Probe-based nucleic acid and protein analysis

Abstract

Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from samples. Methods for processing biomolecules may include hybridizing probe molecules to target regions of nucleic acid molecules (e.g., ribonucleic acid (RNA) molecules), and barcoding the probe-nucleic acid molecule complex or its derivatives. Such methods may include performing nucleic acid reactions, such as extension, denaturation, and amplification. Methods for processing samples may include hybridizing probes to target regions of (i) nucleic acid molecules (e.g., RNA molecules) and (ii) reporter oligonucleotides of characteristic binding groups, and barcoding the molecules associated with the probes. One or more processes of the methods described herein may be performed in a partition such as a droplet or a hole.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 251,446 filed on October 1, 2021, U.S. Provisional Patent Application No. 63 / 235,487 filed on August 20, 2021, U.S. Provisional Patent Application No. 63 / 196,834 filed on June 4, 2021, and U.S. Provisional Patent Application No. 63 / 152,709 filed on February 23, 2021, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Samples can be processed for various purposes, such as identifying a certain type of part within the sample. The sample can be a biological sample. Biological samples can be processed, such as to detect a disease (e.g., cancer) or to identify a specific species. There are many methods for processing samples, such as polymerase chain reaction (PCR) and sequencing.

[0004] Biological samples can be processed in various reaction environments, such as partitions. Partitions can be wells or droplets. Droplets or wells can be used to process biological samples in a manner that enables the biological samples to be separated and processed individually. For example, such droplets can be fluidically isolated from other droplets, thereby achieving accurate control of the corresponding environment in the droplet.

[0005] Various processes, such as chemical processes or physical processes, can be performed on the biological samples in the partitions. The samples in the partitions can be heated or cooled or chemically reacted to produce species that can be qualitatively or quantitatively processed.

[0006] Biomolecules, such as nucleic acids and proteins, within biological samples can be detected and / or processed for quantitative or qualitative assessment. Summary of the invention

[0007] The present disclosure provides methods for sample processing and analysis. The methods provided herein may include hybridizing a probe with a molecule of interest (e.g., a target protein, a target nucleic acid molecule) and treating a probe-molecule complex. Such treatment may 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 treatment may include extension, denaturation, and amplification processes to provide a nucleic acid molecule comprising a sequence identical or substantially identical or complementary to a target region of a nucleic acid molecule of interest (e.g., a target nucleic acid molecule). The method may include hybridizing a first probe and a second probe with a first target region and a second target region of a nucleic acid molecule, connecting the first probe and the second probe to provide a nucleic acid molecule to which the probe is connected, and barcoding the nucleic acid molecule to which the probe is connected. The method may include hybridizing a first probe with a first target region of a nucleic acid molecule, barcoding the probe, and hybridizing a second probe with a second target region of a nucleic acid molecule to produce a nucleic acid molecule to which the probe is connected, which is barcoded. In some aspects, the method may include hybridizing a probe with a nucleic acid molecule attached to a feature binding portion to provide a probe binding portion complex, and barcoding the probe. One or more processes of the method provided herein can be performed in a partition (such as a droplet or a hole). The method disclosed herein can be used for controlled analysis and processing of, for example, analytes (such as biological particles, nucleic acids and proteins). One or more methods described herein can allow for genome, transcriptome or exome spectrum analysis with higher sensitivity. The method disclosed herein can be used for detecting variants and characterizing nucleic acid molecules, for example, for assessing single nucleotide polymorphisms (SNPs), alternative splicing, insertions, deletions, V (D) J rearrangements, etc. The method disclosed herein can be used for multiplex analysis of nucleic acids and proteins, while minimizing the amount of reagents used, for example, by reducing the number of unoccupied partitions used for analysis.

[0008] In one aspect, disclosed herein is a method for multiplex nucleic acid assay, the method comprising: (a) contacting a cell, a cell nucleus or a cell bead with a first probe, a second probe and a third probe under conditions sufficient to produce a first probe-associated molecule and a second probe-associated molecule, wherein the cell, 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 the 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 (i) a third probe sequence complementary to the feature probe binding sequence and (ii) a probe capture sequence; (b) in a first partition of a first group of partitions, associating the first probe-associated molecule and the second probe under conditions sufficient to produce a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule; The method comprises contacting a molecule with a probe binding molecule and a barcode molecule, wherein the barcode molecule comprises (i) a common sequence common to multiple barcode molecules comprising the barcode molecule and (ii) a first barcode sequence common to a first partition of a first group 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 group of partitions, (i) contacting the first barcoded nucleic acid molecule or a derivative thereof with a first capture molecule of a plurality of capture molecules under conditions sufficient to produce a third barcoded nucleic acid molecule, and (ii) contacting the second barcoded nucleic acid molecule or a derivative thereof with a second capture molecule of a plurality of capture molecules under conditions sufficient to produce 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 located on the same strand of the nucleic acid molecule. In some embodiments, the probe capture sequence is shared by a plurality of first probes including the first probe, wherein one or more additional partitions of the first group of partitions include one or more nucleic acid molecules associated with additional probes, wherein each of the nucleic acid molecules associated with the one or more additional probes comprises a probe capture sequence. In some embodiments, the second probe comprises a second probe capture sequence complementary to the capture sequence of a plurality of capture molecules, and wherein (c) comprises hybridizing the second probe capture sequence with the capture sequence. In some embodiments, the barcode molecule comprises a capture binding sequence complementary to the capture sequence of a plurality of capture molecules, and wherein (c) comprises hybridizing the capture binding sequence with the capture sequence. In some embodiments, the first group of partitions is a plurality of holes. In some embodiments, the second group of partitions is a plurality of droplets. In some embodiments, the second group of partitions is a plurality of holes. In some embodiments, a plurality of capture molecules are coupled to particles. In some embodiments, the particles are beads. In some embodiments, the beads are gel beads. In some embodiments, each capture molecule in the plurality of capture molecules coupled to the gel beads comprises a second barcode sequence. In some embodiments, one or more additional partitions of the second group of partitions include one or more additional gel beads in a plurality of gel beads, and wherein the second barcode sequence is unique to the gel beads in the plurality of gel beads. In some embodiments, one capture molecule in a plurality of capture molecules comprises a third barcode sequence unique to the capture molecule in a plurality of capture molecules. In some embodiments, one or more additional partitions of the second group of partitions include one or more additional capture molecules, and wherein the second barcode sequence is unique to the second partition in the second group of partitions. In some embodiments, (a) includes 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 includes subjecting the molecules associated with the first probe to conditions sufficient to produce a probe-connected nucleic acid molecule, the probe-connected nucleic acid molecule comprising the first probe connected to the second probe. In some embodiments, the probe-connected nucleic acid molecule is produced via chemical connection or enzymatic connection of the first probe and the second probe. In some embodiments, the chemical connection or enzymatic connection occurs after (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 not 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, toward the second target region or the first target region, respectively, to produce an extended probe, and (ii) connecting 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 with a nucleic acid molecule within a cell or a cell nucleus. In some embodiments, the first partition comprises a plurality of cells, cell beads, or cell nuclei.In some embodiments, the cell or nucleus is permeabilized. In some embodiments, the cell or nucleus is fixed. In some embodiments, the method also includes releasing the first probe-associated molecule or its derivative from the cell, nucleus or cell bead. In some embodiments, the release includes lysing the cell. In some embodiments, the reporter oligonucleotide comprises a characteristic probe binding sequence. In some embodiments, the method also includes merging the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, the additional first barcoded nucleic acid molecule from the first group of regions, and the additional second barcoded nucleic acid molecule from the first group of regions after (b) and before (c). In some embodiments, the method also includes merging the third barcoded nucleic acid molecule, the fourth barcoded nucleic acid molecule, the additional third barcoded nucleic acid molecule from the second group of regions, and the additional fourth barcoded nucleic acid molecule from the second group of regions after (c) and before sequencing. In some embodiments, the probe capture sequence is 8 to 50bp.

[0010] In another aspect of the present disclosure, a method is provided herein, comprising: (a) contacting a nucleic acid molecule with a first probe to produce 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 produce an extended probe molecule, the extended probe molecule comprising a sequence complementary to the second target region; (c) providing the extended probe molecule, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the second probe, the first ... Barcode molecules and probe binding molecules, wherein the second probe comprises a second probe sequence corresponding to a 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) includes connecting the second probe and the barcode molecule. In some embodiments, the connection includes chemical connection or enzymatic connection. In some embodiments, the particle is a bead. In some embodiments, the bead is a gel bead. In some embodiments, the cell or the nucleus is permeabilized. In some embodiments, the cell or the nucleus is fixed. In some embodiments, the first probe comprises a probe capture sequence. In some embodiments, the method also includes releasing the extended probe molecule from the nucleic acid molecule after (b). In some embodiments, the release includes releasing the ribonuclease (RNA) chain using a ribonuclease. In some embodiments, the release includes thermal cycling. In some embodiments, (c) includes (i) hybridizing the second probe sequence of the second probe with the complementary sequence of 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-annealing complex, wherein the barcode capture sequence anneals to the barcode binding sequence in the pre-annealing complex. In some embodiments, the first probe comprises a probe capture sequence, wherein (c) comprises (i) annealing the probe binding sequence and the barcode binding sequence to the probe capture sequence and the barcode capture sequence, respectively, (ii) connecting the barcode molecule and the extended probe molecule to produce a first barcoded molecule, and (iii) annealing the second probe to the first barcoded molecule and initiating an extension reaction to produce a barcoded molecule. In some embodiments, (ii) and (iii) are performed outside the partition. In some embodiments, the second probe comprises a probe capture sequence, wherein (c) comprises (i) annealing the second probe to the extended probe molecule and initiating an extension reaction to produce an extended molecule, (ii) annealing the probe binding sequence and the barcode binding sequence to the probe capture sequence and the barcode capture sequence, and (iii) connecting the barcode molecule and the extended molecule.

[0012] In another aspect, the present invention provides a method for analyzing a sample, the method comprising: (a) providing: (i) a characteristic binding group bound to at least a portion of the sample, wherein the characteristic 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 arranged on the same chain 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 produce a probe-associated reporter oligonucleotide complex; and (c) subjecting the probe-associated reporter oligonucleotide complex to conditions sufficient to produce a probe-linked nucleic acid molecule, the probe-linked nucleic acid molecule comprising the first probe linked to the second probe.

[0013] In some embodiments, the method further comprises (d) attaching the barcode molecule to the probe-attached 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 a derivative thereof. In some embodiments, the characteristic 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 the barcode sequence to the additional probe sequence. In some embodiments, the method further comprises (d) providing a barcode molecule and a probe binding molecule, the probe binding molecule comprising (i) a first sequence complementary to the additional probe sequence and (ii) a second sequence complementary to the capture sequence of the barcode molecule. In some embodiments, the method further comprises providing conditions sufficient to hybridize the first sequence with the additional probe sequence and hybridize the second sequence with the capture sequence of the barcode molecule, thereby producing a barcoded, probe-associated complex. In some embodiments, (d) occurs in one of the plurality of partitions. In some embodiments, the method further comprises combining the barcoded, probe-associated complexes from the partition with other barcoded, probe-associated complexes from other partitions in the plurality of partitions to produce a combined set of barcoded, probe-associated complexes. In some embodiments, the method further comprises (i) separating the combined set of barcoded, probe-associated complexes into a plurality of additional partitions, wherein an additional partition in the plurality of additional partitions comprises barcoded, probe-associated complexes and an additional barcode molecule having an additional barcode sequence, and (ii) attaching the additional barcode molecule to the barcoded, probe-associated complexes. 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 before (c). In some embodiments, (d) occurs after (c). In some embodiments, at least the portion of the sample comprises the 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 cells or cell beads. 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 one of a plurality of partitions. In some embodiments, (c) comprises enzymatic ligation or chemical ligation. In some embodiments, the connection 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) includes performing an extension reaction to fill the gap region, thereby generating a probe-linked nucleic acid molecule. In some embodiments, (c) includes 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 generate a probe-linked nucleic acid molecule comprising a first probe linked to a second probe via a third probe.

[0014] Another aspect of the present disclosure provides a non-transitory computer-readable medium containing machine-executable code that, when executed by one or more computer processors, implements any of the methods above or elsewhere herein.

[0015] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto. The computer memory comprises machine executable code that, when executed by the one or more computer processors, implements any method above or elsewhere herein.

[0016] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art through the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and description are to be regarded as illustrative in nature, rather than restrictive.

[0017] Incorporated by Reference

[0018] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments that utilize the principles of the present invention and the accompanying drawings (also referred to herein as "Figures"), in which:

[0020] Figure 1An example of a microfluidic channel structure for separating individual bioparticles is shown.

[0021] Figure 2 An example of a microfluidic channel structure for controlled partitioning of beads into discrete droplets is shown.

[0022] Figure 3 Examples of beads carrying barcodes are shown.

[0023] Figure 4 Another example of a barcode-carrying bead is shown.

[0024] Figure 5 An example microwell array is schematically shown.

[0025] Figure 6 An example workflow for processing nucleic acid molecules is schematically shown.

[0026] Figure 7 Another example workflow for processing nucleic acid molecules is schematically shown.

[0027] Figure 8 Another example workflow for processing nucleic acid molecules is schematically shown.

[0028] Fig. 9 Another example workflow for processing nucleic acid molecules is schematically shown.

[0029] Fig.10 An example workflow for analyzing cells, cell nuclei, or cell beads is schematically shown.

[0030] Fig.11 An example labeling agent with attached nucleic acid molecules is schematically shown.

[0031] Fig. 12A Examples of labeling agents are schematically shown. Fig. 12B Another example workflow for processing nucleic acid molecules is schematically shown. Fig. 12C Another example workflow for processing nucleic acid molecules is schematically shown.

[0032] Fig.13 Another example of a barcode-carrying bead is schematically shown.

[0033] Fig.14 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein.

[0034] Fig.15 Example processed nucleic acid molecules described herein are shown.

[0035] Fig.16A An example workflow for processing multiple analytes in one partition is shown. Fig. 16B Another example workflow for processing multiple analytes in one partition is shown.

[0036] Fig.17 Characteristic binding groups described herein are schematically shown.

[0037] Fig.18 Example data from the workflow described herein is shown.

[0038] Fig.19 Additional example data from the workflow described herein is shown.

[0039] Fig. 20 Additional example data from the workflow described herein is shown.

[0040] Fig.21A Example data comparing fixed and non-fixed cells are shown. Fig.21B Additional example data comparing fixed and non-fixed cells are shown. Fig. 21C Additional example data comparing fixed and non-fixed cells are shown.

[0041] Fig. 22 An example workflow for the determination of two different analyte types is schematically shown.

[0042] Fig.23 Example data for the barcoding method described herein is shown.

[0043] Fig.24 Example data for different analyte types using the barcoding methods described herein are shown.

[0044] Fig.25 Example methods for processing nucleic acid molecules are schematically illustrated.

[0045] Fig.26 Another example method for processing nucleic acid molecules is shown.

[0046] Fig. 27 An example workflow for generating probe-ligated nucleic acid molecules is shown.

[0047] Fig.28 Another example workflow for generating probe-ligated nucleic acid molecules is shown.

[0048] Fig.29 An example workflow for processing cells according to the methods described herein is shown.

[0049] Fig. 30AExample protein expression data resulting from barcoding multiple analytes using different sample preparation parameters are shown. Fig. 30B Additional protein expression data obtained by barcoding various analytes using different sample preparation parameters are shown.

[0050] Fig.31 Example gene expression data resulting from barcoding multiple analytes using different sample preparation parameters are shown.

[0051] Fig.32A -C shows example data for multi-analyte probing of a negative control group. Fig.32A Example data showing different immune cell populations are shown. Fig.32B Example data showing gene expression of the GZMB gene. Fig.32C Example data of protein expression obtained by antibody staining are shown.

[0052] Fig.33A -C shows example data of multi-analyte detection of the experimental set. Fig.33A Example data showing different immune cell populations are shown. Fig.33B Example data showing gene expression of the GZMB gene. Fig.33C Example data of protein expression obtained by antibody staining are shown.

[0053] Fig.34A -C shows example data of multi-analyte detection of the experimental set. Fig.34A Example data showing different immune cell populations are shown. Fig.34B Example data showing gene expression of the GZMB gene. Fig.34C Example data of protein expression obtained by antibody staining are shown.

[0054] Fig.35A -C shows example data of multi-analyte detection of the experimental set. Fig.35A Example data showing different immune cell populations are shown. Fig.35B Example data showing gene expression of the GZMB gene. Fig.35C Example data of protein expression obtained by antibody staining are shown.

[0055] Fig.36A -C shows example data of multi-analyte detection of the experimental set. Fig.35A Example data showing different immune cell populations are shown. Fig.36B Example data showing gene expression of the GZMB gene. Fig.36C Example data of protein expression obtained by antibody staining are shown.

[0056] Fig.37A-C shows example data of multi-analyte detection of the experimental set. Fig.37A Example data showing different immune cell populations are shown. Fig.37B Example data showing gene expression of the GZMB gene. Fig.37C Example data of protein expression obtained by antibody staining are shown.

[0057] Fig.38 Another example workflow for assaying two different analyte types is shown. DETAILED DESCRIPTION

[0058] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Without departing from the present invention, those skilled in the art may conceive of many variations, changes and substitutions. It should be understood that various alternative forms of the embodiments of the present invention described herein may be adopted.

[0059] Where values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within such ranges, as well as specific values ​​falling within such ranges, whether or not a specific value or a specific sub-range is explicitly stated.

[0060] As used herein, the terms "a," "an," and "the" are generally intended to refer to both the singular and the plural, unless the context clearly dictates otherwise.

[0061] Whenever the term "at least", "greater than", or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least", "greater than", or "greater than or equal to" applies to each value in the series. 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.

[0062] Whenever the term "not greater than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not greater than," "less than," or "less than or equal to" applies to each value in the series. 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.

[0063] As used herein, the term "barcode" generally refers to a label or identifier that conveys 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 label attached to an analyte (e.g., a nucleic acid molecule) or a combination of the label plus an intrinsic property of the analyte (e.g., the size of the analyte or terminal sequence). A barcode can be unique. A barcode can have a variety of different forms. For example, a barcode can include: a polynucleotide barcode; a random nucleic acid and / or amino acid sequence; and a synthetic nucleic acid and / or amino acid sequence. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to a fragment of, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and / or after sample sequencing. A barcode can allow identification and / or quantification of individual sequencing reads.

[0064] As used herein, the term "real time" may refer to a response time of less than about 1 second, one tenth of a second, one hundredth of a second, one millisecond, or less. The response time may be greater than 1 second. In some cases, real time may refer to simultaneous or substantially simultaneous processing, detection, or identification.

[0065] As used herein, the term "subject" generally refers to an animal such as a mammal (e.g., a human, mouse, rat) or avian (e.g., a bird), or other organisms such as a plant. For example, a subject can be a vertebrate, such as a mammal, a rodent (e.g., a mouse), a primate, an ape, or a human. Animals can include, but are not limited to, farm animals, sports animals, and pets. A subject can be a healthy or asymptomatic individual, an individual suffering from or suspected of suffering from a disease (e.g., cancer) or susceptible to the disease, and / or an individual in need of treatment or suspected of needing treatment. A subject can be a patient. A subject can be a microorganism or a microorganism (e.g., bacteria, fungi, archaea, viruses).

[0066] As used herein, the term "genome" generally refers to genomic information from a subject, which can be, for example, at least a portion or all of the genetic information of a subject. The genome can be encoded in DNA or RNA. The genome can include coding regions (e.g., coding proteins) and non-coding regions. The genome can include the sequence of all chromosomes in an organism. For example, the human genome generally has a total of 46 chromosomes. The sequence of all these chromosomes together can constitute the human genome.

[0067] The terms "linker," "adaptor," and "tag" can be used synonymously. A linker or tag can be coupled to a polynucleotide sequence to be "labeled" by any method, including ligation, hybridization, or other methods.

[0068] As used herein, the term "sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. These 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 a variety of currently available systems, such as, but not limited to, Pacific Biosciences Oxford or Life Technologies The sequencing system of production. Alternatively or in addition, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (for example, digital PCR, quantitative PCR or real-time PCR) or isothermal amplification. Such systems can provide a plurality of original genetic data corresponding to the genetic information of a subject (for example, the human being), as produced by these systems from samples provided by the subject. In some instances, such systems provide sequencing reads (also referred to herein as "reads"). Reads can include a series of nucleic acid bases corresponding to the sequence of the nucleic acid molecule that has been sequenced. In some cases, the system and method provided herein can be used together with proteome information.

[0069] As used herein, the term "bead" generally refers to a particle. A bead can be a solid or semi-solid particle. A bead can be a gel bead. A gel bead can include a polymer matrix (e.g., a matrix formed by polymerization or cross-linking). A polymer matrix can include one or more polymers (e.g., polymers with different functional groups or repeating units). The polymers in the polymer matrix can be randomly arranged, such as in a random copolymer, and / or have an ordered structure, such as in a block copolymer. Crosslinking can be via covalent, ionic or induced interactions or physical entanglement. A bead can be a macromolecule. A bead can be formed by nucleic acid molecules bound together. A bead can be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules) such as monomers or polymers. Such polymers or monomers can be natural or synthetic. Such polymers or monomers can be or include, for example, nucleic acid molecules (e.g., DNA or RNA). A bead can be formed by a polymer material. A bead can be magnetic or non-magnetic. A bead can be rigid. A bead can be flexible and / or compressible. A bead can be destructible or soluble. The beads may be solid particles (eg, metal-based particles including, but not limited to, iron oxide, gold, or silver) covered with a coating comprising one or more polymers. Such coatings may be destructible or dissolvable.

[0070] As used herein, the term "barcoded nucleic acid molecule" generally refers to a nucleic acid molecule produced by, for example, treating a nucleic acid barcode molecule with a nucleic acid sequence (e.g., a nucleic acid sequence complementary to a nucleic acid primer sequence contained in the nucleic acid barcode molecule). The nucleic acid sequence can be a targeted sequence or a non-targeted sequence. For example, in the methods and systems described herein, a nucleic acid molecule (e.g., a messenger RNA (mRNA) molecule) of a cell or a cell nucleus is hybridized and reverse transcribed 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 the nucleic acid sequence of an mRNA molecule) to produce a barcoded nucleic acid molecule having a sequence corresponding to the nucleic acid sequence of the mRNA and a barcode sequence (or its reverse complement). The barcoded nucleic acid molecule can serve as a template, such as a template polynucleotide, which can be further processed (e.g., amplified) and sequenced to obtain a target nucleic acid sequence. For example, in the methods and systems described herein, the barcoded nucleic acid molecule can be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the mRNA.

[0071] As used herein, the term "sample" generally refers to a biological sample of a subject. A biological sample may contain any number of macromolecules, such as cellular macromolecules. A sample may be a cell sample. A sample may be a cell line or a cell culture sample. A sample may contain one or more cells or cell nuclei. A sample may contain one or more microorganisms. A biological sample may be a nucleic acid sample or a protein sample. A biological sample may also be a carbohydrate sample or a lipid sample. A biological sample may be derived from another sample. A sample may be a tissue sample, such as a biopsy sample, a core needle biopsy sample, a needle aspirate, or a fine needle aspirate. A tissue sample may be a fresh tissue sample, a frozen tissue sample (e.g., quick freezing, freeze drying, frozen sections, etc.), or a fixed tissue sample (e.g., a formalin-fixed paraffin-embedded tissue sample). A sample may be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. A sample may be a skin sample. A sample may be a cheek swab. A sample may be a plasma or serum sample. A sample may be a cell-free sample or a cell-free sample. A cell-free sample may contain extracellular polynucleotides. The extracellular polynucleotides may be isolated from a body sample which may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces and tears.

[0072] As used herein, the term "biological particle" generally refers to a discrete biological system derived from a biological sample. Biological particles can be macromolecules. Biological particles can be small molecules. Biological particles can be viruses. Biological particles can be cells or derivatives of cells. Biological particles can be organelles. Examples of organelles from cells include, but are not limited to, nuclei, ribosomes, Golgi bodies, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, and lysosomes. Biological particles can be rare cells from a cell population. Biological particles can be any type of cell, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammals or other animal cell types, mycoplasmas, normal tissue cells, tumor cells, or any other cell type derived from unicellular organisms or multicellular organisms. Biological particles can be components of cells. Biological particles can be or can include DNA, RNA, organelles, proteins, or any combination thereof. Biological particles can be or can include a matrix (e.g., a gel or polymer matrix) that contains cells or one or more components (e.g., cell beads) from cells, such as DNA, RNA, organelles, proteins, or any combination thereof from cells. The bioparticles can be obtained from tissue of a subject (e.g., a human, mouse, rat, or other mammal). The bioparticles can be hardened cells. Such hardened cells may or may not include a cell wall or a cell membrane. The bioparticles can include one or more components of the cell, but may not include other components of the cell. Examples of such components are a nucleus or an organelle. The cell can be a living cell. The living cell can be capable of being cultured, for example, when enclosed in a gel or polymer matrix, or when containing a gel or polymer matrix.

[0073] As used herein, the term "macromolecule component" generally refers to a macromolecule contained in or from a bioparticle. The macromolecule component may include a nucleic acid. In some cases, the bioparticle may be a macromolecule. The macromolecule component may include DNA. The macromolecule component may include RNA. The RNA may be coded or non-coded. The RNA may be, for example, a messenger RNA (mRNA), a ribosomal RNA (rRNA), or a transfer RNA (tRNA). The RNA may be a transcript. The RNA may be a small RNA having a length of less than 200 nucleic acid bases, or a large RNA having a length of greater than 200 nucleic acid bases. The small RNA may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), RNA interacting with Piwi proteins (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA may be a double-stranded RNA or a single-stranded RNA. The RNA may be a circular RNA. The macromolecule component may include a protein. The macromolecule component may include a peptide. The macromolecule component may include a polypeptide.

[0074] As used herein, the term "molecular tag" generally refers to a molecule that can bind to a macromolecular component. A molecular tag can bind to a macromolecular component with high affinity. A molecular tag can bind to a macromolecular component with high specificity. A molecular tag can comprise a nucleotide sequence. A molecular tag can comprise a nucleic acid sequence. A nucleic acid sequence can be at least a portion or all of a molecular tag. A molecular tag can be a nucleic acid molecule or can be a portion of a nucleic acid molecule. A molecular tag can be an oligonucleotide or a polypeptide. A molecular tag can comprise a DNA aptamer. A molecular tag can be or comprise a primer. A molecular tag can be or comprise a protein. A molecular tag can comprise a polypeptide. A molecular tag can be a barcode.

[0075] As used herein, the term "partition" generally refers to a space or volume that can be suitable for containing one or more species or performing one or more reactions. A partition can be a physical compartment, such as a droplet or a hole. A partition can isolate a space or volume from another space or volume. A droplet can be a first phase (e.g., an aqueous phase) in a second phase (e.g., an oil) that is immiscible with the first phase. A droplet can be a first phase in a second phase that is not separated from the first phase, such as a capsule or liposome in an aqueous phase. A partition can include one or more other (internal) partitions. In some cases, a partition can be a virtual compartment, which can be defined and identified by an index (e.g., an index library) across multiple and / or remote physical compartments. For example, a physical compartment can include multiple virtual compartments.

[0076] Provided herein are methods for sample processing and / or analysis. The methods disclosed herein may include barcoding one or more types of biomolecules (e.g., nucleic acid molecules, proteins, lipids, carbohydrates, or combinations thereof). Biomolecules may be, for example, nucleic acid molecules (e.g., ribonucleic acid (RNA) molecules) or proteins. This method may involve attaching one or more probes (e.g., nucleic acid probes) to biomolecules, followed by attaching nucleic acid barcode molecules comprising barcode sequences to one or more probes. For example, nucleic acid barcode molecules may be attached to the overhang sequence of the probe or the end of the probe. Extending from one end of the probe to one end of the nucleic acid barcode molecule may form an extended nucleic acid molecule, the extended nucleic acid molecule comprising a sequence complementary to the barcode sequence and a sequence complementary to the 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 replicated. One or more processes of the method may be performed in a partition (such as a droplet or a hole).

[0077] The present disclosure also provides a method for processing a sample (e.g., a cell sample or a tissue sample), the method providing a barcoded nucleic acid molecule attached with a linked probe molecule. The method may include 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 (i) a first probe sequence complementary to the first target region and (ii) an additional probe sequence; and a second probe having a second probe sequence complementary to the second target region. In some cases, the first target region and the second target region are adjacent. The first probe sequence and the second probe sequence may also include a first reactive portion and a second reactive portion, respectively. When the first probe sequence of the first probe hybridizes with the first target region of the nucleic acid molecule and the second probe sequence of the second probe hybridizes with the second target region of the nucleic acid molecule, the reactive portions may be adjacent to each other. A subsequent reaction between the adjacent reactive portions under sufficient conditions may connect the first probe and the second probe to produce a probe-linked nucleic acid molecule. A probe-linked nucleic acid molecule may also be referred to as a probe-ligated nucleic acid molecule. In other cases, the first target region and the second target region are not adjacent, and a nucleic acid reaction (eg, a nucleic acid extension reaction, a gap-filling reaction) can be performed to produce a probe-ligated nucleic acid molecule.

[0078] The probe-attached nucleic acid molecule can be barcoded with a barcode sequence of a nucleic acid barcode molecule to provide a barcoded, probe-attached nucleic acid molecule. Barcoding can be achieved by hybridizing the binding sequence of the nucleic acid barcode molecule with the additional probe sequence of the first probe of the probe-attached nucleic acid molecule. The barcoded, probe-attached nucleic acid molecule can be subjected to an amplification reaction to produce an amplification product comprising a first target region and a second target region and a barcode sequence or a sequence complementary to these sequences. Therefore, the method can provide an amplification product without using reverse transcription. One or more processes can be performed within a partition (such as a droplet or a well).

[0079] The present disclosure also provides a method for producing a barcoded, probe-connected nucleic acid molecule. The method may include 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 complementary to the first target region and an optional additional probe sequence; and a second probe having a second probe sequence complementary to the second target region. The additional probe sequence of the first probe may include a probe capture sequence. Alternatively or in addition, the second probe may include a probe capture sequence. The first probe sequence of the first probe may hybridize with the first target region of the nucleic acid molecule to produce a probe-associated nucleic acid molecule, and a nucleic acid reaction (e.g., a nucleic acid extension reaction using a polymerase or a reverse transcriptase) may be performed to produce an extended nucleic acid molecule, which includes a sequence complementary to the second target region. Before, during, or after the nucleic acid extension reaction, the second probe may hybridize with the nucleic acid molecule (or the extended nucleic acid molecule or its complementary sequence), and optionally, a nucleic acid extension reaction may be performed. The extended nucleic acid molecule can be barcoded, such as by (a) hybridization of a barcode binding sequence of a nucleic acid barcode molecule with a first probe (e.g., an additional probe sequence of the first probe) or a 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"), wherein the probe binding molecule comprises (i) a probe binding sequence complementary to an additional probe sequence of the first probe (which may comprise a probe capture sequence) and / or a capture sequence of the second probe, and (ii) a barcode binding sequence complementary to a sequence of the barcode molecule (e.g., a common sequence). In some cases, barcoding can be performed prior to hybridization of the second probe to the second target region. In such cases, the barcoded nucleic acid molecule can be subjected to conditions sufficient to hybridize the second probe sequence of the second probe to the second target region of the nucleic acid molecule (or the barcoded nucleic acid molecule). A nucleic acid reaction (e.g., nucleic acid extension) can be performed to produce a barcoded, probe-linked nucleic acid molecule.

[0080] Another aspect of the present disclosure provides a method for barcoding a variety of analytes, such as nucleic acid molecules connected to probes as described herein, and other types of biomolecules (e.g., proteins). The method may include providing (i) a sample comprising a nucleic acid molecule (e.g., an RNA molecule) having a first target region and a second target region, and (ii) a characteristic binding portion comprising a reporter oligonucleotide having a capture sequence; (iii) a first probe having a first probe sequence complementary to the first target region and an additional probe sequence; (iv) a second probe having a second probe sequence complementary to the second target region; and (v) a third probe having a third probe sequence complementary to the sequence of the reporter oligonucleotide. The first probe and the second probe may be subjected to conditions sufficient to hybridize with the first target region and the second target region, respectively, and produce probe-connected nucleic acid molecules. The third probe sequence of the third probe may be subjected to conditions sufficient to hybridize with the capture sequence of the reporter oligonucleotide, thereby producing a probe-binding portion complex. The probe-connected nucleic acid molecule and the probe-binding portion complex may be subjected to conditions sufficient to add barcodes, thereby producing barcoded, probe-connected nucleic acid molecules and barcoded probe-binding portion complexes. The barcoded, probe-connected molecules can be subjected to an amplification reaction to produce an amplification product comprising a first target region and a second target region and a barcode sequence or a sequence complementary to these sequences. The barcoded probe-binding moiety complex can similarly be subjected to an amplification reaction to produce an amplification product comprising a fourth probe sequence and a barcode sequence. One or more processes can be performed in a cell bead and / or a partition (such as a droplet or a well). Advantageously, the methods described herein can be used to index cells, cell nuclei, or cell beads to partitions; such indexing can be used in partitions occupied by more than one cell, and identify the cell, cell nucleus, cell bead, or partition from which the analyte originates.

[0081] Fixed samples

[0082] The sample can be a fixed sample. For example, the sample can include multiple fixed samples, such as multiple fixed cells or fixed nuclei. Alternatively or in addition, the sample can include fixed tissue. The fixation of cells or cell components or tissues containing multiple cells or nuclei can include the application of chemical species or chemical stimulation. The term "fixed" as used herein for biological samples generally refers to a state of preventing decay and / or degradation. "Fixed" generally refers to the process of producing a fixed sample, and in some cases can include contacting the biomolecules in the biological sample with a fixative (or fixative) for a certain amount of time, whereby the fixative causes covalent interactions between the biomolecules in the sample, such as crosslinking. "Fixed biological sample" generally refers to a biological sample that has been contacted with a fixative or fixative. For example, a formaldehyde-fixed biological sample has been contacted with a fixative formaldehyde. "Fixed cells", "fixed nuclei" or "fixed tissue" refers to cells / nuclei or tissues that have been contacted with a fixative under conditions sufficient to allow or cause intramolecular and intermolecular covalent crosslinking between biomolecules in the biological sample. Typically, contact of a biological sample (e.g., a cell or a cell nucleus) with a fixative (e.g., paraformaldehyde or PFA) causes the formation of intramolecular and intermolecular covalent crosslinks between biomolecules in the biological sample. In some cases, the fixative formaldehyde can produce covalent aminal crosslinks within RNA, DNA, and / or protein molecules. For example, the widely used fixative paraformaldehyde or PFA fixes tissue samples by catalyzing the formation of crosslinks between basic amino acids (such as lysine and glutamine) in proteins. Intramolecular and intermolecular crosslinks can be formed in proteins. These crosslinks can preserve the secondary structure of the protein and can also eliminate enzyme activity in the preserved tissue sample. Examples of fixatives include, but are not limited to, aldehyde fixatives (e.g., formaldehyde, also commonly referred to as "paraformaldehyde," "PFA," and "formalin"; glutaraldehyde; etc.), imide esters, NHS (N-hydroxysuccinimide) esters, and the like.

[0083] In some embodiments, the fixative or fixing agent that can be used to fix the sample is formaldehyde. The term "formaldehyde", when used in the context of a fixative, may also refer to "paraformaldehyde" (or "PFA") and "formalin", both of which have specific meanings associated with formaldehyde compositions (e.g., formalin is a mixture of formaldehyde and methanol). Therefore, formaldehyde-fixed biological samples may also be referred to as formalin-fixed or PFA-fixed. The schemes and methods for preparing fixed biological samples using formaldehyde as a fixing agent are well known in the art and can be used in the methods and compositions of the present disclosure. For example, the suitable range of formaldehyde concentration for preparing fixed biological samples is 0.1%-10%, 1%-8%, 1%-4%, 1%-2%, 3%-5% or 3.5%-4.5%. In some embodiments of the present disclosure, a final concentration of 1% formaldehyde, 4% formaldehyde or 10% formaldehyde is used to fix the biological sample. Typically, formaldehyde is diluted from a more concentrated stock solution, such as a 35%, 25%, 15%, 10%, or 5% PFA stock solution.

[0084] 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, crosslinkers can also be used for fixation, including but not limited to disuccinimidyl suberate (DSS), dimethyl suberate (DMS), formalin and dimethyl adipimide (DMA), disulfide-bis (succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis (succinimidyl succinate) (EGS). In some cases, the crosslinker can be a cleavable crosslinker (e.g., thermally cleavable, photocleavable, etc.).

[0085] In some cases, when preparing the biological sample fixed, more than one fixing agent can be used in combination. For example, the first fixing agent (such as an organic solvent) can be used in combination with the second fixing agent (such as a cross-linking agent). The organic solvent can be an alcohol (for example, ethanol or methanol), a ketone (for example, acetone) or an aldehyde (for example, paraformaldehyde, formaldehyde or glutaraldehyde). The cross-linking agent can be selected from the group consisting of disuccinimidyl suberate (DSS), dimethyl suberimide ester (DMS), formalin and dimethyl adipimide ester (DMA), disulfide-bis (succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST) and ethylene glycol bis (succinimidyl succinate) (EGS). In some cases, a first fixative may be provided to a cell or a cell nucleus or the first fixative may be contacted with a cell or a cell nucleus to cause a change in a first characteristic or a characteristic group of a cell / cell nucleus, and a fixative may be provided to a cell or a cell nucleus or a fixative may be contacted with a cell or a cell nucleus to cause a change in a second characteristic or a characteristic group of a cell or a cell nucleus. For example, a first fixative may be provided to a cell or a cell nucleus or the first fixative may be contacted with a cell or a cell nucleus to cause a change in cell size (e.g., a reduction in cross-sectional diameter, see, e.g., U.S. Patent Publication No. 2020 / 0033237, which is incorporated herein by reference in its entirety), and a second fixative may be provided to a cell or a cell nucleus or the second fixative may be contacted with a cell or a cell nucleus to cause a change in a second characteristic or a characteristic group of a cell (e.g., cross-linking is formed in and / or around a cell or a cell nucleus). A first fixative and a second fixative may be provided to a cell or a cell nucleus or the first fixative and a second fixative may be contacted with a cell or a cell nucleus at the same or different times. Other suitable fixatives include those disclosed, for example, in International PCT Application No. PCT / US2020 / 066705, which is incorporated herein by reference in its entirety.

[0086] In one example, a first fixative that is an organic solvent can be provided to the cells to change a first property (e.g., cell size), and a second fixative that is a cross-linking agent can be provided to the cells to change a second property (e.g., cell fluidity or rigidity). The first fixative can be provided to the cells before the second fixative.

[0087] In another embodiment, the biomolecules (e.g., biological samples, such as tissue specimens) are contacted with a fixative reagent containing formaldehyde and glutaraldehyde, so that the contacted biomolecules may include fixed crosslinks produced by formaldehyde-induced fixation and glutaraldehyde-induced fixation. Generally, a suitable concentration of glutaraldehyde used as a fixative reagent may be 0.1% to 1%. Fixing and washing reagents may also include commercially available products, such as Fixation Buffer (420801) and Permeabilization Wash Buffer (421002).

[0088] The change in a property or set of properties of a cell or cell component (e.g., caused upon interaction with one or more fixatives) may be at least partially reversible (e.g., via rehydration or de-crosslinking). Alternatively, the change in a property or set of properties of a cell or cell component (e.g., caused upon interaction with one or more fixatives) may be substantially irreversible.

[0089] Sample (for example, cell sample) can be subjected to fixation process at any useful time point.For example, the cell, nucleus and / or cell / nuclear component of sample can be subjected to the fixation process related to one or more fixatives (for example, as described herein) before any subsequent treatment (such as for storage) starts.The cell, nucleus and / or cell / nuclear component (such as the cell, nucleus and / or cell / nuclear component of tissue sample) subjected to fixation process before storage can be stored in aqueous solution, optionally combined with one or more preservatives, and the one or more preservatives are configured to retain the form, size or other characteristics of cell and / or cell component.Fixed cell, nucleus and / or cell / nuclear component can be stored below room temperature, such as stored in a refrigerator.Alternatively, the cell, nucleus and / or cell / nuclear component of sample can be subjected to the fixation process related to one or more fixatives after one or more other processes (such as filtering, centrifugation, stirring, selective precipitation, purification, permeabilization, separation, heating, etc.). For example, cells, nuclei and / or cell / nuclear components of a given type from a sample can be subjected to a fixation process after separation and / or enrichment procedures (for example, as described herein). In an example, a sample comprising multiple cells (including cells of multiple given types) can be subjected to a forward separation process to provide a sample rich in cells of multiple given types. The enriched sample can then be subjected to a fixation process involving one or more fixatives (for example, as described herein) to provide a sample of the enrichment comprising multiple fixed cells. The fixation process can be performed in a bulk solution. In some cases, a fixed sample (for example, a fixed cell, a fixed nucleus and / or a cell / nuclear component) can be separated in multiple partitions (for example, droplets or holes), and subjected to processing as described elsewhere herein. In some cases, a fixed sample can be subjected to additional processing before separation and any subsequent processing, such as by, for example, rehydration or de-crosslinking to partially or completely reverse the fixation process. In some cases, a fixed sample can be subjected to partial or complete reversal of a fixation process in multiple partitions (for example, before or simultaneously with additional processing described elsewhere herein).

[0090] In some cases, the tissue specimen comprising multiple cells, nuclei and / or cell / nuclear components can be processed to provide formalin-fixed paraffin-embedded (FFPE) tissue. The tissue specimen can be contacted with formalin (for example, soaked with formalin), then embedded in paraffin. The FFPE process can facilitate the preservation of tissue samples (for example, before subsequent treatment and analysis). The tissue sample including the FFPE tissue sample can be stored in a cryogenic refrigerator additionally or alternatively. Cells, nuclei and / or cell / nuclear components can be dissociated from tissue samples (for example, FFPE tissue samples) before experiencing subsequent treatment. In some cases, the individual cells, nuclei and / or cell / nuclear components of tissue samples (such as FFPE tissue samples) can be optically detected, marked or otherwise processed before any such dissociation. This detection, marking or other processing can be performed according to two-dimensional or three-dimensional arrays and optionally according to a predetermined pattern.

[0091] Nucleic acid analysis methods

[0092] In one aspect, the present disclosure provides a method for barcoding a nucleic acid molecule. The method may generally include contacting a nucleic acid molecule with a pair of probes and a barcode molecule to produce a barcoded molecule (e.g., a barcoded, probe-linked molecule). The nucleic acid molecule may include a sequence corresponding to a target sequence or a template sequence. One or more nucleic acid reactions (e.g., ligation, nucleic acid extension reaction, amplification, etc.) may be performed to produce a barcoded molecule. In some aspects, the method includes: contacting a nucleic acid molecule with a first probe to produce a probe-associated nucleic acid molecule, wherein the nucleic acid molecule includes a first target region and a second target region, wherein the first probe includes 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 produce an extended probe molecule, the extended probe molecule including a sequence complementary to the second target region; providing (i) a second probe including 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 a derivative thereof to conditions sufficient to produce a barcoded molecule. The first target area and the second target area can be arranged adjacent to each other, or can be separated from each other (for example, arranged at opposite ends of the gap area). In some cases, barcoding can be facilitated by providing a probe binding molecule (also referred to herein as a "splint molecule" or in some cases a "splint oligonucleotide"). For example, the first probe and / or the second probe may include a probe capture sequence, and the probe binding molecule may include a probe binding sequence complementary to the probe capture sequence. In addition or alternatively, the nucleic acid barcode molecule may include a barcode sequence and a barcode capture sequence, and the probe binding molecule may include a barcode binding sequence complementary to the barcode capture sequence. In some cases, the probe binding molecule may be pre-annealed to the nucleic acid barcode molecule. Barcoding may include hybridization of the probe binding molecule with the probe capture sequence (or its complementary sequence) of the first probe and / or the second probe and with the barcode capture sequence of the nucleic acid barcode molecule. Therefore, the molecule with the barcode may include a sequence corresponding to the first target area, a sequence corresponding to the second target area, 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 (eg, a droplet or a well).

[0093] Methods described herein can use, for example, nucleic acid extension reaction, probe hybridization, chemical connection or enzymatic connection, barcode addition, amplification and sequencing to promote gene expression profile analysis with single cell, single cell nucleus or single cell bead resolution. Methods described herein can allow gene expression analysis while avoiding the use of special imaging equipment, and in some cases, avoid the use of reverse transcription, which may be very error-prone and inefficient. In some cases, the method can be used to analyze a group of predetermined target genes in single cell, cell nucleus or cell bead colonies in a sensitive and accurate manner. Methods described herein can also be used to detect or characterize genetic variants, for example, in the case where the sequence of the region (for example, gap region) arranged between the target regions is unknown. In some cases, methods described herein can be used to analyze single nucleotide polymorphisms (SNPs), alternative splicing, insertion, mutation, deletion, gene rearrangement (for example, V (D) J rearrangement), transposon or other genetic elements or variants. In some cases, the nucleic acid molecules analyzed by the methods described herein can include fusion genes (for example, heterozygous genes produced by translocation, interstitial deletion or chromosome inversion). In some cases, the methods described herein can be used to analyze genomic, transcriptomic, exomic and / or proteomic elements in cells, cell nuclei, cell beads, tissue samples, spatial arrays of cells, cell nuclei or tissues, and the like.

[0094] The nucleic acid molecules analyzed by the methods described herein can be single-stranded or double-stranded nucleic acid molecules. Double-stranded nucleic acid molecules can be completely or partially denatured to approach a target region (e.g., target sequence) of one strand of the nucleic acid molecule. Denaturation can be achieved, for example, by adjusting the temperature or pH of a solution containing nucleic acid molecules; using chemicals such as formamide, guanidine, sodium salicylate, dimethyl sulfoxide, propylene glycol, urea, or alkaline reagents (e.g., NaOH); or using mechanical agitation (e.g., centrifuging or vortexing a solution containing nucleic acid molecules).

[0095] Nucleic acid molecules can be target nucleic acid molecules. Target nucleic acid molecules can be RNA molecules. RNA molecules can be, for example, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules, mitochondrial RNA (mtRNA) molecules, messenger RNA (mRNA) molecules, non-coding RNA molecules, synthetic RNA molecules or another type of RNA molecules. For example, RNA molecules can be mRNA molecules. In some cases, nucleic acid molecules can be viral or pathogenic RNA. In some cases, nucleic acid molecules can be synthetic nucleic acid molecules previously introduced into or on cells. For example, nucleic acid molecules can include multiple barcode sequences, and two or more barcode sequences can be target regions of nucleic acid molecules. In some cases, nucleic acid molecules are guide RNA (gRNA), which can be exogenously introduced into cells or cell beads. In some cases, nucleic acid molecules are RNA molecules derived from exogenously introduced nucleic acid molecules, such as RNA derived from plasmids, integrated DNA sequences (e.g., using viral transduction in cells), gRNAs from CRISPR genetic elements, etc.

[0096] Nucleic acid molecules (e.g., RNA molecules) can include one or more features selected from the group consisting of: 5' cap structure, untranslated region (UTR), 5' triphosphate moiety, 5' hydroxyl moiety, Kozak sequence, Shine-Dalgarno sequence, coding sequence, codon, intron, exon, open reading frame, regulatory sequence, enhancer sequence, silencer sequence, promoter sequence, and poly (A) sequence (e.g., poly (A) tail). For example, nucleic acid molecules can include one or more features selected from the group consisting of: 5' cap structure, untranslated region (UTR), Kozak sequence, Shine-Dalgarno sequence, coding sequence, and poly (A) sequence (e.g., poly (A) tail).

[0097] The characteristics of the nucleic acid molecule can have any useful properties. The 5' cap structure can include one or more nucleoside moieties connected by a linker (such as a triphosphate (ppp) linker). The 5' cap structure can include naturally occurring nucleosides and / or non-naturally occurring (e.g., modified) nucleosides. For example, the 5' cap structure can include a guanine moiety or a modified (e.g., alkylated, reduced, or oxidized) guanine moiety, such as 7-methylguanylate (m 7 G) cap. Examples of 5' cap structures include, but are not limited to m 7 GpppG、m 7 Gpppm 7 G、m 7 GpppA,m 7 GpppC, GpppG, m 2,7 GpppG、m 2,2,7GpppG and anti-reverse cap analogs, such as m 7,2’Ome GpppG、m 7,2’d GpppG、m 7,3’Ome GpppG and m 7,3’d GpppG. The untranslated region (UTR) can be a 5'UTR or a 3'UTR. The UTR can contain any number of nucleotides. For example, the UTR can contain at least 3, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the UTR can contain less than 20 nucleotides. In other cases, the UTR can contain at least 100 nucleotides, such as more than 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides. Similarly, the coding sequence can contain any number of nucleotides, such as at least 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. The UTR, coding sequence or other sequences of the nucleic acid molecule can have any nucleotide or base content or arrangement. For example, the sequence of nucleic acid molecules can include any amount or concentration of guanine, cytosine, uracil and adenine bases. Nucleic acid molecules can also include non-naturally occurring (e.g., modified) nucleosides. Modified nucleosides can include one or more modifications (e.g., alkylation, hydroxylation, oxidation or other modifications) in their core bases and / or sugar moieties.

[0098] Nucleic acid molecules can include one or more target regions. In some cases, the target region can correspond to a gene or a part thereof. Each region can have the same or different sequences. For example, a nucleic acid molecule can include two target regions with the same sequence, which are located at different positions along a chain of nucleic acid molecules. Alternatively, a nucleic acid molecule can include two or more target regions with different sequences. Different target regions can be probed by different probes. The target regions can be adjacent to each other, or can be separated spatially along a chain of nucleic acid molecules. The target region can be located on the same chain or different chains. As used herein with respect to two entities, "adjacent" can mean that entities are directly next to each other (e.g., adjacent) or close to each other. For example, a first target region can be directly next to a second target region (e.g., no other entities are set between the first target region and the second target region) or close to a second target region (e.g., there is an intervening sequence or molecule between the first target region and the second target region). In some cases, a double-stranded nucleic acid molecule can include target regions that may be the same or different in each chain. For nucleic acid molecules comprising multiple target regions, the method described herein can be performed on one or more target regions at a time. For example, a single target region of a plurality of target regions can be analyzed (e.g., as described herein), or two or more target regions can be analyzed simultaneously. Analyzing two or more target regions can involve providing two or more probes, wherein a first probe has a sequence complementary to a first target region, a second probe has a sequence complementary to a second target region, and so on.

[0099] Each probe (e.g., a first probe and a second probe) may also include one or more additional sequences (e.g., additional probe sequences, unique molecular identifiers (UMIs), barcode sequences, primer sequences, capture sequences, or other functional sequences). For example, in some cases, the first probe and / or the second probe may include the same or different barcode sequences. In some instances, the first probe and the second probe may be configured to hybridize with one or more nucleic acid barcode molecules. For example, the first probe and / or the second probe may include a probe capture sequence that may be configured to hybridize with a nucleic acid barcode molecule or a probe binding molecule (e.g., a splint oligonucleotide) that is configured to hybridize with a nucleic acid barcode molecule (e.g., via a barcode binding sequence that is complementary to the capture sequence of the nucleic acid barcode molecule). The probe capture sequence can be any useful length; for example, the probe capture sequence can 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 can 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 length of the probe capture sequence can 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 nucleotides. The length of the probe capture sequence ranges, such as about 8 to about 50 nucleotides in length, etc. In some cases, the probe capture sequence length can vary based on any useful application and properties, such as melting temperature, annealing temperature, annealing strength (e.g., GC content), hybridization stringency, etc.

[0100] Similarly, the probe binding molecules and nucleic acid barcode molecules can also include one or more additional sequences (e.g., unique molecular identifiers (UMIs), barcode sequences, primer sequences, capture sequences, or other functional sequences). For example, in some cases, the probe binding molecules or barcode molecules can include functional sequences, primer sequences (e.g., sequencing primer sequences or partial sequencing primer sequences), UMIs, etc. The probe binding molecules and nucleic acid barcode molecules can be any useful length; for example, the length of either or both can 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. The length of the probe binding molecule or barcode molecule can 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. The length of the probe capture binding molecule or barcode molecule can 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 nucleotides. A range of probe binding molecules or barcode molecules can be used, such as a length of about 16 to about 100 nucleotides, etc. In some cases, the length of the probe binding molecule or barcode molecule can vary based on any useful application and properties, such as melting temperature, annealing temperature, etc. In some cases, the first target region and the second target region of the nucleic acid molecule are not adjacent. For example, the first target region and the second target region can be separated by one or more gap regions arranged between the first target region and the second target region. The gap region can include, 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 contain 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 contain a range of base numbers, such as about 1 to 30 bases.

[0101] The target region of nucleic acid molecules can have one or more useful characteristics. For example, the target region can have any useful length, base content, sequence, melting point or other characteristics. The target region can include, 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. The target region can have any useful base content and any useful base sequence and combination. For example, the target region can include one or more adenine, thymine, uracil, cytosine and / or guanine bases (for example, natural or standard bases). The target region can also comprise one or more derivatives or modified forms of natural or canonical bases, such as oxidized, alkylated (e.g., methylated), hydroxylated or other modified bases. Similarly, the target region can comprise a ribose or deoxyribose moiety and a phosphate moiety or a derivative or modified form thereof.

[0102] The target region of a nucleic acid molecule can comprise one or more sequences or features of a nucleic acid molecule or a portion thereof. For example, the target region can comprise all or part of a UTR (e.g., 3'UTR or 5'UTR), a Kozak sequence, a Shine-Dalgarno sequence, a coding sequence, a poly A sequence, a cap structure, an intron, an exon, or any other sequence or feature of a nucleic acid molecule.

[0103] The nucleic acid molecules (e.g., RNA molecules, such as mRNA molecules) of the sample can be contained in cells, cell nuclei or cell beads. For example, the sample can include cells or cell nuclei with nucleic acid molecules. Cells, cell nuclei or cell beads can include additional nucleic acid molecules that may be the same or different from the nucleic acid molecules of interest. In some cases, the sample can include multiple cells, and each cell can include one or more nucleic acid molecules. The cell can be, for example, a human cell, an animal cell or a plant cell. In some cases, as described herein, the cell can be derived from a tissue or fluid. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be a lymphocyte, such as a B cell or a T cell. The cell can be contained in a bead, such as those disclosed in U.S. Patent No. 10,428,326, which is incorporated herein by reference in its entirety. In some cases, the cell is contained in a tissue sample and can be fixed to a substrate. For example, as described above, the cell can be a cell of a formalin-fixed paraffin-embedded (FFPE) sample. In such cases, the method can include additional operations for preparing the cells or nucleic acid molecules contained therein, such as dewaxing, staining (e.g., using immunogenic agents) or decolorizing, decrosslinking, washing, enzyme treatment, etc. Additional examples of processing FFPE samples before and after probe hybridization are included in PCT / US2020 / 066720, which is incorporated herein by reference in its entirety.

[0104] Can approach the nucleic acid molecule contained in cell, cell nucleus or cell bead by cracking or permeabilizing cell or cell nucleus.Cracked cell, cell nucleus or cell bead can release the nucleic acid molecule contained therein from cell, cell nucleus or cell bead.Cleaning agent (such as bioactivator) cracked cell or cell nucleus can be used.The bioactivator that can be used for cracking cell or cell nucleus can be, for example, enzyme (for example, as described herein).Enzyme for cracking cell or cell nucleus may or may not be able to perform additional functions, such as degradation, extension, reverse transcription or otherwise change nucleic acid molecule.Alternatively, ionic or nonionic surfactants such as TritonX-100, Tween 20, sodium lauryl sarcosine or sodium lauryl sulfate can be used to crack cell or cell nucleus.Cell / cell nuclear cracking can also be realized using cell disruption method, such as electroporation or heat, sound or mechanical disruption method.Alternatively, permeabilized cell or cell nucleus can be used to approach the nucleic acid molecule contained therein.Permeabilization can relate to partial or complete dissolution or destruction of cell membrane / nuclear membrane or a part thereof. Permeabilization can be achieved, for example, by contacting the cell membrane with an organic solvent (eg, methanol) or a detergent (such as Triton X-100 or NP-40). As described elsewhere herein, the cells, cell nuclei or cell beads can be fixed.

[0105] In some cases, cells can be lysed in cell beads, and a subset of intracellular contents can be associated with beads. In some cases, cell beads can include thioacrylamide-modified nucleic acid molecules that can hybridize with nucleic acids from cells. For example, poly-T nucleic acid sequences can be modified with thioacrylamide and incorporated into cell bead matrix. After cell or nuclear lysis, cellular nucleic acid (e.g., mRNA) can hybridize with poly-T sequences. Retained intracellular / nuclear contents can be released, for example, by adding a reducing agent (e.g., DTT, TCEP, etc.). Release can occur in any convenient step, such as before or after separation.

[0106] The nucleic acid molecules of nucleic acid molecules or probe associations can be subjected to conditions sufficient to produce molecules connected by probes. For example, the first target region can be adjacent to the second target region, and the first probe and the second probe can hybridize with the first target region and the second target region, respectively. The first probe can include a first reactive portion, and the second probe can include a second reactive portion. In some cases, the first reactive portion of the first probe is adjacent to the second reactive portion of the second probe. Then, the reactive portion can be subjected to conditions sufficient to react them to produce nucleic acid molecules connected by probes, and the nucleic acid molecules connected by the probes include the first probe connected to the second probe. For example, the reactive portion can be connected together via click chemistry or enzymatic connection, such as those disclosed in U.S. Patent Publication No. 2020 / 0239874, International Publication No. WO 2019 / 165318 and International Patent Publication No. WO2021 / 237087, each of which is incorporated herein by reference in its entirety. In some instances, the first probe or the second probe may comprise an adenylated oligonucleotide or moiety (e.g., an adenylated phosphate group), which may be used to reduce nonspecific ligation reactions. In some cases, the connection of the probes (e.g., via a ligation reaction) may be performed substantially without ATP, and optionally the enzyme (e.g., ligase) used does not require ATP (e.g., truncated T4 RNA ligase) or is preactivated (e.g., preactivated T4 DNA ligase). Additional examples of such connection schemes are found in PCT / US2020 / 066720 and International Patent Application No. PCT / US2021 / 33649 filed May 21, 2021, which are incorporated herein by reference in their entirety.

[0107] In some cases, the first target region of a nucleic acid molecule (e.g., an RNA molecule) may not be adjacent to the second target region. In such cases, the nucleic acid molecule may be subjected to conditions sufficient to hybridize the first probe sequence of the first probe with the first target region to produce probe-associated nucleic acid molecules. The probe-associated nucleic acid molecules may be subjected to a nucleic acid reaction (e.g., a nucleic acid extension reaction, reverse transcription, etc.) to produce an extended probe molecule, which comprises a sequence complementary to the second target region. The second probe comprising the second probe sequence may hybridize with the extended probe molecule (or its complementary sequence) and be subjected to conditions sufficient to produce a probe-connected molecule (e.g., nucleic acid extension, amplification, hybridization of additional probe molecules, connection, etc.), which comprises a sequence corresponding to the first target region and a sequence corresponding to the second target region. Alternatively or in addition, a first probe and a second probe may be provided simultaneously, and after the first probe sequence and the second probe sequence are hybridized to the first target region and the second target region, respectively, to produce a dual-probe associated nucleic acid molecule, the gap (e.g., a region disposed between the first target region and the second region) may be filled (e.g., via a nucleic acid extension or gap filling reaction and / or hybridization of an additional probe molecule hybridized to at least a portion of the gap region). In some cases, one or both probes may include an overhang sequence or a wing sequence (e.g., at the 5' end), which may be recognized or cleaved by an enzyme (e.g., an endonuclease, such as a FEN1 endonuclease). For example, the second probe may include a 5' wing sequence, which may be cleaved by the FEN1 endonuclease if at least one specific portion of the second probe is hybridized to a nucleic acid molecule (e.g., a target molecule). After the second probe is hybridized to the second target sequence of the nucleic acid molecule, the wing sequence may be cleaved using an endonuclease (e.g., FEN1), leaving a ligatable end (e.g., a phosphorylated end) of the second probe. In the case where the first target region is not adjacent to the second target region, the gap region may be filled and then the wing sequence may be cleaved. In some cases, the first probe or the second probe and the gap-filling region can be connected (e.g., chemically connected or enzymatically connected). Additional examples of systems and methods for producing nucleic acid molecules connected to probes and gap-filling reactions can be found in, for example, U.S. Patent Publication No. 2020 / 0239874, International Publication No. WO 2019 / 165318, and International Patent Publication No. WO2021 / 237087, each of which is incorporated herein by reference in its entirety.

[0108] The probe-connected nucleic acid molecule can be barcoded to provide a probe-connected nucleic acid molecule with a barcode, or the barcoding can occur before the probe-connected nucleic acid molecule is generated. Barcoding can be performed using a variety of techniques. For example, the first probe or the second probe can include a probe capture sequence. The nucleic acid barcode molecule can include a barcode capture sequence that can hybridize with the probe capture sequence. Alternatively, barcoding can be mediated by a probe binding molecule (e.g., a splint oligonucleotide), which includes (i) a probe binding sequence that can be complementary to the probe capture sequence of the first probe or the second probe; and (ii) a barcode binding sequence that can be complementary to the barcode capture sequence of the nucleic acid barcode molecule. In some cases, barcoding can be followed by connection, such as chemical or enzyme-mediated connection, to covalently connect the nucleic acid barcode molecule to the probe (or covalently connect to the probe binding sequence, and the probe binding sequence can be connected to the probe). Examples of chemical ligation of nucleic acid molecules can include "click chemistry" methods, such as the reaction of azide and alkyne moieties, as described in U.S. Patent Publication No. 2020 / 0239874, which is incorporated herein by reference in its entirety.

[0109] For example, the first probe may include a first probe sequence and a probe capture sequence, and the first probe may be subjected to conditions sufficient to hybridize the first probe sequence with the first target region, thereby producing probe-associated nucleic acid molecules. In some cases, probe-associated nucleic acid molecules may be subjected to washing or other conditions to remove unannealed probes from the mixture. Probe-associated nucleic acid molecules may extend from one end of the first probe to one end of the nucleic acid molecule hybridized therewith (extending toward one end close to the second target region) to provide extended nucleic acid molecules. Extended nucleic acid barcode molecules may include complementary sequences of the first probe sequence and the second target region. In some cases, extended nucleic acid molecules may be barcoded, for example, by: hybridizing the barcode capture sequence of the nucleic acid barcode molecule with the probe capture sequence, or (i) hybridizing the probe binding molecule comprising the probe binding sequence and the barcode binding sequence with the probe capture sequence, and (ii) hybridizing the barcode capture sequence of the nucleic acid barcode molecule with the barcode binding sequence of the probe binding molecule. In some cases, probe binding molecules pre-annealed to nucleic acid barcode molecules may be provided. Subsequently, a second probe comprising a second probe sequence can be provided. The barcoded, extended nucleic acid molecule can be subjected to conditions sufficient to allow the second probe sequence to hybridize to the second target region or its complement. A nucleic acid extension reaction can be performed to produce a barcoded molecule (e.g., a barcoded, probe-ligated 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.

[0110] Figure 7A method for producing a barcoded nucleic acid molecule as described herein is schematically shown. A nucleic acid molecule (e.g., an RNA molecule) 700 comprising a first target region 702 and a second target region 704 can be provided. The nucleic acid molecule 700 can be contacted with a first probe 706 comprising a first probe sequence 708 and an optional functional sequence 710, thereby producing a probe-associated nucleic acid molecule. The first probe sequence 708 can be complementary to the first target region 702. The functional sequence 710 can include, for example, a probe capture sequence for downstream barcoding, or it can include a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0111] In operation 701, the probe-associated nucleic acid molecule can be subjected to conditions sufficient to extend the first probe 706, thereby generating an extended probe molecule 712, which includes a sequence complementary to the second target region 704. In some cases, the extended probe molecule 712 can be released from the nucleic acid molecule 700, for example, by denaturing and / or degrading the nucleic acid molecule 700 (e.g., using RNase, elevated temperature or thermal cycling, pH, etc.). In operation 703, a nucleic acid barcode molecule can be provided. In some cases, the nucleic acid barcode molecule can be partially double-stranded and can include a first strand 720 having a barcode sequence, and a second strand 722 having a sequence 724 at least partially complementary to the barcode sequence and a probe binding sequence 726, which can be at least partially complementary to the functional sequence (e.g., probe capture sequence) 710 of the first probe 706. In some cases, the nucleic acid barcode molecule is single-stranded and includes only the first strand 720 having the barcode sequence and the barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 722 may be provided that includes a barcode binding sequence 724 that is at least partially complementary to a barcode capture sequence and a probe binding sequence 726. In some cases, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or pre-annealed complex) may be coupled to a bead, such as a gel bead as described herein, and may include additional functional sequences, including but not limited to a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., an R1 / R2 sequence).

[0112] In operation 705, the extended probe molecule can be barcoded by hybridizing the probe binding sequence 726 with a functional sequence (e.g., a probe capture sequence 710). In some cases, the nucleic acid barcode molecule can be covalently attached to the extended probe molecule (e.g., via a probe capture sequence), for example, enzymatically (e.g., using a ligase) or chemically (e.g., using click chemistry). In operation 707, a second probe molecule 716 can be provided. In some cases, operation 707 can also include denaturation of the double-stranded molecule. The second probe molecule 716 can include a second probe sequence 714 corresponding to the second target region 704 and an optional functional sequence 718, which can include a probe capture sequence, a barcode sequence, a primer sequence, a sequencing primer sequence, and the like. In operation 709, a nucleic acid extension reaction can be performed, for example using a polymerase, to extend the second probe 716 along the extended probe molecule, thereby producing 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.

[0113] In another example, the first probe and the second probe can be connected before adding the barcode (for example, by chemical connection or enzymatic extension and / or connection). In such an example, the first probe can be hybridized with a nucleic acid molecule (for example, via hybridization of the first probe sequence with the first target region) to produce a probe-associated nucleic acid molecule. The probe-associated nucleic acid molecule can extend from one end of the first probe to one end of the nucleic acid molecule hybridized therewith to provide an extended nucleic acid molecule. The extended molecule can be subjected to conditions sufficient to hybridize the second probe with the second target region or its complementary sequence (for example, via hybridization of the second probe sequence with the second target region or its complementary sequence). Additional nucleic acid extension reactions can be performed to produce extended molecules, and barcodes can be added to the resulting extended products to produce molecules with barcodes. The molecules with barcodes can include sequences corresponding to the first target region, sequences corresponding to the second target region, sequences corresponding to the probe capture sequence, and sequences corresponding to the barcode sequence. In some cases, nucleic acid barcode molecules (or probe binding molecules) can be chemically connected to the first probe or the second probe, such as by a ligation reaction or click chemistry. For example, a nucleic acid barcode molecule can include a first reactive portion, and the first probe or the second probe can include a second reactive portion; the first reactive portion can be configured to react with the second reactive portion to produce a covalent bond. The barcoded nucleic acid molecule or its derivative can then be further processed and analyzed, optionally by any suitable technique, including nucleic acid sequencing, such as Illumina sequencing.

[0114] Figure 8Another method for producing a barcoded nucleic acid molecule as described herein is schematically shown. A nucleic acid molecule (e.g., an RNA molecule) 800 comprising a first target region 802 and a second target region 804 can be provided. The nucleic acid molecule 800 can be contacted with a first probe 806 comprising a first probe sequence 808 and an optional functional sequence 810, thereby producing a probe-associated nucleic acid molecule. The first probe sequence 808 can be complementary to the first target region 802. The functional sequence 810 can include, for example, a probe capture sequence for downstream barcoding, or it can include a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0115] In operation 801, the probe-associated nucleic acid molecule can be subjected to conditions sufficient to extend the first probe 806, thereby generating an extended probe molecule 812, which includes a sequence complementary to the second target region 804. In some cases, the extended probe molecule 812 can be released from the nucleic acid molecule 800, for example, by denaturing and / or degrading the nucleic acid molecule 800 (e.g., using RNase, elevated temperature or thermal cycling, pH, etc.). In operation 803, a nucleic acid barcode molecule and a second probe 816 can be provided. The second probe 816 can include a second probe sequence 814 corresponding to the second target region 804 and an optional functional sequence 818, which can include a probe capture sequence. In some cases, the nucleic acid barcode molecule can be partially double-stranded and can include a first strand 820 having a barcode sequence, and a second strand 822 having a sequence 824 complementary to the barcode sequence and a probe binding sequence 826, which can be complementary to the functional sequence (e.g., probe capture sequence) 818 of the second probe 816. In some cases, the nucleic acid barcode molecule is single-stranded and comprises only a first strand 820 having a barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 822 may be provided, the probe binding molecule comprising a barcode binding sequence 824 and a probe binding sequence 826 complementary to the barcode capture sequence. In some cases, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. The nucleic acid barcode molecule (or pre-annealed complex) may be coupled to beads, such as gel beads 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., an R1 / R2 sequence). In operation 803, a second probe 816 may be hybridized with the extended probe molecule 812 (e.g., via hybridization of the second probe sequence 814 with the second target region 804 or its complementary sequence), 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 with the probe capture sequence 818). In some cases, a nucleic acid barcode molecule or a probe binding molecule can be linked to the second probe 816 (eg, using a ligase or via chemical ligation, such as click chemistry).

[0116] In operation 805, a nucleic acid extension reaction can be performed, for example, using a polymerase (e.g., a DNA polymerase, a 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. The barcoded nucleic acid molecule or its derivative can then be optionally further processed and analyzed by any suitable technique, including nucleic acid sequencing, such as Illumina sequencing.

[0117] Fig. 9 Similar to Figure 8 The illustrated approach schematically illustrates another method for producing a barcoded nucleic acid molecule. A nucleic acid molecule (e.g., an RNA molecule) 900 comprising a first target region 902 and a second target region 904 can be provided. The nucleic acid molecule 900 can be contacted with a first probe 906 comprising a first probe sequence 908 and an optional functional sequence 910, thereby producing a probe-associated nucleic acid molecule. The first probe sequence 908 can be complementary to the first target region 902. The functional sequence 910 can include, for example, a probe capture sequence, or it can include a different functional sequence, such as a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0118] In operation 901, the probe-associated nucleic acid molecule can be subjected to conditions sufficient to extend the first probe 906, thereby generating an extended probe molecule 912, which comprises a sequence complementary to the second target region 906. In some cases, the extended probe molecule 912 can be released from the nucleic acid molecule 900, for example, by denaturing and / or degrading the nucleic acid molecule 900 (e.g., using RNase, elevated temperature or thermal cycling, pH, etc.). In operation 903, a second probe 916 can be provided. The second probe 916 can include a second probe sequence 914 corresponding to the second target region 904 and an optional functional sequence 918, which can include a probe capture sequence. In operation 905, a nucleic acid extension reaction can be performed, for example, 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, which comprises a sequence corresponding to the first target region 902 and the second target region 904.

[0119] In operation 905, a second probe may also be provided for the nucleic acid barcode molecule. In some cases, the nucleic acid barcode molecule may be partially double-stranded and may include a first strand 920 having a barcode sequence, and a second strand 922 having a sequence 924 complementary to the barcode sequence and a probe binding sequence 926, which may be complementary to a functional sequence (e.g., a probe capture sequence) 918 of the second probe 916. In some cases, the nucleic acid barcode molecule is single-stranded and includes only a first strand 920 having a barcode sequence and a barcode capture sequence. A probe binding molecule (e.g., a splint oligonucleotide) 922 may be provided, which includes a barcode binding sequence 924 complementary to the barcode capture sequence and a probe binding sequence 926. In some cases, the probe binding molecule and the nucleic acid barcode molecule may be provided as a pre-annealed complex. Nucleic acid barcode molecules (or pre-annealed complexes) can be coupled to beads, such as gel beads as described herein, and can include additional functional sequences, including but not limited to unique molecular identifiers (UMIs), capture sequences, primer sequences (e.g., R1 / R2 sequences). In operation 907, the nucleic acid barcode molecules can be attached or coupled to the second probe 916, for example, via hybridization of the probe binding sequence 926 with the probe capture sequence 918. The resulting barcoded product can include sequences 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 cases, the nucleic acid barcode molecules can be covalently linked to the extended probe molecules (e.g., via the probe capture sequence 918), for example, enzymatically (e.g., using a ligase) or chemically (e.g., using click chemistry). The barcoded nucleic acid molecules or their derivatives can then optionally be further processed and analyzed by any suitable technique, including nucleic acid sequencing, such as Illumina sequencing.

[0120] In additional examples, the methods of the present disclosure may include generating probe-associated nucleic acid molecules, barcoding the probe-associated nucleic acid molecules, and optionally performing a ligation operation (e.g., before or after barcoding the probe-associated nucleic acid molecules). For example, a nucleic acid molecule (e.g., an 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 cases, the probe-associated nucleic acid molecule may be subjected to conditions sufficient to connect the first probe to the second probe (e.g., enzymatic ligation, such as with a polymerase, reverse transcriptase and / or ligase, or chemical ligation), thereby generating a probe-linked nucleic acid molecule. The probe-associated nucleic acid molecule or the probe-linked molecule may then be barcoded (e.g., in a partition) to generate a barcoded nucleic acid molecule.

[0121] For example, Fig.25 Schematically illustrated is an example method for producing a probe-connected nucleic acid molecule, which can then be barcoded, for example, in a partition to produce 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 can be provided. In some cases, the first target region is adjacent to the second target region. In operation 2501, the nucleic acid molecule 2500 can be contacted 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 producing a probe-associated nucleic acid molecule. The first probe 2506 and / or the second probe 2516 can include a functional sequence, such as a probe capture sequence, a primer sequence, a partial primer sequence, a barcode sequence, a sequencing primer sequence, etc.

[0122] In some cases, one of the probes (e.g., the second probe 2516) includes a wing sequence or an overhang sequence 2530, which can be recognized by an endonuclease (e.g., FEN1) when the second probe sequence 2514 anneals to the second target region 2504. For example, the second probe 2516 can include a 5' wing sequence 2530, and after the first probe 2506 and the second probe 2516 anneal to the nucleic acid molecule 2500, the wing sequence can be adjacent to one end of the first probe (e.g., 3' end) and one end of the second probe (e.g., 5' end). In operation 2503, an endonuclease (e.g., FEN1) can be used to remove the wing sequence 2530, leaving the ligatable end (e.g., 5' phosphorylated end) of the second probe 2516. In operation 2507, a ligation reaction (e.g., using a ligase) can be performed to connect the first probe to the second probe, thereby generating a probe-connected nucleic acid molecule. Subsequently, the probe-connected nucleic acid molecule can be barcoded, for example, in a partition, as described elsewhere herein. In some cases, probe-associated nucleic acid molecules can be barcoded and linked (eg, in partitions).

[0123] Fig.26 Shown with Fig.25 Another example workflow similar to the workflow shown, wherein the target region of the nucleic acid molecule is not adjacent. This workflow can include additional gap filling reactions to produce probe-associated molecules. In one such example, the first target region 2602 of the nucleic acid molecule 2600 may not be adjacent to the second target region 2604. For example, the gap region may be arranged 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 polymerase, reverse transcriptase, etc.) may be performed to fill the gap region between the first probe 2606 and the second probe 2616, producing a gap-filled nucleic acid molecule. In some cases, the second probe 2616 includes a wing sequence 2630. In such a case, in operation 2605, an endonuclease (e.g., FEN1) may be used to remove the wing sequence 2630, leaving the ligatable end (e.g., 5' phosphorylated end) of the second probe 2616. In operation 2607, a ligation reaction (eg, using a ligase) can be performed to ligate the first probe to the second probe, thereby generating a probe-ligated nucleic acid molecule. The probe-ligated nucleic acid molecule or alternatively the unligated molecule can be barcoded, for example, in one partition.

[0124] Fig. 27 An additional scheme for generating probe-ligated nucleic acid molecules by performing a gap-filling reaction using a third probe is shown. Fig. 27In Figure A, a first probe 2706 and a second probe 2716 are annealed (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 a nucleic acid molecule 2700 to produce a probe-associated nucleic acid molecule. A gap sequence can be provided between the first target region 2702 and the second target region 2704. A third probe molecule 2770 (shown as two different probe molecules that can be used for SNP detection) can be provided, which can anneal to the gap sequence ( Fig. 27 Figure B). Fig. 27 In panel C, the first probe, the third probe, and the second probe can be connected (eg, using a ligase) to produce a probe-connected nucleic acid molecule. The probe-connected nucleic acid molecule or alternatively the probe-associated nucleic acid molecule can be barcoded, for example, in one partition.

[0125] Fig.28 The example of the connection scheme for producing the nucleic acid molecule that probe connects is shown.In such an example, the probe molecule can be hybridized with the nucleic acid molecule.Enzyme can be used, optionally by gap filling operation as above, the first probe is connected to the second probe.In some cases, the enzyme can be a preactivated enzyme, for example preactivated T4 DNA ligase, and connection can occur under the condition that ATP reduces or removes ATP, for example using apyrase.

[0126] Additional examples of methods and systems for generating and barcoding probe-associated nucleic acid molecules can be found in, for example, U.S. Patent Publication No. 2020 / 0239874, International Publication No. WO 2019 / 165318, International Application No. PCT / US2020 / 066720, and International Patent Application No. PCT / US2021 / 33649 filed on May 21, 2021, each of which is incorporated herein by reference in its entirety.

[0127] It should be understood that, for example, see Figures 7 to 9 and Figure 25 to Figure 28 , the nucleic acid barcode molecule can be attached (e.g., via hybridization) to the first probe and / or the second probe (e.g., via a probe capture sequence contained in the first probe or the second probe). Similarly, the first probe and the second probe can contain any useful functional sequence, such as a primer sequence, a barcode sequence, a unique molecular identifier (UMI) sequence, a flow cell attachment sequence, a primer binding sequence, a capture sequence, etc. The first probe can hybridize to the left side (e.g., 3' end) or the right side (e.g., 5' end) of the nucleic acid molecule (e.g., 700, 800, or 900). Similarly, the second probe can hybridize to the left side or the right side of the nucleic acid molecule.

[0128] As described herein, one or more extension reactions can be performed on the nucleic acid molecules hybridized by the probe. For example, the probe can extend from one end of the probe to one end of the nucleic acid barcode molecule, or the second probe can extend from one end of the second probe to one end of the first probe of the nucleic acid molecule associated with the probe. Extension can include adding one or more nucleotides at the end of the probe using an enzyme (e.g., polymerase, reverse transcriptase). Extension can provide an extended nucleic acid molecule, which contains a sequence complementary to the target region of the nucleic acid molecule of interest, a barcode sequence, and one or more additional sequences of the optional nucleic acid barcode molecule, such as one or more binding sequences. In some cases, appropriate conditions and / or chemicals (e.g., as described herein) can be applied to denature the extended nucleic acid molecules from the nucleic acid barcode molecules and the target nucleic acid molecules. In some cases, one or more processes may involve the use of a thermosensitive agent. For example, in some cases, the probe can be annealed or hybridized under a set of temperature conditions, and extension can occur under a set of different temperature conditions. In some cases, a warm start or hot start polymerase can be used. In some cases, hybridization of a nucleic acid barcode molecule with one or more probes (e.g., direct hybridization or via a probe binding molecule such as a splint oligonucleotide) can precede hybridization of the probe with a target region of the nucleic acid molecule. After barcoding, the barcoded nucleic acid molecule can be replicated or amplified, for example, by one or more amplification reactions. The amplification reaction can include a polymerase chain reaction (PCR) and can involve the use of one or more primers or polymerases. Extension, denaturation and / or amplification processes can be performed within a partition or in the body. In some cases, the extended nucleic acid molecule or its derivative (e.g., a barcoded molecule) can be replicated or amplified within a partition to provide an amplified product. The barcoded product or its complementary sequence (e.g., an amplified product) can be detected via sequencing (e.g., as described herein).

[0129] Nucleic acid molecules or derivatives thereof (e.g., probe-linked nucleic acid molecules, nucleic acid molecules with one or more probes hybridized thereto, barcoded, probe-linked nucleic acid molecules or extended nucleic acid molecules or their complementary sequences) or cells or cell beads comprising nucleic acid molecules or derivatives thereof, for example as described herein, can be provided in a partition (such as a well or droplet). One or more reagents can be co-segregated with nucleic acid molecules or derivatives thereof or cells comprising nucleic acid molecules or derivatives thereof. For example, nucleic acid molecules or derivatives thereof or cells comprising nucleic acid molecules or derivatives thereof can be co-segregated with one or more reagents selected from the group consisting of: lysing 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 RNA molecules, permeabilizing or lysing cells, or performing 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, oxidants, nanoparticles, beads, and antibodies. In some cases, nucleic acid molecules or derivatives thereof or cells (for example, cell beads) comprising nucleic acid molecules or derivatives thereof can be separated together with one or more reagents, and the one or more reagents are selected from the group consisting of the following: temperature-sensitive enzyme, pH-sensitive enzyme, light-sensitive enzyme, reverse transcriptase, protease, ligase, polymerase, restriction enzyme, nuclease, protease inhibitor, exonuclease and nuclease inhibitor. For example, nucleic acid molecules or derivatives thereof or cells comprising nucleic acid molecules or derivatives thereof can be separated together with polymerase and nucleotide molecules. Separating nucleic acid molecules or derivatives thereof or cells and one or more reagents comprising nucleic acid molecules or derivatives thereof can include making the first phase comprising aqueous fluid, cell and one or more reagents and the second phase comprising a fluid immiscible with aqueous fluid flow to the connection portion. When the first phase and the second phase interact, discrete droplets of the first phase comprising nucleic acid molecules or derivatives thereof or cells (for example, cell beads) containing nucleic acid molecules or derivatives thereof and one or more reagents can be formed. In some cases, partitions can include single cells. Cells can be cracked or permeabilized in partitions (for example, droplets) to approach the nucleic acid molecules of cells.

[0130] One or more processes can be performed in a partition (e.g., a droplet, a hole, etc.). For example, nucleic acid molecules or cells or cell beads comprising nucleic acid molecules can be co-separated with one or more reagents (e.g., as described herein) at any useful stage of the method. For example, probe-associated nucleic acid molecules (e.g., nucleic acid molecules hybridized with a first probe) can be produced in a body (e.g., in a cell population that can be live or fixed and / or permeabilized, in a tissue sample, etc.) and subjected to conditions sufficient to produce extended probe molecules. The extended probe molecules can then be separated in one of the multiple partitions. The partition can include a second probe and a nucleic acid barcode molecule, as well as an optional probe binding molecule. As described herein, the second probe can hybridize with a second target region of the molecule associated with the probe (e.g., via a second probe sequence) or its complementary sequence. The partition can include additional reagents for performing a nucleic acid reaction (e.g., digestion, connection, extension, amplification). For example, the nucleic acid molecule associated with the probe may include a nucleic acid molecule or hybridize with a nucleic acid molecule, and the partition may include a degradation enzyme (e.g., an RNA enzyme) that can be used to digest or remove a template strand (e.g., a nucleic acid molecule, such as an RNA molecule) from the extended probe molecule. The partition may include a polymerase that can be used to extend a second probe hybridized with the extended probe molecule. In some cases, the partition includes a ligase (e.g., a ligase), which can be used to connect a nucleic acid barcode molecule to a first probe or a second probe (e.g., via a probe capture sequence). In some cases, a ligase can be used to connect a probe binding molecule to a probe capture sequence of a first probe or a second probe. In some cases, the probe binding molecule, the probe capture sequence, and / or the barcode capture sequence include one or more reactive moieties that can be used to chemically or enzymatically connect a nucleic acid barcode molecule to a probe capture sequence or its complementary sequence. The resulting product with a barcode may include 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.

[0131] For example, see again Figure 7 , operation 701 can be performed in the body (e.g., outside the partition), while operations 703 and 705 can be performed in a partition. Operations 707 and 709 can be performed in the body or in the partition. Similarly, see Figure 8 , operation 801 can be performed in the main body, and operation 803 can be performed in a partition. Operation 805 can be performed in the main body or in a partition. Fig. 9, operation 901 can be performed in the body, while operations 903, 905 and 907 can be performed in a partition. It should be understood that any operation can be performed in the body or in the partition at any convenient step, and the order of the operations can be changed for suitable or useful purposes.

[0132] Similarly, nucleic acid molecules or cells or cell beads comprising nucleic acid molecules or derivatives thereof (e.g., probe-associated molecules, extended molecules, molecules with barcodes, etc.) can be released from a partition at any useful stage of the method. For example, the extended probe molecule can be hybridized with the second probe and released from the partition after the barcode capture sequence of the nucleic acid barcode molecule hybridizes with the first probe, the second probe, or the probe-binding molecule. Alternatively, the extended probe molecule can be released from the partition after (i) hybridization of the second probe and the nucleic acid barcode molecule and (ii) extension of the second probe to produce a barcoded molecule, 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. Replication and / or amplification of the extended nucleic acid molecules can be performed within the partition or in the body (e.g., in solution). In some cases, the solution can contain additional extended nucleic acid molecules produced by the same process performed in different partitions. Each extended nucleic acid molecule can comprise a different barcode sequence, and the barcode sequence can be used to identify the partition or cell from which the extended nucleic acid molecule originated. In such cases, the solution can comprise a combined mixture comprising the contents of two or more partitions (e.g., droplets).

[0133] Additional processes or operations may be performed within a partition, including but not limited to: lysis, permeabilization, denaturation, hybridization, extension, replication, and amplification of one or more components of a sample. In some cases, multiple processes are performed within a partition.

[0134] The hybridization of the probe sequence and the target region of the nucleic acid molecule can be performed within a partition or outside the partition. In some cases, the double-stranded nucleic acid molecule can be denatured to provide a single-stranded nucleic acid molecule before hybridization, or the cell can be lysed or permeabilized. In some cases, hybridization can occur in a cell bead containing cells. The probe sequence complementary to the target region can be located at one end of the probe. Alternatively, the sequence can be arranged between other sequences so that when the probe sequence hybridizes with the target region, the additional probe sequence extends beyond the hybridized sequence in one or more directions. The probe sequence hybridized with the target region of the nucleic acid molecule can have the same or different length as the target region. For example, the probe sequence can be shorter than the target region and can only hybridize with a portion of the target region. Alternatively, the probe sequence can be longer than the target region and can hybridize with the entire target region and extend beyond the target region in one or more directions. In addition to the probe sequence complementary to the target region of the nucleic acid molecule, the probe can also include one or more additional probe sequences. For example, the probe can include a probe sequence complementary to the target region and a second probe sequence. The second probe sequence can have any useful length and other characteristics.

[0135] The probe (e.g., the first probe or the second probe) may include one or more additional sequences or portions, such as one or more barcode sequences or unique molecular 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 include a detectable portion, such as a fluorophore or fluorescent portion. In some cases, the first probe or the second probe may include a reactive portion, as described elsewhere herein. For example, the first probe or the second probe may include an azide portion, an alkyne portion, a thiophosphate portion, an iodide portion, an amine portion, a phosphate portion, or a combination thereof. The first probe may include a first reactive portion, and the second probe may include a second reactive portion, and the reaction of the first reactive portion and the second reactive portion may be sufficient to produce a probe-connected molecule, the probe-connected molecule comprising the first probe connected to the second probe. In some cases, the first reactive portion and the second reactive portion are connected via a connection reaction. Thus, the first probe or the second probe may comprise one or more partial or modified nucleotides to facilitate the ligation reaction, for example, one or more ribonucleotides or dideoxynucleotides (ddNTPs), which can be connected to the phosphorylated end of the second probe using a ligase (e.g., T4 DNA ligase, SplintR ligase). In some cases, the probe (e.g., the first probe or the second probe) may comprise an overhang sequence or a wing sequence that can be recognized or cleaved by an endonuclease (e.g., FEN1 endonuclease). Other suitable enzymes, such as ligases, such as enzymes and ligases disclosed in U.S. Provisional Application No. 63 / 171,031, filed April 5, 2021, are incorporated herein by reference in their entirety.

[0136] As described herein, the probe sequence of the probe may be capable of hybridizing with the sequence of a nucleic acid barcode molecule or a probe binding molecule (e.g., a splint oligonucleotide). The nucleic acid barcode molecule may include a first binding sequence (e.g., a barcode capture sequence) that is complementary to the probe sequence of the probe (e.g., a probe capture sequence). The nucleic acid barcode molecule may include one or more additional functional sequences, such as a primer sequence, a primer annealing sequence, and an immobilization sequence. The binding sequence may have any useful length and other characteristics. In some cases, the binding sequence (e.g., a barcode capture sequence) that is complementary to the probe sequence of the probe may be the same length as the probe sequence. Alternatively, the binding sequence may be a probe sequence of different lengths. For example, the binding sequence may be shorter than the probe sequence and may hybridize only with a portion of the probe sequence. Alternatively, the binding sequence may be longer than the probe sequence and may hybridize with the entire probe sequence and extend beyond the probe sequence in one or more directions. Similarly, where a probe binding molecule is used, the binding sequence of the nucleic acid barcode molecule (e.g., barcode capture sequence) can be the same length as the barcode binding sequence of the probe binding molecule, or the binding sequence can be longer or shorter than the barcode binding sequence.

[0137] One or more processes described herein can be carried out in cells, cell nuclei or cell beads. For example, in some embodiments, multiple cells, cell nuclei or cell beads can include multiple nucleic acid molecules. Cells, cell nuclei or cell beads can be live or fixed and / or permeabilized. In some cases, the first probe can be provided to cells, cell nuclei or cell beads, such as in a bulk solution. Optionally, cells, cell nuclei or cell beads can be washed to remove the first unbound probe, and nucleic acid extension reactions as described herein can be performed. Subsequently, cells, cell nuclei or cell beads comprising multiple nucleic acid molecules (or extended probe nucleic acid molecules) can be separated into multiple separate partitions, wherein at least one subset of multiple separate partitions includes single cells, single cell nuclei or single cell beads. Target nucleic acid molecules in cells, cell nuclei or cell beads contained in a partition can be approached by cracking or permeabilizing cell nuclei or cells (for example, as described herein) (this can be performed before or during the separation). Additional probe hybridization (for example, providing a second probe) and / or barcoding can be performed in separate partitions. As described herein, barcoding can include using a nucleic acid barcode molecule to attach or hybridize to a target nucleic acid molecule or a derivative thereof (e.g., an extended probe molecule or its complementary sequence). The nucleic acid barcode molecule provided in each of a plurality of separate partitions can be provided to be attached to a bead. In some cases, as described elsewhere herein, the nucleic acid barcode molecule can be releasably attached to a bead (e.g., via an unstable bond). Each partition (or a subset of partitions) in a plurality of separate partitions can include 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 can include a unique barcode sequence, so that each partition in a plurality of separate partitions includes a different barcode sequence. Upon releasing components from multiple different ones of the multiple individual partitions (e.g., after barcoding), the barcoded molecules originating from a single cell, a single cell nucleus, or a single cell bead can have the same barcode sequence (e.g., a common barcode sequence), such that each barcoded nucleic acid molecule can be traced back to a given partition and / or in some cases, a given cell, cell nucleus, or cell bead.

[0138] The methods described herein may include additional barcoding operations, which can be used, for example, to index nucleic acid molecules to cells, cell nuclei, cell beads, samples, a partition or multiple partitions. This index can be used for situations where a single partition is occupied by multiple cells, cell nuclei or cell beads. In some cases, it may be advantageous to overload the partition so that a partition includes more than a single cell, a single cell nucleus or a single cell bead; for example, in some cases it may be useful to overload the partition, for example, to overcome Poisson load statistics in the partition and / or to prevent reagent waste (e.g., caused by unoccupied partitions). Therefore, this index can be used to attribute cells, cell nuclei or nucleic acid molecules in multiple occupied partitions to starting cells, cell nuclei, cell beads, partitions, samples, etc.

[0139] In one example, barcoded molecules, such as those produced using the methods described herein, can be provided (e.g., in Figures 7 to 9 , Figure 25 to Figure 28 In, and described in U.S. Patent Publication No. 2020 / 0239874 and International Publication No. WO 2019 / 165318, the nucleic acid molecules with barcodes and probes connected, each of which is incorporated herein by reference). As described herein, the molecules with barcodes can include a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence (which can be set on the first probe or the second probe) and a sequence corresponding to the barcode sequence of the nucleic acid barcode molecule. This barcode sequence may be specific to the partition, and may be different from other barcode sequences of other partitions, and therefore can be used to identify the partition from which the nucleic acid molecule (or its derivative) is derived. In some cases, some partitions may include single cells, single cell nuclei or single cell beads, so nucleic acid barcode molecules or barcode sequences can be used to identify cells, cell nuclei or cell beads from which nucleic acid molecules (or its derivatives) are derived.

[0140] In some cases, the barcoded molecules can be subjected to additional barcoding operations (e.g., in a partition or in a body). For example, the barcoded molecules can be re-partitioned in one of a plurality of partitions comprising a plurality of additional nucleic acid barcode molecules. The plurality of additional nucleic acid barcode molecules can contain additional barcode sequences that differ between partitions. The barcoded molecules can be subjected to conditions sufficient to barcode the barcoded molecules to produce a combinatorial barcoded molecule comprising two barcode sequences. Since each barcode sequence belongs to a unique partition, the combination of barcodes can be used to generate more diverse barcoded molecules, as well as to identify the starting partition for the combinatorial barcoded molecules.

[0141] In some cases, combinatorial assembly of barcode fragments can be performed using, for example, a split-pool approach. For example, in some embodiments, a split-pool approach can be used to combinatorially barcode probe-attached nucleic acid molecules. In one such example, a probe-attached nucleic acid molecule (e.g., Figure 7 Operation 709, Figure 8 Operation 805 or Fig. 9 A plurality of permeabilized cells (or permeabilized cell nuclei or cell beads) (product after operation 905 or 907) are separated into a plurality of partitions (e.g., a plurality of wells), wherein each of the plurality of partitions comprises a different (i.e., unique) barcode sequence fragment. Alternatively, a plurality of permeabilized cells (or permeabilized cell nuclei or cell beads) can be separated, and then the different barcode sequence fragments are delivered to the corresponding partitions comprising cells, cell nuclei and / or cell beads. After adding the barcode sequence fragments, cells (or cell nuclei or cell beads) can be collected from the plurality of partitions, merged and separated into additional plurality of partitions (e.g., a plurality of wells), wherein each of the additional plurality of partitions comprises a different (i.e., unique) second barcode sequence fragment. Repeating the split pool process allows the generation of barcodes or molecules with barcodes comprising any suitable amount of barcode sequence fragments. Combinatorial barcoding as described herein can include at least 1, 2, 3, 4, 5, 6, 7, 8 or more operations (e.g., split pool cycles). Combinatorial barcoding including multiple operations can be used, for example, to generate greater barcode diversity and synthesize unique barcode sequences on nucleic acid molecules derived from each single cell, nucleus, or bead in multiple cells, nuclei, or bead. For example, combinatorial barcoding including three operations (each operation including attaching a unique nucleic acid sequence in each of 96 partitions) will generate up to 884,736 unique barcode combinations. In general, when there are M partitions and N split-pool iterations are performed, up to M unique barcode combinations can be generated. NA unique barcode combination. Cells or cell nuclei or cell beads can be separated so that at least one cell (or cell nucleus or cell bead) is present in each partition of multiple partitions. Cells, cell nuclei or cell beads can be separated so 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, cell nuclei or cell beads are present in a single partition. Cells, cell nuclei or cell beads can be separated so 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 cell nucleus or cell bead) is present in a single partition. Cells, cell nuclei and / or cell beads can be separated in a random configuration.

[0142] In some cases, additional barcoding operations may be performed before some operations described herein. For example, it may be advantageous to perform combinatorial barcoding of the first probe in a bulk solution, for example, before or after the generation of extended probe molecules or probe-connected molecules. In such cases, nucleic acid molecules may be contacted with the first probe, for example, in the bulk, to produce probe-associated molecules. The probe-associated molecules may optionally be extended, for example, using the methods described herein, to produce extended probe molecules. Then, the probe-associated molecules or extended probe molecules may be subjected to combinatorial barcoding in partitions, for example, as described above, to produce combinatorial barcoded molecules. The combinatorial barcoded molecules may then be separated together with the second probe and the nucleic acid barcode molecules, which may be attached to the first probe (or combinatorial barcoded probe), the second probe, or both probes, as described herein. Since each partition of the combinatorial barcoding process includes different barcode sequence fragments, multiple combinatorial barcoded molecules may be traced back to the separate partitions from which they originate. In addition, combinatorial barcoding may be used to produce greater probe diversity.

[0143] Advantageously, the combined barcode of the first probe may be particularly useful when combined with a second probe and a nucleic acid barcode molecule, which may contain a barcode sequence specific to the partition. For example, the presence of a probe-specific barcode and a partition-specific barcode sequence may allow for the indexing of individual cells (or cell nuclei or cell beads) within a partition. For example, a partition containing cell / cell nuclei / cell bead multiplicities (e.g., cell doublets, triplets, etc.) may be computationally deconvoluted into a single cell / cell nucleus / cell bead. Therefore, in some cases, conditions may be used to "overload" cells, cell nuclei, or cell beads into a partition so that there is a higher probability of forming cell / cell nuclei / cell bead multiplicities (2, 3, 4, 5+ cells, cell nuclei, or cell beads per partition), wherein the target library of these cell multiplicities may be computationally deconvoluted into a single cell, cell nucleus, or cell bead.

[0144] Fig.10 An example workflow for barcoding nucleic acid molecules in a partition containing a cell / nucleus / cell bead multiplicity is schematically illustrated. 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 Figures 7 to 9 and Figure 15 to Figure 16). For example, a first cell (or cell nucleus or cell bead) population 1002 (comprising a first plurality of nucleic acid molecules) can be subjected to barcoding in a first subset of the first plurality of partitions to produce a first plurality of barcoded nucleic acid molecules comprising a first barcode sequence. A second cell (or cell nucleus or cell bead) population 1004 can be subjected to barcoding in a second subset of the first plurality of partitions to produce a second plurality of barcoded nucleic acid molecules comprising a second barcode sequence. The first barcode sequence may be different from the second barcode sequence. In operation 1020, the first cell (or cell nucleus or cell bead) population 1002 and the second cell (or cell nucleus or cell bead) population 1004 can be merged together to produce a mixture of cells. In operation 1030, the mixture of cells (or cell nuclei or cell beads) can be partitioned into a second plurality of partitions. In some cases, the mixture of cells / cell nuclei / cell beads can be partitioned into a second plurality of partitions such that some of the second plurality of partitions include more than one cell (e.g., cell multisome partitions). For example, partition 1035 of the second plurality of partitions may include cells, cell nuclei, or cell beads ("cell A") from the first cell population 1002 and cells, cell nuclei, or cell beads ("cell B") from the second cell population 1004. Partition 1035 may include an additional barcode sequence, which may be specific to the partition. The cells / cell nuclei / cell beads in each partition may be subjected to additional barcoding operations to add additional barcode sequences to the barcoded nucleic acid molecules. In operation 1040, the barcoded nucleic acid molecules may be deconvoluted using different barcode sequences (e.g., a first barcode sequence, a second barcode sequence, and an additional barcode sequence) to identify the starting cell / cell nucleus / cell bead. For example, a barcoded nucleic acid molecule comprising an additional barcode sequence from partition 1035 and a first barcode sequence from first cell (or cell nuclei or cell beads) population 1002 can be used to identify the barcoded nucleic acid molecule as originating from cell A. Similarly, a barcoded nucleic acid molecule comprising an additional barcode sequence from partition 1035 and a second barcode sequence from second cell (or cell nuclei or cell beads) population 1004 can be used to identify the barcoded nucleic acid molecule as originating from cell B.

[0145] After partition-based barcoding, the contents of the partitions can be combined, and the barcoded molecules (e.g., barcoded, probe-connected nucleic acid molecules) can be replicated or amplified by, for example, one or more amplification reactions, which in some cases can be isothermal. The amplification reaction can include a polymerase chain reaction (PCR) and can involve the use of one or more primers or polymerases. One or more primers can include one or more functional sequences (e.g., primer sequences / primer binding sequences, sequencing primer sequences (e.g., R1 or R2), partial sequencing primer sequences (e.g., partial R1 or partial R2), sequences configured to be attached to a flow cell of a sequencer (e.g., P5 or P7, or partial sequences thereof), etc.) and can facilitate the addition of the one or more functional sequences to the extended nucleic acid molecules. Barcoded molecules or derivatives thereof can be detected via nucleic acid sequencing (e.g., as described herein).

[0146] In some aspects, provided herein is a system for barcoding nucleic acid molecules. The system may include any of the components described herein, such as multiple partitions (e.g., droplets, holes), which may be provided in any useful form, such as a microfluidic device, a porous array or a plate, etc. The system may include a nucleic acid barcode molecule, which is optionally coupled to a support (e.g., a particle, a bead, a gel bead, etc.). In some cases, the system may include any probe described herein, such as a first probe or multiple first probes, a second probe or multiple second probes, and any useful reaction components (e.g., for performing a nucleic acid reaction, such as extension, connection, amplification, etc.). In a non-limiting example, such useful reaction components may include enzymes (e.g., ligases, polymerases, reverse transcriptases, restriction enzymes, etc.), nucleotide bases, etc.

[0147] Also provided herein is a composition useful for systems and methods for barcoding nucleic acid molecules. The composition may include any probe described herein. For example, the composition may include 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 group of probes may be designed to target a specific sequence or a group of specific sequences. Such probes may be designed to have the same or different sequences in different partitions. For example, a first composition may include a first probe and a second probe designed to target two regions of a first gene, and a second composition may include a first probe and a second probe designed to target two regions of a second gene, the second gene being different from the first gene. The composition may include a nucleic acid barcode molecule and / or a probe binding molecule, which may optionally be provided as coupled to a support (e.g., a particle, a bead). The composition may be a part of a reaction mixture or include a reaction mixture, which may include a reaction component or reagent, such as an enzyme, a nucleotide base, a catalyst, a buffer, etc.

[0148] Multiplex analysis of nucleic acids and proteins

[0149] In another aspect, the present disclosure provides a method for performing multiple determinations. Such multiple determinations may include determining or analyzing one or more biomolecules (e.g., nucleic acid molecules, proteins, lipids, carbohydrates, etc.). The method may include barcoding nucleic acid molecules of cells / nuclei / cell beads using one or more probes and nucleic acid barcode molecules to produce a first barcoded nucleic acid molecule; attaching or coupling a characteristic binding group to a feature of a cell / nucleus / cell bead, wherein the characteristic binding group comprises a reporter oligonucleotide, which comprises a reporter sequence that recognizes the characteristic binding group; barcoding the reporter sequence using additional nucleic acid barcode molecules and optional additional probes to produce a second barcoded nucleic acid molecule; and optionally barcoding the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule to produce 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., a droplet or a well).

[0150] The methods described herein can facilitate the profiling of one or more biomolecules at single cell / single cell 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 method can be used to provide genome, transcriptome, proteome, exome, or other "omics" information from a single cell / nucleus / cell bead. As described herein, the method can be used to analyze a set of predetermined target genes and a set of predetermined target features (e.g., proteins, peptides, or other biomolecules) in a sensitive and accurate manner. Alternatively or in addition, the method can be used to analyze the whole genome, whole transcriptome, whole exome, and other characteristics of a cell.

[0151] In some aspects, the method includes contacting a cell / nucleus / cell bead with a first probe, a second probe, and a third probe under conditions sufficient to produce a molecule associated with the first probe and a molecule associated with the second probe. 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., a protein, a peptide, or other biomolecule) coupled to a feature binding group. The feature binding group may comprise or be coupled to: (i) a reporter oligonucleotide comprising a reporter sequence, which may be associated with a feature or may be used to identify a 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 an optional 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 an optional 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 a sequence identical to the probe capture sequence of the first probe and / or the second probe.

[0152] In some cases, the first probe-associated molecule may comprise a nucleic acid molecule, a first probe, a second probe, or a combination or complement thereof. The second probe-associated molecule may comprise a reporter oligonucleotide (which comprises a reporter sequence) and a third probe or a complement thereof.

[0153] In some aspects, the method includes providing a first probe-associated molecule and a second probe-associated molecule, and barcoding the first probe-associated molecule and the second probe-associated molecule. Such barcoding operations can occur in a first set of partitions (e.g., droplets or wells). This example method can include contacting the first probe-associated molecule and the second probe-associated molecule with a probe-binding molecule (e.g., a splint oligonucleotide) and a barcode molecule (e.g., a nucleic acid barcode molecule) under conditions sufficient to produce a first barcoded nucleic acid molecule and a second barcoded nucleic acid molecule. The barcode molecule can include (i) a barcode capture sequence, such as a common sequence shared by multiple barcode molecules, and (ii) a first barcode sequence. In the case of using partitions, the first barcode sequence can be specific to the first partition of the first set of partitions, and the barcode molecules within the first partition can share the same first barcode sequence. The probe binding molecule may comprise (i) a probe binding sequence complementary to a probe capture sequence (of the first probe, the second probe, and / or the third probe), and (ii) a barcode binding sequence complementary to a barcode capture sequence (e.g., a common sequence) of a plurality of barcode molecules. Thus, barcoding of the first probe-associated molecule and the second probe-associated molecule may comprise hybridization of the probe binding molecule with: (i) the probe capture sequence (or its complementary sequence) of the first probe, the second probe, and / or the third probe, and (ii) the barcode capture sequence (or common sequence) of the nucleic acid barcode molecule. In some examples, the first barcoded nucleic acid molecule comprises a sequence corresponding to the first probe sequence, a sequence corresponding to the second probe sequence, and a sequence corresponding to the first barcode sequence. Similarly, the second barcoded nucleic acid molecule may comprise a sequence corresponding to a reporter sequence, a sequence corresponding to a third probe sequence, and a sequence corresponding to the first barcode sequence.

[0154] The method may also include providing a second set of partitions, and in a second partition of the second set of partitions, (i) contacting a first barcoded nucleic acid molecule or a derivative thereof (e.g., a complementary sequence, an amplicon, an extension product thereof) with a first capture molecule of a plurality of capture molecules under conditions sufficient to produce a third barcoded nucleic acid molecule, and (ii) contacting a second barcoded nucleic acid molecule or a derivative thereof with a second capture molecule of a plurality of capture molecules under conditions sufficient to produce 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 from the first barcode sequence from the first set of partitions. The second barcode sequence may be specific to the partition (i.e., different between 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 include a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence, a first barcode sequence, and a second barcode sequence. The fourth barcoded nucleic acid molecule may include a sequence corresponding to a reporter sequence, a sequence corresponding to a characteristic probe binding sequence, a sequence corresponding to a third probe, a first barcode sequence, and a second barcode sequence.

[0155] The characteristic binding group may include a labeling agent, as described elsewhere herein. Thus, in some examples, the characteristic binding group may include an antibody or antibody fragment, an epitope binding portion, a protein, a peptide, a lipophilic portion (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bispecific antibody, a bispecific T cell adapter, a T cell receptor adapter, a B cell receptor adapter, an antibody prodrug, an aptamer, a monoclonal antibody, an affimer, a darpin, and a protein scaffold, or any combination thereof.

[0156] The probe capture sequence of the first probe (or second probe) can be shared by multiple first probes (or second probes), multiple partitions and / or multiple cells / cell nuclei / cell beads. For example, the first set of partitions can include one or more additional partitions, which include nucleic acid molecules associated with additional probes. The nucleic acid molecules associated with the additional probes can contain the same sequence as the nucleic acid molecules associated with the probes of the first partition (e.g., the first probe sequence, the second probe sequence), or the nucleic acid molecules associated with the additional probes of the additional partitions can contain different sequences from the nucleic acid molecules associated with the probes of the first partition (e.g., different probe sequences). In some cases, each of the one or more additional probe-associated nucleic acid molecules contains a probe capture sequence, which can be the same or different in the first set of partitions.

[0157] The probe-associated molecule can be a probe-linked molecule. For example, the probe-associated molecule can be a probe-associated molecule described herein or a barcoded molecule (e.g., Figures 7 to 9 ), or a probe-linked molecule, such as those described in U.S. Patent Publication No. 2020 / 0239874 and International Publication No. WO 2019 / 165318, each of which is incorporated herein by reference in its entirety. In some examples, two sets of probe-associated molecules can be generated, wherein: (i) the first probe-associated molecule comprises a nucleic acid molecule, wherein the first probe and the second probe hybridize to the nucleic acid molecule (e.g., via hybridization of the first probe sequence to the first target region and hybridization of the second probe sequence to the second target region), and (ii) the second probe-associated molecule comprises a reporter oligonucleotide (which comprises a reporter sequence), wherein the third probe hybridizes to the reporter oligonucleotide.

[0158] The first probe, the second probe and / or the third probe may comprise a probe capture sequence. The probe capture sequence on the first probe may be the same or different from 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 from the probe capture sequence of the third probe. Therefore, the barcoding operation 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 a first probe ("probe 1") and a second probe ("probe 2") hybridized therewith, a first barcode molecule comprising a first barcode sequence ("BC1") may hybridize with the first probe (e.g., directly or via a probe binding molecule) to produce a first barcoded nucleic acid molecule, and then a capture molecule comprising a second barcode sequence ("BC2") may anneal to the region of the first barcode molecule, thereby producing a molecule comprising a sequence or complementary sequence of BC2-BC1-probe 1-probe 2. Alternatively or in addition, a first barcode molecule comprising a first barcode sequence ("BC1") can be hybridized with a second probe (e.g., directly or via a probe binding molecule) to produce a first barcoded nucleic acid molecule, and subsequently a capture molecule comprising a second barcode sequence ("BC2") can be annealed to a region of the first barcode molecule, thereby producing a molecule comprising the sequence of probe1-probe2-BC1-BC2. Alternatively or in addition, the barcode molecule and the capture molecule can be annealed to different probes. For example, a first barcode molecule comprising a first barcode sequence ("BC1") can be hybridized with a first probe (e.g., directly or via a probe binding molecule) to produce a first barcoded nucleic acid molecule, and subsequently a capture molecule comprising a second barcode sequence ("BC2") can be annealed to the second probe, thereby producing a molecule comprising the sequence of BC1-probe1-probe2-BC2. Alternatively or in addition, a first barcode molecule comprising a first barcode sequence ("BC1") can be hybridized to a second probe (e.g., directly or via a probe binding molecule) to produce a first barcoded nucleic acid molecule, and then a capture molecule comprising a second barcode sequence ("BC2") can be annealed to the first probe, thereby producing a molecule comprising the BC2-probe1-probe2-BC1 sequence. It should be understood that while several examples of barcoding schemes are described herein, additional combinations and positioning of barcode sequences are possible; for example, combinatorial barcoding can be used to generate greater barcode diversity, as described herein, and such barcoding can occur on any probe molecule (or barcoded molecule).

[0159] In some cases, the barcode molecule may include a capture binding sequence that is complementary to the capture sequence of multiple capture molecules. For example, the first probe may include a probe capture sequence that can hybridize with a probe binding molecule, which can mediate the hybridization of the barcode molecule (e.g., via the hybridization of the barcode binding sequence of the probe binding molecule with the barcode capture sequence (e.g., common sequence) of the barcode molecule). The barcode molecule may additionally include a capture binding sequence, which can allow the capture sequence of the capture molecule to hybridize with the barcode molecule.

[0160] Fig.15 Schematically shows a nucleic acid molecule with a barcode as described herein. Referring to Figure A, a nucleic acid molecule (e.g., RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 can be provided. The nucleic acid molecule 1500 can be contacted with a first probe 1506 comprising a first probe sequence 1508 and an optional first probe capture sequence 1510. The first probe sequence 1508 can be complementary to the first target region 1502. In addition, in some cases, the first probe capture sequence 1510 can include 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 can also be contacted with a second probe 1516 comprising a second probe sequence 1514 and an optional second probe capture sequence 1518. The second probe sequence 1514 can be complementary to the second target region 1504. The second probe capture sequence 1518 can additionally include a functional sequence. The hybridization of the first probe 1506 and the second probe 1516 with the nucleic acid molecule 1500 can produce a probe-associated molecule.

[0161] As described herein, the molecules associated with the probe can be subjected to one or more barcoding operations. This barcoding operation can occur in one or more partitions (e.g., the first group of partitions), and can include hybridizing the probe binding molecule 1517 and the barcode molecule 1519 containing the barcode capture sequence (e.g., common sequence) with the probe-associated molecule. In some cases, the probe binding molecule 1517 and the barcode molecule 1519 can be provided as a pre-annealing complex, or they can be provided as separate molecules. The barcode capture sequence (e.g., common sequence) can be a sequence shared by multiple barcode molecules in the first group of partitions, or the common sequence can be a barcode molecule unique to only a single first partition (i.e., the common sequence is different between the partitions of the first group of partitions). The probe binding molecule 1517 can include a probe binding sequence complementary to the probe capture sequence 1518 of the second probe 1516, and a barcode binding sequence complementary to the sequence of the barcode molecule 1519. The probe-associated molecules can be subjected to conditions sufficient to produce 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., a common sequence) of the barcode molecule 1519. The barcoding process can include additional operations, such as ligation, which can be performed chemically or enzymatically, as described elsewhere herein.

[0162] The first barcoded nucleic acid molecule or a derivative thereof (e.g., a complementary sequence, an amplicon, an extension product, a combinatorial barcoded nucleic acid molecule, as described elsewhere herein) can be subjected to a second barcoding operation. This second barcoding operation can occur in the second set of partitions. For example, the first barcoded nucleic acid molecule can be taken out of the first set of partitions, combined (e.g., combined with other barcoded nucleic acid molecules from other first partitions of the first set of partitions), and separated in the second partition of the second set of partitions. The second partition may include a capture molecule 1520. The capture molecule 1520 may include 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 common to multiple 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., different between the partitions). The capture molecule 1520 may hybridize with the probe capture sequence 1510 to produce an additional barcoded molecule (also referred to herein as a "third barcoded nucleic acid molecule"). The additional barcoded molecules may contain 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).

[0163] Fig.15FIG. B schematically shows another example barcoded molecule, wherein a capture molecule 1520 is hybridized with a barcode molecule 1519. Similar to FIG. A, in FIG. B, a nucleic acid molecule (e.g., an RNA molecule) 1500 comprising a first target region 1502 and a second target region 1504 can be provided. The nucleic acid molecule 1500 can be contacted with a first probe 1506 comprising a first probe sequence 1508 and a probe capture sequence 1510. The first probe sequence 1508 can be complementary to the first target region 1502. In addition, the probe capture sequence 1510 can include 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 can also be contacted with a second probe 1516 comprising a second probe sequence 1514 and an optional additional sequence 1518. The second probe sequence 1514 can be complementary to the second target region 1504. Additional sequence 1518 may include, for example, a probe capture sequence or a functional sequence (eg, a primer, a primer binding site, a sequencing primer sequence, etc.) Hybridization of first probe 1506 and second probe 1516 to nucleic acid molecule 1500 may produce a probe-associated molecule.

[0164] The probe-associated molecules can be contacted with one or more barcode molecules. As described herein, such barcoding operations can occur in multiple partitions (e.g., a first partition of a first group of partitions and / or a second partition of a second group of partitions). The probe-associated molecules can be contacted with probe-binding molecules 1517 and barcode molecules 1519, which can include a first barcode capture sequence (e.g., a common sequence) and a second barcode capture sequence 1521 (also referred to herein as a "capture binding sequence"). In some cases, the probe-binding molecules 1517 and the barcode molecules 1519 can be provided as a pre-annealed complex, or as separate molecules. The first barcode capture sequence (e.g., a common sequence) can be a sequence common to multiple barcode molecules in the first group of partitions, or the common sequence can be unique to the barcode molecules in only the first partition (i.e., different between the partitions). The probe binding molecule 1517 can include a probe binding sequence that is complementary to the probe capture sequence 1510, and a barcode binding sequence that is complementary to a first barcode capture sequence (e.g., a common sequence) of a barcode molecule 1519. The probe-associated molecules can be subjected to conditions sufficient to produce 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., a common sequence) of the barcode molecule 1519. The barcoding process can include additional operations, such as ligation, which can be performed chemically or enzymatically, as described elsewhere herein.

[0165] The first barcoded nucleic acid molecule or its derivative can be subjected to a second barcoding operation. This second barcoding operation can occur in the second group of partitions. For example, the first barcoded nucleic acid molecule can be taken out of the first partition and separated in the second partition of the second group of partitions (e.g., droplets). The second partition may include capture molecules 1520. Capture molecules 1520 may include a second barcode sequence and a sequence complementary to the second barcode capture sequence 1521 of barcode molecule 1519. The second barcode sequence may be a sequence shared by multiple capture molecules in the second group of partitions, or the barcode sequence may be unique to the capture molecules in the second partition only (i.e., different between the partitions). Capture molecules can hybridize with the second barcode capture sequence 1521 to produce additional barcoded molecules (also referred to herein as "third barcoded nucleic acid molecules"). The additional barcoded molecules may contain 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).

[0166] Fig.15 FIG. C of shows another example of a barcoded nucleic acid molecule. 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 an optional first probe capture sequence 1510. The first probe sequence 1508 may be complementary to the first target region 1502. In addition, in some cases, the first probe or the first probe capture sequence 1510 may include 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 an optional 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 include a functional sequence. The hybridization of the first probe 1506 and the second probe 1516 with the nucleic acid molecule 1500 may produce a probe-associated molecule or complex.

[0167] As described herein, probe-associated molecules can be subjected to one or more barcoding operations. Such barcoding operations can occur in one or more partitions (e.g., a first set of partitions), and can include hybridizing probe-binding molecules 1517 and barcode molecules 1519 containing barcode capture sequences (e.g., common sequences) with probe-associated molecules or complexes. In some cases, probe-binding molecules 1517 and barcode molecules 1519 are provided as pre-annealed complexes (e.g., partially double-stranded molecules containing probe-binding molecules 1517 and barcode molecules 1519), or they can be provided as separate molecules that can be annealed separately to probe-associated molecules or complexes (e.g., probe-binding molecules 1517 can be hybridized with probe-associated molecules or complexes, for example, via second probe capture sequences 1518, and barcode molecules 1519 can be hybridized with probe-binding molecules 1517). The barcode capture sequence (e.g., a common sequence) can be a sequence shared by multiple barcode molecules in the first group of partitions, or the common sequence can be unique to the barcode molecules in only a single first partition (i.e., the common sequence is different between the partitions of the first group of partitions). The probe binding molecule 1517 can include a probe binding sequence complementary to the probe capture sequence 1518 of the second probe 1516, and a barcode binding sequence complementary to the sequence of the barcode molecule 1519. In some cases, the probe binding molecule 1517 and / or the barcode molecule 1519 include additional sequences, such as adapter sequences, primer sequences (e.g., sequencing primer sequences or partial sequencing primer sequences), UMIs, sample index sequences, etc. In some cases, the probe binding molecule 1517 includes the complete sequence of the barcode molecule 1519, so no overhangs are left. In some cases, the probe binding molecule 1517 and the barcode molecule 1519 include a sample index sequence, which can be used to identify the partition, cell, cell nucleus, or cell bead from which the target nucleic acid molecule 1500 originates. The probe-associated molecules can be subjected to conditions sufficient to produce 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., a common sequence) of the barcode molecule 1519. The barcoding process can include additional operations, such as ligation (e.g., ligation of the barcode molecule 1519 to the probe capture sequence 1518), which can be performed chemically or enzymatically, as described elsewhere herein.

[0168] The first barcoded nucleic acid molecule or a derivative thereof (e.g., a complementary sequence, an amplicon, an extension product, a combinatorial barcoded nucleic acid molecule, as described elsewhere herein) can be subjected to a second barcoding operation. This second barcoding operation can occur in the second set of partitions. For example, the first barcoded nucleic acid molecule can be taken out of the first set of partitions, combined (e.g., combined with other barcoded nucleic acid molecules from other first partitions of the first set of partitions), and separated in a second partition of the second set of partitions. The second partition may include a capture molecule 1520. The capture molecule 1520 may include 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 common to multiple capture molecules in the second set of partitions, or the barcode sequence may be unique to the capture molecules only in the second partition (i.e., different between the partitions). Capture molecule 1520 can hybridize with probe capture sequence 1510 to produce additional barcoded molecules (also referred to herein as "third barcoded nucleic acid molecules"). The additional barcoded molecules can contain a sequence corresponding to the first barcode sequence (of barcode molecule 1519) and a sequence corresponding to the second barcode sequence (of capture molecule 1520).

[0169] In addition to barcoding nucleic acid molecules, the present disclosure also provides methods for multiplex analysis (e.g., processing additional biomolecule types, such as proteins and peptides). The method may include providing a feature binding group (e.g., an antibody, a protein, a binding moiety, etc.) that can be coupled to or bound to a feature (e.g., a protein, a peptide) of a cell, a cell nucleus, or a cell bead. This method may include providing a cell, a cell nucleus, or a cell bead having a feature of interest (e.g., a protein), and contacting the cell, a cell nucleus, or a cell bead with a feature binding group. The feature binding group may be coupled to a feature of interest. The feature binding group may include a reporter oligonucleotide that includes a reporter sequence coupled thereto that may be specific for a particular feature and is therefore used to identify the feature. For example, the feature binding group may be an antibody, and the reporter oligonucleotide may include a reporter sequence that identifies an antigen or a binding moiety (e.g., an epitope, an epitope fragment) to which the antibody is coupled or bound. Alternatively or in addition, the feature binding group may include a feature probe binding sequence that may be used for downstream probe binding and / or barcoding. After the cell (cell nucleus or cell bead) is contacted with the feature binding group, the cell / cell nucleus / cell bead may comprise the feature coupled to the feature binding group.

[0170] In some cases, the method described herein may further include: providing a nucleic acid molecule comprising (i) a first target region and a second target region and (ii) a cell, a cell nucleus or a cell bead coupled to a feature of a characteristic binding group, and contacting the cell, the cell nucleus or the cell bead with a plurality of probes. Cells / cell nuclei / cell beads can be contacted with the first probe, the second probe and the third probe (for example, in the first partition). As described herein, the first probe and the second probe can be associated with the first target region and the second target region of the nucleic acid molecule to produce a molecule associated with the first probe. Similarly, the third probe can be associated with the characteristic binding group (for example, via hybridization) to produce a molecule associated with the second probe. In some cases, the third probe can include a third probe sequence complementary to the characteristic probe binding sequence, and in some cases, the third probe can additionally include a probe capture sequence. The first probe and / or the second probe can also include a probe capture sequence, which can be the same or different from the probe capture sequence of the third probe.

[0171] In the first group of partitions, a molecule associated with the first probe (e.g., a nucleic acid molecule having the first probe and the second probe associated therewith) and a molecule associated with the second probe (e.g., a characteristic binding group having the third probe associated therewith) can be barcoded. This barcoding operation can include, for example, providing a barcode molecule comprising a first barcode sequence and a barcode capture sequence (such as a common sequence), which can, for example, directly hybridize with the molecule associated with the first probe and the molecule associated with the second probe via the probe capture sequence. Alternatively or in addition, a probe binding molecule can be provided for the barcode molecule, which comprises (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 that can be complementary to the common sequence of the barcode molecule. In some cases, the probe binding molecule and the barcode molecule can be provided as a pre-annealing complex. Barcoding of the first probe-associated molecule and the second probe-associated molecule can include hybridization of a barcode molecule (e.g., a barcode capture sequence such as a common sequence) with a portion of the first probe-associated molecule and the second probe-associated molecule (e.g., a probe capture sequence), or barcoding can include hybridization of a barcode molecule with a probe-binding molecule and hybridization of a probe-binding molecule with the first probe-associated molecule or the second probe-associated molecule. Additional operations such as ligation (e.g., enzymatic ligation or chemical ligation) can be performed to generate the first barcoded molecule and the second barcoded molecule.

[0172] The first barcoded molecule and the second barcoded molecule can be subjected to additional barcoding operations, for example, in a second set of partitions. Such additional barcoding operations can include: contacting the first barcoded nucleic acid molecule or a derivative thereof with a first capture molecule in a plurality of capture molecules to produce a third barcoded nucleic acid molecule, and contacting the second barcoded nucleic acid molecule or a derivative thereof with a second capture molecule in a plurality of capture molecules to produce a fourth barcoded nucleic acid molecule. The capture molecules within a partition can each contain a second barcode sequence, which can be specific to the partition (i.e., different between partitions). Therefore, the third barcoded nucleic acid molecule and the fourth barcoded nucleic acid molecule can both contain a first barcode sequence (or its complementary sequence) and a second barcode sequence (or its complementary sequence).

[0173] Fig.16A An example workflow for barcoding multiple analytes for cells, cell nuclei or cell beads is schematically shown. Cells, cell nuclei or cell beads 1600 may include a nucleic acid molecule (e.g., an RNA molecule or other target nucleic acid molecule) 1601, which includes a first target region 1602 and a second target region 1604. Cells, cell nuclei or cell beads may additionally include features (e.g., proteins, such as cell surface receptors (or nuclear membrane proteins) or intracellular / intranuclear proteins) 1650. In some cases, cells, cell nuclei or cell beads 1600 may be treated, such as fixed, permeabilized, treated with a treatment method, etc. In some cases, such treatment may include providing one or more feature binding groups (e.g., antibodies, antibody fragments, etc.) 1652, which may be coupled to feature 1650. Feature binding group 1652 may include or be coupled to a reporter oligonucleotide 1657, which may include a reporter sequence 1654. The reporter sequence 1654 can be indicative of a feature binding group 1652 or a feature 1650. For example, the reporter sequence 1654 can be pre-indexed or assigned to a specific antibody or other feature binding group, such that the presence of the reporter sequence 1654 indicates the presence of a specific feature 1650 in the sample. The feature binding group 1652 or the reporter oligonucleotide 1657 can also contain or be coupled to a feature probe binding sequence 1656. In some cases, the cell, cell nucleus, or cell bead 1600 can be contacted with the feature binding group 1652 and fixed, for example as a supplement or alternative to a fixation and permeabilization operation prior to contacting.

[0174] In some cases, the analysis of intracellular and / or intranuclear protein and membrane protein of cell (or nucleus) can be performed.In one embodiment, permeabilized (and optionally fixed) cell (or nucleus) can be contacted with the following characteristic binding group: (i) is configured to be coupled to one or more characteristic binding groups (or labeling agents) of intracellular protein (or intranuclear protein), and / or (ii) is configured to be coupled to one or more characteristic binding groups (or labeling agents) of cell membrane protein (or nuclear membrane protein).As further described herein, permeabilization can be related to partially or completely dissolving or destroying cell membrane (or nuclear membrane) or a part thereof.Permeabilization can be realized by, for example, contacting cell membrane (or nuclear membrane) with organic solvent (for example, methanol) or detergent (such as Triton X-100 or NP-40).As described elsewhere herein, cell, nucleus or cell bead can be fixed.

[0175] See again Fig.16A , a second characteristic binding group (or labeling agent) similar to 1652 (not shown) can be used to be coupled to intracellular features, such as intracellular proteins, and includes or is coupled to a second reporting oligonucleotide, which can include a second reporting sequence. The second reporting sequence can indicate a second characteristic binding group or intracellular features. For example, the second reporting sequence can be pre-indexed or assigned to a specific antibody or other characteristic binding group so that the presence of the second reporting sequence indicates the presence of a specific intracellular feature in the sample. The second characteristic binding group or the second reporting oligonucleotide can also include or be coupled to a second characteristic probe binding sequence similar to 1656.

[0176] The cell, cell nucleus or cell bead 1600 can be contacted with a first probe 1606, a second probe 1616 and a third probe 1658 under conditions sufficient to produce a first probe-associated molecule (or a probe-associated complex) 1630 and a second probe-associated molecule (or a probe-associated complex) 1665. As described elsewhere herein, the first probe-associated molecule 1630 can be or include a probe-linked molecule. For example, the first probe-associated molecule 1630 (or a probe-linked molecule) can be any probe-associated molecule or probe-linked molecule described herein (e.g., a molecule generated from an extended probe, an extended probe with a barcode, etc.). The first probe 1606 can include a first probe sequence 1608 and an optional probe capture sequence 1610. The first probe sequence 1608 can be complementary to the first target region 1602. The second probe 1616 can include a second probe sequence 1615 and an optional probe capture sequence 1618. The second probe sequence 1615 can be complementary to the second target region 1604. The third probe 1658 may include a third probe sequence 1660 and a probe capture sequence 1662. The third probe sequence 1660 may be complementary to the characteristic probe binding sequence 1656. In some cases, 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.

[0177] In one embodiment, the cell, cell bead or cell nucleus 1600 may be further contacted with an additional probe under conditions that produce an additional probe-associated molecule or a probe-associated complex. As described elsewhere herein, the additional probe-associated molecule may be or include a probe-attached molecule. For example, the additional probe-associated molecule or the probe-attached molecule may be any probe-associated molecule or probe-attached molecule described herein (e.g., a molecule generated from an extended probe, an extended probe with a barcode, etc.). In one embodiment, the cell (or cell bead or cell nucleus) 1600 may be further contacted with a fourth probe (not shown) similar to 1658, the fourth probe comprising (i) a fourth probe sequence similar to 1660 and (ii) a fourth probe capture sequence similar to 1662. As further described herein, the fourth probe sequence may be complementary to the second characteristic probe binding sequence. In some cases, the fourth probe capture sequence is a probe capture sequence that is identical to the probe capture sequence 1610, 1618 of the first probe and / or the second probe, respectively.

[0178] In one embodiment, cells, cell nuclei or cell beads 1600 can be separated into the first partition of the first set of partitions prior to any of the above-mentioned processing operations, including but not limited to fixation, permeabilization, contact with probes, and production of probe-associated or probe-linked molecules. In another embodiment, cells, cell nuclei or cell beads 1600 can be fixed and optionally permeabilized before separation in the first partition, and then subsequently processed in the first partition, for example, contact with probes and production of molecules.

[0179] In operation 1670, the cell, cell nucleus or cell bead 1600 containing the first probe-associated molecule 1630 and the second probe-associated molecule 1665 can be separated into the first partition of the first group of partitions, or further processed in the first partition. In another embodiment, the cell, cell bead or cell nucleus 1600 can also contain additional probe-associated molecules or complexes. For example, see Fig.16A, 1600 may include a complex (not shown) associated with a third probe similar to 1665, but including (i) a fourth probe having a fourth probe sequence complementary to the second characteristic probe binding sequence, and (ii) a reporter oligonucleotide (similar to 1657), as further described herein. The reporter oligonucleotide may be provided as a part of a second characteristic binding group or coupled to a second characteristic binding group, which is, for example, a characteristic binding group configured to be coupled to an intracellular protein. In some cases, cells, cell nuclei or cell beads 1600 may be processed in the partition, such as cracking, to release cell / nuclear components (e.g., molecules associated with the first probe and molecules associated with the second probe) in the partition. Alternatively, cells, cell nuclei or cell beads 1600 may remain intact. In one embodiment, cell beads are processed to release cell components while keeping cell beads intact. In the first partition, probe binding molecules 1617 and barcode molecules 1619 may be provided. The first probe-associated molecule 1630 and the second probe-associated molecule 1665 can be contacted with one or more probe-binding molecules 1617 and barcode molecules 1619. In some examples, the first partition also contains one or more additional probe-associated molecules or complexes similar to 1665 (not shown). The additional probe-associated complex can include the above-mentioned third probe-associated complex, which includes a fourth probe and a reporter oligonucleotide for a second characteristic binding group, which is, for example, a characteristic binding group configured to be coupled to an intracellular protein. The additional probe-associated complex (such as the third probe-associated complex) can be contacted with one or more probe-binding molecules 1617 and barcode molecules 1619. In one embodiment, the contact of the cells, nuclei or cell beads 1600 in the first partition with the one or more probe-binding molecules can be carried out simultaneously with the contact of the probes (e.g., 1606, 1616, 1658 and the optional fourth probe) as described above. The barcode molecule 1619 may include a barcode capture sequence or a common sequence shared by a plurality of barcode molecules and a first barcode sequence shared by a first partition of the first group of partitions. In some cases, the nucleic acid barcode molecule may be coupled to a bead, such as a gel bead or other support as described herein, and may include additional functional sequences, including but not limited to a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., an R1 / R2 sequence), additional barcode sequence fragments, etc. The probe binding molecule 1617 may include a probe binding sequence complementary to any one or a combination of the probe capture sequences 1610, 1618, 1662, a fourth probe capture sequence, and a barcode binding sequence complementary to the common sequence of the barcode molecule 1619. In some cases, the probe binding molecule 1617 and the barcode molecule 1619 may be provided as a pre-annealed complex.The probe binding molecule 1617 and the barcode molecule 1619 can hybridize with the first probe associated molecule 1630 and the second probe associated molecule 1665 and / or an additional probe associated complex (such as a third probe associated complex) (e.g., via hybridization of the probe binding molecule 1617 with 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 can occur within the first partition, for example, the connection of the barcode molecule 1619 to the probe (1606, 1616, 1658 or the fourth probe). In an additional embodiment, additional probe associated complexes (e.g., a third probe associated complex (not shown)), the probe binding molecule 1617 and the barcode molecule 1619 are used to generate additional barcoded nucleic acid molecules.

[0180] In operation 1680, the contents of each partition or a subset of the first group of partitions can be collected from the first group of partitions, such as from operation 1670, and re-separated into the second group of partitions. The contents of the first group of partitions can include cells, cell nuclei or cell beads 1600 and / or processed cell or cell nuclear components, such as the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and the optional additional barcoded nucleic acid molecule. The contents of each partition of the first group of partitions can be merged together and redistributed to the second group of partitions. Therefore, the second partition of the second group of partitions can include cells, cell nuclei or cell beads 1600 and / or processed cell / cell nuclear components. In some cases, cells, cell nuclei or cell beads 1600 can be subjected to treatment in the second partition, such as lysis, to release cell / cell nuclear components (e.g., the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and the optional additional barcoded nucleic acid molecule) in the second partition. Alternatively, the cell, cell nucleus or cell bead 1600 can remain intact. Within the second partition, a plurality of capture molecules 1620 can be provided. In some cases, the plurality of capture molecules 1620 can be coupled to a support (e.g., a particle, a bead, a gel bead, etc.). In some cases, the plurality of capture molecules 1620 can be releasably coupled to a support, and the plurality of capture molecules 1620 can be released in the second partition. The capture molecules 1620 can each contain a second barcode sequence, which can be a sequence identical to or different from the first barcode sequence (of the barcode molecule 1619). The second barcode sequence can be specific to the second partition and different from the second barcode sequence of the other partitions of the second group of partitions. The first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule can each be contacted with the capture molecule 1620. The capture molecule 1620 can contain a second barcode capture sequence, which can be complementary to the sequence of the barcode molecule 1619. The hybridization of the capture molecule 1620 with the first barcoded molecule and the second barcoded nucleic acid molecule may be sufficient to produce a third barcoded nucleic acid molecule and a fourth barcoded nucleic acid molecule. In addition, the hybridization of the capture molecule 1620 with an additional barcoded nucleic acid molecule (e.g., an additional reporter oligonucleotide 1657 from an additional feature binding group 1652) may be sufficient to produce a fifth barcoded nucleic acid molecule. Alternatively, the hybridization of the capture molecule 1620 with the first barcoded molecule and the second barcoded nucleic acid molecule may be sufficient to couple the capture molecule (containing the second barcode sequence) to both the first barcoded molecule and the second barcoded nucleic acid molecule. In addition, the hybridization of the capture molecule 1620 with the additional barcoded nucleic acid molecule may be sufficient to couple the capture molecule (containing the second barcode sequence) to the additional barcoded nucleic acid molecule.Optionally, further processing can be performed, for example, the capture molecule 1620 is connected to the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule (and optionally additional barcoded nucleic acid molecules). After connection, the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule can include the capture molecule 1620. The third barcoded nucleic acid molecule, the fourth barcoded nucleic acid molecule, and the fifth barcoded nucleic acid molecule can each include a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence. In some cases, an extension reaction (e.g., from the capture molecule 1620 toward the reporter oligonucleotide sequence 1657) is performed to produce a fourth barcoded molecule and / or a fifth barcoded nucleic acid molecule. Fig. 16B Another example workflow for barcoding multiple analytes of cells, cell nuclei, or cell beads is schematically shown. In such an example, the workflow for processing nucleic acid molecules (e.g., RNA molecules) can be substantially similar to Fig.16A The workflow depicted in , but the workflow for processing features (e.g., proteins) can be different. For example, the feature binding group 1652 or the reporter oligonucleotide 1657 can include a binding sequence that can hybridize with the probe binding molecule 1617 and / or the barcode molecule 1619.

[0181] As described herein, permeabilized (and optionally fixed) cells or cell nuclei can be contacted with one or more characteristic binding groups 1652, which can (a) contain a 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 a nuclear membrane protein). In some embodiments, the one or more characteristic binding groups 1652 include (i) a first characteristic binding group, which contains a reporter oligonucleotide 1657 and is configured to couple to an intracellular protein (or an intranuclear protein); and (ii) a second characteristic binding group, which contains a reporter oligonucleotide 1657 and is configured to couple to a cell membrane protein (or a nuclear membrane protein).

[0182] In operation 1670, the cell, cell nucleus or cell bead 1600 containing the first probe-associated molecule 1630 and one or more characteristic binding groups 1652 can be separated into the first partition of the first group of partitions, or further processed in the first partition. Within the first partition, a probe binding molecule 1617 and a barcode molecule 1619 can be provided. The characteristic binding group 1652 coupled to the reporter oligonucleotide 1657 (e.g., one or more characteristic binding groups configured to be coupled to intracellular proteins or intranuclear proteins) can be contacted with the one or more probe binding molecules 1617 and the barcode molecule 1619. The barcode molecule 1619 can contain a barcode capture sequence or a common sequence shared by multiple barcode molecules and a first barcode sequence shared by the first partition of the first group of partitions. In some cases, the nucleic acid barcode molecule can be coupled to a bead, such as a gel bead or other support as described herein, and can include additional functional sequences, including but not limited to a unique molecular identifier (UMI), a capture sequence, a primer sequence (e.g., an R1 / R2 sequence), additional barcode sequence fragments, etc. The probe binding molecule 1617 can include a probe binding sequence that is complementary to the sequence of the reporter oligonucleotide 1657. In some cases, the probe binding molecule 1617 and the barcode molecule 1619 can be provided as a pre-annealed complex. The probe binding molecule 1617 and the barcode molecule 1619 can hybridize with the first probe-associated molecule 1630 (as described above) and the reporter oligonucleotide 1657 (e.g., via the probe binding molecule 1617 hybridizing with the 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 can 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 can occur within the first partition, for example, the connection of barcode molecules 1619 to probes (1606, 1616) or reporter oligonucleotides 1657.

[0183] In operation 1680, the contents of each partition or a subset of the first group of partitions can be collected from the first group of partitions, such as from operation 1670, and re-separated into the second group of partitions. The contents of the first group of partitions can include cells, cell nuclei or cell beads 1600 and / or processed cell / nuclear components, such as the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and the optionally additional barcoded nucleic acid molecule. The contents of each partition of the first group of partitions can be merged together and redistributed to the second group of partitions. Therefore, the second partition of the second group of partitions can include cells, cell nuclei or cell beads 1600 and / or processed cell / nuclear components (e.g., products with barcodes). In some cases, cells, cell nuclei or cell beads 1600 can be treated in the second partition, such as lysis, to release cell / nuclear components (e.g., the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and the optionally additional barcoded nucleic acid molecule) in the second partition. Alternatively, the cell, cell nucleus or cell bead 1600 can remain intact. Within the second partition, a plurality of capture molecules 1620 can be provided. In some cases, the plurality of capture molecules 1620 can be coupled to a support (e.g., a particle, a bead, a gel bead, etc.). In some cases, the plurality of capture molecules 1620 can be releasably coupled to a support, and the plurality of capture molecules 1620 can be released in the second partition. The capture molecules 1620 can each contain a second barcode sequence, which can be a sequence identical to or different from the first barcode sequence (of the barcode molecule 1619). The second barcode sequence can be specific to the second partition and different from the second barcode sequence of the other partitions of the second group of partitions. The first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule can each be contacted with the capture molecule 1620. The capture molecule 1620 can contain a second barcode capture sequence, which can be complementary to the sequence of the barcode molecule 1619. Alternatively, capture molecule 1620 can include a sequence complementary to an additional probe binding molecule (e.g., a splint oligonucleotide, not shown), and the probe binding molecule can include a sequence complementary to the sequence of barcode molecule 1619. Hybridization of capture molecule 1620 with the first barcoded molecule and the second barcoded nucleic acid molecule (or with an additional probe binding molecule that can hybridize with the first barcoded molecule and the second barcoded molecule) can be sufficient to produce a third barcoded nucleic acid molecule and a fourth barcoded nucleic acid molecule. In addition, hybridization of 1620 with an additional barcoded nucleic acid molecule (e.g., an additional reporter oligonucleotide 1657 from an additional feature binding group 1652) can be sufficient to produce a fifth barcoded nucleic acid molecule.Alternatively, hybridization of capture molecule 1620 with 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 with an additional barcoded nucleic acid molecule may be sufficient to couple the capture molecule (comprising the second barcode sequence) to an additional barcoded nucleic acid molecule (e.g., generated by an additional reporter oligonucleotide 1657 on an additional feature binding group 1652). Optionally, further processing may be performed, for example, an extension reaction may be performed to connect capture molecule 1620 to the first barcoded nucleic acid molecule, the second barcoded nucleic acid molecule, and the optional additional barcoded nucleic acid molecule. After connection, the first barcoded nucleic acid molecule and the second barcoded nucleic acid molecule may comprise capture molecule 1620. The third barcoded nucleic acid molecule, the fourth barcoded nucleic acid molecule, and the fifth barcoded nucleic acid molecule can each comprise a sequence corresponding to the first barcode sequence and a sequence corresponding to the second barcode sequence. In some cases, an extension reaction (e.g., from capture molecule 1620 toward reporter oligonucleotide sequence 1657) is performed to generate the fourth barcoded molecule and / or the fifth barcoded nucleic acid molecule.

[0184] In some cases, a reporter oligonucleotide (comprising a reporter sequence) of a characteristic binding group can be contacted with a plurality of probes. For example, it may be advantageous to contact a characteristic binding group with a pair of probes. In some cases, the reporter oligonucleotide comprises one or more characteristic probe binding sequences, which may comprise sequences complementary to the pair of probes. For example, see Fig.17, the cell, cell nucleus or cell bead 1700 can include a feature (e.g., a protein, such as a cell / nuclear membrane protein or an intracellular / intranuclear protein) 1750. A feature binding group 1752 can be coupled to the feature 1750. The feature binding group 1752 can include or be coupled to an oligonucleotide comprising a reporter oligonucleotide (comprising a reporter sequence) 1754, and in some cases, include or be coupled to an additional functional sequence, such as a primer sequence, a sequencing primer sequence, a UMI, etc., as described elsewhere herein. The reporter oligonucleotide 1754 can include any number of target regions. For example, the reporter oligonucleotide 1754 can include two target regions, and the first probe 1757 and the second probe 1758 can hybridize to the two target regions. The two target regions can be adjacent or non-adjacent, and they can be arranged on the same chain of the reporter oligonucleotide 1754. As described herein, the probe can include a sequence complementary to the target region of the reporter oligonucleotide 1754, and each probe can include other useful sequences. For example, a probe (e.g., a first probe 1757 or a second probe 1758) can include (i) a probe sequence (e.g., 1760) that is complementary to the target region of a reporter oligonucleotide 1754, and (ii) a probe capture sequence 1762 that can be complementary to the sequence of a probe binding molecule 1717 (also referred to as a splint or splint oligonucleotide). The probe binding molecule 1717 can also include a sequence that is complementary to the sequence of a barcode molecule 1719 (e.g., a capture sequence). This barcoding (e.g., hybridization of the probe binding molecule 1717 and the barcode molecule 1719 with the probe capture sequence 1762) can occur in the body or in a partition. In some embodiments, barcoding can be performed without a probe binding molecule. For example, the barcode molecule 1719 can include a sequence that is complementary to the probe capture sequence 1762 and anneal directly to the probe.

[0185] In some cases, after the characteristic binding group is contacted with the probe molecules 1757 and 1758 (for example, in the body or in a partition), the characteristic binding group 1752 is subjected to conditions sufficient to hybridize the probe molecules with the reporter oligonucleotide 1754, thereby generating a probe-associated reporter oligonucleotide complex. The coupling of the probe to the reporter oligonucleotide 1754 can occur in the body or in a partition. In some cases, after the probe is coupled or hybridized with the reporter oligonucleotide 1754, the probes can be connected together (for example, enzymatically or chemically) to generate a nucleic acid molecule (or complex) connected to the probe. For example, the first probe 1757 can include a first reactive portion, and the second probe 1758 can include a second reactive portion. The reactive portion can be positioned so that after the first probe 1757 and the second probe 1758 are hybridized with the reporter oligonucleotide 1754, the reactive portion is adjacent. Then, the reactive part can be subjected to conditions sufficient to react them to produce a probe-connected nucleic acid molecule (or complex) comprising a first probe 1757 connected to a second probe 1758. In some cases, the probe comprises a "click chemistry" portion. Alternatively or in addition, the first probe can be enzymatically connected (e.g., via a ligation reaction) to the second probe. In other cases, after the probe is hybridized with the reporter oligonucleotide 1754, a gap region (not shown) can be set between the first probe 1757 and the second probe 1758. In such cases, a gap filling method (such as those described above) can be used to connect the first probe 1757 to the second probe 1758.

[0186] The nucleic acid molecules (or complexes) to which the probe is then connected can be subjected to barcoding (e.g., contact with probe binding molecules 1717 and barcode molecules 1719), which can occur in a partition. Alternatively, barcoding can occur before the probe is connected. For example, the reporter oligonucleotide 1754 can be hybridized with the probe, separated, barcoded, and then the probe can be connected. Alternatively, the reporter oligonucleotide 1754 can be hybridized with the probe, connected, separated, and then barcoded. In another example, the reporter oligonucleotide 1754 can be hybridized with the probe, separated, connected, and then barcoded. It should be understood that the operations described herein (e.g., hybridization, probe connection, barcoding) can occur in any useful process, or in any useful order. In some cases, multiple separation operations can be performed, for example, for combined barcoding.

[0187] The reporter oligonucleotide can comprise the same target sequence as the nucleic acid molecule (e.g., RNA molecule) (e.g., 702, 704, 802, 804, 902, 904, 1502, 1504, 1602, 1604, etc.). For example, see Fig.17, the first probe can have a first sequence that is complementary to both the first target sequence of the nucleic acid molecule (e.g., 702, 802, 902, 1502, 1602) and the first sequence of the reporter oligonucleotide 1754, and the second probe can have a second sequence that is complementary to both the second target sequence of the nucleic acid molecule (e.g., 704, 804, 904, 1504, 1604) and the second sequence of the reporter oligonucleotide 1754. In such a case, providing only two probe types (e.g., the first probe and the second probe) to the cell, cell nucleus, or cell bead can be sufficient to produce a first barcoded molecule (e.g., produced from a nucleic acid molecule such as an RNA molecule), a second barcoded molecule (e.g., produced from a reporter oligonucleotide with a characteristic binding group (such as a group configured to couple to a cell / nuclear membrane protein)), and an additional barcoded molecule (e.g., produced from a reporter oligonucleotide with an additional characteristic binding group (such as a group configured to couple to an intracellular / intranuclear protein)). As described herein, each probe (e.g., a first probe and a second probe) may be capable of or configured to hybridize with a barcode molecule (e.g., in a first partition) and / or a capture molecule. Also as described elsewhere herein, each probe may be multiplexed or combinatorially barcoded so that multiple body partitions (e.g., partitions containing more than one cell, one cell nucleus, or cell bead) can be deconvoluted, for example, to determine the starting partition or sample for each cell, cell nucleus, or cell bead within a partition (see, e.g., Fig.10 ). Similarly, barcoded molecules can be used to determine the origin of different analyte types (e.g., proteins, nucleic acid molecules, etc.); for example, two analyte types can be attributed to the same starting cell, cell nucleus, cell bead, sample or partition.

[0188] In some cases, the reporter oligonucleotide comprises two or more target sequences different from the target sequence of the nucleic acid molecule (e.g., RNA molecule). Therefore, four probe types can be provided for multiple determination; the first probe and the second probe can hybridize with the first target region and the second target region of the nucleic acid molecule, and the third probe and the fourth probe can hybridize with the target region of the reporter oligonucleotide (e.g., a reporter oligonucleotide from a characteristic binding group (such as a characteristic binding group configured to be coupled to a cell / nuclear membrane protein)). Additional probe types, such as the fifth probe and the sixth probe, can be provided, which hybridize with the target region of the additional reporter oligonucleotide (e.g., a reporter oligonucleotide from a characteristic binding group (such as a characteristic binding group configured to be coupled to an intracellular / nuclear protein)). Each probe or a combination of probes can include a probe capture sequence, which can be used for subsequent barcode addition. For example, each probe (e.g., the first probe, the second probe, the third probe, the fourth probe, the fifth probe, the sixth probe, or a combination thereof) may be capable of or configured to hybridize with a barcode molecule (e.g., in the first partition) and / or a capture molecule (e.g., in the second partition). As described elsewhere herein, each probe can be multiplexed or combinatorially barcoded so that multiple body partitions (e.g., partitions containing more than one cell, cell nucleus, or cell bead) can be deconvoluted, for example to determine the starting partition or sample for each cell, cell nucleus, or cell bead within a partition (see, e.g., Fig.10 ). Similarly, barcoded molecules can be used to determine the origin of different analyte types (e.g., proteins, nucleic acid molecules, etc.); for example, two analyte types can be attributed to the same starting cell, cell nucleus, cell bead, sample or partition.

[0189] As described elsewhere herein, nucleic acid molecules (e.g., from cells, nuclei or cell beads or reporter oligonucleotides) can include one or more target regions. One or more target regions can correspond to a gene or a portion thereof, or another known sequence. The target region can have the same or different sequences and can be located in the same chain or on different chains. The target regions can be adjacent to each other, or can be separated spatially along a chain of nucleic acid molecules. The target region can be located on the same chain or on different chains. Analyzing two or more target regions can involve providing two or more probes, wherein the first probe has a sequence complementary to the first target region, the second probe has a sequence complementary to the second target region, and the like. As described elsewhere herein, nucleic acid molecules can be target nucleic acid molecules, and can include any number of nucleic acid features or nucleotides.

[0190] As also described elsewhere herein, any probe (e.g., a first probe, a second probe, a third probe, etc.), a reporter oligonucleotide, or a barcode or a capture molecule can contain any number of additional linkers or functional sequences, such as additional probe sequences, unique molecular identifiers, barcode sequences, primer sequences, capture sequences, sequencing primer sequences, and the like.

[0191] As described herein, one or more operations can be performed in a partition (such as a droplet or a hole). For example, nucleic acid molecules (e.g., RNA molecules) and features (e.g., proteins) or cells, nuclei or cell beads comprising nucleic acid molecules and features can be separated together with one or more reagents (e.g., as described herein) at any useful stage of the method. For example, nucleic acid molecules optionally included in or on probes connected to or probe-associated nucleic acid molecules in cells, nuclei or cell beads can be produced in a bulk solution or in a partition. Similarly, cells, nuclei or cell beads can be contacted with feature binding groups in a bulk solution or a partition. The provision of probes (e.g., the first probe, the second probe and the third probe) can occur in a bulk solution or in a separate partition. In the case of using partitions, a partition (e.g., the first partition of the first group of partitions) can include the first probe, the second probe, the third probe or a combination thereof. Different partitions in the first group of partitions can include the same or different probes (e.g., for different target sequences or different reporter sequences). Alternatively or in addition, probe binding molecules and nucleic acid barcode molecules can be provided in a partition. For example, cells, cell nuclei or cell beads containing the feature and nucleic acid molecules can be contacted with the probe in the body and separated into the first group of partitions. The first group of partitions can include probe binding molecules and nucleic acid barcode molecules containing common sequences. Different partitions in the first group of partitions can include barcode molecules with different barcode sequences; for example, the additional partitions of the first group of partitions can include multiple barcode molecules, each barcode molecule has a barcode sequence that is unique to the partition (i.e., different between each partition). The partition can include additional reagents for performing nucleic acid reactions (e.g., digestion, connection, extension, amplification). For example, the partition can include a ligase (linking enzyme) (e.g., ligase (ligase)), which can be used to connect the nucleic acid barcode molecule to the first probe, the second probe or the third probe (e.g., via the probe capture sequence of each probe). In some cases, the probe binding molecule, the probe capture sequence and / or the barcode capture sequence (e.g., common sequence) include one or more reactive parts, which can be used to chemically connect the nucleic acid barcode molecule to the probe capture sequence. The resulting barcoded product can include: a first barcoded product comprising a sequence corresponding to the first target region, a sequence corresponding to the second target region, a sequence corresponding to the probe capture sequence of the first probe or the second probe, and a sequence corresponding to the barcode sequence; and a second barcoded product comprising a sequence corresponding to the reporter sequence, the probe capture sequence of the third probe (which can be the same or different from the probe capture sequence of the first probe or the second probe), and the barcode sequence.

[0192] As described herein, one or more processes described herein can be performed in cells (e.g., cells in solution, or cells contained in a tissue sample), cell nuclei, or cell beads. For example, multiple cells, cell nuclei, or cell beads can contain multiple nucleic acid molecules and features. Cells, cell nuclei, or cell beads can be alive or fixed and / or permeabilized. In some cases, cells, cell nuclei, or cell beads can be contacted with a feature binding group containing a reporter sequence. The first probe, the second probe, and the third probe can also be provided to cells, cell nuclei, or cell beads in a bulk solution or a partition to produce molecules associated with the first probe and molecules associated with the second probe. Optionally, cells, cell nuclei, or cell beads can be washed to remove unbound probes. Subsequently, cells, cell nuclei, or cell beads containing molecules associated with probes can be separated into multiple separate partitions, wherein at least one subset of multiple separate partitions includes a single cell, a single cell nucleus, or a single cell bead. Barcoding can be performed in separate partitions. As described herein, barcoding can include attaching or hybridizing a nucleic acid barcode molecule to a molecule associated with a first probe and a molecule associated with a second probe. The nucleic acid barcode molecules provided in each partition in a plurality of separate partitions can be provided to be attached to beads. In some cases, as described elsewhere herein, the nucleic acid barcode molecules can be releasably attached to beads (e.g., via unstable bonds). Each partition (or a subset of partitions) in a plurality of separate partitions can include beads, which contain 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 can include a unique barcode sequence, so that each partition in a plurality of separate partitions includes a different barcode sequence. When releasing components from multiple different partitions in multiple individual partitions (e.g., after barcoding), the barcoded molecules originating from a single cell, a single cell nucleus, or a single cell bead can have the same barcode sequence (e.g., a common barcode sequence), so that each barcoded nucleic acid molecule can be traced back to a given partition and / or in some cases, a single cell, a single cell nucleus, or a single cell bead. Then, as described herein, the released components can be separated into a second set of partitions including capture molecules having a second barcode sequence, so that different partitions in the second set of partitions have unique second barcode sequences.

[0193] Cells, nuclei or cell beads as described herein can be processed before, during or after barcoding. For example, cells, nuclei or cell beads can be fixed or permeabilized at any useful time point. In some cases, cells, nuclei or cell beads can be fixed and permeabilized before or after probe hybridization or before or after contacting with a characteristic binding group. In some cases, cells, nuclei or cell beads can be fixed and permeabilized before contacting with a characteristic binding group, and then cells, nuclei or cell beads are contacted with a probe. Fixing or permeabilization processes can be repeated. For example, cells, nuclei or cell beads can be fixed and permeabilized, cells, nuclei or cell beads are contacted with probes and characteristic binding groups (simultaneously or in a stepwise manner), and then cells, nuclei or cell beads are fixed again.

[0194] After fixation and / or permeabilization, the cells, cell nuclei or cell beads can be stored for a period of time before further processing, for example, contacting the cells, cell nuclei or cell beads with probes and / or feature binding groups. For example, the cells, cell nuclei or cell beads can be fixed and / or permeabilized, and then contacting the cells, cell nuclei or cell beads with probes and / or feature binding groups after about 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours or more. Can fix and / or permeabilize cell, cell nucleus or cell bead, then make cell, cell nucleus or cell bead contact with probe and / or feature binding group after about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or longer time.Can fix and / or permeabilize cell, cell nucleus or cell bead, then make cell, cell nucleus or cell bead contact with probe and / or feature binding group after about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 20 weeks, 30 weeks, 40 weeks, 50 weeks or longer time.Can fix and / or permeabilize cell, cell nucleus or cell bead, then make cell, cell nucleus or cell bead contact with probe and / or feature binding group after about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer time. The cells, cell nuclei or cell beads can be fixed and / or permeabilized and then contacted with the probe and / or feature binding group at any useful time, which can be within a certain time range, for example, after about 2-5 weeks, after about 3-6 months, after about 1-2 years, etc.

[0195] In some cases, for example, after fixing and / or permeabilization, cells, nuclei or cell beads can be frozen. This freezing of cells, nuclei or cell beads can be used for longer durations of sample storage (for example, if before sample contacts with probe and / or feature binding group, sample storage is to be more than 1-2 weeks). For example, cells, nuclei or cell beads can be fixed, optionally permeabilized, then frozen any useful duration, and then cells, nuclei or cell beads are contacted with probe and / or feature binding group. Alternatively, before or after cells, nuclei or cell beads contact with probe and / or feature binding group, cells, nuclei or cell beads can be fixed, frozen and permeabilized. It should be understood that freezing operation can be carried out at any useful or convenient time, for example, before, while or after fixing, permeabilization, contacting with probe, contacting with feature binding group, etc.

[0196] Cells, nuclei or cell beads can be contacted with probes and feature binding groups in partitions or in body at any useful time. For example, cells, nuclei or cell beads can be contacted with probes before, during or after contacting with feature binding groups. Contact with probes and / or feature binding groups can occur in body or partitions (e.g., droplets, holes). In some cases, cells, nuclei or cell beads can be contacted with probes and feature binding groups (simultaneously or in a stepwise manner), and then barcodes are added in partitions. In other cases, cells, nuclei or cell beads can be contacted with probes and feature binding groups in partitions.

[0197] Fig.29 An example workflow for processing cells according to the methods described herein is shown. Cells can be fixed, for example, in 4% formaldehyde and 0.01% Tween-20 or a commercially available fixation and permeabilization buffer (e.g., commercially available The cells can be fixed and permeabilized in a fixation and permeabilization buffer. In one example, the fixed and permeabilized cells can be incubated with the first probe and the second probe to produce a molecule associated with the first probe (e.g., a probe-associated RNA molecule). The cells can then be contacted with a feature binding group (e.g., an antibody) comprising a reporter oligonucleotide to produce cells comprising a feature coupled to the feature binding group. Subsequent barcoding can be performed, for example, in partitions.

[0198] In some examples, the fixed and permeabilized cells can be incubated with the characteristic binding group, optionally fixed again, and then contacted with the first probe and the second probe to produce a probe-associated molecule (e.g., a probe-associated RNA molecule). Alternatively, the fixed and permeabilized cells can be incubated with the first probe and the second probe to produce a probe-associated molecule, and then contacted with the characteristic binding group. Subsequent barcoding can be performed, for example, in partitions.

[0199] In some cases, permeabilization cells may be useful (for example, as a negative control) before contacting cells with probes or feature binding groups. Therefore, cells can be fixed, the cells are contacted with probes and / or feature binding groups, and then the cells are permeabilized. It should be understood that any order of operations such as fixing, permeabilization, probe hybridization, contacting with feature binding groups can be performed in any convenient or useful step and in any order, and any process can be repeated. For example, cells, nuclei or cell beads can be contacted with feature binding groups, fixed and / or permeabilized cells, nuclei or cell beads, cells, nuclei or cell beads are contacted with additional feature binding groups (this may be useful for analyzing extracellular and intracellular peptides, polypeptides or proteins), and optionally, fixed cells, nuclei or cell beads again. Alternatively, cells, nuclei or cell beads can be fixed and / or permeabilized, then cells, nuclei or cell beads are contacted with feature binding groups (for example, for intracellular and / or extracellular analytes), and optionally fixed cells, nuclei or cell beads again. Before or after these processes, the cells, cell nuclei or cell beads can be contacted with a probe set (eg, a first probe, a second probe and / or a third probe). See also Examples 8 and 9.

[0200] The methods, compositions, kits and systems of the present disclosure may include providing methods for treating fixed biological particles (e.g., cells, cell nuclei or cell beads). In one embodiment, the method includes a) fixing and permeabilizing biological particles or providing fixed and permeabilized biological particles.

[0201] The method can also include b) contacting the fixed and permeabilized bioparticles with a first reagent, which is configured to be coupled to the analyte of the bioparticles. In one embodiment, the analyte is an intracellular analyte, such as a nucleic acid or a polypeptide, and the bioparticles are cells. In another embodiment, the analyte is an intranuclear analyte, such as a nucleic acid or a polypeptide, and the bioparticles are nuclei. The first reagent configured to be coupled to the analyte can be (i) configured to be coupled to the first reagent of nucleic acid (such as one or more nucleic acid probes as described herein) or (ii) configured to be coupled to the first reagent of a peptide, polypeptide or protein (such as one or more characteristic binding groups as described herein). In another embodiment, b) provides fixed and permeabilized bioparticles, such as cells or nuclei, which include the first reagent coupled to the analyte (for example, nucleic acid or polypeptide) of the bioparticles.

[0202] The method may also include c) additional fixation of the biological particles from b). In one embodiment, c) includes additional fixation of the biological particles from b), wherein the biological particles from b) include a first reagent configured to be coupled to the analyte of the biological particles. The first reagent can be coupled to the analyte (nucleic acid or polypeptide) of the biological particles (e.g., cells or cell nuclei). The first reagent can be a reagent configured to be coupled to a nucleic acid analyte or a reagent configured to be coupled to a polypeptide. In one embodiment, c) includes additional fixation of biological particles (such as cells), wherein the cells include a first reagent coupled to a polypeptide. In another embodiment, the polypeptide is an intracellular polypeptide.

[0203] The method may also include: d) comprising contacting the biological particles (e.g., cells or cell nuclei) from c) (which have been initially fixed and permeabilized, contacted with a first reagent or containing a first reagent, and additionally fixed) with a second reagent configured to couple to an analyte (e.g., a nucleic acid or a polypeptide), wherein the second reagent is different from the first reagent, and / or the second reagent is configured to couple to an analyte different from the analyte to which the first reagent is configured to couple. In one embodiment, the first reagent is configured to couple to a polypeptide (such as one or more characteristic binding groups as described herein), and the second reagent is configured to couple to a nucleic acid (such as one or more nucleic acid probes as described herein). The biological particles of d) may include a first reagent coupled to a polypeptide and a second reagent coupled to a nucleic acid.

[0204] Any number of barcoding operations can be performed for a given nucleic acid molecule and / or feature binding group using, for example, a combined barcoding (e.g., split pool) method. As described herein, additional barcoding operations can be used, for example, to index nucleic acid molecules and features (e.g., proteins) to cells, cell nuclei, cell beads, samples, a partition or multiple partitions. This index can be used for situations where a single partition is occupied by multiple cells, cell nuclei, or cell beads. In some cases, it may be advantageous to overload a partition so that a partition includes more than one cell, cell nucleus, or cell bead; for example, in some cases it may be useful to overload a partition, for example, to overcome Poisson load statistics in a partition and / or to prevent reagent waste (e.g., caused by unoccupied partitions). Therefore, this index can be used to attribute (i) nucleic acid molecules and (ii) features (e.g., proteins) in multiple occupied partitions to starting cells, cell nuclei, cell beads, partitions, samples, etc., as described elsewhere herein.

[0205] For example, nucleic acid molecules and features (e.g., proteins) within a population of cells, cell nuclei, or cell beads can be subjected to Fig.10In such an example, prior to operation 1010, a first cell, cell nucleus, or cell bead population 1002 may be contacted with a first probe, a second probe, and an optional third probe (e.g., Fig.16A and Fig. 16BAs shown). The first probe and the second probe can hybridize with the nucleic acid molecule to produce a molecule (or complex) associated with the first probe, and optionally, the third probe can hybridize with a characteristic probe binding sequence of a reporter oligonucleotide (comprising a reporter sequence) or a characteristic binding group (e.g., a group configured to be coupled to a cell / nuclear membrane protein) to produce a molecule (or complex) associated with the second probe. Additional probes can be provided to hybridize with additional reporter oligonucleotides or characteristic probe binding sequences of additional characteristic binding groups (e.g., a group configured to be coupled to an intracellular / intranuclear protein) of the first cell, cell nucleus, or cell bead population to produce molecules associated with additional probes. The second cell, cell nucleus, or cell bead population 1004 can also be treated in the same manner, for example, with a fourth probe, a fifth probe, and an optional sixth probe. The fourth probe and the fifth probe can be hybridized with the nucleic acid molecules of the second cell, cell nucleus or cell bead population to produce a molecule associated with the third probe, and optionally, the sixth probe can be hybridized with the reporter oligonucleotide or characteristic probe binding sequence of the characteristic binding group of the second cell, cell nucleus or cell bead population to produce a molecule associated with the fourth probe. Additional probes can be provided to hybridize with additional reporter oligonucleotides or characteristic probe binding sequences of additional characteristic binding groups (e.g., groups configured to be coupled to intracellular / intranuclear proteins) of the second cell, cell nucleus or cell bead population to produce molecules associated with the additional probes. As described herein, a first cell (or cell nucleus or cell bead) population 1002 and a second cell (or cell nucleus or cell bead) population 1004 may be barcoded with a first barcode sequence, such that the first cell (or a component therein, such as a molecule associated with a first probe and a molecule associated with a second probe) population 1002 has a first barcode sequence different from that of the second cell (or cell nucleus or cell bead or a component within a cell, cell nucleus or cell bead, such as a molecule associated with a third probe and a molecule associated with a fourth probe) population 1004. In operation 1020, the first cell (or cell nucleus or cell bead) population 1002 and the second cell (or cell nucleus or cell bead) population 1004 may be merged together to produce a mixture of cells (or cell nuclei or cell beads). In operation 1030, the mixture of cells (or cell nuclei or cell beads) may be partitioned into a second plurality of partitions. In some cases, the mixture of cells (or cell nuclei or cell beads) can be separated into the second plurality of partitions so that some of the second plurality of partitions include more than one cell (e.g., cell, cell nuclei or cell bead multimer partitions). For example, partition 1035 in the second plurality of partitions can include cells, cell nuclei or cell beads from the first cell (or cell nuclei or cell beads) population 1002 ("cell A") and cells, cell nuclei or cell beads from the second cell (or cell nuclei or cell beads) population 1004 ("cell B").Partition 1035 may include an additional barcode sequence that may be specific to the partition. The cells (or cell nuclei or cell beads) in each partition may be subjected to additional barcoding operations to add additional barcode sequences to the barcoded nucleic acid molecules. In operation 1040, different barcode sequences (e.g., a first barcode sequence, a second barcode sequence, and an additional barcode sequence) may be used to deconvolute the barcoded nucleic acid molecules to identify the starting cells, cell nuclei, or cell beads. For example, a barcoded nucleic acid molecule comprising an additional barcode sequence from partition 1035 and a first barcode sequence from the first cell population 1002 may be used to identify the barcoded nucleic acid molecule as originating from cell A. Similarly, a barcoded nucleic acid molecule comprising an additional barcode sequence from partition 1035 and a second barcode sequence from the second cell population 1004 may be used to identify the barcoded nucleic acid molecule as originating from cell B.

[0206] In some cases, a feature binding group (e.g., a feature binding group configured to couple to an intracellular / intranuclear protein and / or a feature binding group configured to couple to an intracellular / intranuclear protein) can be pre-indexed to a partition. For example, rather than having a barcode sequence that can be hybridized to a probe (e.g., a third probe) and subsequently used to identify a cell, a cell nucleus, a cell bead, or a partition, a barcode can be added (e.g., Fig.16A -B) instead of the characteristic probe binding sequence, the characteristic binding group can be provided in a partition in a pre-indexed manner, for example, using a barcode sequence that is unique to the partition. For example, after barcoding the nucleic acid molecules in the cell, the characteristic binding group can be provided in a subsequent operation of the method. For example, in a second barcoding operation (e.g., Fig.16A During operation 1680 of step 1670-B), a feature binding group may be provided and contacted with a feature 1650 of a cell, cell nucleus, or cell bead (or released from a cell, cell nucleus, or cell bead in a second partition). The feature binding group may contain or hybridize to a barcode sequence that is specific to the second partition and that differs between the second partitions. Thus, rather than using a first barcode sequence and a second barcode sequence from a first partition and a second partition, respectively, to identify a partition, cell, cell nucleus, or cell bead, the barcode sequence may be used to index the feature binding group to a specific partition and back to the starting cell or cell bead.

[0207] In other examples, the feature binding group can be indexed to the partition by directly attaching or coupling the partition-specific barcode sequence to the feature binding group, thereby avoiding the use of a third probe. In such a case, the feature binding group can include or be coupled to a reporter oligonucleotide comprising a reporter sequence and an attachment sequence, which can be used to directly attach the barcode molecule to the feature binding group. For example, the feature binding group can include a probe capture sequence (e.g., 1662), thereby eliminating the need for a third probe comprising a probe capture sequence. The probe capture sequence can then be barcoded, for example, with the first barcode sequence of the barcode molecule in the first partition and the second barcode sequence of the capture molecule in the second partition. In some cases, the attachment sequence can be used for hybridization probe binding molecules (e.g., splint molecules or splint oligonucleotides), which can be partially complementary to barcode molecules (as described herein). For example, the attachment sequence of the reporter oligonucleotide can be used for hybridization probe binding molecules, which can be hybridized (or pre-annealed) to barcode molecules, for example, in the first partition. A second barcode sequence from a capture molecule can be provided in the first partition or in a different (e.g., second) partition, which can be annealed to a portion of the first barcode molecule. In some cases, additional operations, such as extension, ligation, etc., are performed to generate a barcoded molecule comprising a sequence corresponding to the first barcode sequence, the second barcode sequence, and the reporter sequence.

[0208] After partition-based barcoding, the contents of the partitions can be combined, and the barcoded molecules can be replicated or amplified by, for example, one or more amplification reactions, which in some cases can be isothermal. The amplification reaction can include a polymerase chain reaction (PCR) and can involve the use of one or more primers or polymerases. One or more primers can include one or more functional sequences (e.g., primer sequences / primer binding sequences, sequencing primer sequences (e.g., R1 or R2), partial sequencing primer sequences (e.g., partial R1 or partial R2), sequences configured to be attached to the flow cell of a sequencer (e.g., P5 or P7, or partial sequences thereof), etc.) and can facilitate the addition of the one or more functional sequences to the extended nucleic acid molecules. Barcoded molecules or derivatives thereof can be detected via nucleic acid sequencing (e.g., as described herein).

[0209] In some aspects, the present invention provides a system that can be used to add barcodes to nucleic acid molecules. The system can include any components described herein, such as multiple partitions (e.g., droplets, holes), which can be provided in any useful form, such as microfluidic devices, porous arrays or plates, etc. In some cases, the system can include a first group of partitions and a second group of partitions. The first group of partitions can be partitions of the same or different types as the second group of partitions. For example, the first group of partitions can include micropores, and the second group of partitions can include droplets. As another example, the first group of partitions and the second group of partitions can both include droplets. The system can include nucleic acid barcode molecules, which are optionally coupled to a support (e.g., particles, beads, gel beads, etc.). In some cases, the system can include any probe described herein, such as a first probe or multiple first probes, a second probe or multiple second probes, a third probe or multiple third probes, and any useful reaction components (e.g., for performing nucleic acid reactions, such as extension, connection, amplification, etc.). The system can include one or more feature binding groups. The characteristic binding groups can be the same or different between each partition; for example, the characteristic binding group can include multiple antibodies that bind to different epitopes within a single partition, or the partition can include different characteristic binding groups that bind to different epitopes or moieties. The system can include useful reaction components, such as, in non-limiting examples, enzymes (e.g., ligases, polymerases, reverse transcriptases, restriction enzymes, etc.), nucleotide bases, etc.

[0210] Also provided herein are compositions useful for systems and methods for barcoding multiple analytes (e.g., nucleic acid molecules and proteins) (e.g., via nucleic acid molecules contained in or coupled to a characteristic binding group, such as reporter oligonucleotides). The composition may include any probe described herein. For example, the composition may include a plurality of first probes, a plurality of second probes, a plurality of third probes, and / or a plurality of first probes, a plurality of second probes, and a plurality of third probes. A probe or a group of probes may be designed to target a specific sequence or a group of specific sequences. Such probes may be designed to have the same or different sequences in different partitions. For example, a first composition may include a first probe and a second probe designed to target two regions of a first gene, and a second composition may include a first probe and a second probe designed to target two regions of a second gene, the second gene being different from the first gene. Similarly, a third probe (or probe pair) may be designed to target a reporter oligonucleotide (comprising a reporter sequence) or a region of a characteristic probe binding sequence, which region may be the same or different between each partition. The composition may include a nucleic acid barcode molecule and / or a probe binding molecule, which may optionally be provided to be coupled to a support (e.g., a particle, a bead). The composition may comprise a capture molecule, optionally coupled to a support.The composition may be part of or comprise a reaction mixture, which may comprise reaction components or reagents, such as enzymes, nucleotide bases, catalysts, and the like.

[0211] Systems and methods for sample compartmentalization

[0212] In one aspect, the systems and methods described herein provide for compartmentalization, deposition or separation of one or more particles (e.g., bioparticles, macromolecular components of bioparticles, beads, reagents, etc.) into discrete compartments or partitions (interchangeably referred to herein as partitions), wherein each partition keeps its own contents separate from the contents of other partitions. A partition can be a droplet in an emulsion or a well. A partition can include one or more other partitions.

[0213] The partition may include one or more particles. The partition may include one or more types of particles. For example, the partition of the present disclosure may include one or more biological particles and / or their macromolecular components. The partition may include one or more beads. The partition may include one or more gel beads. The partition may include one or more cell beads. The partition may include a single gel bead, a single cell bead, or both a single cell bead and a single gel bead. The partition may include one or more reagents. Alternatively, the partition may be unoccupied. For example, the partition may not include beads. The cell beads may be one or more of the biological particles and / or their macromolecular components encapsulated in a gel or polymer matrix, such as via polymerization of droplets containing biological particles and precursors capable of polymerization or gelation. A unique identifier such as a barcode may be injected into the droplet, such as via a support (e.g., a bead), before, after, or simultaneously with the droplet generation, as described elsewhere herein.

[0214] The methods and systems of the present disclosure may include methods and systems for generating one or more partitions, such as droplets. The droplets may include multiple droplets in an emulsion. In some instances, the droplets may include droplets in a colloid. In some cases, the emulsion may include a microemulsion or a nanoemulsion. In some instances, the droplets may be generated by means of a microfluidic device and / or by subjecting a mixture of immiscible phases to stirring (e.g., in a container). In some cases, a combination of the methods mentioned may be used to form droplets and / or emulsions.

[0215] Droplets can be formed by mixing and / or stirring immiscible phases to produce an emulsion. Mixing or stirring can include various stirring techniques, such as vortexing, pipetting, gently flicking the tube, or other stirring techniques. In some cases, mixing or stirring can be performed without the use of a microfluidic device. In some instances, droplets can be formed by exposing the mixture to ultrasound or ultrasonic treatment. Systems and methods for producing droplets and / or emulsions by stirring are described in International Application No. PCT / US20 / 17785, which is incorporated herein by reference in its entirety for all purposes.

[0216] A microfluidic device or platform comprising a network of microfluidic channels (e.g., on a chip) can be used to generate partitions (such as droplets and / or emulsions) as described herein. Methods and systems for generating partitions (such as droplets), methods for encapsulating biological particles, methods for increasing droplet generation flux, and various geometries, architectures, and configurations of microfluidic devices and channels are described in U.S. Patent Publication Nos. 2019 / 0367997 and 2019 / 0064173, each of which is incorporated herein by reference in its entirety for all purposes.

[0217] In some examples, individual particles can be separated into discrete partitions by introducing a flowing stream of particles in an aqueous fluid into a flowing stream or reservoir of a non-aqueous fluid such that droplets can be generated at the junction of the two streams / reservoirs (such as at the junction of a microfluidic device provided elsewhere herein).

[0218] The method of the present disclosure can include producing partitions and / or encapsulated particles, such as bioparticles, in some cases independent bioparticles, such as single cells, cell nuclei or cell beads. In some instances, reagents can be encapsulated and / or separated (e.g., separated together with bioparticles) in partitions. Various mechanisms can be used when separating independent particles. An example can include a porous membrane, and the aqueous mixture of cells can be squeezed into a fluid (e.g., a non-aqueous fluid) through the porous membrane.

[0219] Partitions can be flowable in fluid streams. Partitions can include, for example, microcapsules with external barriers around the center or core of the internal fluid. In some cases, partitions can include a porous matrix capable of entraining and / or maintaining materials in its matrix. Partitions can be droplets of the first phase in the second phase, wherein the first phase and the second phase are immiscible. For example, partitions can be droplets of aqueous fluids in a non-aqueous continuous phase (e.g., an oil phase). In another example, partitions can be droplets of non-aqueous fluids in an aqueous phase. In some examples, partitions can be provided in the form of water-in-oil emulsions or oil-in-water emulsions. A variety of different containers are described in, for example, U.S. Patent Application Publication No. 2014 / 0155295, which is incorporated herein by reference in its entirety for all purposes. An emulsion system for producing stable droplets in a non-aqueous or oil continuous phase is described in, for example, U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0220] Fluid properties (e.g., fluid flow rate, fluid viscosity, etc.), particle properties (e.g., volume fraction, particle size, particle concentration, etc.), microfluidic architecture (e.g., channel geometry, etc.), and other parameters can be adjusted to control the occupancy of the resulting partitions (e.g., the number of biological particles in each partition, the number of beads in each partition, etc.). For example, the partition occupancy can be controlled by providing a water flow at a certain concentration and / or flow rate of the particles. In order to produce a single biological particle partition, the relative flow rate of the immiscible fluid can be selected so that each partition in the partition can contain less than one biological particle on average to ensure that the occupied partitions are mainly single occupied. In some cases, a partition in a plurality of partitions can contain at most one biological particle (e.g., bead, DNA, cell or cell material). In some embodiments, various parameters (e.g., fluid properties, particle properties, microfluidic architecture, etc.) can be selected or adjusted so that most of the zones are occupied, such as allowing only a small percentage of unoccupied partitions. The flow and channel architecture can be controlled to ensure that a given number of single occupied partitions is less than a certain level of unoccupied partitions and / or less than a certain level of multi-occupied partitions.

[0221] Figure 1 An example of a microfluidic channel structure 100 for separating individual biological particles is shown. The channel structure 100 may include channel segments 102, 104, 106, and 108 connected at a channel junction 110. In operation, a first aqueous fluid 112 including suspended biological particles (or cells) 114 may be transported along the channel segment 102 into the junction 110, while a second fluid 116 immiscible with the aqueous fluid 112 may be delivered from each of the channel segments 104 and 106 to the junction 110 to produce discrete droplets 118, 120 of the first aqueous fluid 112, which flow into the channel segment 108 and flow away from the junction 110. The channel segment 108 may be fluidically coupled to an outlet reservoir in which the discrete droplets may be stored and / or harvested. The discrete droplets produced may include individual biological particles 114 (such as droplet 118). The discrete droplets produced may include more than one individual biological particle 114 (such as droplet 118). Figure 1 14 ). The discrete droplets may not contain biological particles 114 (such as droplet 120). Each discrete partition may keep its own contents (e.g., individual biological particles 114) separate from the contents of other partitions.

[0222] The second fluid 116 may comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets (e.g., inhibiting subsequent coalescence of the resulting droplets 118, 120). Examples of particularly useful separation fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0223] It should be understood that channel section as described herein can be connected to any one of a variety of different fluid sources or receiving components, including fluid components of reservoirs, pipelines, manifolds or other systems.It should be understood that microfluidic channel structure 100 can have other geometric shapes.For example, microfluidic channel structure can have more than a channel connection.For example, microfluidic channel structure can have 2,3,4 or 5 channel sections that carry particles (for example, bioparticles, cell beads and / or gel beads) respectively, and these channel sections meet at the channel connection.Fluid can be guided to flow along one or more channels or reservoirs via one or more fluid flow units.Fluid flow unit can include compressor (for example, positive pressure is provided), pump (for example, negative pressure is provided), actuator, etc. to control the flow of fluid.It is also possible or separately to control fluid via applied pressure difference, centrifugal force, electric pumping, vacuum, capillary or gravity flow, etc.

[0224] The generated droplets may include two subsets of droplets: (1) occupied droplets 118 that contain one or more biological particles 114, and (2) unoccupied droplets 120 that do not contain any biological particles 114. The occupied droplets 118 may include singly occupied droplets (having one biological particle) and multiply occupied droplets (having more than one biological particle). As described elsewhere herein, in some cases, each occupied partition of a majority of the occupied partitions may include no more than one biological particle, and some of the generated partitions may be unoccupied (by any biological particle). However, in some cases, some of the occupied partitions may include more than one biological particle. In some cases, the partitioning process may be controlled so that less than about 25% of the occupied partitions contain more than one biological particle, and in many cases, less than about 20% of the occupied partitions have more than one biological particle, and in some cases, less than about 10% or even less than about 5% of the occupied partitions include more than one biological particle per partition.

[0225] In some cases, it may be desirable to minimize the creation of an excessive number of empty partitions in order to reduce costs and / or improve efficiency. While such minimization may be achieved by providing a sufficient number of bioparticles (e.g., bioparticles 114) at partition connection 110 to ensure that at least one bioparticle is encapsulated in a partition, a Poisson distribution may be expected to increase the number of partitions that include multiple bioparticles. Thus, where a single occupied partition is to be obtained, a maximum of about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the created partitions may be unoccupied.

[0226] In some cases, the flow of one or more biological particles (e.g., in channel segment 102) or other fluids directed into the partitioning connection (e.g., in channel segments 104, 106) can be controlled so that in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled so as to present a non-Poisson distribution of single occupied partitions while providing a lower level of unoccupied partitions. The above ranges of unoccupied partitions can be achieved while still providing any of the above single occupancies. For example, in many cases, the partitions obtained using the systems and methods described herein can have multiple occupancies of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases less than about 5%, while the unoccupied partitions are less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less percentages.

[0227] It should be understood that the above occupancy ratios also apply to partitions that include both biological particles and additional reagents, including but not limited to supports such as beads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g., oligonucleotides) (relative to Figure 2 The occupied partitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can include both supports (e.g., beads) having barcoded nucleic acid molecules and biological particles.

[0228] In another aspect, as a supplement or alternative to droplet-based separation, the bioparticles can be encapsulated in a support including a shell, layer or porous matrix in which one or more individual bioparticles or a small group of bioparticles are entrained. The support may include other reagents. Encapsulation of bioparticles can be performed by a variety of processes. Such processes can combine aqueous fluids containing bioparticles with polymer precursor materials, which may be able to form gels or other solid or semisolid matrices when specific stimuli are applied to polymer precursors. Such stimuli may include, for example, thermal stimuli (e.g., heating or cooling), light stimuli (e.g., by photocuring), chemical stimuli (e.g., by crosslinking, polymerization initiation of precursors (e.g., by added initiators)), mechanical stimuli or combinations thereof.

[0229] Preparation of a support containing biological particles (e.g., cells) can be performed by a variety of methods. For example, an air knife droplet or aerosol generator can be used to dispense droplets of a precursor fluid into a gelling solution to form beads (e.g., gel beads) comprising individual biological particles or small groups of biological particles. Similarly, membrane-based encapsulation systems can be used to produce beads containing encapsulated biological particles as described herein. Such as Figure 1 The microfluidic system of the present disclosure shown can be readily used to encapsulate biological particles (e.g., cells) as described herein. In particular, and with reference to Figure 1 , an aqueous fluid 112 containing (i) biological particles 114 and (ii) a polymer precursor material (not shown) flows into a channel junction 110, where the aqueous fluid is separated into droplets 118, 120 by the flow of a non-aqueous fluid 116. In the case of an encapsulation method, the non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and / or cross-linking of the polymer precursor to form a porous matrix including the entrained biological particles. Examples of polymer precursor / initiator pairs include those described in U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0230] In some cases, the encapsulated bioparticles can be selectively released from the support, such as by the passage of time or upon application of a specific stimulus, which causes the support to degrade sufficiently to allow the bioparticles (e.g., cells) or its other contents to be released from the support, such as into partitions (e.g., droplets). See, e.g., U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0231] The bioparticles may be subjected to other conditions sufficient to polymerize or gel the precursor. Conditions sufficient to polymerize or gel the precursor may include exposure to heating, cooling, electromagnetic radiation, and / or light. Conditions sufficient to polymerize or gel the precursor may include any conditions sufficient to polymerize or gel the precursor. After polymerization or gelation, a polymer or gel may be formed around the bioparticle. The polymer or gel may be diffusion permeable to chemical or biochemical agents. The polymer or gel may be diffusion impermeable to the macromolecular components of the bioparticle. In this way, the polymer or gel may serve to subject the bioparticle to chemical or biochemical manipulations while spatially confining the macromolecular components to the region of the droplet defined by the polymer or gel. The polymer or gel may include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel may include any other polymer or gel.

[0232] The polymer or gel can be functionalized to be combined with the analyte of the target, such as nucleic acid, protein, carbohydrate, lipid or other analyte. The polymer or gel can be polymerized or gelled via a passive mechanism. The polymer or gel can be stable under alkaline conditions or elevated temperatures. The polymer or gel can have mechanical properties similar to those of beads. For example, the polymer or gel can have a size similar to beads. The polymer or gel can have a mechanical strength (e.g., tensile strength) similar to beads. The density of the polymer or gel can be lower than that of oil. The density of the polymer or gel can be roughly similar to that of a buffer. The polymer or gel can have an adjustable pore size. The pore size can be selected to, for example, retain denatured nucleic acids. The pore size can be selected to maintain diffusion permeability to exogenous chemicals (such as sodium hydroxide (NaOH)) and / or endogenous chemicals (such as inhibitors). The polymer or gel can be biocompatible. The polymer or gel can maintain or enhance cell viability. The polymer or gel can be biochemically compatible. The polymer or gel can be polymerized and / or depolymerized by heat, chemistry, enzymes and / or optical means.

[0233] The polymer may include poly(acrylamide-co-acrylic acid) cross-linked by disulfide bonds. The preparation of the polymer may include a two-step reaction. In the first activation step, the poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent to convert the carboxylic acid into an ester. For example, the poly(acrylamide-co-acrylic acid) may be exposed to 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM). The polyacrylamide-co-acrylic acid may be exposed to other salts of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium. In the second cross-linking step, the ester formed in the first step may be exposed to a disulfide cross-linking agent. For example, the ester may be exposed to cystamine (2,2'-disulfide bis(ethylamine)). After these two steps, the bioparticles may be surrounded by polyacrylamide chains linked together by disulfide bridges. As such, the bioparticles may be encapsulated within or comprise a gel or matrix (eg, a polymer matrix) to form a "cell bead."

[0234] Cell beads can include bioparticles (e.g., cells) or macromolecular components (e.g., RNA, DNA, proteins, etc.) of bioparticles. Cell beads can include single cells or multiple cells, or derivatives of single cells or multiple cells. For example, after cracking and washing cells, the inhibitory components in the cell lysate can be washed away, and the macromolecular components can be combined into cell beads. The system and method disclosed herein can be applicable to cell beads (and / or droplets or other partitions) containing bioparticles and cell beads (and / or droplets or other partitions) containing macromolecular components of bioparticles. Cell beads can be or include cells, cell derivatives, cell materials and / or materials derived from cells in a matrix (such as a polymer matrix), in a matrix or wrapped in a matrix. In some cases, cell beads can include living cells. In some cases, living cells can be cultured when encapsulated in a gel or polymer matrix, or can be cultured when containing a gel or polymer matrix. In some cases, polymers or gels can be diffusible and permeable to certain components, while other components (e.g., macromolecular components) can be non-diffusible and permeable.

[0235] hole

[0236] As described herein, one or more processes can be performed in a partition, which can be a hole. A hole can be a hole in a plurality of holes of a substrate, such as a micropore array or a plate, or a hole can be a micropore or microchamber of a device (e.g., a microfluidic device) comprising a substrate. A hole can be a hole of a hole array or a plate, or a hole can be a hole or chamber of a device (e.g., a fluid device). Accordingly, a hole or micropore can be in an "open" configuration, wherein the hole or micropore is exposed to the environment (e.g., containing an open surface) and is accessible on a planar surface of the substrate, or a hole or micropore can be in a "closed" or "sealed" configuration, wherein the micropore is inaccessible on the planar surface of the substrate. In some cases, a hole or micropore can be configured to switch between an "open" and "closed" configuration. For example, an "open" micropore or a group of micropores can be "closed" or "sealed" using a membrane (e.g., a semipermeable membrane), an oil (e.g., a fluorinated oil covering an aqueous solution) or a lid, as described elsewhere herein.

[0237] The well can have a volume of less than 1 milliliter (mL). For example, the well can be configured to accommodate a volume of up to 1000 microliters (μL), up to 100 μL, up to 10 μL, up to 1 μL, up to 100 nanoliters (nL), up to 10 nL, up to 1 nL, up to 100 picoliters (pL), up to 10 (pL) or less. The well can be configured to accommodate a volume of about 1000 μL, about 100 μL, about 10 μL, about 1 μL, about 100 nL, about 10 nL, about 1 nL, about 100 pL, about 10 pL, etc. The well can be configured to accommodate a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 μL, at least 10 μL, at least 100 μL, at least 1000 μL or more. The wells can be configured to accommodate volumes within the volume ranges listed herein, e.g., about 5 nL to about 20 nL, about 1 nL to about 100 nL, about 500 pL to about 100 μL, etc. The wells can be multiple wells with different volumes and can be configured to accommodate volumes suitable for accommodating any of the partitioned volumes described herein.

[0238] In some cases, micropore array or plate include the micropore of single kind.In some cases, micropore array or plate include various micropores.For example, micropore array or plate may include the micropore of one or more types in single micropore array or plate.The type of micropore may have different sizes (for example, length, width, diameter, depth, cross-sectional area, etc.), shape (for example, circle, triangle, square, rectangle, pentagon, hexagon, heptagon, octagon, enneagon, decagon, etc.), aspect ratio or other physical properties.Micropore array or plate may include any number of different types of micropores.For example, micropore array or plate may include 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900,1000 kinds or more different types of micropores. The holes can have any size (e.g., length, width, diameter, depth, cross-sectional area, volume, etc.), shape (e.g., circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, other polygonal, etc.), aspect ratio, or other physical property described herein with respect to any hole.

[0239] In some cases, microwell arrays or plates include different types of microwells positioned adjacent to each other in arrays or plates. For example, a microwell with a set of sizes can be adjacent to and contact positioned with another microwell with a set of different sizes. Similarly, microwells of different geometries can be placed adjacent to or in contact with each other. Adjacent microwells can be configured to hold different articles; for example, a microwell can be used to hold cells, cell beads or other samples (e.g., cell components, nucleic acid molecules, etc.), and adjacent microwells can be used to hold supports (e.g., beads such as gel beads), droplets or other reagents. In some cases, adjacent microwells can be configured to, for example, fuse the contents held therein when applying a stimulus or spontaneously when contacting the articles in each microwell.

[0240] As described elsewhere herein, multiple partitions can be used in systems, compositions and methods described herein. For example, any suitable number of partitions (e.g., holes or droplets) can be produced or otherwise provided. For example, in the case of using holes, at least about 1,000 holes, at least about 5,000 holes, at least about 10,000 holes, at least about 50,000 holes, at least about 100,000 holes, at least about 500,000 holes, at least about 1,000,000 holes, at least about 5,000,000 holes, at least about 10,000,000 holes, at least about 50,000,000 holes, at least about 100,000,000 holes, at least about 500,000,000 holes, at least about 1,000,000,000 holes or more holes can be produced or otherwise provided. Furthermore, the plurality of pores may include both unoccupied pores (eg, empty pores) and occupied pores.

[0241] Hole can include any reagent as described herein or its combination.These reagents can include, for example, barcode molecules, enzymes, adapters, and combinations thereof.Reagent can be physically separated from the sample (for example, cell, cell bead or cell component, for example, protein, nucleic acid molecule, etc.) placed in the hole.This physical separation can be achieved by including the reagent in the support (for example, bead such as gel bead) placed in the hole or by coupling with the support placed in the hole.This physical separation can also be achieved by distributing reagent in the hole and covering the reagent with, for example, a soluble, meltable or permeable layer before the polynucleotide sample is introduced into the hole.This layer can be, for example, oil, wax, film (for example, semipermeable membrane), etc.The hole can be sealed at any time point, for example, after adding a support (for example, bead), after adding a reagent, or after adding any one of these components.The sealing of the hole can be used for a variety of purposes, including preventing beads or loaded reagents from escaping from the hole, allowing selective delivery of certain reagents (for example, via the use of a semipermeable membrane), storing the hole before or after further processing, etc.

[0242] The wells may contain free reagents, and / or reagents encapsulated in supports (e.g., beads) or droplets, or otherwise coupled or associated with supports (e.g., beads) or droplets. Any reagent described in the present disclosure may be encapsulated in supports (e.g., beads) or droplets or otherwise coupled with supports (e.g., beads) or droplets together with any chemical substance, particle, and element suitable for sample processing reactions involving biomolecules, such as but not limited to nucleic acid molecules and proteins. For example, beads or droplets used in sample preparation reactions for DNA sequencing may contain one or more of the following reagents: enzymes, restriction enzymes (e.g., multi-cutting enzymes), ligases, polymerases, fluorophores, oligonucleotide barcodes, adapters, buffers, nucleotides (e.g., dNTPs, ddNTPs), etc.

[0243] Additional examples of reagents include, but are not limited to, buffers, acidic solutions, alkaline solutions, temperature sensitive enzymes, pH sensitive enzymes, light sensitive enzymes, metals, metal ions, magnesium chloride, sodium chloride, manganese, aqueous buffers, mild buffers, ionic buffers, inhibitors, enzymes, proteins, polynucleotides, antibodies, carbohydrates, lipids, oils, salts, ions, detergents, ionic detergents, non-ionic detergents, oligonucleotides, nucleotides, deoxyribonucleotide triphosphates (dNTPs), dideoxyribonucleotide triphosphates (ddNTPs), DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), plasmid DNA , cosmid DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozymes, riboswitches and viral RNA, polymerases, ligases, restriction enzymes, proteases, nucleases, protease inhibitors, nuclease inhibitors, chelators, reducing agents, oxidants, fluorophores, probes, chromophores, dyes, organic matter, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, drugs, radioactive molecules, preservatives, antibiotics, aptamers and pharmaceutical compounds. As described herein, one or more reagents in the well can be used to perform one or more reactions, including but not limited to: cell lysis, cell fixation, permeabilization, nucleic acid reaction (e.g., nucleic acid extension reaction), amplification, reverse transcription, transposase reaction (e.g., tag fragmentation), etc.

[0244] The wells can be provided as part of a kit. For example, the kit can include instructions for use, a microwell array or device, and reagents (e.g., beads). The kit can include any useful reagents for performing the processes described herein, such as nucleic acid reactions, barcoding nucleic acid molecules, sample processing (e.g., for cell lysis, fixation, and / or permeabilization).

[0245] In some cases, the well includes a support (e.g., a bead) or a droplet that contains a group of reagents with similar properties (e.g., a group of enzymes, a group of minerals, a group of oligonucleotides, a mixture of different barcode molecules, a mixture of the same barcode molecules). In other cases, the support or droplet contains a heterogeneous mixture of reagents. In some cases, the heterogeneous mixture of reagents can contain all the components necessary to react. In some cases, such a mixture can contain all the components necessary to react, except for 1, 2, 3, 4, 5 or more components necessary to react. In some cases, such additional components are contained in different supports or droplets or otherwise coupled to different supports or droplets, or are contained in a solution in a partition (e.g., microwell) of the system.

[0246] Figure 5 An example of a microwell array is schematically shown. The array can be contained in a substrate 500. The substrate 500 includes a plurality of holes 502. The holes 502 can have any size or shape, and the spacing between the holes, the number of holes in each substrate, and the hole density on the substrate 500 can be changed according to the specific application. In one such example application, a sample molecule 506 that can contain a cell or a cell component (e.g., a nucleic acid molecule) is separated from a bead 504, which can include a nucleic acid barcode molecule coupled thereto. The hole 502 can be loaded using gravity or other loading techniques (e.g., centrifugation, a liquid processor, acoustic loading, an optoelectronic device, etc.). In some cases, at least one hole 502 contains a single sample molecule 506 (e.g., a cell) and a single bead 504.

[0247] Reagent can be loaded into hole sequentially or simultaneously.In some cases, reagent is introduced into device before or after specific operation.In some cases, reagent (in some cases, can be provided in support or droplet) is introduced sequentially so that different reactions or operations occur in different steps.Reagent (or support or droplet) can also be loaded in the operation interspersed with reaction or operation steps.For example, support (or droplet) containing reagent (for example, restriction enzyme) and / or other enzyme (for example, transposase, ligase, polymerase, etc.) for making polynucleotide fragmentation can be loaded into one or more holes, and then support or droplet containing reagent for attaching nucleic acid barcode molecule to sample nucleic acid molecule is loaded.Reagent can be provided simultaneously or sequentially with sample such as cell or cell component (for example, organelle, protein, nucleic acid molecule, carbohydrate, lipid, etc.).Therefore, when carrying out multi-step operation or reaction, it may be useful to use hole.

[0248] As described elsewhere herein, nucleic acid barcode molecules and other reagents can be contained in a support (e.g., bead) or droplet. These supports or droplets can be loaded into a partition (e.g., micropore) before, after, or while loading cells, so that each cell is in contact with a different support or droplet. This technology can be used to attach unique nucleic acid barcode molecules to nucleic acid molecules obtained from each cell. Alternatively or in addition, sample nucleic acid molecules can be attached to a support. For example, a partition (e.g., micropore) can include beads having a plurality of nucleic acid barcode molecules coupled thereto. Sample nucleic acid molecules or derivatives thereof can be coupled or attached to nucleic acid barcode molecules on a support. The resulting barcoded nucleic acid molecules can then be taken out of the partition, and in some cases, merged and sequenced. In such cases, nucleic acid barcode sequences can be used to track the source of sample nucleic acid molecules. For example, polynucleotides with the same barcode can be determined to be derived from the same cell or partition, while polynucleotides with different barcodes can be determined to be derived from different cells, cell nuclei, cell beads or partitions.

[0249] Sample or reagent can be loaded into hole or micropore using multiple methods. Sample (for example, cell, cell bead or cell component) or reagent (as described herein) can be loaded into hole or micropore using external force (for example, gravity, electricity, magnetic force) or using mechanism (for example, via pressure driven flow, centrifugal, optoelectronic device, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc.) that drives sample or reagent to enter hole. In some cases, sample or reagent can be loaded into hole using fluid handling system. The loading of sample or reagent can follow Poisson distribution or non-Poisson distribution, such as super Poisson or sub-Poisson. The spacing, density and size between the geometry of micropore, hole can be modified to adapt to useful sample or reagent distribution; For example, the size and spacing of micropore can be adjusted so that sample or reagent can be distributed in super Poisson mode.

[0250] In a specific non-limiting example, a microwell array or microwell plate includes paired microwells, wherein each pair of microwells is configured to accommodate droplets (e.g., containing single cells) and single beads (such as those described herein, which may also be encapsulated in droplets in some cases). Droplets and beads (or droplets containing beads) can be loaded simultaneously or sequentially, and droplets and beads can fuse, for example, when the droplets are in contact with the beads, or when a stimulus (e.g., external force, stirring, heat, light, magnetic force, or electricity, etc.) is applied. In some cases, the loading of droplets and beads is super-Poisson. In other examples of microwell pairs, the holes are configured to accommodate two droplets containing different reagents and / or samples, which fuse when in contact or when a stimulus is applied. In such a case, a droplet of one microwell in the pair may contain a reagent that can react with an agent in a droplet of another microwell in the pair. For example, a droplet may contain a reagent configured to release a nucleic acid barcode molecule of a bead contained in another droplet located in an adjacent microwell. Upon droplet fusion, the nucleic acid barcode molecules can be released from the beads into partitions (e.g., microwells or pairs of contacting microwells) and can undergo further processing (e.g., barcoding, nucleic acid reactions, etc.). In the case where intact or living cells are loaded into microwells, one of the droplets can contain a lysis reagent for lysing the cells upon droplet fusion.

[0251] Droplets or supports (e.g., beads) can be separated into a well. Droplets can be selected or pre-treated before being loaded into the well. For example, droplets can contain cells, and only certain droplets (such as those containing single cells (or at least one cell)) can be selected for loading the well. This pre-selection process can be used to effectively load single cells, such as to obtain a non-Poisson distribution, or to pre-filter cells with selected characteristics before further separation in the well. In addition, the technology can be used to obtain cell doublets or multiplets before or during loading of the microwells, or to prevent the formation of cell doublets or multiplets.

[0252] In some cases, the hole may include a nucleic acid barcode molecule attached thereto. The nucleic acid barcode molecule may be attached to the surface of the hole (e.g., the wall of the hole). The nucleic acid barcode molecule of a hole (e.g., a partition barcode sequence) may be different from the nucleic acid barcode molecule of another hole, which may allow the identification of the contents contained in a single partition or hole. In some cases, the nucleic acid barcode molecule may include a spatial barcode sequence, which may identify the spatial coordinates of the hole, such as in a hole array or a well plate. In some cases, the nucleic acid barcode molecule may include a unique molecular identifier for identifying individual molecules. In some cases, the nucleic acid barcode molecule may be configured to attach to or capture nucleic acid molecules in a sample or cell distributed in a hole. For example, the nucleic acid barcode molecule may include a capture sequence, which may be used to capture nucleic acid molecules (e.g., RNA, DNA) in a sample or hybridize with the nucleic acid molecule. In some cases, the nucleic acid barcode molecule may be released from a micropore. For example, the nucleic acid barcode molecule may include a chemical crosslinker, which may be cut when a stimulus (e.g., light stimulus, magnetic stimulus, chemical stimulus, biological stimulus) is applied. The released nucleic acid barcode molecules (which can hybridize or be configured to hybridize with sample nucleic acid molecules) can be collected and combined for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and / or characterization (e.g., sequencing). In such cases, the unique partition barcode sequence can be used to identify the cell or partition from which the nucleic acid molecule originated.

[0253] The sample in the hole can be characterized. In a non-limiting example, this characterization can include imaging of the sample (e.g., cell, cell bead or cell component) or its derivative. Characterization techniques (such as microscopy or imaging) can be used to measure the sample profile in a fixed spatial position. For example, when cells (nuclei or cell beads) are separated, optionally separated together with beads, imaging of each micropore and the contents contained therein can provide useful information about the following: cell doublet formation (e.g., frequency, spatial position, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, biomarker (e.g., surface markers, fluorescently labeled molecules therein, etc.) expression level, cell or bead loading rate, cell-bead pair quantity, etc. In some cases, imaging can be used to characterize the living cells in the hole, including but not limited to: dynamic living cell tracking, cell-cell interaction (when two or more cells are separated together), cell proliferation, etc. Alternatively or in addition, imaging can be used to characterize the amount of amplification products in the hole.

[0254] In operation, the hole can be loaded with samples and reagents simultaneously or sequentially. When loading cells, nuclei or cell beads, the hole can be washed, for example, to remove excessive cells (or nuclei or cell beads) from holes, microwell arrays or plates. Similarly, cleaning can be performed to remove unnecessary beads or other reagents from holes, microwell arrays or microwell plates. In the case of using living cells, these cells can be cracked in each partition to release intracellular components or cell analytes. Alternatively, cells can be fixed or permeabilized in each partition. Intracellular components or cell analytes can be coupled to a support, for example, on the surface of micropores, on solid supports (for example, beads), or they can be collected for further downstream processing. For example, after cell lysis, intracellular components or cell analytes can be transferred to each droplet or other partitions to add barcodes. Alternatively or in addition, intracellular components or cell analytes (e.g., nucleic acid molecules) can be coupled to beads containing nucleic acid barcode molecules; subsequently, the beads can be collected and further processed, for example, subjected to nucleic acid reactions such as reverse transcription, amplification or extension, and the nucleic acid molecules thereon can be further characterized, for example, via sequencing. Alternatively or in addition, intracellular components or cell analytes can be barcoded in the wells (e.g., using beads containing releasable nucleic acid barcode molecules or on the surface of micropores containing nucleic acid barcode molecules). Barcoded nucleic acid molecules or analytes can be further processed in the wells, or barcoded nucleic acid molecules or analytes can be collected from each partition and subjected to further processing outside the partitions. Further processing can include nucleic acid processing (e.g., performing amplification, extension) or characterization (e.g., fluorescence monitoring of amplified molecules, sequencing). In any convenient or useful step, the wells (or microwell arrays or microwell plates) can be sealed (e.g., using oil, membranes, wax, etc.), which enables storage of assays or selective introduction of additional reagents.

[0255] Once the hole is sealed, it is possible to withstand the conditions for further processing the biological particles (e.g., cells, cell beads or cell nuclei) in the hole. For example, the reagent in the hole can allow the biological particles to be further processed, such as the cell or cell nucleus is lysed, as further described herein. Alternatively, the hole (or multiple holes, such as those in the array based on the hole) containing the biological particles (e.g., cells, cell beads or cell nuclei) can be subjected to a freeze-thaw cycle to process the biological particles, such as the cell or cell nucleus is lysed. The hole containing the biological particles (e.g., cells, cell beads or cell nuclei) can be subjected to a freezing temperature (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C, or -85°C). Freezing can be carried out in a suitable manner, such as a subzero freezer or dry ice / ethanol bath. After initial freezing, the hole (or multiple holes) containing biological particles (e.g., cells, cell beads, one or more nuclei) can be subjected to a freeze-thaw cycle to crack the biological particles. In one embodiment, the initially frozen hole (or multiple holes) is thawed to a temperature (e.g., room temperature or 25°C) higher than the freezing temperature. In another embodiment, the freezing time is less than 10 minutes (e.g., 5 minutes or 7 minutes), and the thawing time at room temperature is less than 10 minutes (e.g., 5 minutes or 7 minutes) afterwards. The freeze-thaw cycle can be repeated multiple times, such as 2,3 or 4 times, to obtain a lysate of biological particles (e.g., cells, cell beads, one or more nuclei) in the hole (or multiple holes). In one embodiment, freezing, thawing and / or freezing / thawing cycles are carried out without lysis buffer.

[0256] Figure 6 An example workflow for processing nucleic acid molecules in a sample is schematically shown. A substrate 600 including a plurality of micropores 602 may be provided. A sample 606 that may include cells, cell beads, cell components, or analytes (e.g., proteins and / or nucleic acid molecules) may be co-separated in a plurality of micropores 602 together with a plurality of beads 604 including nucleic acid barcode molecules. During process 610, sample 606 may be processed within a partition. For example, with respect to living cells, cells may be subjected to conditions sufficient to lyse cells or nuclei and release the analytes contained therein. In process 620, beads 604 may be further processed. For example, processes 620a and 620b schematically illustrate different workflows, depending on the characteristics of beads 604.

[0257] In 620a, beads include nucleic acid barcode molecules attached thereto, and sample nucleic acid molecules (e.g., RNA, DNA) can be attached to nucleic acid barcode molecules, for example, via connection hybridization. This attachment can occur on beads. In process 630, beads 604 can be collected from multiple holes 602 and merged. Further processing can be performed in process 640. For example, one or more nucleic acid reactions, such as reverse transcription, nucleic acid extension, amplification, connection, transposition, etc. can be performed. In some cases, the adapter sequence is connected to the nucleic acid molecule or its derivative, as described elsewhere herein. For example, a sequencing primer sequence can be added to each end of the nucleic acid molecule. In process 650, further characterization (such as sequencing) can be performed to generate sequencing reads. These sequencing reads can generate information about individual cells or cell colonies, which can be presented visually or graphically, for example, in Figure 655.

[0258] In 620b, the beads contain nucleic acid barcode molecules releasably attached thereto, as described below. The beads can degrade or otherwise release nucleic acid barcode molecules into holes 602; then, nucleic acid barcode molecules can be used to barcode nucleic acid molecules in holes 602. Further processing can be performed inside or outside the partition. For example, one or more nucleic acid reactions, such as reverse transcription, nucleic acid extension, amplification, connection, transposition, etc., can be performed. In some cases, the adapter sequence is connected to the nucleic acid molecule or its derivative, as described elsewhere herein. For example, a sequencing primer sequence can be added to each end of the nucleic acid molecule. In process 650, further characterization (such as sequencing) can be performed to generate sequencing reads. These sequencing reads can generate information about individual cells or cell populations, which can be presented visually or graphically, for example, in Figure 655.

[0259] Beads

[0260] Nucleic acid barcode molecules can be delivered to a partition (e.g., a droplet or a well) via a solid support or carrier (e.g., a bead). In some cases, the nucleic acid barcode molecule is initially associated with a solid support and then released from the solid support when a stimulus is applied, which allows the nucleic acid barcode molecule to dissociate or release from the solid support. In a specific example, the nucleic acid barcode molecule is initially associated with a solid support (e.g., a bead) and then released from the solid support when a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, and / or a light stimulus is applied.

[0261] Nucleic acid barcode molecules can comprise a barcode sequence and a functional sequence, such as a nucleic acid primer sequence or a template switch oligonucleotide (TSO) sequence.

[0262] The solid support can be a bead. The solid support (e.g., bead) can be porous, non-porous, hollow (e.g., microcapsule), solid, semi-solid and / or a combination thereof. The bead can be solid, semi-solid, semi-fluid, fluid and / or a combination thereof. In some cases, the solid support (e.g., bead) can be at least partially soluble, rupturable and / or degradable. In some cases, the solid support (e.g., bead) may not be degradable. In some cases, the solid support (e.g., bead) can be a gel bead. The gel bead can be a hydrogel bead. The gel bead can be formed by a molecular precursor (such as a polymer or monomer). The semi-solid support (e.g., bead) can be a liposome bead. The solid support (e.g., bead) can contain a metal, including iron oxide, gold and silver. In some cases, the solid support (e.g., bead) can be a silica bead. In some cases, the solid support (e.g., bead) can be rigid. In other cases, the solid support (eg, beads) can be flexible and / or compressible.

[0263] Partitions can include one or more unique identifiers, such as barcodes. The barcode can be delivered in advance, subsequently or simultaneously to a partition containing compartmentalized or separated bioparticles. For example, the barcode can be injected into a droplet or deposited in a micropore before, after or simultaneously before a reagent is provided in a droplet. The delivery of a barcode to a specific partition allows the characteristics of a separate bioparticle to be attributed to a specific partition later. The barcode can be delivered to the partition via any suitable mechanism, for example, on a nucleic acid molecule (e.g., oligonucleotide). The nucleic acid molecule with a barcode can be delivered to the partition via a support (e.g., bead). In some cases, the support can include beads. Beads are described in further detail below.

[0264] In some cases, the barcoded nucleic acid molecules may initially be associated with a support (e.g., beads) and then released from the support. The release of the barcoded nucleic acid molecules may be passive (e.g., by diffusing out of the support or diffusing outside the support). In addition or alternatively, the release from the support may be performed after applying a stimulus that allows the barcoded nucleic acid molecules to dissociate or release from the support (e.g., beads). This stimulus may destroy the support, i.e., the barcoded nucleic acid molecules are coupled to the support or coupled in the support or an interaction of both. This stimulus may include, for example, thermal stimulation, light stimulation, chemical stimulation (e.g., pH changes or use of a reducing agent), mechanical stimulation, radiation stimulation; biological stimulation (e.g., enzymes) or any combination thereof. Methods and syste...

Claims

1. A method for analyzing cells, comprising: (a) providing a sample comprising cells, wherein the cells comprise: (i) a feature, wherein the feature comprises a protein, a peptide, a lipid or a carbohydrate; and (ii) a nucleic acid molecule; and (b) contacting the cell with a characteristic binding group and coupling the characteristic binding group to the characteristic, wherein the characteristic binding group comprises an oligonucleotide, and the oligonucleotide comprises a characteristic nucleic acid sequence that recognizes the characteristic binding group; (c) hybridizing a first nucleic acid probe to the nucleic acid molecule; wherein the first nucleic acid probe comprises (i) a hybridizing sequence that hybridizes to a first target sequence of the nucleic acid molecule and (ii) a non-hybridizing sequence; (d) generating a first barcoded nucleic acid molecule using the characteristic nucleic acid sequence and a first barcode molecule comprising a first barcode sequence via a nucleic acid ligation reaction or a nucleic acid extension reaction, wherein the first barcoded nucleic acid molecule comprises (i) the characteristic nucleic acid sequence or its reverse complement, and (ii) the first barcode sequence or its reverse complement; and (e) generating a second barcoded nucleic acid molecule using the non-hybridizing sequence and a second barcode molecule comprising a second barcode sequence via a nucleic acid ligation reaction or a nucleic acid extension reaction, wherein the second barcoded nucleic acid molecule comprises (i) the first target sequence or its reverse complement and (ii) the second barcode sequence or its reverse complement.

2. The method of claim 1, wherein the characteristic binding group comprises an antibody.

3. The method of claim 1, wherein the feature comprises a cell surface receptor or an intracellular protein.

4. The method of claim 1, further comprising permeabilizing the cells before or during (c).

5. The method according to claim 1 further comprises providing a splint molecule before (d), wherein the splint molecule comprises a first splint sequence and a second splint sequence, wherein the first portion of the first barcode molecule hybridizes to the first splint sequence in the splint molecule, and the second portion of the oligonucleotide hybridizes to the second splint sequence on the splint molecule. The method of claim 5 , wherein the first splint sequence and the second splint sequence are adjacent on the splint molecule.

7. The method of claim 5, wherein the first splint sequence and the second splint sequence are separated by a gap region of at least 1 nucleotide on the splint molecule.

8. The method of claim 5, wherein (d) comprises ligating the first barcode molecule and the oligonucleotide together to generate the first barcoded nucleic acid molecule.

9. The method of claim 1, wherein the first barcode sequence in the first barcode molecule identifies the sample.

10. The method of claim 1, further comprising (f) using the first barcoded nucleic acid molecule and a third barcode molecule comprising a third barcode sequence to generate a third barcoded nucleic acid molecule comprising (i) the signature nucleic acid sequence or its reverse complement, (ii) the first barcode sequence or its reverse complement; and (iii) the third barcode sequence or its reverse complement.

11. The method of claim 10, wherein (f) occurs in one of a plurality of partitions.

12. The method of claim 11, wherein the third barcode sequence distinguishes the partition from other partitions in the plurality of partitions.

13. The method of claim 10, wherein (f) comprises hybridizing a first capture sequence in the third barcode molecule to a first capture binding sequence in the first barcoded nucleic acid molecule; and extending the third barcode molecule to produce the third barcoded nucleic acid molecule. The method of claim 13 , wherein the third barcode molecule is coupled to beads.

15. The method of claim 10, further comprising detecting the third barcoded nucleic acid molecule to identify (i) the signature nucleic acid sequence, (ii) the first barcode sequence; and (iii) the third barcode sequence.

16. The method of claim 1, wherein the nucleic acid molecule is an RNA transcript.

17. The method of claim 1, wherein (c) further comprises hybridizing a second nucleic acid probe to a second target sequence of the nucleic acid molecule.

18. The method of claim 17, wherein in (c), the first target sequence and the second target sequence are adjacent on the nucleic acid molecule.

19. The method according to claim 17, wherein: In (c), the first target sequence and the second target sequence are separated by a gap region of at least 1 nucleotide on the nucleic acid molecule.

20. The method of claim 17, further comprising ligating the first nucleic acid probe and the second nucleic acid probe together, thereby generating a ligated nucleic acid molecule comprising (i) the first nucleic acid probe and (ii) the second nucleic acid probe.

21. The method of claim 20, wherein the ligated nucleic acid molecule comprises a fourth barcode sequence that identifies the sample.

22. The method of claim 21, wherein the fourth barcode sequence is identical to the first barcode sequence.

23. The method of claim 21, wherein (e) comprises using the non-hybridizing sequence in the ligated nucleic acid molecule and the second barcode molecule to generate the second barcoded nucleic acid molecule, wherein the second barcoded nucleic acid molecule comprises (i) the first target sequence or its reverse complement, (ii) the second barcode sequence or its reverse complement, and (iii) the fourth barcode sequence or its reverse complement.

24. The method of claim 1, wherein (e) occurs in one of a plurality of partitions.

25. The method of claim 24, wherein the second barcode sequence identifies the partition among the plurality of partitions.

26. The method of claim 23, wherein (e) comprises hybridizing a second capture sequence in the second barcode molecule to a second capture binding sequence in the non-hybridized sequence of the first nucleic acid probe; and extending the second barcode molecule to produce the second barcoded nucleic acid molecule.

27. The method of claim 26, wherein the second barcode molecule is coupled to beads.

28. The method of claim 23, further comprising detecting the second barcoded nucleic acid molecule to identify (i) the first target sequence, (ii) the second barcode sequence, and (iii) the fourth barcode sequence.

29. The method of claim 1, wherein (d) is performed in one of a plurality of partitions.

30. The method of claim 29, wherein the first barcode sequence distinguishes the partition from other partitions in the plurality of partitions.

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