Methods of crispr guide detection in single cell workflows via RNA-templated ligation

Abstract

Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.

Description

Attorney Docket No. 43487-1034601 METHODS OF CRISPR GUIDE DETECTION IN SINGLE CELL WORKFLOWS VIA RNA-TEMPLATED LIGATION CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 549,400,filed February 2, 2024, U.S. Provisional Application No.63 / 700,430, filed September 27, 2024, US. Provisional Application No.63 / 740,126, filed December 30, 2024, and U.S. Provisional Application No.63 / 740,918, filed December 31, 2024, the contents of which are incorporated by reference in their entirety. BACKGROUND

[0002] A sample may be processed for various purposes, such as identification of a type ofmoiety within the sample. The sample may be a biological sample. Biological samples may be processed, such as for detection of a disease (e.g., cancer) or identification of a particular species. There are various approaches for processing samples, such as polymerase chain reaction (PCR) and sequencing.

[0003] Biological samples may be processed within various reaction environments, such aspartitions. Partitions may be wells or droplets. Droplets or wells may be employed to process biological samples in a manner that enables the biological samples to be partitioned and processed separately. For example, such droplets may be fluidically isolated from other droplets, enabling accurate control of respective environments in the droplets.

[0004] Biological samples in partitions may be subjected to various processes, such aschemical processes or physical processes. Samples in partitions may be subjected to heating or cooling, or chemical reactions, such as to yield species that may be qualitatively or quantitatively processed.

[0005] Biological molecules, such as nucleic acids and proteins, within biological samplesmay be probed and / or processed for quantitative or qualitative assessment. SUMMARY

[0006] In some aspects, the present disclosure provides methods and compositions foranalyzing (e.g. sequencing) gRNAs, such as from CRISPR / Cas systems. The methods can be performed in single-cell analysis workflows. The methods can be performed alone and are also compatible with analysis, sequencing and / or detection of additional analytes, such as transcripts. In some aspects, the methods comprise single-cell gRNA and transcriptome expression analysis.Attorney Docket No. 43487-1034601 In some aspects, the methods facilitate analysis of gRNA-expressing cells, such as in large-scale CRISPR / Cas screens.

[0007] The present disclosure provides methods for use in sample processing and analysis.The methods provided herein may involve hybridizing a probe to a molecule of interest (e.g., target protein, target nucleic acid molecule) and processing the probe-molecule complex. Such processing can include barcoding the probe, the probe-molecule complex, or the molecule, and / or performing a nucleic acid reaction. The probe may comprise a nucleic acid molecule, and further processing can include extension, denaturation, and amplification processes to provide nucleic acid molecules comprising a sequence the same or substantially the same as or complementary to that of a target region of a nucleic acid molecule of interest (e.g., target nucleic acid molecule). A method may comprise hybridizing a first probe and a second probe to first and second target regions of the nucleic acid molecule, linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. A method may comprise hybridizing a first probe to a first target region of a nucleic acid molecule, barcoding the probe, and hybridizing a second probe to a second target region of the nucleic acid molecule to generate a barcoded, probe-linked nucleic acid molecule. In some aspects, the method may comprise hybridizing a probe to a nucleic acid molecule attached to a feature-binding moiety to provide a probe-binding moiety complex and barcoding the probe. One or more processes of the methods provided herein may be performed within a partition such as a droplet or well. The methods of the present disclosure be useful, for example, in controlled analysis and processing of analytes such as biological particles, nucleic acids, and proteins. One or more of the methods described herein may allow for genomic, transcriptomic, or exomic profiling with higher sensitivity. The methods of the present disclosure may be useful in detecting variants and characterizing nucleic acid molecules, e.g., for assessment of single nucleotide polymorphisms (SNPs), alternative splice junctions, insertions, deletions, V(D)J rearrangements, etc. The methods of the present disclosure may be useful for multiplexed analysis of nucleic acids and proteins while minimizing reagent usage, e.g., by decreasing the number of unoccupied partitions for analysis.

[0008] In an aspect, disclosed herein is a method for multiplexed nucleic acid assays,comprising: (a) contacting a cell, nucleus, or cell bead with a first probe, a second probe, and a third probe under conditions sufficient to generate a first probe-associated molecule and a second probe-associated molecule, wherein the cell, nucleus, or cell bead comprises (i) a nucleic acid molecule comprising a first target region and a second target region and (ii) a feature coupled to a feature binding group, wherein the feature binding group comprises (i) a reporter oligonucleotide associated with the feature and (ii) a feature probe binding sequence, wherein theAttorney Docket No. 43487-1034601 first probe comprises (i) a first probe sequence complementary to the first target region and (ii) a probe capture sequence, wherein the second probe comprises a second probe sequence complementary to the second target region, wherein the third probe comprises a (i) third probe sequence complementary to the feature probe binding sequence and (ii) the probe capture sequence; (b) in a first partition of a first set of partitions, contacting the first probe-associated molecule and the second probe-associated molecule to probe binding molecules and barcode molecules under conditions sufficient to generate a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule, wherein the barcode molecules comprise (i) a common sequence common to a plurality of barcode molecules comprising the barcode molecules and (ii) a first barcode sequence common to the first partition of the first set of partitions, wherein the probe binding molecule comprises (i) a probe binding sequence complementary to the probe capture sequence and (ii) a barcode binding sequence complementary to the common sequence; and (c) in a second partition of a second set of partitions, (i) contacting the first barcoded nucleic acid molecule, or derivative 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, wherein the plurality of capture molecules comprise a second barcode sequence, wherein each of the third barcoded nucleic acid molecule and the fourth barcoded molecule comprises a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence.

[0009] In some embodiments, the first target region and the second target region are on asame strand of the nucleic acid molecule. In some embodiments, the probe capture sequence is common to a plurality of first probes including the first probe, wherein one or more additional partitions of the first set of partitions comprise one or more additional probe-associated nucleic acid molecules, wherein each of the one or more additional probe-associated nucleic acid molecules comprises the probe capture sequence. In some embodiments, the second probe comprises a second probe capture sequence complementary to a capture sequence of the plurality of capture molecules, and wherein (c) comprises hybridizing the second probe capture sequence to the capture sequence. In some embodiments, the barcode molecules comprise a capture binding sequence complementary to a capture sequence of the plurality of capture molecules, and wherein (c) comprises hybridizing the capture binding sequence to the capture sequence. In some embodiments, the first set of partitions are a plurality of wells. In some embodiments, the second set of partitions are a plurality of droplets. In some embodiments, the second set of partitions are a plurality of wells. In some embodiments, the plurality of capture molecules isAttorney Docket No. 43487-1034601 coupled to a particle. In some embodiments, the particle is a bead. In some embodiments, the bead is a gel bead. In some embodiments, each capture molecule of the plurality of capture molecules coupled to the gel bead comprises the second barcode sequence. In some embodiments, one or more additional partitions of the second set of partitions comprise one or more additional gel beads of a plurality of gel beads, and wherein the second barcode sequence is unique to the gel bead among the plurality of gel beads. In some embodiments, a capture molecule of the plurality of capture molecules comprises a third barcode sequence unique to the capture molecule among the plurality of capture molecules. In some embodiments, one or more additional partitions of the second set of partitions comprise one or more additional capture molecules, and wherein the second barcode sequence is unique to the second partition among the second set of partitions. In some embodiments, (a) comprises hybridizing the first probe and the second probe to the first target region and the second target region, respectively. In some embodiments, the method further comprises subjecting the first probe-associated molecule to conditions sufficient to yield a probe-linked nucleic acid molecule comprising the first probe linked to the second probe. In some embodiments, the probe-linked nucleic acid molecule is generated via chemical or enzymatic ligation of the first probe and the second probe. In some embodiments, the chemical or enzymatic ligation occurs subsequent to (b). In some embodiments, the first target region and the second target region are adjacent. In some embodiments, the first target region and the second target region are non-adjacent, and the method further comprises (i) extending the first probe or the second probe annealed to the first target region or the second target region, respectively, towards the second target region or the first target region, respectively, to generate an extended probe, and (ii) ligating the extended probe to the second probe or the first probe, respectively. In some embodiments, (a) comprises contacting the first probe and the second probe to the nucleic acid molecule inside the cell or nucleus. In some embodiments, the first partition comprises a plurality of cells, cell beads, or nuclei. In some embodiments, the cell or nucleus is permeabilized. In some embodiments, the cell or nucleus is fixed. In some embodiments, the method further comprises releasing the first probe-associated molecule, or derivative thereof, from the cell, nucleus, or cell bead. In some embodiments, the releasing comprises lysing the cell. In some embodiments, the reporter oligonucleotide comprises the feature probe binding sequence. In some embodiments, the method further comprises, subsequent to (b) and prior to (c), pooling the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, additional first barcoded nucleic acid molecules from the first set of partitions, and additional second barcoded nucleic acid molecules form the first set of partitions. In some embodiments, the method further comprises subsequent to (c) and prior to sequencing, pooling the third barcoded nucleic acid molecule, the fourthAttorney Docket No. 43487-1034601 barcoded nucleic acid molecule, additional third barcoded nucleic acid molecules from the second set of partitions, and additional fourth barcoded nucleic acid molecules from the second set of partitions. In some embodiments, the probe capture sequence is from 8 to 50 bp.

[0010] In another aspect of the present disclosure, provided herein is a method, comprising:(a) 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 not adjacent to the first target region, wherein the first probe comprises a first probe sequence complementary to the first target region; (b) extending the first probe under conditions sufficient to generate an extended probe molecule comprising a sequence complementary to the second target region; (c) in a partition of a plurality of partitions, providing the extended probe molecule, a second probe, and a barcode molecule and a probe binding molecule, under conditions sufficient to generate a barcoded molecule, wherein the second probe comprises a second probe sequence corresponding to the second target region, wherein the first probe or the second probe comprises a probe capture sequence, wherein the barcode molecule comprises (i) a barcode capture sequence and (ii) a barcode sequence, wherein the probe binding molecule comprises (i) a probe binding sequence complementary to the probe capture sequence and (ii) a barcode binding sequence complementary to the barcode capture sequence, wherein the barcoded molecule comprises 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.

[0011] In some embodiments, (c) comprises ligating the second probe and the barcodemolecule. In some embodiments, the ligating comprises chemical or enzymatic ligation. In some embodiments, the particle is a bead. In some embodiments, the bead is a gel bead. In some embodiments, the cell or nucleus is permeabilized. In some embodiments, the cell or nucleus is fixed. In some embodiments, the first probe comprises the probe capture sequence. In some embodiments, the method further comprises, subsequent to (b), releasing the extended probe molecule from the nucleic acid molecule. In some embodiments, the releasing comprises freeing a ribonucleic acid (RNA) strand using RNase. In some embodiments, the releasing comprises heat cycling. In some embodiments, (c) comprises (i) hybridizing the second probe sequence of the second probe to the sequence complementary to the second target region and (ii) extending the second probe. In some embodiments, in (c) the barcode molecule and the probe binding molecule are provided as a pre-annealed complex, wherein the barcode capture sequence is annealed to the barcode binding sequence in the pre-annealed complex. In some embodiments, the first probe comprises the probe capture sequence, wherein (c) comprises (i) annealing the probe binding sequence and the barcode binding sequence to the probe capture sequence and theAttorney Docket No. 43487-1034601 barcode capture sequence, respectively (ii) ligating the barcode molecule and the extended probe molecule, to generate a first barcoded molecule and (iii) annealing the second probe to the first barcoded molecule and initiating an extension reaction to generate the barcoded molecule. In some embodiments, (ii) and (iii) are preformed outside the partition. In some embodiments, the second probe comprises the probe capture sequence, wherein (c) comprises (i) annealing the second probe to the extended probe molecule and initiating an extension reaction to generate an extension molecule, (ii) annealing the probe binding sequence and the barcode binding sequence to the probe capture sequence and the barcode capture sequence, and (iii) ligating the barcode molecule and the extension molecule.

[0012] In another aspect, provided herein is a method of analyzing a sample, comprising: (a)providing: (i) a feature-binding group bound to at least a portion of the sample, wherein the feature-binding group comprises a reporter oligonucleotide, wherein the reporter oligonucleotide comprises a reporter barcode sequence, a first target region and a second target region, wherein the first target region and the second target region are disposed on a same strand of the reporter oligonucleotide; (ii) a first probe comprising a first probe sequence, wherein the first probe sequence of the first probe is complementary to the first target region of the reporter oligonucleotide; and (iii) a second probe comprising a second probe sequence, wherein the second probe sequence of the second probe is complementary to the second target region of the reporter oligonucleotide; (b) subjecting the sample to conditions sufficient to (i) hybridize the first probe sequence of the first probe to the first target region of the reporter oligonucleotide, and (ii) hybridize the second probe sequence of the second probe to the second target region of the reporter oligonucleotide to yield a probe-associated reporter oligonucleotide complex; and (c) subjecting the probe-associated reporter oligonucleotide complex to conditions sufficient to yield a probe-linked nucleic acid molecule comprising the first probe linked to the second probe.

[0013] In some embodiments, the method further comprises (d) attaching a barcode moleculeto the probe-linked nucleic acid molecule. In some embodiments, (d) occurs in a partition. In some embodiments, the partition is a droplet or a well. In some embodiments, the sample comprises a nucleic acid molecule, and wherein (d) further comprises attaching an additional barcode molecule to the nucleic acid molecule or derivative thereof. In some embodiments, the feature-binding group is an antibody. In some embodiments, the first probe or the second probe comprises an additional probe sequence. In some embodiments, the method further comprises attaching a barcode sequence to the additional probe sequence. In some embodiments, the method further comprises (d) providing a barcode molecule and a probe binding molecule comprising (i) a first sequence complementary to the additional probe sequence and (ii) a second sequence complementary to a capture sequence of the barcode molecule. In some embodiments,Attorney Docket No. 43487-1034601 the method further comprises providing conditions sufficient to hybridize the first sequence to the additional probe sequence and the second sequence to the capture sequence of the barcode molecule, thereby generating a barcoded probe-associated complex. In some embodiments, (d) occurs in a partition among a plurality of partitions. In some embodiments, the method further comprises pooling the barcoded probe-associated complex from the partition with other barcoded probe-associated complexes from other partitions of the plurality of partitions to generate a pooled set of barcoded probe-associated complexes. In some embodiments, the method further comprises (i) partitioning the pooled set of barcoded probe-associated complexes into a plurality of additional partitions, wherein an additional partition of the plurality of additional partitions comprises the barcoded probe-associated complex and an additional barcode molecule comprising an additional barcode sequence and (ii) attaching the additional barcode molecule to the barcoded probe-associated complex. In some embodiments, the additional barcode molecule is coupled to a bead. In some embodiments, the bead is a gel bead. In some embodiments, the additional barcode molecule is releasably coupled to the bead. In some embodiments, (d) occurs prior to (c). In some embodiments, (d) occurs subsequent to (c). In some embodiments, the at least the portion of the sample comprises a feature. In some embodiments, the feature is a protein. In some embodiments, the protein is a cell surface receptor or an intracellular protein. In some embodiments, the sample comprises a cell or cell bead. In some embodiments, the cell is a formalin fixed, paraffin-embedded cell. In some embodiments, (c) occurs in a partition. In some embodiments, the partition is among a plurality of partitions. In some embodiments, (c) comprises enzymatic or chemical ligation. In some embodiments, the ligation is performed in the absence of adenosine triphosphate. In some embodiments, the first probe or the second probe comprises an adenylated end, a phosphorylated end, a ribonucleotide, a dideoxynucleotide, or a flap sequence. In some embodiments, the first target region and the second target region are separated by a gap region disposed between the first target region and the second target region. In some embodiments, (c) comprises performing an extension reaction to fill the gap region to yield the probe-linked nucleic acid molecule. In some embodiments, (c) comprises providing a third probe comprising a third probe sequence complementary to the gap region, hybridizing the third probe sequence to the gap region, and providing conditions sufficient to yield the probe- linked nucleic acid molecule comprising the first probe linked to the second probe via the third probe.

[0014] 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.Attorney Docket No. 43487-1034601

[0015] 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.

[0016] In some aspects, provided herein is a method comprising: providing a gRNA-expressing cell comprising a gRNA having a spacer sequence and a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-specific ligatable probe pair comprising 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence, and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence; ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence using the gRNA as template, thereby generating a gRNA-specific ligated probe pair; generating a partition comprising 1) the gRNA-specific ligatable probe pair or the gRNA-specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof. In some aspects, provided herein is a method comprising: providing a guide ribonucleic acid (gRNA)-expressing cell comprising a gRNA having a spacer sequence and a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-specific ligatable probe pair comprising 1) a scaffold probe having a scaffold- hybridizing sequence that hybridizes to the scaffold sequence, and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence; ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence, thereby generating a gRNA-specific ligated probe pair; partitioning into a partition: 1) the gRNA-specific ligatable probe pair or the gRNA-specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA- specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof.

[0017] In some embodiments, the method comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell. In someAttorney Docket No. 43487-1034601 embodiments, the ligating comprises ligating the scaffold-hybridizing sequence to the spacer- hybridizing sequence. In some embodiments, the ligating comprises ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence using the gRNA as template. In some embodiments, the partitioning into a partition comprises generating a partition comprising: 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the partitioning occurs before ligating the scaffold probe to the spacer probe. In some embodiments, the partitioning occurs after ligating the scaffold probe to the spacer probe. In some embodiments, the partition is generated before ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, the partition is generated after ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, the gRNA-specific ligatable probe pair comprises one or more overhang sequences. In some embodiments, the one or more overhang sequences comprise a barcode sequence. In some embodiments, the barcode sequence is a sample-specific barcode sequence. In some embodiments, the one or more overhang sequences comprise one or more functional sequences. In some embodiments, the one or more functional sequences comprise a primer hybridization sequence or complement thereof, and / or a sequencing primer binding site or complement thereof. In some embodiments, the one or more overhang sequences comprise a capturing sequence that hybridizes to a capture sequence of the nucleic acid barcode molecule. In some embodiments, the method comprises hybridizing the gRNA- specific ligated probe pair to the nucleic acid barcode molecule. In some embodiments, the method comprises extending the gRNA-specific ligated probe pair using the nucleic acid barcode molecule as template, and / or extending the nucleic acid barcode molecule using the gRNA- specific ligated probe pair as template; thereby generating the barcoded spacer oligonucleotide. In some embodiments, the method comprises ligating the gRNA-specific ligated probe pair to the nucleic acid barcode molecule, thereby generating the barcoded spacer oligonucleotide. In some embodiments, the spacer-hybridizing sequence hybridizes to the spacer sequence and a first portion of the scaffold sequence. In some embodiments, the scaffold-hybridizing sequence hybridizes to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence. In some embodiments, the scaffold sequence and the spacer sequence are adjacent. In some embodiments, the scaffold sequence and the spacer sequence are not adjacent and the method comprises extending the scaffold probe or the spacer probe using the gRNA as template prior to ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, the scaffold sequence and the spacer sequence are not adjacent. In some embodiments, the method comprises extending the scaffold probe or the spacer probe using theAttorney Docket No. 43487-1034601 gRNA as template prior to ligating the scaffold probe to the spacer probe. In some embodiments, the gRNA-specific ligatable probe pair comprises one or more locked nucleic acids (LNAs) In some embodiments, the scaffold probe comprises one or more LNAs. In some embodiments, the scaffold-hybridizing sequence comprises one or more LNAs. In some embodiments, the plurality of nucleic acid barcode molecules are coupled to a support. In some embodiments, the support is a particle and / or a bead. In some embodiments, the nucleic acid barcode molecules are released from the support upon generating the partition and / or upon providing a stimulus. In some embodiments, the method comprises contacting the gRNA-expressing cell with an analyte- specific ligatable probe pair comprising a first analyte probe and a second analyte probe that hybridize to sequences of an analyte. In some embodiments, the method comprises ligating the first analyte probe and second analyte probe hybridized to the analyte to generate an analyte- specific ligated probe pair. In some embodiments, the partition comprises the analyte-specific ligatable probe pair or the analyte-specific ligated probe pair. In some embodiments, the method comprises using the analyte-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded analyte oligonucleotide comprising 1) a sequence of the analyte or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof. In some embodiments, the method comprises sequencing the barcoded analyte oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the analyte in the gRNA-expressing cell. In some embodiments, the sequences of the analyte are adjacent. In some embodiments, the sequences of the analyte are not adjacent and the method comprises extending the first or second analyte probe using the analyte as template prior to ligating the first and second analyte probe. In some embodiments, the analyte is a non-gRNA analyte that is not a gRNA. In some embodiments, the analyte is an endogenous cellular transcript. In some embodiments, the analyte is an mRNA. In some embodiments, the partition is a partition of a plurality of partitions. In some embodiments, the partition is a well of a plurality of wells or a droplet of a plurality of droplets. In some embodiments, the providing comprises providing a population of cells comprising the gRNA-expressing cell. In some embodiments, cells of the population of cells comprise gRNAs of a gRNA library, wherein the gRNAs of the gRNA library comprise a scaffold sequence and a spacer sequence. In some embodiments, different cells of the population of cells comprise different gRNAs of a gRNA library, and wherein the different gRNAs comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the differentAttorney Docket No. 43487-1034601 gRNAs. In some embodiments, the contacting comprises contacting the population of cells with the gRNA-specific ligatable probe pair. In some embodiments, the contacting comprises contacting the population of cells with a library of gRNA-specific ligatable probe pairs comprising the gRNA-specific ligatable probe pair. In some embodiments, each gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises 1) a scaffold probe having a scaffold-hybridizing sequence, and 2) a spacer probe having a spacer-hybridizing sequence. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises different gRNA-specific ligatable probe pairs configured to hybridize to different gRNAs of the gRNA library. In some embodiments, the spacer probes of the different gRNA-specific ligatable probe pairs have different spacer-hybridizing sequences. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs have the same scaffold-hybridizing sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs are identical in nucleotide sequence. In some embodiments, the different spacer- hybridizing sequences hybridize to different spacer sequences of the different gRNAs. In some embodiments, the different spacer-hybridizing sequences further hybridize to a first portion of the scaffold sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs hybridize to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs comprise one or more locked nucleic acids (LNAs). In some embodiments, the scaffold-hybridizing sequences of the scaffold probes of the different gRNA- specific ligatable probe pairs comprise one or more LNAs. In some embodiments, the method comprises generating a library of barcoded spacer oligonucleotides, each comprising: 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises hybridizing gRNA-specific ligatable probe pairs of the library of gRNA-specific ligatable probe pairs to gRNAs of the gRNA library in cells of the population of cells; ligating the hybridized gRNA- specific ligatable probe pairs to generate gRNA-specific ligated probes; partitioning the gRNA- specific ligatable probe pairs or gRNA-specific ligated probes from different cells into different partitions comprising nucleic acid barcode molecules comprising partition-specific barcodes; and using the gRNA-specific ligated probes and nucleic acid barcode molecules to generate a library of barcoded spacer oligonucleotides, each comprising 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises sequencing the library of barcoded spacer oligonucleotides. In some embodiments, the method comprises analyzing the results of the sequencing to associate spacer sequences with partition-specific barcodes. In someAttorney Docket No. 43487-1034601 embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of different gRNAs of the gRNA library in different cells of the population of cells. In some embodiments, the method comprises performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in the different cells of the population of cells. In some embodiments, the contacting further comprises contacting the population of cells with a library of analyte-specific ligatable probe pairs configured to hybridize to different non-gRNA analytes. In some embodiments, the method comprises using the library of analyte-specific ligatable probe pairs to generate a library of barcoded analyte oligonucleotides, each comprising 1) a sequence of an analyte of the different analytes or a complement thereof, and 2) a sequence of a partition-specific barcode or a complement thereof. In some embodiments, the method comprises hybridizing analyte-specific ligatable probe pairs of the library of analyte-specific ligatable probe pairs to analytes in cells of the population of cells; ligating the hybridized analyte-specific ligatable probe pairs to generate analyte-specific ligated probes; partitioning the analyte-specific ligatable probe pairs or analyte- specific ligated probes from different cells into different partitions comprising nucleic acid barcode molecules comprising partition-specific barcodes; and using the analyte-specific ligated probes and nucleic acid barcode molecules to generate a library of barcoded analyte oligonucleotides, each comprising 1) a sequence of an analyte or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises sequencing the barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of one or more non-gRNA analytes of the different non-gRNA analytes in the different cells of the population of cells.

[0018] In some aspects, provided herein is a method comprising: providing a population ofcells, wherein different cells of the population of cells comprise different gRNAs of a gRNA library; wherein the different gRNAs of the gRNA library comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs; contacting the population of cells with a library of gRNA-specific ligatable probe pairs, wherein a gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises: 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence of a gRNA of the gRNA library in a gRNA-expressing cell of the population of cells; and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence of the gRNA of the gRNA library in the gRNA-expressing cell of the population of cells; ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence using the gRNA as template, thereby generating a gRNA-specific ligated probe pair;Attorney Docket No. 43487-1034601 generating a partition comprising 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof. In some aspects, provided herein is a method comprising: providing a population of cells, wherein different cells of the population of cells comprise different guide ribonucleic acids (gRNAs) of a guide ribonucleic acid (gRNA) library; wherein the different gRNAs of the gRNA library comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs; contacting the population of cells with a library of gRNA-specific ligatable probe pairs, wherein a gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises: 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence of a gRNA of the gRNA library in a gRNA-expressing cell of the population of cells; and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence of the gRNA of the gRNA library in the gRNA-expressing cell of the population of cells; ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence, thereby generating a gRNA-specific ligated probe pair; partitioning into a partition: 1) the gRNA-specific ligatable probe pair or the gRNA-specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA- specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof. In some aspects, provided herein is a method comprising: providing a population of cells, wherein different cells of the population of cells comprise different guide ribonucleic acids (gRNAs) of a guide ribonucleic acid (gRNA) library; wherein a gRNA of the different gRNAs comprises: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs; contacting the population of cells with a library of gRNA-specific ligatable probe pairs, wherein a gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises: 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence of the gRNA of the different gRNAs in a gRNA-expressing cell of the population of cells; and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence of the gRNA of the different gRNAs in the gRNA-expressing cell of the population of cells; ligating the scaffoldAttorney Docket No. 43487-1034601 probe hybridized to the spacer probe, wherein the scaffold probe and the spacer probe are hybridized to a gRNA of the gRNA library in a gRNA-expressing cell of the population of cells, thereby generating a gRNA-specific ligated probe pair; partitioning, into a partition, 1) the gRNA-specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof.

[0019] In some embodiments, the method comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell. In some embodiments, the ligating comprises ligating the scaffold-hybridizing sequence to the spacer- hybridizing sequence. In some embodiments, the ligating comprises ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence using the gRNA as template. In some embodiments, the partitioning into a partition comprises generating a partition comprising: 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the partitioning occurs before ligating the scaffold probe to the spacer probe. In some embodiments, the partitioning occurs after ligating the scaffold probe to the spacer probe. In some embodiments, the partition is generated before ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, the partition is generated after ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, each gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises 1) a scaffold probe having a scaffold-hybridizing sequence, and 2) a spacer probe having a spacer-hybridizing sequence. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises different gRNA-specific ligatable probe pairs configured to hybridize to different gRNAs of the gRNA library. In some embodiments, the spacer probes of the different gRNA-specific ligatable probe pairs have different spacer-hybridizing sequences. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs have the same scaffold-hybridizing sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs are identical in sequence. In some embodiments, the different spacer-hybridizing sequencesAttorney Docket No. 43487-1034601 hybridize to different spacer sequences of the different gRNAs. In some embodiments, the different spacer-hybridizing sequences further hybridize to a first portion of the scaffold sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs hybridize to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs comprise one or more locked nucleic acids (LNAs), optionally wherein the scaffold-hybridizing sequences of the scaffold probes of the different gRNA-specific ligatable probe pairs comprise one or more LNAs. In some embodiments, the method comprises using the library of gRNA-specific ligatable probe pairs to generate a library of barcoded spacer oligonucleotides, each comprising: 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises sequencing the library of barcoded spacer oligonucleotides. In some embodiments, the method comprises analyzing the results of the sequencing to associate spacer sequences with partition-specific barcodes. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of different gRNAs of the gRNA library in different cells of the population of cells. In some embodiments, the method comprises performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in the different cells of the population of cells.

[0020] 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

[0021] 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.Attorney Docket No. 43487-1034601 BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings illustrate certain features and advantages of this disclosure. Theseembodiments are not intended to limit the scope of the appended claims in any manner. 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:

[0023] FIG. 1 shows an example of a microfluidic channel structure for partitioningindividual biological particles.

[0024] FIG. 2 shows an example of a microfluidic channel structure for the controlledpartitioning of beads into discrete droplets.

[0025] FIG. 3 illustrates an example of a barcode carrying bead.

[0026] FIG. 4 illustrates another example of a barcode carrying bead.

[0027] FIG. 5 schematically illustrates an example microwell array.

[0028] FIG. 6 schematically illustrates an example workflow for processing nucleic acidmolecules.

[0029] FIG. 7 schematically illustrates another example workflow for processing nucleicacid molecules.

[0030] FIG. 8 schematically illustrates another example workflow for processing nucleicacid molecules.

[0031] FIG. 9 schematically illustrates another example workflow for processing nucleicacid molecules.

[0032] FIG. 10 schematically illustrates an example workflow for analyzing cells, nuclei orcell beads.

[0033] FIG. 11 schematically illustrates example labelling agents with nucleic acidmolecules attached thereto.

[0034] FIG. 12A schematically shows an example of labelling agents. FIG. 12Bschematically shows another example workflow for processing nucleic acid molecules. FIG. 12C schematically shows another example workflow for processing nucleic acid molecules.

[0035] FIG. 13 schematically shows another example of a barcode-carrying bead.

[0036] FIG. 14 shows a computer system that is programmed or otherwise configured toimplement methods provided herein.

[0037] FIG. 15 shows an example processed nucleic acid molecule described herein.

[0038] 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 No. 43487-1034601

[0039] FIG. 17 schematically shows a feature-binding group described herein.

[0040] FIG. 18 shows example data from a workflow described herein.

[0041] FIG. 19 shows additional example data from a workflow described herein.

[0042] FIG. 20 shows additional example data from a workflow described herein.

[0043] 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.

[0044] FIG. 22 schematically shows an example workflow for assaying two different analytetypes.

[0045] FIG. 23 shows example data of a barcoding approach described herein.

[0046] FIG. 24 shows example data of different analyte types using the barcodingapproaches described herein.

[0047] FIG. 25 schematically shows an example method for processing nucleic acidmolecules.

[0048] FIG. 26 shows another example method for processing nucleic acid molecules.

[0049] FIG. 27 shows an example workflow for generating probe-linked nucleic acidmolecules.

[0050] FIG. 28 shows another example workflow for generating probe-linked nucleic acidmolecules.

[0051] FIG. 29 shows an example workflow for processing cells according to the methodsdescribed herein.

[0052] 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.

[0053] FIG. 31 shows example gene expression data resulting from barcoding of multipleanalytes using different sample preparation parameters.

[0054] FIGs. 32A-C shows example data of multiple analyte probing for a negative controlgroup. 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.

[0055] FIGs. 33A-C shows example data of multiple analyte probing for an experimentalgroup. FIG.33A shows example data showing different immune cell clusters. FIG.33B shows example data of gene expression of GZMB gene. FIG.33C shows example data of protein expression resulting from antibody staining.Attorney Docket No. 43487-1034601

[0056] FIGs. 34A-C 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.

[0057] FIGs. 35A-C 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.

[0058] FIGs. 36A-C 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.

[0059] FIGs. 37A-C 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.

[0060] FIG. 38 shows another example workflow for assaying two different analyte types.

[0061] FIG. 39 shows a Human K562 cell line, stably expressing KRAB-dCas9, which wastransduced with a pooled lentiviral CRISPRi lncRNA library. sgRNA vectors including fluorescent protein BFP, and antibiotic resistance to puromycin, enabling a double positive selection strategy.

[0062] FIGs. 40A-B show (A) custom probes which were designed to capture individualsgRNA protospacer sequences contained within the CRISPRi library. LHS probe has a TruSeq R2, a probe barcode, and a sequence that is the reverse-complement of the guide scaffold. Incorporating the probe barcode into the LHS probe allows the same pool of protospacer- targeting RHS probes to be used in all multiplexing reactions. RHS probe contains a sequence that is the reverse-complement of the protospacer being targeted, and partial capture sequence 1 (pCS1). (B) Custom probes were also designed to detect the ~700 lncRNA transcripts targeted by the CRISPRi library.

[0063] FIGs. 41A-C show UMAPs showing (A) gene expression, (B) antibody, and (C)CRISPR (sgRNA) based clustering of a single 16-plex replicate, recovering 138,218 cells from a single GEM reaction.

[0064] FIGs. 42A-C show UMAPs showing (A) gene expression, (B) antibody, and (C)CRISPR (sgRNA) based clustering of a single 96-plex replicate, recovering 936,395 cells from a single GEM reaction.Attorney Docket No. 43487-1034601

[0065] FIGs. 43A-D show (A) protospacer-based clustering of cells containing LINC00963-targeting protospacers, non-targeting protospacers, multiple protospacers, or no protospacer. Log normalized (B) LINC00963 expression, (C) five unique sgRNAs targeting LINC00963, and (D) LINC00963 expression in cells containing protospacers targeting LINC00963, non-targeting protospacers, multiple protospacers, or no protospacer.

[0066] FIGs. 44A-B show cells plotted in Loupe using the corresponding UMAPcoordinates. UMAP plots showing Log2 expression of (A) transferrin receptor (TFRC) and its protein product, (B) CD71, a cell surface marker expressed in erythroid lineage cells. Automated cell-type annotation was performed using the Human Bone Marrow reference in Azimuth.

[0067] FIG. 45 shows key metrics of sequencing results from 16-plex and 96-plex assays asdescribed in Example 11.

[0068] FIG. 46 shows key metrics of sequencing results as described in Example 11.

[0069] FIG. 47 shows an exemplary workflow for analyzing gRNAs and non-gRNAanalytes in cells according to the methods provided herein.

[0070] FIG. 48 shows an exemplary workflow for detecting a gRNA using a gRNA-specificligatable probe pair according to the methods provided herein. DETAILED DESCRIPTION

[0071] While various embodiments of the invention have been shown and described herein,it will be apparent 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. OVERVIEW

[0072] Large-scale screens using CRISPR / Cas systems to identify how specific genetic andepigenetic perturbations affect cellular phenotypes such as gene expression have the potential to provide transformative insights for biology and disease. However, the ability to gain insight from such screens is limited by the quality and resolution of data that is acquired.

[0073] In certain common CRISPR / Cas screening strategies, cells are transduced with alibrary of gRNAs that complex with a Cas protein to target different loci and mediate a genetic or epigenetic effect. Subsequently, cells are selected or enriched in bulk for a particular pre- defined phenotype (e.g. using flow cytometry for expression of a marker), and the cells with the particular phenotype are sequenced to identify gRNAs that are enriched, thereby revealing specific targets and / or perturbations that affect the particular phenotype. These and otherAttorney Docket No. 43487-1034601 methods for leveraging CRISPR / Cas screening strategies are inherently limited in their ability to facilitate discovery of new or unexpected phenotypes, and to probe CRISPR / Cas-mediated perturbations in an unbiased manner.

[0074] A potentially much more powerful approach for generating biological insights can beachieved with the ability to analyze a large number of cells at the single cell level in order to associate the expression of specific gRNAs with specific phenotypes, such as gene expression, in an unbiased manner. For example, the ability to perform both transcriptome sequencing and gRNA sequencing at the single cell level, and in the same single cells, in the context of a large- scale CRISPR / Cas screen could provide insight into how various different genes or pathways affected by different gRNAs contribute to any number of different phenotypes, all within a single experiment. However, there is a paucity of available methods available that facilitate such an approach. The methods provided herein address these and other challenges.

[0075] Provided herein are methods for analyzing (e.g. detecting, and / or sequencing)gRNAs. In some aspects, the methods are compatible and can be performed in parallel with single-cell transcriptome sequencing assays, such as any described herein, thereby facilitating powerful insights, for example as described above in combination with CRISPR / Cas perturbations and screens. In some aspects, the methods can be performed in a variety of tissue types. In some aspects, the methods can be performed in fixed cells, thereby providing increased power to analyze a large number of samples collected at any number of time points. The methods may also be performed in suitable alternatives to cells, such as cell nuclei (e.g. for analysis of a gRNA-expressing nucleus, which may be a nucleus of a gRNA-expressing cell).

[0076] In some aspects, provided herein are methods for analyzing gRNA-expressing cells.In some aspects, a method described herein can be described with reference to a single gRNA in a single cell (e.g. a single gRNA-expressing cell). However, it is to be understood that for all such described methods, it is envisioned that the method can be performed in parallel for a plurality of gRNAs (e.g. gRNAs of a gRNA library) expressed in a plurality of single cells. For example, for each individual cell of a plurality of cells, the methods can be employed to analyze the presence and / or abundance of one or more gRNAs therein. Similarly, the methods can be employed to sequence a plurality of gRNAs and a plurality of analytes (e.g. cellular transcripts) in each of a plurality of cells, at the single-cell level.

[0077] In some aspects, ligatable probe pairs (e.g. comprising a first probe and second probeas described herein) can facilitate nucleic acid analysis (e.g. detection and / or sequencing), such as single-cell transcriptome sequencing, for example according to any of the methods described herein. In some aspects, the methods provided herein utilize ligatable probe pairs to facilitate cellular transcript (e.g. transcriptome) sequencing and / or gRNA sequencing. In someAttorney Docket No. 43487-1034601 embodiments, the ligatable probe pairs provided herein facilitate single-cell cellular transcript and gRNA sequencing in the same single cells. Thus, the methods and compositions provided herein can facilitate powerful approaches for analyzing populations of cells at the single cell level in the context of large-scale CRISPR screens, both in terms of phenotype (e.g. based on endogenous expression of non-gRNA analytes such as mRNA transcripts) and gRNA expression, thereby unlocking the potential to survey unexpected phenotypes resulting from specific genetic perturbations in an unbiased manner.

[0078] In some aspects, the methods provided herein facilitate analysis (e.g. detection and / orsequencing) of a gRNA using a gRNA-specific ligatable probe pair. In some aspects, the gRNA- specific ligatable probe pair facilitates the detection, quantification, and / or sequencing of a gRNA, such as within a cell. In some aspects, the gRNA-specific ligatable probe pair comprises a scaffold probe and a spacer probe that hybridize to scaffold and spacer sequences, respectively, of a target gRNA. In some embodiments, the scaffold probe and spacer probe hybridize to the gRNA such that the scaffold probe and spacer probe are configured to be ligated (e.g. as exemplified in FIG.40A). In some embodiments, the scaffold probe and spacer probe are ligated to generate a gRNA-specific ligated probe pair. In some embodiments, ligation of the gRNA- specific ligatable probe pair results in detection of the gRNA via downstream processing and sequencing steps, such as by capture and barcoding of the gRNA-specific ligated probe pair, followed by amplification and sequencing, e.g. as described in detail herein for various probe pairs that are ligated. In some aspects, the method comprises determining a sequence of the gRNA. In some embodiments, the method comprises determining all or a portion of the spacer sequence of the gRNA. In some embodiments, determining a sequence of the gRNA comprises sequencing the gRNA-specific ligated probe pair or a derivative thereof, such as a barcoded spacer oligonucleotide as described herein. In some embodiments, the method comprises determining the presence and / or abundance of the gRNA in a cell (e.g. a gRNA-expressing cell). In some embodiments, the gRNA-specific ligatable probe pair is hybridized to the gRNA within a cell and / or a partition, and the method comprises partitioning the cell for partition-specific (e.g. cell-specific) barcoding of the ligated probe. In some embodiments, the method comprises generating a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof. In some embodiments, the method comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell. In someAttorney Docket No. 43487-1034601 aspects, the gRNA-specific ligatable probe pair can be used in methods for gRNA sequencing, such as in a single-cell sequencing workflow, as described herein. In some aspects, a plurality of gRNA-specific ligatable probe pairs can be used to analyze (e.g. determine and / or quantify the presence of) a plurality of different gRNAs (e.g. different gRNAs of a gRNA library) in a plurality of single cells. In some embodiments, gRNA-specific ligatable probe pairs facilitate single-cell gRNA sequencing, for example in the context of CRISPR screens.

[0079] In some aspects, the methods provided herein comprise both analysis of gRNAs incell(s) and analysis of one or more non-gRNA analytes in the same cell(s) (e.g. any of the non- gRNA analytes provided herein, such as mRNAs or proteins). In some aspects, the methods comprise performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in a cell or a plurality of cells. In some aspects, the methods provided herein comprise both gRNA sequencing and transcript (e.g. transcriptome) sequencing. In some aspects, the methods and compositions provided herein facilitate detection of a target analyte, such as a transcript or other nucleic acid, with the use of an analyte-specific ligatable probe pair (e.g. comprising a first probe and second probe as described herein, such as a first analyte probe and second analyte probe). In some aspects, the analyte-specific ligatable probe pair comprises a first analyte probe and second analyte probe that hybridize to sequences on a target analyte such that the first analyte probe and second analyte probe are configured to be ligated with or without a gap filling step (e.g. extension of the first analyte probe or second analyte probe using the analyte as template) prior to ligation. In some embodiments, the first analyte probe and second analyte probe hybridize to adjacent sequences on the target analyte such that the first probe and second probe are configured to be ligated without a gap filling step prior to ligation. (e.g. as exemplified in FIG.40B). In some embodiments, the first and second analyte probe are ligated to generate an analyte-specific ligated probe pair. In some embodiments, ligation of the analyte-specific ligatable probe pair results in detection of the analyte via downstream processing and sequencing steps, such as by capture and barcoding of the ligated probe, followed by amplification and sequencing, for example as described in detail herein. In some embodiments, the analyte-specific ligatable probe pair is hybridized to the analyte within a cell and / or a partition, and the method comprises partitioning the cell for partition-specific (e.g. cell-specific) barcoding of the analyte-specific ligated probe pair. Thus, in some aspects, the analyte-specific ligatable probe pair can be used in methods for nucleic acid analysis (e.g. sequencing), such as single-cell sequencing, as described herein. In some aspects, a plurality of analyte-specific ligatable probe pairs can be used to analyze (e.g. determine and / or quantify the presence of) a plurality of different analytes (e.g. transcripts) in a plurality of single cells, as described herein, such as in a plurality of single cells expressing gRNAs. In someAttorney Docket No. 43487-1034601 embodiments, analyte-specific ligatable probe pairs facilitate single cell analysis, such as single- cell transcriptome analysis (e.g. sequencing). Various methods and workflows for nucleic acid sequencing using analyte-specific ligatable probe pairs are described herein, for example as illustrated in FIG.38.

[0080] The gRNA-specific ligatable probe pair-based workflows provided herein providesignificant advantages in terms of modularity, flexibility, and scalability. In some aspects, the methods provided herein allow for robust detection of gRNAs, such as in cells. The approach of using ligatable probe pairs can facilitate robust detection of gRNAs, including in fixed cells. Because the gRNA detection approaches are compatible with scalable methods for detecting target nucleic acids, the methods are advantageous because they facilitate parallel analysis of both gRNAs and cellular transcripts in the same single cells, effectively enabling parallel gRNA and non-gRNA analyte single-cell sequencing.

[0081] In some aspects, certain other methods of sequencing that may be applied to detectgRNAs in sequencing workflows can require up-front gRNA design considerations, such as the inclusion of certain custom sequences being included in the gRNA scaffold, or in the expression vector on which a gRNA is encoded. Consequently, gRNA libraries that do not fit such design considerations (including various gRNA libraries that have already been synthesized and are available to researchers) may not be compatible with such certain other methods of sequencing. In contrast, the gRNA-specific ligatable probe pairs provided herein are readily designed and scaled to accommodate any custom gRNA library, without specific up-front design considerations. This can allow researchers greatly increased flexibility to select any suitable gRNA or gRNA library for inclusion in a CRISPR screen experiment in which single-cell gRNA sequencing is to be performed. Furthermore, the custom gRNA-specific ligatable probe pairs provided herein are compatible with and can be spiked in to existing libraries of analyte-specific ligatable probe pairs, e.g. for assaying transcript or protein expression in parallel with gRNA analysis.

[0082] In some aspects, the methods provided herein are scalable approaches for analyzing(e.g. sequencing) a large number of gRNAs having different spacer sequences (e.g. from a gRNA library). In some aspects, the methods are scalable because gRNA-specific ligatable probe pairs can be readily synthesized for detection of any gRNA library (e.g. as demonstrated in the working examples of the current application). In some embodiments, a gRNA-specific ligatable probe pair comprises a scaffold probe that hybridizes to the scaffold sequence of a gRNA, and a spacer probe that hybridizes to the spacer sequence of the gRNA (e.g. as shown in FIG.40A). In such an arrangement, it can be seen that for a library of gRNA-specific ligatable probe pairs for analyzing a library of gRNAs sharing a constant scaffold sequence, the scaffold probe targetingAttorney Docket No. 43487-1034601 the constant region can remain the same (e.g. have an identical sequence) among the different gRNA-specific ligatable probe pairs of the library, whereas the spacer probe providing specificity for the spacer will vary among the different gRNA-specific ligatable probe pairs. Thus, in some aspects, the number of unique molecules that need to be synthesized for a library of gRNA-specific ligatable probe pairs is only about half of the number of molecules that need to be synthesized for a library of analyte-specific ligatable probe pairs targeting, for example, different transcripts. Thus, scalability of the methods provided herein can provide the flexibility to assay various customized gRNA libraries while minimizing new library synthesis costs.

[0083] 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 acid 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.

[0084] 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 aAttorney Docket No. 43487-1034601 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.

[0085] 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.

[0086] 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 first 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 captureAttorney Docket No. 43487-1034601 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.

[0087] 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 the 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. TERMINOLOGYAttorney Docket No. 43487-1034601

[0088] 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.

[0089] The terms “a,” “an,” and “the,” as used herein, generally refers to singular and pluralreferences unless the context clearly dictates otherwise.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 simianAttorney Docket No. 43487-1034601 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).

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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,Attorney Docket No. 43487-1034601 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.

[0099] 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. In some embodiments, a barcoded nucleic acid molecule is a barcoded spacer oligonucleotide, such as any described herein. In some embodiments, a barcoded nucleic acid molecule is a barcoded analyte oligonucleotide, such as any described herein.

[0100] 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 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 biologicalAttorney Docket No. 43487-1034601 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.

[0101] 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.

[0102] 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.Attorney Docket No. 43487-1034601 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. The macromolecular constituent may comprise a protein. The macromolecular constituent may comprise a peptide. The macromolecular constituent may comprise a polypeptide.

[0103] 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.

[0104] 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. gRNAs AND CRISPR / CAS SYSTEMS

[0105] Clustered regularly interspaced short palindromic repeats (CRISPR) / Cas (CRISPR-associated proteins) systems are a component of prokaryotic adaptive immune systemsAttorney Docket No. 43487-1034601 represented in archaea and bacteria. Various naturally occurring CRISPR / Cas systems from different species have been engineered to allow sequence-specific targeting for genetic and epigenetic perturbations in a wide variety of contexts. CRISPR / Cas systems are composed of a Cas protein component and a guide RNA (gRNA) component. When co-expressed, a Cas protein and gRNA form a Cas / gRNA complex in which the Cas protein binds to a region of the gRNA known as the scaffold. The gRNA further includes a spacer sequence, which provides specificity by hybridizing to a specific target site (e.g. genomic locus), at which the complexed Cas protein mediates a genetic or epigenetic effect. gRNAs from different CRISPR / Cas systems include different scaffolds that allow them to complex with a particular Cas protein. In general, for a particular CRISPR / Cas system, the sequence of the scaffold remains constant, whereas the spacer sequence varies according to the target site. Naturally occurring Cas proteins can mediate DNA cleavage (such as a double-stranded break) at the target site. Engineered Cas proteins and CRISPR / Cas systems can be used to mediate a variety of different effects at the target site, including double-stranded breaks and single-stranded breaks. In addition, certain engineered Cas proteins, commonly referred to as dCas proteins, have been engineered to lack nuclease activity. dCas proteins (e.g. dCas9) can be recruited to a locus alone, for example to repress gene expression, or can be fused to epigenetic effectors, for example to repress (e.g. dCas-KRAB) or activate (e.g. dCas-VP64) gene expression. A variety of engineered CRISPR / Cas systems can be leveraged for large-scale CRISPR / Cas perturbation screens. Naturally occurring and engineered CRISPR / Cas systems, and methods of their use, are described in detail elsewhere, for example in Bock et al., “High-content CRISPR screening” Nat. Rev. Methods Primers, 2022, 2(1):9; and in Liu et al., “The CRISPR-Cas toolbox and gene editing technologies” Mol. Cell, 2022, 82(2):333- 347; each of which is incorporated by reference herein in its entirety.

[0106] The methods provided herein can be readily applied for sequencing gRNAs from anysuitable CRISPR / Cas system, including in single-cell workflows, as described herein. In addition, one of skill in the art would readily be able to apply the methods provided herein for sequencing gRNAs from any suitable CRISPR / Cas system, including within the context of a large-scale CRISPR / Cas screen involving the generation of a plurality of gRNA-expressing cells expressing different gRNAs (e.g. from a gRNA library).

[0107] In some aspects, the methods provided herein facilitate sequencing gRNAs, forexample in single-cell sequencing workflows. A gRNA provided herein can be any suitable gRNA to be sequenced in accordance with the provided methods. gRNAs can from any suitable CRISPR / Cas system, and / or can be compatible with (e.g. capable of complexing with) any suitable Cas protein. For example, in some embodiments, the gRNA is capable of complexing with Cas9 (e.g. a Cas9-compatible gRNA). In some embodiments, the gRNA is capable ofAttorney Docket No. 43487-1034601 complexing with a Cas12 (e.g. Cas12a or Cpf1). In some embodiments, the gRNA is capable of complexing with Cas12a). A gRNA provided herein can be from any other CRISPR / Cas system, such as those described in detail elsewhere.

[0108] In some aspects, a gRNA can be composed of more than one RNA molecule (e.g. asin naturally occurring CRISPR / Cas systems). For example, Cas9 system gRNAs include a crRNA that includes a spacer sequence, and a tracrRNA that hybridizes to the crRNA and facilitates complexing with the Cas9 via a scaffold. In contrast, the gRNAs of many engineered CRISPR / Cas systems have been engineered to comprise a single gRNA that includes both the spacer and the full scaffold in a single molecule. The methods provided herein can be readily adapted to sequence any suitable gRNA, including those composed of a single RNA molecule or those composed of more than one RNA molecule (e.g. a gRNA consisting of a crRNA / tracrRNA duplex).

[0109] In some aspects, the gRNA comprises a spacer. In some embodiments, the spacer ofthe gRNA is at the 5’ end of the gRNA (e.g. as in gRNAs from Cas9 CRISPR / Cas systems). In some embodiments, the gRNA is from a Cas9 CRISPR / Cas system, e.g. is Cas9-compatible. In some embodiments, the spacer of the gRNA is at the 3’ end of the gRNA (e.g. as in gRNAs from Cas12a (Cpf1) CRISPR / Cas systems). In some embodiments, the gRNA is from a Cas12 CRISPR / Cas system, e.g. is Cas12-compatible. In some embodiments, the gRNA is from a Cas12a (i.e. Cpf1) CRISPR / Cas system, e.g. is Cas12a-compatible. In some embodiments, the gRNA spacer is flanked by non-spacer sequences.

[0110] In some embodiments, the gRNA comprises a constant region. In some embodiments,the gRNA comprises a scaffold sequence. In some embodiments, the gRNA comprises a scaffold region. In some embodiments, the gRNA comprises a scaffold. In some embodiments, the constant region is a scaffold sequence. In some embodiments, the constant region comprises a scaffold sequence. In some embodiments, the constant region comprises a scaffold sequence and an additional sequence. In some embodiments, the constant region comprises a scaffold sequence and a functional sequence. In some embodiments, the functional sequence is a primer hybridization sequence, a sequencing primer binding site, or complement thereof. In some embodiments, the constant region comprises a capturing sequence. The constant region of the gRNA can be designed to include any suitable additional sequence in accordance with the methods described herein.

[0111] In some aspects, provided herein are methods for analyzing (e.g. sequencing) guideRNAs (gRNAs). In some aspects, gRNA sequencing may refer to the detection of a gRNA in a sample by a method involving nucleic acid sequencing, such as according to any of the methods provided herein. In some embodiments, the sequencing comprises determining and / or detecting aAttorney Docket No. 43487-1034601 sequence of a gRNA, a product of the gRNA, a probe bound to the gRNA or product thereof (e.g. a gRNA-specific ligatable probe pair), or a product of the probe. In some aspects, provided herein are methods for analyzing a gRNA-expressing cell. In some aspects, provided herein are methods for analyzing a plurality of gRNA-expressing cells. In some aspects, provided herein are methods for analyzing (e.g. detecting and / or sequencing) gRNAs in a plurality of cells. In some aspects, provided herein are methods for detecting, quantifying, and / or sequencing gRNAs in a plurality of cells.

[0112] In some aspects, the gRNA analysis can be performed at the single-cell level (e.g.single-cell gRNA sequencing). In some embodiments, the methods for analyzing a gRNA- expressing cell can be performed in parallel for a plurality of gRNA-expressing cells. In some embodiments, the methods can be performed in a single-cell gRNA analysis (e.g. sequencing) workflow. In some embodiments, cells (e.g. gRNA-expressing cells) are partitioned into different partitions to facilitate single-cell analysis. The partitions can be any suitable partition, such as a droplet or a well. The partitions can comprise nucleic acid barcode molecules having partition-specific barcodes. In some embodiments, the nucleic acid barcode molecules and gRNA-specific ligatable probe pairs are used to generate nucleic acids (e.g. barcoded spacer oligonucleotides as described herein) comprising both a gRNA spacer sequence (or complement thereof) and a partition-specific barcode (or complement thereof). Sequencing the barcoded spacer oligonucleotides can thus reveal the presence and / or abundance of a gRNA having a particular spacer sequence, as well as the partition (e.g. single-cell) that the gRNA was present in. This analysis can be readily performed for a plurality of gRNAs collectively having a plurality of different spacer sequences in a plurality of single cells according to the methods provided herein. In some aspects, the methods for analyzing a gRNA-expressing cell are compatible with analyzing (e.g. sequencing) additional analytes, such as non-gRNA analytes (e.g. endogenous mRNA transcripts), in the same single cells, as described herein. The additional analytes may be any suitable analyte, including endogenous transcripts, proteins, other cellular components, and / or nucleic acids associated therewith. In some aspects, provided herein are workflows for combined gRNA and non-gRNA analyte (e.g. endogenous cellular transcript) analysis in the same single cell(s).

[0113] In some aspects, the methods herein are described for analysis of a gRNA-expressingcell. In some embodiments, the gRNA-expressing cell is a cell comprising a gRNA. The gRNA- expressing cell can be any cell comprising a gRNA, regardless of how the gRNA was generated (e.g. transcribed within the cell or directly transduced into the cell). In some embodiments, the gRNA is transcribed in the cell (e.g. from an expression construct). In some embodiments, the gRNA is not transcribed in the cell. For example, the gRNA can be transduced directly into theAttorney Docket No. 43487-1034601 cell without needing to be transcribed within the cell. The gRNA-expressing cell can be any suitable cell or derivative or product thereof. In some embodiments, the gRNA-expressing cell is a fixed cell, such as any fixed cell described herein or any cell fixed or prepared according to the methods provided herein. In some embodiments, the methods can be applied to cell derivatives or components thereof. For example, in some embodiments, any of the methods provided herein can be performed to analyze a gRNA-expressing nucleus. gRNA analysis with gRNA-specific ligatable probe pairs

[0114] In some aspects, provided herein is a method for detecting a gRNA. In some aspects,provided herein is a method for analyzing a cell. In some aspects, provided herein is a method for analyzing a gRNA-expressing cell. In some aspects, provided herein is a method for analyzing the presence and / or abundance of a gRNA. In some aspects, provided herein is a method for analyzing the presence and / or abundance of a gRNA in a cell. In some aspects, provided herein is a method for analyzing the presence and / or abundance of a plurality of gRNAs in a plurality of cells.

[0115] In some embodiments, the method comprises providing a gRNA-expressing cellcomprising a gRNA having a spacer sequence and a scaffold sequence. In some embodiments, the scaffold sequence is a target scaffold sequence. In some embodiments, the method comprises contacting the gRNA-expressing cell with a gRNA-specific ligatable probe pair. In some embodiments, the gRNA-specific ligatable probe pair comprises 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence, and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence. In some embodiments, the method comprises ligating the scaffold-hybridizing sequence to the spacer- hybridizing sequence using the gRNA as template, thereby generating a gRNA-specific ligated probe pair. In some embodiments, the method comprises ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence, thereby generating a gRNA-specific ligated probe pair. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the method comprises partitioning into a partition 1) the gRNA-specific ligatable probe pair or the gRNA-specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the method comprises generating the partition. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-specific ligatable probe pair hybridized to the gRNA or the gRNA-specific ligated probe pair hybridized to the gRNA, and 2)Attorney Docket No. 43487-1034601 a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the method comprises using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide. In some embodiments, the barcoded spacer oligonucleotide comprises 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition- specific barcode or a complement thereof. In some embodiments, the spacer-hybridizing sequence is in a spacer-hybridizing region of the spacer probe. In some embodiments, the scaffold-hybridizing sequence is in a scaffold-hybridizing region of the scaffold probe. In some embodiments, the barcoded spacer oligonucleotide (such as any barcoded spacer oligonucleotide provided herein) can, instead of the spacer sequence or complement thereof, comprise a sequence that is indicative of the spacer sequence. For example, the gRNA-specific ligatable probe pair can comprise a sequence corresponding to the spacer sequence (e.g. a spacer-specific barcode) that is incorporated into the barcoded spacer oligonucleotide. Similarly, the barcoded analyte oligonucleotide (such as any barcoded analyte oligonucleotide provided herein) can comprise a sequence that is indicative of the target nucleic acid (e.g. a target nucleic acid- specific barcode) that is incorporated into the barcoded analyte oligonucleotide.

[0116] In some embodiments, the scaffold-hybridizing sequence hybridizes to the scaffoldsequence (e.g. a target scaffold sequence). In some embodiments, the scaffold sequence is in the gRNA scaffold. In some embodiments, the scaffold sequence is in the constant region of the gRNA. In some embodiments, the scaffold sequence is a sequence of the gRNA scaffold that is hybridized by the scaffold-hybridizing sequence (and, in some embodiments, by a portion of the spacer-hybridizing sequence, as described herein). In some embodiments, the scaffold sequence can be any portion of the gRNA that is not the spacer sequence. In some embodiments, the scaffold sequence is adjacent to the spacer sequence. In some embodiments, the scaffold sequence is a sequence of the gRNA that is adjacent to the spacer sequence. In some embodiments, the scaffold sequence is not adjacent to the spacer sequence (e.g. is separated from the spacer sequence by one or more nucleotides on the gRNA). In some embodiments, the scaffold-hybridizing sequence hybridizes to both the gRNA scaffold and a portion of the spacer sequence.

[0117] In some embodiments, the spacer-hybridizing sequence hybridizes to the spacer ofthe gRNA. In some embodiments, the spacer-hybridizing sequence hybridizes to the entire spacer sequence or a portion thereof. In some embodiments, the spacer-hybridizing sequence hybridizes to a portion of the spacer sequence. In some embodiments, the spacer-hybridizing sequence hybridizes to both a sequence of the spacer and a portion of the gRNA that is not the spacer (e.g. the scaffold). In some embodiments, the spacer-hybridizing sequence hybridizes toAttorney Docket No. 43487-1034601 both the spacer (e.g. the entire spacer or a portion thereof) and a portion of the scaffold. The spacer-hybridizing sequence hybridizing to both the spacer and a portion of the scaffold sequence (e.g. a first portion) can provide certain advantages for assay design and gRNA detection, as described herein. For example, in a multiplexed assay, such an arrangement can allow a consistent ligation site among different gRNA-specific ligatable probe pairs, providing consistent detection sensitivity.

[0118] While in some embodiments the gRNA-specific ligatable probe pairs provided hereincomprise a spacer probe and a scaffold probe, methods are also contemplated in which a gRNA- specific ligatable probe pair comprises two probes that both hybridize to the spacer, and optionally exclusively to the spacer. For example, a first and second spacer probe of a gRNA- specific ligatable probe pair can hybridize to a first and second portion of the gRNA spacer, respectively. In some embodiments, the first and second probe hybridized to the gRNA spacer are configured to be ligated by a ligase, e.g. without a gap filling step prior to ligation. A first and second spacer probe of a gRNA-specific ligatable probe pair need not hybridize to a portion of the gRNA scaffold at all, although in some embodiments the first or second spacer probe can hybridize to a portion of the gRNA scaffold. In some embodiments, the first and second spacer probe do not hybridize to the gRNA scaffold. In some aspects, use of gRNA-specific ligatable probe pairs comprising a first and second spacer probe may increase the need for unique probe synthesis in comparison to methods involving gRNA-specific ligatable probe pairs comprising a spacer probe and a scaffold probe. However, gRNA-specific ligatable probe pairs comprising a first and second spacer probe may provide other advantages, such as increased specificity. In some aspects, a gRNA-specific ligatable probe pair comprising a first and second spacer probe is compatible with the remaining steps of the methods described with respect to methods involving a gRNA-specific ligatable probe pair comprising a spacer probe and a scaffold probe. Thus, in some aspects, a gRNA-specific ligatable probe pair comprising a spacer probe and a scaffold probe is interchangeable with a gRNA-specific ligatable probe pair comprising a first and second spacer probe in any of the methods provided herein.

[0119] In some embodiments, the method comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell.

[0120] In some embodiments, the partition is generated before ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, the partition is generated after ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. InAttorney Docket No. 43487-1034601 some embodiments, the partitioning occurs before ligating the scaffold probe to the spacer probe. In some embodiments, the partitioning occurs after ligating the scaffold probe to the spacer probe.

[0121] In some embodiments, the gRNA-specific ligatable probe pair comprises one or moreoverhang sequences. In some embodiments, the one or more overhang sequences comprise a barcode sequence. In some embodiments, the barcode sequence is a sample-specific barcode sequence. In some embodiments, the one or more overhang sequences comprise one or more functional sequences. In some embodiments, the one or more functional sequences comprise a primer hybridization sequence or complement thereof, and / or a sequencing primer binding site or complement thereof. In some embodiments, the one or more overhang sequences comprise a capturing sequence that hybridizes to a capture sequence of the nucleic acid barcode molecule. In some embodiments, the method comprises hybridizing the gRNA-specific ligated probe pair to the nucleic acid barcode molecule. In some embodiments, the method comprises extending the gRNA-specific ligated probe pair using the nucleic acid barcode molecule as template, and / or extending the nucleic acid barcode molecule using the gRNA-specific ligated probe pair as template; thereby generating the barcoded spacer oligonucleotide. In some embodiments, the method comprises ligating the gRNA-specific ligated probe pair to the nucleic acid barcode molecule, thereby generating the barcoded spacer oligonucleotide.

[0122] In some embodiments, the spacer-hybridizing sequence hybridizes to the spacersequence and a first portion of the scaffold sequence. In some embodiments, the scaffold- hybridizing sequence hybridizes to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence. In some aspects, this arrangement can be advantageous, particularly in multiplexed assays in which multiple different gRNA-specific ligatable probe pairs are used to detect different gRNAs having different spacers. This is because the arrangement can allow the ligation junction between the scaffold probe and the spacer probe to remain uniform among gRNA-specific ligatable probe pairs targeting different gRNAs, since the ligation will always occur between the same two nucleotides hybridized to the constant scaffold sequence. However, such an arrangement is not required to perform the assays provided herein.

[0123] In some embodiments, the scaffold sequence and the spacer sequence are adjacent. Insome embodiments, the scaffold sequence and the spacer sequence are not adjacent and the method comprises extending the scaffold probe or the spacer probe using the gRNA as template prior to ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence.

[0124] In some embodiments, the plurality of nucleic acid barcode molecules are coupled toa support. In some embodiments, the support is a particle. In some embodiments, the support is a bead. In some embodiments, the nucleic acid barcode molecules are released from the supportAttorney Docket No. 43487-1034601 upon generating the partition. In some embodiments, the nucleic acid barcode molecules are released from the support after generating the partition. In some embodiments, the nucleic acid barcode molecules are released from the support upon providing a stimulus.

[0125] In some embodiments, the method comprises contacting the gRNA-expressing cellwith an analyte-specific ligatable probe pair comprising a first analyte probe and a second analyte probe that hybridize to sequences of an analyte. In some embodiments, the method comprises ligating the first analyte probe and second analyte probe hybridized to the analyte to generate an analyte-specific ligated probe pair. In some embodiments, the partition comprises the analyte-specific ligatable probe pair or the analyte-specific ligated probe pair. In some embodiments, the method comprises using the analyte-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded analyte oligonucleotide comprising 1) a sequence of the analyte or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof. In some embodiments, the method comprises sequencing the barcoded analyte oligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the analyte in the gRNA-expressing cell. In some embodiments, the sequences of the analyte are adjacent. In some embodiments, the sequences of the analyte are not adjacent and the method comprises extending the first or second analyte probe using the analyte as template prior to ligating the first and second analyte probe. In some embodiments, the analyte is a non-gRNA analyte that is not a gRNA. In some embodiments, the analyte is an endogenous cellular transcript. In some embodiments, the analyte is an mRNA.

[0126] In some aspects, provided herein is a method for analyzing the presence and / orabundance of a plurality of gRNAs in a plurality of cells. In some embodiments, the partition is a partition of a plurality of partitions. In some embodiments, the partition is a well of a plurality of wells or a droplet of a plurality of droplets. In some embodiments, the providing comprises providing a population of cells comprising the gRNA-expressing cell. In some embodiments, cells of the population of cells comprise gRNAs of a gRNA library, wherein the gRNAs of the gRNA library comprise a scaffold sequence and a spacer sequence. In some embodiments, different cells of the population of cells comprise different gRNAs of a gRNA library. In some embodiments, the different gRNAs comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs. In some embodiments, the contacting comprises contacting the population of cells with the gRNA- specific ligatable probe pair. In some embodiments, the contacting comprises contacting theAttorney Docket No. 43487-1034601 population of cells with a library of gRNA-specific ligatable probe pairs comprising the gRNA- specific ligatable probe pair. In some embodiments, each gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises 1) a scaffold probe having a scaffold-hybridizing sequence, and 2) a spacer probe having a spacer-hybridizing sequence. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises different gRNA-specific ligatable probe pairs configured to hybridize to different gRNAs of the gRNA library. In some embodiments, the spacer probes of the different gRNA-specific ligatable probe pairs have different spacer-hybridizing sequences. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs have the same scaffold-hybridizing sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs are identical in nucleotide sequence. In some embodiments, the different spacer-hybridizing sequences hybridize to different spacer sequences of the different gRNAs. In some embodiments, the different spacer-hybridizing sequences further hybridize to a first portion of the scaffold sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs hybridize to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence.

[0127] In some embodiments, the method comprises generating a library of barcoded spaceroligonucleotides, each comprising: 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises hybridizing gRNA-specific ligatable probe pairs of the library of gRNA-specific ligatable probe pairs to gRNAs of the gRNA library in cells of the population of cells; ligating the hybridized gRNA-specific ligatable probe pairs to generate gRNA-specific ligated probes; partitioning the gRNA-specific ligatable probe pairs or gRNA- specific ligated probes from different cells into different partitions comprising nucleic acid barcode molecules comprising partition-specific barcodes; and using the gRNA-specific ligated probes and nucleic acid barcode molecules to generate a library of barcoded spacer oligonucleotides, each comprising 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof.

[0128] In some embodiments, the method comprises sequencing the library of barcodedspacer oligonucleotides. In some embodiments, the method comprises analyzing the results of the sequencing to associate spacer sequences with partition-specific barcodes. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of different gRNAs of the gRNA library in different cells of the population of cells.Attorney Docket No. 43487-1034601

[0129] In some embodiments, the method comprises performing nucleic acid sequencing todetermining the presence and / or abundance of one or more non-gRNA analytes in the different cells of the population of cells. In some embodiments, the contacting further comprises contacting the population of cells with a library of analyte-specific ligatable probe pairs configured to hybridize to different non-gRNA analytes. In some embodiments, the method comprises using the library of analyte-specific ligatable probe pairs to generate a library of barcoded analyte oligonucleotides, each comprising 1) a sequence of an analyte of the different analytes or a complement thereof, and 2) a sequence of a partition-specific barcode or a complement thereof. In some embodiments, the method comprises hybridizing analyte-specific ligatable probe pairs of the library of analyte-specific ligatable probe pairs to analytes in cells of the population of cells; ligating the hybridized analyte-specific ligatable probe pairs to generate analyte-specific ligated probes; partitioning the analyte-specific ligatable probe pairs or analyte- specific ligated probes from different cells into different partitions comprising nucleic acid barcode molecules comprising partition-specific barcodes; and using the analyte-specific ligated probes and nucleic acid barcode molecules to generate a library of barcoded analyte oligonucleotides, each comprising 1) a sequence of an analyte or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises sequencing the barcoded analyte oligonucleotides or derivatives thereof. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of one or more non-gRNA analytes of the different non-gRNA analytes in the different cells of the population of cells.

[0130] In some aspects, provided herein is a method for analyzing the presence and / orabundance of a plurality of gRNAs in a plurality of cells. In some aspects, the method comprises providing a population of cells. In some embodiments, different cells of the population of cells comprise different gRNAs of a gRNA library. In some embodiments, the different gRNAs of the gRNA library comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs. In some embodiments, the method comprises contacting the population of cells with a library of gRNA-specific ligatable probe pairs. In some embodiments, a gRNA-specific ligatable probe pair of the library of gRNA- specific ligatable probe pairs comprises: 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence of a gRNA of the gRNA library in a gRNA- expressing cell of the population of cells; and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence of the gRNA of the gRNA library in the gRNA- expressing cell of the population of cells. In some embodiments, the method comprises ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence using the gRNA asAttorney Docket No. 43487-1034601 template, thereby generating a gRNA-specific ligated probe pair. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-specific ligatable probe pair or the gRNA-specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the method comprises generating a partition comprising 1) the gRNA-specific ligatable probe pair hybridized to the gRNA or the gRNA-specific ligated probe pair hybridized to the gRNA, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode. In some embodiments, the method comprises using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide. In some embodiments, the barcoded spacer oligonucleotide comprises 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof.

[0131] In some embodiments, the method comprises sequencing the barcoded spaceroligonucleotide or a derivative thereof. In some embodiments, the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode. In some embodiments, the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell. In some embodiments, the partition is generated before ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, the partition is generated after ligating the scaffold-hybridizing sequence to the spacer-hybridizing sequence. In some embodiments, each gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises 1) a scaffold probe having a scaffold-hybridizing sequence, and 2) a spacer probe having a spacer-hybridizing sequence. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises different gRNA-specific ligatable probe pairs configured to hybridize to different gRNAs of the gRNA library. In some embodiments, the spacer probes of the different gRNA-specific ligatable probe pairs have different spacer-hybridizing sequences. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs have the same scaffold-hybridizing sequence. In some embodiments, the scaffold probes of the different gRNA-specific ligatable probe pairs are identical in sequence. In some embodiments, the method comprises using the library of gRNA-specific ligatable probe pairs to generate a library of barcoded spacer oligonucleotides, each comprising: 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof. In some embodiments, the method comprises sequencing the library of barcoded spacer oligonucleotides.Attorney Docket No. 43487-1034601

[0132] In some embodiments, the method comprises analyzing the results of the sequencingto associate spacer sequences with partition-specific barcodes. In some embodiments, the method comprises analyzing the results of the sequencing to analyze and / or determine the presence and / or abundance of different gRNAs of the gRNA library in different cells of the population of cells. In some embodiments, the method comprises performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in the different cells of the population of cells.

[0133] In some embodiments, the gRNA library comprises at least 2 different gRNAs. Insome embodiments, the gRNA library comprises at least 5 different gRNAs. In some embodiments, the gRNA library comprises at least 10 different gRNAs. In some embodiments, the gRNA library comprises at least 50 different gRNAs. In some embodiments, the gRNA library comprises at least 100 different gRNAs. In some embodiments, the gRNA library comprises at least 500 different gRNAs. In some embodiments, the gRNA library comprises at least 1000 different gRNAs. In some embodiments, the gRNA library comprises at least 10,000 different gRNAs.

[0134] In some embodiments, the method comprises analyzing the presence and / orabundance of at least 2 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 5 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 10 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 50 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 100 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 500 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 1000 different gRNAs. In some embodiments, the method comprises analyzing the presence and / or abundance of at least 10,000 different gRNAs.

[0135] In some embodiments, the method comprises contacting a cell or a plurality of cellswith at least 2 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 5 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 10 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 50 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 100 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 500Attorney Docket No. 43487-1034601 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 1000 different gRNA-specific ligatable probe pairs. In some embodiments, the method comprises contacting a cell or a plurality of cells with at least 10,000 different gRNA-specific ligatable probe pairs.

[0136] In some embodiments, the library of gRNA-specific ligatable probe pairs comprisesat least 2 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 5 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 10 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 50 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 100 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 500 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 1000 different gRNA-specific ligatable probe pairs. In some embodiments, the library of gRNA-specific ligatable probe pairs comprises at least 10,000 different gRNA-specific ligatable probe pairs.

[0137] In some embodiments, provided herein is a composition or kit. In some embodiments,the composition or kit comprises a gRNA-specific ligatable probe pair, such as any described in connection with the methods provided herein. In some embodiments, the gRNA-specific ligatable probe pair is any of the gRNA-specific ligatable probe pairs provided herein. In some embodiments, the kit further comprises an analyte-specific ligatable probe pair, such as any described in connection with the methods provided herein.. The analyte-specific ligatable probe pair can comprise any of the analyte-specific ligatable probe pairs provided herein. In some embodiments, provided herein is a composition or kit comprising a gRNA-specific ligatable probe pair and an analyte-specific ligatable probe pair. In some embodiments, the composition or kit comprises a plurality (e.g. library) of gRNA-specific ligatable probe pairs, such as any described in connection with the methods provided herein. In some embodiments, the composition or kit further comprises a plurality (e.g. library) of analyte-specific ligatable probe pairs, such as any described in connection with the methods provided herein. In some embodiments, the composition or kit further comprises a ligase. In some embodiments, the composition or kit comprises a plurality of barcoded nucleic acid molecules, such as any described in connection with the methods provided herein.

[0138] In some embodiments, provided herein are systems for analyzing gRNA-expressingcells according to any of the methods provided herein. In some embodiments, the systemsAttorney Docket No. 43487-1034601 comprise any of the compositions or kits provided herein. In some embodiments, the system further comprises one or more components for performing the methods. In some embodiments, the system comprises a partition or a plurality of partitions. In some embodiments, the system comprises a device for generating partitions, such as wells or droplets. In some embodiments, the system comprises wells for the partitioning. In some embodiments, the system comprises means for sequencing the barcoded spacer oligonucleotides and / or the barcoded analyte oligonucleotides. In some embodiments, the system comprises a sequencer. In some embodiments, the system comprises one or more devices, processors, and / or computers for analyzing the results of the sequencing. Exemplary gRNA and non-gRNA analyte analysis workflows

[0139] In some aspects, any of the workflows for analyzing and / or sequencing gRNAs canbe performed in combination with analysis of additional analytes, such as non-gRNA analytes. For example, FIG.47 shows an exemplary workflow in which gRNA-expressing cells expressing gRNAs and other analytes (e.g. cellular transcripts) are analyzed. Barcoded spacer oligonucleotides and barcoded analyte oligonucleotides can be generated from the same single cells, for example as described herein. The barcoded spacer oligonucleotides and barcoded analyte oligonucleotides can be amplified and sequenced to determine the presence and / or abundance of gRNAs and non-gRNA analytes at the single-cell level in a plurality of single cells.

[0140] In some aspects, a workflow for detecting and / or sequencing a gRNA using a gRNA-specific ligatable probe pair as described herein is shown in FIG.48. Part 1 of the figure shows a spacer probe and a scaffold probe of a gRNA-specific ligatable probe pair hybridized to the spacer and scaffold sequence, respectively, of a gRNA, which may be in a cell. Part 2 of the figure shows the gRNA-specific ligatable probe pair is ligated to generate a gRNA-specific ligated probe pair. Part 3 shows that the cell comprising the gRNA (black circle) can be partitioned with a plurality of nucleic acid barcode molecules having partition-specific barcodes. For simplicity, the figure shows only a single nucleic acid barcode molecule coupled to a support (e.g. bead), shown as a white circle. Part 4 shows that the gRNA-specific ligatable probe pair and a nucleic acid barcode molecule can be used to generate a barcoded spacer oligonucleotide, for example via hybridization and extension. The barcoded spacer oligonucleotide can be sequenced to determine the presence and / or abundance of the gRNA in the cell. As described herein, the method can be performed in parallel using any number of different gRNA-specific ligatable probe pairs for analyzing and / or determining the presence and / or abundance of any number of different gRNAs in one or more cells (e.g. in a plurality of single cells). In any of the methodsAttorney Docket No. 43487-1034601 provided herein, the method can further comprise performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in the same cell(s). For example, analyte-specific ligatable probe pairs may also be used in parallel to analyze the presence and / or abundance of any number of additional non-gRNA analytes in the same cell(s). CRISPR and cellular perturbations

[0141] In some embodiments, the present disclosure is further drawn to assaying not just thetranscriptome and proteome, but also cellular perturbations. In some embodiments, the probe- based approach described herein is further adapted to assay cellular perturbations by utilizing the probe-based approach – the LHS and RHS hybridize to the guide scaffold (LHS or RHS) and the protospacer (LHS or RHS) of sgRNA. The sequences of the LHS and RHS regions that hybridize to the guide scaffold and the protospacer can be designed to target specific sequences, thus allowing a high degree of modularity to design probe pairs to identify the sgRNA of interest for each experiment.

[0142] In some embodiments, the probe-based approach described herein is applied only toassaying the transcriptome and perturbome of the cells of the sample. In some embodiments, the probe-based approach described herein is applied to assaying both the transcriptome and perturbome of the cells of the sample. In further embodiments, the proteome is assayed, as described herein to identify and discern intra- and extra-cellular features, along with the transcriptome and / or proteome.

[0143] In some embodiments, the cells of the sample comprise a dCas9, which is a modifiedCas9 whose endonucleoase activity is removed or substantially impaired. In some embodiments, each cell of the sample comprises at least two different sgRNAs. In some embodiments, each cell of the sample comprises at least three different, at least four different, at least five different, at least six different, at least seven different, at least eight different, at least nine different, at least ten different, at least eleven different, at least twelve different, at least thirteen different, at least fourteen different, or at least fifteen different sgRNAs. SAMPLES, COMPOSITIONS, SYSTEMS, AND ANALYSIS Fixed Samples

[0144] 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. Alternatively or in addition, a sample may comprise a fixed tissue. Fixation of cell or cellular constituent, or a tissue comprising a plurality of cells or nuclei, may comprise application of a chemical species orAttorney Docket No. 43487-1034601 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 to 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.

[0145] 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 are well known in the art and 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. Typically, the formaldehyde is diluted from a more concentrated stock solution – e.g., a 35%, 25%, 15%, 10%, 5% PFA stock solution.Attorney Docket No. 43487-1034601

[0146] 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.).

[0147] 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.

[0148] 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.Attorney Docket No. 43487-1034601

[0149] 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. Typically, a suitable concentration of glutaraldehyde 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).

[0150] 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 partially 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.

[0151] 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 asAttorney Docket No. 43487-1034601 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).

[0152] 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 paraffin wax. FFPE processing may facilitate preservation of a tissue sample (e.g., prior to subsequent processing 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. Methods of Nucleic Acid Analysis

[0153] 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 reaction (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 ofAttorney Docket No. 43487-1034601 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).

[0154] 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. 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.

[0155] 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 moleculeAttorney Docket No. 43487-1034601 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).

[0156] 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. See also US20240002901.

[0157] 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 Kozak sequence, a Shine-Dalgarno sequence, a coding sequence, and a poly(A) sequence (e.g., a poly(A) tail).

[0158] 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,Attorney Docket No. 43487-1034601 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.

[0159] 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). 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.Attorney Docket No. 43487-1034601

[0160] 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.

[0161] 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 functional 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 probeAttorney Docket No. 43487-1034601 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.

[0162] 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, 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.

[0163] 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-Attorney Docket No. 43487-1034601 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.

[0164] 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.

[0165] 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 acid 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 membraneAttorney Docket No. 43487-1034601 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.

[0166] 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.

[0167] 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 in PCT / US2020 / 066720 and International Pat. App. No. PCT / US2021 / 33649, filed May 21, 2021, which is incorporated by reference herein in its entirety.

[0168] 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., aAttorney Docket No. 43487-1034601 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.

[0169] 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) aAttorney Docket No. 43487-1034601 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.

[0170] 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 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.

[0171] 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 acidAttorney Docket No. 43487-1034601 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.

[0172] 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).

[0173] 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). In 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.,Attorney Docket No. 43487-1034601 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.

[0174] 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).

[0175] 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 a 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.Attorney Docket No. 43487-1034601

[0176] 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 probe binding molecule may be ligated to the second probe 816, e.g., using a ligase or via chemical linkage, such as click chemistry.

[0177] 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 orAttorney Docket No. 43487-1034601 derivatives thereof may then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing (e.g., Illumina sequencing).

[0178] 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.

[0179] 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.

[0180] 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 probe 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 aAttorney Docket No. 43487-1034601 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).

[0181] 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.

[0182] 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 theAttorney Docket No. 43487-1034601 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.

[0183] 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).

[0184] 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, the 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 probe2616 comprises a flap sequence 2630. In such instances, in operation 2605, an endonuclease,e.g., FEN1 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.

[0185] 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 nucleicAttorney Docket No. 43487-1034601 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.

[0186] 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.

[0187] 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.

[0188] 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. 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.

[0189] 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 anAttorney Docket No. 43487-1034601 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).

[0190] 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 cell 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,Attorney Docket No. 43487-1034601 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.

[0191] 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 and 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 someAttorney Docket No. 43487-1034601 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.

[0192] 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.

[0193] 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 partition 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 cellAttorney Docket No. 43487-1034601 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).

[0194] 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.

[0195] 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.

[0196] 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 molecule 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 moleculeAttorney Docket No. 43487-1034601 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.

[0197] 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 barcode 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.

[0001] 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 aAttorney Docket No. 43487-1034601 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. 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.

[0198] 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.

[0199] 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 firstAttorney Docket No. 43487-1034601 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.

[0200] 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.

[0201] 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 (i.e., 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 (i.e., 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. CombinatorialAttorney Docket No. 43487-1034601 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.

[0202] 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 be 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.

[0203] 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 comprisingAttorney Docket No. 43487-1034601 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.

[0204] 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 operation 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 theAttorney Docket No. 43487-1034601 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.

[0205] 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).

[0206] 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.). In 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 transciptases, restriction enzymes, etc.), nucleotides bases, etc.

[0207] 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,Attorney Docket No. 43487-1034601 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

[0208] 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).

[0209] 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.

[0210] 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,Attorney Docket No. 43487-1034601 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.

[0211] 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.

[0212] 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 binding 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, theAttorney Docket No. 43487-1034601 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.

[0213] 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 (i.e., 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 second 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.

[0214] 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.

[0215] 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 theAttorney Docket No. 43487-1034601 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.

[0216] 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.

[0217] 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 probe 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 aAttorney Docket No. 43487-1034601 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).

[0218] 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 the capture-binding sequence, which may allow for hybridization of the capture sequence of the capture molecules to the barcode molecule.

[0219] FIG. 15 schematically illustrates an example barcoded nucleic acid molecule asdescribed herein. Referring to Panel A, 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.Attorney Docket No. 43487-1034601

[0220] 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 (i.e., 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.

[0221] 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 (i.e., 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).Attorney Docket No. 43487-1034601

[0222] Panel B of FIG. 15 schematically illustrates another example barcoded molecule inwhich the capture molecule 1520 is hybridized to the barcode molecule 1519. Similar to Panel A, in Panel B, 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.

[0223] 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 of 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 (i.e., 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.Attorney Docket No. 43487-1034601

[0224] 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 (i.e., 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).

[0225] Panel C of FIG. 15 illustrates another example barcoded nucleic acid molecule. Anucleic 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 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.

[0226] 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 theyAttorney Docket No. 43487-1034601 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 (i.e., 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 capture 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.

[0227] 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 barcodeAttorney Docket No. 43487-1034601 sequence may be unique to the capture molecules in only the second partition (i.e., 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).

[0228] 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 the 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.

[0229] 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 compriseAttorney Docket No. 43487-1034601 may also comprise a probe capture sequence, which may be the same or different than the probe capture sequence of the third probe.

[0230] 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. 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.

[0231] 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 (i.e., 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).

[0232] 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 aAttorney Docket No. 43487-1034601 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.

[0233] 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 binding 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.

[0234] 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 particularAttorney Docket No. 43487-1034601 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.

[0235] 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.

[0236] 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.

[0237] 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-Attorney Docket No. 43487-1034601 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.

[0238] 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 probe 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 describedAttorney Docket No. 43487-1034601 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.

[0239] 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 inAttorney Docket No. 43487-1034601 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 the 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.Attorney Docket No. 43487-1034601

[0240] 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).

[0241] 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, 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.Attorney Docket No. 43487-1034601

[0242] 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 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. 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 captureAttorney Docket No. 43487-1034601 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.

[0243] 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, 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) aAttorney Docket No. 43487-1034601 probe 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.

[0244] 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 oligonucleotide 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.

[0245] 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 oligonucleotid...

Claims

Attorney Docket No. 43487-1034601 CLAIMS WHAT IS CLAIMED IS:

1. A method comprising: providing a guide ribonucleic acid (gRNA)-expressing cell comprising a gRNA having a spacer sequence and a scaffold sequence; contacting the gRNA-expressing cell with a gRNA-specific ligatable probe pair comprising 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence, and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence; ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence, thereby generating a gRNA-specific ligated probe pair; partitioning into a partition: 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof.

2. The method of claim 1, wherein the method comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof.

3. The method of claim 2, wherein the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode.

4. The method of claim 2 or 3, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell.

5. The method of any of claims 1-4, wherein the ligating comprises ligating the scaffold- hybridizing sequence to the spacer-hybridizing sequence.Attorney Docket No. 43487-1034601 6. The method of any of claims 1-5, wherein the ligating comprises ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence using the gRNA as template.

7. The method of any of claims 1-6, wherein the partitioning into a partition comprises generating a partition comprising: 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode.

8. The method of any of claims 1-7, wherein the partitioning occurs before ligating the scaffold probe to the spacer probe..

9. The method of any of claims 1-7, wherein the partitioning occurs after ligating the scaffold probe to the spacer probe.

10. The method of any of claims 1-9, wherein the gRNA-specific ligatable probe pair comprises one or more overhang sequences.

11. The method of claim 10, wherein the one or more overhang sequences comprise a barcode sequence.

12. The method of claim 11, wherein the barcode sequence is a sample-specific barcode sequence.

13. The method of any of claims 10-12, wherein the one or more overhang sequences comprise one or more functional sequences.

14. The method of claim 13, wherein the one or more functional sequences comprise a primer hybridization sequence or complement thereof, and / or a sequencing primer binding site or complement thereof.

15. The method of any of claims 10-14, wherein the one or more overhang sequences comprise a capturing sequence that hybridizes to a capture sequence of the nucleic acid barcode molecule.Attorney Docket No. 43487-1034601 16. The method of any of claims 1-15, wherein the method comprises hybridizing the gRNA- specific ligated probe pair to the nucleic acid barcode molecule.

17. The method of any of claims 1-16, wherein the method comprises extending the gRNA- specific ligated probe pair using the nucleic acid barcode molecule as template, and / or extending the nucleic acid barcode molecule using the gRNA-specific ligated probe pair as template; thereby generating the barcoded spacer oligonucleotide.

18. The method of any of claims 1-17, wherein the method comprises ligating the gRNA- specific ligated probe pair to the nucleic acid barcode molecule, thereby generating the barcoded spacer oligonucleotide.

19. The method of any of claims 1-18, wherein the spacer-hybridizing sequence hybridizes to the spacer sequence and a first portion of the scaffold sequence.

20. The method of claim 19, wherein the scaffold-hybridizing sequence hybridizes to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence.

21. The method of any of claims 1-20, wherein the scaffold sequence and the spacer sequence are adjacent.

22. The method of any of claims 1-20, wherein the scaffold sequence and the spacer sequence are not adjacent.

23. The method of claim 22, wherein the method comprises extending the scaffold probe or the spacer probe using the gRNA as template prior to ligating the scaffold probe to the spacer probe.

24. The method of any of claims 1-23, wherein the plurality of nucleic acid barcode molecules are coupled to a support.

25. The method of claim 24, wherein the support is a particle and / or a bead.Attorney Docket No. 43487-1034601 26. The method of claim 24 or 25, wherein the nucleic acid barcode molecules are released from the support upon generating the partition and / or upon providing a stimulus.

27. The method of any of claims 1-26, wherein the method comprises contacting the gRNA- expressing cell with an analyte-specific ligatable probe pair comprising a first analyte probe and a second analyte probe that hybridize to sequences of an analyte.

28. The method of claim 27, wherein the method comprises ligating the first analyte probe and second analyte probe hybridized to the analyte to generate an analyte-specific ligated probe pair.

29. The method of claim 27 or 28, wherein the partition comprises the analyte-specific ligatable probe pair or the analyte-specific ligated probe pair.

30. The method of claim 28 or 29, wherein the method comprises using the analyte-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded analyte oligonucleotide comprising 1) a sequence of the analyte or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof.

31. The method of claim 30, wherein the method comprises sequencing the barcoded analyte oligonucleotide or a derivative thereof.

32. The method of claim 31, wherein the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode.

33. The method of claim 31 or 32, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the analyte in the gRNA-expressing cell.

34. The method of any of claims 27-33, wherein the sequences of the analyte are adjacent.

35. The method of any of claims 27-33, wherein the sequences of the analyte are not adjacent and the method comprises extending the first or second analyte probe using the analyte as template prior to ligating the first and second analyte probe.Attorney Docket No. 43487-1034601 36. The method of any of claims 27-35, wherein the analyte is a non-gRNA analyte that is not a gRNA.

37. The method of any of claims 27-36, wherein the analyte is an endogenous cellular transcript.

38. The method of any of claims 27-37, wherein the analyte is an mRNA.

39. The method of any of claims 1-38, wherein the partition is a partition of a plurality of partitions.

40. The method of any of claims 1-39, wherein the partition is a well of a plurality of wells or a droplet of a plurality of droplets.

41. The method of any of claims 1-40, wherein the providing comprises providing a population of cells comprising the gRNA-expressing cell.

42. The method of claim 41, wherein cells of the population of cells comprise gRNAs of a gRNA library, wherein the gRNAs of the gRNA library comprise a scaffold sequence and a spacer sequence.

43. The method of claim 41 or 42, wherein different cells of the population of cells comprise different gRNAs of a gRNA library, and wherein the different gRNAs comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs.

44. The method of any of claims 41-43, wherein the contacting comprises contacting the population of cells with the gRNA-specific ligatable probe pair.

45. The method of any of claims 41-44, wherein the contacting comprises contacting the population of cells with a library of gRNA-specific ligatable probe pairs comprising the gRNA- specific ligatable probe pair.Attorney Docket No. 43487-1034601 46. The method of claim 45, wherein each gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises 1) a scaffold probe having a scaffold- hybridizing sequence, and 2) a spacer probe having a spacer-hybridizing sequence.

47. The method of claim 45 or 46, wherein the library of gRNA-specific ligatable probe pairs comprises different gRNA-specific ligatable probe pairs configured to hybridize to different gRNAs of the gRNA library.

48. The method of claim 47, wherein the spacer probes of the different gRNA-specific ligatable probe pairs have different spacer-hybridizing sequences.

49. The method of claim 47 or 48, wherein the scaffold probes of the different gRNA- specific ligatable probe pairs have the same scaffold-hybridizing sequence.

50. The method of any of claims 47-49, wherein the scaffold probes of the different gRNA- specific ligatable probe pairs are identical in nucleotide sequence.

51. The method of any of claims 48-50, wherein the different spacer-hybridizing sequences hybridize to different spacer sequences of the different gRNAs.

52. The method of claim 51, wherein the different spacer-hybridizing sequences further hybridize to a first portion of the scaffold sequence.

53. The method of claim 52, wherein the scaffold probes of the different gRNA-specific ligatable probe pairs hybridize to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence.

54. The method of any of claims 42-53, wherein the method comprises generating a library of barcoded spacer oligonucleotides, each comprising: 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof.

55. The method of claim 54, wherein the method comprises hybridizing gRNA-specific ligatable probe pairs of the library of gRNA-specific ligatable probe pairs to gRNAs of the gRNA library in cells of the population of cells; ligating the hybridized gRNA-specific ligatable probe pairs to generate gRNA-specific ligated probes; partitioning the gRNA-specific ligatableAttorney Docket No. 43487-1034601 probe pairs or gRNA-specific ligated probes from different cells into different partitions comprising nucleic acid barcode molecules comprising partition-specific barcodes; and using the gRNA-specific ligated probes and nucleic acid barcode molecules to generate a library of barcoded spacer oligonucleotides, each comprising 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof.

56. The method of claim 54 or 55, wherein the method comprises sequencing the library of barcoded spacer oligonucleotides.

57. The method of claim 56, wherein the method comprises analyzing the results of the sequencing to associate spacer sequences with partition-specific barcodes.

58. The method of claim 56 or 57, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of different gRNAs of the gRNA library in different cells of the population of cells.

59. The method of any of claims 43-58, wherein the method comprises performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in the different cells of the population of cells.

60. The method of any of claims 41-59, wherein the contacting further comprises contacting the population of cells with a library of analyte-specific ligatable probe pairs configured to hybridize to different non-gRNA analytes.

61. The method of claim 60, wherein the method comprises using the library of analyte- specific ligatable probe pairs to generate a library of barcoded analyte oligonucleotides, each comprising 1) a sequence of an analyte of the different analytes or a complement thereof, and 2) a sequence of a partition-specific barcode or a complement thereof.

62. The method of claim 61, wherein the method comprises hybridizing analyte-specific ligatable probe pairs of the library of analyte-specific ligatable probe pairs to analytes in cells of the population of cells; ligating the hybridized analyte-specific ligatable probe pairs to generate analyte-specific ligated probes; partitioning the analyte-specific ligatable probe pairs or analyte- specific ligated probes from different cells into different partitions comprising nucleic acid barcode molecules comprising partition-specific barcodes; and using the analyte-specific ligatedAttorney Docket No. 43487-1034601 probes and nucleic acid barcode molecules to generate a library of barcoded analyte oligonucleotides, each comprising 1) a sequence of an analyte or complement thereof; and 2) a partition-specific barcode or complement thereof.

63. The method of claim 61 or 62, wherein the method comprises sequencing the barcoded analyte oligonucleotides or derivatives thereof.

64. The method of claim 63, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of one or more non-gRNA analytes of the different non-gRNA analytes in the different cells of the population of cells.

65. A method comprising: providing a population of cells, wherein different cells of the population of cells comprise different guide ribonucleic acids (gRNAs) of a guide ribonucleic acid (gRNA) library; wherein the different gRNAs of the gRNA library comprise: 1) a scaffold sequence that is the same among the different gRNAs, and 2) a spacer sequence that is different among the different gRNAs; contacting the population of cells with a library of gRNA-specific ligatable probe pairs, wherein a gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises: 1) a scaffold probe having a scaffold-hybridizing sequence that hybridizes to the scaffold sequence of a gRNA of the gRNA library in a gRNA-expressing cell of the population of cells; and 2) a spacer probe having a spacer-hybridizing sequence that hybridizes to the spacer sequence of the gRNA of the gRNA library in the gRNA-expressing cell of the population of cells; ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence, thereby generating a gRNA-specific ligated probe pair; partitioning into a partition: 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode; and using the gRNA-specific ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules to generate a barcoded spacer oligonucleotide comprising 1) a sequence of the spacer sequence or a complement thereof, and 2) a sequence of the partition-specific barcode or a complement thereof.Attorney Docket No. 43487-1034601 66. The method of claim 65, wherein the method comprises sequencing the barcoded spacer oligonucleotide or a derivative thereof.

67. The method of claim 66, wherein the method comprises analyzing the results of the sequencing to associate the spacer sequence with the partition-specific barcode.

68. The method of claim 66 or 67, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of the gRNA in the gRNA-expressing cell.

69. The method of any of claims 65-68, wherein the ligating comprises ligating the scaffold- hybridizing sequence to the spacer-hybridizing sequence.

70. The method of any of claims 65-69, wherein the ligating comprises ligating the scaffold probe hybridized to the scaffold sequence to the spacer probe hybridized to the spacer sequence using the gRNA as template.

71. The method of any of claims 65-70, wherein the partitioning into a partition comprises generating a partition comprising: 1) the gRNA-specific ligatable probe pair or the gRNA- specific ligated probe pair, and 2) a plurality of nucleic acid barcode molecules comprising a partition-specific barcode.

72. The method of any of claims 65-71, wherein the partitioning occurs before ligating the scaffold probe to the spacer probe.

73. The method of any of claims 65-71, wherein the partitioning occurs after ligating the scaffold probe to the spacer probe.

74. The method of any of claims 65-73, wherein each gRNA-specific ligatable probe pair of the library of gRNA-specific ligatable probe pairs comprises 1) a scaffold probe having a scaffold-hybridizing sequence, and 2) a spacer probe having a spacer-hybridizing sequence.

75. The method of any of claims 65-74, wherein the library of gRNA-specific ligatable probe pairs comprises different gRNA-specific ligatable probe pairs configured to hybridize to different gRNAs of the gRNA library.Attorney Docket No. 43487-1034601 76. The method of claim 75, wherein the spacer probes of the different gRNA-specific ligatable probe pairs have different spacer-hybridizing sequences.

77. The method of claim 75 or 76, wherein the scaffold probes of the different gRNA- specific ligatable probe pairs have the same scaffold-hybridizing sequence.

78. The method of any of claims 75-77, wherein the scaffold probes of the different gRNA- specific ligatable probe pairs are identical in sequence.

79. The method of any of claims 76-78, wherein the different spacer-hybridizing sequences hybridize to different spacer sequences of the different gRNAs.

80. The method of claim 79, wherein the different spacer-hybridizing sequences further hybridize to a first portion of the scaffold sequence.

81. The method of claim 80, wherein the scaffold probes of the different gRNA-specific ligatable probe pairs hybridize to a second portion of the scaffold sequence that is adjacent to the first portion of the scaffold sequence.

82. The method of any of claims 65-81, wherein the method comprises using the library of gRNA-specific ligatable probe pairs to generate a library of barcoded spacer oligonucleotides, each comprising: 1) a spacer sequence of a gRNA of the gRNA library or complement thereof; and 2) a partition-specific barcode or complement thereof.

83. The method of claim 82, wherein the method comprises sequencing the library of barcoded spacer oligonucleotides.

84. The method of claim 83, wherein the method comprises analyzing the results of the sequencing to associate spacer sequences with partition-specific barcodes.

85. The method of claim 83 or 84, wherein the method comprises analyzing the results of the sequencing to determine the presence and / or abundance of different gRNAs of the gRNA library in different cells of the population of cells.Attorney Docket No. 43487-1034601 86. The method of any of claims 65-85, wherein the method comprises performing nucleic acid sequencing to determining the presence and / or abundance of one or more non-gRNA analytes in the different cells of the population of cells.

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