial analysis include a first step of providing a blocked sample.

[0251] In some forms, a blocked sample is provided in the same form after blocking as prior to the described methods for blocking. In some forms, the blocked sample is a tissue sample, having at least one dimension having a thickness of between about 1 pm and about 20 pm, inclusive. In some forms, a blocked sample is provided on a substrate (e.g., a slide). In some forms, where a spatial analysis methodology includes a previously blocked sample, such as a frozen or other archival blocked sample, the methods include one or more steps for disposing the blocked sample onto a first substrate. An exemplary substrate includes a metal, plastic, ceramic, glass, rubber or silicon substrate. A preferred substrate is a slide, such as a glass slide. In some forms, the methods include one or more steps of staining and / or labelling a blocked sample prior to spatial analysis. In some forms, the methods include one or more steps of staining and / or labelling a blocked sample after contacting the sample with one or more probes to detect a target analyte e.g., mRNA). In some instances, a blocked sample is stained to detect double- stranded DNA. For example, in some forms, the methods include one or more steps of hematoxylin and / or eosin (H and E) staining of a blocked sample prior to spatial analysis. In some forms, the methods include one or more steps of imaging a blocked sample prior to spatial analysis. In some forms, the methods include one or more steps of de-staining a stained, blocked sample prior to spatial analysis.

[0252] 2. Blocked Samples as Reference or Control Samples

[0253] In some forms, a blocked sample is used as a reference for an amount of single stranded and / or double stranded DNA in a subset of samples.

[0254] In some forms, a blocked sample includes no ssDNA, or no accessible ssDNA. In some forms, the methods include one or more steps for quantifying the amount of ssDNA in a sample, for use as a reference value for subsequent or consecutive assays, such as spatial analysis assays. For example, it is contemplated that first and second assays are carried out, including a first blocked sample, and then including a second, non-blocked (i.e., “native”) sample. The first and second samples can be obtained from the same or different sources and obtained and / or processed according to the same or different methods. In some forms, a first sample is blocked to reduce or remove ssDNA accessible to probes for hybridization in the sample. In some forms, the amount of ssDNA in the first sample is determined, for example, using block primers associated or bound to one or more labels or dyes. In some forms, a reference sample includes a known proportion, for example, from about 0% up to about 100%, inclusive, such as 1%, 2%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of ssDNA or dsDNA. The fluorescence or other label or dye obtained from the reference sample(s), having known proportion(s) of dsDNA or ssDNA can be used to estimate the amount of ssDNA that is present in a first and / or second test sample(s), by comparison with the amount of label or other marker present in these samples before and after the blocking process, as described. In some forms, for quantitation of the proportion or amount of ssDNA and / or dsDNA in a sample, it is necessary to know the total amount of all DNA in the sample. In other forms, it is necessary only to estimate the amount of ssDNA in the sample, for example, by quantitation of the amount of a label or marker associated with a block primer.

[0255] Therefore, in some forms, the methods include one or more steps to quantitate the amount or proportion of ssDNA and / or dsDNA present in a sample by extrapolation of the relative presence of a marker or label in a blocked sample relative to a reference sample having a known amount or proportion of ssDNA and / or dsDNA. In some forms, when the methods include quantitation of ssDNA and / or dsDNA in a blocked or, by extrapolation, a non-blocked sample, the methods include one or more steps to optimize the blocking process for a subset of non-blocked sample, such as a multiplicity of samples from the same tissue or organ or subject, and / or processed, or stored, or otherwise modified according to the same or different methods. Exemplary parameters for optimization of the described blocking methods include the amount and species and diversity of different block primers that are used when contacting the sample with block primers.

[0256] In other forms, the extent of the improved resolution of spatial analysis data for one or more target analytes using a single blocked sample is used to inform a coefficient value for modifying data obtained using a similar or different non-blocked sample. Therefore, in some forms, the methods include spatial analysis of one or more blocked sample, as a means for informing the accuracy or precision of spatial analyses for one or more non-blocked samples.

[0257] In some forms, further non-blocked samples that are used as a reference include samples prepared with a method that will not generate ssDNA or that will degrade it (e.g., upon DNAse treatment). In some forms, further non-blocked samples that are used as a reference include unfixed samples that did not undergo de-crosslinking.

[0258] E. Additional Steps

[0259] Any of the described methods can include one or more additional steps to further enhance or otherwise facilitate methods for spatial profiling of one or more target analytes in a blocked sample.

[0260] For example, as described herein, in some forms the methods include one or more steps of washing a sample before and / or after blocking, labeling and / or imaging a sample before and / or after blocking, permeabilizing a sample before and / or after blocking, etc. Each of these processes can be carried out as part of one or more of the described method steps. For example, the additional methodologies can be applied during, before or after any one or more of the described method steps (a), (b), (c), (d), (e), (f), (g) or (h). Additional details of these steps are provided below.

[0261] 1. Preparation of the Sample for Block Primers and / or Target Analyte-Binding Probes

[0262] In some forms, the methods include one or more steps for preparation of the biological sample for application of block primers and / or probes. For example, in some forms, the biological sample or blocked sample is de-paraffinized and / or de-crosslinked. In some forms, one or more steps to de-paraffinize a biological sample or blocked sample is carried out prior to contacting the biological sample with one or more block primers or first and / or second probes (e.g., prior to step (a) or step (d)). a. De-paraffinizing a Sample

[0263] In some forms, the methods include one or more steps to de-paraffinize a biological sample or blocked sample that includes paraffin wax. For example, in some forms, the biological sample or blocked sample is treated with a series of washes that include xylene and various concentrations of ethanol. In some forms, methods of deparaffinization include treatment of xylene e.g., three washes at 5 minutes each). In some forms, the methods further include treatment with ethanol (e.g., 100% ethanol, two washes 10 minutes each; 95% ethanol, two washes 20 minutes each; 70% ethanol, two washes 10 minutes each; 50% ethanol, two washes 10 minutes each). In some forms, after ethanol washes, the biological sample or blocked sample is washed with deionized water (e.g., two washes for 5 minutes each). In some embodiments, the biological sample (e.g., FFPE sample) is permeable after deparaffinization. In some embodiments, processing of the biological sample, such as de-waxing, allows the biological sample to become permeabilized. It is appreciated that one skilled in the art can adjust these methods to optimize deparaffinization. b. De-crosslinking a Sample

[0264] In some forms, the biological sample or blocked sample is de-crosslinked. In some forms, the biological sample is de-crosslinked, then blocked. In some instances, an extension reaction as described herein is performed in a de-crosslinked sample to generate a blocked sample. For example, in some forms, the biological sample or blocked sample is de-crosslinked in a solution containing TE buffer (including Tris and EDTA). In some forms, the TE buffer is basic (e.g., at a pH of about 9). In some forms, de-crosslinking occurs at about 50°C to about 80°C. In some forms, de-crosslinking occurs at about 70°C. In some forms, de-crosslinking is performed at a temperature of at least about 70°C. In some forms, de-crosslinking is performed at a temperature of at least about 75°C. In some forms, de-crosslinking is performed at a temperature of at least about 80°C. In some forms, de-crosslinking is performed at a temperature of at least about 85 °C. In some forms, de-crosslinking is performed at a temperature of at least about 90°C. In some forms, de-crosslinking is performed at a temperature of at least about 95 °C. In some forms, de-crosslinking occurs for about 1 hour at 70°C. For example, in some forms, just prior to de-crosslinking, the biological sample or blocked sample is treated with an acid (e.g., 0.1 M HC1 for about 1 minute). After the decrosslinking step, the biological sample or blocked sample can be washed (e.g., with lx PBST). c. Permeabilizing a Sample

[0265] In some forms, the biological sample or blocked sample is permeabilized before, during or after one or more of steps of the methods, e.g., steps (a), (b), (c), (d) or (e). In some forms, the methods of preparing a biological sample or blocked sample for probe or block primer application include permeabilizing the sample or blocked sample. In some forms, the biological sample or blocked sample is permeabilized using a phosphate buffer. In some forms, the phosphate buffer is PBS (e.g., lx PBS). In some forms, the phosphate buffer is PBST (e.g., lx PBST). In some forms, the permeabilization step is performed multiple times (e.g., 3 times at 5 minutes each).

[0266] In some forms, permeabilization occurs using a protease. In some forms, the protease is an endopeptidase. Endopeptidases that can be used include but are not limited to trypsin, chymotrypsin, elastase, thermolysin, pepsin, clostripan, glutamyl endopeptidase (GluC), ArgC, peptidyl-asp endopeptidase (ApsN), endopeptidase LysC and endopeptidase LysN. In some forms, the endopeptidase is pepsin. In some forms, the protease is proteinase K.

[0267] In some forms, after creating a connected probe (e.g., by ligating a first probe and / or a second probe that are hybridized to adjacent sequences in a target RNA or ligating a circularizable probe such as a padlock probe), the biological sample or blocked sample is permeabilized. In some forms, the biological sample or blocked sample is permeabilized contemporaneously with, or prior to, contacting the biological sample or blocked sample with a first probe and / or a second probe, e.g., hybridizing and coupling the first probe and the second probe to the provide a combined probe, and then permeabilizing the biological sample or blocked sample to release the connected probe from the analytes in the blocked sample.

[0268] In some forms, methods provided herein include permeabilization of the biological sample or blocked sample such that a probe / bound analyte can more easily hybridize to the immobilized capture probe (e.g., compared to no permeabilization).

[0269] In some forms, the permeabilization step includes application of a permeabilization buffer to the biological sample or blocked sample. In some forms, the permeabilization buffer includes a buffer e.g., Tris pH 7.5), MgCh, a detergent (e.g., sarkosyl detergent, also known as sodium lauroyl sarcosinate), enzyme (e.g., proteinase K), and nuclease free water. In some forms, the permeabilization step is performed at 37°C. In some forms, the permeabilization step is performed 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 forms, the releasing step is performed for about 40 minutes.

[0270] In some forms, after generating a connected probe / ligation product, the connected probe / ligation product is released from the analyte. In some forms, a connected probe / ligation product is released from the analyte using an endoribonuclease. In some forms, the endoribonuclease is RNase H, RNase A, RNase C, or RNase I. In some forms, the endoribonuclease is RNase H. RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA, when hybridized to DNA.

[0271] RNase H is part of a conserved family of ribonucleases which are present in many different organisms. There are two primary classes of RNase H: RNase Hl and RNase H2. Retroviral RNase H enzymes are similar to the prokaryotic RNase Hl. All of these enzymes share the characteristic that they are able to cleave the RNA component of an RNA:DNA heteroduplex. In some forms, the RNase His RNase Hl, RNase H2, or RNase Hl, or RNase H2. In some forms, the RNase H includes but is not limited to RNase HII from Pyrococcus furiosus, RNase HII from Pyrococcus horikoshi, RNase HI from Thermococcus litoralis, RNase HI from Thermus thermophilus, RNAse HI from E. coli, or RNase HII from E. coli. In some forms, the releasing step is performed using a releasing buffer. In some forms, the release buffer includes one or more of a buffer (e.g., Tris pH 7.5), enzyme (e.g., RNAse H) and nuclease-free water.

[0272] In some forms, the releasing step is performed at 37°C. In some forms, the releasing step is performed 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 forms, the releasing step is performed for about 30 mins. In some forms, the releasing step occurs before the permeabilization step. In some forms, the releasing step occurs after the permeabilization step. In some forms, the releasing step occurs at the same time as the permeabilization step (e.g., in the same buffer). d. Pre-Hybridization Processing of a Sample

[0273] In some forms, the methods of preparing a biological sample or blocked sample for target analyte binding probe application include steps of equilibrating and treating the biological sample or blocked sample. In some forms, equilibrating is performed using a pre-hybridization (pre-Hyb) buffer. In some forms, the pre-Hyb buffer is RNase-free. In some forms, the pre-Hyb buffer contains no bovine serum albumin (BSA), solutions like Denhardt's, or other potentially nuclease-contaminated biological materials.

[0274] In some forms, the equilibrating step is performed multiple times (e.g., 2 times at 5 minutes each; 3 times at 5 minutes each). In some forms, the biological sample or blocked sample is treated with a buffer. In some forms, the buffer includes a carrier such as tRNA, for example yeast tRNA such as from brewer's yeast (e.g., at a final concentration of 10-20 pg / mL). In some forms, treating with a buffer including tRNA can be performed for 5, 10, 15, 20, 25, or 30 minutes. In some forms, equilibrating is performed using a pre-hybridization (pre-Hyb) buffer. In some forms, the pre-Hyb buffer is RNase-free. In some forms, the pre-Hyb buffer contains no bovine serum albumin (BSA), solutions like Denhardt's, or other potentially nuclease-contaminated biological materials.

[0275] In some forms, the equilibrating step is performed multiple times (e.g., 2 times at 5 minutes each; 3 times at 5 minutes each).

[0276] Any of the foregoing steps can be optimized for performance. For example, one can vary the temperature. In some forms, the pre-hybridization methods are performed at room temperature. In some forms, the pre -hybridization methods are performed at 4°C (in some forms, varying the timeframes provided herein).

[0277] In some forms, the capture domain of a capture probe or the capture domain binding site of a first / second probe is deliberately occluded prior to binding and / or adding a first probe or second probe to a biological sample or blocked sample. This prevents the first and / or second probe capture domain binding sequences from prematurely hybridizing, for example, prior to analyte binding.

[0278] Therefore, in some forms, an occluding probe is used to occlude or modify the free 3' end of the capture domain of a capture probe, or the capture domain binding site of a second probe. In some forms, an occluding probe can be hybridized to the capture domain of the second probe to mask the free 3' end of the capture domain. In some forms, an occluding probe can be a hairpin probe or partially double stranded probe. In some forms, the free 3' end of the capture domain, or of the second probe capture domain binding sequence can be occluded by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture domains do not include a free 3' end.

[0279] Occluding or modifying the capture domain or the capture domain binding site of a second probe, particularly at the free 3' end of the capture domain, prior to contacting with a first, second or combined probe, prevents undesired or premature hybridization of the second probe capture domain binding sequence to the capture domain (e.g., prevents the capture of a poly(A) of a capture domain to a poly(T) capture domain). In some forms, an occluding probe can be referred to as a capture domain occluding moiety.

[0280] In some forms, an occluding probe can be reversibly removed. For example, an occluding probe can be applied to occlude the free 3' end of either or both the capture domain or second probe capture domain binding sequence. Occluding interaction between the capture domains can reduce non-specific capture to the capture probes. After the second probe hybridizes to an analyte (e.g., a nucleic acid) (and is optionally docked and / or ligated to a first probe), an occluding probe can be removed from the 3' end of the capture domain and / or the capture probe, and the combined probe / ligation product can migrate to and become bound by a capture probe (e.g., immobilized on a substrate). In some forms, the removal includes denaturing an occluding probe from capture domain and / or first oligonucleotide or second oligonucleotide capture domain binding sequence. In some forms, the removal includes removing a chemically reversible capping moiety. In some forms, the removal includes digesting the blocking probe with an RNase (e.g., RNase H).

[0281] In some forms, an occluding probe is an oligo (dT) occluding probe. In some forms, the oligo (dT) occluding probes have a length of 15-30 nucleotides. In some forms, the oligo (dT) occluding 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 forms, the capture domain or capture domain binding site can be blocked at different temperatures (e.g., 4°C and 37°C). e. Washing a Sample

[0282] In some forms, the methods disclosed herein also include a wash step. In some forms, a wash step removes any unbound oligonucleotides, and / or any unbound probes. Wash steps could be performed between any of the steps in the methods disclosed herein. For example, a wash step can be performed after adding block primers, target analytebinding probes or oligonucleotides to the biological sample or blocked sample. As such, free / unbound probes or oligonucleotides are washed away, leaving only probes or oligonucleotides that have hybridized to an analyte. In some forms, multiple e.g., at least 2, 3, 4, 5, or more) wash steps occur between the methods disclosed herein. Wash steps can be performed at times (e.g., 1, 2, 3, 4, or 5 minutes) and temperatures (e.g., room temperature; 4°C) known in the art and determined by a person of skill in the art. In some forms, wash steps are performed using a wash buffer. In some forms, the wash buffer includes SSC (e.g., lx SSC). In some forms, the wash buffer includes PBS (e.g., lx PBS). In some forms, the wash buffer includes PBST (e.g., lx PBST). In some forms, the wash buffer can also include formamide or be formamide free. f. Processing for Sequence Determination

[0283] In some forms, the step of determining a sequence for two or more components of a captured probe (e.g. , as in step (h)) includes amplifying all or part of the connected probe / ligation product bound to the capture domain of a capture probe. In some forms, the connected probe is extended using the capture probe as a template and / or the capture probe is extended using the connected probe as a template. In some aspects, provided herein are methods comprising in situ assays using microscopy as a readout for sequence determination, e.g., nucleic acid sequencing, hybridization, or other detection or determination methods involving an optical readout. In some aspects, detection or determination of a sequence of one, two, three, four, five, or more nucleotides of an analyte nucleic acid is performed in situ in a cell in a biological sample, e.g., tissue sample or tissue section.

[0284] Therefore, in some forms, a step of determining includes amplifying a captured connected probe (e.g., after extension using the capture probe as a template) to produce an amplified product. In some forms, a step of determining includes amplifying a capture probe (e.g., after extension using the connected probe as a template) to produce an amplified product. An exemplary amplified product includes (i) all or part of a sequence of the connected probe / ligation product bound to the capture domain, or a complement thereof, and (ii) the spatial barcode, or a complement thereof.

[0285] In some forms, the determining step includes sequencing. In some forms, the sequencing step includes in situ sequencing, Sanger sequencing, next-generation sequencing, and / or nanopore sequencing.

[0286] In some forms, the methods further include subjecting a region of interest in the biological sample or blocked sample to spatial transcriptomic analysis. In some forms, one or more of the capture probes includes a unique molecular identifier (UMI). In some forms, one or more of the capture probes includes a cleavage domain. In some forms, the cleavage domain includes a sequence recognized and cleaved by a uracil-DNA glycosylase, apurinic / apyrimidinic (AP) endonuclease (APE1), U uracil-specific excision reagent (USER), and / or an endonuclease VIII. In some forms, one or more capture probes do not include a cleavage domain and therefore, for example, is not cleaved from an array.

[0287] In some forms, a capture probe bound to a connected probe (“captured probe”) can be extended (an "extended captured probe," e.g., as described herein). Therefore, in some forms, the methods include extending a capture probe bound to a connected probe to provide an extended captured probe. For example, extending a capture probe bound to a connected probe can include generating cDNA from a captured (hybridized) nucleic acid hybridized to the complementary region of the second probe.

[0288] An exemplary captured nucleic acid is an RNA, such as an mRNA. This process typically involves synthesis of a complementary strand of the hybridized nucleic acid, e.g., generating cDNA based on the captured RNA template (the RNA hybridized to the capture domain of the capture probe). Thus, in an initial step of extending a captured probe, e.g., the cDNA generation, the captured (hybridized) nucleic acid, e.g., RNA, acts as a template for the extension, e.g., reverse transcription, step. In some forms, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can produce spatially-barcoded fuller partial-length cDNA from the captured analytes (e.g., polyadenylated mRNA). Second strand reagents e.g., second strand primers, enzymes) can be added to the biological sample to initiate second strand synthesis.

[0289] In some forms, the extension includes reverse transcription. For example, reverse transcription includes synthesizing cDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), using a reverse transcriptase. In some forms, reverse transcription is performed while the tissue is still in place, generating an analyte library, where the analyte library includes the spatial barcodes from the associated capture probes. In some forms, a captured probe is extended using one or more DNA polymerases. In some forms, a capture probe binding domain of a first probe, or a second probe, and / or a capture domain of a capture probe includes a primer for producing the complementary strand of a nucleic acid hybridized to the probe, e.g., a primer for DNA polymerase and / or reverse transcription. The nucleic acid, e.g., DNA and / or cDNA, molecules generated by the extension reaction incorporate the sequence of the first probe, second probe or capture probe, respectively. The extension of the captured probe, e.g., a DNA polymerase and / or reverse transcription reaction, can be performed using a variety of suitable enzymes and protocols.

[0290] In some forms, a full-length DNA e.g., cDNA) molecule is generated. In some forms, a "full-length" DNA molecule refers to the whole of a “captured” nucleic acid molecule. However, if a nucleic acid (e.g., RNA) was partially degraded in the tissue sample, then the captured nucleic acid molecules will not be the same length as the initial RNA in the tissue sample. In some forms, the 3' end of an extended captured probe, e.g., first strand cDNA molecules, is modified. For example, a linker or adaptor can be ligated to the 3' end of the extended captured probes. This can be achieved using single stranded ligation enzymes such as T4 RNA ligase or CIRCLIGASE™ (available from Lucigen, Middleton, WI).

[0291] In some forms, template switching oligonucleotides are used to extend cDNA in order to generate a full-length cDNA (or as close to a full-length cDNA as possible). In some forms, a second strand synthesis helper probe (a partially double stranded DNA molecule capable of hybridizing to the 3' end of the extended captured probe), can be ligated to the 3' end of the extended captured probe, e.g., first strand cDNA, molecule using a double stranded ligation enzyme such as T4 DNA ligase. Other enzymes appropriate for the ligation step are known in the art and include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA ligase (9°N™ DNA ligase, New England Biolabs), AMPLIGASE™ (available from Lucigen, Middleton, WI), and SplintR (available from New England Biolabs, Ipswich, MA). In some forms, a polynucleotide tail, e.g., a poly(A) tail, is incorporated at the 3' end of the extended captured probe / cDNA molecules. In some forms, the polynucleotide tail is incorporated using a terminal transferase active enzyme. i. Sequence Amplification

[0292] In some forms, double- stranded extended captured probes are treated to remove any un-extended captured probes prior to amplification and / or analysis, e.g., sequence analysis. This can be achieved by a variety of methods, e.g., using an enzyme to degrade the un-extended captured probes, such as an exonuclease enzyme, or purification columns.

[0293] In some forms, extended captured probes, or complements thereof, are amplified to yield quantities that are sufficient for analysis, e.g., via DNA sequencing. In some forms, the first strand of the extended captured probes (e.g., DNA and / or cDNA molecules) acts as a template for the amplification reaction (e.g., a polymerase chain reaction).

[0294] In some forms, the amplification reaction incorporates an affinity group onto the extended capture probe (e.g., RNA-cDNA hybrid) using a primer including the affinity group. In some forms, the primer includes an affinity group and the extended captured probes includes the affinity group.

[0295] In some forms, amplifying the extended captured probes can function, e.g., to release the extended captured probes from the surface of a substrate, insofar as copies of the extended probes are not immobilized on the substrate.

[0296] In some forms, the extended capture probe or complement or amplicon thereof is released. The step of releasing the extended captured probe or complement or amplicon thereof from the surface of the substrate can be achieved in a number of ways. In some forms, an extended captured probe or a complement thereof is released from the array by nucleic acid cleavage and / or by denaturation (e.g., by heating to denature a double stranded molecule).

[0297] In some forms, the extended captured probe or complement or amplicon thereof is released from a surface, e.g., of a substrate (e.g., array) by physical means. For example, where the extended captured probe is indirectly immobilized on an array substrate, e.g., via hybridization to a surface probe, it can be sufficient to disrupt the interaction between the extended captured probe and the surface probe. One method for releasing the DNA molecules e.g. , of stripping the array of extended probes) is to use a solution that interferes with the hydrogen bonds of the double stranded molecules. In some forms, the extended captured probe is released by an applying heated solution, such as water or buffer, of at least 85°C, e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99°C. In some forms, a solution including salts, surfactants, etc. that can further destabilize the interaction between the nucleic acid molecules is added to release the extended captured probe from a substrate.

[0298] In some forms, where the extended captured probe includes a cleavage domain, the extended capture probe is released from a surface of a substrate by cleavage. For example, the cleavage domain of the extended captured probe can be cleaved by any of the methods described herein, e.g., via enzymatic cleavage. In some forms, the extended captured probe is released from the surface of the substrate, e.g., via cleavage of a cleavage domain in the extended captured probe, prior to the step of amplifying the extended captured probe. ii. Template Switching Oligonucleotides

[0299] In some forms, the captured analytes can be spatially-barcoded by performing a reverse transcriptase first strand cDNA reaction using template switching oligonucleotides. For example, a template switching oligonucleotide (TSO) can hybridize to a poly(C) tail added to a 3 'end of the cDNA by a reverse transcriptase enzyme in a template-independent manner. The hybridized TSO is used to further extend the first strand cDNA such that it includes a complement of the TSO. The original nucleic acid template and template switching oligonucleotide can then be denatured from the extended cDNA and the spatially-barcoded capture probe can then hybridize with the cDNA and a complement of the cDNA can be generated. In some forms, the TSO (or a primer having a similar sequence thereto) can be used to prime synthesis of a second strand cDNA templated from the first strand cDNA. The first strand cDNA can then be purified and collected for downstream amplification steps. The first strand cDNA can be amplified using PCR, where the forward and reverse primers flank the spatial barcode and analyte regions of interest, generating a library associated with a particular spatial barcode. In some forms, the library preparation can be quantitated and / or quality controlled to verify the success of the library preparation steps. A "template switching oligonucleotide" is an oligonucleotide that hybridizes to untemplated nucleotides added by a reverse transcriptase (e.g., enzyme with terminal transferase activity) during reverse transcription. In some forms, a template switching oligonucleotide hybridizes to untemplated poly(C) nucleotides added by a reverse transcriptase. In some forms, the template switching oligonucleotide adds a common 5' sequence to full-length cDNA that is used for cDNA amplification.

[0300] In some forms, the template switching oligonucleotide adds a common sequence onto the 5' end of an RNA being reverse transcribed. For example, a template switching oligonucleotide can hybridize to untemplated poly(C) nucleotides added onto the end of a cDNA molecule and provide a template for the reverse transcriptase to continue replication to the 5' end of the template switching oligonucleotide, thereby generating full length cDNA ready for further amplification.

[0301] In some forms, once a full-length cDNA molecule is generated, the template switching oligonucleotide can serve as a primer in a cDNA amplification reaction. In some forms, a template switching oligonucleotide is added before, contemporaneously with, or after a reverse transcription, or other terminal transferase-based reaction. In some forms, a template switching oligonucleotide is included in the capture probe. In certain forms, methods of sample analysis using template switching oligonucleotides can involve the generation of nucleic acid products from analytes of the biological sample, followed by further processing of the nucleic acid products with the template switching oligonucleotide.

[0302] Template switching oligonucleotides can include a hybridization region and a template region. The hybridization region can include any sequence capable of hybridizing to the target. In some forms, the hybridization region can, e.g., include a series of G bases to complement the overhanging C bases at the 3' end of a cDNA molecule. The series of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. The template sequence can include any sequence to be incorporated into the cDNA.

[0303] In other forms, the hybridization region can include at least one base in addition to at least one G base. In other forms, the hybridization can include bases that are not a G base. In some forms, the template region includes at least 1 (e.g., at least 2, 3, 4, 5 or 10 more) tag sequences and / or functional sequences. In some forms, the template region and hybridization region are separated by a spacer. In some forms, the template regions include a barcode sequence. The barcode sequence can act as a spatial barcode and / or as a unique molecular identifier. Template switching oligonucleotides can include deoxyribonucleic acids; ribonucleic acids; modified nucleic acids including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT, 5-methyl dC, 2'-deoxyinosine, Super T (5-hydroxybutynl-2'-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,

[0304] UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2' fluoro bases e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or any combination of the foregoing.

[0305] In some forms, the length of a template switching oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides or longer. In some forms, the length of a template switching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides or longer.

[0306] F. Additional Reagents

[0307] Any of the described methods for using blocking primers for enhanced resolution of spatial profiling provided herein can include one or more of the following reagents, such as biological samples, reagents for sample preparation, probe hybridization and sequence analysis.

[0308] 1. Biological Samples Prior to Blocking

[0309] In some forms, a biological sample prior to blocking according to the described methods is provided as a tissue section. In some forms, the biological sample is a tissue sample. In some forms, the biological sample (e.g., tissue sample) is a tissue microarray (TMA). A tissue microarray contains multiple representative tissue samples - which can be from different tissues or organisms - assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time. Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates. In some forms, the biological sample is a fresh frozen sample (e.g., tissue sample). In some forms, the biological sample (e.g., tissue sample) was previously frozen.

[0310] In some forms, a biological sample can be a fixed and / or stained biological sample (e.g., a fixed and / or stained tissue section). Non-limiting examples of stains include histological stains (e.g., hematoxylin and / or eosin) and immunological stains (e.g., fluorescent stains). In some forms, a biological sample can be fixed with a fixative selected from ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, and combinations thereof. Thus, in some forms, the biological sample is a formalin-fixed paraffin embedded tissue sample, a paraformaldehyde fixed tissue sample, a methanol fixed tissue sample, or an acetone fixed tissue sample. In some instances, the biological sample is fixed using PAXgene. PAXgene is a formalin-free, non-cross-linking fixative that preserves morphology and biomolecules. It is a mixture of different alcohols, acid, and a soluble organic compound. Ergin B. et al., J Proteome Res. 2010 Oct 1 ;9(10):5188-96 describes the development of PAXgene. Kap M. etal., PLoS One. ', 6(1 l):e27704 (2011) and Mathieson W. et al., Am J Clin Pathol.; 146(l):25-40 (2016) both describe and evaluate PAXgene for tissue fixation.

[0311] In some forms, a biological sample (e.g., a fixed and / or stained biological sample) can be imaged. Biological samples are also described in Section (I)(d) of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0312] Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease e.g., cancer) or a pre-disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy. In some forms, the biological sample can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. In some forms, the biological sample includes cancer or tumor cells. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. In some forms, the biological sample is a heterogenous sample. In some forms, the biological sample is a heterogenous sample that includes tumor or cancer cells and / or stromal cells.

[0313] In some forms, the cancer is breast cancer. In some forms, the breast cancer is triple positive breast cancer (TPBC). In some forms, the breast cancer is triple negative breast cancer (TNBC). In some forms, the cancer is colorectal cancer. In some forms, the cancer is ovarian cancer. In certain forms, the cancer is squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's or nonHodgkin’s lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, myeloma, salivary gland carcinoma, kidney cancer, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, or a type of head or neck cancer. In certain forms, the cancer treated is desmoplastic melanoma, inflammatory breast cancer, thymoma, rectal cancer, anal cancer, or surgically treatable or non-surgically treatable brain stem glioma. In some forms, the subject is a human. a. Target Nucleic Acid Analytes

[0314] Compositions for use with the described methods can include one or more analyte-binding probes configured to bind one or more classes of nucleic acid analytes.

[0315] Typically, when the methods include DNA-blocking primers to provide a blocked sample, the methods for spatial analysis are suitable for spatial profiling of non-DNA nucleic acids present in a biological sample. Exemplary classes of target nucleic acids include biological RNAs, as well as synthetic RNA. Exemplary classes of target RNAs include small interfering RNA (siRNA), microRNA (miRNA), P-element-induced wimpy testis (PlWI)-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), ribosomal RNA (rRNA), long noncoding RNAs (incRNA), and transfer RNA (tRNA). In certain forms, the target RNA is transcriptomic RNA, such as mRNA.

[0316] 2. Sample Permeabilization Reagents

[0317] In some forms, a biological sample or blocked sample is permeabilized with one or more permeabilization reagents. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)( 13) or the Exemplary Forms Section of WO 2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. In some forms, permeabilizing includes the use of an organic solvent, a detergent, an enzyme, or a combination thereof. In some forms, permeabilizing includes the use of an endopeptidase (e.g., pepsin or proteinase K), a protease, sodium dodecyl sulfate, polyethylene glycol tert-octylphenyl ether, polysorbate 80, polysorbate 20, N- lauroylsarcosine sodium salt solution, saponin, Triton X-100™, Tween-20™, or combinations thereof.

[0318] Array-based spatial analysis methods involve the transfer of one or more analytes, or proxies thereof, from a biological sample or blocked sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes / proxies includes determining the identity of the analytes and the spatial location of the analytes within the biological sample or blocked sample. The spatial location of an analyte within the biological sample or blocked sample is determined based on the feature to which the analyte is bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array.

[0319] III. Spatial Analysis with In situ Detection

[0320] In some aspects, provided herein are methods comprising in situ assays using microscopy as a readout, e.g., nucleic acid sequencing, hybridization, or other detection or determination methods involving an optical readout. In some aspects, detection or determination of a sequence of one, two, three, four, five, or more nucleotides of an analyte nucleic acid is performed in situ in a cell in a biological sample, e.g., tissue sample or tissue section. In some instances, the methods provided herein decrease off- target recognition of ssDNA in a sample by target analyte-binding oligonucleotides e.g., probes) and thereby reduce inflated counts of one or more target analytes in a sample.

[0321] In some embodiments, the methods for reducing ssDNA e.g., as described in Section II) are performed prior to performing an assay for detecting a target analyte. In some embodiments, the methods for reducing ssDNA (e.g., as described in Section II) are performed prior to contacting the biological sample with a probe or probe set for detecting a target analyte. Provided herein is a method including contacting a biological sample including a nucleic acid with a polymerase, whereby the nucleic acid includes one or more regions of ssDNA; performing an extension using the one or more regions of ssDNA as template to generate double stranded DNA (dsDNA), thereby providing a blocked sample; and contacting the blocked sample with a probe or probe set to detect one or more target analyte(s) in the blocked sample. In some embodiments, the probe or probe set or an amplification product thereof is detected in the biological sample.

[0322] In some embodiments, the assay includes detecting the presence or absence of an amplification product (e.g., RCA product). In some embodiments, the present disclosure provides methods for high-throughput profiling of a large number of analytes in situ, such as transcripts, e.g., for detecting and / or quantifying nucleic acids and / or proteins in cells, tissues, organs or organisms. In some embodiments, the hybridization of oligonucleotides (e.g., probes or sequencing primer) with the sample and / or detection steps during the in situ assay is performed on analytes after the blocked sample is provided.

[0323] In some forms, an in situ assay is performed in addition or alternatively to the connected probe (e.g., RTL) approach described in the methods disclosed herein. Thus, in some forms, the in situ assay is performed on a blocked sample as described herein. In some embodiments of the methods including in situ analysis, hybridization to capture probes on an array is not included. In some forms, the biological sample, e.g., the tissue sample is fixed, for example in methanol, acetone, acetone-methanol, PFA, PAXgene or is formalin-fixed and paraffin-embedded (FFPE). In some forms, the biological sample includes intact cells. In some forms, the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet. In some embodiments, the biological sample is an intact tissue sample. In some embodiment, the biological sample comprises a cell or tissue sample comprising blocked DNA processed as described herein. In some embodiments, the biological sample is an FFPE sample, e.g., a human FFPE sample. In some embodiments, the biological sample comprises genomic DNA e.g., open and / or fragmented DNA. In some aspects, it is beneficial for assay performance (e.g., an assay for RNA detection) to treat the biological sample to provide a blocked sample (e.g., with reduced gDNA). In some embodiments, one or more target RNA analytes are detected in a cell or in a tissue section of a blocked sample. In some embodiments, one or more target RNA analytes are detected at a location in a cell or tissue section of a blocked sample.

[0324] In some examples, the methods comprises:

[0325] (a) contacting a biological sample including a ssDNA region with a DNA polymerase to extend a free 3’ end of a DNA using the one or more regions of ssDNA present in the biological sample as template to provide a blocked sample; and

[0326] (b) contacting the blocked sample with one or more nucleic acid probes that directly or indirectly hybridize to a set of analytes or complements or amplification products thereof in the blocked sample; and

[0327] (c) detecting the one or more nucleic acid probes at a spatial location of the blocked sample.

[0328] In some examples, the methods comprises:

[0329] (b) contacting a biological sample including a ssDNA region with a DNA polymerase to extend a free 3’ end of a DNA using the one or more regions of ssDNA present in the biological sample as template to provide a blocked sample; and

[0330] (b) contacting the blocked sample with one or more nucleic acid primers that directly or indirectly hybridize to a set of analytes or complements or amplification products thereof in the blocked sample; and (c) performing base-by-base sequencing to detect the set of analytes or complements or amplification products thereof at a spatial location of the blocked sample.

[0331] In some aspects, disclosed herein a method for processing a biological sample comprising single- stranded DNA (ssDNA), comprising:

[0332] (a) contacting a biological sample comprising ssDNA with a multiplicity of block primers configured to hybridize with one or more regions of ssDNA present in the biological sample;

[0333] (b) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample, wherein the blocked sample comprises less ssDNA than the biological sample prior to step (a);

[0334] (c) optionally washing the blocked sample to remove any unbound (or unhybridized) block primers and / or block primer dimers;

[0335] (d) contacting the blocked sample with one or more nucleic acid probes that directly or indirectly hybridize to a set of analytes or complements or amplification products thereof in the blocked sample; and

[0336] (e) detecting the one or more nucleic acid probes at a spatial location of the blocked sample.

[0337] In some aspects, disclosed herein a method for processing a biological sample comprising single- stranded DNA (ssDNA), comprising:

[0338] (a) contacting a biological sample comprising ssDNA with a multiplicity of block primers configured to hybridize with one or more regions of ssDNA present in the biological sample;

[0339] (b) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample, wherein the blocked sample comprises less ssDNA than the biological sample prior to step (a);

[0340] (c) optionally washing the blocked sample to remove any unbound (or unhybridized) block primers and / or block primer dimers;

[0341] (d) contacting the blocked sample with one or more nucleic acid primers that directly or indirectly hybridize to a set of analytes or complements or amplification products thereof in the blocked sample; and (c) performing base-by-base sequencing to detect the set of analytes or complements or amplification products thereof at a spatial location of the blocked sample.

[0342] Also disclosed herein is a method for analyzing an RNA analyte in a biological sample, the method comprising:

[0343] (a) providing the biological sample disposed on a substrate;

[0344] (b) contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of single-stranded DNA (ssDNA) present in the biological sample;

[0345] (c) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample, optionally wherein the blocked sample comprises less ssDNA than the biological sample prior to step (b);

[0346] (d) contacting the blocked sample with one or more nucleic acid probes that directly or indirectly hybridize to a set of analytes or complements or amplification products thereof in the blocked sample; and

[0347] (e) detecting in the blocked sample the one or more nucleic acid probes at a spatial location of the blocked sample.

[0348] In some forms of the foregoing methods, the analytes comprises an RNA analyte. In some forms, the RNA analyte is an mRNA analyte. In some forms the set of first analytes comprises a DNA analyte. In some forms, the DNA analyte is genomic DNA. In some forms, the one or more nucleic acid probes directly hybridize to a first analyte in the set of first analytes or the complement or the amplification product thereof. In some forms, the one or more nucleic acid primers for base-by-base sequencing directly hybridize to a first analyte in the set of first analytes or the complement or the amplification product thereof. In some forms, the one or more nucleic acid probes indirectly hybridize to a first analyte in the set of first analytes or the complement or the amplification product thereof. In some embodiments, one analyte is bound by two or more nucleic acid probes or primers for sequence detection.

[0349] In some forms, a nucleic acid probe of the one or more nucleic acid probes comprises a padlock probe, a circular probe, or a circularized probe. In some forms, a nucleic acid probe of the one or more nucleic acid probes is part of a probe set, e.g., a circularizable probe set. In some forms, detecting the one or more nucleic acid probes comprises imaging the blocked sample to detect fluorescent signal. In some forms, detecting the one or more nucleic acid probes comprises in situ sequencing and / or sequential hybridization of a plurality of probes.

[0350] In some forms, the one or more nucleic acid probes comprise a nucleic acid probe that directly hybridizes to a first analyte in the set of first analytes or the complement or the amplification product thereof. In preferred forms, the first analyte is an mRNA, the complement is a cDNA, and / or the amplification product is a rolling circle amplification (RCA) product. In some examples, a probe is a padlock probe, a circular probe, or a circularized probe. In some forms, the probe comprises one or more barcode sequences that uniquely identify the first analyte.

[0351] In some forms, detecting the one or more nucleic acid probes comprises contacting the biological sample with one or more detectably labelled probes capable of directly or indirectly hybridizing to the primary probe or a complement or amplification product thereof, optionally wherein the one or more detectably labelled probes hybridize to one or more barcode sequences of the primary probe or the complement or amplification product thereof.

[0352] In some forms, detecting the one or more nucleic acid probes comprises contacting the biological sample with one or more detectable probes capable of directly or indirectly hybridizing to the nucleic acid probe or the complement or amplification product thereof, optionally wherein the one or more detectable probes hybridize to one or more barcode sequences of the nucleic acid probe or the complement or amplification product thereof. In some forms, detecting the one or more nucleic acid probes comprises contacting the biological sample with one or more detectably labelled probes capable of directly or indirectly hybridizing to the one or more detectable probes or the complement or amplification product thereof, optionally wherein the one or more detectably labelled probes hybridize to one or more barcode sequences of the one or more secondary probes or the complement or amplification product thereof.

[0353] In some forms, the method further comprises imaging the biological sample or blocked sample to detect the one or more barcode sequences of the primary probe or the complement or amplification product thereof, and / or the one or more barcode sequences of the one or more detectable probes or the complement or amplification product thereof, optionally wherein determining the sequences comprises base-by-base sequencing (e.g., sequencing by ligation) or sequencing by hybridization.

[0354] In some forms, the method further includes generating an amplification product comprising a sequence of the barcode sequence or a complementary sequence thereof. In some forms, the amplification product is generated by RCA. In some forms, the amplification product includes one or more modified nucleotides.

[0355] In some forms, the in situ detection includes sequencing by ligation. In some forms, the in situ sequencing includes sequencing by hybridization. In some forms, the in situ sequencing includes sequencing by synthesis. In some forms, the wherein the detecting step includes generating a RCA product in situ in the biological sample, the RCA product including a sequence of the first nucleic acid probe or complement thereof; and detecting a signal (e.g., a fluorescent signal) associated with the RCA product at a spatial location of the biological sample or blocked sample.

[0356] A. in situ Analysis

[0357] In some aspects, provided herein is a method comprising analyzing biological analytes based on in situ hybridization of probes comprising nucleic acid sequences. In some embodiments, the method comprises sequential hybridization of detectably- labelled oligonucleotides to barcoded probes that directly or indirectly bind to biological analytes in a sample. In some embodiments, a detectably-labelled oligonucleotide directly binds to one or more barcoded probes. In some embodiments, a detectably- labelled oligonucleotide indirectly binds to one or more barcoded probes, e.g., via one or more intermediate nucleic acid molecules.

[0358] In some aspects, an in situ hybridization based assay is used to localize and analyze nucleic acid sequences (e.g., a DNA or RNA molecule comprising one or more specific sequences of interest) within a native biological sample, e.g., a portion or section of tissue. In some embodiments, the in situ assay is used to analyze the presence, absence, an amount or level of mRNA transcripts (e.g., a transcriptome or a subset thereof, or mRNA molecules of interest) in a biological sample, while preserving spatial context. In some embodiments, the present disclosure provides compositions and methods for in situ hybridization using directly or indirectly labeled molecules, e.g., complementary DNA or RNA or modified nucleic acids, as probes that bind or hybridize to analyte nucleic acids within a biological sample of interest.

[0359] Nucleic acid probes, in some examples, may be labelled with radioisotopes, epitopes, hapten, biotin, or fluorophores, to enable detection of the location of specific nucleic acid sequences on chromosomes or in tissues. In some embodiments, probes are locus specific (e.g., gene specific) and bind or couple to specific regions of a chromosome. In alternative embodiments, probes are alphoid or centromeric repeat probes that bind or couple to repetitive sequences within each chromosome. Probes may also be whole chromosome probes (e.g., multiple smaller probes) that bind or couple to sequences along an entire chromosome.

[0360] In some embodiments, provided herein is a method comprising RNA in situ hybridization to measure and localize RNAs e.g., mRNAs, IncRNAs, and miRNAs) within a biological sample e.g., a fixed tissue sample). In some embodiments, RNA in situ hybridization involves single-molecule RNA fluorescence in situ hybridization (FISH). In some embodiments, fluorescently labelled nucleic acid probes are hybridized to pre-determined RNA analytes, to visualize gene expression in a biological sample. In some embodiments, a FISH method comprises using a single nucleic acid probe specific to each analyte, e.g., single-molecule FISH (smFISH). The use of smFISH may produce a fluorescence signal that allows for quantitative measurement of RNA transcripts. In some embodiments, smFISH comprises a set of nucleic acid probes, about 50 nucleotides in length, wherein each probe is coupled to a set of fluorophores. For example, the set of nucleic acid probes may comprise five probes, wherein each probe is coupled to five fluorophores. In some embodiments, said nucleic acid probes are instead each coupled to one fluorophore. For example, a smFISH protocol may use a set of about 40 nucleic acid probes, about 20 nucleotides in length, each coupled to a single fluorophore. In some embodiments, the length of the nucleic acid probes varies, comprising 10 to 100 nucleotides, such as 30 to 60 nucleotides. Alternatively, a plurality of nucleic acid probes targeting different regions of the same RNA transcript may be used. It will be appreciated by those skilled in the art that the type of nucleic acid probes, the number of nucleic acid probes, the number of fluorophores coupled to said probes, and the length of said probes, may be varied to fit the specifications of the individual assay.

[0361] In further embodiments smFISH is applied to a multiplexed workflow, wherein consecutive / sequential hybridizations are used (e.g., as in seqFISH or seqFISH+) to impart a temporal barcode on analyte transcripts. Sequential rounds of fluorescence in situ hybridization may be accompanied by imaging and probe stripping, detecting individual transcripts (e.g., RNA transcripts) within a biological sample (e.g., a tissue sample). In some embodiments, each round of hybridization comprises a pre-defined set of probes (e.g., between about 10 and about 50 probes such as 24 to 32 probes) that target unique RNA transcripts. In some examples, the pre-defined set of probes is multicolored. Optionally, multiple nucleic acid probes are attached onto the sample, wherein each probe comprises an initiation sequence for amplification, allowing for decreased autofluorescence (e.g., as in single- molecule hybridization chain reaction (smHCR)). In some embodiments, a multiplexed smFISH method described herein may multiplex from 10s to over 10,000 mRNAs, optionally accompanied by imaging, to efficiently and accurately profile the entire transcriptome. In situ hybridization methods may further comprise using two probes to bind analyte transcripts (e.g., RNA transcripts), that serve as binding targets for amplification primers. In some embodiments, this process results in signal amplification (e. ., as in RNAscope). In some embodiments, in situ hybridization methods may employ metal tags instead of fluorophores (e.g., imaging mass cytometry). Metal-conjugated antibodies may couple to the metal tags hybridized to transcripts on a biological sample. In some embodiments, mass-cytometry may be used to quantify metal abundances, allowing the concurrent evaluation of RNA and protein within a biological sample.

[0362] In some embodiments, a method described herein comprises a multiplexed FISH protocol that is error-robust (e.g., MERFISH). In some embodiments, said protocol comprises non-readout nucleic acid probes (e.g., primary probes) comprising a binding region (e.g., a region that binds to a target such as RNA transcripts) coupled to one or more flanking regions. In some embodiments, each non-readout nucleic acid probe is coupled to two flanking regions. The non-readout nucleic acid probes may hybridize to a transcript (e.g., RNA transcript) within a biological sample (e.g., tissue sample or a single cell), such that florescent readout nucleic acid probes may subsequently serially hybridize to the flanking region(s) of the non-readout nucleic acid probes. In some embodiments, each round of hybridization comprises successive imaging and probe stripping to quench signals from readout nucleic acid probes from previous rounds. RNAs may be imaged by FISH, and errors accumulated during multiple imaging rounds (e.g., imperfect hybridizations) are detected and / or corrected. In some embodiments, expansion microscopy is employed to increase the number of detected RNA analytes without signal overlap. In similar embodiments, non-readout nucleic acid probes are cross-linked to analyte transcripts prior to imaging. Cross-linking may be performed by any method known in the art. In preferred embodiments, cross-linking is performed using hydrogel tissue embedding. Following said cross-linking steps, barcoding may be performed, comprising sequential hybridizations using readout probes coupled to predetermined colors to generate unique barcodes (e.g., generating pseudocolors from consecutive hybridizations).

[0363] In some embodiments, one or more barcodes of a probe are targeted by detectably labeled detection oligonucleotides, such as fluorescently labeled oligonucleotides. In some embodiments, one or more decoding schemes are used to decode the signals, such as fluorescence, for sequence determination. In any of the embodiments herein, barcodes (e.g., primary and / or secondary barcode sequences) can be analyzed using any suitable methods or techniques, comprising those described herein, such as RNA sequential probing of analytes (RNA SPOTs), sequential fluorescent in situ hybridization (seqFISH or seqFISH+), single-molecule fluorescent in situ hybridization (smFISH), or multiplexed error-robust fluorescence in situ hybridization (MERFISH). In some embodiments, the methods provided herein comprise analyzing the barcodes by sequential hybridization and detection with a plurality of labelled probes (e.g., detection oligonucleotides). Exemplary decoding schemes are described in Eng et al., “Transcriptome-scale Super- Resolved Imaging in Tissues by RNA SeqFISH+,” Nature 568(7751):235-239 (2019); Chen et al., “Spatially resolved, highly multiplexed RNA profiling in single cells,” Science 348(6233 ):aaa6090 (2015); US 10,457,980 B2; US 2016 / 0369329 Al; WO 2018 / 026873 Al ; and US 2017 / 0220733 Al, all of which are incorporated by reference in their entirety. In some embodiments, these assays enable signal amplification, combinatorial decoding, and error correction schemes at the same time.

[0364] Similar strategies of in situ hybridization using variations of FISH techniques may also be adopted by methods described herein. In some embodiments, a method comprises non-barcoding multiplexed FISH protocols (e.g., ouroboros sm-FISH (osmFISH)). Non-barcoding methods may be limited to detecting a specific number of analytes, defined by the number of hybridization rounds performed. In some embodiments, imaging is performed following each hybridization round, wherein the probe is stripped after imaging, allowing for subsequent hybridization and imaging rounds.

[0365] B. Probes

[0366] Disclosed herein in some aspects are nucleic acid probes and / or probe sets (e.g., circular probes or circularizable probes or probe sets) that are introduced into a cell or used to otherwise contact a biological sample such as a tissue sample. In some instances, the probes are contacted with the blocked sample after extension is performed using the ssDNA as template. The probes may comprise any of a variety of entities that can hybridize to a nucleic acid, typically by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc. The nucleic acid probe typically contains a sequence (e.g., hybridization region such as a target recognition sequence) that can directly or indirectly bind to at least a portion of a target nucleic acid. The nucleic acid probe or probe set may be able to bind to a specific target nucleic acid (e.g., an mRNA, or other nucleic acids as discussed herein). In some embodiments, RCA products of the circular probes or circularized probes generated from the circularizable probes or probe sets are detected using a detectable label, and / or by using secondary nucleic acid probes able to bind to the RCA products or sequences thereof

[0367] In some embodiments, more than one type of nucleic acid probes are contacted with a sample. In some embodiments, the nucleic acid probes comprise circular probes and / or circularizable probes (such as padlock probes) or circularizable probe sets. In some embodiments, more than one type of detectable nucleic acid probes may be contacted with a sample, e.g., simultaneously or sequentially in any suitable order, such as in sequential probe hybridization / unhybridization cycles. In some embodiments, the detectable probes may comprise probes that bind to a product (e.g., an RCA product) of a primary probe targeting an analyte e.g., an RNA molecule). In some embodiments, more than one type of higher order nucleic acid probes may be contacted with a sample, e.g., simultaneously or sequentially in any suitable order, such as in sequential probe hybridization / unhybridization cycles. In some embodiments, more than one type of detectably labeled nucleic acid probes may be contacted with a sample, e.g., simultaneously or sequentially in any suitable order, such as in sequential probe hybridization / unhybridization cycles. In some embodiments, the detectably labeled probes may comprise probes that bind to one or more primary probes, one or more secondary probes, one or more higher order probes, one or more intermediate probes between a primary / second / higher order probes, and / or one or more detectably or non- detectably labeled probes. In some embodiments, at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 250,000, at least 500,000, or at least 1,000,000 distinguishable nucleic acid probes (e.g., primary, secondary, higher order probes, and / or detectably labeled probes) can be contacted with a sample, e.g., simultaneously or sequentially in any suitable order. In some embodiments, at least 500, at least 1,000, at least 2,000, at least 3,000 distinguishable nucleic acid probes (e.g., primary circular or circularizable probes) can be contacted with a sample. In some embodiments, a plurality of distinguishable nucleic acid probes may be complementary to different sequences of the same target RNA. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 distinguishable nucleic acid probes may each have different target recognition sequences complementary to non-overlapping target sequences of the same target RNA.

[0368] Between any of the probe contacting steps disclosed herein, the method may comprise one or more intervening reactions and / or processing steps, such as modifications of a target nucleic acid, modifications of a probe or product thereof (e.g., via hybridization, ligation, extension, amplification, cleavage, digestion, branch migration, primer exchange reaction, click chemistry reaction, crosslinking, attachment of a detectable label, activating photo-reactive moieties, etc.), removal of a probe or product thereof (e.g., cleaving off a portion of a probe and / or unhybridizing the entire probe), signal modifications (e.g., quenching, masking, photo-bleaching, signal enhancement (e.g., via FRET), signal amplification, etc.), signal removal (e.g., cleaving off or permanently inactivating a detectable label), crosslinking, de-crosslinking, and / or signal detection.

[0369] The target recognition sequence (e.g., hybridization region) of a probe may be positioned anywhere within the probe. For instance, the target recognition sequence of a primary probe such as a circularizable probe that binds to a target nucleic acid can be 5’ or 3’ to any barcode sequence in the primary probe. Likewise, the target recognition sequence of a secondary probe (which binds to an RCA product of a circular or circularized primary probe) can be 5’ or 3’ to any barcode sequence in the secondary probe. In some embodiments, the target recognition sequence comprises a sequence that is substantially complementary to a portion of a target nucleic acid (a target sequence). In some embodiments, the target recognition sequence and the target sequence are at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.

[0370] The target recognition sequence of a nucleic acid probe may be designed with reference to a target nucleic acid (e.g., a cellular RNA such as an mRNA) that is present or suspected of being present in a sample. In some embodiments, more than one target recognition sequence can be used to identify a particular target RNA. The more than one target-binding sequence can be in the same probe or in different probes. For instance, multiple probes can be used, sequentially and / or simultaneously, that can bind to (e.g., hybridize to) different regions of the same target RNA. In some embodiments, a single RCA product is associated with a particular target RNA (e.g., by providing a panel of circular probes or circularizable probes or probe sets, wherein each probe or probe set is designed to hybridize to a different target RNA in the biological sample). In some embodiments, the probe or probe set is configured to bind a single stranded target nucleic acid sequence (e.g., an mRNA). In some instances, the target RNA analyte is in a cell or tissue sample, such as a tissue section.

[0371] In some embodiments, a circular probe is a probe that is pre-circularized prior to hybridization to a target RNA. In some embodiments, a circularizable probe is a probe that can be circularized upon hybridization to a target RNA and / or one or more other probes such as a splint. In some embodiments, a circularizable probe set comprises at least a first nucleic acid probe and a second nucleic acid probe that can be circularized upon hybridization to a target RNA and another probe such as a splint (e.g., the first and second nucleic acid probes are ligated to each other, optionally using the target RNA and a separate nucleic acid splint to form a circularized probe).

[0372] In some embodiments, the method comprises detecting the RCA product by hybridizing one or more linear probes to the RCA product. In some embodiments, a linear probe is one that comprises a target recognition sequence (e.g., a sequence complementary to a barcode sequence or subunit thereof in the RCA product) and a sequence that does not hybridize to a target nucleic acid, such as a 5’ overhang, a 3’ overhang, and / or a linker or spacer (which may comprise a nucleic acid sequence or a non-nucleic acid moiety). In some embodiments, the sequence (e.g., the 5’ overhang, 3’ overhang, and / or linker or spacer) is non-hybridizing to the target nucleic acid but may hybridize to one another and / or one or more other probes, such as detec tably labeled probes. In some embodiments, a linear probe is one that comprises a target recognition sequence (e.g., a sequence complementary to a barcode sequence or subunit thereof in the RCA product) and an optically detectable label.

[0373] In any of the embodiments herein, the circularizable probe or probe set can comprise one, two, three, four, or more ribonucleotides. In some embodiments, a circularizable probe or probe set disclosed herein can comprise one, two, three, four, or more ribonucleotides in a DNA backbone. In any of the embodiments herein, the one or more ribonucleotides can be at and / or near a ligatable 3’ end of the circularizable probe or probe set. In some embodiments, a circularizable probe disclosed herein can comprise one, two, three, four, or more ribonucleotides in a DNA backbone, wherein the one or more ribonucleotides are at a ligatable 3’ end of the circularizable probe (e.g., a ligatable 3’ end in a target recognition sequence of the circularizable probe, wherein the ligatable 3’ end can be ligated to a ligatable 5’ end in a target recognition sequence of the circularizable probe to generate a circularized probe). In some embodiments, a 3’ terminal nucleotide of the circularizable probe hybridized to the target RNA is a ribonucleotide. In some embodiments, a 3’ terminal nucleotide of the circularizable probe set hybridized to the target RNA is a ribonucleotide. In some embodiments, a 3’ end and a 5’ end of the circularizable probe or probe set are ligated using the target RNA as a template.

[0374] In some embodiments, a probe disclosed herein (e.g., circularizable probe or probe set) can comprise a 5' flap which may be recognized by a structure- specific cleavage enzyme, e.g., an enzyme capable of recognizing the junction between singlestranded 5' overhang and a DNA duplex, and cleaving the single- stranded overhang. It will be understood that the branched three-strand structure which is the substrate for the structure-specific cleavage enzyme may be formed by 5' end of one probe part and the 3' end of another probe part when both have hybridized to the target nucleic acid molecule, as well as by the 5' and 3' ends of a one-part probe. Enzymes suitable for such cleavage include Flap endonucleases (FENS), which are a class of enzymes having endonucleolytic activity and being capable of catalyzing the hydrolytic cleavage of the phosphodiester bond at the junction of single- and double- stranded DNA. Thus, in some embodiment, cleavage of the additional sequence 5' to the first target- specific binding site is performed by a structure- specific cleavage enzyme, e.g., a Flap endonuclease. Suitable Flap endonucleases are described in Ma et al. 2000. JBC 275, 24693- 24700 and in US 2020 / 0224244 (herein incorporated by reference in their entireties) may include P. furiosus (Pfu), A. fulgidus (Afu), M. jannaschii (Mja) or M. thermoautotrophicum (Mth). In other embodiments an enzyme capable of recognizing and degrading a single-stranded oligonucleotide having a free 5' end may be used to cleave an additional sequence (5' flap) from a structure as described above. Thus, an enzyme having 5' nuclease activity may be used to cleave a 5' additional sequence. Such 5' nuclease activity may be 5' exonuclease and / or 5' endonuclease activity. A 5' nuclease enzyme is capable of recognizing a free 5' end of a single- stranded oligonucleotide and degrading said singlestranded oligonucleotide. A 5' exonuclease degrades a single-stranded oligonucleotide having a free 5' end by degrading the oligonucleotide into constituent mononucleotides from its 5' end. A 5' endonuclease activity may cleave the 5' flap sequence internally at one or more nucleotides. Further, a 5’ nuclease activity may take place by the enzyme traversing the single- stranded oligonucleotide to a region of duplex once it has recognized the free 5' end, and cleaving the single-stranded region into larger constituent nucleotides (e.g., dinucleotides or trinucleotides), or cleaving the entire 5' singlestranded region, e.g., as described in Lyamichev et al. 1999. PNAS 96, 6143-6148 for Taq DNA polymerase and the 5' nuclease thereof. Preferred enzymes having 5' nuclease activity include Exonuclease VIII, or a native or recombinant DNA polymerase enzyme from Thermits aquaticiis (Taq), Thermits thernwphilus or Thermusflavus, or the nuclease domain therefrom.

[0375] Any suitable circularizable probe or probe set may be used to generate the RCA template which is used to generate the RCA product. In some embodiments, a circularizable probe is in the form of a linear molecule having ligatable ends which may be circularized by ligating the ends together directly or indirectly, e.g., to each other, or to the respective ends of an intervening (“gap”) oligonucleotide or to an extended 3' end of the circularizable probe. A circularizable probe may also be provided in two or more parts, namely two or more molecules (e.g., oligonucleotides) which may be ligated together to form a circle. When said RCA template is circularizable it is circularized by ligation prior to RCA. Ligation may be templated using a ligation template, and in the case of padlock and molecular inversion probes and such like the target analyte may provide the ligation template, or it may be separately provided. The circularizable RCA template (or template part or portion) will comprise at its respective 3' and 5' ends regions of complementarity to corresponding cognate complementary regions (or binding sites) in the ligation template, which may be adjacent where the ends are directly ligated to each other, or non-adjacent, with an intervening “gap” sequence, where indirect ligation is to take place.

[0376] In some embodiments (e.g., wherein the circularizable probe is a padlock probe) the ends of the circularizable probe may be brought into proximity to each other by hybridization to adjacent sequences on a target nucleic acid molecule (such as a target analyte), which acts as a ligation template, thus allowing the ends to be ligated together to form a circular nucleic acid molecule, allowing the circularized circularizable probe to act as template for an RCA reaction. In such an example the terminal sequences of the circularizable probe which hybridize to the target nucleic acid molecule will be specific to the target analyte in question, and will be replicated repeatedly in the RCA product. They may therefore act as a marker sequence indicative of that target analyte. Accordingly, it can be seen that the marker sequence in the RCA product may be equivalent to a sequence present in the target analyte itself. Alternatively, a marker sequence (e.g., tag or barcode sequence) may be provided in the non-target complementary parts of the circularizable probe. In still a further embodiment, the marker sequence may be present in the gap oligonucleotide which is hybridized between the respective hybridized ends of the circularizable probe, where they are hybridized to non-adjacent sequences in the target molecule. Such gap-filling padlock probes are akin to molecular inversion probes.

[0377] In some embodiments, similar circular RCA template molecules can be generated using molecular inversion probes. Like padlock probes, these are also typically linear nucleic acid molecules capable of hybridizing to a target nucleic acid molecule (such as a target analyte) and being circularized. The two ends of the molecular inversion probe may hybridize to the target nucleic acid molecule at sites which are proximate but not directly adjacent to each other, resulting in a gap between the two ends. The size of this gap may range from only a single nucleotide in some embodiments, to larger gaps of 100 to 500 nucleotides, or longer, in other embodiments. Accordingly, it is necessary to supply a polymerase and a source of nucleotides, or an additional gap-filling oligonucleotide, in order to fill the gap between the two ends of the molecular inversion probe, such that it can be circularized.

[0378] As with the circularizable probe, the terminal sequences of the molecular inversion probe which hybridize to the target nucleic acid molecule, and the sequence between them, will be specific to the target analyte in question, and will be replicated repeatedly in the RCA product. They may therefore act as a marker sequence indicative of that target analyte. Alternatively, a marker sequence (e.g., tag or barcode sequence) may be provided in the non-target complementary parts of the molecular inversion probe.

[0379] In some embodiments, the probes disclosed herein may be invader probes, e.g., for generating a circular nucleic acid such as a circularized probe. Such probes are of particular utility in the detection of single nucleotide polymorphisms. The detection method of the present disclosure may, therefore, be used in the detection of a single nucleotide polymorphism, or indeed any variant base, in the target nucleic acid sequence. Probes for use in such a method may be designed such that the 3' ligatable end of the probe is complementary to and capable of hybridizing to the nucleotide in the target molecule which is of interest (the variant nucleotide), and the nucleotide at the 3' end of the 5' additional sequence at the 5' end of the probe or at the 5' end of another, different, probe part is complementary to the same said nucleotide, but is prevented from hybridizing thereto by a 3' ligatable end (e.g., it is a displaced nucleotide). Cleavage of the probe to remove the additional sequence provides a 5' ligatable end, which may be ligated to the 3' ligatable end of the probe or probe part if the 3' ligatable end is hybridized correctly to (e.g. is complementary to) the target nucleic acid molecule. Probes designed according to this principle provide a high degree of discrimination between different variants at the position of interest, as only probes in which the 3' ligatable end is complementary to the nucleotide at the position of interest may participate in a ligation reaction. In one embodiment, the probe is provided in a single part, and the 3' and 5' ligatable ends are provided by the same probe. In some embodiments, an invader probe is a padlock probe (an invader padlock or “iLock”), e.g., as described in Krzywkowski et al., Nucleic Acids Research 45, el 61 , 2017, and US 2020 / 0224244, which are incorporated herein by reference in their entirety.

[0380] Other types of probe which result in circular molecules which can be detected by RCA and which comprise either a target analyte sequence or a complement thereof include selector-type probes described in US 2019 / 0144940 (herein incorporated by reference in its entirety), which comprise sequences capable of directing the cleavage of a target nucleic acid molecule (e.g. a target analyte) so as to release a fragment comprising a target sequence from the target analyte and sequences capable of templating the circularization and ligation of the fragment. US 2018 / 0327818, the content of which is herein incorporated by reference in its entirety, describes probes which comprise a 3’ sequence capable of hybridizing to a target nucleic acid molecule (e.g. a target analyte) and acting as a primer for the production of a complement of a target sequence within the target nucleic acid molecule (e.g. by target templated extension of the primer), and an internal sequence capable of templating the circularization and ligation of the extended probe comprising the reverse complement of the target sequence within the target analyte and a portion of the probe. In the case of both such probes, target sequences or complements thereof are incorporated into a circularized molecule which acts as the template for the RCA reaction to generate the RCA product, which consequently comprises concatenated repeats of said target sequence. In some embodiments, said target sequence may act as, or may comprise a marker sequence within the RCA product indicative of the target analyte in question. Alternatively, a marker sequence (e.g., tag or barcode sequence) may be provided in the non-target complementary parts of the probes.

[0381] In some embodiments, a nucleic acid probe disclosed herein can be preassembled from multiple components, e.g., prior to contacting the nucleic acid probe with a target nucleic acid or a sample. In some embodiments, a nucleic acid probe disclosed herein can be assembled during and / or after contacting a target nucleic acid or a sample with multiple components. In some embodiments, a nucleic acid probe disclosed herein is assembled in situ in a sample. In some embodiments, the multiple components can be contacted with a target nucleic acid or a sample in any suitable order and any suitable combination. For instance, a first component and a second component can be contacted with a target nucleic acid, to allow binding between the components and / or binding between the first and / or second components with the target nucleic acid. Optionally a reaction involving either or both components and / or the target nucleic acid, between the components, and / or between either one or both components and the target nucleic acid can be performed, such as hybridization, ligation, primer extension and / or amplification, chemical or enzymatic cleavage, click chemistry, or any combination thereof. In some embodiments, a third component can be added prior to, during, or after the reaction. In some embodiments, a third component can be added prior to, during, or after contacting the sample with the first and / or second components. In some embodiments, the first, second, and third components can be contacted with the sample in any suitable combination, sequentially or simultaneously. In some embodiments, the nucleic acid probe can be assembled in situ in a stepwise manner, each step with the addition of one or more components, or in a dynamic process where all components are assembled together. One or more removing steps, e.g., by washing the sample such as under stringent conditions, may be performed at any point during the assembling process to remove or destabilize undesired intermediates and / or components at that point and increase the chance of accurate probe assembly and specific target binding of the assembled probe.

[0382] In some embodiments, a nucleic acid probe disclosed herein can be preassembled from multiple components, e.g., prior to contacting the nucleic acid probe with a target nucleic acid or a sample. In some embodiments, a nucleic acid probe disclosed herein is assembled in vitro prior to contacting with the sample. For example, a circular probe disclosed herein can be ligated and purified prior to contacting with the sample. In some embodiments, the 3’ and 5’ ends of a linear nucleic acid molecule can be ligated to form a circular probe (e.g., using a nucleic acid splint that hybridizes to sequences at the 3’ and 5’ ends of a linear nucleic acid molecule). In some embodiments, a common splint can be used to ligate a plurality of different linear nucleic acid molecules to generate a plurality of different circular probes for different target RNAs. In some embodiments, different linear nucleic acid molecules may hybridize to a corresponding different splint for ligation. In some embodiments, the 3’ and 5’ ends of a linear nucleic acid molecule can be ligated to form a circular probe without the use of a splint. In some embodiments, to generate a plurality of different circular probes for different target RNAs, the different circular probes may be generated separately (e.g., in individual reactions) and then purified and pooled with other circular probes targeting different target RNAs to generate a pool of circular probes prior to contacting with the sample.

[0383] In some embodiments, the hybridization conditions include salt concentrations of approximately less than 1 M, e.g. less than about 500 mM and or less than about 200 mM. In some embodiments, hybridization is performed in a hybridization buffer that includes a buffered salt solution such as 5% SSPE, or other such buffers known in the art. Hybridization temperatures can be as low as 5 °C, but are typically greater than 22°C, and more typically greater than about 30°C, and typically in excess of 37°C. Hybridizations are often performed under stringent conditions, e.g., conditions under which a sequence will hybridize to its target sequence but will not hybridize to other, non-complementary sequences. Stringent conditions are sequence-dependent and are different in different circumstances. For example, longer fragments may require higher hybridization temperatures for specific hybridization than short fragments. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one parameter alone. Generally stringent conditions are selected to be about 5 °C lower than the Tm for the specific sequence at a defined ionic strength and pH. The melting temperature Tm can be the temperature at which a population of double- stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation, Tm =81.5 + 0.41 (% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references (e.g., Allawi and SantaLucia, Jr., Biochemistry, 36:10581-94 (1997)) include alternative methods of computation which take structural and environmental, as well as sequence characteristics into account for the calculation of Tm. In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency.

[0384] In some instances, the circular or circularizable probe is hybridized to the target nucleic acid (e.g., target RNA) and ligated to form a circular template for RCA. In some embodiments, the ligation comprises RNA-templated ligation using the target RNA as a template. In some embodiments, the ligation involves chemical ligation. In some embodiments, the ligation involves template dependent ligation. In some embodiments, the ligation involves template independent ligation. In some embodiments, the ligation involves enzymatic ligation. In some embodiments, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases comprise ATP-dependent doublestrand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD-i-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, AMPLIGASE™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some embodiments, the ligase is a T4 RNA ligase or derivative thereof. In some embodiments, the ligase is a T4 RNA ligase 2 (Rnl2) or derivative thereof. In some embodiments, the ligase is a splintR ligase. In some embodiments, the ligase is a Chlorella virus DNA Ligase (PBCV-1 DNA ligase) or derivative thereof, in some embodiments, the ligase is a single stranded DNA ligase. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some embodiments, the ligase is a ligase that has an RNA-splinted DNA ligase activity. In some embodiments, the ligase is selected from the group consisting of a Chlorella virus DNA ligase (PBCV DNA ligase), a T4 RNA ligase, a T4 DNA ligase, and a single-stranded DNA (ssDNA) ligase. In some embodiments, the DNA ligase is SPLINTR® ligase (also known as Chlorella virus DNA ligase or PBCV-1 DNA ligase), T4 DNA ligase or T4 RNA ligase 2.

[0385] In some embodiments, a circular probe, circularizable probe, or circularizable probe set disclosed herein comprises a barcode sequence or complement thereof (e.g., such that the RCA product produced using the circular probe or circularized probe as a template comprises the barcode sequence). In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., subbarcodes) that are separated by one or more non-barcode sequences. In some embodiments, the one or more barcode(s) can also provide a platform for targeting functionalities, such as oligonucleotides, oligonucleotide- antibody conjugates, oligonucleotide-streptavidin conjugates, modified oligonucleotides, affinity purification, detectable moieties, enzymes, enzymes for detection assays or other functionalities, and / or for detection and identification of the polynucleotide. In any of the preceding embodiments, the methods provided herein can include analyzing the barcodes by sequential hybridization and detection with a plurality of labelled probes (e.g., detection oligos).

[0386] In some embodiments, in a barcode sequencing method, barcode sequences are detected for identification of other molecules including nucleic acid molecules (DNA or RNA) longer than the barcode sequences themselves, as opposed to direct sequencing of the longer nucleic acid molecules. In some embodiments, a N-mer barcode sequence comprises 4N complexity given a sequencing read of N bases, and a much shorter sequencing read may be required for molecular identification compared to nonbarcode sequencing methods such as direct sequencing. For example, 1024 molecular species may be identified using a 5-nucleotide barcode sequence (45=1024), whereas 8 nucleotide barcodes can be used to identify up to 65,536 molecular species, a number greater than the total number of distinct genes in the human genome. In some embodiments, the barcode sequences contained in the probes or RCPs are detected, rather than endogenous sequences, which can be an efficient read-out in terms of information per cycle of sequencing. Because the barcode sequences are pre-determined, they can also be designed to feature error detection and correction mechanisms, see, e.g., U.S. Pat. Pub. 20190055594 and U.S. Pat. Pub 20210164039, which are hereby incorporated by reference in their entirety.

[0387] In some embodiments, the ligation involves chemical ligation (e.g., click chemistry ligation). In some embodiments, the chemical ligation involves template dependent ligation. In some embodiments, the chemical ligation involves template independent ligation. In some embodiments, the click reaction is a template-independent reaction (see, e.g., Xiong and Seela (2011), J. Org. Chem. 76(14): 5584-5597, incorporated by reference herein in its entirety). In some embodiments, the click reaction is a template-dependent reaction or template-directed reaction. In some embodiments, the template-dependent reaction is sensitive to base pair mismatches such that reaction rate is significantly higher for matched versus unmatched templates. In some embodiments, the click reaction is a nucleophilic addition template-dependent reaction. In some embodiments, the click reaction is a cyclopropane-tetrazine templatedependent reaction.

[0388] In some embodiments, the ligation involves enzymatic ligation. In some embodiments, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases comprise ATP-dependent double-strand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and single-strand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP-dependent ligases), EC 6.5.1.2 (NAD-i-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, AMPLIGASE™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some embodiments, the ligase is a T4 RNA ligase. In some embodiments, the ligase is a splintR ligase. In some embodiments, the ligase is a single stranded DNA ligase. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some embodiments, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0389] In some embodiments, the ligation herein is a direct ligation. In some embodiments, the ligation herein is an indirect ligation. "Direct ligation" means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize non- adjacently to one another, i.e., separated by one or more intervening nucleotides or "gaps". In some embodiments, said ends are not ligated directly to each other, but instead occurs either via the intermediacy of one or more intervening (so-called "gap" or "gapfilling" (oligo)nucleotides) or by the extension of the 3' end of a probe to "fill" the "gap" corresponding to said intervening nucleotides (intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be "filled" by one or more "gap" (oligo)nucleotide(s) which are complementary to a splint, padlock probe, or target nucleic acid. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific embodiments, the gap may be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap between said terminal regions may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some embodiments, the ligation herein is preceded by gap filling. In other embodiments, the ligation herein does not require gap filling.

[0390] In some embodiments, ligation of the polynucleotides produces polynucleotides with melting temperature higher than that of unligated polynucleotides. Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotides prior to subsequent steps, comprising amplification and detection.

[0391] In some aspects, a high fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tmaround the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0392] In some embodiments, the ligation herein is a proximity ligation of ligating two (or more) nucleic acid sequences that are in proximity with each other, e.g., through enzymatic means (e.g., a ligase). In some embodiments, proximity ligation can include a “gap-filling” step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic acid molecules of interest (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference). A wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt-end” ligations. Additionally, single- stranded ligation can be used to perform proximity ligation on a single-stranded nucleic acid molecule. Sticky-end proximity ligations involve the hybridization of complementary single- stranded sequences between the two nucleic acid molecules to be joined, prior to the ligation event itself. Blunt-end proximity ligations generally do not include hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack a single-stranded overhang at the site of ligation.

[0393] The target recognition sequences may be of any length, and multiple recognition sequences in the same or different circular probes or circularizable probes or probe sets may be of the same or different lengths. For instance, the target recognition sequence may be at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 nucleotides in length. In some embodiments, the target recognition sequence may be no more than 48, no more than 45, or no more than 40 nucleotides in length. Combinations of any of these are also possible, e.g., the recognition sequence may have a length of between 25 and 40, between 30 and 45, or between 20 and 48 nucleotides, etc. In some embodiments, the target recognition sequence is at least 95%, at least 98%, at least 99%, or at least 100% complementary to the target sequence in the target RNA.

[0394] In some embodiments, the ligation herein is a direct ligation. In some embodiments, the ligation herein is an indirect ligation. "Direct ligation" means that the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (intramolecular ligation). Alternatively, "indirect" means that the ends of the polynucleotides hybridize non- adjacently to one another, e.g., separated by one or more intervening nucleotides or "gaps". In some embodiments, said ends are not ligated directly to each other, but instead occurs either via the intermediacy of one or more intervening (so-called "gap" or "gapfilling" (oligo)nucleotides) or by the extension of the 3' end of a probe to "fill" the "gap" corresponding to said intervening nucleotides (intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be "filled" by one or more "gap" (oligo)nucleotide(s) which are complementary to a splint, a circularizable probe or probe set (e.g., padlock probe), or target nucleic acid. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific embodiments, the gap may be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap between said terminal regions may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some embodiments, the ligation herein is preceded by gap filling. In other embodiments, the ligation herein does not require gap filling.

[0395] In some embodiments, ligation of the polynucleotides produces polynucleotides with melting temperature higher than that of unligated polynucleotides. Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotides prior to subsequent steps, comprising amplification and detection.

[0396] In some aspects, a high-fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used, for example, for ligating two or more probes to form a circular probe disclosed herein. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (Tm) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tmaround the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0397] In some embodiments, a ligation herein comprises ligating two (or more) nucleic acid termini that are in proximity with each other, e.g., that are brought into proximity upon hybridization to the target RNA and / or to a separate nucleic acid molecule (e.g., a splint oligonucleotide). In some embodiments, the circularizable probe comprises a 3’ end and a 5’ end that are brought into proximity upon hybridization to the target RNA. In some embodiments, the circularizable probe is a padlock probe. In some embodiments, the 3’ end and the 5’ end of the circularizable probe do not hybridize to the target RNA (e.g., the target recognition sequence is in an internal region of the circularizable probe), and the 3’ end and 5’ end optionally hybridize to a separate nucleic acid molecule (e.g., a splint oligonucleotide) to bring the ends in proximity for ligation. In some embodiments, the ligation is with a ligase. In some embodiments, ligation includes a gap- filling step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule (e.g., a nucleic acid molecule such as a DNA splint). C. Amplification

[0398] In some embodiments, the methods herein comprise the step of amplifying one or more polynucleotides, for instance the circularizable probe such as a padlock probe or a circular probe formed from the padlock probe. In some instances, an amplification reaction is performed in the blocked sample (e.g., generated as described in Section II). In some embodiments, the amplifying is achieved by performing rolling circle amplification (RCA). In other embodiments, a primer that hybridizes to the padlock probe is added and used as such for amplification. In some embodiments, a removing step is performed to remove molecules that are not specifically hybridized to the analyte nucleic acid and / or the circular probe. In some embodiments, the removing step is performed to remove unligated probes. In some embodiments, the removing step is performed after ligation and prior to amplification. In some embodiments, the amplification is performed at a temperature between or between about 20°C and about 60°C. In some embodiments, the amplification is performed at a temperature between or between about 30°C and about 40°C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41 °C, 43°C, 45°C, 47°C, or 49°C. In some embodiments, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, a primer is elongated to produce multiple copies of the circular template. This amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after the formation of the hybridization complex and association of the amplification probe, the hybridization complex is rolling-circle amplified to generate a cDNA nanoball (e.g. or i.e., amplicon) containing multiple copies of the cDNA. Techniques for rolling circle amplification (RCA) are known in the art such as linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. (See, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 November 15; 49(11): 2540-2550; Schweitzer et al. Proc. Natl Acad. Sci. USA 97:101 13- 1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el 18, 2001; Dean et al. Genome Res. 1 1 :1095- 1099, 2001 ; Schweitzer et al, Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). Exemplary polymerases for use in RCA comprise DNA polymerase such phi29 (cp29) polymerase, Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some aspects, DNA polymerases that have been engineered or mutated to have desirable characteristics can be employed. In some embodiments, the polymerase is phi29 DNA polymerase.

[0399] In some instances, the biological sample is contacted with a polymerase for performing an extension reaction using the one or more regions of ssDNA as template to generate dsDNA and an additional polymerase is provided for an amplification reaction of the one or more probes or probe sets. In some instances, the polymerase used for generating the blocked sample and the additional polymerase for amplifying probes are different. In some instances, the polymerase used for generating the blocked sample and the additional polymerase for amplifying probes are the same and are provided at different steps in the workflow. In some instances, the polymerase used for generating the blocked sample is a T4 DNA polymerase. In some instances, the polymerase used for generating the blocked sample is a primer-free polymerase. In some instances, the polymerase used for generating the blocked sample is a Phi29 DNA polymerase. In some embodiments, the additional polymerase used for amplifying probes (e. ., using RCA) is a Phi29 DNA polymerase.

[0400] In some aspects, during the amplification step, modified nucleotides can be added to the reaction to incorporate the modified nucleotides in the amplification product (e.g., nanoball). Exemplary of the modified nucleotides comprise amine- modified nucleotides. In some aspects of the methods, for example, for anchoring or cross-linking of the generated amplification product (e.g., nanoball) to a scaffold, to cellular structures and / or to other amplification products e.g., other nanoballs). In some aspects, the amplification products comprise a modified nucleotide, such as an amine-modified nucleotide. In some embodiments, the amine-modified nucleotide comprises an acrylic acid N- hydroxysuccinimide moiety modification. Examples of other amine-modified nucleotides comprise, but are not limited to, a 5-Aminoallyl-dUTP moiety modification, a 5-Propargylamino-dCTP moiety modification, a N6-6-Aminohexyl-dATP moiety modification, or a 7-Deaza-7-Propargylamino-dATP moiety modification. In some aspects, the polynucleotides and / or amplification product e.g., amplicon) can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more of the polynucleotide probe(s) can be modified to contain functional groups that can be used as an anchoring site to attach the polynucleotide probes and / or amplification product to a polymer matrix. Exemplary modification and polymer matrix that can be employed in accordance with the provided embodiments comprise those described in, for example, WO 2014 / 163886, WO 2017 / 079406, US 2016 / 0024555, US 2018 / 0251833 and WO2014 / 025392. In some examples, the scaffold also contains modifications or functional groups that can react with or incorporate the modifications or functional groups of the probe set or amplification product. In some examples, the scaffold can comprise oligonucleotides, polymers or chemical groups, to provide a matrix and / or support structures.

[0401] The amplification products may be immobilized within the matrix generally at the location of the nucleic acid being amplified, thereby creating a localized colony of amplicons. The amplification products may be immobilized within the matrix by steric factors. The amplification products may also be immobilized within the matrix by covalent or noncovalent bonding. In this manner, the amplification products may be considered to be attached to the matrix. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the size and spatial relationship of the original amplicons is maintained. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the amplification products are resistant to movement or unraveling under mechanical stress.

[0402] In some aspects, the amplification products are copolymerized and / or covalently attached to the surrounding matrix thereby preserving their spatial relationship and any information inherent thereto. For example, if the amplification products are those generated from DNA or RNA within a cell embedded in the matrix, the amplification products can also be functionalized to form covalent attachment to the matrix preserving their spatial information within the cell thereby providing a subcellular localization distribution pattern. In some embodiments, the provided methods involve embedding the one or more polynucleotide probe sets and / or the amplification products in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, the hydrogel-tissue chemistry described comprises covalently attaching nucleic acids to in situ synthesized hydrogel for tissue clearing, enzyme diffusion, and multiple-cycle sequencing while an existing hydrogel-tissue chemistry method cannot. In some embodiments, to enable amplification product embedding in the tissue-hydrogel setting, amine-modified nucleotides are comprised in the amplification step (e.g., RCA), functionalized with an acrylamide moiety using acrylic acid N- hydroxy succinimide esters, and copolymerized with acrylamide monomers to form a hydrogel. D. Detection and Analysis

[0403] In some aspects, the provided methods involve analyzing, e.g., detecting or determining, one or more sequences present in the probes or probe sets or products thereof (e.g., rolling circle amplification products thereof). In some embodiments, the detecting is performed at one or more locations in the biological sample. In some embodiments, the locations are the locations of RNA transcripts in the biological sample. In some embodiments, the locations are the locations at which the probes or probe sets hybridize to the RNA transcripts in the biological sample, and are optionally ligated and amplified by rolling circle amplification. In some embodiments, the detecting comprises a plurality of repeated cycles of hybridization and removal of probes (e.g., detectably labeled probes, or intermediate probes that bind to detectably labeled probes) to the primary probe or probe set hybridized to the target nucleic acid, or to a rolling circle amplification product generated from the probe or probe set hybridized to the target nucleic acid. Methods for binding and identifying a target nucleic acid that uses various probes or oligonucleotides have been described in, e.g., US2003 / 0013091, US2007 / 0166708, US2010 / 0015607, US2010 / 0261026, US2010 / 0262374, US2010 / 0112710, US2010 / 0047924, and US2014 / 0371088, each of which is incorporated herein by reference in its entirety. Detectably-labeled probes can be useful for detecting multiple target nucleic acids and be detected in one or more hybridization cycles (e.g., sequential hybridization assays, or sequencing by hybridization). In some embodiments, the detecting can comprise binding an intermediate probe directly or indirectly to the nucleic acid probe or probe set, binding a detectably labeled probe directly or indirectly to a detection region of the intermediate probe, and detecting a signal associated with the detectably labeled probe. In some embodiments, the method comprises detecting a rolling circle amplification product (RCP) generated using a circular or circularized primary probe or probe set as a template. In some embodiments, the method comprises detecting a rolling circle amplification product (RCP) generated using a circular or circularized probe or probe that binds to a primary probe or probe set as a template. In some embodiments, detecting the RCP comprises binding an intermediate probe directly or indirectly to the RCP, binding a detectably labeled probe directly or indirectly to a detection region of the intermediate probe, and detecting a signal associated with the detectably labeled probe. In some embodiments, the method can comprise performing one or more wash steps to remove unbound and / or nonspecifically bound intermediate probe molecules from the primary probes or the products of the primary probes. In some embodiments, the detecting comprises: detecting signals associated with detectably labeled probes that are hybridized to barcode regions or complements thereof in the primary probe or probe set or a product thereof (e.g., an RCP); and / or detecting signals associated with detectably labeled probes that are hybridized to intermediate probes which are in turn hybridized to the barcode regions or complements thereof. In some embodiments, the detectably labeled probes is fluorescently labeled. In some embodiments, the methods include detecting the sequence in all or a portion of a nucleic acid probe or probe set or an RCP, or detecting a sequence of the nucleic acid probe or probe set or RCP, such as one or more barcode sequences present in the nucleic acid probe or probe set or RCP. In some embodiments, the sequence of the RCP, or barcode thereof, is indicative of a sequence of the target nucleic acid to which the RCP is hybridized. In some embodiments, the analysis and / or sequence determination comprises detecting a sequence in all or a portion of the nucleic acid concatemer and / or in situ hybridization to the RCP. In some embodiments, the detection step involves sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, and / or fluorescent in situ sequencing (FISSEQ), and / or hybridization-based in situ sequencing. In some embodiments, the detection step is by sequential fluorescent in situ hybridization (e.g., for combinatorial decoding of the barcode sequence or complement thereof). In some embodiments, the detection or determination includes hybridizing to the probe directly or indirectly a detection oligonucleotide labeled with a fluorophore, an isotope, a mass tag, or a combination thereof. In some embodiments, the detection or determination comprises imaging the probe hybridized to the target nucleic acid (e.g., imaging one or more detectably labeled probes hybridized thereto). In some embodiments, the target nucleic acid is an mRNA in a tissue sample, and the detection or determination is performed when the target nucleic acid and / or the amplification product is in situ in the tissue sample. In some embodiments, the target nucleic acid is an amplification product (e.g., a rolling circle amplification product). In some instances, the disclosed methods may include the use of a branched DNA (bDNA) amplification approach to amplify signals. In branched DNA (bDNA) amplification, primary and secondary amplifier oligonucleotides, each containing multiple replicate binding sites, are assembled on an individual nucleic acid probe. In some embodiments, the degree of amplification in bDNA amplification is controlled by the design of the amplification reaction, i.e., the assembled bDNA structures cannot grow indefinitely even in the presence of excess reagents, which may be used to control spot size or limit the variability in brightness from molecule to molecule. In some instances, the disclosed methods comprise the use of a hybridization chain reaction (HCR) approach to amplify signals. In a hybridization chain reaction, two fluorescently-labeled metastable hairpin oligonucleotides self-assemble into long fluorescent polymers starting from an initiator sequence present on each probe molecule (Xia, et al. (2019), ibid.). The degree of amplification achieved through HCR can be tuned by changing the hybridization or polymerization times, and can be adjusted to achieve highly amplified signals (which may, however, increase the size of the fluorescent spots generated and / or lead to variable degrees of amplification for different copies of the same target molecule).

[0404] In some embodiments, provided herein are methods and compositions for analyzing analytes in a sample using concatemer primers and labeling agents. In various embodiments, a primer with domain on its 3’ end binds to a catalytic hairpin, and is extended with a new domain by a strand displacing polymerase. For example, a primer with domain 1 on its 3 ends binds to a catalytic hairpin, and is extended with a new domain 1 by a strand displacing polymerase, with repeated cycles generating a concatemer of repeated domain 1 sequences. In various embodiments, the strand displacing polymerase is Bst. In various embodiments, the catalytic hairpin includes a stopper which releases the strand displacing polymerase. In various embodiments, branch migration displaces the extended primer, which can then dissociate. In various embodiments, the primer undergoes repeated cycles to form a concatemer primer. In various embodiments, a plurality of concatemer primers is contacted with a sample. In various embodiments, an assembly include a plurality of concatemer primers, a plurality of labeled probes, and a sample including nucleic acids. In various embodiments, each the plurality of concatemer primers each includes domain 1, 2, 3, etc. In various embodiments, each the plurality of labeled probes each include domain 1 ’, 2’, 3’, etc., with each corresponding domain 1 ’, 2’, 3’ being complementary to domain 1, 2, 3, etc., respectively. In various embodiments, the assembly includes the plurality of concatemer primers, which are capable of hybridizing to target nucleic acid sequences in the sample. Described herein is a method using the aforementioned assembly, including contacting the sample including target nucleic acids with the plurality of concatemer primers, then contacting the sample and plurality of concatemer primers with the plurality of labeled probes, thereby labeling the target nucleic acid sequences with a plurality of labeled probes. See e.g., U.S. Pat. Pub. No. 2021 / 0147902 and 2020 / 0362398, each of which is fully incorporated by reference herein.

[0405] In some aspects, the provided methods comprise imaging a detectably labeled probe bound directly or indirectly to the primary probe or probe set or product thereof and detecting the detectable label. In some embodiments, the detectably labeled probe comprises a detectable label that can be measured and quantitated. The label or detectable label can comprise a directly or indirectly detectable moiety, e.g., any fluorophores, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like. Fluorescence detection in tissue samples can often be hindered by the presence of strong background fluorescence. Background fluorescence can include autofluorescence (that can arise from a variety of sources, including aldehyde fixation, extracellular matrix components, red blood cells, lipofuscin, and the like), as opposed to the desired immunofluorescence from the fluorescently labeled antibodies or probes. Tissue autofluorescence can lead to difficulties in distinguishing the signals due to fluorescent antibodies or probes from the general background. In some embodiments, a method disclosed herein utilizes one or more agents to reduce tissue autofluorescence, for example, Autofluorescence Eliminator (Sigma / EMD Millipore), True Black Lipofuscin Autofluorescence Quencher (Biotium), MaxBlock Autofluorescence Reducing Reagent Kit (MaxVision Biosciences), and / or a very intense black dye (e.g., Sudan Black, or comparable dark chromophore). Examples of detectable labels comprise but are not limited to various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs and protein- antibody binding pairs. Examples of fluorescent proteins and fluorescent dyes comprise, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.

[0406] Examples of bioluminescent markers comprise, but are not limited to, luciferase (e.g., bacterial, firefly and click beetle), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals comprise, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases and cholinesterases. Identifiable markers also comprise radioactive compounds such as1251,35S,14C, or3H. Identifiable markers are commercially available from a variety of sources. In some embodiments, one or more fluorescent dyes are used as detectable labels. Commercially available fluorescent dyes include, but are not limited to 4,7-dichlorofluorescein dyes, spectrally resolvable rhodamine dyes, 4,7- dichlororhodamine dyes, cyanine dyes, ethersubstituted fluorescein dyes, energy transfer dyes, and xanthine dyes. Labeling can also be carried out with quantum dots. In some embodiments, a fluorescent label comprises a signaling moiety that conveys information through the fluorescent absorption and / or emission properties of one or more molecules. Exemplary fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer. Examples of commercially available fluorescent nucleotide analogues readily incorporated into nucleotide and / or polynucleotide sequences comprise, but are not limited to, Cy™3-dCTP, Cy™3-dUTP, Cy™5-dCTP, Cy™5- dUTP (Amersham Biosciences, Piscataway, N.J.), fluorescein- 12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14-dUTP, RHOD AMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™ 630 / 650- 14-dUTP, BODIPY™ 650 / 665 -14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546-14-dUTP, fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5- UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR- 14-UTP, BODIPY™ TR-14-UTP, RHOD AMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5- UTP, and ALEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg.). Methods are known for custom synthesis of nucleotides having other fluorophores (See, Henegariu, et al. (2000) Nature Biotechnol. 18:345). Fluorophores available for postsynthetic attachment comprise, but are not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY® FL (4,4-difluoro-4-bora-3a,4a-diaza-s- indacene FL), BODIPY® TMR (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene TMR), BODIPY® TR-X (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene TR-X), BODIPY® 530 / 550 (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 530 / 550), BODIPY® 558 / 568 (4,4- difluoro-4-bora-3a,4a-diaza-s-indacene 558 / 550), BODIPY® 564 / 570 (4,4-difluoro-4- bora-3a,4a-diaza-s-indacene 564 / 570), BODIPY® 581 / 591 (4,4-difluoro-4-bora-3a,4a- diaza-s-indacene 581 / 591), BODIPY® 630 / 650-X (4,4-difluoro-4-bora-3a,4a-diaza-s- indacene 630 / 650-X), BODIPY® 650-665-X (4,4-difluoro-4-bora-3a,4a-diaza-s- indacene 650 / 650-X), Cascade Blue® (N-(4-{ [4-(diethylamino)phenyl][4-(ethylamino)- 2-naphthyl]methylene } cyclohexa-2,5-dien- 1 -ylidene)-N-ethylethanaminium), Cascade Yellow™ (5-{2-[l-(3-{[(2,5-dioxopyrrolidin-l-yl)oxy]carbonyl}benzyl)pyridinium-4- yl]-l,3-oxazol-5-yl}-2-methoxybenzenesulfonate), Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red® (sulforhodamine 101 acid chloride, available from Molecular Probes, Inc., Eugene, Oreg.), Cy2 (cyanine 2), Cy3.5™ (cyanine 3.5) , Cy5.5™ (cyanine 5.5), and Cy7™ (cyanine 7) (Amersham Biosciences, Piscataway, N.J.). FRET tandem fluorophores may also be used, comprising, but not limited to, PerCP-Cy5.5™ (PerCP-cyanine 5.5), PE-Cy5™ (R- phycoerythrin (PE) coupled to cyanine 5), PE-Cy5.5™ (PE-cyanine 5.5), PE-Cy7™ (PE- cyanine 7), PE-Texas Red® (PE-sulforhodamine 101), APC-Cy7™ (APC-cyanine 7), PE-Alexa dyes (610, 647, 680), and APC-Alexa dyes. Biotin, or a derivative thereof, may also be used as a label on a nucleotide and / or a polynucleotide sequence, and subsequently bound by a detectably labeled avidin / streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin). An aminoallyl-dUTP residue may be incorporated into a polynucleotide sequence and subsequently coupled to an N-hydroxy succinimide (NHS) derivatized fluorescent dye. In general, any member of a conjugate pair may be incorporated into a detection polynucleotide provided that a detectably labeled conjugate partner can be bound to permit detection. Other suitable labels for a polynucleotide sequence may comprise fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and phosphor-amino acids (e.g., P- tyr, P-ser, P-thr). In some embodiments the following hapten / antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin / a- biotin, digoxigenin / a- digoxigenin, dinitrophenol (DNP)Za-DNP, 5 -Carboxy fluorescein (FAM) / a-FAM. In some embodiments, a nucleotide and / or a oligonucleotide sequence is indirectly labeled, such as with a hapten that is then bound by a capture agent. Many different hapten-capture agent pairs are available for use. Exemplary haptens comprise, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, cyanine dyes (e.g. , Cy5™, and digoxigenin. For biotin, a capture agent may be avidin, streptavidin, or antibodies. Antibodies may be used as capture agents for the other haptens (many dye-antibody pairs being commercia...

Claims

We claim:

1. A method for processing a biological sample, comprising:(a) contacting a biological sample comprising a nucleic acid with a polymerase, wherein the nucleic acid comprises one or more regions of ssDNA;(b) performing an extension reaction in the biological sample using the one or more regions of ssDNA as template to generate double stranded DNA (dsDNA), thereby providing a blocked sample; and(c) contacting the blocked sample with a probe or probe set to detect one or more target analytes in the blocked sample.

2. The method of claim 1 , further comprising:(d) detecting the probe or probe set in the biological sample.

3. The method of claim 1 or 2, wherein the polymerase is a Phi29 DNA polymerase.

4. The method of any one of claims 1-3, wherein the polymerase is provided in a reaction mixture that comprises deoxynucleotide triphosphates (dNTPs) and / or nucleotide triphosphates (NTPs).

5. The method of any one of claims 1-4, wherein the reaction mixture comprises a cofactor of the polymerase.

6. The method of claim 5, wherein the cofactor of the polymerase is a di-cation selected from the group consisting of Mg2+, Co2+, and Mn2+.

7. The method of any one of claims 1-6, wherein the blocked sample comprises less ssDNA than the biological sample prior to step (a).

8. The method of any one of claims 1-7, wherein the method further comprises, prior to step (a), providing the biological sample disposed on a first substrate.

9. The method of any one of claims 1-8, wherein the probe set comprises a first probe and a second probe, and wherein the first probe and the second probe each comprise sequences that are substantially complementary to sequences of an RNA analyte present within the blocked sample.

10. The method of claim 9, wherein the first probe or the second probe comprises a capture probe binding domain.

11. The method of claim 10, further comprising coupling the first probe and the second probe and ligating the probes to form a connected probe.

12. The method of any one of claims 1-11, wherein a sequence of the probe or probe set, or a complement thereof, is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

13. The method of claim 12, wherein the probe or probe set comprises a circular probe or a circularizable probe or probe set, wherein the circular probe or the circularizable probe or probe set comprises a target recognition sequence complementary to a target sequence in a target RNA in the blocked sample and, wherein the circular probe or the circularizable probe or probe set hybridizes to the target RNA.

14. The method of claim 13, further comprising performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP).

15. The method of claim 14, further comprising detecting the RCP at a location in the blocked sample.

16. The method of any one of claims 13-15, wherein the circularizable probe or probe set comprises one or more ribonucleotides.

17. The method of claim 15, wherein a 3’ end and a 5’ end of the circularizable probe or probe set are ligated using the target RNA as a template.

18. The method of any one of claims 2-17, wherein step (d) comprises imaging the blocked sample to detect the probe or probe set or a product thereof.

19. The method of claim 18, wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to a RCP.

20. The method of any of claims 14-19, wherein a sequence of the RCP is analyzed at a location in the blocked sample or a matrix embedding the blocked sample.

21. The method of claim 20, wherein the sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

22. The method of claim 21, wherein the sequence of the RCP product comprises one or more barcode sequences or complements thereof.

23. The method of claim 22, wherein the one or more barcode sequences or complements thereof correspond to the target RNA.

24. The method of any one of claims 14-23, wherein the RCP is immobilized in the biological sample and / or crosslinked to one or more other molecules in the biological sample.

25. The method of any one of claims 22-24, wherein detecting the probe or probe set in the biological sample comprises: contacting the blocked sample with a universal pool of detectably labeled probes and a first pool of intermediate probes, wherein the intermediate probes of the first pool of intermediate probes comprise hybridization regions complementary to the barcode sequence or complement thereof and reporter regions complementary to a detectably labeled probe of the universal pool of detectably labeled probes; detecting complexes formed between the barcode sequence or complement thereof, the intermediate probes of the first pool of intermediate probes, and the detectably labeled probes; and removing the intermediate probes of the first pool of intermediate probes and the detectably labeled probes.

26. The method of any one of claims 22-25, wherein the barcode sequence or complement thereof is assigned a series of signal codes that identifies the barcode sequence or complement thereof, and wherein detecting the barcode sequences or complements thereof comprises decoding the barcode sequence or complement thereof by detecting the corresponding sequence of signal codes detected from sequential hybridization, detection, and removal of sequential pools of intermediate probes and the universal pool of detectably labeled probes.

27. The method of claim 26, wherein the series of signal codes are fluorophore sequences assigned to the corresponding barcode sequence or complement thereof.

28. The method of any one of claims 1-27, wherein the one or more regions of ssDNA comprises genomic ssDNA.

29. The method of any one of claims 1-28, wherein the biological sample comprises a tissue sample.

30. The method of claim 29, further comprising permeabilizing the tissue sample, optionally wherein permeabilizing the tissue sample is performed prior to (a).

31. The method of claim 29 or 30, wherein the tissue sample comprises a frozen and / or lyophilized tissue sample.

32. The method of claim 31, wherein the frozen tissue sample comprises a fresh frozen tissue sample or a fresh frozen tissue section,optionally wherein the fresh frozen sample or fresh frozen tissue section is frozen following removal from a biological source, optionally wherein the fresh frozen sample or fresh frozen tissue section is removed and frozen without further processing.

33. The method of claim 29 or 30, wherein the tissue sample comprises a fixed tissue sample or a fixed tissue section.

34. The method of claim 33, wherein the fixed tissue sample or fixed tissue section is fixed with a fixative selected from the group consisting of ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, and glutaraldehyde, or combinations thereof.

35. The method of claim 34, wherein the fixed tissue sample comprises a Paraffin- Embedded (PE) tissue sample or the fixed tissue section comprises an PE tissue section, and wherein the method further comprises deparaffinizing the PE tissue sample or PE tissue section.

36. The method of any one of claims 33-35, wherein the fixed tissue sample comprises a Formaldehyde-Fixed Paraffin-Embedded (FFPE) tissue sample or the fixed tissue section comprises an FFPE tissue section.

37. The method of claim 35 or 36, wherein the deparaffinizing comprises contacting the (FF)PE tissue sample or (FF)PE tissue section with a solvent, optionally(i) a solvent comprising xylene; or(ii) a solvent comprising ethanol; or(iii) a solvent comprising xylene, followed by a solvent comprising ethanol.

38. The method of any one of claims 33-37, further comprising de-crosslinking the tissue sample, optionally wherein de-crosslinking the tissue sample is prior to step (a).

39. The method of claim 38, wherein the de-crosslinking comprises heating the biological sample and / or contacting the biological sample with an alkaline solvent and / or an acidic solvent.

40. The method of claim 39, wherein the heating comprises exposing the biological sample to a temperature of between about 70 °C and about 99 °C, inclusive.

41. The method of claim 39 or 40, wherein the heating comprises exposing the biological sample to a temperature of about 95 °C.

42. The method of any one of claims 38-41, wherein the de-crosslinking comprises contacting the tissue sample with de-crosslinking buffer.

43. The method of any of claims 38-42, wherein the tissue sample is a tissue section between about 1 pm and about 50 pm in thickness,optionally wherein the tissue slice is between about 5 pm and about 35 pm in thickness.

44. A method for processing a biological sample, comprising:(a) performing an extension reaction in a biological sample, wherein the extension reaction comprises using one or more regions of ssDNA in the biological sample as template to generate double stranded DNA (dsDNA), thereby providing a blocked sample;(b) contacting the blocked sample with a probe or probe set to detect a target RNA analyte in the blocked sample; and(c) performing an amplification reaction using the probe or probe set and detecting a product of the amplification reaction in the blocked sample.

45. The method of claim 44, wherein the biological sample is contacted with a polymerase in (a) and an additional polymerase in (c), wherein the polymerase and the additional polymerase are different.

46. The method of claim 45, wherein the additional polymerase is a Phi29 DNA polymerase.

47. The method of any one of claims 44-46, wherein the one or more region(s) of ssDNA comprises genomic ssDNA.

48. The method of any one of claims 44-47, wherein the biological sample is decrosslinked prior to performing the extension reaction.

49. The method of any one of claims 44-48, wherein a sequence of the probe or probe set, or a complement thereof, is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

50. The method of any one of claims 44-49, wherein the product of the amplification reaction comprises one or more barcode sequences corresponding to the target RNA analyte or complements thereof.

51. The method of any one of claims 44-50, wherein the amplification reaction comprises rolling circle amplification (RCA).

52. The method of any one of claims 44-51, wherein the biological sample is a Formaldehyde-Fixed Paraffin-Embedded (FFPE) tissue sample or tissue section.

52. The method of any one of claims 48-51, wherein the de-crosslinking comprises heating the biological sample.

53. The method of claim 52, wherein the heating comprises exposing the biological sample to a temperature of at least about 70 °C.

54. The method of any one of claims 1-53, wherein the probe or probe set comprises a circularizable probe or probe set.

55. The method of claim 54, wherein the circularizable probe is a padlock probe.

56. The method of claim 54 or claim 55, further comprising ligating the circularizable probe or probe set to form a circular nucleic acid.

57. The method of claim 56, wherein the ligating comprises performing an enzymatic ligation to connect a 5’ end of the circularizable probe to a 3’ end of the circularizable probe.

58. The method of any one of claims 1-57, wherein the blocked sample is contacted with a plurality of probes or probe sets configured for detecting a plurality of target RNA analytes.

59. The method of any one of claims 1-58, wherein the one or more target analyte or the target RNA analyte comprises mRNA.

60. A method for processing a biological sample, comprising:(a) contacting a biological sample comprising a nucleic acid molecule comprising one or more regions of ssDNA with a multiplicity of block primers configured to hybridize with the one or more regions of ssDNA present in the biological sample;(b) extending the block primers in the biological sample using the one or more regions of ssDNA to provide a blocked sample; and(c) optionally washing the blocked sample to remove any unhybridized block primers and / or block primer dimers.

61. The method of claim 60, wherein the method further comprises, prior to step (a), providing the biological sample disposed on a first substrate.

62. The method of claim 60 or 61, further comprising:(d) contacting the biological sample with a first probe and optionally a second probe, wherein the first probe and optionally the second probe each comprise sequences that are substantially complementary to sequences of an RNA analyte present within the blocked sample, and wherein the first probe or optionally the second probe comprises a capture probe binding domain.

63. The method of claim 62, further comprising:(e) hybridizing the first probe and optionally the second probe to the RNA analyte, coupling the first probe and optionally the second probe, and ligating the probes to form a connected probe.

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

65. The method of claim 64, further comprising:(g) releasing the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array to provide a released probe.

66. The method of claim 65, further comprising:(h) hybridizing the released probe to the capture domain of the capture probe, optionally wherein the capture probe binding domain is substantially complementary to the capture domain of the capture probe.

67. The method of any one of claims 60-66 wherein the blocked sample comprises less ssDNA than the biological sample prior to step (a).

68. A method for analyzing an RNA analyte in a biological sample, the method comprising:(a) providing the biological sample disposed on a first substrate;(b) contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of single-stranded DNA (ssDNA) present in the biological sample;(c) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample, optionally wherein the blocked sample comprises less ssDNA than the biological sample prior to step (b);(d) hybridizing a first probe and a second probe to the RNA analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to sequences of the RNA analyte,wherein the second probe comprises a capture probe binding domain;(e) coupling the first probe and the second probe, thereby generating a connected probe;(f) aligning the first substrate with a second substrate comprising an array, such that at least a portion of the biological sample is aligned with at least a portion of the array, wherein the array comprises 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;(g) releasing the connected probe from the RNA analyte when at least a portion of the biological sample is aligned with at least a portion of the array; and(h) hybridizing the connected probe via the capture probe binding domain to the capture domain of the capture probe, optionally wherein the capture probe binding domain is substantially complementary to the capture domain of the capture probe.

69. The method of claim 67 or 68, further comprising(i) determiningI. all or a part of the sequence of the connected probe, or a complement thereof, and / orII. the sequence of the spatial barcode, or a complement thereof.

70. The method of claim 69, further comprising(j) using the determined sequences of I and II to provide the sequence and / or location of the RNA analyte in the biological sample.

71. The method of any one of claims 60-70, wherein extending the block primers across the length of the one or more regions of ssDNA comprises contacting the biological sample with a polymerase enzyme.

72. The method of claim 71, wherein the polymerase enzyme is selected from the group consisting of DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V.

73. The method of any one of claims 62-72, wherein the RNA analyte comprises mRNA.

74. The method of any one of claims 60-73, wherein the one or more regions of ssDNA comprises genomic ssDNA.

75. The method of any one of claims 60-74, wherein the biological sample comprises a tissue sample.

76. The method of claim 75, further comprising permeabilizing the tissue sample, optionally permeabilizing the tissue sample prior to step (a).

77. The method of claim 75 or 76, wherein the tissue sample comprises a frozen and / or lyophilized tissue sample.

78. The method of claim 77, wherein the frozen tissue sample comprises a fresh frozen tissue sample or a fresh frozen tissue section, optionally wherein the fresh frozen sample or fresh frozen tissue section is frozen following removal from a biological source, preferably without further processing.

79. The method of any one of claims 75-77, wherein the tissue sample comprises a fixed tissue sample or a fixed tissue section.

80. The method of claim 79, wherein the fixed tissue sample or fixed tissue section is fixed with a fixative selected from the group consisting of ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, and glutaraldehyde, or combinations thereof.

81. The method of claim 80, wherein the fixed tissue sample comprises a Paraffin- Embedded (PE) tissue sample or the fixed tissue section comprises an PE tissue section; and wherein the method further comprises deparaffinizing the PE tissue sample or PE tissue section.

82. The method of any one of claims 80-81, wherein the fixed tissue sample comprises a Formaldehyde-Fixed Paraffin-Embedded ((FF)PE) tissue sample or the fixed tissue section comprises an (FF)PE tissue section.

83. The method of claim 81 or 82, wherein the deparaffinizing comprises contacting the (FF)PE tissue sample or (FF)PE tissue section with a solvent, optionally(i) a solvent comprising xylene; or(ii) a solvent comprising ethanol; or(iii) a solvent comprising xylene, followed by a solvent comprising ethanol.

84. The method of any one of claims 75-83, further comprising de-crosslinking the tissue sample, optionally de-crosslinking the tissue sample prior to step (a).

85. The method of claim 84, wherein the de-crosslinking comprises heating the biological sample and / or contacting the biological sample with an alkaline solvent and / or an acidic solvent.

86. The method of claim 85, wherein the heating comprises exposing the biological sample to a temperature of between about 70 °C and about 99 °C, inclusive.

87. The method of claim 85 or 86, wherein the heating comprises exposing the biological sample to a temperature of about 95 °C.

88. The method of any one of claims 84-87, wherein the de-crosslinking comprises contacting the tissue sample with de-crosslinking buffer.

89. The method of any one of claims 75-88, wherein the tissue sample comprises at least one dimension having a thickness of between about 1 pm and about 20 pm, inclusive; about 5 pm and about 15 pm, inclusive; or about 10 pm and about 12 pm, inclusive.

90. The method of any one of claims 75-89, wherein the tissue sample is disposed on a first substrate comprising a glass slide.

91. The method of any one of claims 75-90, further comprising staining and / or labelling the tissue sample, optionally before or after step (a).

92. The method of claim 91, wherein staining the tissue sample comprises hematoxylin and / or eosin (H and E) staining.

93. The method of claim 91 or 92, further comprising imaging the tissue sample.

94. The method of any one of claims 91-93, further comprising de-staining the tissue sample.

95. The method of any one of claims 60-94, wherein contacting the biological sample with a multiplicity of block primers occurs under conditions suitable for hybridizing the block primers to the one or more regions of ssDNA.

96. The method of any one of claims 60-95, wherein the multiplicity of block primers comprises between one and one hundred million species of oligonucleotide sequences.

97. The method of claim 96, wherein the oligonucleotide sequences comprise fixed sequence(s) and / or partially or completely randomly -generated sequences of between about 6 nucleotides and about 40 nucleotides, inclusive.

98. The method of claim 97, wherein the sequence of one or more of the oligonucleotide sequences comprises GAGAATGTGAGTGAAGATGTATGGTGANNNNNNN (SEQ ID NO:1), wherein each N is, independently, A, G, T, or C.

99. The method of any one of claims 60-98, wherein the multiplicity of block primers comprises between one and one hundred thousand species of fixed oligonucleotide sequences of about 6 nucleotides and about 40 nucleotides, inclusive.

100. The method of claim 96 or 97, wherein the multiplicity of block primers comprise a set of random hexamers comprising 4,096 species of oligonucleotide sequences.

101. The method of any one of claims 95-100, wherein a block primer of the multiplicity of block primers comprises a guanine / cytosine (GC) content of from about 30% to about 70%, inclusive.

102. The method of claim 101 , wherein the 3’ terminus of the block primer comprises guanine (G) or cytosine (C).

103. The method of any one of claims 60-102, wherein a block primer of the multiplicity of block primers comprises one or more of DNA, LNA, PNA, UNA, TNA phosphorothioate and P5' phosphoramidate.

104. The method of any one of claims 60-103, wherein the step of contacting the biological sample with a multiplicity of block primers further comprises contacting the biological sample with one or more reagents for block primer extension.

105. The method of claim 104, wherein the one or more reagents for block primer extension comprises a polymerase enzyme, deoxynucleotide triphosphates (dNTPs), and / or a suitable reaction buffer for block primer extension.

106. The method of claim 105, wherein the polymerase enzyme comprises a DNA polymerase that catalyzes synthesis of DNA from a DNA template, optionally wherein the DNA polymerase enzyme does not catalyze the synthesis of DNA from an RNA template.

107. The method of claim 106, wherein the polymerase enzyme is selected from DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA polymerase IV and DNA polymerase V.

108. The method of claim 106 or 107, wherein the polymerase enzyme is a thermostable DNA polymerase I.

109. The method of claim 108, wherein the thermostable DNA polymerase I is derived from Thermus aquaticus.

110. The method of any one of claims 60-109, wherein the method further comprises, following the step of contacting the biological sample with a multiplicity of block primers, one or more steps for washing the biological sample to remove unbound / unhybridized block primers and / or primer extension reagents from the blocked sample, optionally wherein the washing comprises(i) contacting the blocked sample with a suitable solvent in an amount and time effective to solubilize the block primers and / or primer extension reagents; and(ii) removing the blocked sample from the solvent.

111. The method of claim 110, wherein the solvent comprises an aqueous buffer and optionally one or more additional reagents.

112. The method of claim 111, wherein the aqueous buffer comprises PBS and a polysorbate.

113. The method of any one of claims 110-112, comprising repeating steps (i) and (ii) one or more times.

114. The method of any one of claims 60-113, further comprising contacting the blocked sample with one or more additives in an amount and time effective to stabilize one or more regions of double strand DNA (dsDNA) formed by extension of one or more blocked primers in the blocked sample.

115. The method of claim 114, wherein the additive comprises spermine and / or methylene blue.

116. The method of any one of claims 60-115, wherein the amount of ssDNA within the blocked sample is less than 50%, 40%, 30% 20%, 10%, 5%, 2%, 1%, or 0.1% of the amount of ssDNA within the biological sample prior to contacting the biological sample with the multiplicity of block primers.

117. The method of any one of claims 60- 116, wherein the sequence of RNAs within the blocked sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of RNAs within the biological sample prior to contacting the biological sample with the multiplicity of block primers.

118. The method of any one of claims 60-117, wherein the abundance of RNAs within the blocked sample is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the abundance of RNAs within the tissue sample prior to contacting the biological sample with the multiplicity of block primers.

119. The method of any one of claims 63 to 118, wherein the first probe hybridizes to a first sequence in the RNA analyte, wherein the second probe hybridizes to a second sequence in the RNA analyte, wherein the first and second sequences are contiguous, and / or wherein one or more ends of the hybridized first and second probes are adjacent to one another.

120. The method of any one of claims 63 to 119, wherein the first probe hybridizes to a first sequence in the RNA analyte, wherein the second probe hybridizes to a second sequence in the RNA analyte, wherein the first and second sequences are not contiguous, and / orwherein a 3’ end of the hybridized first probe and a 5 ’end of the hybridized second probes are separated by a gap or a 5’ end of the hybridized first probe and a 3 ’end of the hybridized second probes are separated by a gap.

121. The method of claim 120, further comprising filling the gap between the hybridized first and second probes.

122. The method of claim 121, further comprising contacting the biological sample with a polymerase enzyme to extend the first and / or second probes using the RNA analyte as a template to fill in the gap between the hybridized first and second probes.

123. The method of any one of claims 119, or 121-122, further comprising ligating the first and second probes to form the connected probe.

124. The method of claim 123, wherein the ligating comprises chemical ligation.

125. The method of claim 123, wherein the ligating comprises contacting the biological sample with a ligase enzyme.

126. The method of claim 71, wherein the polymerase enzyme is a DNA polymerase I derived from Thermus aquaticus.

127. The method of any one of claims 75 to 126, wherein the tissue sample comprises tissue from a human cancer selected from the group consisting of ovarian cancer, lung cancer, tonsil cancer, glioblastoma, and prostate cancer.

128. The method of claim 60 or 61 , further comprising:(d) contacting the blocked sample with a circularizable probe or probe set comprising sequences that are substantially complementary to sequences of an RNA analyte present within the blocked sample.

129. The method of claim 128, further comprising:(e) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and(f) detecting the RCP in the biological sample.

130. The method of claim 129, wherein detecting RCP in the sample comprises detecting a barcode sequence or a complement thereof in the RCP.

131. The method of claim 130, wherein detecting the barcode sequence or complement thereof comprises: contacting the biological sample with a universal pool of detectably labeled probes and a first pool of intermediate probes, wherein the intermediate probes of the first pool of intermediate probes comprise hybridization regions complementary to the barcode sequenceor complement thereof and reporter regions complementary to a detectably labeled probe of the universal pool of detectably labeled probes; detecting complexes formed between the barcode sequence or complement thereof, the intermediate probes of the first pool of intermediate probes, and the detectably labeled probes; and removing the intermediate probes of the first pool of intermediate probes and the detectably labeled probes.

132. The method of claim 131, wherein detecting the barcode sequences or complements thereof further comprises: contacting the biological sample with the universal pool of detectably labeled probes and a second pool of intermediate probes, wherein the intermediate probes of the second pool of intermediate probes comprise hybridization regions complementary to the barcode sequence or complement thereof and reporter regions complementary to a detectably labeled probe of the universal pool of detectably labeled probes; and detecting complexes formed between the barcode sequence or complement thereof, the intermediate probes of the second pool of intermediate probes, and the detectably labeled probes.

133. The method of any of claims 130-132, wherein the barcode sequence or complement thereof is assigned a series of signal code that identifies the barcode sequence or complement thereof, and wherein detecting the barcode sequence or a complement thereof comprises decoding the barcode sequence or complement thereof by detecting the corresponding sequences of signal code detected from sequential hybridization, detection, and removal of sequential pools of intermediate probes and the universal pool of detectably labeled probes.

134. The method of claim 133, wherein the series of signal code is a fluorophore sequence assigned to the corresponding barcode sequence or complement thereof.

135. The method of any of claims 130-134, wherein the detectably labeled probes are fluorescently labeled.

136. The method of any of claims 130-135, wherein the method comprises imaging the blocked sample to detect the RCP.

137. The method of claim 136, wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCP.

138. The method of any one of claims 128-137, wherein a sequence of the RCP is analyzed at a location in the biological sample or a matrix embedding the biological sample.

139. The method of claim 138, wherein the sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

140. A kit comprising a multiplicity of block primers configured to hybridize with single-stranded DNA (ssDNA), and / or a polymerase enzyme, and instructions for performing the method of any one of claims 60-139.

141. The kit of claim 140, wherein the multiplicity of block primers comprises between one and one hundred million species of fixed oligonucleotide sequence(s) and / or partially or completely randomly generated sequences each comprising about 6 nucleotides to about 40 nucleotides, inclusive, optionally wherein the sequence of one or more of the block primers comprises GAGAATGTGAGTGAAGATGTATGGTGANNNNNNN (SEQ ID NO:1), wherein N is A, G, T, or C.

142. The kit of claim 141, wherein the multiplicity of block primers comprises between one and one hundred thousand species of oligonucleotide sequences comprising from about 6 nucleotides and about 40 nucleotides, inclusive, optionally wherein the multiplicity of block primers comprises a set of random hexamers comprising 4,096 species of oligonucleotide sequences.

143. The kit of any one of claims 139 to 142, further comprising one or more of(i) a solvent suitable to solubilize the oligonucleotides and / or polymerase enzyme;(ii) deoxynucleotide triphosphates (dNTPs);(iii) a suitable reaction buffer for primer extension; and(iv) one or more inhibitors of the polymerase enzyme.

144. A method for analyzing an RNA analyte in a biological sample, the method comprising:(a) contacting the biological sample with a multiplicity of block primers configured to hybridize with one or more regions of single-stranded DNA (ssDNA) present in the biological sample;(b) extending the block primers across the length of the one or more regions of ssDNA to provide a blocked sample, optionally wherein the blocked sample comprises less ssDNA than the biological sample prior to step (b);(c) hybridizing a first probe and a second probe to the RNA analyte, wherein the first probe and the second probe each comprise a sequence that is substantially complementary to sequences of the RNA analyte, andwherein the second probe comprises a capture probe binding domain;(d) coupling the first probe and the second probe, thereby generating a connected probe;(e) releasing the connected probe from the RNA analyte;(f) hybridizing the connected probe to a capture domain of a capture probe on an array, wherein the array comprises a plurality of capture probes, wherein the capture probe is comprised in the plurality of capture probes, and wherein the capture probe comprises: i. a spatial barcode; and ii. the capture domain.

145. The method of claim 144, wherein the biological sample is disposed on a substrate comprising the array comprising the plurality of capture probes.

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