SNP detection with RNA-templated ligation

Abstract

The present disclosure relates to methods for analyzing target nucleic acids in a biological sample using Argonaute proteins and single cell RNA-templated ligation probe chemistry. Variant sequences (e.g., single nucleotide variations such as SNPs or point mutations) in a plurality of target nucleic acids in a cell or tissue sample are analyzed in the sample. The application is drawn to assay methods, and further drawn to compositions and kits for use in accordance with the methods. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.

Description

Attorney Docket: 43487-1036601 SNP DETECTION WITH RNA-TEMPLATED LIGATION CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 661,533, filed June 18, 2024, which is incorporated herein by reference in its entirety for allpurposes. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submittedelectronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 10, 2025, is named 43487-1036_601_SL.xml and is 23,442 bytes in size. BACKGROUND

[0003] Methods are available for detecting nucleic acids present in a biological sample.However, analysis of short sequences (e.g., single nucleotide differences such as single nucleotide polymorphisms (SNPs) or point mutations) on individual transcripts in samples (e.g., a tissue section) has remained challenging. Improved methods for identifying short variant sequences and analyzing single cell profiles (e.g., sequence identity and / or abundance) in cells or tissue samples are needed. Provided herein are methods, compositions, and kits that address such and other needs. SUMMARY

[0004] Argonaute proteins are a large family of proteins that use nucleic acid guides to targetother nucleic acids for binding and / or cutting. Family members are derived from prokaryotic and eukaryotic organisms. Some Argonaute family members use RNA guide nucleic acids, and some use DNA guide nucleic acids. The guide nucleic acid can direct Argonaute binding to a target sequence complementary to the guide nucleic acid or a portion thereof (e.g., complementary to a seed sequence of the guide nucleic acid) with high sensitivity. In the case of nuclease-active Argonaute proteins, the Argonaute and guide nucleic acid cut the complementary target nucleic acid sequence. Argonaute proteins include family members capable of binding and cutting RNA, as well as family members capable of binding and cutting DNA. Some Argonaute proteins lack nuclease activity. For such nuclease-deficient Argonaute proteins, the guide nucleic acid directs Argonaute binding to a specific nucleic acid sequence complementary to the guide nucleic acid sequence. In certain cases, Argonaute proteins are engineered to be nuclease deficient.

[0005] The present application harnesses the sequence-specific binding or binding andcutting activities of Argonaute proteins for improved methods of detection of variant sequences. In some aspects, the guide nucleic acid-mediated sequence-specific binding properties ofAttorney Docket: 43487-1036601 nuclease-deficient Argonaute are used to provide improved methods of detecting a variant sequence in a target nucleic acid.

[0006] In some aspects, the present disclosure provides a method for analyzing a biologicalsample, comprising (a) contacting the biological sample with: (i) a complex comprising a nuclease-deficient Argonaute protein and a first probe of a probe set, wherein: (I) the first probe comprises a first probe region that hybridizes to a first target sequence in a target nucleic acid in the biological sample and (II) the first target sequence comprises a variant sequence; and (ii) a second probe of the probe set, wherein the second probe comprises a second probe region that hybridizes to a second target sequence in the target nucleic acid in the biological sample, (b) coupling the first probe and second probe to generate a first-second probe product; (c) detecting the first-second probe product or derivative thereof.

[0007] In some aspects, the present disclosure is generally drawn to methods for analyzing abiological sample, (a) contacting the biological sample with: (i) a complex comprising a nuclease-deficient Argonaute protein and a first probe of a probe set, wherein: (I) the first probe comprises a first probe region that hybridizes to a first target sequence in a target nucleic acid in the biological sample and (II) the first target sequence comprises a variant sequence; and (ii) a second probe of the probe set, wherein the second probe comprises a second probe region that hybridizes to a second target sequence in the target nucleic acid in the biological sample, (b) coupling the first probe and second probe to generate a first-second probe product; (c) detecting the first-second probe product or a derivative thereof. In some aspects, the Argonaute protein is an RNA-guided Argonaute, and the first probe is an RNA molecule.

[0008] In some aspects, the Argonaute protein is a eukaryotic Argonaute protein. In someaspects, the Argonaute protein is a DNA-guided Argonaute, and the first probe is a DNA molecule. In some aspects, the Argonaute protein is a prokaryotic Argonaute protein. In some aspects, the first probe comprises a 5’-phosphate or a 5’-OH. In some aspects, the nuclease- deficient Argonaute protein is Ago1 or Ago4. In some aspects, the nuclease-deficient Argonaute protein is a Drosophila Argonaute protein or a derivative or variant thereof. In some aspects, the nuclease-deficient Argonaute protein is a nuclease-deficient Argonaute derived from Thermus thermophilus (dTtAgo). In some aspects, the nuclease-deficient Argonaute protein comprises one or more inactivating mutations in a PIWI and / or PAZ domain of the Argonaute protein.

[0009] In some aspects, the first probe and the Argonaute protein are bound in the complexbefore contacting the biological sample. In some aspects, the first probe and the Argonaute protein are contacted with the biological sample simultaneously. Accordingly, in some aspects, the method for analyzing a biological sample comprises: (a) contacting the biological sample with: (i) a nuclease-deficient Argonaute protein and a first probe of a probe set, wherein theAttorney Docket: 43487-1036601 Argonaute protein and the first probe of a probe set form a complex, and wherein the first probe comprises a first probe region that hybridizes to a first target sequence in a target nucleic acid in the biological sample and the first target sequence comprises a variant sequence; and (ii) a second probe of the probe set, wherein the second probe comprises a second probe region that hybridizes to a second target sequence in the target nucleic acid in the biological sample, (b) coupling the first probe and second probe to generate a first-second probe product; and (c) detecting the first-second probe product. In some aspects, the nuclease-deficient Argonaute protein and the first probe of the probe set contact the biological sample separately. In some embodiments, the probe (e.g., first probe) and the Argonaute protein form a complex in the biological sample In some aspects, the first probe comprises one or more ribonucleotides. In some aspects, the first probe comprises a sequence complementary to the variant sequence, wherein the sequence comprises one or more nucleotides between positions 2 and 8 from the 5’ end of the first probe. In some aspects, the nuclease-deficient Argonaute protein or the first probe is labeled with a detectable moiety, optionally wherein the detectable moiety is a fluorescent dye. In some aspects, the first target sequence in the target nucleic acid bound to the first probe is 15- 35 nucleotides. In some aspects, a nucleotide at position 6 and / or position 7 from the 5’ end of the first probe region is complementary to the variant sequence. In some aspects, the first target sequence and the second target sequence are each about 15 to about 40 nucleotides in length.

[0010] In some aspects, in (a) the biological sample is contacted with a plurality of differentfirst probes comprising a plurality of different sequences complementary to a plurality of different variant sequences. In some aspects, (a) further comprises contacting the biological sample with a plurality of different first probes comprising a plurality of different sequences complementary to a plurality of different variant sequences, wherein the plurality of different first probes comprises the first probe. Accordingly, in some aspects the method for analyzing a biological sample comprises: (a) contacting the biological sample with: (i) a plurality of complexes, wherein each complex comprises a nuclease-deficient Argonaute protein and a first probe of a probe set, wherein the first probe comprises a first probe region that hybridizes to a first target sequence in a target nucleic acid in the biological sample, wherein the first target sequence comprises a variant sequence, and wherein the first probe in different complexes may hybridize to a different target nucleic acid or different variant sequence; and (ii) a second probe of the probe set that hybridizes to a second target sequence in the target nucleic acid in the biological sample, (b) ligating the first probe and second probe to generate a ligated first-second probe product; and (c) detecting the ligated first-second probe product. In some aspects, the method for analyzing a biological sample comprises: (a) contacting the biological sample with (i) a nuclease-deficient Argonaute protein and a plurality of different first probes of a probe set,Attorney Docket: 43487-1036601 wherein the Argonaute protein and each of the first probes of the plurality of first probes forms a complex, wherein each of the first probes comprises a first probe region that hybridizes to a first target sequence in a target nucleic acid in the biological sample, wherein the first target sequence comprises a variant sequence and wherein the first probe in different complexes may hybridize to a different target nucleic acid or different variant sequence, and (ii) a second probe of the probe set to a second target sequence in the target nucleic acid in the biological sample, (b) ligating the first probe and second probe to generate a ligated first-second probe product; and (c) detecting the ligated first-second probe product. In some aspects, the nuclease-deficient Argonaute protein and the plurality of different first probes of a first probe set first contact the biological sample separately. In some embodiments, the probe (e.g., first probe) and the Argonaute protein form a complex in the biological sample In some aspects, the second probe is complementary to a common second target sequence among a plurality of different target nucleic acids comprising different variant sequences.

[0011] In some aspects, the first probe comprises a sequence complementary to the variantsequence, wherein the sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 5’ or 3’ end of the first probe. In some aspects, the sequence complementary to the variant sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 5’ or 3’ end of the first probe. In some aspects, the variant sequence is at or near the central nucleotide(s) of the first target sequence in the target nucleic acid. In some aspects, the first probe comprises a sequence complementary to the variant sequence, wherein the sequence is at or near the central nucleotide(s) of the first probe region. In some aspects, the sequence complementary to the variant sequence is at or near the central nucleotide(s) of the first probe region.

[0012] In some aspects, the variant sequence comprises a nucleotide variation, a nucleotidepolymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, and / or a repetitive sequence. In some aspects, the variant sequence comprises two or more nucleotide residues. In some aspects, the variant sequence is a single nucleotide. In some aspects, the variant sequence comprises a single nucleotide variation (SNV), a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion.

[0013] In some aspects, (b) comprises performing a ligation reaction. In some aspects, (b)comprises performing a nucleic acid extension reaction. In some aspects, (b) comprises ligating a first end of the first probe to a second end of the second probe. In some aspects, the first target sequence and the second target sequence are separated by a gap region of at least 1 nucleotide on the target nucleic acid, and (b) comprises performing a gap-fill reaction to generate the first- second probe product. In some aspects, the target nucleic acid is RNA and the first probe andAttorney Docket: 43487-1036601 second probe are ligated using a ligase having an RNA-templated DNA or RNA ligase activity. In some aspects, the target nucleic acid is RNA, and the method further comprises ligating the first probe and the second probe using a ligase having an RNA-templated DNA or RNA ligase activity. In some aspects, the ligase is a PBCV-1 DNA ligase or a T4 RNA ligase 2. In some aspects, the ligase is a Chlorella virus DNA ligase (PBCV DNA ligase) or a T4 RNA ligase. In some aspects, the method further comprises contacting biological sample with a library of first probes that comprises at least or about 2, at least or about 3, at least or about 4, or more different first probes comprising different first probe region sequences. In some aspects, the biological sample after contacting with the library of first probes.

[0014] In some aspects, the first probe comprises a detection region that does not hybridizeto the first target sequence in a target nucleic acid. In some aspects, detecting the first-second probe product or derivative thereof comprises detecting a sequence of the detection region in the first-second probe product or derivative thereof. In some aspects, detecting the ligated first- second probe product comprises detecting a sequence of the detection region. In some aspects, the methods further comprise binding a circular or circularizable probe to a sequence in the detection region of the ligated first-second probe product. In some aspects, the detecting comprises imaging the biological sample to detect the first-second probe product or derivative thereof. In some aspects, the detecting comprises imaging the biological sample to detect ligated first-second probe product. In some aspects, the imaging is performed over a plurality of sequential cycles. In some aspects, the method further comprises identifying a sequence of the first-second probe product by sequencing. In some aspects, the sequence of the ligated first- second probe product is detected by sequencing. In some aspects, the methods further comprise releasing the first-second probe product from the biological sample before (c). In some aspects, the methods further comprise releasing the ligated probe from the sample before (c). In some aspects, the variant sequence is among a plurality of different sequences. In some aspects, the variant sequence is a mutant sequence or a minor variant among a plurality of different variant sequences. In some aspects, the variant sequence is a wildtype sequence or a major variant among a plurality of different variant sequences. In some aspects, the first target sequence comprises a genetic hotspot, optionally wherein the first target sequence comprises two or more hotspot mutations.

[0015] In some aspects, the target nucleic acid is a cellular nucleic acid analyte or a productthereof. In some aspects, the cellular nucleic acid analyte is an RNA, and the product thereof is a cDNA. In some aspects, the target nucleic acid is mRNA. In some aspects, the target nucleic acid is a cDNA. In some aspects, the biological sample is a tissue section. In some aspects, the biological sample is a formalin-fixed, paraffin-embedded (FFPE) sample or a fresh frozen tissueAttorney Docket: 43487-1036601 sample. In some aspects, the biological sample is fixed and / or permeabilized. In some aspects, the biological sample is crosslinked and / or embedded in a matrix, optionally wherein the matrix comprises a hydrogel. In some aspects, the biological sample is cleared. In some aspects, the biological sample comprises a plurality of cells or nuclei. In some aspects, the biological sample comprises a plurality of cells. In some aspects, the biological sample comprises a plurality of nuclei. In some aspects, the biological sample is a single cell or nucleus. In some aspects, the biological sample is a single cell. In some aspects, the biological sample is a single nucleus.

[0016] In some aspects, the present disclosure provides a method for analyzing a biologicalsample, comprising: (a) contacting the biological sample with a complex comprising a nuclease- deficient Argonaute protein and a probe, wherein the probe comprises a probe region and a detection region, wherein: (i) the probe region hybridizes to a target sequence in a target nucleic acid in the biological sample and the target sequence comprises a variant sequence; and (ii) the detection region does not hybridize to the target nucleic acid; and (b) detecting the probe or derivative thereof. In some aspects, the present disclosure is drawn to methods for analyzing a biological sample, (a) contacting the biological sample with a complex comprising a nuclease- deficient Argonaute protein and a probe, wherein the probe comprises: (i) a probe region that hybridizes to a target sequence in a target nucleic acid in the biological sample and the target sequence comprises a variant sequence; and (ii) a detection region that does not hybridize to the target nucleic acid; and (b) detecting the bound probe in the biological sample. In some aspects, the Argonaute protein is an RNA-guided Argonaute, and the probe is an RNA molecule. In some aspects, the Argonaute protein is a eukaryotic Argonaute protein. In some aspects, the Argonaute protein is a DNA-guided Argonaute, and the first probe is a DNA molecule. In some aspects, the Argonaute protein is a prokaryotic Argonaute protein. In some aspects, the nuclease-deficient Argonaute protein is Ago1 or Ago4.

[0017] In some aspects, the nuclease-deficient Argonaute protein is a Drosophila Argonauteprotein or a derivative or variant thereof. In some aspects, the nuclease-deficient Argonaute protein is a nuclease-deficient Argonaute derived from Thermus thermophilus (dTtAgo). In some aspects, the nuclease-deficient Argonaute protein comprises one or more inactivating mutations in a PIWI and / or PAZ domain of the Argonaute protein. In some aspects, the probe and the Argonaute protein are bound in the complex before contacting the biological sample. In some aspects, the probe and the Argonaute protein are contacted with the biological sample simultaneously. Accordingly, in some aspects, the method for analyzing a biological sample comprises: (a) contacting the biological sample with a nuclease-deficient Argonaute protein and a probe, wherein the nuclease-deficient Argonaute protein and the probe form a complex, and wherein the probe comprises: (i) a probe region that hybridizes to a target sequence in a targetAttorney Docket: 43487-1036601 nucleic acid in the biological sample and the target sequence comprises a variant sequence; and (ii) a detection region that does not hybridize to the target nucleic acid; and (b) detecting the bound probe in the biological sample. In some aspects, the nuclease-deficient Argonaute protein and the probe contact the biological sample separately. In some embodiments, the probe (e.g., first probe) and the Argonaute protein form a complex in the biological sample. In some aspects, the probe comprises a sequence complementary to the variant sequence, wherein the sequence comprises one or more nucleotides between positions 2 and 8 from the 5’ end of the probe. In some aspects, a nucleotide at position 6 and / or position 7 from the 5’ end of the first probe region is complementary to the variant sequence. In some aspects, in (a), the biological sample is contacted with a plurality of different probes comprising a plurality of different sequences complementary to a plurality of different variant sequences. In some aspects, the method for analyzing a biological sample comprises, (a) contacting the biological sample with a plurality of complexes, wherein each complex comprises a nuclease-deficient Argonaute protein and a probe, wherein the probe comprises: (i) a probe region that hybridizes to a target sequence in a target nucleic acid in the biological sample and the target sequence comprises a variant sequence; and (ii) a detection region that does not hybridize to the target nucleic acid, and wherein the probe in different complexes may hybridize to a different target nucleic acid or different variant sequence, and (b) detecting the bound probe in the biological sample. In some aspects, the method for analyzing a biological sample comprises, (a) contacting the biological sample with a nuclease-deficient Argonaute protein and a plurality of probes, wherein the Argonaute protein and each of the probes of the plurality of probes forms a complex, wherein the probe comprises: (i) a probe region that hybridizes to a target sequence in a target nucleic acid in the biological sample and the target sequence comprises a variant sequence; and (ii) a detection region that does not hybridize to the target nucleic acid, and wherein the probe in different complexes may hybridize to a different target nucleic acid or different variant sequence, and wherein the probe in different complexes may hybridize to a different target nucleic acid or different variant sequence and (b) detecting the bound probe in the biological sample. In some aspects, the nuclease-deficient Argonaute protein and the plurality of different probes contact the biological sample separately. In some embodiments, the probe (e.g., first probe) and the Argonaute protein form a complex in the biological sample

[0018] In some aspects, the methods further comprise binding an additional probe to anadditional (e.g., second) target sequence in the target nucleic acid in the biological sample, wherein the additional probe is complementary to a common additional (e.g., second) target sequence among a plurality of different target nucleic acids comprising different variant sequences. In some aspects, the probe comprises a sequence complementary to the variantAttorney Docket: 43487-1036601 sequence, wherein the sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 5’ or 3’ end of the probe. In some aspects, the variant sequence is at or near the central nucleotide(s) of the target sequence in the target nucleic acid. In some aspects, the variant sequence comprises a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, and / or a repetitive sequence. In some aspects, the variant sequence comprises two or more nucleotide residues. In some aspects, the variant sequence is a single nucleotide.

[0019] In some aspects, the variant sequence comprises a single nucleotide variation (SNV),a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion. In some aspects, the biological sample is contacted with a library of probes that comprises at least or about 2, at least or about 3, at least or about 4, or more different probes comprising different probe region sequences. In some aspects, the biological sample after contacting with the library of probes. In some aspects, the methods further comprise binding a circular or circularizable probe to a sequence in the detection region of the probe or derivative thereof prior to (b). In some aspects, the detecting comprises imaging the biological sample to detect the probe or derivative thereof. In some aspects, the detecting comprises imaging the biological sample to detect bound probe. In some aspects, the imaging is performed over a plurality of sequential cycles. In some aspects, (b) comprises detecting the probe or derivative thereof in the biological sample.

[0020] In some aspects, the present disclosure provides a kit, comprising: a probe setcomprising a first probe and a second probe, wherein the first probe of the probe set comprises a first probe region that is configured to hybridize to a first target in a target nucleic acid in the biological sample, and wherein the second probe of the probe set comprises a capture region and a second probe region that is configured to hybridize a second target sequence in the target nucleic acid in the biological sample, wherein the first probe is in a complex with a nuclease- deficient Argonaute protein; and a ligase configured to ligate the first probe to the second probe to generate a ligated first-second probe product. In some aspects, the disclosure is generally drawn to a kit, comprising: a probe set comprising a first probe and a second probe, wherein the first probe of the probe set comprises a first probe region that hybridize to a first target in a target nucleic acid in the biological sample and the second probe of the probe set comprises a capture region and a second probe region that hybridize a second target sequence in the target nucleic acid in the biological sample, wherein the first probe is in a complex with a nuclease-deficient Argonaute protein; and a ligase for ligating the first probe to the second probe to generate a ligated first-second probe product. In some aspects, the disclosure is generally drawn to a kit, comprising: a probe set comprising a first probe and a second probe, wherein the first probe ofAttorney Docket: 43487-1036601 the probe set comprises a first probe region that is configured to hybridize to a first target in a target nucleic acid in the biological sample and the second probe of the probe set comprises a capture region and a second probe region that is configured to hybridize to a second target sequence in the target nucleic acid in the biological sample; a nuclease-deficient Argonaute protein; and a ligase for ligating the first probe to the second probe to generate a ligated first- second probe product.

[0021] Another aspect of the present disclosure provides a non-transitory computer readablemedium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0022] Another aspect of the present disclosure provides a system comprising one or morecomputer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0023] Additional aspects and advantages of the present disclosure will become readilyapparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0024] All publications, patents, and patent applications mentioned in this specification areherein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The novel features of the invention are set forth with particularity in the appendedclaims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:Attorney Docket: 43487-1036601

[0026] FIG. 1 shows an example of a microfluidic channel structure for partitioningindividual biological particles.

[0027] FIG. 2 shows an example of a microfluidic channel structure for the controlledpartitioning of beads into discrete droplets.

[0028] FIG. 3 illustrates an example of a barcode carrying bead.

[0029] FIG. 4 illustrates another example of a barcode carrying bead.

[0030] FIG. 5 schematically illustrates an example microwell array.

[0031] FIG. 6 schematically illustrates an example workflow for processing nucleic acidmolecules.

[0032] FIG. 7 schematically illustrates another example workflow for processing nucleicacid molecules.

[0033] FIG. 8 schematically illustrates another example workflow for processing nucleicacid molecules.

[0034] FIG. 9 schematically illustrates another example workflow for processing nucleicacid molecules.

[0035] FIG. 10 schematically illustrates an example workflow for analyzing cells, nuclei orcell beads.

[0036] FIG. 11 schematically illustrates example labelling agents with nucleic acidmolecules attached thereto.

[0037] FIG. 12A schematically shows an example of labelling agents. FIG. 12Bschematically shows another example workflow for processing nucleic acid molecules. FIG. 12B shows “AAAAAAAAAAA” (SEQ ID NO: 1) and “TTTTTTTTTTTTT” (SEQ ID NO: 2). FIG.12C schematically shows another example workflow for processing nucleic acid molecules. FIG.12C shows “AAAAAAAAAAA” (SEQ ID NO: 1).

[0038] FIG. 13 schematically shows another example of a barcode-carrying bead.

[0039] FIG. 14 shows a computer system that is programmed or otherwise configured toimplement methods provided herein.

[0040] FIGs. 15A-15C shows an example processed nucleic acid molecule described herein.FIG.15A shows 1506 (SEQ ID NO: 3), 1516 (SEQ ID NO: 4), 1519 (SEQ ID NO: 5), 1520 (SEQ ID NO: 6), 1500 (SEQ ID NO: 7), and 1517 (SEQ ID NO: 8). FIG.15B shows 1520 (SEQ ID NO: 6), 1519 (SEQ ID NO: 9), 1506 (SEQ ID NO: 10), 1516 (SEQ ID NO: 11), 1517 (SEQ ID NO: 12), and 1500 (SEQ ID NO: 13). FIG.15C discloses 1506 (SEQ ID NO: 3), 1520 (SEQ ID NO: 6), 1500 (SEQ ID NO: 7), 1516 (SEQ ID NO: 11), 1517 (SEQ ID NO: 14).

[0041] FIG. 16A shows an example workflow for processing multiple analytes in a partition.FIG.16B shows another example workflow for processing multiple analytes in a partition.Attorney Docket: 43487-1036601

[0042] FIG. 17 schematically shows a feature-binding group described herein. FIG. 17shows 1719 (SEQ ID NO: 5) and 1717 (SEQ ID NO: 8).

[0043] FIG. 18 shows example data from a workflow described herein.

[0044] FIG. 19 shows additional example data from a workflow described herein.

[0045] FIG. 20 shows additional example data from a workflow described herein.

[0046] FIG. 21A shows example data comparing fixed cells and unfixed cells. FIG. 21Bshows additional example data comparing fixed cells and unfixed cells. FIG.21C shows additional example data comparing fixed cells and unfixed cells.

[0047] FIG. 22 schematically shows an example workflow for assaying two different analytetypes.

[0048] FIG. 23 shows example data of a barcoding approach described herein.

[0049] FIG. 24 shows example data of different analyte types using the barcodingapproaches described herein.

[0050] FIG. 25 schematically shows an example method for processing nucleic acidmolecules.

[0051] FIG. 26 shows another example method for processing nucleic acid molecules.

[0052] FIG. 27 shows an example workflow for generating probe-linked nucleic acidmolecules. FIG.27 shows “AAAAAAAAAA” (SEQ ID NO: 15).

[0053] FIG. 28 shows another example workflow for generating probe-linked nucleic acidmolecules.

[0054] FIG. 29 shows an example workflow for processing cells according to the methodsdescribed herein.

[0055] FIG. 30A shows example protein expression data resulting from barcoding ofmultiple analytes using different sample preparation parameters. FIG.30B shows additional protein expression data resulting from barcoding of multiple analytes using different sample preparation parameters.

[0056] FIG. 31 shows example gene expression data resulting from barcoding of multipleanalytes using different sample preparation parameters.

[0057] FIGs. 32A-32C shows example data of multiple analyte probing for a negativecontrol group. FIG.32A shows example data showing different immune cell clusters. FIG.32B shows example data of gene expression of GZMB gene. FIG.32C shows example data of protein expression resulting from antibody staining.

[0058] FIGs. 33A-33C shows example data of multiple analyte probing for an experimentalgroup. FIG.33A shows example data showing different immune cell clusters. FIG.33B showsAttorney Docket: 43487-1036601 example data of gene expression of GZMB gene. FIG.33C shows example data of protein expression resulting from antibody staining.

[0059] FIGs. 34A-34C shows example data of multiple analyte probing for an experimentalgroup. FIG.34A shows example data showing different immune cell clusters. FIG.34B shows example data of gene expression of GZMB gene. FIG.34C shows example data of protein expression resulting from antibody staining.

[0060] FIGs. 35A-35C shows example data of multiple analyte probing for an experimentalgroup. FIG.35A shows example data showing different immune cell clusters. FIG.35B shows example data of gene expression of GZMB gene. FIG.35C shows example data of protein expression resulting from antibody staining.

[0061] FIGs. 36A-36C shows example data of multiple analyte probing for an experimentalgroup. FIG.35A shows example data showing different immune cell clusters. FIG.36B shows example data of gene expression of GZMB gene. FIG.36C shows example data of protein expression resulting from antibody staining.

[0062] FIGs. 37A-37C shows example data of multiple analyte probing for an experimentalgroup. FIG.37A shows example data showing different immune cell clusters. FIG.37B shows example data of gene expression of GZMB gene. FIG.37C shows example data of protein expression resulting from antibody staining.

[0063] FIG. 38 shows another example workflow for assaying two different analyte types.

[0064] FIGs. 39A-39E are an example of time series data for fixed samples.

[0065] FIG. 40 shows a flowchart of a method for analyzing a nucleic acid molecule of anembedded, fixed tissue, according to some embodiments.

[0066] FIG. 41 shows an example of a nucleic acid profiling workflow, according to someembodiments.

[0067] FIG. 42 shows an example of a nucleic acid profiling workflow, according to someembodiments.

[0068] FIG. 43 show an example of a plurality of probe molecules, according to someembodiments.

[0069] FIGs. 44 - 46 show examples of methods of preparation for fixed samples, accordingto some embodiments.

[0070] FIG. 47 provides a table of the pass percentages and other properties for the variousprocedures of the present example, according to some embodiments.

[0071] FIG. 48, shows examples of various tissues processed by the workflows of FIGs. 44– 46, according to some embodiments.

[0072] FIGs. 49A-49D show example cell type clustering for single cell cancer fixed tissues.Attorney Docket: 43487-1036601

[0073] FIGs. 50A and 50B show example tissue dissociation using pestle dissociation.

[0074] FIG. 51 shows an example gentleMACSTM program for tissue dissociation.

[0075] FIG. 52A shows an example of representative cells after dissociation. FIG. 52Bshows an example of representative cells after post-hybridization wash.

[0076] FIG. 53 shows example cell yield from fixed tissue sections.

[0077] FIG. 54A and 54B depict using nuclease-deficient Argonaute protein assisted probehybridization (e.g., a first probe and a second probe of a probe set) to target a variant sequence and detect single-nucleotide variants.

[0078] FIG. 55 shows an example method of capturing ligated first-second probe productswith oligonucleotides on a support (identified as a substrate), such as a bead. DETAILED DESCRIPTION

[0079] While various embodiments of the invention have been shown and described herein,it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0080] Where values are described as ranges, it will be understood that such disclosureincludes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0081] The terms “a,” “an,” and “the,” as used herein, generally refers to singular and pluralreferences unless the context clearly dictates otherwise.

[0082] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedesthe first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0083] Whenever the term “no more than,” “less than,” or “less than or equal to” precedesthe first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0084] The terms “about” and “approximately,” as used herein, when preceding a numericalvalue indicates the value plus or minus a range of 10%. For example, about 10 may beAttorney Docket: 43487-1036601 reasonably understood to convey 9, 10, or 11, or a range of numerical values spanning from 9 to 11. Whenever “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, the term “about” or “approximately” applies to each of the numerical values in that series of numerical values.

[0085] The term “barcode,” as used herein, generally refers to a label, or identifier, thatconveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. A barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique. Barcodes can have a variety of different formats. For example, barcodes can include: polynucleotide barcodes; random nucleic acid and / or amino acid sequences; and synthetic nucleic acid and / or amino acid sequences. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and / or after sequencing of the sample. Barcodes can allow for identification and / or quantification of individual sequencing-reads.

[0086] The term “real time,” as used herein, can refer to a response time of less than about 1second, a tenth of a second, a hundredth of a second, a millisecond, or less. The response time may be greater than 1 second. In some instances, real time can refer to simultaneous or substantially simultaneous processing, detection or identification.

[0087] The term “subject,” as used herein, generally refers to an animal, such as a mammal(e.g., human, mouse, rat) or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, such as a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and / or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient. A subject can be a microorganism or microbe (e.g., bacteria, fungi, archaea, viruses).

[0088] The term “genome,” as used herein, generally refers to genomic information from asubject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information. A genome can be encoded either in DNA or in RNA. A genome can comprise coding regions (e.g., that code for proteins) as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome ordinarily has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome.Attorney Docket: 43487-1036601

[0089] The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be used synonymously. Anadaptor or tag can be coupled to a polynucleotide sequence to be “tagged” by any approach, including ligation, hybridization, or other approaches.

[0090] The term “sequencing,” as used herein, generally refers to methods and technologiesfor determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or Life Technologies (Ion Torrent®). Alternatively or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject. In some examples, such systems provide sequencing reads (also “reads” herein). A read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced. In some situations, systems and methods provided herein may be used with proteomic information.

[0091] The term “bead,” as used herein, generally refers to a particle. The bead may be asolid or semi-solid particle. The bead may be a gel bead. The gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and / or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductive, interactions, or physical entanglement. The bead may be a macromolecule. The bead may be formed of nucleic acid molecules bound together. The bead may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of a polymeric material. The bead may be magnetic or non-magnetic. The bead may be rigid. The bead may be flexible and / or compressible. The bead may be disruptable or dissolvable. The bead may be a solid particle (e.g., a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable.Attorney Docket: 43487-1036601

[0092] As used herein, the term “barcoded nucleic acid molecule” generally refers to anucleic acid molecule that results from, for example, the processing of a nucleic acid barcode molecule with a nucleic acid sequence (e.g., nucleic acid sequence complementary to a nucleic acid primer sequence encompassed by the nucleic acid barcode molecule). The nucleic acid sequence may be a targeted sequence or a non-targeted sequence. For example, in the methods and systems described herein, hybridization and reverse transcription of a nucleic acid molecule (e.g., a messenger RNA (mRNA) molecule) of a cell or nucleus with a nucleic acid barcode molecule (e.g., a nucleic acid barcode molecule containing a barcode sequence and a nucleic acid primer sequence complementary to a nucleic acid sequence of the mRNA molecule) results in a barcoded nucleic acid molecule that has a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or a reverse complement thereof). A barcoded nucleic acid molecule may serve as a template, such as a template polynucleotide, that can be further processed (e.g., amplified) and sequenced to obtain the target nucleic acid sequence. For example, in the methods and systems described herein, a barcoded nucleic acid molecule may be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the mRNA.

[0093] The term “sample,” as used herein, generally refers to a biological sample of asubject. The biological sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample can include one or more cells or nuclei. The sample can include one or more cells. The sample can include one or more nuclei. The sample can be a single cell. The sample can be a single nucleus. The sample can include one or more microbes. The biological sample may be a nucleic acid sample or protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The tissue sample may be a fresh tissue sample, a frozen tissue sample (e.g., flash frozen, lyophilized, cryo-sectioned, etc.), or a fixed tissue sample (e.g., a formalin-fixed and paraffin-embedded tissue sample). The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be a cell- free or cell free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.Attorney Docket: 43487-1036601

[0094] The term “biological particle,” as used herein, generally refers to a discrete biologicalsystem derived from a biological sample. The biological particle may be a macromolecule. The biological particle may be a small molecule. The biological particle may be a virus. The biological particle may be a cell or derivative of a cell. The biological particle may be an organelle. Examples of an organelle from a cell include, without limitation, a nucleus, a ribosome, a Golgi apparatus, an endoplasmic reticulum, a chloroplast, an endocytic vesicle, an exocytic vesicle, a vacuole, and a lysosome. The biological particle may be a rare cell from a population of cells. The biological particle may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The biological particle may be a constituent of a cell. The biological particle may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particle may be or may include a matrix (e.g., a gel or polymer matrix) comprising a cell or one or more constituents from a cell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The biological particle may be obtained from a tissue of a subject (e.g., a human, a mouse, a rat, or other mammal). The biological particle may be a hardened cell. Such hardened cell may or may not include a cell wall or cell membrane. The biological particle may include one or more constituents of a cell, but may not include other constituents of the cell. An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix, or cultured when comprising a gel or polymer matrix.

[0095] The term “macromolecular constituent,” as used herein, generally refers to amacromolecule contained within or from a biological particle. The macromolecular constituent may comprise a nucleic acid. In some cases, the biological particle may be a macromolecule. The macromolecular constituent may comprise DNA. The macromolecular constituent may comprise RNA. The RNA may be coding or non-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), for example. The RNA may be a transcript. The RNA may be small RNA that are less than 200 nucleic acid bases in length, or large RNA that are greater than 200 nucleic acid bases in length. Small RNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). The RNA may be double-stranded RNA or single-stranded RNA. The RNA may be circular RNA. TheAttorney Docket: 43487-1036601 macromolecular constituent may comprise a protein. The macromolecular constituent may comprise a peptide. The macromolecular constituent may comprise a polypeptide.

[0096] The term “molecular tag,” as used herein, generally refers to a molecule capable ofbinding to a macromolecular constituent. The molecular tag may bind to the macromolecular constituent with high affinity. The molecular tag may bind to the macromolecular constituent with high specificity. The molecular tag may comprise a nucleotide sequence. The molecular tag may comprise a nucleic acid sequence. The nucleic acid sequence may be at least a portion or an entirety of the molecular tag. The molecular tag may be a nucleic acid molecule or may be part of a nucleic acid molecule. The molecular tag may be an oligonucleotide or a polypeptide. The molecular tag may comprise a DNA aptamer. The molecular tag may be or comprise a primer. The molecular tag may be, or comprise, a protein. The molecular tag may comprise a polypeptide. The molecular tag may be a barcode.

[0097] The term “partition,” as used herein, generally, refers to a space or volume that maybe suitable to contain one or more species or conduct one or more reactions. A partition may be a physical compartment, such as a droplet or well. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and / or remote physical compartments. For example, a physical compartment may comprise a plurality of virtual compartments.

[0098] The term “solid medium,” as used herein, generally refers to a non-gaseous or non-liquid material. The solid medium may comprise a long chain hydrophobic compound, glycerin, gels, hydrogels, epoxy, resin, or any combination thereof. A long chain hydrophobic compound may include a wax, such as paraffin wax.

[0099] Provided herein are methods for sample processing and / or analysis. A method of thepresent disclosure may comprise barcoding one or more types of biomolecules (e.g., a nucleic acid molecule, a protein, a lipid, a carbohydrate, or a combination thereof). The biomolecule may be, for instance, a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) or a protein. Such a method may involve attaching one or more probes (e.g., nucleic acid probes) to the biomolecules and subsequently attaching a nucleic acid barcode molecule comprising a barcode sequence to the one or more probes. For example, the nucleic acid barcode molecule may attach to an overhanging sequence of a probe or to the end of a probe. Extension from an end of the probe to an end of the nucleic acid barcode molecule may form an extended nucleic acidAttorney Docket: 43487-1036601 molecule comprising both a sequence complementary to the barcode sequence and a sequence complementary to a target region of the nucleic acid molecule. The extended nucleic acid molecule may then be denatured from the nucleic acid barcode molecule and the nucleic acid molecule may be duplicated. One or more processes of the method may be carried out within a partition such as a droplet or well.

[0100] The present disclosure also provides a method of processing a sample (e.g., a cellsample or a tissue sample) that provides a barcoded nucleic acid molecule having linked probe molecules attached thereto. The method may comprise providing a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having a first and second target region; a first probe having a (i) first probe sequence that is complementary to the first target region and (ii) an additional probe sequence; and a second probe having a second probe sequence that is complementary to the second target region. In some instances, the first target region and the second target region are adjacent. The first and second probe sequences may also comprise first and second reactive moieties, respectively. Upon hybridization of the first probe sequence of the first probe to the first target region of the nucleic acid molecule, and hybridization of the second probe sequence of the second probe to the second target region of the nucleic acid molecule, the reactive moieties may be adjacent to one another. Subsequent reaction between the adjacent reactive moieties under sufficient conditions may link the first and second probes to yield a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule may also be referred to as a probe-ligated nucleic acid molecule. In other instances, the first target region and the second target region are not adjacent, and a nucleic acid reaction (e.g., a nucleic acid extension reaction, a gap-filling reaction) may be performed to yield a probe-linked nucleic acid molecule.

[0101] The probe-linked nucleic acid molecule may be barcoded with a barcode sequence ofa nucleic acid barcode molecule to provide a barcoded probe-linked nucleic acid molecule. Barcoding may be achieved by hybridizing a binding sequence of the nucleic acid barcode molecule to the additional probe sequence of the first probe of the probe-linked nucleic acid molecule. The barcoded probe linked-nucleic acid molecule may be subjected to amplification reactions to yield an amplified product comprising the first and second target regions and the barcode sequence or sequences complementary to these sequences. Accordingly, the method may provide amplified products without the use of reverse transcription. One or more processes may be performed within a partition such as a droplet or well.

[0102] The present disclosure also provides a method of generating barcoded, probe-linkednucleic acid molecules. The method may comprise providing a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having a first target region and a second target region; a firstAttorney Docket: 43487-1036601 probe having a first probe sequence that is complementary to the first target region and optionally an additional probe sequence; and a second probe having a second probe sequence that is complementary to the second target region. The additional probe sequence of the first probe may comprise a probe capture sequence. Alternatively, or in addition to, the second probe may comprise a probe capture sequence. The first probe sequence of the first probe may hybridize to the first target region of the nucleic acid molecule, generating a probe-associated nucleic acid molecule, and a nucleic acid reaction (e.g., a nucleic acid extension reaction using a polymerase or reverse transcriptase) may be performed to generate an extended nucleic acid molecule comprising a sequence complementary to the second target region. Prior to, during, or subsequent to the nucleic acid extension reaction, the second probe may hybridize to the nucleic acid molecule (or extended nucleic acid molecule, or complement thereof), and optionally, a nucleic acid extension reaction may be performed. The extended nucleic acid molecule may be barcoded, such as by (a) hybridization of a barcode binding sequence of the nucleic acid barcode molecule to the first probe (e.g., the additional probe sequence of the first probe) or the second probe (e.g., a probe capture sequence of the second probe), or (b) via a probe binding molecule (also referred to herein as a “splint molecule” or “splint oligonucleotide”), in which the probe binding molecule comprises (i) a probe binding sequence complementary to the additional probe sequence of the first probe (which may comprise the probe capture sequence) and / or a capture sequence of the second probe and a (ii) barcode binding sequence complementary to a sequence (e.g., a common sequence) of the barcode molecule. In some instances, the barcoding may be performed prior to hybridization of the second probe to the second target region. In such cases, the barcoded nucleic acid molecule may be subjected to conditions sufficient for hybridization of the second probe sequence of the second probe to the second target region of the nucleic acid molecule (or barcoded nucleic acid molecule). A nucleic acid reaction (e.g., nucleic acid extension) may be performed, thereby generating a barcoded, probe-linked nucleic acid molecule.

[0103] Another aspect of the present disclosure provides a method of barcoding multipleanalytes, such as the probe-linked nucleic acid molecules described herein, as well as other types of biomolecules (e.g., proteins). The method may comprise providing (i) a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having first and second target regions and (ii) a feature-binding moiety comprising a reporter oligonucleotide comprising a capture sequence; (iii) a first probe having a first probe sequence that is complementary to the first target region and an additional probe sequence; (iv) a second probe having a second probe sequence that is complementary to the second target region; and (v) a third probe having a third probe sequence that is complementary to a sequence of the reporter oligonucleotide. The first probe and theAttorney Docket: 43487-1036601 second probe may be subjected to conditions sufficient to hybridize to the first target region and the second target region, respectively, and to generate a probe-linked nucleic acid molecule. The third probe sequence of the third probe may be subjected to conditions sufficient to hybridize to the capture sequence of the reporter oligonucleotide, generating a probe-binding moiety complex. The probe-linked nucleic acid molecule and the probe-binding moiety complex may be subjected to conditions sufficient for barcoding, thereby generating a barcoded probe-linked nucleic acid molecule and a barcoded probe-binding moiety complex. The barcoded probe- linked molecule may be subjected to amplification reactions to yield an amplified product comprising the first and second target regions and the barcode sequence or sequences complementary to these sequences. The barcoded probe-binding moiety complex may similarly be subjected to amplification reactions to yield an amplified product comprising the fourth probe sequence and the barcode sequence. One or more processes may be performed within a cell bead and / or a partition, such as a droplet or well. Beneficially, the methods described herein may be useful in indexing cells, nuclei, or cell beads to partitions; such indexing may be useful in partitions occupied by more than one cell and identifying the cell, nucleus, cell bead or partition from which an analyte was derived. SNP / SNV Determination

[0104] Single nucleotide variants (SNVs) are the most common genetic variants andubiquitous in genomes and transcriptomes. The ability to identify and quantify SNVs in cells and tissue samples has the potential to significantly propel our understanding of biology. Transcriptomic SNV detection with current technologies is challenging due to the biochemistry involved. Some detection methods depend on the RNA-templated ligation of probes using a ligase. However, options of RNA-templated ligases are more limited (compared to DNA- templated ligases), and ligases used for RNA-templated ligation can suffer from ligation junction biases as well as poor ability to discriminate between matched and mismatched nucleotides at the ligation junctions.

[0105] Argonaute proteins are a large family of proteins that use nucleic acid guides to targetother nucleic acids and either bind or cut at a defined location in a target sequence in the target nucleic acid. Argonaute family members are derived from prokaryotic and eukaryotic organisms. Some Argonaute family members use RNA guides as guide nucleic acids. Some Argonaute family members use DNA guides as guide nucleic acids. Some Argonaute family members bind RNA. Some Argonaute family members bind and cut RNA. Some Argonaute family members bind, but do not cut, RNA. Some Argonaute family members bind DNA. Some Argonaute family members bind and cut DNA. Some Argonaute family members bind, but do not cut,Attorney Docket: 43487-1036601 DNA. Argonaute proteins that cut a target nucleic acid are said to have slicer activity. Not all Argonaute proteins have slicer activity; for example, Argonaute proteins involved in miRNA- mediated post-transcriptional regulation are slicer-dead (e.g., the Argonaute-guide nucleic acid binds, but does not cut, at the target sequence). While Argonaute proteins are endogenously involved in gene regulation and defense from pathogenic sequences, Argonaute proteins have been demonstrated to be useful tools for molecular biology.

[0106] In some aspects, slicer-dead (e.g., catalytically inert or nuclease-dead) Argonauteproteins in a complex with a nucleic acid molecule (e.g., a probe) binds at a SNP (e.g., a single nucleotide polymorphism) with better success than hybridization of free oligonucleotides not in a complex with an Argonaute protein. In some instances, probes in complex with nuclease- deficient Argonaute proteins bind with target sites faster than free oligonucleotides and also have a very low rate of off-target binding. For some Argonaute proteins, binding accuracy is due to the high sensitivity of the guide nucleic acid seed region (e.g., the seed region comprising nucleotides 2-8 at the 5’ end of the guide nucleic acid) to single-nucleotide mismatches. In some cases, the guide nucleic acid requires full sequence complementarity to the target strand throughout the seed region. For some Argonaute proteins (e.g., such as non-cutting Argonaute proteins involved in regulation of miRNAs), sequence complementarity of a supplementary 3’ region with the nucleic acid target is also required for successful binding of the Argonaute-guide nucleic acid complex in addition to complementarity of the seed sequence.

[0107] Certain methods for detecting variant sequences aim to discriminate a variantsequence (e.g., a particular SNP or point mutation) at the probe hybridization / ligation step. For instance, a first probe of a probe set with only one base difference in the probe region (e.g., at the 3’ or 5’ end of the probe) targeting a single nucleotide difference in target nucleic acids can compete with each other for hybridization to a target sequence. In some instances, a second probe of the probe set binds to a second target sequence in the target nucleic acid. The best matching first probe can outcompete the other probes, become more stably hybridized to a target molecule, and can then be ligated using a ligase to the second probe using the target sequence as a ligation template, thereby forming a probe-linked molecule (e.g., a ligated probe product). Alternatively, the first probe can be linked to a second probe using a gap-fill reaction, as described elsewhere herein, thereby forming a probe-linked molecule. A gap-fill reaction can comprise nucleic acid extension and ligation to link the first probe and the second probe. In certain embodiments, the probe-linked product or a derivative thereof is detected by various readout methods. In some instances, the probe linked molecule or a derivative thereof is sequenced to determine the variant sequence. In some instances, a sequence of the probe-linked molecule or a derivative thereof (e.g., with a complementary sequence of the ligated probe set) isAttorney Docket: 43487-1036601 determined by sequencing. In some aspects, the probe-linked molecule or a derivative thereof is detected in a single cell analysis platform, such as those that utilize partition barcoding schemes and those that utilize split-seq barcoding schemes, among others.

[0108] Assays using probe hybridization / ligation to discriminate variant sequences maysuffer from low specificity for multiple reasons, including properties of the ligase and / or the target nucleic acid. Low ligase fidelity can result in formation and detection of a ligation product even when the sequence of interest does not match the region where the probe binds the target sequence comprising a variant sequence, producing a high level of background or false positive results. For example, RNA templated ligases can tolerate some mismatches. In addition, ligases can have a strong base preference and probe end bias. Moreover, in the case of a probe set comprising a first and second probe, a first probe of the probe set is targeting a single nucleotide difference, while the second probe of the probe set covers a common region (e.g., a conserved region) among nucleic acid molecules containing different bases at the single nucleotide position of interest. As a result, on the one hand, the second probe can hybridize to the common region in a particular target nucleic acid molecule, but the first probe of the probe set binds to the particular target nucleic acid molecule at some rate even if it does not fully match the single nucleotide position in the particular target nucleic acid molecule. Due to low ligase fidelity on RNA templates, the nonspecifically hybridized first probes can nevertheless be ligated. Improved methods for analyzing nucleic acids present in a biological sample, such as for detecting a single nucleotide of interest are needed.

[0109] In some embodiments, provided herein are methods and compositions that reduceerrors in detecting a variant nucleotide or short variant sequence (e.g., an SNV) using an Argonaute / probe complex to detect a variant nucleotide or short variant sequence. As shown in FIG.54A, provided herein is an approach to detect a short variant nucleotide or sequence with a high level of specificity by performing detection using an Argonaute protein in complex with a first probe of a probe set, wherein the first probe has a first probe region complementary to the short variant nucleotide. In some embodiments, a second probe of the probe set binds to a second target sequence in the target nucleic acid (e.g., adjacent to the first target sequence comprising the variant sequence) and the first and second probes are ligated in the biological sample to form a ligated first-second probe product. In some embodiments, the Argonaute protein complex with the first probe of a probe set comprises a detectable label and / or a detectable region. In some embodiments, the Argonaute protein complex with the first probe of a probe set does not comprise a detectable label (e.g., a fluorophore). In some embodiments, the ligated first-second probe product is captured on an array and further processed for analysis. In some embodiments, the ligated first-second probe product is used to generate a nucleic acid molecule for downstreamAttorney Docket: 43487-1036601 sequencing. As shown in FIG.54B, provided herein is an approach to capture a short variant nucleotide or sequence with a high level of specificity by performing detection using an Argonaute protein in complex with a probe comprising (i) a probe region complementary to the short variant nucleotide in the target nucleic and a (ii) detection region.

[0110] In some embodiments, detection of a variant sequence with an Argonaute proteincomplex with the probe (e.g., probe or first probe of a probe set) is highly specific due to the requirement for exact sequence complementarity within the region of the first probe that binds to its target sequence. In some embodiments, within the probe region, at least one of the nucleotides at positions 2-8 from the 5’ end of the probe is complementary to a variant sequence. In some embodiments, nucleotides 6 and / or 7 of the region of the probe (e.g., first probe) comprise the interrogatory nucleotides. In some embodiments, most or all of the region of the probe (e.g., first probe) are complementary to the target nucleic acid in order for target recognition and binding of the Argonaute protein complex with the probe (e.g., first probe) to the target nucleic acid to occur.

[0111] In some embodiments, the methods and compositions herein can be used in singlecell based assays. In some embodiments, provided herein are methods and compositions that reduce false positive signals (e.g., false positive signals due to incorrect probe ligation) for the identification of variant sequences in cells or tissue samples (e.g., SNPs) using single cell based assays. In some embodiments, a method of SNV detection disclosed herein uses a probe set which is to generate a ligated first-second probe product comprising a sequence of a SNV or a complement thereof (or a hotspot region containing the SNV). In some embodiments, a method of SNV detection disclosed herein uses a probe set is used to detect a sequence of a SNV or a complement thereof (or a hotspot region containing the SNV), wherein binding of the probe to the target nucleic acid is detectable via a sequence in the detection region of the probe. A genetic hotspot can include a region of a nucleic acid in which a mutation is more likely occur. A hotspot mutation is such a mutation of in a genetic hotspot.

[0112] Argonaute-mediated hybridization of a probe to a target sequence may offer severaladvantages for accurate detection and discrimination of variant sequences such as single nucleotide polymorphisms (SNPs). Argonaute-mediated hybridization of a probe to a target nucleic acid may occur more rapidly than probe hybridization in the absence of an Argonaute protein. Requirements for complementarity of the binding region at the 5’ end of the probe in the complex with the Argonaute protein and / or 3’ supplementary region may provide more stringent matching criteria than hybridization, allowing for precise detection and discrimination of single- nucleotide differences within these regions of the probe (e.g., first probe of a probe set).Attorney Docket: 43487-1036601

[0113] Argonaute proteins can be nuclease-active (e.g., have slicer activity) or nuclease-deficient (e.g., lack slicer activity). In some embodiments, provided herein is a method comprising contacting a biological sample with a nuclease-deficient Argonaute protein and a first probe of a probe set, wherein the nuclease-deficient Argonaute protein and the first probe form a complex with a complementary target sequence in a target nucleic acid. In some embodiments, the nuclease-deficient Argonaute protein comprise a detectable moiety such as a fluorescent label. In some embodiments, the Argonaute protein complex with the first probe does not comprise a detectable moiety. In some embodiments, the method comprises detecting the Argonaute protein complex with the first probe bound to a variant sequence.

[0114] In some embodiments, the first target sequence in a target nucleic acidcomplementary to the first probe region is a variant sequence of interest (e.g., a single nucleotide variant or single nucleotide polymorphism). In certain embodiments, a complement of the variant sequence is in the ligated first-second probe product.

[0115] FIG. 54A shows scenarios in detecting single-nucleotide variants using probehybridization and ligation to discriminate among single nucleotides of interest. For instance, a first probe of a probe set in complex with a nuclease-deficient Argonaute protein hybridizes to an RNA sequence such that an interrogatory nucleotide in the first probe region (for example, X’ which is complementary to nucleotide X) is at a ligation junction and the hybridized first probe is ligated to a second probe of the probe pair. The first probe has an optional tail, sometimes referred to a 5’ tail, which may comprise one or more of the sequences described herein, such as, but not limited to, a barcode (1st barcode, 2nd barcode, 3rd barcode, etc.), a unique molecular identifier (UMI), a capture sequence, a primer binding sequence, a sequencing adaptor, etc. The second probe has an optional tail, sometimes referred to a 3’ tail, which may comprise one or more of the sequences described herein, such as, but not limited to, a barcode (1st barcode, 2nd barcode, 3rd barcode, etc.), a unique molecular identifier (UMI), a capture sequence, a primer binding sequence, a sequencing adaptor, etc. FIG.54B shows scenarios in detecting single- nucleotide variants using probe hybridization to discriminate among single nucleotides of interest. For instance, a probe with an overhang in complex with a nuclease-deficient Argonaute protein hybridizes to an RNA sequence such that an interrogatory nucleotide in the first probe region (for example, X’ which is complementary to nucleotide X) and the bound probe comprises a detection region. The left panel of FIG.54B depicts a probe with an overhang that does not hybridize to the target nucleic acid comprising a detection region for binding to a circular or circularizable probe for performing rolling circular amplification. In some aspects, the overhang that does not hybridize to the target nucleic acid comprises a detection region that is used as an additional probe target sequence, for which a first probe and a second probe (or anyAttorney Docket: 43487-1036601 subsequent numbering scheme) can target. Such first and second probes are described herein for identifying the presence of a sequence of interest in a cell, plurality of cells, or tissue sample. The right panel of FIG.54B depicts a probe with a loop detection region (that does not hybridize to the target nucleic acid) for binding to a circular or circularizable probe for performing rolling circular amplification. In some instances, the probe comprises a toehold region at the 3’ end of the probe that hybridizes to the target nucleic acid. In some examples, the toehold region is 3’ to the loop detection region. In some embodiments, the probe comprising the detection region is optionally ligated to an additional probe. In some aspects, the loop comprises a detection region that is used as an additional probe target sequence, for which a first probe and a second probe (or any subsequent numbering scheme) can target. Such first and second probes are described herein for identifying the presence of a sequence of interest in a cell, plurality of cells, or tissue sample.

[0116] An embodiment of a method for detecting a variant sequence of interest at a locationin a biological sample is shown in FIG.55. This embodiment comprises contacting the biological sample with molecules of first probes and second probes of probe sets, wherein some first probes have an interrogatory nucleotide in the first probe region (for example, X’) which is complementary to nucleotide X in the first target sequence in a target nucleic acid and some first probes have an interrogatory nucleotide in the first probe region (for example, Y’) which is complementary to alternative variant nucleotide Y in the first target sequence in a target nucleic acid. The various first probes for targeting variant sequences X and Y are added to the sample, and complexes with Argonaute proteins and first probes bind the corresponding variant sequence of interest. The variant sequence X or an alternative variant sequence Y when bound by a complex comprising an Argonaute protein with the matching first probes results in ligation to generate a ligated first-second probe product. The ligated first-second probe product comprises the complement sequence X’ complementary to the variant sequence of interest X or alternative complement sequence Y’ complementary to the alternative variant sequence Y. The ligated first-second probe product can be captured by a capture probe or capture oligonucleotide.

[0117] In some embodiments, the method presented herein comprises contacting a biologicalsample with an Argonaute protein in complex with a first probe of a probe set. In some embodiments, the method presented herein comprises contacting a biological sample with an Argonaute protein in complex with a probe that is not part of a probe set. In some embodiments, the probe (e.g., first probe) and the Argonaute protein form a complex prior to contacting the biological sample. In some embodiments, the probe (e.g., first probe) and the Argonaute protein form a complex in the biological sample. In some embodiments, the Argonaute protein in a complex with the probe (e.g., first probe) does not cut the target nucleic acid after binding to the target nucleic acid.Attorney Docket: 43487-1036601

[0118] In some aspects, the present application provides designs for probes (e.g., probe setscomprising and first probe and a second probe or a probe not in a probe set), wherein the probe is capable of forming DNA-RNA hybrid duplexes or RNA duplexes for binding the Argonaute protein enzyme to at least a portion of a target sequence in a target RNA nucleic acid. In some embodiments, the probe may be bind to the target nucleic acid with high sensitivity and allow for detection of the variant sequence with improved accuracy of localization and / or reduced false positive detection compared to detection methods using probes without Argonaute protein assistance. In some embodiments, the probe may be bind to the target nucleic acid with higher compared to detection methods using probes without Argonaute protein assistance.

[0119] In some embodiments, the probe or probe set (e.g., first probe and second probe)comprises RNA. In some embodiments, the probe or probe set (e.g., first probe and second probe) comprises DNA. In some embodiments, the probe or probe set (e.g., first probe and second probe) comprises cDNA. In some embodiments, the probe or probe set (e.g., first probe and second probe) comprises both DNA and RNA. In some embodiments, the probe or first probe is single-stranded. In some cases, the probe or first probe is a single-stranded DNA (ssDNA) oligonucleotide. In some embodiments, the probe or first probe comprises one or more synthetic nucleotides and / or one or more synthetic nucleosides. In some embodiments, the one or more synthetic nucleosides comprise bromodeoxyuridine (BrdU). In some embodiments, a first probe in a probe set is in a complex with a nuclease-deficient Argonaute protein. In some embodiments, the first probe hybridizes to a first target sequence in a target nucleic acid. The first probe can comprise a first probe region (e.g., a target-hybridizing region) that hybridizes to the first target sequence. The first probe can further comprise a non-target-hybridizing region that does not hybridize to the first target sequence. In some cases, a second probe in the probe set hybridizes to a second target sequence in the target nucleic acid. The second probe can comprise a target-hybridizing region that hybridizes to the second target sequence. The second probe can further comprise a non-target-hybridizing region that does not hybridize to the second target sequence. The non-target-hybridizing region of the first probe or the non-target-hybridizing region of second probe can comprise a detection region. In some cases, the detection region is used for binding to a circular or circularizable probe for performing rolling circular amplification. In some cases, the detection region is used as an additional probe target sequence that additional probes can target. The first probe can have a tail, sometimes referred to a 5’ tail, which may comprise one or more of the sequences described herein, such as, but not limited to, a barcode (1st barcode, 2nd barcode, 3rd barcode, etc.), a unique molecular identifier (UMI), a capture sequence, a primer binding sequence, a sequencing adaptor, etc. The second probe can have a tail, sometimes referred to a 3’ tail, which may comprise one or more of the sequencesAttorney Docket: 43487-1036601 described herein, such as, but not limited to, a barcode (1st barcode, 2nd barcode, 3rd barcode, etc.), a unique molecular identifier (UMI), a capture sequence, a primer binding sequence, a sequencing adaptor, etc. In some cases, the first probe comprises an interrogatory nucleotide in a first probe region at a ligation junction, and the hybridized first probe is ligated to the second probe of the probe set.

[0120] In some embodiments, the probe set comprises at least one barcode that as a partition-specific barcode. A partition-specific barcode is one that identifies the probe sets of that specific partition, and in instances of a plurality of partitions, each partition will have a different barcode. The partition specific barcode can be designed as a part of the sequence of the first probe or the second probe, or it may be introduced as an adaptor that is added to the probe set, or it may be introduced via an oligonucleotide sequence that comprises the barcode and a capture sequence that hybridizes to (captures) the probe set via a complementary capture sequence, and an amplification / extension reaction is performed to create a subsequent molecule that comprises the probe set (or a complement thereof) and the partition-specific barcode (or a complement thereof). This subsequent molecule can then be subjected to sequence determination.

[0121] In some embodiments, the probe set comprises at least one probe set-specific barcodesequence that identifies the particular probe pair for a given target sequence or target sequences. In some aspects, the probe set comprises at least one sample-specific barcode sequence that allows for multiplex analysis of multiple samples at the same time (samples can be pooled together and analyses as described herein). After the addition or integration of the probe set- specific barcode sequence or the sample-specific barcode sequence with the partition-specific barcode sequence, the sequence determination process, such as sequencing, can be demultiplexed to determine the specific sample / probe set / partition associated with the cell or cells of interest.

[0122] The present disclosure describes at least a first probe and a second probe, and itfurther describes a left hand sequence (LHS) and a right hand sequence (RHS). These may be used interchangeably. In some aspects, the LHS, or the first prove, or the probe found closest to the 5’ side of the target strand comprising the target region(s) is where SNP detection is occurring. In some aspects, the probe pair comprises a RHS with an overhang on the 3’ end that does not hybridize to a target sequence on the strand of interest, and further comprises a LHS that does not have an overhang on the 5’ end of the probe that does not hybridize to a target sequence on the strand of interest. In some aspects, the LHS is where SNP detection occurs. In some aspects, the LHS has an overhang on the 3’ end of the probe to allow for ligation to occur with the 5’ end of the RHS. In some aspects, the LHS with the overhang on the 3’ end of the probe comprises an overhang of only 1 or 2 nucleic acid residues that do not hybridize to theAttorney Docket: 43487-1036601 target sequence of the target region. In some aspects, the LHS probe or the first probe is in a complex with a nuclease-deficient Argonaute protein.

[0123] In some embodiments, the probe or first probe is an RNA molecule, and theArgonaute protein is an RNA-guided Argonaute. In some embodiments, the probe or first probe is a DNA molecule, and the Argonaute protein is a DNA-guided Argonaute. In some embodiments, the first probe comprises a first probe region that binds to a first target sequence in a target nucleic acid. In some embodiments, the first probe comprises a 5’-phosphate or a 5’- OH. In some embodiments, the probe or first probe is at least about 5, at least about 8, at least about 10, at least about 12, at least about 15, at least about 20, or at least about 30 nucleotides in length. In some embodiments, the probe or first probe is between about 10 and about 30, about 15 and about 25, about 14 and about 20, about 16 and about 20, about 20 and about 30 nucleotides, or about 25 and about 35 nucleotides in length. In some embodiments, the first probe region of the first probe is at least about 5, at least about 8, at least about 10, at least about 12, at least about 15, at least about 20, or at least about 30 nucleotides in length. In some embodiments, the first probe region is between about 10 and about 30, about 15 and about 25, about 14 and about 20, about 16 and about 20, about 20 and about 30 nucleotides, or about 25 and about 35 nucleotides in length. In some embodiments, the first probe is 10 to 35 nucleotides in length, 20 to 35 nucleotides in length, 20 to 31 nucleotides in length, 20 to 25 nucleotides in length, 25-35 nucleotides in length, or 26 to 31 nucleotides in length. In some embodiments, the first probe is 20 to 30 nucleotides in length. In some embodiments, the first probe is 20 to 25 nucleotides in length. In some embodiments, the first probe is 26 to 31 nucleotides in length. In some embodiments, the first probe region of the first probe is fully complementary to the first target sequence. In some embodiments, the first probe region of the first probe is partially complementary to the guide target sequence. In some embodiments, the first probe region of the first probe is at least about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 95, or about 100% complementary to the first target sequence.

[0124] In some embodiments, the 5’ end of the first probe comprises a modification. In someembodiments, the first probe comprises a modified 5’ residue. In some embodiments, the modified 5’ residue is advantageous for the first probe region to bind to target nucleic acids or for the first probe to have binding stability over a nucleic acid comprising a non-modified 5’ residue. In some embodiments, the 5’ end of the first comprises a phosphoryl group. In some embodiments, the 5’ end of the first probe comprises a hydroxyl group.

[0125] In some embodiments, the first probe or second probe comprises one or moremodified synthetic nucleoside analogues and / or one or more modified synthetic nucleotides. In some embodiments, the 5’ end of the probe comprises a modified synthetic nucleoside analogue.Attorney Docket: 43487-1036601 In some embodiments, the 5’ end of the probe comprises a bromodeoxyuridine (BrdU) nucleoside. In some embodiments, the 5’-BrdU nucleoside increases binding stability of the probe compared to the binding stability of a probe that does not comprise a 5’-BrdU (e.g., a first probe with the same sequence of bases, but without BrdU.

[0126] In some embodiments, the probe (e.g., first probe) comprises a sequencecomplementary to a variant sequence of a target nucleic acid, such as a variant sequence of interest or an alternative sequence. FIG.55 shows an example first probe with a nucleotide X’ or Y’ complementary to the variant sequence of interest X or Y. In some embodiments, the probe region is located central in the probe for detecting the variant sequence of interest (e.g., at nucleotide positions 2-8 from the 5’ end of the probe). In some embodiments, the first probe region is located central in the first probe. In some embodiments, the first probe region comprises nucleotide positions 2-8 of the first probe. In some embodiments, the first probe region of the first probe comprises one or more nucleotides between positions 2 and 8 from the 5’ end of the first probe. In some embodiments, the first probe region binds to the variant sequence in the first target sequence at an internal position of the first probe. In some embodiments, the first probe region binds to the variant sequence in the first target sequence at least 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from the 5’ or 3’ end of the first probe. In some embodiments, the first probe region comprises a 6-7 nucleotide region. In some embodiments, the first probe region binds to the variant sequence in the first target sequence at 6-7 nucleotides of the first probe. In some embodiments, the first probe region binds to the variant sequence in the first target sequence at the 3’ end of the first probe.

[0127] In some embodiments, the probe that comprises a sequence complementary to avariant sequence of a target nucleic acid, such as a variant sequence of interest, is between about 14 and 35 nucleotides in length. In some embodiments, the first probe is between about 14 and 20 nucleotides in length. Optionally, in some embodiments the first probe is between about 16 and 20 nucleotides in length. Optionally, in some embodiments, the first probe and / or the second probe of the probe set is between 20 and 35 nucleotides in length. In some embodiments, the first probe and the second probe of the probe set is contacted with the biological sample simultaneously and the Argonaute protein and the first probe are bound in a complex before contacting the biological sample.

[0128] In some embodiments, the Argonaute protein and the probe that comprises a sequencecomplementary to a variant sequence of a target nucleic acid are bound in a complex before contacting the biological sample. In some embodiments, the first probe and the second probe of the probe set is contacted with the biological sample separately, and the Argonaute protein and the first probe are bound in a complex before contacting the biological sample. In someAttorney Docket: 43487-1036601 embodiments, the Argonaute protein and the first probe are contacted the biological sample before the second probe is contacted with the biological sample. In some embodiments, the Argonaute protein and the first probe are contacted the biological sample after the second probe is contacted with the biological sample.

[0129] In some embodiments, the second probe comprises a capture region and a secondprobe region that hybridizes to a second target sequence in a target nucleic acid. In some embodiments, the capture region can be found on the first probe. In some instances, the capture region of the second probe does not hybridize to a sequence of the target nucleic acid (e.g., is in an overhang of the first or second probe as shown in FIG.54A and FIG.54B). In some embodiments, the capture region of the first probe or second probe is or includes a sequence that is complementary to a particular capture domain present in a capture probe or present in a capture oligonucleotide, such as an oligonucleotide associated with a support that may comprise a plurality of such oligonucleotides. In some embodiments, the capture region includes a poly- uridine sequence, a poly-thymidine sequence, or a combination thereof. In some embodiments, the capture region of the first probe or the second probe includes a poly(A) sequence or a complement thereof. In some embodiments, the poly(A) sequence or a complement thereof is on the 3’ end of the second probe. In some embodiments, the capture region is at the 3’ end of the second probe. In some embodiments, the second probe comprises from 5’ to 3’, a second probe region and a capture region. In some embodiments, the poly(A) sequence or a complement thereof is on the 5’ end of the first probe. In some embodiments, the capture region is at the 5’ end of the first probe. In some embodiments, the capture region is for binding to a capture domain of a capture probe on a substrate (e.g., the capture array). In some embodiments, the first probe or the second probe includes a degenerate sequence (e.g., a UMI sequence). For example, in some embodiments the capture region of the first probe or the second probe includes a UMI sequence. In some embodiments, the UMI sequence is specific to a particular target (e.g., a particular SNV). In some embodiments, the second probe additionally or alternatively includes a functional sequence. In some embodiments, the functional sequence is a sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence) or a primer sequence / primer binding site (e.g., a sequencing primer sequence). In some embodiments, the first probe or the second probe includes a primer-binding site, such as for next-generation sequencing (NGS). The primer- binding site can include all or a part of a sequencing primer sequence (e.g., R1 or a partial R1 (“pR1”), R2, etc.), or combinations thereof. In some embodiments, the capture region of the first probe or the second probe includes the functional sequence (e.g., primer-binding site).

[0130] In some embodiments, the second probe comprises a sequence complementary to asecond target sequence of the target nucleic acid. FIG.55 shows an example second probe thatAttorney Docket: 43487-1036601 binds to a second target sequence that is adjacent to the first target sequence bound by the first probe in a complex with the Argonaute protein. In some embodiments, the second probe is between about 5 and 50 nucleotides in length. In some embodiments, the second probe is between about 10 and 50 nucleotides in length. In some embodiments, the second probe is between about 10 and 40 nucleotides in length. In some embodiments, the second probe is between about 10 and 30 nucleotides in length. In some embodiments, the second probe is between about 10 and 20 nucleotides in length. In some embodiments, the first probe and the second probe of the probe set is contacted with the biological sample simultaneously. In some embodiments, the first probe and the second probe of the probe set is contacted with the biological sample separately, and the Argonaute protein and the first probe are contacted first with the biological sample before the second probe is contacted with the biological sample. In some embodiments, the Argonaute protein and the first probe are contacted first with the biological sample before the second probe is contacted with the biological sample, wherein the second probe binds to the target nucleic acid under conditions in which the Argonaute protein and first probe remain bound to the target nucleic acid. In some embodiments, the first probe and the second probe of the probe set is contacted with the biological sample in the same buffer. In some embodiments, the first probe and the second probe of the probe set is contacted with the biological sample in different buffers.

[0131] In some embodiments, the method presented herein further comprises contacting thetarget nucleic acid in the biological sample with an Argonaute protein in complex with a probe (e.g., first probe) and the Argonaute protein does not have cutting activity (e.g., an Argonaute that is slicer-dead). Any suitable Argonaute protein for binding a nucleic acid in a nucleic acid duplex without cutting can be used. Generally, Argonaute proteins contain 6 main domains (N- terminal, L1 (Linker 1), PAZ (Piwi-Argonaute-Zwille), L2 (Linker 2), MID (Middle) and PIWI (P-element induced wimpy testis) responsible for binding of a guide nucleic acid and recognition of a target sequence. In some embodiments, the Argonaute protein is an RNA-guided Argonaute, and the probe (e.g., used as a guide nucleic acid) is an RNA molecule. In some embodiments, the Argonaute protein is a DNA-guided Argonaute, and the probe (e.g., used as a guide nucleic acid) is a DNA molecule.

[0132] In some embodiments, the Argonaute protein is a naturally-occurring protein (e.g.,naturally occurs in prokaryotic or eukaryotic cells). In some embodiments, the Argonaute protein is not a naturally-occurring protein (e.g., a variant or mutant protein). In some embodiments, the Argonaute protein is a recombinant protein. In some embodiments, the Argonaute protein is genetically engineered (such as an Argonaute protein described in WO 2019 / 222036, which is hereby incorporated by reference in its entirety). In some embodiments, the Argonaute protein isAttorney Docket: 43487-1036601 a slicer-dead Argonaute protein, meaning that it lacks cutting activity or is nuclease-dead. In some embodiments, the Argonaute protein has been modified (e.g., genetically engineered or mutated) to lack cutting activity. In some embodiments, lacking cutting activity means that the Argonaute protein is not capable of cutting a target nucleic acid. In some embodiments, lacking cutting activity means that the Argonaute protein does not cut the target nucleic acid. In some embodiments, an Argonaute protein that naturally lacks cutting activity or that has been modified to lack cutting activity is a slicer-dead Argonaute.

[0133] In some embodiments, the Argonaute protein is a eukaryotic Argonaute protein.Generally, eukaryotic Argonaute proteins can mediate binding of a target RNA with a guide nucleic acid of RNA. In some embodiments, an Argonaute protein is of plant, algal, fungal (e.g., yeast), or animal (e.g., human, rodent, fruit fly, cnidarian, echinoderm, nematode, fish, amphibian, reptile, bird, etc.) origin. In some embodiments, the Argonaute protein is a eukaryotic Argonaute protein that has been modified to lack cutting activity.

[0134] In some embodiments, the Argonaute protein is a slicer-dead Ago1, Ago2, Ago3,Ago4, PIWI 1, PIWIL 2, PIWI 3, or PIWI 4 (such as the Argonaute proteins described in WO 2007 / 048629, which is hereby incorporated by reference in its entirety). In some embodiments, the Argonaute protein is Ago2. In some embodiments, the Ago2 is Drosophila Ago2. In some embodiments, the Argonaute protein is a recombinant Drosophila Argonaute protein. In some embodiments, the Argonaute protein is expressed in a mammalian cell line. In some embodiments, the Argonaute protein is a Drosophila Argonaute protein expressed in a mammalian cell line. In some embodiments, a Drosophila Argonaute protein is expressed using a method such that a loading complex specific to Drosophila species is not provided to obtain guide-free proteins. In some embodiments, the Argonaute protein is a purified recombinant Drosophila Argonaute protein. In some embodiments, the Argonaute protein is expressed in an insect cell line, such as a Schneider 2 (S2) cell line. In some embodiments, the Argonaute protein is a Drosophila Argonaute protein expressed in an insect cell line, such as a S2 cell line. In some embodiments, the Drosophila Argonaute protein is loaded with the first probe prior to contacting the biological sample.

[0135] In some embodiments, the slicer-dead Argonaute protein is a eukaryotic Argonauteprotein from a mammalian organism. In some embodiments, the mammalian Argonaute protein is selected from mammalian AGO1, AGO2, AGO3, and AGO4. In some embodiments, the mammalian Argonaute protein is a human Argonaute protein. In some embodiments, the human Argonaute protein is a human AGO1 or AGO4 protein which naturally lacks slicer activity (See Faehnle et al. The making of a slicer: activation of a human Argonaute-1. Cell Reports 2015 June 27, 3(6): 1901-1909, which is hereby incorporated by reference in its entirety). In someAttorney Docket: 43487-1036601 embodiments, the human Argonaute protein is a human AGO2 protein that has been modified to lack slicer activity (See McGeary et al., The Biochemical Basis of microRNA Targeting Efficacy. Science 2019 Dec 20; 366(6472): eaav1741., which is hereby incorporated by reference in its entirety). In some embodiments, the human Argonaute protein is a human AGO3 protein that has been modified to lack slicer activity.

[0136] In some embodiments, the Argonaute protein is used as described herein for binding atarget sequence (e.g., comprising the variant sequence) at a temperature at which slicer activity of the Argonaute protein is not active. In some examples, an Argonaute protein derived from Thermus thermophilus (dTtAgo) is used to bind a target sequence at about 30ºC. (See Shin et al, “Quantification of purified endogenous miRNAs with high sensitivity and specificity.” Nature Commun 11:6033 (2020), which is herein incorporated by reference in its entirety).

[0137] In some embodiments, the slicer-dead Argonaute protein is a prokaryotic Argonauteprotein or a variant thereof. Generally, prokaryotic Argonaute proteins can mediate binding of a target RNA with a guide oligonucleotide. In some cases, the prokaryotic Argonaute protein uses RNA as a guide oligonucleotide. In some cases, the prokaryotic Argonaute protein uses DNA as a guide oligonucleotide. In some embodiments, the slicer-dead Argonaute protein is a prokaryotic Argonaute protein that has been modified to lack cutting activity.

[0138] In some embodiments, the slicer-dead Argonaute protein is a modified Nitratireductor(optionally Nitratirereductor sp. XY-223), Enhydrobacter (optionally Enhydrobacter aerosaccus), Mesorhizobium (optionally Mesorhizobium sp. CNPSo 3140), Hyphomonas (optionally Hyphomonas sp. T16B2), Pseudooceanicola (optionally Pseudooceanicola lipolyticus), Tateyamaria (optionally Tateyamaria omphalii), Bradyrhizobium (optionally Bradyrhizobium sp. ORS 3257), Dehalococcoides (optionally Dehalococcoides mccartyi), Chroococcidiopsis (optionally Chroococcidiopsis cubana), Runella (optionally Runella slithyformis), Roseivirga (optionally Rosevirga seohaensis), Spirosoma (optionally Spirosoma endophyticum), Pedobacter (optionally Pedobacter yonginense, Pedobacter insulae, or Pedobacter nyackensis), Planctomycetes bacterium (optionally Planctomycetes bacterium TBK1r or Planctomycetes bacterium V6), Dyadobacter (optionally Dyadobacter sp. QTA69), Mucilaginibacter (optionally Mucilaginibacter gotjawali, Mucilaginibacter polytichastri or Mucilaginibacter paludis), Hydrobacter (optionally Hydrobacter penzbergensis), Chitinophaga (optionally Chitinophaga costaii), Cytophagaceae bacterium (optionally Cytophagaceae bacterium SJW1-29), Emticicia (optionally Emticicia oligotrophica), Runella (optionally Runella sp. YX9), or Spirosoma (optionally Spirosoma pollinicola) Argonaute protein (See Li et al., “A programmable pAgo nuclease with RNA target preference from the psychrotolerant bacterium Mucilaginibacter paludis” Nucleic Acids Res.2022 May 20;50(9):5226-5238; Lisitskaya et al., “ProgrammableAttorney Docket: 43487-1036601 RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity.” Nat Commun.2022 Aug 8;13(1):4624; Sun et al., “An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.” Acta Biochim Biophys Sin (Shanghai).2022 May 25;54(5):686-695; U.S. Patent No. 10,253,311; US15 / 089,243; US17 / 575,957; US17 / 854,897; and WO 2022 / 222920 each of which is herein incorporated by reference in their entireties) that has been modified to lack cutting activity. In some embodiments, the slicer-dead Argonaute protein is from Thermus thermophilus. In some embodiments, the slicer-dead Argonaute protein is from Marinitoga piezophile (See Lapinaite et al, “Programmable RNA recognition by a CRISPR-associated Argonaute.” PNAS 2018 Mar 27;115(13):3368-3373, which is herein incorporated by reference in its entirety). In some embodiments, the slicer-dead Argonaute protein is from Rhobacter sphaeroidis (See Miyoshi et al, “Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.” Nature Comm 2016; 7: 11846, which is herein incorporated by reference in its entirety). In some embodiments, the slicer-dead Argonaute protein is from Thermomyces thermophilus (such as an Argonaute protein described in patent application no. CN202210082114, the content of which is herein incorporated by reference in its entirety). In some embodiments, the slicer-dead Argonaute protein is from Vanderwaltozyma polyspora (also known as Kluyveromyces polysporus) (such as an Argonaute protein described in WO 2018 / 112336, the content of which is herein incorporated by reference in its entirety). In some embodiments, the slicer-dead Argonaute protein is a modified Argonaute protein from Clostridium perfringens (CpAgo) or an Argonaute protein from Intestinibacter bartlettii (IbAgo) that lacks cutting activity (See Cao et al, Argonaute proteins from human gastrointestinal bacteria catalyze DNA-guided cleavage of single- and double-stranded DNA at 37 C. Cell Discovery 20195(38), which is hereby incorporated by reference in its entirety). In some embodiments, the slicer-dead Argonaute protein is a modified Argonaute protein from Clostridium butyricum (CbAgo) that lacks cutting activity (See Hegge et al. DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute. BioRXIV 2019, https: / / doi.org / / 10.1101 / 534206 and Kuzmenko et al. Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea. BioRXIV 2019, https: / / doi.org / / 10.1101 / 558684, both of which are hereby incorporated by reference in their entirety).

[0139] In some embodiments, the slicer-dead Argonaute protein is a variant of a DNA-binding Argonaute protein. In some embodiments, the Argonaute is a DNA-guided Pyrococcus furiosus (PfAgo) that binds single- and / or double-stranded DNA (See Swarts et al, “Argonaute of the archaeon Pyrocuccus furiosus is a DNA-guided nuclease that targets cognate DNA.”Attorney Docket: 43487-1036601 Nucleic Acids Research Volume 43, Issue 10, 26 May 2015, Pages 5120–5129, which is herein incorporated by reference in its entirety). In some embodiments, the Argonaute protein has been modified to lack cutting activity of an RNA substrate via selection and / or directed evolution.

[0140] In some embodiments, the slicer-dead Argonaute protein comprises one or moreamino acid substitutions compared to any of the species of Argonaute protein described herein. In certain embodiments, the one or more amino acid substitutions are conservative substitutions. In some aspects, conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK’s of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g., US8562989; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 8910915- 10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

[0141] An amino acid substitution may include replacement of one amino acid in apolypeptide with another amino acid. Amino acid substitutions may be introduced to generate a modified Argonaute protein as described herein.

[0142] Amino acids generally can be grouped according to the following common side-chainproperties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0143] In some contexts, conservative substitutions can involve the exchange of a member ofone of these classes for another member of the same class. In some contexts, non-conservativeAttorney Docket: 43487-1036601 amino acid substitutions can involve exchanging a member of one of these classes for another class. In some contexts, particular substitutions can be considered “conservative” or “non- conservative” depending on the stringency and context and environment of the particular residue in primary, secondary and / or tertiary structure of the protein.

[0144] In some embodiments, the Argonaute is nuclease-deficient (e.g., lacks slicer activity).In some embodiments, the Argonaute is not capable of cutting the target nucleic acid. In some embodiments, the Argonaute protein does not cut the target nucleic acid. In some embodiments, the Argonaute protein is an Argonaute protein that lacks slicer activity. In some embodiments, the Argonaute protein is an Argonaute protein that has been modified (e.g., selectively mutated) to lack slicer activity. In some embodiments, the modified, nuclease-deficient Argonaute protein comprises one or more inactivating mutations in a PIWI and / or PAZ domain of the Argonaute protein.

[0145] In some embodiments, the slicer-dead Argonaute protein is an RNA-guidedArgonaute, and the first probe is an RNA molecule. In some embodiments, the slicer-dead Argonaute protein is a eukaryotic Argonaute protein. In some embodiments, the slicer-dead Argonaute protein is a DNA-guided Argonaute, and the first probe is a DNA molecule. In some embodiments, the slicer-dead Argonaute protein is a prokaryotic Argonaute protein. In some embodiments, the first probe comprises a 5’-phosphate or a 5’-OH. In some embodiments, the nuclease-deficient Argonaute protein is Ago1, Ago3, or Ago4. In some embodiments, the nuclease-deficient Argonaute protein is a Drosophila Argonaute protein or a derivative or variant thereof. In some embodiments, the nuclease-deficient Argonaute protein is a nuclease-deficient Argonaute derived from Thermus thermophilus (dTtAgo).

[0146] In some embodiments, the nuclease-deficient Argonaute is a nuclease-deficientArgonaute derived from Marinitoga piezophile (MpAgo). In some embodiments, the nuclease- deficient Argonaute is a MpAgo that has been additionally modified to lack slicer activity. In some embodiments, the MpAgo protein forms a complex with a 5’-hydroxylated first probe to form a MpAgo-guide nucleic acid complex. In some embodiments, the first probe in complex with the MpAgo protein comprises a 5’-BrdU (e.g., as described in Lapinaite et al., “Programmable RNA recognition using a CRISPR-associated Argonaute”, PNAS 2018) for increased binding stability the complex. In some embodiments, nucleotides 5-15 of the first probe in complex with a MpAgo protein has full complementarity to the target nucleic acid sequence. In some embodiments, the irst probe in complex with a MpAgo protein is used to detect a SNV at the position of the target RNA that is complementary to nucleic acids 6 or 7 of first probe. In some embodiments, the first probe in complex with a MpAgo protein detects nucleotides in the target RNA at positions complementary to nucleotides 6-7 of the first probe.Attorney Docket: 43487-1036601 In some embodiments, the method disclosed herein comprises detecting a probe assisted by an Argonaute protein bound to a target sequence comprising a variant sequence. In some embodiments, the method disclosed herein comprises detecting a generated ligated first-second probe product or a sequence thereof. In some embodiments, the biological sample is contacted with a plurality of nuclease-deficient Argonaute and first probe complexes wherein the Argonaute-proteins comprise distinct first probes with distinct first probe regions corresponding to a plurality of variant sequences, and each different Argonaute in a complex with a first probe comprises a different detectable label corresponding to the first probe. In some embodiments, the binding of the Argonaute and first probe complex with the variant sequence enables detection of the variant sequence in the biological sample. In some embodiments, the binding of the Argonaute and first probe complex with the variant sequence generates a ligated first-second probe product that is further processed for downstream analysis. In some embodiments, the Argonaute protein remains bound to the target sequence during the ligation of the first probe to the second probe. In some embodiments, the method disclosed herein comprises detecting a probe assisted by an Argonaute protein bound to a target sequence comprising a variant sequence. Fixed Samples

[0147] A sample may be a fixed sample. For example, a sample may comprise a plurality offixed samples, such as a plurality of fixed cells or fixed nuclei. In some aspects, the fixed sample comprises a plurality of cells or nuclei. In some aspects, the fixed sample comprises a plurality of cells. In some aspects, the fixed sample comprises a plurality of nuclei. In some aspects, the fixed sample is a single fixed cell or fixed nucleus. In some aspects, the biological sample is a single fixed cell. In some aspects, the biological sample is a single fixed nucleus.

[0148] Alternatively, or in addition, a sample may comprise a fixed tissue. Fixation of cellor cellular constituent, or a tissue comprising a plurality of cells or nuclei, may comprise application of a chemical species or chemical stimulus. The term “fixed” as used herein with regard to biological samples generally refers to the state of being preserved from decay and / or degradation. “Fixation” generally refers to a process that results in a fixed sample, and in some instances can include contacting the biomolecules within a biological sample with a fixative (or fixation reagent) for some amount of time, whereby the fixative results in covalent bonding interactions such as crosslinks between biomolecules in the sample. A “fixed biological sample” may generally refer to a biological sample that has been contacted with a fixation reagent or fixative. For example, a formaldehyde-fixed biological sample has been contacted with the fixation reagent formaldehyde. “Fixed cells”, “fixed nuclei” or “fixed tissues” refer toAttorney Docket: 43487-1036601 cells / nuclei or tissues that have been in contact with a fixative under conditions sufficient to allow or result in the formation of intra- and inter-molecular covalent crosslinks between biomolecules in the biological sample. Generally, contact of biological sample (e.g., a cell or nucleus) with a fixation reagent (e.g., paraformaldehyde or PFA) results in the formation of intra- and inter-molecular covalent crosslinks between biomolecules in the biological sample. In some cases, the fixation reagent, formaldehyde, may result in covalent aminal crosslinks within RNA, DNA, and / or protein molecules. For example, the widely used fixative reagent, paraformaldehyde or PFA, fixes tissue samples by catalyzing crosslink formation between basic amino acids in proteins, such as lysine and glutamine. Both intra-molecular and inter-molecular crosslinks can form in the protein. These crosslinks can preserve protein secondary structure and also eliminate enzymatic activity in the preserved tissue sample. Examples of fixation reagents include but are not limited to aldehyde fixatives (e.g., formaldehyde, also commonly referred to as “paraformaldehyde,” “PFA,” and “formalin”; glutaraldehyde; etc.), imidoesters, NHS (N- Hydroxysuccinimide) esters, and the like.

[0149] In some embodiments, the fixative or fixation reagent useful for fixing samples isformaldehyde. The term “formaldehyde” when used in the context of a fixative may also refer to “paraformaldehyde” (or “PFA”) and “formalin”, both of which are terms with specific meanings related to the formaldehyde composition (e.g., formalin is a mixture of formaldehyde and methanol). Thus, a formaldehyde-fixed biological sample may also be referred to as formalin- fixed or PFA-fixed. Protocols and methods for the use of formaldehyde as a fixation reagent to prepare fixed biological samples can be used in the methods and compositions of the present disclosure. For example, suitable ranges of formaldehyde concentrations for use in preparing a fixed biological sample is 0.1 to 10%, 1-8%, 1-4%, 1-2%, 3-5%, or 3.5-4.5%. In some embodiments of the present disclosure the biological sample is fixed using a final concentration of 1% formaldehyde, 4% formaldehyde, or 10% formaldehyde. The formaldehyde can be diluted from a more concentrated stock solution – e.g., a 35%, 25%, 15%, 10%, 5% PFA stock solution.

[0150] Other examples of fixatives include, for example, organic solvents such as alcohols(e.g., methanol or ethanol), ketones (e.g., acetone), and aldehydes (e.g., paraformaldehyde, formaldehyde (e.g., formalin), or glutaraldehyde). As described herein, cross-linking agents may also be used for fixation including, without limitation, disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA), dithio-bis(- succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In some cases, a cross-linking agent may be a cleavable cross-linking agent (e.g., thermally cleavable, photocleavable, etc.).Attorney Docket: 43487-1036601

[0151] In some cases, more than one fixation reagent can be used in combination whenpreparing a fixed biological sample. For example, a first fixation agent, such as an organic solvent, may be used in combination with a second fixation agent, such as a cross-linking agent. The organic solvent may be an alcohol (e.g., ethanol or methanol), ketone (e.g., acetone), or aldehyde (e.g., paraformaldehyde, formaldehyde, or glutaraldehyde). The cross-linking agent may be selected from the group consisting of disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA), dithio-bis(- succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS). In some cases, a first fixation agent may be provided to or brought into contact with the cell or nucleus to bring about a change in a first characteristic or set of characteristics of the cell / nucleus, and a fixation agent may be provided to or brought into contact with the cell or nucleus to bring about a change in a second characteristic or set of characteristics of the cell or nucleus. For example, a first fixation agent may be provided to or brought into contact with a cell or nucleus to bring about a change in a dimension of the cell (e.g., a reduction in cross-sectional diameter, see, e.g., U.S. Pat. Pub. No.2020 / 0033237, which is incorporated herein by reference in its entirety), and a second fixation agent may be provided to or brought into contact with a cell or nucleus to bring about a change in a second characteristic or set of characteristics of the cell (e.g., forming crosslinks within and / or surrounding the cell or nucleus). The first and second fixation agents may be provided to or brought into contact with the cell or nucleus at the same or different times. Other suitable fixing agents include those disclosed in, e.g., International PCT App. No. PCT / US2020 / 066705, which is incorporated herein by reference in its entirety.

[0152] In an example, a first fixation agent that is an organic solvent may be provided to acell to change a first characteristic (e.g., cell size) and a second fixation agent that is a cross- linking agent may be provided to a cell to change a second characteristic (e.g., cell fluidity or rigidity). The first fixation agent may be provided to the cell before the second fixation agent.

[0153] In another embodiment, biomolecules (e.g., biological samples such as tissuespecimens) are contacted with a fixation reagent containing both formaldehyde and glutaraldehyde, and thus the contacted biomolecules can include fixation crosslinks resulting both from formaldehyde induced fixation and glutaraldehyde induced fixation. A suitable concentration of glutaraldehyde can be used for use as a fixation reagent can be 0.1 to 1%. Fixation and wash reagents may also include commercially available products, e.g., BioLegend® Fixation Buffer (420801) and Permeabilization Wash Buffer (421002).

[0154] Changes to a characteristic or a set of characteristics of a cell or cellular constituents(e.g., incurred upon interaction with one or more fixation agents) may be at least partiallyAttorney Docket: 43487-1036601 reversible (e.g., via rehydration or de-crosslinking). Alternatively, changes to a characteristic or set of characteristics of a cell or cellular constituents (e.g., incurred upon interaction with one or more fixation agents) may be substantially irreversible.

[0155] A sample (e.g., a cell sample) may be subjected to a fixation process at any usefulpoint in time. For example, cells, nuclei and / or cellular / nuclear constituents of a sample may be subjected to a fixation process involving one or more fixation agents (e.g., as described herein) prior to commencement of any subsequent processing, such as for storage. Cells, nuclei and / or cellular / nuclear constituents, such as cells, nuclei and / or cellular / nuclear constituents of a tissue sample, subjected to a fixation process prior to storage, may be stored in an aqueous solution, optionally in combination with one or more preserving agents configured to preserve morphology, size, or other features of the cells and / or cellular components. Fixed cells, nuclei and / or cellular / nuclear constituents may be stored below room temperature, such as in a freezer. Alternatively, cells, nuclei and / or cellular / nuclear constituents of a sample may be subjected to a fixation process involving one or more fixation agents subsequent to one or more other processes, such as filtration, centrifugation, agitation, selective precipitation, purification, permeabilization, isolation, heating, etc. For example, cells, nuclei, and / or cellular / nuclear constituents of a given type from a sample may be subjected to a fixation process following a separation and / or enrichment procedure (e.g., as described herein). In an example, a sample comprising a plurality of cells including a plurality of cells of a given type may be subjected to a positive separation process to provide a sample enriched in the plurality of cells of the given type. The enriched sample may then be subjected to a fixation process involving one or more fixation agents (e.g., as described herein) to provide an enriched sample comprising a plurality of fixed cells. A fixation process may be performed in a bulk solution. In some cases, fixed samples (e.g., fixed cells, fixed nuclei, and / or cellular / nuclear constituents) may be partitioned amongst a plurality of partitions (e.g., droplets or wells) and subjected to processing as described elsewhere herein. In some cases, fixed samples may undergo additional processing, such as partial or complete reversal of a fixation process by, for example, rehydration or de-crosslinking, prior to partitioning and any subsequent processing. In some cases, fixed samples may undergo partial or complete reversal of a fixation process within a plurality of partitions (e.g., prior to or concurrent with additional processing described elsewhere herein).

[0156] In some cases, a tissue specimen comprising a plurality of cells, nuclei and / orcellular / nuclear constituents may be processed to provide formalin-fixed paraffin-embedded (FFPE) tissue. A tissue specimen may be contacted (e.g., saturated) with formalin and then embedded in a solid medium. In an example, the solid medium may be paraffin wax. FFPE processing may facilitate preservation of a tissue sample (e.g., prior to subsequent processingAttorney Docket: 43487-1036601 and analysis). A tissue sample, including an FFPE tissue sample, may additionally or alternatively be subjected to storage in a low-temperature freezer. Cells, nuclei and / or cellular / nuclear constituents may be dissociated from a tissue sample (e.g., FFPE tissue sample) prior to undergoing subsequent processing. In some cases, individual cells, nuclei and / or cellular / nuclear constituents of a tissue sample such as an FFPE tissue sample may be optically detected, labeled, or otherwise processed prior to any such dissociation. Such detection, labeling, or other processing may be performed according to a 2- or 3-dimensional array and optionally according to a pre-determined pattern. In some cases, a tissue specimen may be embedded in other materials such as, for example, optimal cutting temperature (OCT) compound, crosslinking-based supports (e.g., polymers), or the like.

[0157] The tissue specimen may have been fixed at least about 1 day (d), 2 d, 3 d, 4 d, 5 d, 6d, 1 week (wk), 2 wk, 3 wk, 1 month (m), 2 m, 3 m, 4 m, 5 m, 6 m, 7 m, 8 m, 9 m, 10 m, 11 m, 1 year (y), 2 y, 3 y, 4 y, 5 y, 6 y, 7 y, 8 y, 9 y, 10 y, 15 y, 20 y, 25 y, 30 y, 35 y, 40 y, 45 y, 50 y, or longer before use in the methods and systems described elsewhere herein.

[0158] In some cases, preparation of a fixed sample may comprise securing and sectioning atleast a portion of a fixed sample (e.g., via microtomy, ultramicrotomy, etc.). The fixed sample may be sectioned into one or more (e.g., a plurality) of scrolls. The fixed tissue (e.g., a scroll) may be at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more micrometers in thickness. The sectioning and / or securing may be performed at ambient temperature. The sectioning and / or securing may be performed at temperatures above or below ambient temperature. The fixed tissue (e.g., a scroll) may have a mass of at least about 100 micrograms (µg), 150 µg, 200 µg 250 µg, 300 µg, 350 µg, 400 µg, 450 µg, 500 µg, 550 µg, 600 µg, 650 µg, 700 µg, 750 µg, 800 µg, 850 µg, 900 µg, 950 µg, 1 milligram (mg), 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 gram (g), 1.25 g, 1.5 g, 1.75 g, 2 g, 2.25 g, 2.5 g, 2.75 g, 3 g, 3.25 g, 3.5 g, 3.75 g, 4 g, 4.25 g, 4.5 g, 4.75 g, 5 g, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 40 g, 45 g, 50 g, or more. The fixed tissue (e.g., a scroll) may have a mass of at most about 50 g, 45 g, 40 g, 35 g, 30 g, 25 g, 20 g, 15 g, 10 g, 5 g, 4.75 g, 4.5 g, 4.25 g, 4 g, 3.75 g, 3.5 g, 3.25 g, 3 g, 2.75 g, 2.5 g, 2.25 g, 2 g, 1.75 g, 1.5 g, 1.25 g, 1 g, 950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg, 500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, 100 mg, 95 mg, 90 mg, 85 mg, 80 mg, 75 mg, 70 mg, 65 mg, 60 mg, 55 mg, 50 mg, 45 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 9 mg, 8 mg, 7 mg, 6 mg, 5 mg, 4 mg, 3 mg, 2 mg, 1 mg, 950 µg, 900 µg, 850 µg, 800 µg, 750 µg, 700 µg, 650 µg, 600 µg, 550 µg, 500 µg,Attorney Docket: 43487-1036601 450 µg, 400 µg, 350 µg, 300 µg, 250 µg, 200 µg, 150 µg, 100 µg, or less. The fixed tissue (e.g., a scroll) may have a mass in a range as defined by any two of the preceding values. For example, a fixed tissue or a scroll can have a mass from about 100 micrograms to about 5 grams.

[0159] In some cases, the scroll can be mechanically and / or enzymatically dissociated. Themechanical dissociation may comprise sonication (e.g., sonication at below ambient temperatures). The sonication may comprise a sonication at a power of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 65, 80, 85, 90, 95, or 100 percent power. The sonication may comprise sonication at a power of at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less percent power. The sonication may be for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 30, 45, 60, or more minutes. The sonication may be for at most about 60, 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or fewer minutes. The mechanical dissociation may comprise use of a shake plate. For example, the sample can be placed in a sample tube, and the sample tube can be shaken on a shake plate. The mechanical dissociation may comprise stirring the sample.

[0160] In some optional cases, the fixed sample may be processed to remove one or morefixatives and / or supports (e.g., solid medium). For example, the fixed sample can be deparaffinized. In some cases, the fixed sample may not be processed to remove the one or more fixatives and / or supports (e.g., solid medium). For example, a fixed sample can be used as sectioned (e.g., a scroll). Examples of processes include use of one or more non-polar solvents (e.g., linear alkanes, cyclic alkanes, benzene, xylenes, neo-clear, orange oil, other substituted or non-substituted alkanes, or the like, or any combination thereof). In an example, the non-polar solvent is xylene. The removal of the one or more fixatives and / or supports (e.g., solid medium) may be repeated (e.g., for more complete removal of the one or more fixatives and / or supports). In some optional cases, the sample may be rehydrated (e.g., via water addition, ethanol rehydration, gaseous rehydration, or the like, or any combination thereof). For example, ethanolic solutions of water with increasing water concentration can be used to rehydrate the sample. In some cases, the sample can be analyzed without rehydration. The sample may be washed using a polar (e.g., aqueous) solution to remove additional impurities. For example, an aqueous solution of phosphate buffered saline can be used to remove impurities from a sample.

[0161] One or more dissociation solutions comprising, for example, liberase with lowthermolysis (TL), liberase with medium thermolysis (TM), liberase with high thermolysis (TH), collagenases, or the like, or any combination thereof may be added to process a sample. The dissociation sample may be added at ambient (e.g., room) temperature. The dissociation solution be heated prior to the addition. The dissociation solution may be cooled prior to the addition. The dissociation sample may be at a temperature of at least about 20, 21, 22, 23, 24, 25, 26, 27, 28,Attorney Docket: 43487-1036601 29, 30, 31, 32, 33, 34, 34, 36, 37, 38, 39, 40, or more degrees Celsius when added. The dissociation solution may be at a temperature of at most about 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or fewer degrees Celsius when added. The dissociation solution may be added at a temperature in a range as defined by any two of the proceeding values. The sample may be titrated at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more times to form a cellular suspension. In some cases, impurities (e.g., paraffin) can be removed by allowing the suspension to rest, and the impurities can precipitate from the solution, and the purified solution can be removed and further processed.

[0162] A liberase can comprise one or more enzymes configured to degrade at least a portionof a biological molecule. For example, the liberase can comprise one or more collagenases. The collagenases can comprise one or more isoforms of collagenase, collagenase I, collagenase II, or the like, or any combination thereof. The collagenase isoforms may be present in ratios between the various isoforms of at least about 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, or more. In some cases, a composition comprising the one or more enzymes can comprise a dispase. The dispase may be a protease (e.g., a neutral protease, etc.). The dispase may be a non-clostridial dispase. A composition comprising the one or more enzymes may comprise thermolysin. The thermolysin can be a protease (e.g., a neutral protease, etc.). The thermolysin can be a non-clostridial thermolysin. A composition comprising the one or more enzymes may comprise a plurality of additional components as described elsewhere herein (e.g., a dispase, a thermolysin, etc.).

[0163] The sample may be filtered one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)times. The filtration may comprise use of one or more different sizes (e.g., pore sizes) of filter. The filter may comprise a pore size of at most about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1, or less micrometers. For example, a first filtration with a 70 micrometer pore size filter can be performed. In this example, a second filtration with a 30 micrometer filtration can be performed, which can reduce debris (e.g., undissolved paraffin or other support) without reducing cellular recovery from the sample. The filtrate and the cellular suspension may be combined and subsequently centrifuged. The centrifugation may occur at a value of at least about 100, 200, 300, 400, 500, 600, 700, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 3,000, or more reciprocal centrifugal force (rcf). The centrifugation may occur at a value of at most about 3,000, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,200, 1,100, 1,000, 950, 900, 850, 800, 700, 600 ,500, 400, 300, 200, 100, or fewer rcf. The centrifugationAttorney Docket: 43487-1036601 may be performed at a value within a range as defined by any two of the proceeding values. For example, the centrifugation may be performed at a value of about 850 to about 2,000 rcf.

[0164] In some cases, the solution can be removed from the centrifuged pellet (e.g., withoutdisturbing the pellet). In some cases, the pellet can be resuspended into solution. For example, the pellet can be resuspended into a buffer solution.

[0165] The resuspended solution can then be analyzed (e.g., to determine cellularconcentration). Examples of cellular concentration determination systems include, but are not limited to the Countess II FL Automated Cell Counter, Cellaca MX High-Throughput Automated Cell Counter, or the like using a fluorescent dye (e.g., ethidium homodimer-1, ect.) or AO / PI staining solution, or the like. The resuspended solution may then be used as a sample for the methods and systems described elsewhere herein (e.g., RNA profiling, etc.).

[0166] In some cases, use of a fixed sample (e.g., an FFPE sample) with the methods andsystems described elsewhere herein may provide different information as compared to use of a fresh sample. For example, the fixation process may capture ephemeral states of the cells of the sample and / or ephemeral types of cells in the sample that may not be captured in fresh samples. In this way, cellular processes can be investigated in different ways by use of fixed samples. In some cases, use of the methods and systems described elsewhere herein on fixed samples may provide unexpected improvements to the analysis of the fixed samples, such as improved sensitivity versus other analysis methods as well as the aforementioned analysis of different ephemeral states / types within the sample. In some cases, the use of fixed samples may permit time series analysis of samples (e.g., samples can be fixed at different times and later analyzed). Such a time series analysis may provide information related to the evolution of states and / or cell types within the samples. Methods of Nucleic Acid Analysis

[0167] In an aspect, the present disclosure provides a method for barcoding nucleic acidmolecules. The method may generally comprise contacting a nucleic acid molecule with a pair of probes and a barcode molecule to generate a barcoded molecule (e.g., a barcoded probe-linked molecule). The nucleic acid molecule may comprise a sequence corresponding to a target sequence or a template sequence. One or more nucleic acid reactions (e.g., a ligation, a nucleic acid extension reaction, amplification, etc.) may be performed to generate the barcoded molecule. In some aspects, the method comprises: contacting a nucleic acid molecule with a first probe to generate a probe-associated nucleic acid molecule, wherein the nucleic acid molecule comprises a first target region and a second target region, wherein the first probe comprises a first probe sequence complementary to the first target region; performing a nucleic acid reactionAttorney Docket: 43487-1036601 (e.g., a nucleic acid extension reaction, e.g., by using a polymerase or reverse transcriptase, etc.) to generate an extended probe molecule comprising a sequence complementary to the second target region; providing (i) a second probe comprising a second probe sequence corresponding to or complementary to the second target region and (ii) a nucleic acid barcode molecule; and subjecting the extended probe molecule or derivative thereof to conditions sufficient to generate a barcoded molecule. The first target region and the second target region may be disposed adjacent to one another or may be separate from one another (e.g., disposed on opposite ends of a gap region). In some instances, barcoding may be facilitated by providing a probe binding molecule (also referred to herein as a “splint molecule” or in some instances, a “splint oligonucleotide”). For example, the first probe and / or the second probe may comprise a probe capture sequence, and the probe-binding molecule may comprise a probe-binding sequence complementary to the probe capture sequence. In addition to or alternatively, the nucleic acid barcode molecule may comprise a barcode sequence and a barcode capture sequence, and the probe-binding molecule may comprise a barcode binding sequence complementary to the barcode capture sequence. In some instances, the probe-binding molecule may be pre-annealed to the nucleic acid barcode molecule. Barcoding may comprise hybridization of the probe binding molecule to the probe capture sequence (or complement thereof) of the first probe and / or second probe and to the barcode capture sequence of the nucleic acid barcode molecule. Accordingly, the barcoded molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. One or more operations may be performed within a partition (e.g., droplet or well).

[0168] The methods described herein may facilitate gene expression profiling with single-cell, single-nucleus or single-cell bead resolution using, for example, nucleic acid extension reactions, probe hybridization, chemical or enzymatic ligation, barcoding, amplification, and sequencing. The methods described herein may allow for gene expression analysis while avoiding the use of specialized imaging equipment and, in certain instances, reverse transcription, which may be highly error prone and inefficient. In some instances, the methods may be used to analyze a pre-determined panel of target genes in a population of single cells, nuclei, or cell beads in a sensitive and accurate manner. The methods described herein may also be useful in detecting or characterizing genetic variants, for example, in instances where the sequence of a region disposed between the target regions (e.g., a gap region) is not known. In some cases, the methods described herein may be useful in analyzing a single nucleotide polymorphism (SNP), an alternative-spliced junction, an insertion, a mutation, a deletion, a gene rearrangement (e.g., V(D)J rearrangements), a transposon, or other genetic element or variants.Attorney Docket: 43487-1036601 In some cases, the nucleic acid molecule analyzed by the methods described herein may comprise a fusion gene (e.g., a hybrid gene generated via translocation, interstitial deletion, or chromosomal inversion). In some cases, the methods described herein may be useful in analyzing genomic, transcriptomic, exomic and / or proteomic elements in cells, nuclei, cell beads, tissue samples, spatial arrays of cells, nuclei or tissues, etc.

[0169] The nucleic acid molecule analyzed by the methods described herein may be a single-stranded or a double-stranded nucleic acid molecule. A double-stranded nucleic acid molecule may be completely or partially denatured to provide access to a target region (e.g., a target sequence) of a strand of the nucleic acid molecule. Denaturation may be achieved by, for example, adjusting the temperature or pH of a solution comprising the nucleic acid molecule; using a chemical agent such as formamide, guanidine, sodium salicylate, dimethyl sulfoxide, propylene glycol, urea, or an alkaline agent (e.g., NaOH); or using mechanical agitation (e.g., centrifuging or vortexing a solution including the nucleic acid molecule).

[0170] The nucleic acid molecule may be a target nucleic acid molecule. The target nucleicacid molecule may be an RNA molecule. The RNA molecule may be, for example, a transfer RNA (tRNA) molecule, ribosomal RNA (rRNA) molecule, mitochondrial RNA (mtRNA) molecule, messenger RNA (mRNA) molecule, non-coding RNA molecule, synthetic RNA molecule, or another type of RNA molecule. For example, the RNA molecule may be an mRNA molecule. In some cases, the nucleic acid molecule may be a viral or pathogenic RNA. In some cases, the nucleic acid molecule may be a synthetic nucleic acid molecule previously introduced into or onto a cell. For example, the nucleic acid molecule may comprise a plurality of barcode sequences, and two or more barcode sequences may be target regions of the nucleic acid molecule. In some instances, the nucleic acid molecule is a guide RNA (gRNA), which may be exogenously introduced in a cell or cell bead. In some instances, the nucleic acid molecule is an RNA molecule derived from an exogenously introduced nucleic acid molecule, e.g., an RNA derived from a plasmid, an integrated DNA sequence (e.g. using viral transduction in a cell), a gRNA from a CRISPR genetic element, etc.

[0171] The nucleic acid molecule (e.g., RNA molecule) may comprise one or more featuresselected from the group consisting of a 5’ cap structure, an untranslated region (UTR), a 5’ triphosphate moiety, a 5’ hydroxyl moiety, a Kozak sequence, a Shine-Dalgarno sequence, a coding sequence, a codon, an intron, an exon, an open reading frame, a regulatory sequence, an enhancer sequence, a silencer sequence, a promoter sequence, and a poly(A) sequence (e.g., a poly(A) tail). For example, the nucleic acid molecule may comprise one or more features selected from the group consisting of a 5’ cap structure, an untranslated region (UTR), a KozakAttorney Docket: 43487-1036601 sequence, a Shine-Dalgarno sequence, a coding sequence, and a poly(A) sequence (e.g., a poly(A) tail).

[0172] Features of the nucleic acid molecule may have any useful characteristics. A 5’ capstructure may comprise one or more nucleoside moieties joined by a linker such as a triphosphate (ppp) linker. A 5’ cap structure may comprise naturally occurring nucleoside and / or non- naturally occurring (e.g., modified) nucleosides. For example, a 5’ cap structure may comprise a guanine moiety or a modified (e.g., alkylated, reduced, or oxidized) guanine moiety such as a 7- methylguanylate (m7G) cap. Examples of 5’ cap structures include, but are not limited to, m7GpppG, m7Gpppm7G, m7GpppA, m7GpppC, GpppG, m2,7GpppG, m2,2,7GpppG, and anti- reverse cap analogs such as m7,2’OmeGpppG, m7,2’dGpppG, m7,3’OmeGpppG, and m7,3’dGpppG. An untranslated region (UTR) may be a 5’ UTR or a 3’ UTR. A UTR may include any number of nucleotides. For example, a UTR may comprise at least 3, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides. In some cases, a UTR may comprise fewer than 20 nucleotides. In other cases, a UTR may comprise at least 100 nucleotides, such as more than 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides. Similarly, a coding sequence may include any number of nucleotides, such as at least 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides. A UTR, coding sequence, or other sequence of a nucleic acid molecule may have any nucleotide or base content or arrangement. For example, a sequence of a nucleic acid molecule may comprise any number or concentration of guanine, cytosine, uracil, and adenine bases. A nucleic acid molecule may also include non-naturally occurring (e.g., modified) nucleosides. A modified nucleoside may comprise one or more modifications (e.g., alkylations, hydroxylation, oxidation, or other modification) in its nucleobase and / or sugar moieties.

[0173] The nucleic acid molecule may comprise one or more target regions. In some cases, atarget region may correspond to a gene or a portion thereof. Each region may have the same or different sequences. For example, the nucleic acid molecule may comprise two target regions having the same sequence located at different positions along a strand of the nucleic acid molecule. Alternatively, the nucleic acid molecule may comprise two or more target regions having different sequences. Different target regions may be interrogated by different probes. Target regions may be located adjacent to one another or may be spatially separated along a strand of the nucleic acid molecule. The target regions may be located on the same strand or different strands. As used herein with regard to two entities, “adjacent,” may mean that the entities directly next to one other (e.g., contiguous) or in proximity to one another. For example, a first target region may be directly next to a second target region (e.g., having no other entity disposed between the first and second target regions) or in proximity to a second target region (e.g., having an intervening sequence or molecule between the first and second target regions).Attorney Docket: 43487-1036601 In some cases, a double-stranded nucleic acid molecule may comprise a target region in each strand that may be the same or different. For a nucleic acid molecule comprising multiple target regions, the methods described herein may be performed for one or more target regions at a time. For example, a single target region of the multiple target regions may be analyzed (e.g., as described herein) or two or more target regions may be analyzed at the same time. Analyzing two or more target regions may involve providing two or more probes, where a first probe has a sequence that is complementary to the first target region, a second probe has a sequence that is complementary to the second target region, etc.

[0174] Each probe (e.g., the first probe and the second probe) may further comprise one ormore additional sequences (e.g., additional probe sequences, unique molecular identifiers (UMIs), a barcode sequence, a primer sequence, a capture sequence, or other functional sequence). For example, in some instances, the first probe and / or the second probe may comprise the same or different barcode sequences. In some examples, the first probe and the second probe may be configured to hybridize to one or more nucleic acid barcode molecules. For example, the first probe and / or the second probe may comprise a probe capture sequence, which may be configured to hybridize to a nucleic acid barcode molecule or to a probe binding molecule (e.g., a splint oligonucleotide) that is configured to hybridize to a nucleic acid barcode molecule (e.g., via a barcode binding sequence that is complementary to a capture sequence of the nucleic acid barcode molecule). The probe capture sequence may be any useful length; for example, the probe capture sequence may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more nucleotides in length. The probe capture sequence may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more nucleotides in length. The probe capture sequence may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 nucleotide in length. A range of lengths of the probe capture sequence, such as from about 8 to about 50 nucleotides in length, etc. In some instances, the probe capture sequence length may be varied based on any useful application and properties, e.g., melting temperature, annealing temperature, annealing strength (e.g., GC content), hybridization stringency, etc.

[0175] Similarly, the probe binding molecule and nucleic acid barcode molecule may furthercomprise one or more additional sequences (e.g., unique molecular identifiers (UMIs), a barcode sequence, a primer sequence, a capture sequence, or other functional sequence). For example, in some instances, the probe binding molecule or barcode molecule may comprise a functionalAttorney Docket: 43487-1036601 sequence, a primer sequence (e.g., sequencing primer sequence or partial sequencing primer sequence), a UMI, etc. The probe binding molecule and the nucleic acid barcode molecule may be any useful length; for example, either or both may be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more nucleotides in length. The probe binding molecule or the barcode molecule may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 or more nucleotides in length. The probe capture binding molecule or the barcode molecule may be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 nucleotide in length. A range of lengths of the probe binding molecule or barcode molecule may be used, such as from about 16 to about 100 nucleotides in length, etc. In some instances, the probe binding molecule or barcode molecule length may be varied based on any useful application and properties, e.g., melting temperature, annealing temperature, etc. In some instances, the first target region and the second target region of the nucleic acid molecule are not adjacent. For instance, the first target region and the second target region may be separated by one or more gap regions disposed between the first target region and the second target region. The gap region may comprise, for example, at least one nucleotide base, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or more bases. The gap region may comprise at most about 1000, at most about 500, at most about 400, at most about 300, at most about 200, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 10, or at most about 5 bases. The gap region may comprise a range of number of bases, such as between about 1 and 30 bases.

[0176] A target region of the nucleic acid molecule may have one or more usefulcharacteristics. For example, a target region may have any useful length, base content, sequence, melting point, or other characteristic. A target region may comprise, for example, at least 10 bases, such as at least about 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or more bases. A target region may have any useful base content and any useful sequence and combination of bases. For example, a target region may comprise one or more adenine, thymine, uracil,Attorney Docket: 43487-1036601 cytosine, and / or guanine bases (e.g., natural or canonical bases). A target region may also comprise one or more derivatives or modified versions of a natural or canonical base, such as an oxidized, alkylated (e.g., methylated), hydroxylated, or otherwise modified base. Similarly, a target region may comprise ribose or deoxyribose moieties and phosphate moieties or derivatives or modified versions thereof.

[0177] A target region of the nucleic acid molecule may comprise one or more sequences orfeatures, or portions thereof, of the nucleic acid molecule. For example, a target region may comprise all or a portion of a UTR (e.g., a 3’ UTR or a 5’ UTR), a Kozak sequence, a Shine- Dalgarno sequence, a coding sequence, a polyA sequence, a cap structure, an intron, an exon, or any other sequence or feature of the nucleic acid molecule.

[0178] The nucleic acid molecule (e.g., RNA molecule, such as an mRNA molecule) of asample may be included within a cell, nucleus or cell bead. For example, the sample may comprise a cell or nucleus comprising the nucleic acid molecule. The cell, nucleus, or cell bead may comprise additional nucleic acid molecules that may be the same as or different from the nucleic acid molecule of interest. In some cases, the sample may comprise a plurality of cells, and each cell may contain one or more nucleic acid molecules. The cell may be, for example, a human cell, an animal cell, or a plant cell. In some cases, the cell may be derived from a tissue or fluid, as described herein. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a lymphocyte such as a B cell or T cell. The cell may be comprised within a bead, such as those disclosed in U.S. Pat. No.10,428,326, which is incorporated by reference herein in its entirety. In some instances, the cell is comprised within a tissue sample and may be fixed to a substrate. For example, the cell may be a cell of a formalin-fixed, paraffin-embedded (FFPE) sample, as described above. In such instances, the method may comprise additional operations for preparing the cell or nucleic acid molecule comprised therein, e.g., deparaffinization, staining (e.g., using immunological agents) or destaining, decrosslinking, washing, enzymatic treatment, etc. Additional examples of treating FFPE samples prior to and following hybridization of probes are included in PCT / US2020 / 066720, which is included by reference herein in its entirety.

[0179] Access to a nucleic acid molecule included in a cell, nucleus or cell bead may beprovided by lysing or permeabilizing the cell or nucleus. Lysing the cell, nucleus or cell bead may release the nucleic acid molecule contained therein from the cell, nucleus or cell bead. A cell or nucleus may be lysed using a lysis agent such as a bioactive agent. A bioactive agent useful for lysing a cell or nucleus may be, for example, an enzyme (e.g., as described herein). An enzyme used to lyse a cell or nucleus may or may not be capable of carrying out additional functions such as degrading, extending, reverse transcribing, or otherwise altering a nucleic acidAttorney Docket: 43487-1036601 molecule. Alternatively, an ionic or non-ionic surfactant such as TritonX-100, Tween 20, sarcosyl, or sodium dodecyl sulfate may be used to lyse a cell or nucleus. Cell / nucleus lysis may also be achieved using a cellular disruption method such as an electroporation or a thermal, acoustic, or mechanical disruption method. Alternatively, a cell or nucleus may be permeabilized to provide access to a nucleic acid molecule included therein. Permeabilization may involve partially or completely dissolving or disrupting a cell / nuclear membrane or a portion thereof. Permeabilization may be achieved by, for example, contacting a cell membrane with an organic solvent (e.g., methanol) or a detergent such as Triton X-100 or NP-40. The cell, nucleus or cell bead may be fixed, as described elsewhere herein.

[0180] In some cases, the cell may be lysed within the cell bead, and a subset of theintracellular contents may associate with the bead. In some cases, the cell bead may comprise thioacrydite-modified nucleic acid molecules that can hybridize with nucleic acids from the cell. For example, a poly-T nucleic acid sequence may be thioacrydite-modified and bound to the cell bead matrix. Upon cell or nucleus lysis, the cellular nucleic acids (e.g., mRNA) may hybridize with the poly-T sequence. The retained intracellular / intranuclear contents may be released, for example, by addition of a reducing agent, e.g., DTT, TCEP, etc. The release may occur at any convenient step, such as before or after partitioning.

[0181] The nucleic acid molecule or probe-associated nucleic acid molecule may besubjected to conditions sufficient to generate a probe-linked molecule. For instance, the first target region may be adjacent to the second target region, and the first probe and the second probe may hybridize to the first target region and the second target region, respectively. The first probe may comprise a first reactive moiety, and the second probe may comprise a second reactive moiety. In some instances, the first reactive moiety of the first probe is adjacent to the second reactive moiety of the second probe. The reactive moieties may then be subjected to conditions sufficient to cause them to react to yield a probe-linked nucleic acid molecule comprising the first probe linked to the second probe. For example, the reactive moieties may be joined together via click chemistry or enzymatic ligation, such as those disclosed in in U.S. Pat. Pub. No.2020 / 0239874, International Pub. No. WO 2019 / 165318, and International Pat. Pub. No. WO2021 / 237087, each of which is incorporated by reference herein in its entirety. In some examples, the first probe or the second probe may comprise an adenylated oligonucleotide or moiety (e.g., an adenylated phosphate group), which may be useful in reducing non-specific ligation reactions. In some instances, the linking of the probes (e.g., via ligation) may be performed in substantially ATP-free conditions, optionally using an enzyme (e.g., ligase) that does not require ATP (e.g., truncated T4 RNA ligase) or that is pre-activated (e.g., a preactivated T4 DNA ligase). Additional examples of such ligation schemes can be found inAttorney Docket: 43487-1036601 PCT / US2020 / 066720 and International Pat. App. No. PCT / US2021 / 33649, filed May 21, 2021, which is incorporated by reference herein in its entirety.

[0182] In some instances, the first target region of the nucleic acid molecule (e.g., RNAmolecule) may not be adjacent to the second target region. In such cases, the nucleic acid molecule may be subjected to conditions sufficient for hybridization of the first probe sequence of the first probe to the first target region to generate a probe-associated nucleic acid molecule. The probe-associated nucleic acid molecule may be subjected to a nucleic acid reaction (e.g., a nucleic acid extension reaction, reverse transcription, etc.) to generate an extended probe molecule comprising a sequence complementary to the second target region. A second probe comprising a second probe sequence may hybridize to the extended probe molecule (or complement thereof) and subjected to conditions sufficient (e.g., nucleic acid extension, amplification, hybridization of additional probe molecules, ligation, etc.) to generate a probe- linked molecule comprising a sequence corresponding to the first target region and a sequence corresponding to the second target region. Alternatively or in addition to, the first probe and the second probe may be provided simultaneously, and following hybridization of the first probe sequence and the second probe sequence to the first target region and the second target region, respectively, to generate a dual-probe-associated nucleic acid molecule, the gap (e.g., the region disposed between the first target region and the second region) may be filled (e.g., via a nucleic acid extension or gap-fill reaction and / or hybridization of additional probe molecules that hybridize to at least a portion of the gap region). In some instances, one or both probes may comprise an overhang or flap sequence (e.g., at a 5’ end) that is recognizable or cleavable by an enzyme (e.g., an endonuclease such as FEN1 endonuclease). For example, the second probe may comprise a 5’ flap sequence that is cleaved by FEN1 endonuclease if at least a specific portion of the second probe hybridizes to the nucleic acid molecule (e.g., target molecule). Subsequent to hybridization of the second probe to the second target sequence of the nucleic acid molecule, an endonuclease (e.g., FEN1) may be used to cleave the flap sequence and leave a ligatable end (e.g., a phosphorylated end) of the second probe. In instances in which the first target region is not adjacent to the second target region, the gap region may be filled, followed by cleavage of the flap sequence. In some instances, the first probe or the second probe and the gap-filled region may be ligated, e.g., chemically or enzymatically. Additional examples of systems and methods for generating probe-linked nucleic acid molecules and gap-filling reactions can be found, for example in U.S. Pat. Pub. No.2020 / 0239874, International Pub. No. WO 2019 / 165318, and International Pat. Pub. No. WO2021 / 237087, each of which is incorporated by reference herein in its entirety.Attorney Docket: 43487-1036601

[0183] The probe-linked nucleic acid molecule may be barcoded to provide a barcodedprobe-linked nucleic acid molecule, or barcoding may occur prior to generation of the probe- linked nucleic acid molecule. Barcoding may be performed using a variety of techniques. For example, the first probe or the second probe may comprise a probe capture sequence. The nucleic acid barcode molecule may comprise a barcode capture sequence capable of hybridizing to the probe capture sequence. Alternatively, barcoding may be mediated by a probe binding molecule (e.g., a splint oligonucleotide) comprising (i) a probe binding sequence, which may be complementary to the probe capture sequence of the first probe or the second probe, and (ii) a barcode binding sequence, which may be complementary to the barcode capture sequence of the nucleic acid barcode molecule. In some instances, the barcoding may be followed by ligation, e.g., chemically or enzyme-mediated, to covalently link the nucleic acid barcode molecule to the probe (or to the probe binding sequence, and the probe binding sequence may be ligated to the probe). Examples of chemical ligation of nucleic acid molecules may include “click chemistry” approaches, e.g., reaction of azide and alkyne moieties, as described in U.S. Pat. Pub. No. 2020 / 0239874, which is incorporated by reference herein in its entirety.

[0184] By way of example, the first probe may comprise a first probe sequence and a probecapture sequence, and the first probe may be subjected to conditions sufficient to hybridize the first probe sequence to the first target region, thereby generating a probe-associated nucleic acid molecule. In some instances, the probe-associated nucleic acid molecule may be subjected to washing or other conditions to remove unannealed probes from a mixture. The probe-associated nucleic acid molecule may be extended from an end of the first probe towards an end of the nucleic acid molecule to which it is hybridized (towards the end which is proximal to the second target region) to provide an extended nucleic acid molecule. The extended nucleic acid barcode molecule may comprise the first probe sequence and a complement to the second target region. In some instances, the extended nucleic acid molecule may be barcoded, e.g., by hybridizing the barcode capture sequence of the nucleic acid barcode molecule to the probe capture sequence, or by hybridizing (i) a probe-binding molecule comprising a probe binding sequence and a barcode binding sequence to the probe capture sequence and (ii) the barcode capture sequence of the nucleic acid barcode molecule to the barcode binding sequence of the probe binding molecule. In some instances, the probe-binding molecule may be provided pre-annealed to the nucleic acid barcode molecule. Subsequently, a second probe comprising a second probe sequence may be provided. The barcoded, extended nucleic acid molecule may be subjected to conditions sufficient to hybridize the second probe sequence to the second target region or complement thereof. A nucleic acid extension reaction may be performed, thereby generating a barcoded molecule (e.g., barcoded probe-linked molecule) comprising a sequence corresponding to theAttorney Docket: 43487-1036601 first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence.

[0185] FIG. 7 schematically shows a method for generating a barcoded nucleic acidmolecule, as described herein. A nucleic acid molecule (e.g., RNA molecule) 700 comprising a first target region 702 and a second target region 704 may be provided. The nucleic acid molecule 700 may be contacted with a first probe 706 comprising a first probe sequence 708 and, optionally, a functional sequence 710, thereby generating a probe-associated nucleic acid molecule. The first probe sequence 708 may be complementary to the first target region 702. The functional sequence 710 may comprise, for instance, a probe capture sequence used for downstream barcoding, or it may comprise a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0186] In operation 701, the probe-associated nucleic acid molecule may be subjected toconditions sufficient to extend the first probe 706, thereby generating an extended probe molecule 712 comprising a sequence complementary to the second target region 704. In some instances, the extended probe molecule 712 may be released from the nucleic acid molecule 700, e.g., via denaturing and / or degrading the nucleic acid molecule 700 (e.g., using an RNAse, increased temperature or heat cycling, pH, etc.). In operation 703, a nucleic acid barcode molecule may be provided. In some instances, the nucleic acid barcode molecule may be partially double-stranded and may comprise a first strand 720 comprising a barcode sequence, and a second strand 722 comprising a sequence 724 at least partially complementary to the barcode sequence and a probe binding sequence 726, which may be at least partially complementary to the functional sequence (e.g., probe capture sequence) 710 of the first probe 706. In some instances, the nucleic acid barcode molecule is single-stranded and comprises only first strand 720 comprising the barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 722 may be provided, comprising barcode- binding sequence 724, which is at least partially complementary to the barcode capture sequence, and the probe binding sequence 726. In some instances, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or the pre-annealed complex) may be coupled to a bead, such as a gel bead, as described herein, and may comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence).

[0187] In operation 705, the extended probe molecule may be barcoded by hybridizing theprobe binding sequence 726 to the functional sequence (e.g., probe capture sequence 710). InAttorney Docket: 43487-1036601 some instances, the nucleic acid barcode molecule may be covalently linked to the extended probe molecule (e.g., via the probe capture sequence), e.g., enzymatically (e.g., using a ligase) or chemically (e.g., using click chemistry). In operation 707, a second probe molecule 716 may be provided. In some instances, operation 707 may also include a denaturation of the double- stranded molecule. The second probe molecule 716 may comprise a second probe sequence 714 corresponding to the second target region 704 and optionally a functional sequence 718, which may comprise a probe capture sequence, a barcode sequence, a primer sequence, a sequencing primer sequence, etc. In operation 709, a nucleic acid extension reaction may be performed, e.g., using a polymerase, to extend the second probe 716 along the extended probe molecule, thereby generating a barcoded molecule comprising a sequence corresponding to the first target region 702, the second target region 704, a sequence corresponding to the probe capture sequence 710, and a sequence corresponding to the barcode sequence 720.

[0188] In another example, the first probe and the second probe may be linked (e.g., bychemical ligation or enzymatic extension and / or ligation) prior to barcoding. In such an example, the first probe may be hybridized to the nucleic acid molecule (e.g., via hybridization of the first probe sequence to the first target region) to generate a probe-associated nucleic acid molecule. The probe-associated nucleic acid molecule may be extended from an end of the first probe to an end of the nucleic acid molecule to which it is hybridized, to provide an extended nucleic acid molecule. The extended molecule may be subjected to conditions sufficient to hybridize the second probe to the second target region or complement thereof (e.g., via hybridization of the second probe sequence to the second target region or complement thereof). An additional nucleic acid extension reaction may be performed, to generate an extended, and the resultant extension product may be barcoded, generating a barcoded molecule. The barcoded molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. In some instances, the nucleic acid barcode molecule (or the probe binding molecule) may be chemically linked to the first probe or the second probe, such as by ligation or click chemistry. For example, the nucleic acid barcode molecule may comprise a first reactive moiety, and the first or the second probe may comprise a second reactive moiety; the first reactive moiety may be configured to react with the second reactive moiety to generate a covalent linkage. Barcoded nucleic acid molecules or derivatives thereof may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0189] FIG. 8 schematically shows another method for generating a barcoded nucleic acidmolecule, as described herein. A nucleic acid molecule (e.g., RNA molecule) 800 comprising aAttorney Docket: 43487-1036601 first target region 802 and a second target region 804 may be provided. The nucleic acid molecule 800 may be contacted with a first probe 806 comprising a first probe sequence 808 and, optionally, a functional sequence 810, thereby generating a probe-associated nucleic acid molecule. The first probe sequence 808 may be complementary to the first target region 802. The functional sequence 810 may comprise, for instance, a probe capture sequence used for downstream barcoding, or it may comprise a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0190] In operation 801, the probe-associated nucleic acid molecule may be subjected toconditions sufficient to extend the first probe 806, thereby generating an extended probe molecule 812 comprising a sequence complementary to the second target region 804. In some instances, the extended probe molecule 812 may be released from the nucleic acid molecule 800, e.g., via denaturing and / or degrading the nucleic acid molecule 800 (e.g., using an RNAse, increased temperature or heat cycling, pH, etc.). In operation 803, a nucleic acid barcode molecule and a second probe 816 may be provided. The second probe 816 may comprise a second probe sequence 814 corresponding to the second target region 804 and optionally a functional sequence 818, which may comprise a probe capture sequence. In some instances, the nucleic acid barcode molecule may be partially double-stranded and may comprise a first strand 820 comprising a barcode sequence, and a second strand 822 comprising a sequence 824 complementary to the barcode sequence and a probe binding sequence 826, which may be complementary to the functional sequence (e.g., probe capture sequence) 818 of the second probe 816. In some instances, the nucleic acid barcode molecule is single-stranded and comprises only first strand 820 comprising the barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 822 may be provided, comprising barcode-binding sequence 824 that is complementary to the barcode capture sequence, and the probe binding sequence 826. In some instances, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or the pre-annealed complex) may be coupled to a bead, such as a gel bead, as described herein, and may comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence). In operation 803, the second probe 816 may hybridize to the extended probe molecule 812 (e.g., via hybridization of the second probe sequence 814 to the second target region 804 or complement thereof), and the nucleic acid barcode molecule may be attached or coupled to the second probe 816, e.g., via hybridization of the probe binding sequence 826 to the probe capture sequence 818. In some instances, the nucleic acid barcode molecule or the probeAttorney Docket: 43487-1036601 binding molecule may be ligated to the second probe 816, e.g., using a ligase or via chemical linkage, such as click chemistry.

[0191] In operation 805, a nucleic acid extension reaction may be performed, e.g., using apolymerase (e.g., DNA polymerase, Hot Start polymerase, etc.), to extend the nucleic acid barcode molecule and the second probe 816 along the extended probe molecule, thereby generating a barcoded molecule comprising a sequence corresponding to the first target region 802, the second target region 804, a sequence corresponding to the probe capture sequence 818, and a sequence corresponding to the barcode sequence 820. Barcoded nucleic acid molecules or derivatives thereof may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0192] FIG. 9 schematically shows another method for generating a barcoded nucleic acidmolecule, similar to that shown in FIG.8. A nucleic acid molecule (e.g., RNA molecule) 900 comprising a first target region 902 and a second target region 904 may be provided. The nucleic acid molecule 900 may be contacted with a first probe 906 comprising a first probe sequence 908 and, optionally, a functional sequence 910, thereby generating a probe-associated nucleic acid molecule. The first probe sequence 908 may be complementary to the first target region 902. The functional sequence 910 may comprise, for instance, a probe capture sequence, or it may comprise a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0193] In operation 901, the probe-associated nucleic acid molecule may be subjected toconditions sufficient to extend the first probe 906, thereby generating an extended probe molecule 912 comprising a sequence complementary to the second target region 906. In some instances, the extended probe molecule 912 may be released from the nucleic acid molecule 900, e.g., via denaturing and / or degrading the nucleic acid molecule 900 (e.g., using an RNAse, increased temperature or heat cycling, pH, etc.). In operation 903, a second probe 916 may be provided. The second probe 916 may comprise a second probe sequence 914 corresponding to the second target region 904 and optionally a functional sequence 918, which may comprise a probe capture sequence. In operation 905, a nucleic acid extension reaction may be performed, e.g., using a polymerase, to extend the nucleic acid barcode molecule and the second probe 916 along the extended probe molecule, thereby generating a probe-linked molecule comprising a sequence corresponding to the first target region 902 and the second target region 904.

[0194] In operation 905, a nucleic acid barcode molecule may also be provided with thesecond probe. In some instances, the nucleic acid barcode molecule may be partially double- stranded and may comprise a first strand 920 comprising a barcode sequence, and a second strand 922 comprising a sequence 924 complementary to the barcode sequence and a probeAttorney Docket: 43487-1036601 binding sequence 926, which may be complementary to the functional sequence (e.g., probe capture sequence) 918 of the second probe 916. In some instances, the nucleic acid barcode molecule is single-stranded and comprises only first strand 920 comprising the barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 922 may be provided, comprising barcode-binding sequence 924 that is complementary to the barcode capture sequence, and the probe binding sequence 926. In some instances, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or the pre-annealed complex) may be coupled to a bead, such as a gel bead, as described herein, and may comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence). In operation 907, the nucleic acid barcode molecule may be attached or coupled to the second probe 916, e.g., via hybridization of the probe binding sequence 926 to the probe capture sequence 918. The resultant barcoded product may comprise a sequence corresponding to the first target region 902, the second target region 904, a sequence corresponding to the probe capture sequence 918, and a sequence corresponding to the barcode sequence 920. In some instances, the nucleic acid barcode molecule may be covalently linked to the extended probe molecule (e.g., via the probe capture sequence 918), e.g., enzymatically (e.g., using a ligase) or chemically (e.g., using click chemistry). Barcoded nucleic acid molecules or derivatives thereof may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0195] In additional examples, the methods of the present disclosure may comprisegenerating probe-associated nucleic acid molecules, and barcoding the probe-associated nucleic acid molecules, optionally with a linking operation (e.g., prior to or subsequent to barcoding of the probe-associated nucleic acid molecules). For example, a nucleic acid molecule (e.g., RNA molecule) comprising a first target region and a second target region may be provided. The nucleic acid molecule may be contacted with (i) a first probe comprising a first probe sequence complementary to the first target region and (ii) a second probe comprising a second probe sequence complementary to the second target region, thereby generating a probe-associated nucleic acid molecule. In some instances, the probe-associated nucleic acid molecule may be subjected to conditions sufficient to link the first probe to the second probe (e.g., enzymatically, such as with a polymerase, reverse transcriptase, and / or ligase, or chemically), thereby generating a probe-linked nucleic acid molecule. The probe-associated nucleic acid molecule or the probe-linked molecule may subsequently be barcoded (e.g., in a partition) to generate a barcoded nucleic acid molecule.Attorney Docket: 43487-1036601

[0196] For example, FIG. 25 schematically shows an example method for generating aprobe-linked nucleic acid molecule, which may subsequently be barcoded, e.g., in a partition, to generate a barcoded nucleic acid molecule. A nucleic acid molecule (e.g., RNA molecule) 2500 comprising a first target region 2502 and a second target region 2504 may be provided. In some instances, the first target region is adjacent to the second target region. The nucleic acid molecule 2500 may be contacted, in operation 2501, with a first probe 2506 comprising a first probe sequence 2508 complementary to the first target region 2502 and a second probe 2516 comprising a second probe sequence 2514 complementary to the second target region 2504, thereby generating a probe-associated nucleic acid molecule. The first probe 2506 and / or the second probe 2516 may comprise a functional sequence, e.g., a probe capture sequence, a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0197] In some instances, one of the probes (e.g., the second probe 2516) comprises a flap oroverhang sequence 2530, which may be recognized by an endonuclease (e.g., FEN1) upon annealing of the second probe sequence 2514 to the second target region 2504. For example, the second probe 2516 may comprise a 5’ flap sequence 2530, and subsequent to annealing of the first probe 2506 and the second probe 2516 to the nucleic acid molecule 2500, the flap sequence may be adjacent to an end of the first probe (e.g., a 3’ end) as well as an end of the second probe (e.g., a 5’ end). In operation 2503, an endonuclease, e.g., FEN1 may be used to remove the flapsequence 2530 ļeaving a ligatable end (e.g., 5’phosphorylated end) of the second probe 2516. Inoperation 2507, a ligation reaction may be performed (e.g., using a ligase) to link the first probe to the second probe, thereby generating a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule may subsequently be barcoded, e.g., in partitions, as is described elsewhere herein. In some instances, the probe-associated nucleic acid molecules may be barcoded and linked (e.g., in partitions).

[0198] FIG. 26 shows another example workflow, similar to that shown in FIG. 25, inwhich the target regions of the nucleic acid molecule are not adjacent. Such a workflow may comprise an additional gap-fill reaction to generate the probe-associated molecule. In one such example, the first target region 2602 of nucleic acid molecule 2600 may not be adjacent to the second target region 2604. For example, a gap region may be disposed between the first target region and the second target region. In operation 2601, the first probe 2606 may anneal to the first target region 2602 and the second probe 2616 may anneal to the second target region 2604. In operation 2603, an extension reaction (e.g., using a polymerase, reverse transcriptase, etc.) may be performed to fill in the gap region between the first probe 2606 and the second probe 2616, yielding a gap-filled nucleic acid molecule. In some instances, the second probe 2616comprises a flap sequence 2630. In such instances, in operation 2605, an endonuclease, e.g.,Attorney Docket: 43487-1036601FEN1 may be used to remove the flap sequence 2630 ̧leaving a ligatable end (e.g.,5’phosphorylated end) of the second probe 2616. In operation 2607, a ligation reaction may be performed (e.g., using a ligase) to link the first probe to the second probe, thereby generating a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule, or alternatively, the un-linked molecule, may be barcoded, e.g., in a partition.

[0199] FIG. 27 shows an additional scheme of generating a probe-linked nucleic acidmolecule by performing a gap-filling reaction using a third probe. In FIG.27 Panel A, a first probe 2706 and a second probe 2716 anneal (e.g., via a first probe sequence and a second probe sequence, respectively) to a first target region 2702 and a second target region 2704 of nucleic acid molecule 2700 to generate a probe-associated nucleic acid molecule. A gap sequence may be disposed between the first target region 2702 and the second target region 2704. Third probe molecules 2770 may be provided (illustrated as two different probe molecules, which may be used for SNP detection), which may anneal to the gap sequence (FIG.27 Panel B). In FIG.27 Panel C, the first probe, the third probe, and the second probe may be ligated (e.g., using a ligase) to generate a probe-linked nucleic acid molecule. The probe-linked nucleic acid molecule, or alternatively, the probe-associated nucleic acid molecule, may be barcoded, e.g., in a partition.

[0200] FIG. 28 shows an example of a ligation scheme used to generate probe-linked nucleicacid molecules. In such an example, the probe molecules may hybridize to the nucleic acid molecule. The first probe may be ligated to the second probe, optionally with a gap-fill operation, as described above, using an enzyme. In some instances, the enzyme may be a pre- activated enzyme, e.g., a preactivated T4 DNA ligase, and the ligation may occur under ATP- reduced or ATP-removed conditions, e.g. using Apyrase.

[0201] Additional examples of methods and systems for generating probe-associated nucleicacid molecules, and barcoding the probe-associated nucleic acid molecules, can be found in, for example U.S. Pat. Pub. No.2020 / 0239874, International Pub. No. WO 2019 / 165318, International App. No. PCT / US2020 / 066720, and International Pat. App. No. PCT / US2021 / 33649, filed May 21, 2021, each of which is incorporated by reference herein in its entirety.

[0202] It will be appreciated that, e.g., referring to FIGs. 7-9 and FIGs. 25-28, the nucleicacid barcode molecule may be attached (e.g., via hybridization) to either the first probe and / or the second probe (e.g., via a probe capture sequence comprised in the first probe or the second probe). Similarly, the first probe and the second probe may comprise any useful functional sequences, such as primer sequences, barcode sequences, unique molecular identifier (UMI) sequences, flow cell attachment sequences, primer-binding sequences, capture sequences, etc.Attorney Docket: 43487-1036601 The first probe may hybridize to the left-hand side (e.g., a 3’ end) of a nucleic acid molecule (e.g., 700, 800, or 900) or to the right-hand side (e.g., a 5’ end). Similarly, the second probe may hybridize to the left-hand side or to the right-hand side of the nucleic acid molecule.

[0203] As described herein, one or more extension reactions may be performed on the probe-hybridized nucleic acid molecules. For example, the probe may be extended from an end of the probe to an end of the nucleic acid barcode molecule, or a second probe may be extended from an end of the second probe to an end of the first probe of a probe-associated nucleic acid molecule. Extension may comprise the use of an enzyme (e.g., a polymerase, reverse transcriptase) to add one or more nucleotides to the end of the probe. Extension may provide an extended nucleic acid molecule comprising sequences complementary to the target region of the nucleic acid molecule of interest, the barcode sequence, and optionally, one or more additional sequences of the nucleic acid barcode molecule such as one or more binding sequences. In some instances, appropriate conditions and or chemical agents (e.g., as described herein) may be applied to denature the extended nucleic acid molecule from the nucleic acid barcode molecule and the target nucleic acid molecule. In some cases, one or more processes may involve the use of thermosensitive agents. For example, in some cases, probes may be annealed or hybridized under one set of temperature conditions, and extension may occur under a different set of temperature conditions. In some cases, a Warm or Hot Start polymerase may be used. In some cases, hybridization of the nucleic acid barcode molecule to one or more of the probes (e.g., directly hybridizing or via a probe binding molecule such as a splint oligonucleotide) may precede hybridization of the probe to the target region of the nucleic acid molecule. Following barcoding, the barcoded nucleic acid molecule may be duplicated or amplified by, for example, one or more amplification reactions. The amplification reactions may comprise polymerase chain reactions (PCR) and may involve the use of one or more primers or polymerases. The extension, denaturation, and / or amplification processes may take place within a partition, or in bulk. In some cases, the extended nucleic acid molecule or derivatives thereof (e.g., the barcoded molecule) may be duplicated or amplified within a partition to provide an amplified product. The barcoded product, or a complement thereof (e.g., an amplified product), may be detected via sequencing (e.g., as described herein).

[0204] The nucleic acid molecule or a derivative thereof (e.g., a probe-linked nucleic acidmolecule, a nucleic acid molecule having one or more probes hybridized thereto, a barcoded probe-linked nucleic acid molecule, or an extended nucleic acid molecule or complement thereof) or a cell or cell bead comprising the nucleic acid molecule or a derivative thereof may be provided within a partition such as a well or droplet, e.g., as described herein. One or more reagents may be co-partitioned with a nucleic acid molecule or a derivative thereof or a cellAttorney Docket: 43487-1036601 comprising the nucleic acid molecule or a derivative thereof. For example, a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof may be co-partitioned with one or more reagents selected from the group consisting of lysis agents or buffers, permeabilizing agents, enzymes (e.g., enzymes capable of digesting one or more RNA molecules, extending one or more nucleic acid molecules, reverse transcribing an RNA molecule, permeabilizing or lysing a cell, or carrying out other actions), fluorophores, oligonucleotides, primers, probes, barcodes, nucleic acid barcode molecules (e.g., nucleic acid barcode molecules comprising one or more barcode sequences), buffers, deoxynucleotide triphosphates, detergents, reducing agents, chelating agents, oxidizing agents, nanoparticles, beads, and antibodies. In some cases, a nucleic acid molecule or a derivative thereof, or a cell comprising the nucleic acid molecule or a derivative thereof (e.g., a cell bead), may be co- partitioned with one or more reagents selected from the group consisting of temperature- sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, reverse transcriptases, proteases, ligase, polymerases, restriction enzymes, nucleases, protease inhibitors, exonucleases, and nuclease inhibitors. For example, a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof may be co-partitioned with a polymerase and nucleotide molecules. Partitioning a nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof and one or more reagents may comprise flowing a first phase comprising an aqueous fluid, the cell, and the one or more reagents and a second phase comprising a fluid that is immiscible with the aqueous fluid toward a junction. Upon interaction of the first and second phases, a discrete droplet of the first phase comprising the nucleic acid molecule or a derivative thereof or a cell comprising the nucleic acid molecule or a derivative thereof (e.g., a cell bead) and the one or more reagents may be formed. In some cases, the partition may comprise a single cell. The cell may be lysed or permeabilized within the partition (e.g., droplet) to provide access to the nucleic acid molecule of the cell.

[0205] One or more processes may be carried out within a partition (e.g., droplet, well, etc.).For instance, the nucleic acid molecule, or a cell or cell bead comprising the nucleic acid molecule, may be co-partitioned with one or more reagents (e.g., as described herein) at any useful stage of the method. For example, the probe-associated nucleic acid molecule (e.g., the nucleic acid molecule with the first probe hybridized thereto) may be generated in bulk (e.g., in a population of cells, which may be alive or fixed and / or permeabilized, in a tissue sample, etc.) and subjected to conditions sufficient for generating for generating an extended probe molecule. The extended probe molecule may be subsequently partitioned in a partition among a plurality of partitions. The partition may comprise the second probe and a nucleic acid barcode molecule andAttorney Docket: 43487-1036601 optionally, a probe binding molecule. As described herein, the second probe may hybridize (e.g., via the second probe sequence) to the second target region or complement thereof of the probe- associated molecule. The partition may comprise additional reagents for performing a nucleic acid reaction (e.g., digestion, ligation, extension, amplification). For instance, the probe- associated nucleic acid molecule may comprise or be hybridized to the nucleic acid molecule, and the partition may comprise a degrading enzyme (e.g., RNAse), which may be useful in digesting or removing the template strand (e.g., the nucleic acid molecule, such as an RNA molecule) from the extended probe molecule. The partition may comprise a polymerase, which may be used to extend the second probe hybridized to the extended probe molecule. In some instances, the partition comprises a linking enzyme (e.g., ligase), which may be used to ligate the nucleic acid barcode molecule to the first probe or the second probe (e.g., via a probe capture sequence). The ligase may in some instances be used to ligate the probe binding molecule to the probe capture sequence of the first probe or the second probe. In some instances, the probe binding molecule, the probe capture sequence, and / or the barcode capture sequence comprises one or more reactive moieties, which may be used to chemically or enzymatically link the nucleic acid barcode molecule to the probe capture sequence, or complement thereof. The resultant barcoded product may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence.

[0206] For example, referring again to FIG. 7, operation 701 may be performed in bulk (e.g.,outside a partition), while operations 703, 705 may be performed in a partition. Operations 707 and 709 may be performed in bulk or within the partition. Similarly, referring to FIG.8, operation 801 may be performed in bulk, while operation 803 may be performed in a partition. Operation 805 may be performed in bulk or in a partition. Referring to FIG.9, operation 901 may be performed in bulk, while operations 903, 905, and 907 may be performed in a partition. It will be appreciated that any of the operations may be performed in bulk or in partitions at any convenient step and that the order of the operations may be changed for a suitable or useful purpose.

[0207] Similarly, the nucleic acid molecule or the cell or cell bead comprising the nucleicacid molecule, or derivatives thereof (e.g., the probe-associated molecule, the extended molecule, the barcoded molecule, etc.) may be released from a partition at any useful stage of the method. For example, the extended probe molecule may be hybridized to the second probe and released from the partition subsequent to hybridization of the barcode capture sequence of the nucleic acid barcode molecule to the first probe, the second probe, or the probe binding molecule. Alternatively, the extended probe molecule may be released from the partitionAttorney Docket: 43487-1036601 subsequent to (i) hybridization of the second probe and nucleic acid barcode molecule and (ii) extension of the second probe to generate the barcoded molecule comprising a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, and a sequence corresponding to the barcode sequence. Duplication and / or amplification of the extended nucleic acid molecule may be carried out within the partition or in bulk, e.g., within a solution. In some cases, the solution may comprise additional extended nucleic acid molecules generated through the same process carried out in different partitions. Each extended nucleic acid molecule may comprise a different barcode sequence, and the barcode sequence may be useful in identifying the partition or cell from whence the extended nucleic acid molecules originated. In such cases, the solution may comprise a pooled mixture comprising the contents of two or more partitions (e.g., droplets).

[0208] Additional processes or operations may be performed within a partition, including,but not limited to: lysis, permeabilization, denaturation, hybridization, extension, duplication, and amplification of one or more components of a sample. In some cases, multiple processes are carried out within a partition.

[0209] Hybridization of the probe sequences to the target regions of the nucleic acidmolecule may be performed within or outside of a partition. In some cases, hybridization may be preceded by denaturation of a double-stranded nucleic acid molecule to provide a single- stranded nucleic acid molecule or by lysis or permeabilization of a cell. In some cases, the hybridization may occur in a cell bead comprising a cell. The sequence of the probe that is complementary to the target region may be situated at an end of the probe. Alternatively, this sequence may be disposed between other sequences such that when the probe sequence is hybridized to the target region, additional probe sequences extend beyond the hybridized sequence in one or more directions. The probe sequence that hybridizes to the target region of the nucleic acid molecule may be of the same or different length as the target region. For example, the probe sequence may be shorter than the target region and may only hybridize to a portion of the target region. Alternatively, the probe sequence may be longer than the target region and may hybridize to the entirety of the target region and extend beyond the target region in one or more directions. In addition to a probe sequence complementary to a target region of the nucleic acid molecule, the probe may comprise one or more additional probe sequences. For example, the probe may comprise the probe sequence complementary to the target region and a second probe sequence. The second probe sequence may have any useful length and other characteristics.

[0210] The probe (e.g., the first probe or the second probe) may comprise one or moreadditional sequences or moieties, such as one or more barcode sequences or unique moleculeAttorney Docket: 43487-1036601 identifier (UMI) sequences, adapter sequences, functional sequences (e.g., primer sequences, sequencing primer sequences, etc.). In some cases, one or more probe sequences of the probe may comprise a detectable moiety such as a fluorophore or a fluorescent moiety. In some instances, the first probe or the second probe may comprise a reactive moiety, as described elsewhere herein. For example, the first probe or the second probe may comprise an azide moiety, an alkyne moiety, a phosphorothioate moiety, an iodide moiety, an amine moiety, a phosphate moiety, or a combination thereof. The first probe may comprise a first reactive moiety and the second probe may comprise a second reactive moiety, and reaction of the first reactive moiety and the second reactive moiety may be sufficient to yield a probe-linked molecule comprising the first probe linked to the second probe. In some instances, the first reactive moiety and the second reactive moiety is linked via ligation. Accordingly, the first probe or the second probe may comprise one or more moieties or modified nucleotides to facilitate ligation, e.g., one or more ribonucleotides or dideoxynucleotides (ddNTPs), which may be ligated to a phosphorylated end of the second probe using a ligase (e.g., T4 DNA ligase, SplintR ligase). In some instances, the probe (e.g., the first probe or the second probe) may comprise an overhang or flap sequence which is recognizable or cleavable by an endonuclease (e.g., FEN1 endonuclease). Other suitable enzymes, e.g., ligases, may be used, for example, the enzymes and ligases disclosed in U.S. Provisional App. No.63 / 171,031, filed April 5, 2021, which is incorporated herein by reference in its entirety.

[0211] As described herein, a probe sequence of the probe may be capable of hybridizingwith a sequence of a nucleic acid barcode molecule or a probe binding molecule (e.g., splint oligonucleotide). A nucleic acid barcode molecule may comprise a first binding sequence (e.g., a barcode capture sequence) that is complementary to a probe sequence of the probe (e.g., a probe capture sequence). The nucleic acid barcode molecule may comprise one or more additional functional sequences , e.g., primer sequences, primer annealing sequences, and immobilization sequences. The binding sequences may have any useful length and other characteristics. In some cases, the binding sequence (e.g., barcode capture sequence) that is complementary to a probe sequence of the probe may be the same length as the probe sequence. Alternatively, the binding sequence may be a different length of the probe sequence. For example, the binding sequence may be shorter than the probe sequence and may only hybridize to a portion of the probe sequence. Alternatively, the binding sequence may be longer than the probe sequence and may hybridize to the entirety of the probe sequence and extend beyond the probe sequence in one or more directions. Similarly, in instances when a probe-binding molecule is used, the binding sequence (e.g., barcode capture sequence) of the nucleic acid barcodeAttorney Docket: 43487-1036601 molecule may be the same length as the barcode binding sequence of the probe-binding molecule, or the binding sequence may be longer or shorter than the barcode binding sequence.

[0212] One or more processes described herein may be performed in a cell, nucleus or cellbead. For example, in some embodiments, a plurality of cells, nuclei or cell beads may comprise a plurality of nucleic acid molecules. The cells, nuclei or cell beads may be alive or fixed and / or permeabilized. In some instances, the first probes may be provided to the cells, nuclei or cell beads, such as in a bulk solution. Optionally, the cells, nuclei or cell beads may be washed to remove unbound first probes, and the nucleic acid extension reaction, as described herein, may be performed. Subsequently, the cells, nuclei or cell beads comprising the plurality of nucleic acid molecules (or the extended, probe nucleic acid molecules) may be partitioned into a plurality of separate partitions, where at least a subset of the plurality of separate partitions comprises a single cell, single nucleus, or single cell bead. Access to a target nucleic acid molecule contained within a cell, nucleus or cell bead in a partition may be provided by lysing or permeabilizing the nucleus or cell (e.g., as described herein), which may be performed prior to or during partitioning. Alternatively, access to a target nucleic acid molecule contained within a cell, nucleus, or cell bead may be performed in a bulk solution using the methods described herein. Barcoding of the target nucleic acid may be performed within a cell, within a cell bead, or within a partition comprising the cell or cell bead. In an example, the target nucleic acid molecule may be barcoded in a partition and outside the cell, cell nucleus, or cell bead. Additional probe hybridization (e.g., providing of the second probe) and / or barcoding may be performed within the separate partitions. Barcoding, as described herein, may comprise using a nucleic acid barcode molecule to attach or hybridize to the target nucleic acid molecule or derivative thereof (e.g., the extended probe molecule, or complement thereof). Nucleic acid barcode molecules provided within each partition of the plurality of separate partitions may be provided attached to beads. In some instances, as described elsewhere herein, the nucleic acid barcode molecule may be releasably attached to a bead (e.g., via a labile bond). Each partition (or a subset of partitions) of the plurality of separate partitions may comprise a bead comprising a plurality of nucleic acid barcode molecules attached thereto (e.g., as described herein). The plurality of nucleic acid barcode molecules attached to each bead may comprise a unique barcode sequence, such that each partition of the plurality of separate partitions comprises a different barcode sequence. Upon release of components from the plurality of different partitions of the plurality of separate partitions (e.g., following barcoding), the barcoded molecules arising from a single cell, single nucleus, or single cell bead may have a same barcode sequence (e.g., a common barcode sequence), such that each barcoded nucleic acid molecule can be traced to a given partition and / or, in some instances, a given cell, nucleus or cell bead.Attorney Docket: 43487-1036601

[0213] The methods described herein may comprise additional barcoding operations, whichmay be useful, for example, in indexing nucleic acid molecules to a cell, nucleus, cell bead, a sample, a partition, or a plurality of partitions. Such indexing may be useful in situations when a single partition is occupied by multiple cells, nuclei, or cell beads. In some instances, it may be beneficial to overload partitions such that a partition comprises more than a single cell, single nucleus, or single cell bead; for example, it may be useful in certain situations to overload partitions, e.g., to overcome Poisson loading statistics in partitions and / or to prevent reagent waste (e.g., from unoccupied partitions). Accordingly, such indexing may be useful in attributing cells, nuclei or nucleic acid molecules in multiply-occupied partitions to the originating cell, nucleus, cell bead, partition, sample, etc.

[0214] In an example, a barcoded molecule, such as the barcoded molecules generated usingthe methods described herein (e.g., in FIGs.7-9, FIGs.25-28, as well as barcoded, probe-linked nucleic acid molecules described in U.S. Pat. Pub. No.2020 / 0239874 and International Pub. No. WO 2019 / 165318, each of which is incorporated by reference herein) may be provided. The barcoded molecule may comprise, as described herein, a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence (which may be disposed on the first probe or the second probe), and a sequence corresponding to the barcode sequence of the nucleic acid barcode molecule. Such a barcode sequence may be specific to the partition and may differ from other barcode sequences of other partitions and thus may be used to identify a partition from which a nucleic acid molecule (or derivative thereof) originated. In some instances, some of the partitions may comprise a single cell, single nucleus, or single cell bead and thus the nucleic acid barcode molecule or barcode sequence may be used to identify a cell, nucleus, or cell bead from which a nucleic acid molecule (or derivative thereof) originated.

[0215] In some instances, the barcoded molecule may be subjected to an additionalbarcoding operation, e.g., in partitions or in bulk. For example, the barcoded molecule may be re-partitioned in a partition among a plurality of partitions comprising a plurality of additional nucleic acid barcode molecules. The plurality of additional nucleic acid barcode molecules may comprise additional barcode sequences that differ across the partitions. The barcoded molecules may be subjected to conditions sufficient to barcode the barcoded molecules to generate a combinatorially barcoded molecule comprising two barcode sequences. As each barcode sequence pertains to a unique partition, the combination of barcodes may be useful in generating a greater diversity of barcoded molecules, as well as for identifying the originating partitions of the combinatorially barcoded molecule.Attorney Docket: 43487-1036601

[0216] In some cases, combinatorial assembly of barcode segments may be performed using,e.g., a split-pool approach. For example, in some embodiments, the probe-linked nucleic acidmolecules may be combinatorially barcoded using a split pool approach. In one such example, aplurality of permeabilized cells (or permeabilized nuclei or cell beads) comprising, e.g., probe- linked nucleic acid molecule, which may optionally be barcoded (e.g., the product following operation 709 of FIG.7, 805 of FIG.8, or 905 or 907 of FIG.9) may be partitioned into a plurality of partitions (e.g., a plurality of wells), wherein each partition of the plurality of partitions comprises a different (e.g., unique) barcode sequence segment. Alternatively, the plurality of permeabilized cells (or permeabilized nuclei or cell beads) may be partitioned, and then the different barcode sequence segments delivered to the respective partitions containing the cells, nuclei, and / or cell beads. After addition of the barcode sequence segment, cells (or nuclei or cell beads) can be collected from the plurality of partitions, pooled, and partitioned into an additional plurality of partitions (e.g., a plurality of wells) wherein each partition of the additional plurality of partitions comprises a different (e.g., unique) second barcode sequence segment. Repeating this split-pool process allows the generation of barcodes or barcoded molecules comprising any suitable amount of barcode sequence segments. Combinatorial barcoding as described herein may comprise at least 1, 2, 3, 4, 5, 6, 7, 8 or more operations (e.g., split-pool cycles). Combinatorial barcoding comprising multiple operations may be useful, for example, in generation of greater barcode diversity and to synthesize a unique barcode sequence on nucleic acid molecules derived from each single cell, nucleus, or cell bead of a plurality of cells, nuclei, cell beads. For example, combinatorial barcoding comprising three operations, each comprising attachment of a unique nucleic acid sequence in each of 96 partitions, will yield up to 884,736 unique barcode combinations. Generally, where there are M partitions, and N number of split-pool iterations are performed, up to MNunique barcode combinations may be generated. Cells or nuclei or cell beads may be partitioned such that at least one cell (or nuclei or cell bead) is present in each partition of a plurality of partitions. Cells, nuclei, or cell beads may be partitioned such that at least 1; 2; 3; 4; 5; 10; 20; 50; 100; 500; 1,000; 5,000; 10,000; 100,000; 1,000,000; or more cells, nuclei, or cell beads are present in a single partition. Cells, nuclei, or cell beads may be partitioned such that at most 1,000,000; 100,000; 10,000; 5,000; 1,000; 500; 100; 50; 20; 10; 5; 4; 3; 2; or 1 cell (or nucleus or cell bead) is present in a single partition. Cells, nuclei, and / or cell beads may be partitioned in a random configuration.

[0217] In some instances, the additional barcoding operations may be performed prior tosome of the operations described herein. For example, it may be beneficial to combinatorially barcode the first probe in a bulk solution, e.g., prior to or following generation of the extended probe molecule or probe-linked molecule. In such cases, the nucleic acid molecule may beAttorney Docket: 43487-1036601 contacted, e.g., in bulk, with a first probe to generate a probe-associated molecule. The probe- associated molecule may optionally be extended, e.g., using the methods described herein, to generate an extended probe molecule. The probe-associated molecule or the extended probe molecule may then be subjected to combinatorial barcoding, e.g., in partitions, as described above, to generate a combinatorially barcoded molecule. The combinatorially barcoded molecule may then be partitioned with a second probe and a nucleic acid barcode molecule, which, as described herein, may attach to either the first probe (or combinatorially barcoded probe), the second probe, or both probes. As each partition of the combinatorial barcoding process comprises a different barcode sequence segment, a plurality of the combinatorially barcoded molecules may be traced back to the individual partitions from which they originated. Moreover, the combinatorial barcoding may be useful in generating greater probe diversity.

[0218] Beneficially, the combinatorial barcoding of the first probe may be particularly usefulwhen combined with the second probe and nucleic acid barcode molecule, which may comprise a barcode sequence that is specific to the partition. For example, the presence of the probe- specific barcode(s) and the partition-specific barcode sequence may allow for indexing of individual cells (or nuclei or cell beads) within a partition. For instance, partitions comprising cell / nucleus / cell bead multiplets (e.g., cell doublets, triplets, etc.) can be computationally deconvolved into single cells / nuclei / cell beads. Thus, in some instances, cells, nuclei, or cell beads may be “overloaded” into partitions using conditions such that a higher probability of cell / nucleus / cell bead multiplets (2,3,4,5+ cells, nuclei, or cell beads per partition) are formed, wherein target libraries of these cell multiplets may be computationally deconvolved into single cells, nuclei, or cell beads.

[0219] FIG. 10 schematically shows an example workflow of barcoding nucleic acidmolecules in partitions comprising cell / nucleus / cell bead multiplets. In operation 1010, one or more populations of cells / nuclei / cell beads (or nucleic acid molecules contained therein) may be subjected to barcoding, as described herein (e.g., using processes shown and described in FIGs. 7-9 and FIGs.15-16). For example, a first population of cells (or nuclei or cell beads) 1002 (comprising a first plurality of nucleic acid molecules) may be subjected to barcoding in a first subset of a first plurality of partitions, generating a first plurality of barcoded nucleic acid molecules comprising a first barcode sequence. A second population of cells (or nuclei or cell beads) 1004 may be barcoded in a second subset of the first plurality of partitions, generating a second plurality of barcoded nucleic acid molecules comprising a second barcode sequence. The first barcode sequence may be different than the second barcode sequence. In operation 1020, the first population of cells (or nuclei or cell beads) 1002 may be pooled together with the second population of cells (or nuclei or cell beads) 1004 to generate a mixture of cells. In operationAttorney Docket: 43487-1036601 1030, the mixture of cells (or nuclei or cell beads) may be partitioned into a second plurality of partitions. In some instances, the mixture of cells / nuclei / cell beads may be partitioned into the second plurality of partitions such that some partitions of the second plurality of partitions comprises more than one cell (e.g., a cell multiplet partition). For example, a partition 1035 of the second plurality of partitions may comprise a cell, nucleus, or cell bead (“Cell A”) from the first population of cells 1002 and a cell, nucleus, or cell bead (“Cell B”) from the second population of cells 1004. The partition 1035 may comprise an additional barcode sequence, which may be unique to the partition. The cells / nuclei / cell beads in each partition may be subjected to an additional barcoding operation to append the additional barcode sequence on the barcoded nucleic acid molecules. In operation 1040, the barcoded nucleic acid molecules may be deconvolved, using the different barcode sequences (e.g., the first barcode sequence, the second barcode sequence, and the additional barcode sequences), to identify the originating cell / nucleus / cell bead. For instance, a barcoded nucleic acid molecule comprising the additional barcode sequence from partition 1035 and the first barcode sequence from the first population of cells (or nuclei or cell beads) 1002 may be used to identify that barcoded nucleic acid molecule as originating from Cell A. Similarly, a barcoded nucleic acid molecule comprising the additional barcode sequence from partition 1035 and the second barcode sequence from the second populations of cells (or nuclei or cell beads) 1004 may be used to identify that barcoded nucleic acid molecule as originating from Cell B.

[0220] Following partition-based barcoding, the contents of the partitions may be pooled andthe barcoded molecules (e.g., barcoded probe-linked nucleic acid molecules) may be duplicatedor amplified by, for example, one or more amplification reactions, which may in some instances be isothermal. The amplification reactions may comprise polymerase chain reactions (PCR) and may involve the use of one or more primers or polymerases. The one or more primers may comprise one or more functional sequences (e.g., a primer sequence / primer binding sequence, a sequencing primer sequence (e.g., R1 or R2), a partial sequencing primer sequence (e.g., partial R1 or partial R2), a sequence configured to attach to the flow cell of a sequencer (e.g., P5 or P7, or partial sequences thereof), etc.) and may facilitate addition of said one or more functional sequences to the extended nucleic acid molecule. The barcoded molecules, or derivatives thereof, may be detected via nucleic acid sequencing (e.g., as described herein).

[0221] In some aspects, provided herein are systems useful for barcoding nucleic acidmolecules. The systems may comprise any of the components described herein, e.g., a plurality of partitions (e.g., droplets, wells), which may be provided in any useful format, e.g., a microfluidic device, a multi-well array or plate, etc. The systems may include nucleic acid barcode molecules, optionally coupled to supports (e.g., particles, beads, gel beads, etc.). InAttorney Docket: 43487-1036601 some instances, the systems may comprise any of the probes described herein, such as a first probe or plurality of first probes, a second probe or plurality of second probes, and any useful reaction components (e.g., for performing a nucleic acid reaction, e.g., extension, ligation, amplification, etc.). Such useful reaction components can include, in non-limiting examples, enzymes (e.g., ligases, polymerases, reverse transcriptases, restriction enzymes, etc.), nucleotides bases, etc.

[0222] Also provided herein are compositions useful for systems and methods for barcodingnucleic acid molecules. A composition may comprise any of the probes described herein. For example, a composition may comprise a plurality of first probes, a plurality of second probes, and / or a plurality of first probes and a plurality of second probes. A probe or a set of probes may be designed to target a specific sequence or a set of specific sequences. Such probes may be designed to have the same or different sequences within different partitions. For example, a first composition may comprise a first probe and a second probe designed to target two regions of a first gene, and a second composition may comprise a first probe and a second probe designed to target two regions of a second gene, which second gene is different than the first gene. A composition may comprise nucleic acid barcode molecules, and / or probe binding molecules, which may optionally be provided coupled to a support (e.g., particle, bead). A composition may be a part of or comprise a reaction mixture, which can include reaction components or reagents, e.g., enzymes, nucleotide bases, catalysts, buffers etc. Multiplexed analysis of nucleic acids and proteins

[0223] In another aspect, the present disclosure provides methods for performingmultiplexed assays. Such a multiplexed assay may comprise assaying or analyzing one or more biomolecules (e.g., nucleic acid molecules, proteins, lipids, carbohydrates, etc.). A method may comprise using one or more probes and a nucleic acid barcode molecule to barcode a nucleic acid molecule of a cell / nucleus / cell bead, thereby generating a first barcoded nucleic acid molecule; attaching or coupling a feature-binding group to a feature of the cell / nucleus / cell bead, wherein the feature-binding group comprises a reporter oligonucleotide comprising a reporter sequence that identifies the feature-binding group; using an additional nucleic acid barcode molecule, and optionally, an additional probe, to barcode the reporter sequence to generate a second barcoded nucleic acid molecule; and optionally barcoding the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule to generate a third barcoded nucleic acid molecule and a fourth barcoded nucleic acid molecule. One or more operations may be performed within a partition (e.g., droplet or well).Attorney Docket: 43487-1036601

[0224] The methods described herein may facilitate profiling of one or more biomoleculeswith single-cell / single nucleus / single cell bead resolution, using, for example, probe hybridization, feature binding groups (e.g., antibodies, antibody fragments, epitope-binding groups, etc.), barcoding, amplification, and sequencing. The methods may be useful in providing genomic, transcriptomic, proteomic, exomic, or other “-omic” information from a single cell / nucleus / cell bead. As described herein, the methods may be used to analyze a pre- determined panel of target genes and a pre-determined panel of target features (e.g., proteins, peptides, or other biomolecules) in a sensitive and accurate manner. Alternatively, or in addition to, the methods may be used to analyze whole genomic, whole transcriptomic, whole exomic, etc. characteristics of a cell.

[0225] In some aspects, the methods comprise contacting a cell / nucleus / cell bead with a firstprobe, a second probe, and a third probe under conditions sufficient to generate a first probe- associated molecule and a second probe-associated molecule. The cell / nucleus / cell bead may comprise (i) a nucleic acid molecule (e.g., a target nucleic acid molecule such as RNA or DNA) comprising a first target region and a second target region and (ii) a feature (e.g., protein, peptide, or other biomolecule) coupled to a feature-binding group. The feature binding group may comprise or be coupled to (i) a reporter oligonucleotide comprising a reporter sequence, which may be associated with the feature or may be used to identify the feature, and (ii) a feature probe-binding sequence. The first probe may comprise a first probe sequence complementary to the first target region of the nucleic acid molecule and, optionally, an additional probe sequence, such as a probe capture sequence or other functional sequence. The second probe may comprise a second probe sequence complementary to the second target region and, optionally, a probe capture sequence or functional sequence. The third probe may comprise (i) a third probe sequence complementary to the feature probe-binding sequence and (ii) a probe capture sequence or functional sequence, which may be the same sequence as the probe capture sequence of the first probe and / or second probe.

[0226] In some instances, the first probe-associated molecule may comprise the nucleic acidmolecule, the first probe, the second probe, or combinations or complements thereof. The second probe-associated molecule may comprise the reporter oligonucleotide (which comprises the reporter sequence) and the third probe, or complements thereof.

[0227] In some aspects, the method comprises providing the first probe-associated moleculeand the second probe-associated molecule, and barcoding the first probe-associated molecule and the second probe-associated molecules. Such barcoding operations may occur in a first set of partitions (e.g., droplets or wells). Such an example method may comprise contacting the first probe-associated molecule and the second-probe-associated molecule with probe bindingAttorney Docket: 43487-1036601 molecules (e.g., a splint oligonucleotide) and barcode molecules (e.g., nucleic acid barcode molecules) under conditions sufficient to generate a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule. The barcode molecules may comprise (i) a barcode capture sequence, e.g., a common sequence that is common to a plurality of barcode molecules and (ii) a first barcode sequence. In instances where partitions are used, the first barcode sequence may be unique to a first partition of a first set of partitions, and the barcode molecules within the first partition may share the same first barcode sequence. The probe-binding molecule may comprise (i) a probe-binding sequence complementary to the probe capture sequence (of the first probe, the second probe, and / or the third probe) and (ii) a barcode binding sequence complementary to the barcode capture sequence (e.g., common sequence) of the plurality of barcode molecules. As such, barcoding of the first probe-associated molecule and the second probe-associated molecule may comprise hybridization of the probe binding molecule to (i) the probe capture sequence (or complement thereof) of the first probe, the second probe, and / or the third probe, and (ii) the barcode capture sequence (or common sequence) of the nucleic acid barcode molecule. In some examples, the first barcoded nucleic acid molecule comprises a sequence corresponding to the first probe sequence, a sequence corresponding to the second probe sequence, and a sequence corresponding to the first barcode sequence. Similarly, the second barcoded nucleic acid molecule may comprise a sequence corresponding to the reporter sequence, a sequence corresponding to the third probe sequence, and a sequence corresponding to the first barcode sequence.

[0228] The method may further comprise providing a second set of partitions, and in asecond partition of the second set of partitions, (i) contacting the first barcoded nucleic acid molecule, or derivative thereof (e.g., complements, amplicons, extension products thereof), to a first capture molecule of a plurality of capture molecules under conditions sufficient to generate a third barcoded nucleic acid molecule, and (ii) contacting the second barcoded nucleic acid molecule, or derivative thereof, to a second capture molecule of the plurality of capture molecules under conditions sufficient to generate a fourth barcoded nucleic acid molecule. The plurality of capture molecules may each comprise a second barcode sequence, which may be the same or different than the first barcode sequence from the first set of partitions. The second barcode sequence may be unique to the partition (e.g., differ across partitions). The third barcoded nucleic acid molecule and the fourth barcoded molecule may each comprise a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence. For example, the third barcoded nucleic acid molecule may comprise a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to a probe capture sequence, the first barcode sequence and the secondAttorney Docket: 43487-1036601 barcode sequence. The fourth barcoded nucleic acid molecule may comprise a sequence corresponding to the reporter sequence, a sequence corresponding to the feature probe binding sequence, a sequence corresponding to the third probe, the first barcode sequence and the second barcode sequence.

[0229] The feature binding group may comprise a labelling agent, as described elsewhereherein. Accordingly, the feature binding group may comprise, in some examples, an antibody or antibody fragment, an epitope binding moiety, a protein, a peptide, a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi- specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof.

[0230] The probe capture sequence of the first probe (or the second probe) may be commonto a plurality of first probes (or second probes), a plurality of partitions, and / or a plurality of cells / nuclei / cell beads. For instance, the first set of partitions may comprise one or more additional partitions that comprise additional probe-associated nucleic acid molecules. The additional probe-associated nucleic acid molecules may comprise identical sequences (e.g., first probe sequence, second probe sequence) to the probe-associated nucleic acid molecule of the first partition, or the additional probe-associated nucleic acid molecules of the additional partitions may comprise different sequences (e.g., different probe sequences) than the probe- associated nucleic acid molecule of the first partition. In some instances, each of the one or more additional probe-associated nucleic acid molecules comprises a probe capture sequence, which may be identical or different across the first set of partitions.

[0231] The probe-associated molecules may be a probe-linked molecule. For example, theprobe-associated molecules may be the probe-associated molecules or barcoded molecules described herein (e.g., in FIGS.7-9), or a probe-linked molecule, such as those described in U.S. Pat. Pub. No.2020 / 0239874 and International Pub. No. WO 2019 / 165318, each of which is incorporated by reference herein in its entirety. In some examples, two sets of probe-associated molecules may be generated, in which: (i) a first probe-associated molecule comprises the nucleic acid molecule, with the first probe and the second probe hybridized thereto (e.g., via hybridization of the first probe sequence to the first target region and the second probe sequence to the second target region) and (ii) a second probe-associated molecule comprises the reporter oligonucleotide (which comprises the reporter sequence), with the third probe hybridized thereto.

[0232] The first probe, the second probe, and / or the third probe may comprise a probecapture sequence. The probe capture sequence on the first probe may be the same or different than the probe capture sequence of the second probe or the third probe. Similarly, the probeAttorney Docket: 43487-1036601 capture sequence of the second probe may be the same or different than the probe capture sequence of the third probe. Accordingly, the barcoding operations described herein may occur on the first probe, the second probe, the third probe, or any combination thereof. For example, for a probe-associated molecule comprising a nucleic acid molecule and the first probe (“probe 1”) and second probe (“probe 2”) hybridized thereto, a first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the first probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising a second barcode sequence (“BC2”) may be annealed to a region of the first barcode molecule, thereby generating a molecule comprising a sequence, or complementary sequences, of BC2-BC1-probe 1-probe 2. Alternatively or in addition to, the first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the second probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising the second barcode sequence (“BC2”) may be annealed to a region of the first barcode molecule, thereby generating a molecule comprising a sequence of probe 1-probe 2-BC1-BC2. Alternatively, or in addition to, the barcode molecules and the capture molecules may be annealed to different probes. For example, the first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the first probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising the second barcode sequence (“BC2”) may be annealed to the second probe, thereby generating a molecule comprising a sequence of BC1- probe 1-probe 2-BC2. Alternatively or in addition to, the first barcode molecule comprising the first barcode sequence (“BC1”) may hybridize (e.g., directly or via a probe-binding molecule) to the second probe to generate a first barcoded nucleic acid molecule, and subsequently, a capture molecule comprising the second barcode sequence (“BC2”) may be annealed to the first probe, thereby generating a molecule comprising a sequence of BC2-probe 1-probe 2-BC1. It will be appreciated that while several examples of barcoding schemes are described herein, additional combinations and positioning of barcode sequences are possible; for example, combinatorial barcoding may be used to generate greater barcode diversity, as described herein, and such barcoding may occur on any of the probe molecules (or already barcoded molecules).

[0233] In some instances, the barcode molecules may comprise a capture-binding sequencecomplementary to a capture sequence of the plurality of capture molecules. For example, the first probe may comprise a probe capture sequence which may hybridize to a probe binding molecule, which may mediate hybridization of the barcode molecule (e.g., via hybridization of the barcode binding sequence of the probe binding molecule to the barcode capture sequence (e.g., common sequence) of the barcode molecule). The barcode molecule may additionally comprise theAttorney Docket: 43487-1036601 capture-binding sequence, which may allow for hybridization of the capture sequence of the capture molecules to the barcode molecule.

[0234] FIGs. 15A-15C schematically illustrate an example barcoded nucleic acid moleculeas described herein. Referring to FIG.15A, a nucleic acid molecule (e.g., RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 may be provided. The nucleic acid molecule 1500 may be contacted with a first probe 1506 comprising a first probe sequence 1508 and, optionally, a first probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target region 1502. The first probe capture sequence 1510 may additionally, in some instances, comprise a functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc. The nucleic acid molecule 1500 may also be contacted with a second probe 1516 comprising a second probe sequence 1514 and, optionally, a second probe capture sequence 1518. The second probe sequence 1514 may be complementary to the second target region 1504. The second probe capture sequence 1518 may additionally comprise a functional sequence. Hybridization of the first probe 1506 and the second probe 1516 to the nucleic acid molecule 1500 may generate a probe-associated molecule.

[0235] As described herein, the probe-associated molecule may be subjected to one or morebarcoding operations. Such a barcoding operation may occur in one or more partitions (e.g., a first set of partitions) and may include hybridizing a probe binding molecule 1517 and a barcode molecule 1519 comprising a barcode capture sequence (e.g., a common sequence), to the probe- associated molecule. In some instances, the probe binding molecule 1517 and the barcode molecule 1519 may be provided as a pre-annealed complex, or they may be provided as separate molecules. The barcode capture sequence (e.g., common sequence) may be a sequence that is common to the plurality of barcode molecules in the first set of partitions, or the common sequence may be unique to the barcode molecules in only a single first partition (e.g., the common sequence differs across partitions of the first set of partitions). The probe binding molecule 1517 may comprise a probe binding sequence complementary to the probe capture sequence 1518 of the second probe 1516, as well as a barcode binding sequence complementary to a sequence of the barcode molecule 1519. The probe-associated molecule may be subjected to conditions sufficient to generate a first barcoded nucleic acid molecule, which can include annealing of the probe-binding molecule 1517 to (i) the probe capture sequence 1518 and (ii) the barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The barcoding process may comprise additional operations, such as ligation, which may be performed chemically or enzymatically, as described elsewhere herein.Attorney Docket: 43487-1036601

[0236] The first barcoded nucleic acid molecule or derivatives thereof (e.g., a complement,an amplicon, an extension product, a combinatorially barcoded nucleic acid molecule, as described elsewhere herein), may be subjected to a second barcoding operation. Such a second barcoding operation may occur in a second set of partitions. For example, the first barcoded nucleic acid molecule may be removed from the first set of partitions, pooled (e.g., with other barcoded nucleic acid molecules from other first partitions of the first set of partitions), and partitioned in a second partition of a second set of partitions. The second partition may comprise a capture molecule 1520. The capture molecule 1520 may comprise a second barcode sequence and a sequence complementary to the probe capture sequence 1510 of the first probe 1506. The second barcode sequence may be a sequence that is common to the plurality of capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules in only the second partition (e.g., differ across partitions). The capture molecule 1520 may hybridize to the probe capture sequence 1510 to generate an additional barcoded molecule (also referred to herein as a “third barcoded nucleic acid molecule”). The additional barcoded molecule may comprise a sequence corresponding to the first barcode sequence (of the barcode molecule 1519), and a sequence corresponding to the second barcode sequence (of the capture molecule 1520).

[0237] FIG. 15B schematically illustrates another example barcoded molecule in which thecapture molecule 1520 is hybridized to the barcode molecule 1519. Similar to FIG.15A, in FIG. 15B, the nucleic acid molecule (e.g., RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 may be provided. The nucleic acid molecule 1500 may be contacted with a first probe 1506 comprising a first probe sequence 1508 and a probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target region 1502. The probe capture sequence 1510 may additionally comprise a functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc. The nucleic acid molecule 1500 may also be contacted with a second probe 1516 comprising a second probe sequence 1514 and, optionally, an additional sequence 1518. The second probe sequence 1514 may be complementary to the second target region 1504. The additional sequence 1518 may comprise, for instance, a probe capture sequence, or a functional sequence (e.g., primer, primer binding site, sequencing primer sequence, etc.). Hybridization of the first probe 1506 and the second probe 1516 to the nucleic acid molecule 1500 may generate a probe-associated molecule.

[0238] The probe-associated molecule may be contacted with one or more barcodemolecules. Such barcoding operations, as described herein, may occur in a plurality of partitions (e.g., a first partition of a first set of partitions and / or a second partition of a second set ofAttorney Docket: 43487-1036601 partitions). The probe-associated molecule may be contacted with a probe binding molecule 1517 and a barcode molecule 1519, which may comprise a first barcode capture sequence (e.g., a common sequence) and a second barcode capture sequence 1521 (also referred to herein as “capture binding sequence”). In some instances, the probe binding molecule 1517 and the barcode molecule 1519 may be provided as a pre-annealed complex or as separate molecules. The first barcode capture sequence (e.g., common sequence) may be a sequence that is common to the plurality of barcode molecules in the first set of partitions, or the common sequence may be unique to the barcode molecules in only the first partition (e.g., differ across partitions). The probe binding molecule 1517 may comprise a probe binding sequence complementary to the probe capture sequence 1510 as well as a barcode binding sequence complementary to the first barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The probe- associated molecule may be subjected to conditions sufficient to generate a first barcoded nucleic acid molecule, which can include annealing of the probe-binding molecule 1517 to (i) the probe capture sequence 1510 and (ii) the first barcode capture sequence (e.g., common sequence) of the barcode molecule 1519. The barcoding process may comprise additional operations, such as ligation, which may be performed chemically or enzymatically, as described elsewhere herein.

[0239] The first barcoded nucleic acid molecule or derivatives thereof, may be subjected to asecond barcoding operation. Such a second barcoding operation may occur in a second set of partitions. For example, the first barcoded nucleic acid molecule may be removed from the first partition and partitioned in a second partition of a second set of partitions (e.g., droplets). The second partition may comprise a capture molecule 1520. The capture molecule 1520 may comprise a second barcode sequence and a sequence complementary to the second barcode capture sequence 1521 of the barcode molecule 1519. The second barcode sequence may be a sequence that is common to the plurality of capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules in only the second partition (e.g., differ across partitions). The capture molecule may hybridize to the second barcode capture sequence 1521 to generate an additional barcoded molecule (also referred to herein as a “third barcoded nucleic acid molecule”). The additional barcoded molecule may comprise a sequence corresponding to the first barcode sequence (of the barcode molecule 1519), and a sequence corresponding to the second barcode sequence (of the capture molecule 1520).

[0240] FIG. 15C illustrates another example barcoded nucleic acid molecule. A nucleic acidmolecule (e.g., RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 may be provided. The nucleic acid molecule 1500 may be contacted with a first probe 1506 comprising a first probe sequence 1508 and, optionally, a first probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target regionAttorney Docket: 43487-1036601 1502. The first probe or first probe capture sequence 1510 may additionally, in some instances, comprise a functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc. The nucleic acid molecule 1500 may also be contacted with a second probe 1516 comprising a second probe sequence 1514 and, optionally, a second probe capture sequence 1518. The second probe sequence 1514 may be complementary to the second target region 1504. The second probe capture sequence 1518 may additionally comprise a functional sequence. Hybridization of the first probe 1506 and the second probe 1516 to the nucleic acid molecule 1500 may generate a probe-associated molecule or complex.

[0241] As described herein, the probe-associated molecule may be subjected to one or morebarcoding operations. Such a barcoding operation may occur in one or more partitions (e.g., a first set of partitions) and may include hybridizing a probe binding molecule 1517 and a barcode molecule 1519 comprising a barcode capture sequence (e.g., a common sequence), to the probe- associated molecule or complex. In some instances, the probe binding molecule 1517 and the barcode molecule 1519 are provided as a pre-annealed complex (e.g., a partially double-stranded molecule comprising the probe binding molecule 1517 and the barcode molecule 1519), or they may be provided as separate molecules, which may separately anneal to the probe-associated molecule or complex (e.g., the probe binding molecule 1517 may hybridize to the probe- associated molecule or complex, e.g., via the second probe capture sequence 1518, and the barcode molecule 1519 may hybridize to the probe binding molecule 1517). The barcode capture sequence (e.g., common sequence) may be a sequence that is common to the plurality of barcode molecules in the first set of partitions, or the common sequence may be unique to the barcode molecules in only a single first partition (e.g., the common sequence differs across partitions of the first set of partitions). The probe binding molecule 1517 may comprise a probe binding sequence complementary to the probe capture sequence 1518 of the second probe 1516, as well as a barcode binding sequence complementary to a sequence of the barcode molecule 1519. In some instances, the probe binding molecule 1517 and / or the barcode molecule 1519 comprise an additional sequence, e.g., an adapter sequence, a primer sequence (e.g., sequencing primer sequence or partial sequencing primer sequence), a UMI, a sample index sequence, etc. In some instances, the probe binding molecule 1517 comprises the entire sequence of the barcode molecule 1519, such that no overhang remains. In some instances, the probe binding molecule 1517 and barcode molecule 1519 comprise a sample index sequence, which may be useful in identifying the partition, cell, nucleus, or cell bead from which the target nucleic acid molecule 1500 originates. The probe-associated molecule may be subjected to conditions sufficient to generate a first barcoded nucleic acid molecule, which can include annealing of the probe- binding molecule 1517 to (i) the probe capture sequence 1518 and (ii) the barcode captureAttorney Docket: 43487-1036601 sequence (e.g., common sequence) of the barcode molecule 1519. The barcoding process may comprise additional operations, such as ligation (e.g., ligation of the barcode molecule 1519 to the probe capture sequence 1518), which may be performed chemically or enzymatically, as described elsewhere herein.

[0242] The first barcoded nucleic acid molecule or derivatives thereof (e.g., a complement,an amplicon, an extension product, a combinatorially barcoded nucleic acid molecule, as described elsewhere herein), may be subjected to a second barcoding operation. Such a second barcoding operation may occur in a second set of partitions. For example, the first barcoded nucleic acid molecule may be removed from the first set of partitions, pooled (e.g., with other barcoded nucleic acid molecules from other first partitions of the first set of partitions), and partitioned in a second partition of a second set of partitions. The second partition may comprise a capture molecule 1520. The capture molecule 1520 may comprise a second barcode sequence and a sequence complementary to the probe capture sequence 1510 of the first probe 1506 (and / or the second probe 1516). The second barcode sequence may be a sequence that is common to the plurality of capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules in only the second partition (e.g., differ across partitions). The capture molecule 1520 may hybridize to the probe capture sequence 1510 to generate an additional barcoded molecule (also referred to herein as a “third barcoded nucleic acid molecule”). The additional barcoded molecule may comprise a sequence corresponding to the first barcode sequence (of the barcode molecule 1519), and a sequence corresponding to the second barcode sequence (of the capture molecule 1520).

[0243] In addition to barcoding of nucleic acid molecules, the present disclosure provides formethods of multiplexed analysis, e.g., processing of additional biomolecule types, such as proteins and peptides. The method may comprise providing a feature-binding group (e.g., antibody, protein, binding moiety, etc.), which may couple to or bind to a feature (e.g., protein, peptide) of a cell, nucleus or cell bead. Such a method may comprise providing a cell, nucleus or cell bead having a feature of interest (e.g., protein) and contacting the cell, nucleus or cell bead with the feature-binding group. The feature-binding group may couple to the feature of interest. The feature-binding group may comprise a reporter oligonucleotide comprising a reporter sequence coupled thereto, which may be specific for a particular feature and thus be used to identify the feature. For example, the feature-binding group may be an antibody and the reporter oligonucleotide may comprise a reporter sequence that identifies the antigen or binding moiety (e.g., epitope, epitope fragment) to which the antibody couples or binds. Alternatively, or in addition to, the feature binding group may comprise a feature probe binding sequence, which may be used for downstream probe-binding and / or barcoding. Following the contacting of theAttorney Docket: 43487-1036601 cell (nucleus or cell bead) with the feature binding group, the cell / nucleus / cell bead may comprise the feature coupled to the feature binding group.

[0244] In some instances, the methods described herein may additionally comprise:providing a cell, nucleus or cell bead comprising (i) the nucleic acid molecule comprising the first target region and the second target region and (ii) the feature coupled to the feature binding group and contacting the cell, nucleus or cell bead with a plurality of probes. The cell / nucleus / cell bead may be contacted (e.g., in a first partition) with a first probe, a second probe, and a third probe. As described herein, the first probe and the second probe may associate with the first target region and the second target region of the nucleic acid molecule, thereby generating a first probe-associated molecule. Similarly, the third probe may associate with (e.g., via hybridization) with the feature binding group, thereby generating a second probe-associated molecule. In some instances, the third probe may comprise a third probe sequence that is complementary to the feature probe binding sequence, and in some instances, the third probe may additionally comprise a probe capture sequence. The first probe and / or the second comprise may also comprise a probe capture sequence, which may be the same or different than the probe capture sequence of the third probe.

[0245] In the first set of partitions, the first probe-associated molecule (e.g., the nucleic acidmolecule with the first probe and the second probe associated therewith) and the second-probe- associated molecule (e.g., the feature binding group with the third probe associated therewith) may be barcoded. Such a barcoding operation may comprise, for example, providing barcode molecules comprising a first barcode sequence and a barcode-capture sequence such as a common sequence, which may hybridize directly with the first probe-associated molecule and the second probe-associated molecule, e.g., via the probe capture sequences. Alternatively or in addition to, the barcode molecules may be provided with probe-binding molecules which comprise (i) a probe binding sequence complementary to the probe capture sequence of the first probe, the second probe, and / or the third probe and (ii) a barcode binding sequence, which may be complementary to the common sequence of the barcode molecules. In some instances, the probe binding molecules and the barcode molecules may be provided as a pre-annealed complex. Barcoding of the first probe-associated molecule and the second probe-associated molecule may include hybridization of the barcode molecules (e.g., the barcode capture sequence such as a common sequence) to a portion (e.g., the probe capture sequence) of the first probe-associated molecule and the second probe-associated molecule, or the barcoding may include hybridization of the barcode molecules to the probe binding molecule and hybridization of the probe binding molecule to the first probe-associated molecule or the second probe-associated molecule.Attorney Docket: 43487-1036601 Additional operations such as ligation (e.g., enzymatic or chemical ligation) may be performed to generate the first barcoded molecule and the second barcoded molecule.

[0246] The first barcoded molecule and the second barcoded molecule may be subjected toadditional barcoding operations, e.g., in a second set of partitions. Such additional barcoding operations may include: contacting the first barcoded nucleic acid molecule or derivative thereof to a first capture molecule of a plurality of capture molecules to generate a third barcoded nucleic acid molecule and contacting the second barcoded nucleic acid molecule or derivative thereof to a second capture molecule of the plurality of capture molecules to generate a fourth barcoded nucleic acid molecule. The capture molecules within a partition may each comprise a second barcode sequence, which may be unique to the partition (e.g., differ across partitions). Accordingly, both the third barcoded nucleic acid molecule and the fourth barcoded nucleic acid molecule may comprise a first barcode sequence (or complement thereof) and a second barcode sequence (or complement thereof).

[0247] FIG. 16A schematically illustrates an example workflow for barcoding multipleanalytes of a cell, nucleus or cell bead. The cell, nucleus or cell bead 1600 may comprise a nucleic acid molecule (e.g., RNA molecule or other target nucleic acid molecule) 1601 comprising a first target region 1602 and a second target region 1604. The cell, nucleus or cell bead may additionally comprise a feature (e.g., a protein, such as a cell surface receptor (or nuclear membrane protein) or an intracellular / intranuclear protein) 1650. In some instances, the cell, nucleus or cell bead 1600 may be processed, e.g., fixed, permeabilized, treated with a treatment, etc. In some instances, such processing may include providing one or more feature binding groups (e.g., antibodies, antibody fragments, etc.) 1652, which may couple to the feature 1650. The feature binding group 1652 may comprise or be coupled to a reporter oligonucleotide 1657, which may comprise a reporter sequence 1654. The reporter sequence 1654 may be indicative of the feature binding group 1652 or feature 1650. For instance, the reporter sequence 1654 may be pre-indexed or assigned to a particular antibody or other feature binding group, such that presence of the reporter sequence 1654 indicates presence of the particular feature 1650 in a sample. The feature binding group 1652 or the reporter oligonucleotide 1657 may also comprise or be coupled to feature probe binding sequence 1656. In some instances, the cell, nucleus or cell bead 1600 may be contacted with the feature binding group 1652 and fixed, e.g., either in addition to or alternatively to a fixation and permeabilization operation before the contacting.

[0248] In some cases, the analysis of both intracellular and / or intranuclear proteins andmembrane proteins of a cell (or nucleus) can be performed. In one embodiment, a permeabilized (and optionally fixed) cell (or nucleus) may be contacted with (i) one or more feature bindingAttorney Docket: 43487-1036601 groups (or labeling agents) that are configured to couple to intracellular proteins (or intranuclear proteins) and / or (ii) one or more feature binding groups (or labeling agents) that are configured to couple to cell membrane proteins (or nuclear membrane proteins). As further described herein, permeabilization may involve partially or completely dissolving or disrupting a cell membrane (or nuclear membrane) or a portion thereof. Permeabilization may be achieved by, for example, contacting a cell membrane (or a nuclear membrane) with an organic solvent (e.g., methanol) or a detergent such as Triton X-100 or NP-40. The cell, nucleus, or cell bead may be fixed, as described elsewhere herein.

[0249] Referring again to FIG. 16A, a second feature binding group (or labeling agent)similar to 1652 (not shown) can be used to couple to an intracellular feature, such as an intracellular protein, and comprise or be coupled to a second reporter oligonucleotide, which may comprise a second reporter sequence. The second reporter sequence may be indicative ofthe second feature binding group or the intracellular feature. For instance, the second reportersequence may be pre-indexed or assigned to a particular antibody or other feature binding group, such that presence of the second reporter sequence indicates presence of the particular intracellular feature in a sample. The second feature binding group or the second reporter oligonucleotide may also comprise or be coupled to a second feature probe binding sequence, similar to that of 1656.

[0250] The cell, nucleus or cell bead 1600 may be contacted with a first probe 1606, asecond probe 1616, and a third probe 1658, under conditions sufficient to generate a first probe- associated molecule (or probe-associated complex) 1630 and a second probe-associated molecule (or probe-associated complex) 1665. The first probe-associated molecule 1630 may be or comprise a probe-linked molecule, as described elsewhere herein. For example, the first probe-associated molecule 1630 (or probe-linked molecule) may be any of the probe-associated molecules or probe-linked molecules described herein (e.g., generated from an extended probe, a barcoded extended probe, etc.). The first probe 1606 may comprise a first probe sequence 1608 and, optionally, a probe capture sequence 1610. The first probe sequence 1608 may be complementary to the first target region 1602. The second probe 1616 may comprise a second probe sequence 1615 and, optionally, a probe capture sequence 1618. The second probe sequence 1615 may be complementary to the second target region 1604. The third probe 1658 may comprise a third probe sequence 1660 and a probe capture sequence 1662. The third probe sequence 1660 may be complementary to the feature probe binding sequence 1656. In some instances, the probe capture sequence 1662 is the same probe capture sequence as the probe capture sequences 1610, 1618 of the first probe and / or the second probe, respectively.Attorney Docket: 43487-1036601

[0251] In one embodiment, the cell, cell bead or nucleus 1600 may be further contacted withadditional probes under conditions to generate additional probe-associated molecules or probe-associated complexes. The additional probe-associated molecule(s) may be or comprise a probe-linked molecule, as described elsewhere herein. For example, the additional probe-associatedmolecule(s) or probe-linked molecule(s) may be any of the probe-associated molecules or probe-linked molecules described herein (e.g., generated from an extended probe, a barcoded extended probe, etc.). In one embodiment, the cell (or cell bead or nucleus) 1600 may be further contacted with a fourth probe (not shown) similar to 1658 which comprises (i) a fourth probe sequence similar to 1660 and (ii) a fourth probe capture sequence similar to 1662. The fourth probe sequence may be complementary to the second feature probe binding sequence, as further described herein. In some instances, the fourth probe capture sequence is the same probe capture sequence as the probe capture sequences 1610, 1618 of the first probe and / or the second probe, respectively.

[0252] In one embodiment, the cell, nucleus or cell bead 1600 may be partitioned into a firstpartition of a first set of partitions prior to any processing operations described above including, without limitation, fixing, permeabilizing, contacting with probes, and generating probe- associated or probe-linked molecules. In another embodiment, the cell, nucleus or cell bead 1600 may be fixed and optionally permeabilized prior to partitioning in the first partition and then subsequently processed in the first partition, e.g., contacting with probes and generating molecules.

[0253] In operation 1670, the cell, nucleus or cell bead 1600 comprising the first probe-associated molecule 1630 and the second probe-associated molecule 1665 may be partitioned into a first partition of a first set of partitions or further processed in the first partition. In another embodiment, the cell, cell bead or nucleus 1600 may further comprise additional probe- associated molecules or complexes. For instance, referring to FIG.16A, 1600 may comprise a third probe-associated complex (not shown) that is similar to 1665 but comprises (i) a fourth probe comprising a fourth probe sequence complementary to the second feature probe binding sequence and (ii) a reporter oligonucleotide (similar to 1657) as further described herein. The reporter oligonucleotide may be provided as part of or coupled to the second feature binding group, e.g., a feature binding group configured to couple to an intracellular protein. In some instances, the cell, nucleus or cell bead 1600 may be subjected to processing within the partition, such as lysis, to release the cellular / nuclear components (e.g., the first probe-associated molecule and the second probe-associated molecule) within the partition. Alternatively, the cell, nucleus or cell bead 1600 may remain intact. In one embodiment, the cell bead is processed to release cellular components while keeping the cell bead intact. Within the first partition, a probeAttorney Docket: 43487-1036601 binding molecule 1617 and a barcode molecule 1619 may be provided. The first probe- associated molecule 1630 and the second probe-associated molecule 1665 may be contacted with one or more probe binding molecules 1617 and barcode molecules 1619. In some examples, the first partition further comprises one or more additional probe-associated molecules or complexes similar to 1665 (not shown). The additional probe-associated complex may comprise the third probe-associated complex described above, which comprises a fourth probe and a reporter oligonucleotide for a second feature binding group, e.g., a feature binding group configured to couple to an intracellular protein. Additional probe-associated complexes, such as the third probe-associated complex, may be contacted with one or more probe binding molecules 1617 and barcode molecules 1619. In one embodiment, the contacting of a cell, nucleus or cell bead 1600 in the first partition with one or more probe binding molecules may be simultaneously as the contacting with the probes (e.g., 1606, 1616, 1658 and optionally the fourth probe) as described above. The barcode molecules 1619 may comprise a barcode capture sequence or a common sequence common to a plurality of barcode molecules and a first barcode sequence common to the first partition of the first set of partitions. The nucleic acid barcode molecule may, in some instances, be coupled to a bead, such as a gel bead, or other support, as described herein, and can comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence), additional barcode sequence segments, etc.. The probe binding molecules 1617 may comprise a probe binding sequence complementary to any or a combination of the probe capture sequences1610, 1618, 1662, a fourth probe capture sequence, and a barcode binding sequencecomplementary to the common sequence of the barcode molecule 1619. In some instances, the probe binding molecules 1617 and the barcode molecules 1619 may be provided as a pre- annealed complex. The probe binding molecules 1617 and the barcode molecules 1619 may hybridize to the first probe-associated molecule 1630 and the second probe-associated molecule 1665 and / or an additional probe-associated complex, such as the third probe-associated complex (e.g., via hybridization of the probe binding molecules 1617 to the probe capture sequences 1610, 1618, 1662, and the fourth probe capture sequence), thereby generating a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule, and optionally additional barcoded nucleic acid molecules. Additional processing may occur within the first partition, e.g., ligation of the barcode molecules 1619 to the probes (1606, 1616, 1658 or the fourth probe). In one additional embodiment, the additional barcoded nucleic acid molecule is generating using an additional probe-associated complex, e.g., the third probe-associated complex (not shown), probe binding molecules 1617 and barcode molecules 1619.Attorney Docket: 43487-1036601

[0254] In operation 1680, the contents of each partition or a subset of the first set ofpartitions may be collected from the first set of partitions, e.g., from operation 1670, and re- partitioned into a second set of partitions. The contents of the first set of partitions may comprise the cell, nucleus or cell bead 1600 and / or the processed cellular or nuclear components, e.g., the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and optionally the additional barcoded nucleic acid molecule(s). The contents of the partitions of the first set of partitions may be pooled together and re-distributed to a second set of partitions. Accordingly, a second partition of the second set of partitions may comprise the cell, nucleus or cell bead 1600 and / or the processed cellular / nuclear components. In some instances, the cell, nucleus or cell bead 1600 may be subjected to processing within the second partition, such as lysis, to release the cellular / nuclear components (e.g., the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and optionally the additional barcoded nucleic acid molecule(s)) within the second partition. Alternatively, the cell, nucleus or cell bead 1600 may remain intact. Within the second partition, a plurality of capture molecules 1620 may be provided. In some instances, the plurality of capture molecules 1620 may be coupled to a support (e.g., a particle, bead, gel bead, etc.). In some instances, the plurality of capture molecules 1620 may be releasably coupled to the support and the plurality of capture molecules 1620 may be released in the second partition. The capture molecules 1620 may each comprise a second barcode sequence, which may be the same sequence or a different sequence as the first barcode sequence (of the barcode molecule 1619). The second barcode sequence may be unique to the second partition and differ from the second barcode sequences of other partitions of the second set of partitions. The first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule may each be contacted with a capture molecule 1620. The capture molecules 1620 may comprise a second barcode capture sequence, which may be complementary to a sequence of the barcode molecule 1619. Hybridization of the capture molecules 1620 to the first barcoded molecule and the second barcoded nucleic acid molecule may be sufficient to generate a third barcoded nucleic acid molecule and a fourth barcoded nucleic acid molecule. In addition, hybridization of capture molecules 1620 to the additional barcoded nucleic acid molecule(s), e.g., from additional reporter oligonucleotides 1657 on additional feature binding groups 1652, may be sufficient to generate a fifth barcoded nucleic acid molecule. Alternatively, hybridization of the capture molecules 1620 to the first barcoded molecule and the second barcoded nucleic acid molecule may be sufficient to couple the capture molecule (comprising the second barcode sequence) to both the first barcoded molecule and the second barcoded nucleic acid molecule. In addition, hybridization of a capture molecule 1620 to the additional barcoded nucleic acid molecule may be sufficient to couple the capture molecule (comprising the second barcode sequence) to theAttorney Docket: 43487-1036601 additional barcoded nucleic acid molecule. Optionally, further processing may be performed, e.g., ligation of the capture molecules 1620 to the first barcoded nucleic acid molecule and the second barcode nucleic acid molecule (and optionally the additional barcoded nucleic acid molecule). Following ligation, the first and second barcoded nucleic acid molecule may comprise the capture molecule 1620. The third barcoded nucleic acid molecule, the fourth barcoded nucleic acid molecule, and the fifth barcoded nucleic acid molecule may each comprise a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence. In some instances, an extension reaction is performed (e.g., from the capture molecule 1620 toward the reporter oligonucleotide sequence 1657) to generate the fourth barcoded molecule and / or the fifth barcoded nucleic acid molecule. FIG.16B schematically illustrates another example workflow for barcoding multiple analytes of a cell, nucleus or cell bead. In such an example, the workflow for processing a nucleic acid molecule (e.g., RNA molecule) may be substantially similar to that depicted in FIG.16A, but the workflow for processing a feature (e.g., protein) may differ. For instance, the feature binding group 1652 or the reporter oligonucleotide 1657 may comprise a binding sequence that is capable of hybridizing to a probe binding molecule 1617 and / or barcode molecule 1619.

[0255] As described herein, a permeabilized (and optionally fixed) cell or nucleus may becontacted with one or more feature binding groups 1652, which may (a) comprise the reporter oligonucleotide 1657 and (b) be configured to couple to (i) an intracellular protein (or an intranuclear protein) or (ii) a cell membrane protein (or nuclear membrane protein). In some embodiments, the one or more feature binding groups 1652 includes (i) a first feature binding group that comprises the reporter oligonucleotide 1657 and is configured to couple to an intracellular (or an intranuclear protein) and (ii) a second feature binding group that comprises the reporter oligonucleotide 1657 and is configured to couple to a cell membrane protein (or a nuclear membrane protein).

[0256] In operation 1670, the cell, nucleus or cell bead 1600 comprising the first probe-associated molecule 1630 and the one or more feature binding group 1652 may be partitioned into a first partition of a first set of partitions or further processed in the first partition. Within the first partition, a probe binding molecule 1617 and a barcode molecule 1619 may be provided. The feature binding group 1652 (e.g., one or more feature binding groups configured to couple to an intracellular protein or an intranuclear protein) coupled to the reporter oligonucleotide 1657 may be contacted with one or more probe binding molecules 1617 and barcode molecules 1619. A barcode molecule 1619 may comprise a barcode capture sequence or a common sequence common to a plurality of barcode molecules and a first barcode sequence common to the first partition of the first set of partitions. The nucleic acid barcode molecule may, in some instances,Attorney Docket: 43487-1036601 be coupled to a bead, such as a gel bead, or other support, as described herein, and can comprise additional functional sequences, including, but not limited to, a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., a R1 / R2 sequence), additional barcode sequence segments, etc.. The probe binding molecules 1617 may comprise a probe binding sequence complementary to a sequence of the reporter oligonucleotide 1657. In some instances, the probe binding molecules 1617 and the barcode molecules 1619 may be provided as a pre- annealed complex. The probe binding molecules 1617 and the barcode molecules 1619 may hybridize to the first probe-associated molecule 1630 (as described above) and the reporter oligonucleotide 1657 (e.g., via hybridization of the probe binding molecules 1617 to a sequence of the reporter oligonucleotide 1657), thereby generating a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule. Additional barcoded nucleic acid molecules may be generated using additional reporter oligonucleotides 1657 from additional feature binding groups 1652 (e.g., configured to couple to cell or nuclear membrane proteins and / or intracellular or intranuclear proteins). Additional processing may occur within the first partition, e.g., ligation of the barcode molecules 1619 to the probes (1606, 1616) or to the reporter oligonucleotide 1657.

[0257] In operation 1680, the contents of each partition or a subset of the first set ofpartitions may be collected from the first set of partitions, e.g., from operation 1670, and re- partitioned into a second set of partitions. The contents of the first set of partitions may comprise the cell, nucleus or cell bead 1600 and / or the processed cellular / nuclear components, e.g., the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and optionally the additional barcoded nucleic acid molecule(s). The contents of the partitions of the first set of partitions may be pooled together and re-distributed to a second set of partitions. Accordingly, a second partition of the second set of partitions may comprise the cell, nucleus or cell bead 1600 and / or the processed cellular / nuclear components (e.g., barcoded products). In some instances, the cell, nucleus or cell bead 1600 may be subjected to processing within the second partition, such as lysis, to release the cellular / nuclear components (e.g., the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and optionally the additional barcoded nucleic acid molecule(s)) within the second partition. Alternatively, the cell, nucleus or cell bead 1600 may remain intact. Within the second partition, a plurality of capture molecules 1620 may be provided. In some instances, the plurality of capture molecules 1620 may be coupled to a support (e.g., a particle, bead, gel bead, etc.). In some instances, the plurality of capture molecules 1620 may be releasably coupled to the support and the plurality of capture molecules 1620 may be released in the second partition. The capture molecules 1620 may each comprise a second barcode sequence, which may be the same sequence or a different sequence as the firstAttorney Docket: 43487-1036601 barcode sequence (of the barcode molecule 1619). The second barcode sequence may be unique to the second partition and differ from the second barcode sequences of other partitions of the second set of partitions. The first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule may each be contacted with a capture molecule 1620. The capture molecules 1620 may comprise a second barcode capture sequence, which may be complementary to a sequence of the barcode molecule 1619. Alternatively, the capture molecules 1620 may comprise a sequence complementary to an additional probe-binding molecule (e.g., splint oligonucleotide, not shown), and the probe-binding molecule may comprise a sequence complementary to a sequence of the barcode molecule 1619. Hybridization of the capture molecules 1620 to the first barcoded molecule and the second barcoded nucleic acid molecule (or to the additional probe-binding molecule, which may hybridize to the first barcoded molecule and the second barcoded molecule) may be sufficient to generate a third barcoded nucleic acid molecule and a fourth barcoded nucleic acid molecule. In addition, hybridization of 1620 to the additional barcoded nucleic acid molecule(s), e.g., from additional reporter oligonucleotides 1657 on additional feature binding groups 1652, may be sufficient to generate a fifth barcoded nucleic acid molecule. Alternatively, hybridization of the capture molecules 1620 to the first barcoded molecule and the second barcoded nucleic acid molecule may be sufficient to couple the capture molecule (comprising the second barcode sequence) to both the first barcoded molecule and the second barcoded nucleic acid molecule. In addition, hybridization of 1620 to the additional barcoded nucleic acid molecule may be sufficient to couple the capture molecule (comprising the second barcode sequence) to the additional barcoded nucleic acid molecule e.g., generated from additional reporter oligonucleotides 1657 on additional feature binding groups 1652. Optionally, further processing may be performed, e.g., performing an extension reaction, ligation of the capture molecules 1620 to the first barcoded nucleic acid molecule, the second barcode nucleic acid molecule, and optionally the additional barcoded nucleic acid molecule. Following ligation, the first and second barcoded nucleic acid molecule may comprise the capture molecule 1620. The third barcoded nucleic acid molecule, the fourth barcoded nucleic acid molecule, and the fifth barcoded nucleic acid molecule may each comprise a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence. In some instances, an extension reaction is performed (e.g., from the capture molecule 1620 toward the reporter oligonucleotide sequence 1657) to generate the fourth barcoded molecule and / or the fifth barcoded nucleic acid molecule.

[0258] In some instances, the reporter oligonucleotide (comprising the reporter sequence) ofthe feature binding group may be contacted with a plurality of probes. For example, it may be beneficial for the feature binding group to be contacted with a pair of probes. In some instances,Attorney Docket: 43487-1036601 the reporter oligonucleotide comprises one or more feature probe binding sequences, which may comprise sequences complementary to the pair of probes. For example, referring to FIG.17, a cell, nucleus or cell bead 1700 may comprise a feature (e.g., a protein such as a cell / nuclear membrane protein or an intracellular / intranuclear protein) 1750. A feature binding group 1752 may be coupled to the feature 1750. The feature binding group 1752 may comprise or be coupled to an oligonucleotide comprising a reporter oligonucleotide (comprising a reporter sequence) 1754 and, in some instances, additional functional sequences, such as primer sequences, sequencing primer sequences, UMIs, etc., as described elsewhere herein. The reporter oligonucleotide 1754 may comprise any number of target regions. For example, the reporter oligonucleotide 1754 may comprise two target regions to which a first probe 1757 and a second probe 1758 may hybridize. The two target regions may be adjacent or non-adjacent, and they may be disposed on the same strand of the reporter oligonucleotide 1754. As described herein, the probes may comprise sequences that are complementary to the target regions of the reporter oligonucleotide 1754, and each probe may comprise other useful sequences. For example, a probe (e.g., the first probe 1757 or the second probe 1758) may comprise (i) a probe sequence(e.g., 1760) complementary to a target region of the reporter oligonucleotide 1754, and (ii) aprobe capture sequence 1762, which may be complementary to a sequence of a probe binding molecule 1717 (also referred to as a splint or splint oligonucleotide). The probe binding molecule 1717 may also comprise a sequence complementary to a sequence (e.g., capture sequence) of a barcode molecule 1719. Such barcoding (e.g. hybridization of the probe binding molecule 1717 and barcode molecule 1719 to the probe capture sequence 1762) may occur in bulk or in a partition. In some embodiments, barcoding may be performed without a probe binding molecule. For example, the barcode molecule 1719 may comprise a sequence complementary to the probe capture sequence 1762 and directly anneal to the probe.

[0259] In some instances, after contacting the feature binding group with the probemolecules 1757 and 1758 (e.g., in bulk or in a partition), the feature binding group 1752 is subjected to conditions sufficient for hybridization of the probe molecules to the reporter oligonucleotide 1754, thereby generating a probe-associated reporter oligonucleotide complex. The coupling of the probes to the reporter oligonucleotide 1754 may occur in bulk or in a partition. In some instances, following coupling or hybridization of the probes to the reporter oligonucleotide 1754, the probes may be linked together (e.g., enzymatically or chemically), thereby generating a probe-linked nucleic acid molecule (or complex). For example, the first probe 1757 may comprise a first reactive moiety and the second probe 1758 may comprise a second reactive moiety. The reactive moieties may be positioned such that, following hybridization of the first probe 1757 and the second probe 1758 to the reporter oligonucleotideAttorney Docket: 43487-1036601 1754, the reactive moieties are adjacent. The reactive moieties may then be subjected to conditions sufficient to cause them to react to yield a probe-linked nucleic acid molecule (or complex) comprising the first probe 1757 linked to the second probe 1758. In some instances, the probes comprise “click chemistry” moieties. Alternatively, or in addition to, the first probe may be enzymatically linked (e.g., via ligation) to the second probe. In other instances, a gap region (not shown) may be disposed between the first probe 1757 and the second probe 1758, following hybridization of the probes to the reporter oligonucleotide 1754. In such cases, the first probe 1757 may be linked to the second probe 1758 using a gap-fill approach, such as those described above.

[0260] The probe-linked nucleic acid molecule (or complex) may then be subjected tobarcoding (e.g., contacting with the probe binding molecule 1717 and the barcode molecule 1719), which may occur in a partition. Alternatively, the barcoding may occur prior to the linking of the probes. For example, the reporter oligonucleotide 1754 may be hybridized to the probes, partitioned, barcoded, and then the probes may be linked. Alternatively, the reporter oligonucleotide 1754 may be hybridized to the probes, linked, partitioned, then barcoded. In yet another example, the reporter oligonucleotide 1754 may be hybridized to the probes, partitioned, linked, then barcoded. As will be appreciated, the operations described herein (e.g., hybridization, probe-linking, barcoding) may occur at any useful process, or in any useful order. In some instances, multiple partitioning operations maybe performed, e.g., for combinatorial barcoding.

[0261] The reporter oligonucleotide may comprise the same target sequences (e.g., 702, 704,802, 804, 902, 904, 1502, 1504, 1602, 1604, etc.) as the nucleic acid molecule (e.g., RNA molecule). For example, referring to FIG.17, the first probe may have a first sequence that is complementary to both the first target sequence of a nucleic acid molecule (e.g., 702, 802, 902, 1502, 1602) and a first sequence of the reporter oligonucleotide 1754, and the second probe may have a second sequence that is complementary to both the second target sequence of a nucleic acid molecule (e.g., 704, 804, 904, 1504, and 1604) and a second sequence of the reporter oligonucleotide 1754. In such instances, the provision of just two probe types (e.g., a first probe and a second probe) to a cell, nucleus or cell bead may be sufficient to generate the first barcoded molecule (e.g., generated from the nucleic acid molecule, e.g., RNA molecule), the second barcoded molecule (e.g., generated from the reporter oligonucleotide of the feature binding group, such as a group configured to couple to a cell / nuclear membrane protein), and additional barcoded molecules (e.g., generated from the reporter oligonucleotide of an additional feature binding group, such as a group configured to couple to an intracellular / intranuclear protein). As described herein, each of the probes (e.g., the first probe and the second probe) mayAttorney Docket: 43487-1036601 be capable of or configured to hybridize to a barcode molecule (e.g., in the first partition) and / or a capture molecule. As is also described elsewhere herein, each of the probes may be multiplexed or combinatorially barcoded, such that multiplet partitions (e.g., partitions comprising more than one cell, one nucleus or cell bead) may be deconvolved, for example to determine the originating partition or sample of each cell, nucleus or cell bead within a partition (see, e.g., FIG.10). Similarly, the barcoded molecules may be used to determine the origin of different analyte types (e.g., proteins, nucleic acid molecule, etc.); for example, two analyte types may be attributed to the same originating cell, nucleus, cell bead, sample, or partition(s).

[0262] In some instances, the reporter oligonucleotide comprises two or more targetsequences which are different than the target sequences of the nucleic acid molecule (e.g., RNA molecule). Accordingly, four probe types may be provided for performing multiplexed assays; a first probe and a second probe may hybridize to a first target region and a second target region of a nucleic acid molecule, and a third probe and a fourth probe may hybridize to target regions of a reporter oligonucleotide (e.g., a reporter oligonucleotide from a feature binding group, such as a feature binding group configured to couple to a cell / nuclear membrane protein). Additional probe types may be provided, such as a fifth probe and a sixth probe, that hybridize to target regions of an additional reporter oligonucleotide (e.g., a reporter oligonucleotide from a feature binding group, such as a feature binding group configured to couple to an intracellular / intranuclear protein). Each of the probes or a combination of the probes may comprise probe capture sequences, which may be used for subsequent barcoding. For example, each of the probes (e.g., the first probe, the second probe, the third probe, the fourth probe, fifth probe, sixth probe, or a combination thereof) may be capable of or configured to hybridize to a barcode molecule (e.g., in the first partition) and / or a capture molecule (e.g., in a second partition). As is described elsewhere herein, each of the probes may be multiplexed or combinatorially barcoded, such that multiplet partitions (e.g., partitions comprising more than one cell, nucleus or cell bead) may be deconvolved, for example to determine the originating partition or sample of each cell, nucleus or cell bead within a partition (see, e.g., FIG.10). Similarly, the barcoded molecules may be used to determine the origin of different analyte types (e.g., proteins, nucleic acid molecules, etc.); for example, two analyte types may be attributed to the same originating cell, nucleus, cell bead, sample, or partition(s).

[0263] As described elsewhere herein, the nucleic acid molecules (e.g., from a cell, a nucleusor cell bead, or a reporter oligonucleotide) may comprise one or more target regions. The one or more target regions may correspond to a gene or a portion thereof, or another known sequence. The target regions may have the same or different sequences, and may be located within the same strand or on different strands. The target regions may be located adjacent to one another orAttorney Docket: 43487-1036601 may be spatially separated along a strand of the nucleic acid molecule. The target regions may be located on the same strand or different strands. Analyzing two or more target regions may involve providing two or more probes, where a first probe has a sequence that is complementary to the first target region, a second probe has a sequence that is complementary to the second target region, etc. As described elsewhere herein, the nucleic acid molecule may be a target nucleic acid molecule and may comprise any number of nucleic acid features or nucleotides.

[0264] As is also described elsewhere herein, any of the probes (e.g., the first probe, thesecond probe, the third probe, etc.), reporter oligonucleotides, or the barcode or capture molecules, may comprise any number of additional adaptor or functional sequences, such as an additional probe sequence, a unique molecule identifier, a barcode sequence, a primer sequence, a capture sequence, a sequencing primer sequence, etc.

[0265] As described herein, one or more operations may be performed within a partition,such as a droplet or well. For instance, the nucleic acid molecule (e.g., RNA molecule) and the feature (e.g., protein), or a cell, nucleus or cell bead comprising the nucleic acid molecule and feature, may be co-partitioned with one or more reagents (e.g., as described herein) at any useful stage of the method. For example, the probe-linked or probe-associated nucleic acid molecule, optionally comprised within or on a cell, nucleus or cell bead, may be generated in a bulk solution or in a partition. Similarly, the cell, nucleus or cell bead may be contacted with a feature binding group in a bulk solution or in a partition. Provision of the probes (e.g., the first probe, the second probe, and the third probe) may occur in the bulk solution or in individual partitions. In the instances where partitions are used, a partition (e.g., a first partition of a first set of partitions) may comprise the first probe, the second probe, the third probe, or a combination thereof. Different partitions within the first set of partitions may comprise the same or different probes (e.g., for different target sequences or different reporter sequences). Alternatively, or in addition to, the probe binding molecules and the nucleic acid barcode molecules may be provided in a partition. For example, the cell, nucleus or cell bead comprising the feature and the nucleic acid molecule may be contacted with the probes in bulk, and partitioned into a first set of partitions. The first set of partitions may comprise the probe binding molecule and the nucleic acid barcode molecules comprising a common sequence. Different partitions among the first set of partitions may comprise barcode molecules with different barcode sequences; for instance, an additional partition of the first set of partitions may comprise numerous barcode molecules that each have a barcode sequence that is unique to the partition (e.g. differs across partitions). The partition may comprise additional reagents for performing a nucleic acid reaction (e.g., digestion, ligation, extension, amplification). For example, the partition may comprise a linking enzyme (e.g., ligase), which may be used to ligate the nucleic acid barcode molecule to the first probe, theAttorney Docket: 43487-1036601 second probe, or the third probe (e.g., via the probe capture sequence of each probe). In some instances, the probe binding molecule, the probe capture sequence, and / or the barcode capture sequence (e.g., common sequence) comprises one or more reactive moieties, which may be used to chemically link the nucleic acid barcode molecule to the probe capture sequence. The resultant barcoded products may comprise: a first barcoded product comprising a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence of the first probe or the second probe, and a sequence corresponding to the barcode sequence; and a second barcoded product comprising a sequence corresponding to the reporter sequence, the probe capture sequence of the third probe (which may be the same or different than that of the first probe or second probe), and the barcode sequence.

[0266] As described herein, one or more processes described herein may be performed in acell (e.g., a cell in solution, or a cell comprised within a tissue sample), nucleus or cell bead. For example, a plurality of cells, nuclei or cell beads may comprise a plurality of nucleic acid molecules and features. The cells, nuclei or cell beads may be alive or fixed and / or permeabilized. In some instances, the cells, nuclei or cell beads may be contacted with a feature binding group comprising a reporter sequence. The first probe, the second probe, and the third probe may also be provided to the cells, nuclei or cell beads, in bulk solution or in a partition to generate the first probe-associated molecule and the second probe-associated molecule. Optionally, the cells, nuclei or cell beads may be washed to remove unbound probes. Subsequently, the cells, nuclei or cell beads comprising the probe-associated molecules may be partitioned into a plurality of separate partitions, where at least a subset of the plurality of separate partitions comprises a single cell, single nucleus, or single cell bead. Barcoding may be performed within the separate partitions. Barcoding, as described herein, may comprise attaching or hybridizing a nucleic acid barcode molecule to the first probe-associated molecule and the second probe-associated molecule. The nucleic acid barcode molecules provided within each partition of the plurality of separate partitions may be provided attached to beads. In some instances, as described elsewhere herein, the nucleic acid barcode molecule may be releasably attached to a bead (e.g., via a labile bond). Each partition (or a subset of partitions) of the plurality of separate partitions may comprise a bead comprising a plurality of nucleic acid barcode molecules attached thereto (e.g., as described herein). The plurality of nucleic acid barcode molecules attached to each bead may comprise a unique barcode sequence, such that each partition of the plurality of separate partitions comprises a different barcode sequence. Upon release of components from the plurality of different partitions of the plurality of separate partitions (e.g., following barcoding), the barcoded molecules arising from a single cell, singleAttorney Docket: 43487-1036601 nucleus, or single cell bead may have a same barcode sequence (e.g., a common barcode sequence), such that each barcoded nucleic acid molecule can be traced to a given partition and / or, in some instances, a single cell, a single nucleus, or a single cell bead. The released components may then be partitioned, as described herein, in a second set of partitions comprising capture molecules with a second barcode sequence, such that different partitions of the second set of partitions have a unique second barcode sequence.

[0267] The cells, nuclei, or cell beads described herein may be processed either prior to,during, or following barcoding. For example, the cells, nuclei, or cell beads may be fixed or permeabilized at any useful point in time. In some instances, the cells, nuclei, or cell beads may be fixed and permeabilized prior to or following hybridization of the probes, or prior to or following contact with the feature binding groups. In some instances, the cells, nuclei, or cell beads may be fixed and permeabilized prior to contact with the feature binding groups, and then contacted with the probes. The fixation or permeabilization process may be repeated. For example, a cell, nucleus, or cell bead may be fixed and permeabilized, contacted with the probes and the feature binding groups (either simultaneously or in a step-wise fashion), and then fixed again.

[0268] Following fixation and / or permeabilization, the cells, nuclei, or cell beads may bestored for a duration of time prior to further processing, e.g., contacting the cells, nuclei, or cell beads with the probes and / or feature binding groups. For example, the cells, nuclei, or cell beads may be fixed and / or permeabilized and then contacted with the probes and / or feature binding groups after about 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more. The cells, nuclei, or cell beads may be fixed and / or permeabilized and then contacted with the probes and / or feature binding groups after about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more. The cells, nuclei, or cell beads may be fixed and / or permeabilized and then contacted with the probes and / or feature binding groups after about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks...

Claims

Attorney Docket: 43487-1036601 CLAIMS WHAT IS CLAIMED IS:

1. A method for analyzing a biological sample, comprising:(a) contacting the biological sample with: (i) a complex comprising a nuclease-deficient Argonaute protein and a first probe of a probe set, wherein: (I) the first probe comprises a first probe region that hybridizes to a first target sequence in a target nucleic acid in the biological sample and (II) the first target sequence comprises a variant sequence; and (ii) a second probe of the probe set, wherein the second probe comprises a second probe region that hybridizes to a second target sequence in the target nucleic acid in the biological sample, (b) coupling the first probe and second probe to generate a first-second probe product; (c) detecting the first-second probe product or derivative thereof.

2. The method of claim 1, wherein the nuclease-deficient Argonaute protein is anRNA-guided Argonaute, and the first probe is an RNA molecule.

3. The method of claim 1 or claim 2, wherein the nuclease-deficient Argonauteprotein is a eukaryotic Argonaute protein.

4. The method of claim 1 or claim 3, wherein the nuclease-deficient Argonauteprotein is a DNA-guided Argonaute, and the first probe is a DNA molecule.

5. The method of any one of claims 1, 2, and 4, wherein the nuclease-deficientArgonaute protein is a prokaryotic Argonaute protein.

6. The method of any one of claims 1-5, wherein the first probe comprises a 5’-phosphate or a 5’-OH.

7. The method of claim 1 or claim 6, wherein the nuclease-deficient Argonauteprotein is Ago1 or Ago4.

8. The method of any one of claims 1, 6, or 7, wherein the nuclease-deficientArgonaute protein is a Drosophila Argonaute protein or a derivative or variant thereof.

9. The method of claim 1 or claim 6, wherein the nuclease-deficient Argonauteprotein is a nuclease-deficient Argonaute derived from Thermus thermophilus (dTtAgo).

10. The method of any one of claims 1-9, wherein the nuclease-deficient Argonauteprotein comprises one or more inactivating mutations in a PIWI or PAZ domain of the Argonaute protein.Attorney Docket: 43487-1036601 11. The method of any one of claims 1-10, wherein the first probe and the nuclease-deficient Argonaute protein are bound in the complex before contacting the biological sample.

12. The method of any one of claims 1-10, wherein the first probe and the nuclease-deficient Argonaute protein are contacted with the biological sample simultaneously.

13. The method of any one of claims 1-12, wherein the first probe comprises one ormore ribonucleotides.

14. The method of any one of claims 1-13, wherein the first probe comprises asequence complementary to the variant sequence, wherein the sequence comprises one or more nucleotides between positions 2 and 8 from the 5’ end of the first probe.

15. The method of any of one claims 1-14, wherein the nuclease-deficient Argonauteprotein or the first probe is labeled with a detectable moiety, optionally wherein the detectable moiety is a fluorescent dye.

16. The method of any one of claims 1-15, wherein the first target sequence in thetarget nucleic acid bound to the first probe is 15-35 nucleotides.

17. The method of claim 16, wherein a nucleotide at position 6 or position 7 from the5’ end of the first probe region is complementary to the variant sequence.

18. The method of any one of claims 1-17, wherein the first target sequence and thesecond target sequence are each about 15 to about 40 nucleotides in length.

19. The method of any one of claims 1-18, wherein (a) further comprises contactingthe biological sample with a plurality of different first probes comprising a plurality of different sequences complementary to a plurality of different variant sequences, wherein the plurality of different first probes comprises the first probe.

20. The method of any one of claims 1-19, wherein the second probe iscomplementary to a common second target sequence among a plurality of different target nucleic acids comprising different variant sequences.

21. The method of any one of claims 1-20, wherein the first probe comprises asequence complementary to the variant sequence, wherein the sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 5’ or 3’ end of the first probe.

22. The method of any one of claims 1-21, wherein the variant sequence is at or nearthe central nucleotide(s) of the first target sequence in the target nucleic acid.

23. The method of any one of claims 1-22, wherein the first probe comprises asequence complementary to the variant sequence, wherein the sequence is at or near the central nucleotide(s) of the first probe region.Attorney Docket: 43487-1036601 24. The method of any one of claims 1-23, wherein the variant sequence comprises anucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, or a repetitive sequence.

25. The method of any one of claims 1-24, wherein the variant sequence comprisestwo or more nucleotide residues.

26. The method of any one of claims 1-24, wherein the variant sequence is a singlenucleotide.

27. The method of any one of claims 1-26, wherein the variant sequence comprises asingle nucleotide variation (SNV), a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion.

28. The method of any one of claims 1-27, wherein (b) comprises performing aligation reaction.

29. The method of any one of claims 1-28, wherein (b) comprises performing anucleic acid extension reaction.

30. The method of any one of claims 1-28, wherein (b) comprises ligating a first endof the first probe to a second end of the second probe.

31. The method of any one of claims 1-29, wherein the first target sequence and thesecond target sequence are separated by a gap region of at least 1 nucleotide on the target nucleic acid, and (b) comprises performing a gap-fill reaction to generate the first-second probe product.

32. The method of any one of claims 1-28, wherein the target nucleic acid is RNA,and wherein the method further comprises ligating the first probe and the second probe using a ligase having an RNA-templated DNA or RNA ligase activity.

33. The method of claim 32, wherein the ligase is a PBCV-1 DNA ligase or a T4RNA ligase 2.

34. The method of claim 32, wherein the ligase is a Chlorella virus DNA ligase(PBCV DNA ligase) or a T4 RNA ligase.

35. The method of any one of claims 1-34, further comprising contacting thebiological sample with a library of first probes that comprises at least or about 2, at least or about 3, at least or about 4, or more different first probes comprising different first probe region sequences.

36. The method of claim 35, comprising washing the biological sample aftercontacting with the library of first probes.

37. The method of any one of claims 1-36, wherein the first probe comprises adetection region that does not hybridize to the first target sequence in the target nucleic acid.Attorney Docket: 43487-1036601 38. The method of claim 37, wherein detecting the first-second probe product orderivative thereof comprises detecting a sequence of the detection region in the first-second probe product or derivative thereof.

39. The method of claim 37 or claim 38, further comprising binding a circular orcircularizable probe to a sequence in the detection region of the first-second probe product or derivative thereof.

40. The method one of claims 1-39, wherein the detecting comprises imaging thebiological sample to detect the first-second probe product or derivative thereof.

41. The method of claim 40, wherein the imaging is performed over a plurality ofsequential cycles.

42. The method of claim 38, further comprising identifying a sequence of the first-second probe product by sequencing.

43. The method of claim 44, further comprising releasing the first-second probeproduct from the biological sample before (c).

44. The method of any one of claims 1-43, wherein the variant sequence is among a plurality of different sequences.

45. The method of any one of claims 1-44, wherein the variant sequence is a mutant sequence or a minor variant among a plurality of different variant sequences.

46. The method of any one of claims 1-44, wherein the variant sequence is a wildtype sequence or a major variant among a plurality of different variant sequences.

47. The method of any one of claims 1-44, wherein the first target sequence comprises a genetic hotspot, optionally wherein the first target sequence comprises two or more hotspot mutations.

48. The method of any one of claims 1-47, wherein the target nucleic acid is a cellular nucleic acid analyte or a product thereof.

49. The method of claim 48, wherein the cellular nucleic acid analyte is an RNA and the product thereof is a cDNA.

50. The method of any one of claims 1-47, wherein the target nucleic acid is mRNA.

51. The method of any one of claims 1-47, wherein the target nucleic acid is a cDNA.

52. The method of any one of claims 1-51, wherein the biological sample is a tissue section.Attorney Docket: 43487-1036601 53. The method of any one of claims 1-52, wherein the biological sample is a formalin-fixed, paraffin-embedded (FFPE) sample or a fresh frozen tissue sample.

54. The method of any one of claims 1-52, wherein the biological sample is fixed or permeabilized.

55. The method of any one of claims 1-52, wherein the biological sample is crosslinked or embedded in a matrix, optionally wherein the matrix comprises a hydrogel.

56. The method of any one of claims 1-55, wherein the biological sample is cleared.

57. A method for analyzing a biological sample, comprising: (a) contacting the biological sample with a complex comprising a nuclease-deficient Argonaute protein and a probe, wherein the probe comprises a probe region and a detection region, wherein: (i) the probe region hybridizes to a target sequence in a target nucleic acid in the biological sample and the target sequence comprises a variant sequence; and (ii) the detection region does not hybridize to the target nucleic acid; and (b) detecting the probe or derivative thereof.

58. The method of claim 57, wherein the nuclease-deficient Argonaute protein is an RNA-guided Argonaute, and the first probe is an RNA molecule.

59. The method of claim 57 or claim 58, wherein the nuclease-deficient Argonaute protein is a eukaryotic Argonaute protein.

60. The method of claim 57 or claim 59, wherein the nuclease-deficient Argonaute protein is a DNA-guided Argonaute, and the first probe is a DNA molecule.

61. The method of any one of claims 57, 58, or 60, wherein the nuclease-deficient Argonaute protein is a prokaryotic Argonaute protein.

62. The method of claim 57 or claim 61, wherein the nuclease-deficient Argonaute protein is Ago1 or Ago4.

63. The method of any one of claims 57, 61, or 62, wherein the nuclease-deficient Argonaute protein is a Drosophila Argonaute protein or a derivative or variant thereof.

64. The method of claim 57 or claim 62, wherein the nuclease-deficient Argonaute protein is a nuclease-deficient Argonaute derived from Thermus thermophilus (dTtAgo) 65. The method of any one of claims 57-64, wherein the nuclease-deficient Argonaute protein comprises one or more inactivating mutations in a PIWI or PAZ domain of the Argonaute protein.Attorney Docket: 43487-1036601 66. The method of any one of claims 57-65, wherein the probe and the nuclease- deficient Argonaute protein are bound in the complex before contacting the biological sample.

67. The method of any one of claims 57-66, wherein the probe and the Argonaute protein are contacted with the biological sample simultaneously.

68. The method of any one of claims 57-67, wherein the probe comprises a sequence that is complementary to the variant sequence, wherein the sequence comprises one or more nucleotides between positions 2 and 8 from the 5’ end of the probe.

69. The method of claim 68, wherein a nucleotide at position 6 or position 7 from the 5’ end of the first probe region is complementary to the variant sequence.

70. The method of any one of claims 57-69, wherein (a) further comprises contacting the biological sample with a plurality of different probes comprising a plurality of different sequences complementary to a plurality of different variant sequences.

71. The method of any one of claims 57-70, further comprising binding an additional probe to an additional target sequence in the target nucleic acid in the biological sample, wherein the additional probe is complementary to a common additional target sequence among a plurality of different target nucleic acids comprising different variant sequences.

72. The method of any one of claims 57-71, wherein the probe comprises a sequence complementary to the variant sequence, wherein the sequence is at least 2, 3, 4, 5, 6, 7, 8, 9, or more phosphodiester bonds from the 5’ or 3’ end of the probe.

73. The method of any one of claims 57-72, wherein the variant sequence is at or near the central nucleotide(s) of the target sequence in the target nucleic acid.

74. The method of any one of claims 57-73, wherein the variant sequence comprises a nucleotide variation, a nucleotide polymorphism, a mutation, a substitution, an insertion, a deletion, a translocation, a duplication, an inversion, or a repetitive sequence.

75. The method of any one of claims 57-74, wherein the variant sequence comprises two or more nucleotide residues.

76. The method of any one of claims 57-74, wherein the variant sequence is a single nucleotide.

77. The method of any one of claims 57-76, wherein the variant sequence comprises a single nucleotide variation (SNV), a single nucleotide polymorphism (SNP), a point mutation, a single nucleotide substitution, a single nucleotide insertion, or a single nucleotide deletion.

78. The method of any one of claims 57-77, further comprising contacting the biological sample with a library of probes that comprises at least or about 2, at least or about 3, at least or about 4, or more different probes comprising different probe region sequences.Attorney Docket: 43487-1036601 79. The method of claim 78, comprising washing the biological sample after contacting with the library of probes.

80. The method of any one of claims 57-79, further comprising binding a circular or circularizable probe to a sequence in the detection region of the probe or derivative thereof prior to (b).

81. The method one of claims 57-80, wherein the detecting comprises imaging the biological sample to detect the probe or derivative thereof.

82. The method of claim 81, wherein the imaging is performed over a plurality of sequential cycles.

83. The method of any one of claims 57-82, wherein (b) comprises detecting the probe or derivative thereof in the biological sample.

84. A kit, comprising: a probe set comprising a first probe and a second probe, wherein the first probe of the probe set comprises a first probe region that is configured to hybridize to a first target in a target nucleic acid in the biological sample, and wherein the second probe of the probe set comprises a capture region and a second probe region that is configured to hybridize a second target sequence in the target nucleic acid in the biological sample, wherein the first probe is in a complex with a nuclease-deficient Argonaute protein; and a ligase configured to ligate the first probe to the second probe to generate a ligated first- second probe product.

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