Systems and methods for analysis of cells

Abstract

Provided herein are methods and systems for preparing a sequencing library from nucleic acids such as messenger ribonucleic acids (mRNA). The nucleic acids may be derived from immune cells.

Description

Attorney Docket No. 59528-731601SYSTEMS AND METHODS FOR ANALYSIS OF CELLSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 740,767 filed December 31, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Immune cells express various adaptive immunological receptors relating to immune function, such as T cell receptors and B cell receptors, which play a part in the immune response by specifically recognizing and binding to antigens and aiding in their destruction. T cell receptors and B cell receptors possess an N-terminal variable domain and a C-terminal constant domain. When traditional 3’ barcoding methods are applied to long transcripts, valuable information at the 5’ end of the transcripts can be lost.SUMMARY

[0003] Described herein are systems, devices, and methods for preparing a sequencing library from nucleic acid molecules. The systems, devices, and methods described herein can make many copies of captured nucleic acid molecules (e.g., RNA molecules) such that important sequence information located on the 5’ end of the nucleic acid molecules is retained during sequencing.

[0004] In one aspect, the present disclosure provides a method for processing a biological material, comprising: (a) introducing the biological material into a fluidic device, wherein the biological material comprises a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, wherein a surface of the fluidic device comprises (i) one or more capture probes, and (ii) one or more barcode primers comprising one or more barcode sequences; (b) hybridizing a 3’ end of the RNA molecule to a capture probe of the one or more capture probes; (c) extending the capture probe to generate a complementary deoxyribonucleic acid (cDNA) molecule comprising a sequence complementary to the nucleic acid sequence of the RNA molecule; (d) hybridizing at least a portion of the cDNA molecule generated in (c) to a barcode primer of the one or more barcode primers; and (e) extending: the barcode primer using at least a portion of the cDNA molecule generated in (c) as a template, thereby obtaining a tagged molecule complementary to the cDNA molecule generated in (c),Attorney Docket No. 59528-731601the cDNA molecule generated in (c) using at least a portion of the barcode primer as a template, or a combination thereof.

[0005] In some aspects, the method further comprises releasing the RNA molecule from the biological material prior to (b). In certain aspects, in (d), the cDNA molecule is simultaneously coupled to the capture probe and hybridized to the barcode primer of the one or more barcode primers. In additional aspects, the method further comprises incubating the biological material in the fluidic device prior to (b). In some particular aspects, the method comprises quantitating an amount of the RNA molecule in the biological material. In additional aspects, the quantitating comprises comparing a relative amount of the RNA molecule to an amount of a second RNA molecule. In further aspects, the capture probe comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to a sequence of the one or more barcode primers. In certain aspects, the tagged molecule is configured to self-hybridize.

[0006] In a particular aspect, the method further comprises cleaving the cDNA, the tagged molecule, or the cDNA and the tagged molecule from the surface of the fluidic device. In another aspect, the method further comprises pooling the cDNA, the tagged molecule, or the cDNA and the tagged molecule subsequent to the cleaving.

[0007] In a further aspect, the surface comprises a planar surface. In a specific aspect, the planar surface is a surface of a flow cell. In another aspect, the planar surface is a top surface of the flow cell. In an additional aspect, the planar surface is a bottom surface of the flow cell.

[0008] In a certain aspect, the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material. In one aspect, the capture element comprises a physical trap. In a further aspect, the capture element comprises one or more functional groups capable of interacting with the biological material. In another aspect, the one or more functional groups comprise an integrin-binding group. In certain aspects, the integrin-binding group comprises fibronectin. In some aspects, the integrin-binding group comprises laminin. In additional aspects, the integrin-binding group comprises an arginylglycylaspartic acid (RGD) peptide. In some aspects, the one or more functional groups comprise antibodies. In a particular aspect, the fluidic device further comprises a top surface opposite of the bottom surface, and wherein the top surface comprises a capture element configured to capture the biological material.Attorney Docket No. 59528-731601In one aspect, the surface is a top surface of the fluidic device. In a particular aspect, the fluidic device further comprises a bottom surface opposite of the top surface, and wherein the bottom surface comprises a capture element configured to capture the biological material.

[0009] In some aspects, the capture probe comprises a poly(T) tail. In particular aspects, the poly(T) tail is located at a 3’ end of the capture probe. In certain aspects, the poly(T) tail comprises a sequence of three or more thymine bases. In some aspects, in (c), the 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe. In additional aspects, the poly(T) tail comprises a reverse transcriptase primer. In further aspects, the capture probe comprises a T30VN reverse transcriptase primer. In some such aspects, the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe.

[0010] In one aspect, the one or more capture probes do not include barcode sequences. In another aspect, wherein in (e), the extending is performed using an enzyme. In a particular aspect, the enzyme incorporates, at the 3’ end of the cDNA molecule, a sequence that is complementary to the at least a portion of the barcode primer. In a further aspect, the method further comprises, subsequent to (c), incorporating a sequence complementary to a template switch oligonucleotide (TSO) at a 3’ end of the cDNA molecule. In another aspect, the incorporating the sequence complementary to the template switch oligonucleotide at the 3’ end of the cDNA molecule comprises hybridizing the template switch oligonucleotide to the cDNA molecule and extending the cDNA molecule over at least a portion of the template switch oligonucleotide. In a specific aspect, the sequence complementary to the TSO comprises a portion that is complementary to at least a portion of the barcode primer. In an additional aspect, the TSO comprises a complement of a unique molecular identifier or a unique molecular identifier.

[0011] In some aspects, the method further comprises, subsequent to (d), incorporating at a 3' end of the cDNA molecule, a sequence that is complementary to at least a portion of the barcode primer. In some aspects, the method comprises extending the cDNA molecule using the barcode primer or the portion of the barcode primer as a template. In some aspects, the incorporating comprises extending the cDNA molecule over the at least the portion of the barcode primer. In certain aspects, the method comprises extending the barcode primer using the cDNA molecule or the portion of the cDNA molecule as a template.Attorney Docket No. 59528-731601

[0012] In certain aspects, the one or more barcode primers comprise a unique molecular identifier (UMI) or a complement thereof. In some such aspects, the extending in (e) comprises incorporating the unique molecular identifier (UMI) or a complement thereof into the cDNA molecule.

[0013] In an aspect, the method further comprises sequencing the tagged molecule, the cDNA molecule, an amplicon generated therefrom, or a combination thereof. In another aspect, the method further comprises sequencing a subset of the nucleic acid sequence of the RNA molecule or a sequence complementary thereto. In a particular aspect, the subset of the nucleic acid sequence of the RNA molecule comprises a 5’ end of the nucleic acid sequence of the RNA molecule. In another aspect, the subset of the nucleic acid sequence of the RNA molecule does not comprise a 3’ end of the nucleic acid sequence of the RNA molecule.

[0014] In one aspect, the method further comprises generating an amplicon of the tagged molecule. In a particular aspect, the generating the amplicon uses a genespecific primer. In an additional aspect, the gene-specific primer binds to an internal position of the cDNA or the tagged molecule. In a further aspect, the generating the amplicon uses exponential amplification. In another aspect, the generating the amplicon uses nested amplification. In a certain aspect, the generating the amplicon uses any combination of i) a gene-specific primer, (ii) exponential amplification, and (iii) nested amplification. In an aspect, the amplicon comprises a nucleic acid sequence that corresponds to at least a portion of a sequence of the RNA molecule or to at least a portion of a complementary sequence of the RNA molecule. In one such aspect, the nucleic acid sequence that corresponds to at least a portion of the sequence of the RNA molecule comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity to the RNA molecule except that all uracil bases are replaced with thymine bases. In another such aspect, the nucleic acid sequence that corresponds to at least a portion of the complementary sequence of the RNA molecule comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity to the complementary sequence of the RNA molecule except that all uracil bases are replaced with thymine bases. In a certain aspect, the nucleic acid sequence corresponds to at least a portion of a 5’ end of the nucleic acid sequence of the RNA molecule. In another aspect, the nucleic acid sequence does not correspond to a 3’ end of the nucleic acid sequence of the RNA molecule. In a further aspect, theAttorney Docket No. 59528-731601amplicon does not comprise a 3’ end of the nucleic acid sequence of the RNA molecule. In an additional aspect, the amplicon comprises (i) the one or more barcode sequences or (ii) complements to the barcode sequences. In another aspect, the amplicon comprises a double stranded deoxyribonucleic acid molecule that corresponds to a subset of the nucleic acid sequence of the RNA molecule.

[0015] In a particular aspect, the tagged molecule comprises, from a 5’ end to a 3’ end, (i) the one or more barcode sequences and (ii) a modified nucleic acid sequence, wherein the modified nucleic acid sequence corresponds to at least a portion of the nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases. In a further aspect, the tagged molecule further comprises an additional sequence that is 3’ to the modified nucleic acid sequence and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to a barcode sequence of the one or more barcode sequences.

[0016] In one aspect, the barcode primer further comprises one or more unique molecular identifiers (UMIs). In another aspect, the one or more barcode sequences comprise a barcode which indicates a position of the capture probe within the fluidic device.

[0017] In a further aspect, the method further comprises, subsequent to (e), hybridizing the 3’ end of the cDNA molecule to an additional barcode primer of the one or more barcode primers. In another aspect, the method further comprises extending the additional barcode primer using the cDNA molecule as a template, thereby obtaining an additional tagged molecule complementary to the cDNA molecule. In an additional aspect, the additional tagged molecule comprises, from a 5’ end to a 3’ end, (i) the one or more barcode sequences and (ii) an additional modified nucleic acid sequence, wherein the additional modified nucleic acid sequence corresponds to the nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases. In a certain aspect, the additional tagged molecule further comprises a complement to at least a portion of the capture probe.

[0018] In particular aspects, the nucleic acid sequence of the RNA molecule comprises, from a 5’ end to a 3’ end, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence. In one such aspect, the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) the one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the modified first nucleic acid sequence is identical to the firstAttorney Docket No. 59528-731601nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the modified second nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases. In a further aspect, the method further comprises sequencing the one or more barcode sequences and the modified first nucleic acid sequence of the tagged molecule. In another aspect, the modified second nucleic acid sequence of the tagged molecule is not sequenced. In an additional aspect, the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of an antigen binding molecule of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the antigen binding molecule. In a further aspect, the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR. In a specific aspect, the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

[0019] In certain aspects, (a) comprises introducing a plurality of biological materials into the fluidic device, and wherein the plurality of biological materials comprises the biological material. In some aspects, the method further comprises selectively encapsulating the biological material. In specific aspects, the biological material is selectively encapsulated prior to (b). In additional aspects, the method further comprises, prior to the selective encapsulation of the biological material, determining a location of the biological material within the fluidic device.

[0020] In one aspect, the biological material is encapsulated within a chamber comprising one or more polymer matrix walls. In another aspect, the one or more polymer matrix walls extend from the surface to an additional surface opposite of the surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material. In an additional aspect, the one or more polymer matrix walls extend partially from the surface towards an additional surface opposite of the surface. In a further aspect, the chamber comprises a gap between the polymer matrix wall and the additional surface. In another aspect, the method further comprises introducing one or more polymer precursors into the fluidic device. In a particular aspect, the one or more polymer precursors are polymerized, therebyAttorney Docket No. 59528-731601forming the one or more polymer matrix walls. In another aspect, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photoinitiator. In a certain aspect, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen. In a further aspect, the one or more polymer precursors are polymerized by light. In select aspects, the method further comprises determining a location of the biological material within the fluidic device and selectively polymerizing the one or more polymer precursors to generate the one or more polymer matrix walls. In some aspects, the determining of the location of the biological material within the fluidic device comprises using a detector to image the fluidic device. In one aspect, the detector is coupled to an energy source configured to emit energy. In another aspect, the energy source is a light generating device, and wherein the energy comprises light. In certain aspects, the method further comprises generating a virtual mask based on the location of the biological material within the fluidic device and projecting the virtual mask using the light emitted from the light generating device. In an additional aspect, the virtual mask is generated from a spatial light modulator (SLM). In further aspects, the SLM is a digital micromirror device (DMD). In one aspect, the energy source is in optical communication with the fluidic device. In a particular aspect, the chamber has an annular-like cross section.

[0021] In another embodiment, the present disclosure provides a method for biological analysis, comprising: (a) providing a fluidic device comprising a surface, wherein the surface comprises (i) one or more capture probes, and (ii) one or more barcode primers separate from said one or more capture probes; (b) introducing a biological material comprising a ribonucleic acid (RNA) molecule into the fluidic device, wherein the RNA molecule comprises, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence; and (c) on the surface, generating a tagged molecule from the RNA molecule, wherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the modified second nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.Attorney Docket No. 59528-731601

[0022] In one aspect, the method further comprises, prior to (c), hybridizing a 3’ end of the RNA molecule to a capture probe of the one or more capture probes. In another aspect, the method further comprises extending the capture probe to generate a complementary deoxyribonucleic acid (cDNA) molecule comprising (i) a sequence complementary to the first nucleic acid sequence of the RNA molecule and (ii) a sequence complementary to the second nucleic acid sequence of the RNA molecule. In an additional aspect, the method further comprises hybridizing the cDNA molecule generated to a barcode primer of the one or more barcode primers. In a particular aspect, the method further comprises extending the barcode primer using the cDNA molecule generated in (c) as a template, thereby obtaining the tagged molecule, wherein the tagged molecule is complementary to the cDNA molecule.

[0023] In some aspects, the surface comprises a planar surface. In one such aspect, the planar surface is a top surface of a flow cell. In certain aspects, the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material. In some aspects, the capture element comprises a physical trap. In further aspects, the capture element comprises one or more functional groups capable of interacting with the biological material. In additional aspects, the one or more functional groups comprise an integrin-binding group. In certain aspects, the integrin-binding group comprises fibronectin. In particular aspects, the integrin-binding group comprises an arginylglycylaspartic acid (RGD) peptide. In further aspects, the one or more functional groups comprise antibodies. In further aspects, the surface is a top surface of the fluidic device. In one such aspect, the fluidic device further comprises a bottom surface opposite of the top surface, and wherein the bottom surface comprises a capture element configured to capture the biological material. In some aspects, the surface is a bottom surface of the fluidic device. In some such aspects, the fluidic device further comprises a top surface opposite of the bottom surface, and wherein the top surface comprises a capture element configured to capture the biological material.

[0024] In some aspects, the capture probe comprises a poly(T) tail. In a particular aspect, the poly(T) tail is located at a 3’ end of the capture probe. In a certain aspect, the poly(T) tail comprises a sequence of three or more thymine bases. In a further aspect, in (c), the 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe. In another aspect, the poly(T) tail comprises a reverse transcriptase primer. In an additional aspect, the capture probe comprises a T30VN reverseAttorney Docket No. 59528-731601transcriptase primer. In a certain aspect, the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe. In one aspect, the one or more capture probes do not include barcode sequences.

[0025] In some aspects, the extending is performed using an enzyme. In certain aspects, the enzyme incorporates, at the 3’ end of the cDNA molecule, a sequence that is complementary to the at least a portion of the barcode primer. In particular aspects, the method further comprises incorporating a sequence complementary to a template switch oligonucleotide (TSO) at a 3’ end of the cDNA molecule. In one aspect, the sequence complementary to the TSO comprises a portion that is complementary to at least a portion of the barcode primer. In another aspect, the method further comprises incorporating, at a 3' end of the cDNA molecule, a sequence that is complementary to at least a portion of the barcode primer. In a particular aspect, the incorporating comprises ligating the sequence that is complementary to at least the portion of the barcode primer to the 3’ end of the cDNA molecule.

[0026] In certain aspects, the one or more barcode primers comprise a unique molecular identifier (UMI). In particular aspects, the extending comprises incorporating the unique molecular identifier (UMI) or a complement thereof into the cDNA molecule.

[0027] In one aspect, the method further comprises sequencing the tagged molecule or an amplicon generated therefrom, or a combination thereof. In another aspect, the method further comprises sequencing the one or more barcode primers and the modified first nucleic acid sequence. In a particular aspect, the modified second nucleic acid is not sequenced. In certain aspects, the method further comprises generating an amplicon of at least a portion of the tagged molecule. In specific aspects, the generating the amplicon uses a gene-specific primer. In select aspects, the gene-specific primer binds to an internal position of the tagged molecule. In particular aspects, the generating the amplicon uses exponential amplification. In additional aspects, the generating the amplicon uses nested amplification. In further aspects, the generating the amplicon uses any combination of (i) a gene-specific primer, (ii) exponential amplification, and (iii) nested amplification. In certain aspects, the amplicon comprises the modified first nucleic acid sequence. In some aspects, wherein the amplicon does not comprise the modified second nucleic acid sequence. In further aspects, the amplicon comprises the one or more barcode sequences.Attorney Docket No. 59528-731601

[0028] In an aspect, the barcode primer further comprises one or more unique molecular identifiers (UMIs). In a further aspect, the one or more barcode sequences comprise a barcode which indicates a position of the capture probe within the fluidic device. In a particular aspect, the method further comprises hybridizing a 3’ end of the cDNA molecule to an additional barcode primer of the one or more barcode primers. In a certain aspect, the method further comprises extending the additional barcode primer using the cDNA molecule as a template, thereby obtaining an additional tagged molecule complementary to the cDNA molecule. In select aspects, the additional tagged molecule comprises, from a 5’ end to a 3’ end, (i) the one or more barcode sequences, (ii) an additional modified first nucleic acid sequence, and (iii) an additional modified second nucleic acid sequence, wherein the additional modified first nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the additional modified second nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases. In some aspects, the additional tagged molecule further comprises a complement to at least a portion of the additional capture probe.

[0029] In one aspect, the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of an antigen binding molecule of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the antigen binding molecule. In another aspect, the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR. In an additional aspect, the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

[0030] In certain aspects, (a) comprises introducing a plurality of biological materials into the fluidic device, and wherein the plurality of biological materials comprises the biological material. In further aspects, the method further comprises selectively encapsulating the biological material. In some aspects, the biological material is selectively encapsulated prior to (c). In particular aspects, the methodAttorney Docket No. 59528-731601further comprises, prior to the selective encapsulation of the biological material, determining a location of the biological material within the fluidic device.

[0031] In certain aspects, the biological material is encapsulated within a chamber comprising one or more polymer matrix walls. In some aspects, the one or more polymer matrix walls extend from the surface to an additional surface opposite of the surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material. In further aspects, the one or more polymer matrix walls extend partially from the surface towards an additional surface opposite of the surface. In select aspects, the chamber comprises a gap between the polymer matrix wall and the additional surface. In additional aspects, the method further comprises introducing one or more polymer precursors into the fluidic device. In further aspects, the one or more polymer precursors are polymerized, thereby forming the one or more polymer matrix walls. In some aspects, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photoinitiator. In certain aspects, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen. In select aspects, the one or more polymer precursors are polymerized by light. In additional aspects, the method further comprises determining a location of the biological material within the fluidic device and selectively polymerizing the one or more polymer precursors to generate the one or more polymer matrix walls. In further aspects, the determining of the location of the biological material within the fluidic device comprises using a detector to image the fluidic device. In a particular aspect, the detector is coupled to an energy source configured to emit energy. In a specific aspect, the energy source is a light generating device, and wherein the energy comprises light. In one aspect, the method further comprises generating a virtual mask based on the location of the biological material within the fluidic device and projecting the virtual mask using the light emitted from the light generating device. In another aspect, the virtual mask is generated from a spatial light modulator (SLM). In an additional aspect, the SLM is a digital micromirror device (DMD). In one aspect, the energy source is in optical communication with the fluidic device. In a specific aspect, the chamber has an annular-like cross section.

[0032] In an additional embodiment, the present disclosure method for processing a biological sample, comprising: (a) introducing a biological material into a fluidic device, wherein the biological material comprises a ribonucleic acid (RNA) moleculeAttorney Docket No. 59528-731601comprising, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence; (b) introducing one or more polymer precursors into the fluidic device; (c) determining a location of the biological material within the fluidic device; (d) selectively polymerizing the polymer precursors to generate a polymer matrix from the polymer precursors within the fluidic device, wherein the polymer matrix at least partially encapsulates the biological material; and (e) generating a tagged molecule from the RNA molecule, wherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) a modified first nucleic acid sequence and (ii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the second modified nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

[0033] In another embodiment, the present disclosure provides a fluidic device, comprising: a planar surface comprising a tagged molecule generated from a ribonucleic acid (RNA) molecule, wherein the RNA molecule comprises, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence, and wherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the second modified nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

[0034] In one aspect, the planar surface is a bottom surface of a flow cell. In another aspect, the RNA molecule is released from a cell, wherein the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material. In a further aspect, the capture element comprises a physical trap. In one aspect, the capture element comprises one or more functional groups capable of interacting with the biological material. In a particular aspect, the one or more functional groups comprise an integrin-binding group. In a further aspect, the integrin-binding groupAttorney Docket No. 59528-731601comprises fibronectin. In a specific aspect, the integrin-binding group comprises an arginylglycylaspartic acid (RGD) peptide. In a select aspect, the one or more functional groups comprise antibodies.

[0035] In some aspects, the planar surface is a bottom surface of the fluidic device. In further aspects, the fluidic device further comprises a top surface opposite of the bottom surface, wherein the RNA molecule is released from a biological material, and wherein the top surface comprises a capture element configured to capture the biological material. In certain aspects, the planar surface is a top surface of the fluidic device. In select aspects, the fluidic device further comprises a bottom surface opposite of the top surface, wherein the RNA molecule is released from a biological material, and wherein the bottom surface comprises a capture element configured to capture the biological material.

[0036] In some aspects, the planar surface further comprises one or more capture probes. In certain aspects, the capture probe comprises a poly(T) tail. In additional aspects, the poly(T) tail is located at a 3’ end of the capture probe. In particular aspects, the poly(T) tail comprises a sequence of three or more thymine bases. In specific aspects, a 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe. In further aspects, the poly(T) tail comprises a reverse transcriptase primer. In some aspects, the capture probe comprises a T30VN reverse transcriptase primer. In one aspect, the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe. In certain aspects, the one or more capture probes do not include barcode sequences.

[0037] In an aspect, the planar surface further comprises one or more barcode primers, and wherein the one or more barcode primers comprise the one or more barcode sequences. In another aspect, the one or more barcode primers comprise a unique molecular identifier (UMI). In a select aspect, the one or more barcode sequences comprise a barcode which indicates a position of the RNA molecule within the fluidic device.

[0038] In a certain aspect, the RNA molecule is released from a biological material. In some aspects, the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the immune cell. In further aspects, the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region ofAttorney Docket No. 59528-731601the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR. In additional aspects, the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR. In select aspects, the biological material is encapsulated within a chamber comprising one or more polymer matrix walls. In some such aspects, the one or more polymer matrix walls extend from the planar surface to an additional surface opposite of the planar surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material. In further aspects, the one or more polymer matrix walls extend partially from the planar surface towards an additional surface opposite of the planar surface. In select aspects, the chamber comprises a gap between the polymer matrix wall and the additional surface. In specific aspects, the one or more polymer matrix walls are formed by polymerization of one of more polymer precursors. In certain aspects, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photo-initiator. In some aspects, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen. In certain aspects, the one or more polymer precursors are polymerized by light. In further aspects, the chamber has an annular-like cross section.

[0039] In a further embodiment, the present disclosure provides a fluidic device, comprising: a hydrogel chamber comprising a tagged molecule generated from a ribonucleic acid (RNA) molecule, wherein the RNA molecule comprises, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence, and wherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the second modified nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

[0040] In an aspect, the hydrogel chamber is located on a planar surface of the fluidic device. In another aspect, the RNA molecule is released from a biologicalAttorney Docket No. 59528-731601material, wherein the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material. In a further aspect, the capture element comprises a physical trap. In a select aspect, the capture element comprises one or more functional groups capable of interacting with the biological material. In a particular aspect, the one or more functional groups comprise an integrin-binding group. In an additional aspect, the integrin-binding group comprises fibronectin. In a further aspect, the integrin-binding group comprises laminin. In an additional aspect, the integrin-binding group comprises an arginylglycylaspartic acid (RGD) peptide. In a certain aspect, the one or more functional groups comprise antibodies.

[0041] In some aspects, the planar surface is a bottom surface of the fluidic device. In one such aspect, the fluidic device further comprises a top surface opposite of the bottom surface, wherein the RNA molecule is released from a biological material, and wherein the bottom surface comprises a capture element configured to capture the biological material. In some aspects, the planar surface is a top surface of the fluidic device. In one such aspect, the fluidic device further comprises a bottom surface opposite of the top surface, wherein the RNA molecule is released from a biological material, and wherein the bottom surface comprises a capture element configured to capture the biological material.

[0042] In certain aspects, the fluidic device further comprises one or more capture probes. In some aspects, a capture probe of the one or more capture probes comprises a poly(T) tail. In one aspect, the poly(T) tail is located at a 3’ end of the capture probe. In another aspect, the poly(T) tail comprises a sequence of three or more thymine bases. In a further aspect, a 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe. In a certain aspect, the poly(T) tail comprises a reverse transcriptase primer. In a specific aspect, the capture probe comprises a T30VN reverse transcriptase primer. In a select aspect, the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe. In another aspect, the one or more capture probes do not include barcode sequences.

[0043] In some aspects, the fluidic device further comprises one or more barcode primers, and wherein the one or more barcode primers comprise the one or more barcode sequences. In other aspects, the one or more barcode primers comprise a unique molecular identifier (UMI). In certain aspects, the one or more barcode sequences comprise a barcode which indicates a position of the RNA molecule withinAttorney Docket No. 59528-731601the fluidic device. In select aspects, the RNA molecule is released from a biological material. In one aspect, the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the immune cell. In another aspect, the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR. In a further aspect, the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

[0044] In certain aspects, the hydrogel chamber comprises one or more polymer matrix walls. In one such aspect, the one or more polymer matrix walls extend from the planar surface to an additional surface opposite of the surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material. In another aspect, the one or more polymer matrix walls extend partially from the planar surface towards an additional surface opposite of the surface. In select aspects, the hydrogel chamber comprises a gap between the polymer matrix wall and the additional surface. In further aspects, the one or more polymer matrix walls are formed by polymerization of one of more polymer precursors. In some aspects, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photo-initiator. In additional aspects, the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen. In further aspects, the one or more polymer precursors are polymerized by light. In select aspects, the hydrogel chamber has an annular-like cross section.

[0045] In an additional embodiment, the present disclosure provides a method for processing a ribonucleic acid (RNA) molecule, comprising: introducing the RNA molecule into a fluidic device, wherein the fluidic device comprises (i) a plurality of capture probes and (ii) a plurality of barcode primers comprising one or more barcode sequences; hybridizing a 3’ end of the RNA molecule to a capture probe of the plurality of capture probes; extending the capture probe using at least a portion of the RNA molecule as a template, thereby generating a complementary deoxyribonucleic acid (cDNA) molecule comprising a sequence complementary to the nucleic acid sequence of the RNA molecule; hybridizing the cDNA molecule to a first barcodeAttorney Docket No. 59528-731601primer of the plurality of barcode primers; extending the first barcode primer using at least a portion of the cDNA molecule as a template, thereby obtaining a first tagged molecule complementary to the cDNA molecule; hybridizing the cDNA molecule to a second barcode primer of the plurality of barcode primers; and extending the second barcode primer using the cDNA molecule or the portion of the cDNA molecule as a template, thereby obtaining a second tagged molecule complementary to the cDNA molecule. In one such aspect, the method further comprises hybridizing the cDNA molecule to additional barcode primers of the plurality of barcode primers, and extending the additional barcode primers using the cDNA molecule, the portion of the cDNA molecule, or an additional portion of the cDNA molecule as a template.

[0046] In another embodiment, the present disclosure provides a method for processing a ribonucleic acid (RNA) molecule, comprising: introducing the RNA molecule into a fluidic device, wherein the fluidic device comprises (i) a capture probe coupled to a surface and (ii) a barcode primer comprising one or more barcode sequences; hybridizing a 3’ end of the RNA molecule to the capture probe; extending the capture probe using at least a portion of the RNA molecule as a template to generate a complementary deoxyribonucleic acid (cDNA) molecule coupled to the capture probe and comprising a sequence complementary to the nucleic acid sequence of the RNA molecule; hybridizing the cDNA molecule to the barcode primer; extending: the cDNA molecule using at least a portion of the barcode primer as a template, thereby incorporating a complement of a barcode sequence of the one or more barcode sequences to the cDNA molecule, the barcode primer using at least a portion of the cDNA molecule as a template, thereby incorporating a complement of the cDNA molecule or the portion of the cDNA molecule to the barcode primer, or a combination thereof; and cleaving: the capture probe from the surface, the cDNA molecule from the capture probe, the barcode primer from the surface, or a combination thereof.

[0047] In a further embodiment, the present disclosure provides a method for processing ribonucleic acid (RNA) molecules, comprising: introducing the RNA molecules into a fluidic device, wherein the fluidic device comprises (i) a plurality of capture probes coupled to a surface and (ii) a plurality of barcode primers comprising one or more barcode sequences; hybridizing 3’ ends of the RNA molecules to capture probes of the plurality of capture probes; extending the capture probes using the RNA molecules or portions of the RNA molecules as templates to generate complementaryAttorney Docket No. 59528-731601deoxyribonucleic acid (cDNA) molecules coupled to the capture probes and comprising sequences complementary to nucleic acid sequences of the RNA molecules; hybridizing the cDNA molecules to barcode primers of the plurality of barcode primers; extending the cDNA molecules using at least portions of the barcode primers as templates, thereby incorporating complements of barcode sequences of the one or more barcode sequences to the cDNA molecules; cleaving the capture probes from the surface or cleaving the cDNA molecules from the capture probe, thereby releasing the cDNA molecules from the surface; amplifying a first portion of the cDNA molecules using a first amplification reaction; and amplifying a second portion of the cDNA molecules using a second amplification reaction; and sequencing amplicons generated from the first and second amplification reaction.

[0048] In one aspect, the first amplification reaction comprises a whole transcriptome amplification method. In another aspect, the whole transcriptome amplification method comprises tagmentation, fragmentation, adapter ligation, a random primer, or a combination thereof. In a further aspect, the first amplification reaction comprises amplification with a gene-specific primer. In a certain aspect, the second amplification reaction comprises a whole transcriptome amplification method. In a particular aspect, the whole transcriptome amplification method comprises tagmentation, fragmentation, adapter ligation, a random primer, or a combination thereof. In another aspect, the second amplification reaction comprises amplification with a gene-specific primer. In an additional aspect, the first amplification reaction comprises amplification with a first gene-specific primer and the second amplification reaction comprises amplification with a second gene-specific primer. In a further aspect, during the incorporating the complements of barcode sequences of the one or more barcode sequences to the cDNA molecules, the barcode primers are extended over the cDNA molecules, thereby generating tagged molecules, and wherein the tagged molecules are amplified with the cDNA molecules in the first amplification reaction and the second amplification reaction. In a particular aspect, the incorporating the complements of barcode sequences of the one or more barcode sequences to the cDNA molecules, the method further comprises melting the cDNA molecules from the barcode primers, hybridizing the cDNA molecules to additional barcode primers of the plurality of barcode primers; and extending the additional barcode primers over the cDNA molecules, thereby generating additional tagged molecules, wherein the additional tagged molecules are amplified with the cDNAAttorney Docket No. 59528-731601molecules in the first amplification reaction and the second amplification reaction. In a specific aspect, the first amplification reaction and the second amplification reaction are different. In another aspect, the first amplification reaction and the second amplification reaction are the same.

[0049] In another embodiment, the present disclosure provides a method for processing a ribonucleic acid (RNA) molecule, comprising: introducing the RNA molecule into a fluidic device, wherein the fluidic device comprises (i) a capture probe comprising a first primer binding site and (ii) a barcode primer comprising one or more barcode sequences and a second primer binding site; hybridizing a 3’ end of the RNA molecule to the capture probe; extending the capture probe using at least a portion of the RNA molecule as a template to generate a complementary deoxyribonucleic acid (cDNA) molecule coupled to the capture probe, wherein the cDNA comprises a sequence complementary to the RNA molecule or a portion of the RNA molecule; incorporating a sequence that is complementary to a template switch oligonucleotide (TSO) at a 3’ end of the cDNA molecule; hybridizing the cDNA molecule to the barcode primer; and extending the cDNA molecule using the barcode primer as a template, thereby incorporating complements of the one or more barcode sequences and the second primer binding site 3’ to the sequence that is complementary to the template switch oligonucleotide, thereby generating a product strand comprising the first primer binding site and a complement of the second primer binding site, wherein the first primer binding site is configured to hybridize to the complement of the second primer binding site.

[0050] In an additional embodiment, the present disclosure provides a fluidic device comprising: i) a surface; ii) a capture probe coupled to the surface, wherein the capture probe comprises, in a direction from 5’ to 3’ : a) a first primer binding site, and b) a nucleic acid capture sequence; and iii) a barcode primer coupled to the surface, wherein the barcode primer comprises, in a direction from 5’ to 3’ : a) a second primer binding site, b) one or more barcode sequences, and c) a known sequence.

[0051] In one aspect, the nucleic acid capture sequence comprises a poly(T) sequence. In another aspect, the capture probe comprises a cleavage domain at a 5’ end of the first primer binding site; the barcode primer comprises a cleavage domain at a 5’ end of the second primer binding site; or the capture probe comprises a cleavage domain at a 5’ end of the first primer binding site and the barcode primer comprises a cleavage domain at a 5’ end of the second primer binding site.Attorney Docket No. 59528-731601

[0052] In some aspects, the first primer binding site comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 99%, or 100% sequence homology with the second primer binding site. In further aspects, the known sequence comprises about 10 to 100 nucleotides or about 15 to 40 nucleotides. In certain aspects, the one or more barcode sequences comprise a sequence associated with a location on the surface. In particular aspects, a spot on the surface of the fluidic device comprises a plurality of instances of the capture probe and a plurality of instances of the barcode primer. In a select aspect, the one or more barcode sequences in each instance of the barcode primer comprise a sequence associated with a location of the spot on the surface. In further aspect, the spot has a diameter ranging from about 80 microns to about 140 microns. In another aspect, the fluidic device comprises a plurality of spots each comprising a plurality of instances of the capture probe and a plurality of instances of the barcode primer, wherein the one or more barcode sequences in each instance of the barcode primer comprises a sequence uniquely associated with the spot of the plurality of spots in which the barcode primer is located. In a specific aspect, a 5’ end of the first primer binding site and a 5’ end of the second primer binding site are each coupled to the surface. In one aspect, the surface comprises a first surface, wherein the fluidic device further comprises a second surface, wherein the first surface is opposite of the second surface, and wherein the second surface comprises a cell adherent support. In another aspect, the cell adherent support comprises a material selected from the group consisting of actinin, collagen, fibrinogen, fibronectin, gelatin, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, poly-l-lysine (PLL), poly-d-lysine (PDL), poly-l-ornithine, talin, VCAM-1, vinculin, vitronectin, a cell adherent peptide, or a combination thereof.

[0053] In certain aspects, the biological material comprises a cell, a cell aggregate, a spheroid, an organoid, an assembloid, an organelle, a vesicle, a virus, a virus-like particle, or a combination thereof. In particular aspects, the biological material is a cell.

[0054] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, where only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obviousAttorney Docket No. 59528-731601respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0055] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0056] FIG. 1 illustrates a capture probe with a poly(T) tail that hybridizes to a poly(A) tail of a ribonucleic acid (RNA) molecule, according to some embodiments.

[0057] FIG. 2A illustrates a template switch oligonucleotide (TSO) that can be added to a 5’ end of a captured RNA molecule by a reverse transcriptase, according to some embodiments.

[0058] FIG. 2B illustrates a barcode primer, that can be coupled to a surface, with one or more barcode sequences, a unique molecular identifier (UMI), and a known sequence that that represents a type of TSO as indicated in FIG. 2A, according to some embodiments.

[0059] FIG. 3 A illustrates a TSO with a unique molecule identifier (UMI) that can be added to a 5’ end of a captured RNA molecule by a reverse transcriptase, according to some embodiments.

[0060] FIG. 3B illustrates an embodiment of a barcode primer, that can be coupled to a surface, with one or more barcode sequences and a known sequence that represents another type of TSO as indicated in FIG. 3 A, according to some embodiments.

[0061] FIG. 4A illustrates a surface with a capture probe and a barcode primer disposed thereon, in accordance with some embodiments.

[0062] FIG. 4B illustrates a messenger RNA (mRNA) molecule with a poly(A) tail hybridized to a poly(T) tail of the capture probe shown in FIG. 4A, in accordance with some embodiments.

[0063] FIG. 4C illustrates cDNA generated by extension of a capture probe over an mRNA molecule.Attorney Docket No. 59528-731601

[0064] FIG. 4D illustrates a template switching oligonucleotide (TSO) that is hybridized to a 3’ overhang of the cDNA of FIG. 4C and ligated to the mRNA molecule.

[0065] FIG. 4E illustrates the complementary deoxyribonucleic acid (cDNA) sequence extended over a template switch oligonucleotide, in accordance with some embodiments.

[0066] FIG. 4F illustrates a cDNA sequence with a 3’ sequence that is a complement of a template switch oligonucleotide, in accordance with some embodiments.

[0067] FIG. 4G illustrates the nucleic acid of FIG. 4F hybridizing to a portion of a barcode primer, in accordance with some embodiments.

[0068] FIG. 4H illustrates a first product strand generated by extending the nucleic acid of FIG. 4F over the barcode primer, and a second product strand generated by extending the barcode primer over the nucleic acid of FIG. 4F, in accordance with some embodiments.

[0069] FIG. 41 illustrates the first and second product strands of FIG. 4H following dehybridization.

[0070] FIG. 4J illustrates the first product strand hybridizing to an additional barcode primer on the surface, in accordance with some embodiments.

[0071] FIG. 4K illustrates an additional second product strand generated by extending the additional barcode primer over the first product strand, in accordance with some embodiments.

[0072] FIG. 5 A illustrates an exploded view of the first product strand (top) hybridized to the second product strand (bottom) of FIG. 4H, in accordance with some embodiments.

[0073] FIG. 5B illustrates self-hybridization by first and second product strands, in accordance with some embodiments.

[0074] FIG. 6A illustrates a gene-specific amplification method on a DNA complex of first and second product strands, in accordance with some embodiments.

[0075] FIG. 6B illustrates a transposase-mediated amplification method on a DNA complex of first and second product strands described herein.

[0076] FIG. 6C illustrates a fragmentation and ligation method on a DNA complex of first and second product strands described herein.

[0077] FIG. 7 shows a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments.Attorney Docket No. 59528-731601

[0078] FIG. 8A shows a portion of a system as provided herein including an energy source, according to some embodiments.

[0079] FIG. 8B is an illustration of a polymer matrix being formed around a biological component in a portion of a system as provided herein, according to some embodiments.

[0080] FIG. 8C is an illustration of a method of forming a polymer matrix around a biological component in a system as provided herein, according to some embodiments.

[0081] FIG. 9A shows a higher yield of amplicons can be achieved with two turnaround cycles than one turnaround cycle, without changing the expression profile.

[0082] FIG. 9B shows that higher UMI counts were achieved with a 2X turnaround than with a IX turnaround.

[0083] FIG. 9C shows that there is minimal impact on gene expression between a IX and 2X turnaround.

[0084] FIGs. 9D and 9E show that UMIs can become saturated at around six turnaround cycles.

[0085] FIG. 9F is a plot of gene counts of DNA generated in 5’-barcoding assays that utilized different numbers of turnaround steps.

[0086] FIG. 9G is a plot of read lengths of DNA generated in 5 ’-barcoding assays that utilized different numbers of turnaround steps.

[0087] FIG. 9H is a plot of GC content of DNA generated in 5’-barcoding assays that utilized different numbers of turnaround steps.

[0088] FIG. 91 is a plot of DNA product as a function of turnaround steps in a 5’-barcoding assay disclosed herein.

[0089] FIG. 10A illustrates a portion of a surface of a fluidic device, spanning a width of 15 fields in a given lane.

[0090] FIGs. 10B and 11 A illustrate an exploded view of one of the fields shown in FIG. 10 A.

[0091] FIG. 1 IB illustrates a zoomed in view of the field of 11 A.

[0092] FIG. 12 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0093] FIG. 13 A illustrates a tissue sample disposed on a surface that contains capture probes and barcode primers for generating 5 ’-barcoded cDNA for spatial transcriptomic analysis of the tissue sample, in accordance with some embodiments.Attorney Docket No. 59528-731601

[0094] FIG. 13B depicts a process of mapping nucleic acid sequences to specific barcoded regions of a substrate, in accordance with some embodiments.

[0095] FIG. 13C depicts a process of overlaying spatial sequencing and imaging data on a tissue sample, in accordance with some embodiments.

[0096] FIG. 14A is a plot of total DNA yield per fluidic channel in a 5’-barcoding assay.

[0097] FIG. 14B is a plot of nucleotide frequencies as a function of position in DNA generated from a 5 ’-barcoding assay.

[0098] FIG. 14C is a plot of nucleotide frequencies as a function of position in the coding region of DNA generated from a 5’-barcoding assay.

[0099] FIG. 14D is a plot of gene coverage as a function of transcript position in DNA generated from a 5 ’-barcoding assay.

[0100] FIG. 15A is a plot of DNA yield per fluidic channel in a 5 ’-barcoding assay.

[0101] FIG. 15B is a plot of nucleotide frequencies as a function of position in DNA generated from a 5 ’-barcoding assay.

[0102] FIG. 16A is an illustration of a fluidic device surface, a capture probe, and a barcode primer prior to coupling the capture probe and barcode primer to the fluidic device surface.

[0103] FIG. 16B is an illustration of the fluidic device surface of FIG. 16A following affixation of the capture probe and barcode primer to the fluidic device surface.DETAILED DESCRIPTION

[0104] The present disclosure provides systems and methods for generating nucleic acids that contain 5’ barcode sequences, differing from many conventional nucleic acid capture and library preparation methods that generate 3 ’-barcoded nucleic acids. The barcode sequences can identify the multiple types of information associated with a cell, including where the cell was located within a fluidic device. These systems and methods are amenable to a wide range of assay formats, including nucleic acid analysis following single-cell partitioning within photopolymerized chambers and in multiplexed biomolecular assay formats.

[0105] A polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of one or more biological components in a fluidic deviceAttorney Docket No. 59528-731601to isolate selected biological components. The polymer matrix may be selectively generated to surround a component. One or more hydrogel or polymer matrix walls can be used to physically separate one or more biological components from one another.

[0106] In order to compartmentalize individual components of a biological sample, a polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of an individual component in a fluidic device. The hydrogel matrix may be selectively generated to surround a component after the system detects the component or hydrogel matrices can be generated according to a predefined pattern in a fluidic device. The hydrogel matrix may allow reagents and smaller entities to pass while retaining the individual component of the biological sample in place. Because one or more individual components can be localized within a fluidic device (e.g., encapsulated) and the localized components be exposed to one or more reagents and / or washing solutions during and / or in between analyses, multiple assays can be performed within the compartments (e.g., simultaneously, substantially simultaneously, serially, etc.).

[0107] Different assays may be performed in different locations of the fluidic device, for example, to test the effects of distinct treatment conditions. By having two or more components within a compartment, interactions between components can be studied as well. The polymer matrix can be degradable “on demand” allowing for controlled localization and release mechanisms. The solutions provided herein can retain spatial information of the components and generate data on a cellular, proteomic, transcriptomic, or genomic level. Since spatial information is retained, the data can be associated (e.g., linked) with phenotypic data. Further, the solutions provided herein can retain spatial information of the components and link data (e.g., phenotypic data) on a cellular, proteomic, transcriptomic, or genomic level.

[0108] Whenever the term “at least” precedes the first numerical value in a series of two or more numerical values, the term “at least” applies to each of the numerical values in that series of numerical values. For example, at least 1, 2, or 3 is equivalent to at least 1, at least 2, or at least 3.

[0109] Whenever the term “less than” precedes the first numerical value in a series of two or more numerical values, the term “less than” applies to each of the numerical values in that series of numerical values. For example, less than 3, 2, or 1 is equivalent to less than 3, less than 2, or less than 1.Attorney Docket No. 59528-731601

[0110] The terms “coupled to,” “connected to,” and “in communication with,” as used herein, generally refer to any form of interaction between two or more entities, including mechanical, electrical, magnetic, electromagnetic, fluid, biological, and thermal interaction. Two components may be coupled to each other even though they are not in direct contact with each other.

[0111] The terms “polypeptide” and “peptide,” as used interchangeably herein, generally refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (e.g., an amino acid analogue). The polypeptide can be linear or branched. The polypeptide can include modified amino acids. The polypeptide may be interrupted by non-amino acids. A polypeptide can occur as a single chain or an associated chain. The polypeptide may include a plurality of amino acids. The polypeptide may have a secondary and tertiary structure (e.g., the polypeptide may be a protein). In some examples, the polypeptide can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1,000, 10,000, or more amino acids. The polypeptide may be a fragment of a larger polymer. In some examples, the polypeptide can be a fragment of a larger polypeptide, such as a fragment of a protein.

[0112] The term “amino acid,” as used herein, generally refers to a naturally occurring or non-naturally occurring amino acid (e.g., an amino acid analogue). The non-naturally occurring amino acid may be an engineered or synthesized amino acid.

[0113] The term “sample,” as used herein, generally refers to a chemical or biological sample containing a biological component. The biological component may comprise a cell, a nucleic acid, a microbiome, a protein, a combination of cells, a metabolite, a combination thereof, or any other suitable component of a biological sample. The biological component may also be a biological material, which may be a cell; a multicellular structure such as a cell aggregate, a spheroid, an organoid, or an assembloid; a subcellular structure such as an organelle (e.g., mitochondria or nucleus), or a vesicle; a virus or a virus-like particle, or a combination thereof. A sample can be a biological sample including one or more cells. For another example, a sample can be a biological sample including one or more polypeptides. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. The biological sample can be a fluid or tissue sample (e.g., skin sample). In someAttorney Docket No. 59528-731601instances, the sample may be derived from a homogenized tissue sample (e.g., brain homogenate, liver homogenate, or kidney homogenate). In certain embodiments, the sample may include a specific type of cell (e.g., a neuronal cell, muscle cell, liver cell, or kidney cell,). The sample may comprise or be acquired from a diseased cell or tissue (e.g., a tumor cell or a necrotic cell), In some embodiments, the sample may include or may be from a disease-associated inclusion (e.g., a plaque, a biofilm, a tumor, or a non-cancerous growth). In certain embodiments, the sample may include or may be obtained from a cell-free bodily fluid, such as whole blood, saliva, or urine. In various embodiments, the sample can include circulating tumor cells. In some cases, the sample may include or may be an environmental sample (e.g., soil, waste, or ambient air), industrial sample (e.g., samples from any industrial processes), or a food sample (e.g., dairy product, vegetable product, or meat product). The sample may be processed prior to loading into a microfluidic device. For example, the sample may be processed to purify a certain cell type or polypeptide and / or to include reagents.

[0114] As used herein, the term “polymer matrix” generally refers to a phase material (e.g., continuous phase material) that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the interstitial space not occupied by the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also contain non-polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term “polymer matrix” may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. A polymer matrix may comprise one or more polymer precursors in a polymerized form, which generally refers to one or more molecules that upon activation can trigger or initiate a polymeric reaction. A polymer precursor can be activated by electrochemical energy, photochemical energy, a photon, magnetic energy, or any other suitable energy. As used herein, the term “polymer precursor” includes monomers (that are polymerized to produce a polymer matrix), porogens, and crosslinking compounds, which may include photo-initiators, other compounds necessary or useful for generating polymer matrices, and the like. A polymer matrix may be semi permeable so that cells and beads (ranging from 3 to 50 microns) are too big to pass through, but smaller reagents can pass through such asAttorney Docket No. 59528-731601antibodies, buffering salts, cellular media, lysing agents (e.g., SDS, Triton-X, Triton-Xi 00, Tween 20, Sarkosyl).

[0115] In some embodiments, as used herein, the term “local parameter” means a value of a parameter (such as, pH) in or immediately adjacent to a chamber formed by polymer matrix walls.

[0116] As used herein, the term “on demand” means an operation may be directed to individual, discrete, selected locations (e.g. a spatial location of polymer precursor solution; or a selected polymer matrix chamber). Such selection may be based on manual observation of optical signals or data collected by a detector, or such selection may be based on a computer algorithm operating on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include either real-time detection or detection at a time period prior to modulating a unit of energy to polymerize polymer precursors or degrading a chamber. For example, a subset of chambers (all formed with photo-degradable polymer matrix walls) may be pre-selected for releasing and removing their contents based on position information and the values of optical signals from an analytical assay carried out in the chambers. The pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) to degrade the polymer matrix walls of the pre-selected chambers. In another embodiment, the pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) in the presence of a photoinitiator to degrade the polymer matrix walls of the preselected chambers. Examples of a photoinitiator includes one of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) nanoparticles, 2,2’-azobis[2-methyl-N-(2-hydroxy ethyl) promionamide] (VA-086), BAPO-Oli, BAPO-Ona, Eosin- Y, Riboflavin, and combination thereof. In another example, a plurality of chambers may be observed in real-time (e.g. via fluorescent microscopy) for detection of an analyte of interest and one or more chambers of the plurality of chambers is selected, in real-time, upon detection of the analyte of interest, for degradation.

[0117] As used herein, the term “analyte” generally refers to a discrete biological or chemical entity to be measured, detected, and / or distinguished using theAttorney Docket No. 59528-731601methods and systems described herein. In some embodiments, an analyte may be a biological component as described herein.

[0118] As used herein, the term “morphology” generally refers to the study of the size, shape, and structure of biological components (i.e., cells) and of the relationships of constituent parts thereof. As used herein, “morphological assays” generally refer to assays used to analyze morphological features of the biological components. Morphological assays can utilize microscopy to identify the shape, structure, form, color, texture, pattern, and size of a biological component. In some embodiments, the shape is circular, oval, or oblong. In some embodiments, the size is an area, number of pixels, diameter, eccentricity (e.g., shortest diameter and longest diameter), perimeter, or texture of the biological material.

[0119] The present disclosure provides systems for compartmentalizing or isolating one or more biological components. The system can include a fluidic device containing or including one or more biological components. The fluidic device may contain or include one or more polymer precursors. In some cases, the fluidic device can comprise a first surface configured to couple or receive at least one of the one or more biological components to form a coupled biological component. The systems may also include at least one energy source, wherein the energy source is in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In various embodiments, the at least one energy source may form a polymer matrix on or adjacent to at least a portion of the one or more biological components.

[0120] In some cases, a sample may be introduced or provided to the system. In certain cases, the sample may comprise one or more biological components. In various cases, the biological components may be physically separated. In some cases, the biological components may be physically separated but in fluidic communication with one another. In certain cases, the biological components may be in chemical communication with one another. The system may be used for single-cell analysis. In some embodiments, the system may be used for single-cell analysis on a genome level. For example, the system may be used for genome sequencing. For another example, the system may be used for deoxyribonucleic acid (DNA) sequencing. As non-limiting examples, the system may be used for DNA sequencing of cell-free DNA, whole genome sequencing, whole exome sequencing, whole transcriptome analysis, targeted sequencing, or 16S sequencing. The system may be used forAttorney Docket No. 59528-731601studying DNA tags attached to biomolecules of interest. The biomolecules may comprise proteins, metabolites, etc. In some cases, the DNA may be a nuclear DNA or a mitochondrial DNA. The system may be used for single-cell or bulk analysis on a transcriptome level. For example, the system may be used for ribonucleic acid (RNA) sequencing. For example, the system may be used for 3’ or 5’ gene expression analysis, immune repertoire study of a cell, or full-length mRNA analysis. In some embodiments, the system may be used for single-cell analysis on a proteome level. The system may be used for functional assay(s) of a biological component. The system may be used for studying surface proteins, secreted proteins, or metabolites of a biological component. In some cases, the system may be used to measure a quality of a biological component. In some cases, the measured quality may be the size or shape of a biological component. In some cases, the system may be used to study epigenomics, DNA methylation, or chromatin accessibility in a biological component. The system may be used for other suitable assays, experiments, and processes.

[0121] In certain embodiments, the system may be used for single-cell analysis on an indirect cell-cell interaction level. For example, an effect of one or more molecules produced from a first cell on a second cell can be analyzed using the system as provided herein. In various embodiments, the system may be used for analyzing direct cell-cell interactions. For example, two or more cells (e.g., a first cell and a second cell) can be in physical contact and the effect or effects of the first cell on the second cell, or vice versa, can be analyzed using the system as disclosed herein. In some embodiments, the system may be used for drug response analysis in a biological component. In certain embodiments, the system may be used for analyzing a biological component’s response to various physiological conditions (e.g., various media, temperature, mechanical stimuli, etc.). In some embodiments the analyte is selected from a plurality of analytes introduced into the fluidic device.

[0122] In certain embodiments, one or more polymer precursors may be added to or included with the biological sample. One or more biological samples and one or more polymer precursors may be introduced into the system (e.g., into the fluidic device of the system). The one or more biological samples and the one or more polymer precursors may be introduced into the fluidic device in any order (e.g., in parallel, sequentially, etc.). For example, the biological sample(s) may be introduced prior to the polymer precursor(s), the polymer precursor(s) may be introduced prior to the biological sample(s), the biological sample(s) and polymer precursor(s) may beAttorney Docket No. 59528-731601introduced simultaneously (or substantially simultaneously), or in any other suitable manner or order. In some embodiments, a polymer precursor may include one or more hydrogel precursors, a porogen, and a photoinitiator. Examples of a photoinitiator includes one of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) nanoparticles, 2,2’-azobis[2-methyl-N-(2 -hydroxyethyl) promionamide] (VA-086), BAPO-Oli, BAPO-Ona, Eosin- Y, Riboflavin, and combination thereof. Examples of a porogen can be polyethylene glycol (PEG, molecular weight from 1 kDa to 1000 kDa), 8 arm PEG, 4 arm PEG, 3 arm PEG, and combinations thereof. The one or more polymer precursors may be stored and / or introduced separately into the system. In some cases, the one or more polymer precursors may be mixed with the one or more biological components prior to introduction into the system. In various cases, the one or more polymer precursors may be mixed with the one or more biological components after introduction into the system.

[0123] The system may comprise a fluidic device. In some embodiments, the fluidic device may include one or more polymer precursors. In other words, one or more polymer precursors may be disposed within at least a portion of the fluidic device (e.g., within at least a portion of a channel of the fluidic device). In some embodiments, the fluidic device may comprise one or more channels or chambers. In some embodiments, the fluidic device may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000 channels or chambers, or any number of channels or chambers between any of the two numbers mentioned herein. In some embodiments, the fluidic device comprises more than 10,000 channels or chambers. As described herein, the fluidic device may include one or more channels. The fluidic device may also, or alternatively, include one or more chambers. The terms channel and chamber may be used interchangeably in the disclosure herein unless indicated otherwise. For example, a channel or a chamber of the fluidic device may comprise a first surface, a second surface, or more surfaces.

[0124] A channel or chamber of a fluidic device (also sometimes referred to as a “flow chamber,” “flow channel,” or “reaction chamber,” as opposed to a chamber that is formed from polymer matrix walls within a channel) may receive or be configured to receive a biological sample. FIG. 7 shows a simplified schematic cross-sectional side view illustration of a portion of a channel 800 that may be disposed in at least a portion of a fluidic device of a system as provided herein. The fluidic deviceAttorney Docket No. 59528-731601may comprise a channel 800. The channel 800 may comprise a first surface 801. Further, the channel 800 may comprise a second surface 802. In some embodiments, the first surface 801 and the second surface 802 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 7). In some embodiments, a middle spacer layer of double-sided adhesive with a cut-out portion can be used to position the first surface 801 and second surface 802 in a facing relationship to at least partly form the flow channel. In some embodiments, the first surface and second surface are substantially parallel, so that the perpendicular distance between them is substantially the same throughout the channel, for example, where chambers are formed. In some embodiments, the perpendicular distance between a first surface and a second surface depends in part and the nature and size of the biological components to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the largest biological component in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the first surface 801 may be a lower surface. In certain embodiments, the second surface 802 may be an upper surface. The channel 800 may receive a biological sample comprising one or more biological components 850, 851. The channel 800 may receive one or more polymer precursors. As illustrated in FIG. 7, the biological components 850, 851 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 801, the second surface 802, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 850, 851. In some cases, the first surface 801 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 850, 851). In certain cases, the second surface, 802 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 850, 851). In some embodiments, the first surface and / or second surface can be optically transmissive soAttorney Docket No. 59528-731601that visible and UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the flowcell, and the measurement of the analyte and biological components.

[0125] In certain cases, a channel may have a cross-sectional area that is rectangular, circular, semi-circular, or oval. Accordingly, the channel may have a single, internal surface. In some cases, a channel may have a triangular, square, rectangular, polygonal, or other cross-section. Accordingly, the channel may have three or more internal surfaces. One or more of the internal surfaces may be couple or receive, or be configured to couple or receive, the one or more biological components.

[0126] In some cases, the first surface 801, the second surface 802, or both surfaces 801, 802 may be functionalized, for example, with a coating (e.g., a surface coating). In some embodiments, the surface coating may be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), a functional group to capture one or more moieties (e.g., a chemical moiety), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, an aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group to capture one or more moieties. For example, the acrylamide, the agarose, etc. may include such a functional group. In certain embodiments, the surface polymer may comprise polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or combinations thereof. In various embodiments, the surface polymer may comprise a silane polymer. In some embodiments, the surface polymer may be functionalized with at least one of an oligonucleotide, an antibody, a cytokine, a chemokine, a protein, an antibody derivative, an antibody fragment, a carbohydrate, a toxin, or an aptamer.

[0127] In some cases, the first surface 801, the second surface 802, or both surfaces 801, 802 may comprise one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the first surface 801, the second surface 802, or both surfaces 801, 802 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein. The barcodes may cover an area of about 500 nm2to about 100,000 pm2and preferably 500 nm2to about 5000 pm2. InAttorney Docket No. 59528-731601some embodiments, the first surface 801, the second surface 802, or both surfaces 801, 802 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodes between any of the two numbers mentioned herein. In some cases, a barcode (or a portion / subset of barcodes) may be associated with the location of the barcode on a surface (location coordinates (e.g., x-, y-coordinates) on a surface of a channel). A barcode may be attached to or coupled to the captured biological component. In some embodiments, the barcode may be a unique identifier that distinguishes a biological component from other biological components (e.g., that identifies a first biological component versus a second biological component). In some embodiments, a barcode may comprise a nucleic acid sequence (e.g., common sequence) to capture a biological component, or used in amplification. In some embodiments, a barcode may comprise a unique identifier comprising a unique nucleic acid sequence (e.g., DNA sequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. In some embodiments the barcode may comprise a fluorescent molecule. In some embodiments, a location of the captured biological component may be associated with the unique identifier to, for example, retain spatial information of a biological component.

[0128] In some embodiments, the fluidic device may be a flow cell. For example, the fluidic device may be used for sequencing (e.g., DNA or RNA sequencing). In some embodiments, the fluidic device may be a microfluidic device. In certain embodiments, the fluidic device may be a nanofluidic device.

[0129] The system disclosed herein may comprise one or more energy sources. The energy source may be in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In some cases, the energy source can be used to form one or more polymer matrices in the fluidic device (e.g., on or adjacent to a surface of a channel or chamber of the fluidic device). In some embodiments, the energy source may comprise a light generating device, a heat generating device, an electrochemical reaction generating device, an electrode, or a microwave device. A polymer matrix may be formed in a channel of the fluidic device. The energy source may direct or transfer energy to aAttorney Docket No. 59528-731601predetermined position in the fluidic device. The energy may cause or activate the one or more polymer precursors to form a polymer matrix (e.g., to polymerize) in the predetermined position.

[0130] In some embodiments, the polymer matrix may comprise a hydrogel. In some embodiments, the hydrogel may be porous enough, or have pores of a suitable size, to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule, an antibody, etc.) through the polymer matrix, while the hydrogel may not allow movement or transfer of the biological component (e.g., DNA, RNA, a protein, a cell, etc.) through the polymer matrix. In some embodiments, the pores may have a diameter from 5 nm to 100 nm. In some embodiments, the pores may have a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores may have a diameter smaller than 5 nm. The reagent may comprise an enzyme or a primer having a size of less than 50 base pairs (bp). A primer may comprise a single-stranded DNA (ssDNA). In some embodiments, a primer may have a size from 5 bp to 50 bp. In some embodiments, a primer may have a size from 5 bp to 10 bp, 10 bp to 20 bp, from 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, a primer may have a size of more than 50 bp. In certain cases, a primer may have a size of less than 5 bp. A reagent may comprise a lysozyme, a proteinase K, hexamers (e.g., random hexamers), a polymerase, a transposase, a ligase, a catalyzing enzyme, a deoxyribonuclease, a deoxyribonuclease inhibitor, a ribonuclease, a ribonuclease inhibitor, DNA oligos, deoxynucleotide triphosphates, buffers, detergents, salts, divalent cations, or any other suitable reagent.

[0131] FIG. 8 shows a portion of a system as provided herein including an energy source 803. The embodiment of FIG. 8 may include components that resemble components of FIG. 7 in some respects. For example, the embodiment of FIG.8 includes a channel 800 that may resemble the channel 800 of FIG. 7. With continued reference to FIG. 8, the channel 800 of the system may include a first surface 801 and a second surface 802. In some embodiments, the energy source 803 may comprise one or more energy emitting portions (e.g., an energy emitting portion 805). In some embodiments, the energy source 803 may comprise an energy one or more non-emitting portions (e.g., a non-emitting portion 804). The non-emitting portion 804 may not emit, or be configured to emit, energy. In some embodiments, theAttorney Docket No. 59528-731601emitting portion 805 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic device. In certain embodiments, the emitting portion 805 can emit energy to the fluidic device. In some embodiments, the fluidic channel may be coupled to and / or disposed on a movable stage. In other embodiments, light may be projected to or onto at least a portion of the fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the fluidic channel. In some embodiments, the emitting portion 805 may be coupled to an objective (e.g., a microscope objective or lens), where the objective may be moved to different portions of the fluidic device. The objective may provide a shape (e.g., virtual mask) to allow light to form a pattern on the fluidic device, in order to form a polymer matrix similar or complementary to the pattern. In various embodiments, the one or more polymer precursors in the fluidic device or mixed with the biological sample can absorb emitted energy 806. In some embodiments, the emitted energy 806 can form, or be sufficient to form, a polymer matrix from the one or more polymer precursors. For example, a portion of the one or more polymer precursors within the channel 800 of the fluidic device may be activated by the emitted energy and a polymerization reaction may be initiated to form a polymer matrix.

[0132] The energy source (e.g., light source) may be coupled to the fluidic device via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and / or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on at least a portion of a surface of the fluidic device. The polymer matrix may be formed similarly or complementary to the pattern of energy emission.

[0133] In some embodiments, a first polymer matrix 808 can be formed on or adjacent to a biological component 850. In certain embodiments, the first polymer matrix 808 can form a cylindrical analysis chamber or compartment 820 that separates (e.g., physically separates) the biological component 850 from other biological components (e.g., biological components 851, 852, or 853) in the fluidic device. Stated another way, the polymer matrix may compartmentalize the channel (e.g., channel 800) in cooperation with the first surface 801 and the second surface 802. In various embodiments, the polymer matrix may partially surround a biological component. For example, a polymer structure fully surrounding a biologicalAttorney Docket No. 59528-731601component may form a closed structure (e.g., a hollow cylinder-shaped polymeric structure) or a partially open structure (e.g., a crescent-shaped polymeric structure). In some embodiments, two or more polymer matrices may be formed adjacent to a biological component forming a compartment separating the biological component from other biological components. In certain embodiments, the polymer matrix may comprise or form a wall (e.g., a polymer matrix wall).

[0134] With continued reference to FIG. 8A, in some cases, the energy source 803 can, or be configured to, form or produce one or more emitting portions 805 and one or more non-emitting portions 804. In particular, a surface of the energy source 810 can include a plurality of micromirrors, pixels, or other structures configured to spatially modulate light output. The systems disclosed herein may further include a spatial energy modulating element to direct energy from the energy source to one or more targeted portions of the fluidic device. For example, the spatial energy modulating element may be configured to selectively direct the energy from the energy source to form a polymer matrix in a discrete area of the fluidic device. In some embodiments, the discrete area is chosen based on the location of a biological component. In some embodiments, the area of the discrete area is less than the area of the fluidic device. In some embodiments, a biological component is captured within the discrete area. In some embodiments, the size and shape of the discrete area is adjustable according to the size, shape, or other properties of the biological component. In some embodiments, an algorithm is used to determine the shape and size of the discrete area. In some embodiments, the algorithm is a supervised, a selfsupervised, or an unsupervised learning algorithm. The spatial energy modulating element may be configured to selectively direct the energy by, for example, inhibiting or preventing energy from being directed to one or more portions other than the one or more targeted portions of the fluidic device. In some embodiments, the spatial energy modulating element may comprise a physical mask. In some cases, the spatial energy modulating element may comprise a virtual mask. In some cases, the spatial energy modulating element may be a spatial light modulator (SLM). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid-crystal based.

[0135] In some embodiments, the first surface 801 or the second surface 802 may comprise a detector that detects, or is configured to detect, one or more locationsAttorney Docket No. 59528-731601of one or more biological components in the fluidic device (e.g., in the channel 800). In certain embodiments, the energy source 803 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In some embodiments, the detector may be a microscope objective for imaging the fluidic device. In various embodiments, a mask may be generated using an image obtained from at least a portion of the fluidic device. The mask may allow or permit the energy source 803 to emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 801. The mask may inhibit or prevent the energy source 803 from emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 801. In some embodiments, the image may be obtained from a camera (e.g., a digital camera, fluorescent imaging camera, etc.). In some embodiments, the imaging is bright-field imaging, phase-contrast imaging, or fluorescence imaging, or any combination thereof. In some embodiments, the camera may be coupled to, connected to, or in communication with the energy source 803. For example, the camera (not shown) may be in electrical communication with the energy source 803. In some embodiments, the energy source 803 may comprise the camera. In various embodiments, the energy source 803 may comprise a microscope (e.g., a fluorescence microscope, a confocal microscope, lens-free imaging system, a transmission electron microscopy (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used to detect one or more positions of one or more biological components (e.g., in combination with the detector).

[0136] In some embodiments, an algorithm is used to determine where a biological component or analyte is located based on the imaging. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. In some embodiments, the objective is coupled to an energy source to emit energy to the predetermined portion in the fluidic channel.

[0137] FIG. 8B shows polymer matrices 858, 859 formed surrounding the biological component 852 after being separated from the biological component 853. FIG. 8C shows a process, according to various embodiments, of separating the two biological components 850, 851, which are in close proximity. That is, by agitating or shaking the fluidic device the biological components 850 and 851 can be separated. In some embodiments, separation of the biological components is achieved through fluidic pressure, flow pulsation, dielectrophoresis,Attorney Docket No. 59528-731601optothermal flow, or some combination thereof. In some cases, separation of the biological components is achieved through acoustic vibration. FIG. 8C also shows a polymer matrix being formed to generate a compartment 822 surrounding the biological component 850 after the separation of the biological components 850, 851.

[0138] Methods and compositions disclosed herein can be utilized for sequence analysis of the immune repertoire. Analysis of sequence information underlying the immune repertoire can provide a significant improvement in understanding the status and function of the immune system.

[0139] Non-limiting examples of immune cells which can be analyzed utilizing the methods described herein include B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells), natural killer cells, cytokine induced killer (CIK) cells; myeloid cells, such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes / hypersegmented neutrophils), monocytes / macrophages, mast cell, thrombocytes / megakaryocytes, and dendritic cells. In some embodiments, individual T cells are analyzed using the methods disclosed herein. In some embodiments, individual B cells are analyzed using the methods disclosed herein.

[0140] Immune cells express various adaptive immunological receptors relating to immune function, such as T cell receptors and B cell receptors. T cell receptors and B cells receptors play a part in the immune response by specifically recognizing and binding to antigens and aiding in their destruction.

[0141] The T cell receptor, or TCR, is a molecule found on the surface of T cells that is generally responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is generally a heterodimer of two chains, each of which is a member of the immunoglobulin superfamily, possessing an N-terminal variable (V) domain, and a C terminal constant domain. In humans, in 95% of T cells the TCR consists of an alpha (a) and beta (P) chain, whereas in 5% of T cells the TCR consists of gamma and delta (y / 8) chains. This ratio can change during ontogeny and in diseased states as well as in different species. When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction.

[0142] Each of the two chains of a TCR contains multiple copies of gene segments — a variable ‘V’ gene segment, a diversity ‘D’ gene segment, and a joining T gene segment. The TCR alpha chain is generated by recombination of V and JAttorney Docket No. 59528-731601segments, while the beta chain is generated by recombination of V, D, and J segments. Similarly, generation of the TCR gamma chain involves recombination of V and J gene segments, while generation of the TCR delta chain occurs by recombination of V, D, and J gene segments. The intersection of these specific regions (V and J for the alpha or gamma chain, or V, D and J for the beta or delta chain) corresponds to the CDR3 region that is important for antigen-MHC recognition. Complementarity determining regions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, are sequences in the variable domains of antigen receptors (e.g., T cell receptor and immunoglobulin) that can complement an antigen. Most of the diversity of CDRs is found in CDR3, with the diversity being generated by somatic recombination events during the development of T lymphocytes. A unique nucleotide sequence that arises during the gene arrangement process can be referred to as a clonotype.

[0143] The B cell receptor, or BCR, is a molecule found on the surface of B cells. The antigen binding portion of a BCR is composed of a membrane-bound antibody that, like most antibodies (e.g., immunoglobulins), has a unique and randomly determined antigen-binding site. The antigen binding portion of a BCR includes membrane-bound immunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, or IgE). When a B cell is activated by its first encounter with a cognate antigen, the cell proliferates and differentiates to generate a population of antibodysecreting plasma B cells and memory B cells. The various immunoglobulin isotypes differ in their biological features, structure, target specificity and distribution. A variety of molecular mechanisms exist to generate initial diversity, including genetic recombination at multiple sites.

[0144] The BCR is composed of two genes IgH and IgK (or IgL) coding for antibody heavy and light chains. Immunoglobulins are formed by recombination among gene segments, sequence diversification at the junctions of these segments, and point mutations throughout the gene. Each heavy chain gene contains multiple copies of three different gene segments — a variable ‘V’ gene segment, a diversity ‘D’ gene segment, and a joining T gene segment. Each light chain gene contains multiple copies of two different gene segments for the variable region of the protein — a variable ‘V’ gene segment and a joining T gene segment. The recombination can generate a molecule with one of each of the V, D, and J segments. Furthermore, several bases may be deleted and others added (called N and P nucleotides) at each ofAttorney Docket No. 59528-731601the two junctions, thereby generating further diversity. After B cell activation, a process of affinity maturation through somatic hypermutation occurs. In this process progeny cells of the activated B cells accumulate distinct somatic mutations throughout the gene with higher mutation concentration in the CDR regions leading to the generation of antibodies with higher affinity to the antigens. In addition to somatic hypermutation activated B cells undergo the process of isotype switching. Antibodies with the same variable segments can have different forms (isotypes) depending on the constant segment. Whereas all naive B cells express IgM (or IgD), activated B cells mostly express IgGbut also IgM, IgA and IgE. This expression switching from IgM (and / or IgD) to IgG, IgA, or IgE occurs through a recombination event causing one cell to specialize in producing a specific isotype. A unique nucleotide sequence that arises during the gene arrangement process can similarly be referred to as a clonotype.

[0145] In some embodiments, the methods, compositions and systems disclosed herein are utilized to analyze the various sequences of TCRs and BCRs from immune cells, for example various clonotypes. In some embodiments, methods, compositions and systems disclosed herein are used to analyze the sequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain, a TCR gamma chain, or any fragment thereof (e.g., variable regions including VDJ or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof). In some embodiments, methods, compositions and systems disclosed herein are used to analyze the sequence of a B cell receptor heavy chain, B cell receptor light chain, or any fragment thereof (e.g., variable regions including VDJ or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof).

[0146] Where immune cells are to be analyzed, primer sequences useful in any of the various operations for attaching barcode sequences and / or amplification reactions may comprise gene specific sequences which target genes or regions of genes of immune cell proteins, for example immune receptors. Such gene sequences include, but are not limited to, sequences of various T cell receptor alpha variable genes (TRAV genes), T cell receptor alpha joining genes (TRAJ genes), T cell receptor alpha constant genes (TRAC genes), T cell receptor beta variable genes (TRB V genes), T cell receptor beta diversity genes (TRBD genes), T cell receptor beta joining genes (TRB J genes), T cell receptor beta constant genes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), T cell receptor gamma joiningAttorney Docket No. 59528-731601genes (TRGJ genes), T cell receptor gamma constant genes (TRGC genes), T cell receptor delta variable genes (TRDV genes), T cell receptor delta diversity genes (TRDD genes), T cell receptor delta joining genes (TRDJ genes), and T cell receptor delta constant genes (TRDC genes).

[0147] The present disclosure also provides methods of enriching cDNA sequences. Enrichment may be useful for TCR, BCR, and immunoglobulin gene analysis since these genes may possess similar yet polymorphic variable region sequences. These sequences can be responsible for antigen binding and peptide-MHC interactions. For example, due to gene recombination events in individual developing T cells, a single human or mouse can naturally express many thousands of different TCR genes. This T cell repertoire can exceed 100,000 or more different TCR rearrangements occurring during T cell development, yielding a total T cell population that is highly polymorphic with respect to its TCR gene sequences especially for the variable region. For immunoglobulin genes, the same may apply, except even greater diversity may be present. As previously noted, each distinct sequence may correspond to a clonotype. In certain embodiments, enrichment increases accuracy and sensitivity of methods for sequencing TCR, BCR and immunoglobulin genes at a single cell level. In certain embodiments, enrichment increases the number of sequencing reads that map to a TCR, BCR, or immunoglobulin gene.

[0148] Surfaces of fluidic devices or flow cells can be functionalized with one or more types of oligonucleotides. In some cases, a surface of a flow cell (or a hydrogel) is functionalized with at least two distinct types of oligonucleotides.

[0149] A first type of oligonucleotide can be a hybridization domain complementary to a target on interest. This first type of oligonucleotide can be a capture probe that hybridizes to a cellular species, such as a nucleic acid. In particular, a capture probe can couple to a messenger ribonucleic acid (mRNA) molecule. In some cases, a capture probe has a poly(T) tail that hybridizes to a poly(A) tail of an mRNA molecule. The poly(A) tail can be located on a 3’ end of the mRNA molecule. FIG. 1 illustrates a capture probe 100 with a poly(T) tail 110 that hybridizes to a poly(A) tail of a ribonucleic acid (RNA) molecule. The poly(T) tail 110 may comprise any number of thymine (T) bases and optionally may end with VN (where V = A or G or C and N = A or G or C or T). In some cases, the poly(T) tail 110 has between 20-40 T bases. The poly(T) tail 110 can be T30VN. The capture probe 100 can alsoAttorney Docket No. 59528-731601include a handle 105 (e.g., a PCR handle complementary to a particular primer sequence). The handle 105 can be a common sequence across all capture probes that enables PCR amplification. Alternatively, the handle 105 can be a PCR suppression sequence to facilitate suppressive or semi-suppressive PCR. For example, the handle 105 can include an incomplete TSR1 sequence complementary to a portion of a capture probe adapter sequence 305. As depicted in FIG. 5B, this intrastrand complementarity can allow first and second product strands generated according to the method FIGS. 4A-4K to self-hybridize, which can suppress amplification of spurious reverse transcription and extension products during downstream analysis. It is worth noting that the capture probe 100 may include capture sequences other than poly(T) tails 110, such as gene-specific primers, random hexamers, and the like. Handle 105 may also optionally be absent, such that the capture probe 100 consists of a capture sequence such as a poly(T) sequence 100 or gene-specific primer.

[0150] The capture probe and barcode primer can also comprise cleavage domains that allow for controlled release of first and second product strands from a flow cell surface or other substrate. Following first and second product strand generation, these molecules can be cleaved from a surface, eluted from a fluidic device, and collected for downstream analysis.

[0151] Once hybridized to the capture probe, the target of interest (e.g., a messenger RNA) can be reverse transcribed onto a 3’ end of the capture probe, thereby generating a cDNA sequence complementary to at least a portion of the mRNA on the capture probe. This process can generate an oligocytosine overhang 3’ to the cDNA sequence.

[0152] As illustrated in FIG. 4D, a template switching oligonucleotide (TSO, 207A) can be coupled to a 3 ’-end of the cDNA 500 (e.g., hybridized to a terminal oligocytosine overhang). The TSO 207A can comprise the known sequence 200 (FIGS. 2A, 3 A), which can serve as a template for cDNA extension. The template switching oligonucleotide (TSO) 207A can include a targeting sequence, such as an rGrGrG or rNrNrN terminal sequence, that can be hybridized to a terminal overhang 501 of the cDNA and used as a template for amplification or reverse transcription. The TSO 207A, and in particular cases an rGrGrG region 201 of the TSO 207A, can include one or more non-canonical or non-naturally occurring nucleotides, including one or more locked nucleotides (LNAs), 2’ O-Me nucleotides, 2’ O-Et nucleotides, 2’ S-Me nucleotides, fluoroarabino (FANA) nucleotides, dideoxynucleotides,Attorney Docket No. 59528-731601isonucleotides (e.g., isoguanosine or isocytosine), 2'-04'-C-ethylene-bridged nucleotides (ENAs), 4’-thio nucleotides, and the like. The TSO 207A, and in particular cases an rGrGrG region of the TSO 207A, can also include one or more modified linkages, including phosphorothioate linkages, pho sphoroami date linkages, phosphorodiamidate morpholino oligomer (PMO) linkages, thiophosphoramidate morpholino (TMO) linkages, borano-phosphate linkages, 3 ’3 ’-reversed linkages, or 5 ’-5 ’-reversed linkages. The TSO 207A can also include a 3’-end such as 3’-phosphate, 3’-amino, 3’-O-alkyl, 2’,3’-cyclic nucleotides, dideoxynucleotides, and the like; a 5’-end blocking group such as 5’-O-NH2, 5’-O-alkyl, 5’-0-DMTr, and the like; or a 3 ’-end blocking group and a 5 ’-end blocking group. Once the TSO 207A and the cDNA sequence 500 are hybridized, the TSO 207A can optionally be ligated to the mRNA 400. For example, as shown in FIG. 4D, the TSO 207A can be ligated to the mRNA 400 after hybridizing to a cDNA 500 terminal overhang 501.

[0153] In some cases, the TSO 207A includes a unique molecular identifier. The presence of a unique molecular identifier on the TSO 207A and optionally on the capture probe 100 or the barcode primer 300 can prevent UMI count inflation during multiple turnaround steps, wherein multiple barcode primers can extend over a single first product strand and multiple capture probes can extend over a single second product strand. Accordingly, in some aspects disclosed herein, the TSO 207A comprises a first UMI and the barcode primer 300 comprises a second UMI. In other aspects, the TSO 207A comprises a first UMI and the capture probe 100 comprises a second UMI. In further aspects, the capture probe 100 comprises a first UMI and the barcode primer 300 comprises a second UMI.

[0154] The cDNA sequence 500 can then be extended over the TSO 207A. Alternatively, the complement of the known sequence 600A can be added to the cDNA sequence without extension over a TSO, for example using ligation (e.g., Single Reaction Single-stranded LibrarY preparation as disclosed in Troll etal., BMC Genomics (2019) 20:1023; terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation as disclosed in Miura etal., Nucleic Acids Research, 2019; 47(15):e85; transposase-mediated ligation as disclosed in Hennig et al., G3 (Bethesda), 2018; S(l): 79-89) or amplification (e.g., amplification with random primers as disclosed in Grothues etal., Nucleic Acids Research, 1993; 27(5): 1321-1322). The known sequence 200 (‘SS’) and / or theAttorney Docket No. 59528-731601complement of the known sequence 600A (‘cSS’) can be around 20-40 nucleotides long.

[0155] In some cases, a unique molecular identifier (UMI) 205 or a complement of the UMI can be added to the cDNA sequence 500 coupled to a 3’ end of the capture probe along with the complement of the known sequence. FIG. 3 A illustrates TSO 207B that contains a known sequence 200 and a unique molecule identifier (UMI) 205. Complements of these sequences can then be added to the 3’ end of cDNA sequence 500 by extension. It is worth noting that when the templateswitch oligonucleotide 207A includes a unique molecular identifier 205, the barcode primer 300 may not include a unique molecular identifier 205. In such cases, the barcode primer 300 may include, in order, the adapter sequence 305, the one or more barcode sequences 310, and the at least a portion of the known sequence 200A.

[0156] A second type of oligonucleotide in the compositions, devices, systems, and methods disclosed herein can be a barcode primer. The barcode primer can include one or more barcode sequences. The barcode sequences can add additional information to a cDNA sequence (e.g., a cDNA sequence 500 comprising a complement of a known sequence 600A derived from a TSO 207A). The barcode primer and capture probe can be coupled to a single substrate, such as a surface of a flow cell. In such cases, the barcode primer can be in sufficient proximity to the capture probe to allow a cDNA sequence 500 (or a complement of a known sequence 600A or another extension of the cDNA) that is coupled to the capture probe to hybridize to the barcode primer. In some cases, the one or more barcode sequences comprise a spatial location tag corresponding to a unique location of the barcode primer on a surface of the fluidic device.

[0157] As shown in FIG. 4G, at least a portion of the complement of the known sequence 600A, which can be 3’ to the cDNA sequence 500, can hybridize to the known sequence or the portion of the known sequence (e.g., 200A) of the barcode primer 300. The complement of the known sequence 600A on the extended capture probe may be shorter, equal in length to, or longer than the known sequence 200A on the barcode primer 300. In some cases (for example as depicted in subsequent FIG.4H), the 3’ end of the complement of the known sequence 600A may be hybridized to the known sequence 200A, and the 3’ end of the known sequence 200A may be hybridized to the complement of the known sequence 600A, such that the complement of the known sequence 600A and the known sequence 200A may beAttorney Docket No. 59528-731601extended. However, it is worth noting that in some implementations of the disclosed methods, only one of the complement of the known sequence 600A and the known sequence 200A are configured for further extension. For example, in some cases, the complement of the known sequence 600A includes a 3 ’-terminal sequence that does not hybridize to the known sequence 200A and thus prevents or limits extension from the 3’ end of the complement of the known sequence 600A using the barcode primer (or a portion of the barcode primer) 300 as a template. The barcode primer can also include a UMI and / or an adapter sequence. As non-limiting examples, the adapter sequence 305 can be TruSeq Read 1, TruSeq Read 2, Nextera Read 1, or Nextera Read 2 sequence. However, any sequence-complement pair that facilitates hybridization between the known sequence (e.g. 200A) in the barcode primer 300 and the known sequence 600A at the 3’ end of the cDNA 500 may be utilized herein. A barcode primer can comprise an attachment or immobilization sequence for a given sequencing system, e.g., a P5 sequence used for attachment in flow cells of an Illumina Hiseq® or Miseq® system.

[0158] FIG. 2B illustrates a barcode primer 300 with an adapter sequence 305, one or more barcode sequences 310, a unique molecular identifier (UMI) 205, and at least a portion of known sequence 200A that is configured to hybridize to at least a portion of a sequence 600A located at or near a 3’ end of the cDNA sequence 500. Since a UMI is not added to the end of the cDNA sequence 600A by the TSO 207A depicted in FIG. 2A, which includes known sequence 200 and a terminal rGrGrG sequence 201, the barcode primer 300A in FIG. 2B includes a UMI 205. The barcode primer can include the following sequences, listed from 5’ to 3’: a known adapter sequence, one or more barcode sequences, a UMI, and at least a portion of the known sequence of which a complement was added to the 3’ end of the cDNA sequence 500 (e.g., using a TSO). The 5’ end of the barcode primer can be attached to a surface of the fluidic device. The adapter sequence 305 can be identical or highly homologous to the handle 105 on the capture probe 100 to facilitate suppressive or semi-suppressive amplification (e.g., PCR) of cDNA products. Such designs can suppress amplification of spurious products, such as DNA generated from self-hybridization between two capture probes 100 or between a barcode primer 300A and a capture probe 100.

[0159] FIG. 3B illustrates a barcode primer 300B with an adapter sequence 305, one or more barcode sequences 310, and at least a portion of known sequence 200B used as a template for cDNA extension (e.g., as shown in FIG. 3 A). BecauseAttorney Docket No. 59528-731601FIG. 3 A depicts a TSO 207B that contains a UMI 205, of which a complement can be added to the 3’ end of the cDNA sequence 500 coupled to the capture probe 100, the barcode primer shown in FIG. 3B does not require an additional UMI.

[0160] The barcode primer can include the following sequences, listed from 5’ to 3’ : a known adapter sequence, one or more barcode sequences, and at least a portion of the known sequence of the TSO, for example). The 5’ end of the barcode primer can be attached to a surface of the fluidic device. In some cases, the barcode primer and capture probe are coupled to the same surface within a microfluidic device, and are in sufficient proximity to allow a cDNA sequence 500 or an extension of the cDNA sequence 500 that is coupled to the capture probe to hybridize to the barcode primer.

[0161] FIGS. 4A-K depict a method for mRNA molecule 400 capture on a capture probe 100; extension of the capture probe 100 using at least a portion of an mRNA molecule 400 and at least a portion of a template-switch oligonucleotide 207A as templates; coupling the resultant extended nucleic acid capture probe to a barcode primer 300; and extension of the barcode primer 300 using at least a portion of the extended nucleic acid capture probe as a template, extension of the extended nucleic acid capture probe using at least a portion of the barcode primer 300 as a template, or a combination thereof. One or more of these steps may be performed in a compartment such as a hydrogel chamber. In some cases, the mRNA molecule 400 is released from a biological material such as a cell inside of a compartment that coencloses the biological material and the capture probe 100. In some cases, the compartment is degraded after the mRNA molecule 400 is released from the biological material and couples to the capture probe 100. In such cases, compartment degradation facilitates reagent flow to the mRNA molecule 400, the capture probe 100, and the barcode primer 300 by removing portions of the compartment (e.g., polymer matrix walls) that may block the flow of the reagents. In other cases, one or more subsequent steps may be performed while the compartment is intact. In some cases, extension of the capture probe 100 using at least a portion of an mRNA molecule 400 and at least a portion of a tempi ate- switch oligonucleotide 207A as templates are performed in the compartment. In some cases, coupling the resultant extended nucleic acid capture probe to a barcode primer 300 is performed in the compartment. In some cases, extension of the barcode primer 300 using at least a portion of the extended nucleic acid capture probe as a template, extension of theAttorney Docket No. 59528-731601extended nucleic acid capture probe using at least a portion of the barcode primer 300 as a template, or the combination thereof are performed in the compartment. In such cases, reagents such as reverse transcriptases, DNA polymerases, small molecules and the like may flow through openings or pores in polymer matrix walls of the compartment. In some cases, a product strand (e.g., 719A and 719B in FIG. 41) is released from a surface and eluted from a fluidic device for analysis. In some such methods, a compartment, when present, may be degraded prior to the release and / or eluting. In other such methods, the compartment may not be degraded prior to the release and / or elution.

[0162] FIG. 4A illustrates a surface with a capture probe 100 and a barcode primer 300 disposed thereon. The surface can comprise a plurality of capture probes and a plurality of barcode primers. The capture probe 100 can be similar to that described in FIG. 1. The barcode primer 300 can be similar to those described in FIG.2B (300A) or FIG. 3B (300B).

[0163] A cell can be introduced into the fluidic device. One or more intercellular components can be released from the cell, including messenger RNA (mRNA). FIG. 4B illustrates an mRNA molecule 400 with a poly(A) tail hybridized to a poly(T) tail of the capture probe 100. In some cases, the mRNA molecule 400 includes a first nucleic acid sequence 405 and a second nucleic acid sequence 410. The first nucleic acid sequence 405 can be located closer to the 5’ end of the mRNA molecule as compared to the second nucleic acid sequence 410. In some cases, the first nucleic acid sequence 405 comprises a variable region. The variable region can be a T cell receptor variable region sequence, a B cell receptor variable region sequence, or an immunoglobulin variable region sequence, for example. In some cases, the second nucleic acid sequence 410 comprises a constant region. The constant region can be a constant region of a T cell receptor nucleic acid sequence, a constant region of a B cell receptor nucleic acid sequence, or a constant region of an immunoglobulin nucleic acid sequence, for example.

[0164] The mRNA 400 can be reverse transcribed onto the capture probe 100, producing a cDNA sequence 500 coupled to the capture probe 100 and comprising a sequence that is complementary to the nucleic acid sequence of the RNA molecule 400. The cDNA sequence 500 can then be further extended to include a complement of the known sequence 600A (and optionally further sequences). For example, an oligocytosine overhang may be 3’ to the cDNA sequence 500. A tempi ate- switchingAttorney Docket No. 59528-731601oligonucleotide can be hybridized to the overhang and optionally coupled (e.g., ligated) to the mRNA. The cDNA sequence 500 can then be further extended using the template-switching oligonucleotide. An example of a product generated by this process is illustrated in FIG. 4E, which shows the capture probe 100 coupled to cDNA comprising a sequence 500 that is complementary to the nucleic acid sequence of the RNA molecule 400 and a sequence 600A that is complementary to the known sequence 200 of the template-switching oligonucleotide. FIG. 4F illustrates the extended capture probe 100 comprising the cDNA sequence 500 and the complement of the known sequence 600A after the captured mRNA molecule and TSO 207A has been removed from the fluidic device, in accordance with some embodiments.

[0165] Alternatively, as shown in FIG. 4E, a known sequence 200 can be inserted at a 5’ end of the mRNA molecule prior to capture probe 100 extension. The known sequence 200 can be similar to that described in FIG. 2A and FIG. 3 A. The capture probe 100 can be extended, using the mRNA molecule as a template, to generate a complementary deoxyribonucleic acid (cDNA) sequence 500 that is complementary to the nucleic acid sequence of the RNA molecule 400. Extension reaction reagents, e.g., DNA polymerase, nucleoside triphosphates, co-factors (e.g., Mg2+or Mn2+) can be used to extend the primer sequence using the mRNA template to produce a complementary cDNA sequence. The extension may proceed to the known sequence 200, thereby resulting in the cDNA sequence also having a sequence 600A that is complementary to the known sequence 200. FIG. 4F illustrates the cDNA sequence after the captured mRNA molecule has been removed from the fluidic device, in accordance with some embodiments.

[0166] As described in reference to FIGS. 2B and 3B, a barcode primer can include at least a portion of the known sequence 200A, 200B that is complementary to sequence 600A. That is, the barcode primer can comprise a portion of the TSO known sequence 200. Alternatively or in addition thereto, the barcode primer can include the full length of the known sequence 200A, 200B. Therefore, as shown in FIG. 4G, the 3’ end of the extended capture probe 100 can include sequence 600A, which can hybridize to at least a portion of a barcode primer 300.

[0167] As illustrated in FIG. 4H,the extension step illustrated in FIG. 4H can result in the extended capture probe comprising the cDNA sequence 500 and the complement of the known sequence 600A extending over the capture probe 300, thereby generating a first product strand that includes a 3 ’ sequence 600BAttorney Docket No. 59528-731601complementary to a 5’ sequence of the barcode primer 300. This sequence can be complementary to the UMI 205, barcode sequences 310, and adapter sequence 305 of the barcode primer 300. The barcode primer 300 can also be extended using the extended capture probe 100 comprising the cDNA sequence 500 as a template, thereby generating a second product strand (also referred to as a “tagged molecule” herein) comprising a sequence 700 that is complementary to cDNA sequence 500 and a sequence 790 that is complementary to the capture probe 100. Sequence 700 may include a portion that is identical (or has at least 75%, 85%, 90%, 95%, or 99% sequence homology) to sequence 400 of the mRNA molecule except that the uracil bases are replaced with thymine bases. The second product strand can also include a sequence 790 that is complementary to sequence 100 of the capture probe.

[0168] FIG. 41 illustrates the first product strand 719A (i.e., the extended form of the capture probe 100 illustrated in FIG. 4H), the second product strand 719B (i.e., the extended form of the barcode primer 300 illustrated in FIG. 4H), an additional capture probe, and an additional barcode primer 350 on the surface. The second product strand can include, from a 5’ to a 3’ end: one or more barcode sequences from the barcode primer 300, a modified first nucleic acid sequence 705, and a modified second nucleic acid sequence 710. The first modified nucleic acid sequence 705 can be identical (or have at least 75%, 85%, 90%, 95%, or 99% sequence homology) to the first nucleic acid sequence of the RNA molecule 405 except that all uracil bases are replaced with thymine bases. The modified second nucleic acid sequence 710 can be identical (or have at least 75%, 85%, 90%, 95%, or 99% sequence homology) to the second nucleic acid sequence of the RNA molecule 410 except that all uracil bases are replaced with thymine bases.

[0169] As shown in FIG. 4J, the surface can include an additional barcode primer 350. The additional barcode primer 350 can be identical to the barcode primer 300. The additional barcode primer can include at least a portion of the known sequence 200, which is complementary to sequence 600. Therefore, as shown in FIG.4J, the 3’ end of the first product strand 719A, which can include sequence 600A as well as a sequence complementary to 5’ sequences of the primer 600B, can hybridize to at least a portion of additional barcode primer 350. This hybridization step or a melting and rehybridization step (Fig. 4J) followed by subsequent extension (Fig 4K) may be referred to as a “turnaround.” In various embodiments, it is worthwhile to note that after the “turnaround”, the 5’ end of the complement to the cDNA is coupled toAttorney Docket No. 59528-731601the barcoded primer 300. A method may utilize any number of turnaround steps, including about 1 to 5, about 1 to 10, about 1 to 15, about 1 to 20, about 1 to 25, about 1 to 30, about 1 to 50, about 5 to 10, about 5 to 15, about 5 to 20, about 5 to 25, about 5 to 30, about 5 to 50, about 10 to 15, about 10 to 20, about 10 to 25, about 10 to 30, about 10 to 50, about 15 to 20, about 15 to 25, about 15 to 30, about 15 to 50, about 20 to 25, about 20 to 30, about 20 to 50, or about 25 to 50.

[0170] As illustrated in FIG. 4K, the additional barcode primer 350 can then be extended, using the first product strand 719A as a template and, for example, a strand-displacing DNA polymerase, thereby generating an additional second product strand 719C comprising a sequence 700 that is complementary to sequence 500 of the first product strand 719A. Sequence 700 may include a portion that is identical or similar to (or has at least 75%, 85%, 90%, 95%, or 99% sequence homology) to sequence 400 of the mRNA molecule except that the uracil bases are replaced with thymine bases. The additional second product strand 719C can also include a sequence 790 that is complementary to sequence 100 of the capture probe. Sequence 700 can be identical (or have at least 75%, 85%, 90%, 95%, or 99% sequence homology) to sequence 700. The second product strand 719B and additional second product strand 719C may be identical.

[0171] In one embodiment, the surface comprises equal or approximately equal numbers of the capture probe and barcode primer. In other instances, the surface comprises a greater number of capture probes than barcode primers. For example, the surface can comprise about 1.5 to 5, about 5 to 50, about 25 to 250, or about 100 to 500 times the number of capture probes as barcode primers. Such designs may be suitable, for example, for capturing and tagging target nucleic acids with low captureprobe binding affinities, such as target nucleic acids with high degrees of secondary and tertiary structure. Alternatively, the surface can comprise a greater number of barcode primers than capture probes. For example, the surface can comprise about 1.5 to 5, about 5 to 50, about 25 to 250, or about 100 to 500 times the number of capture probes as barcode primers. Excess barcode primers can favor second product strand generation through multiple turnaround steps.

[0172] The first product strand 719A and / or second product strand 719B can be detached from the surface. For example, the capture probe and barcode primer can each comprise a nuclease recognition sequence, a photocleavable moiety, a pH cleavable moiety, or a chemically cleavable moiety that allows them to be selectivelyAttorney Docket No. 59528-731601detached from the surface. The capture probe and barcode primer can alternatively or additionally be coupled to one or more species on the surface, and the one or more species can be detached from the surface of the flow cell. As an example, capture probes and barcode primers may be coupled to a protein or oligopeptide, wherein the protein or oligopeptide is coupled to a surface of a fluidic device, and the protein or oligopeptide may be hydrolyzed or otherwise released from the surface using a basic buffer and / or a protease.

[0173] FIG. 5 A illustrates an exploded view of the 5’ end of a double-stranded complex of the first product strand 719A and the second product strand 719B of FIG.41. Sequence 600B of the first product strand can hybridize to sequences 305 and 310 of the second product strand, sequence 600A of the first product strand can hybridize to 200B of the second product strand, cDNA sequence 500 of the first product strand can hybridize to cDNA complement sequence 700 of the second product strand, and barcode primer sequence 100 of the first product strand can hybridize to complementary sequence 790 of the second product strand.

[0174] As illustrated in FIGS. 4H-4K, the barcode primer 300 can be extended from a 5’ end to a 3’ end using the first product strand 719A (or an extended capture probe 100 comprising a cDNA sequence 500 and a complement of a known sequence 600A) as a template to generate a second product strand 719B with sequence 700 that is complementary to sequence 500 (i.e., a complement of the mRNA sequence that served as a template for sequence 500) of the cDNA sequence. First and second product strands may then rehybridize to unexentended capture probes 100 and barcode primers 300, and undergo further extension reactions to continue this turnaround process (see FIGS. 4I-4K), thereby generating a number of copies of the first and second product strands. The process can be repeated until the desired number of copies of the first and second product strands are generated. One of the first or second product strand, once generated, may be disfavored from performing turnaround relative to the other product strand due to the AT content of sequence 790.

[0175] Once sufficient copies of first and / or second product strands are generated, the first and / or second can be sequenced. Prior to sequencing, the first and / or second product strands can optionally be amplified and further tagged. FIGS.6A-6C depict methods for tagging first and / or second product strands for downstream sequencing, such as Illumina next generation sequencing. FIG. 6A illustrates a genespecific tagging and amplification method for generating tagged amplicons thatAttorney Docket No. 59528-731601contain a predetermined sequence. FIGS. 6B-6C illustrate sequence non-specific tagging and amplification methods that cleave and affix adapters at random locations along cDNA molecules (e.g., a double stranded cDNA molecule that includes first and second product strands). As used herein, a sequence non-specific tagging and amplification method may be a method that acts randomly along a nucleic acid, or may be a method that acts on short nucleic acid sequences that are typically present throughout a transcriptome or genome. A sequence non-specific method may include minor biases for particular sequence motifs, epigenomic modifications, GC-content, or other nucleic acid features. While FIGS. 6A-C are provided as examples of methods consistent with the present disclosure, additional gene-specific and sequence non-specific tagging and amplification methods are contemplated herein, including homologous recombination, 5 ’-RACE, nickase-mediated, Cas nuclease-mediated, and random priming methods. In addition to the steps depicted in FIGS. 6A-6C, first and / or second product strands may be amplified (e.g., using capture probe and 5’-barcode sequences as priming sites or using random primers).

[0176] FIG. 6 A illustrates gene-specific amplification of a 5’ portion of a double-stranded nucleic acid 719 of a second product strand 719B that includes, from 5’ to 3’, a barcode primer 300, a complement of a cDNA molecule 700 with a 5’ sequence of interest 705 and an additional 3’ sequence 710, and a complement of the capture probe 790; and a first product strand 719A that can include, from 3’ to 5’, a complement of the barcode primer 600, cDNA 500 comprising a sequence of interest 505 (a complement of cDNA sequence 705) and an additional sequence 510 (a complement of cDNA sequence 710), and a capture probe 100. It is worthwhile to note that sequence 405 of mRNA 400 corresponds to the reverse transcription cDNA product sequence 505 and that sequence 705 corresponds to a complement of the cDNA product sequence 505. Similarly, sequence 410 of mRNA corresponds to the reverse transcription cDNA product sequence 510 and that sequence 710 corresponds to a complement of the cDNA product sequence 510. In various embodiments, 5’ sequence 705 can encode the variable region of a T-cell receptor (TCR), and a 3’ sequence 710 can encode the constant region of the TCR. A first set of primers 701A and 701B can be hybridized at step 701 to barcode primer 300 and the additional sequence 510, respectively. In this example, primer 701B is a gene-specific primer, while primer 701A primes off of the barcode primer 300. The first set of primers 701A, 701B can cause the formation of cDNA 702A that includes at least a portion ofAttorney Docket No. 59528-731601the barcode primer 300, the 5’ sequence of interest 705, and optionally a portion of the additional 3’ sequence 710. The double-stranded nucleic acid 719 can then be amplified at step 702 using the first set of primers 701A, 701B. The resulting amplicons 702A can then be subject to a second amplification step with a set of tagged primers 702B, 702E. In this example, tagged primer 702E is a gene-specific primer, while tagged primer 702B primes off of the barcode primer 300. Tagged primer 702B, can include a sequence 702C and a primer tag 702D. Tagged primer 702E, can include a sequence 702F and a primer tag 702G. Sequence 702C and 702F are both complementary to separate portions of the amplicon 702A. The second amplification step 703 can generate tagged amplicons 703A that include, from 5’ to 3’ along a first strand, a first primer tag 702D, at least a portion of the barcode primer 300, the 5’ sequence of interest 705, optionally a portion of the additional 3’ sequence 710, and a complement of a second primer tag 702G’. The primer tags 702D, 702G can comprise sequencing adapters, for example for next-generation sequencing (NGS) on an Illumina NextSeq instrument, such that the amplification product 703A is suitable for sequencing. In some cases, the primer 702B does not comprise the first primer tag 702D, and the capture probe 300 serves as a sequencing adaptor. While FIG. 6A illustrates an amplification method with two sets of primers, methods that utilize a single set of gene-specific primers (e.g., 702B and 702E but not 701A and 701B) are also applicable to the disclosed DNA products (e.g., first and second product strands).

[0177] FIG. 6B illustrates a sequence non-specific tagmentation method utilizable for whole-transcriptome analysis (WTA). In this example, a doublestranded nucleic acid 719 that includes a first product strand 719A and a second product strand 719B. Double-stranded nucleic acid 719 is coupled to a transposase configured for sequence non-specific tagmentation in step 720, for example with a Tn5 transposase 721 (e.g., tagmentation enzyme). The second product strand 719B of the double-stranded nucleic acid 719 includes a barcode primer 300 and a complement of a cDNA molecule 700 with a 5’ sequence of interest 705, an additional 3’ sequence 710, and a sequence 790 to the capture probe. The first product strand 719A includes a complement of the barcode primer 600, cDNA 500 (corresponding to the reverse transcription of mRNA sequence portion 400), and the capture probe 100. The ends of the double-stranded nucleic acid molecule may be truncated during cleavage from a surface (e.g., a top or bottom surface of a channel inAttorney Docket No. 59528-731601a fluidic device). For step 730, tagmentation enzyme 721 cleaves and incorporates barcodes 722 and 722’, at a random internal position along the double-stranded nucleic acid, thereby generating a double-stranded nucleic acid fragment 724 with barcodes 722 and 722’ a at terminal end opposite the barcode primer 300 and complement of the barcode primer 600. The double-stranded nucleic acid fragment 724 is then amplified using primers 731 and 732, as illustrated in steps 740 and 745.These primers are targeted to the barcode primer 300 and barcode 722 by complementary sequences 731A and 732A, respectively, and optionally contain additional barcodes 731B and 732B. When the primers 731 and 732 contain additional barcodes 731B and 732B, a first strand of the resultant amplicon 741 includes, in order from 5’ to 3’, primer barcode 731B’, the barcode primer 300, a fragment of the complement of the cDNA sequence 741A’, tagmentation barcode 722’, and primer barcode 732B. A second strand of the resultant amplicon includes, in order from 3’ to 5’, primer barcode 731B, the complement of the barcode primer 600, a fragment of the cDNA sequence 741A’, tagmentation barcode 722, and primer barcode 732B’. The tagmentation barcodes (722, 722’), and primer barcodes (731B, 732B), as well as the barcode primer 300, may be used for downstream sequencing, for example for next-generation sequencing (NGS) on an Illumina NextSeq instrument.

[0178] FIG. 6C illustrates a sequence non-specific fragmentation and ligation method utilizable for whole-transcriptome analysis. In this example, a doublestranded nucleic acid 719 comprised of a first product strand 719A and a second product strand 719B is subjected to sequence non-specific fragmentation. The second product strand 719B of the double-stranded nucleic acid 719 includes a barcode primer 300 and a complement of a cDNA molecule 700 with a 5’ sequence of interest 705 and an additional 3’ sequence 710, and sequence 790 that is complementary to the capture probe. The first product strand 719A includes a complement of the barcode primer 600, cDNA 500 comprising a sequence of interest 505 (a complement of cDNA sequence 705) and an additional sequence 510 (a complement of cDNA sequence 710), and a capture probe 100. . The ends of the double-stranded nucleic acid molecule may be truncated during cleavage from a surface (e.g., a top or bottom surface of a channel in a fluidic device). In an initial step 750, a nuclease 751 is contacted to a portion of a cDNA molecule to cleave 760 the double-stranded nucleic acid at an internal location, thereby generating a DNA fragment 761. The cleaved endAttorney Docket No. 59528-731601of the DNA fragment 761 includes an activated group 762 (also labeled with a circle A), such as a sticky end or 5’-phosphate, to facilitate ligation. Barcoded DNA molecule 761 can then be ligated 770 at the cleaved end of the DNA fragment 761 to form a tagged DNA fragment 771 with barcodes 771A and 771B (and the respective complements 771A’ and 771B’). The tagged DNA fragment 771 can optionally be amplified and subjected to downstream analysis 780 (e.g., sequencing).

[0179] In many aspects disclosed herein, cDNA molecules (e.g., first and / or second product strands) are pooled, amplified, and then divided into multiple pools for multiple forms of sequencing analysis. For example, first and / or second product strands generated from multiple single cell analyses may be pooled, amplified, and then split into two pools that are separately subjected to (i) TCR variable region analysis enabled by a TCR-specific primer and a 5’ barcode-specific primer, and (ii) whole transcriptome analysis enabled by random tagmentation. In general, first and / or second product strands may be pooled, amplified, and then split into two, three, four, five, six, eight, ten, twelve or more pools for separate forms of sequencing analysis. The amplification may generate full-length copies of the first and / or second product strands. For example, the amplification may utilize primers targeted to capture probe and barcode primer sequences.

[0180] Similarly, in certain aspects, first and / or second product strands are pooled and then divided into multiple pools for multiple forms of sequencing analysis without a prior amplification step. For example, first and / or second product strands (e.g., generated from multiple single cell analyses in a single fluidic device) may be pooled and then split into two, three, four, five, six, eight, ten, twelve or more pools for separate forms of sequencing analysis.

[0181] Amplicons can be generated using exponential amplification. As used herein, the term “exponential amplification” can refer to a nucleic acid amplification method that approximately doubles the amount of product nucleic acid during each cycle. Exponential amplification methods can be distinguished from linear amplification methods, which generate approximately equal amounts of product nucleic acid during each cycle. Amplicons can be generated using nested amplification. In some cases, full-length amplicons of the first product strand, the second product strand, and / or a double-stranded nucleic acid complex comprising the first and second product strands are generated in an initial amplification step. These full-length amplicons may then be subjected to tagging and / or cleavage methods (e.g.,Attorney Docket No. 59528-731601a method depicted in FIG. 6A-6C) to prepare the amplicons for downstream sequencing analysis. A population of amplicons may be divided into multiple pools that are subjected to different tagging and / or cleavage methods. For example, a first portion of a population of amplicons may be subjected to amplification with a genespecific primer (e.g., a TCR constant region-targeted primer) for gene-specific analysis, while a second portion of the population of amplicons may be subjected to random tagmentation for whole transcriptome analysis. In some cases, tagged and / or fragmented nucleic acids (e.g., nucleic acid molecules 703A, 741, and 771 generated in the methods shown in FIGS. 6A-6C, respectively), may be amplified prior to analysis (e.g., sequencing).

[0182] An amplification method can be suppressive or semi-suppressive. In particular, the adapter sequence 305 (e.g., see FIG. 2B) can be identical or highly homologous to the handle 105 (e.g., see FIG. 1) on the capture probe 100 to promote self-hybridization in nucleic acid segments generated from side-reactions (e.g., from two barcode primers annealing and extending during reverse transcription). As depicted in FIG. 5B, this intrastrand complementarity within the first product strand, the second product strand, or the first and second product strands (i.e., the first product strand comprises self-complementarity and the second product strand comprises self-complementarity) can promote self-hybridization by the first product strand, the second product strand, or the first and second product strands.

[0183] Encoding self-complementarity into the first and / or second product strands can facilitate suppressive or semi-suppressive amplification of the first and / or second product strands, which exploit the relationship between self-hybridization probability and length. Long nucleic acids such as full-length first and second product strands have relatively lower probabilities for self-hybridization than short nucleic acids. Accordingly, during the annealing phase of amplification, for example at about 50-72°C, a higher percentage of short nucleic acid side products (e.g., an extension product generated from a TSO spuriously hybridizing to and extending over a capture probe) can be self-hybridized and therefore inaccessible to primers than the first and / or second product strands. Over multiple amplification cycles, the full-length first and / or second product strands can thus amplify exponentially higher relative to short, spuriously generated nucleic acids. The amplification can thus be selective for the full-length first and / or second product strands.Attorney Docket No. 59528-731601

[0184] In some cases, amplicons are generated using a polymerase chain reaction (PCR) and one or more gene-specific primers (GSP)s and / or sequencing adapters. GSPs or sequencing adapters can be added to the first and / or second product strands. GSPs or sequencing adapters can be added to an internal position of the first and / or second product strand. Similarly, one or more GSPs and / or sequencing adapters can be added to the first and / or second product strand. GSPs can be used to amplify genes or gene-specific sequences.

[0185] As shown in FIGS. 6A-6C, the methods described herein can result in the barcode sequences of the barcoding primer 300 and the variable region of the second product strand 705 being located on the 5’ end of the second product strand . Amplicons that are suitable for sequencing analysis can then be generated from the 5’ end of the second product strand. Short-read next generation sequencing (NGS) methods - examples of which include ion torrent sequencing (e.g., Merriman et al., Electrophoresis, 2012; 33:3397), Illumina sequencing (e.g., Illumina. Benchtop Sequencers, https: / / www.illumina.com / systems / sequencing-platforms.html; Illumina. Bringing next-generation sequencing to clinical labs. https: / / www. illumina.com / systems / ivd-instruments.html; Illumina. Faster sequencing and data processing. https: / / www. illumina.com / science / technology / next-generation-sequencing / sequencingtechnology / 2-channel-sbs.html), 454 pyrosequencing (e.g., Rothberg and Leamon, Nat. Biotechnol., 2008; 26(10): 1117), SOLiD sequencing (e.g., Valouev et al., Genome Res., 2008; 75(7): 1051 ; Tang and Wang, Nat. Methods, 2009; 6(5):377), and cP AL sequencing (e.g., Drmanac et al., Science, 2010; 327:78) -typically generate and then sequence sub- 1000 base pair clonal amplicons of target nucleic acids. When 3 ’-barcoded nucleic acids are subjected to short-read NGS, barcode information is often not captured in 5 ’-oriented sequencing reads, preventing this sequence information from being barcode-indexable (and thus, e.g., preventing this information from being associated with specific cells, chambers, regions within a fluidic device, cell treatment conditions, etc.). For example, short-read NGS methods applied to 3 ’-barcoded TCR and BCR nucleic acid sequences often fail to capture TCR and BCR variable domain sequences. Conversely, while long-read NGS methods - for example SMRT sequencing (e.g., PACBIO. SEQUENCE WITH CONFIDENCE, https: / / www.pacb.com / wp-content / uploads / SMRT-Sequencing-Brochure-Delivering-highly-accurate-long-readsto-drive-discovery -in-lifescience. pdf; PACBIO. PacBio RS II Sequencing System.Attorney Docket No. 59528-731601https: / / www.mscience.com.au / upload / pages / pacbio / pacbio_rs_ii_brochure.pdf) and Oxford Nanopore Technology (e.g., Miga et al., bioRxiv 2019:735928) - can often sequence more than 105consecutive base pairs from target nucleic acids, and thus can fully sequence a larger percentage of genomic sequences and transcripts, long-read NGS methods typically have lower accuracies and sensitivities than short-read NGS methods, and therefore can be unsuitable for sequencing low copy number target nucleic acids (e.g., a single copy of an mRNA sequence from a single cell).

[0186] The methods and systems described herein allow the generated amplicons to retain valuable information on the 5’ end of the second product strand (including the barcoding information from the barcode primer 300 and the sequence of the variable region 705). If any information is lost (due to a long transcript), it will be at the 3’ end (which contains the constant region 710). In some embodiments, amplification results in amplicons that comprise (a) at least one of a T cell receptor variable region sequence, a B cell receptor variable region sequence, and an immunoglobulin variable region sequence, and (b) at least one of an adaptor sequence, a barcode sequence, a unique molecular identifier sequence, a primer binding site, and a sequencing primer binding site. In such cases, amplicon sequencing can yield clonotype information for one or more cells.

[0187] Multiple rounds of turnarounds can improve sensitivity and yield. In some cases, one or more turnarounds are used. For example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more turnarounds can be completed. In general, the first turnaround can melt first product strands from mRNA (see FIGS. 4E and 4F) and hybridize the cDNA molecules to barcode primers (see FIG. 4G) , while subsequent turnarounds can melt the first product strands from second product strands (see FIGS. 4H and 41), allowing the first product strands to hybridize to nonextended barcode primers (see FIG. 4J). A turnaround or multiple rounds of turnarounds can then be followed by an extension step to form an additional second product strand 719C that includes a complement to cDNA 700 and a sequence 790 complementary to the capture probe (see FIG. 4K). Although FIGS. 41 and 4 J illustrate a turnaround hybridization of a complement to the barcode primer 600 of the first product strand 719A hybridizing to non-extended barcode primer 350, it is also possible for a portion of the first product strand 719A to alternatively hybridize to a portion of the second product strand 719B. More particularly, this alternative hybridization can include the binding of sequence 600 to barcode primer sequence 300 of 719B, which does not allow for an extension reactionAttorney Docket No. 59528-731601and can be represented by the first and second product strand (719A and 719B) transitioning from the orientation in FIG. 41 to FIG. 4H, instead of the desired transitioning from the orientation in FIG. 41 to FIG. 4J. The occurrence of this alternative hybridization can decrease the likelihood of the turnaround hybridization. As this turnaround hybridization is typically stochastic, it is worthwhile to note that increasing the number of turnaround cycle steps can in turn increase the probability of an occurrence of a first product strand 719A hybridizing to a non-extended barcode primer 350. In particular, during a turnaround step, a first product strand 719A can hybridize to a non-extended barcode primer 350 or to a fully extended second product strand 719B. When the first product strand hybridizes to a non-extended barcode primer, the barcode primer can extend over the first product strand to generate an additional second product strand 719C. However, when the first product strand 719A hybridizes to a second product strand 719B, no extension reaction occurs, and no new second product strand 719C is generated. Similarly, during a turnaround step, a second product strand 719B may hybridize to a non-extended capture probe 350 or to a full-length first product strand 719A. Accordingly, the number of first and second product strands generated during a turnaround step is often less than the number of first and second product strands present prior to the turnaround step. Accordingly, in order to favor the formation of additional first and second product strands (copies of first and second product strands generated during turnaround steps), a method disclosed herein may utilize 2 or more turnaround steps. For example, the method may utilize 2 to 5 turnaround steps, 2 to 8 turnaround steps, 2 to 10 turnaround steps, 2 to 12 turnaround steps, 2 to 15 turnaround steps, 2 to 20 turnaround steps, 5 to 8 turnaround steps, 5 to 10 turnaround steps, 5 to 12 turnaround steps, 5 to 15 turnaround steps, 5 to 20 turnaround steps, 8 to 12 turnaround steps, 8 to 15 turnaround steps, 8 to 20 turnaround steps, 10 to 15 turnaround steps, 10 to 20 turnaround steps, or 15 to 30 turnaround steps. In some cases, a method may utilize greater than 30 turnaround steps.

[0188] As disclosed herein, increasing the number of turnaround steps can increase the yield without introducing sequencing biases into DNA product molecules. FIG. 91 shows increasing DNA product yield in going from 1 to 9 turnaround steps (0.8, 1.5, 2.8, and 4.1 ng / pl product for 1, 3, 6, and 9 turnaround steps, respectively). As shown in FIG. 9B, higher UMI counts were achieved with a 2 turnaround steps than with a 1 turnaround. FIG. 9C shows that there is minimalAttorney Docket No. 59528-731601impact on gene expression between a 1 and 2 turnaround since the scatterplot approximates the unity line. However, as shown in the Qualimap chart of FIG. 14D, which shows read counts (y-axis) as a function of transcript position (x-axis, % of distance from 5’ to 3’ end), the disclosed sequencing methods can be biased towards 5’-sequences within individual genes. As a note, the bases of each sequence fragment are normalized for the % distance from the 5’ to the 3’ end. FIG. 14D indicates a significantly higher bias in read count towards the 5’ end of the sequence. For example, random tagmentation (e.g., as depicted in FIG. 6B) or fragmentation (as depicted in FIG. 6C) of first and / or second product strands according to FIGS. 4A-4K can generate nucleic acid fragments that include the barcode primer and a 5’ portion of a reverse transcribed mRNA sequence, such that 5’ sequences are primarily captured and sequenced. The results shown in FIG. 14D contrast sequence coverage from 3 ’-barcoding methods, which exhibit sequencing bias towards the 3’ ends of transcripts, and sequence coverage from double fragmentation methods, which exhibit sequencing bias towards the middle of transcripts and low 5’ and 3’ coverage. Thus, the presently disclosed methods can generate sequencing information that would not be captured by other barcoding and sequencing methods.

[0189] The presently disclosed methods are also applicable for capturing, barcoding, and sequencing guide ribonucleic acids (RNA), for example guide RNA associated with a genetic modification of the cell. DNA encoding the guide RNA may be stably or transiently introduced into a cell. The DNA may include multiple promoters, such that the guide RNA is expressed as a short transcript and within a longer transcript that includes a capturable sequence such as a polyA tail and optionally one or more exogenous messenger RNA (mRNA) sequences, such as a survival marker. The guide RNA transcript may also be coupled to a barcode. The cell may thus express an RNA transcript that includes the guide RNA, the capturable sequence, and optional additional sequences. This transcript may be captured and reverse transcribed to generate barcoded first and / or second product strands as disclosed herein. Accordingly, a method disclosed herein can include generating a first product strand comprising a complement of a guide RNA sequence, an exogenous mRNA, a barcode, or a combination thereof, and similarly can generate a second product strand comprising a DNA analogue of the guide RNA sequence, the exogenous mRNA, the barcode, or the combination thereof. Detection of the guide RNA (e.g., through sequencing) may then be associated with one or more geneticAttorney Docket No. 59528-731601modifications of a cell, including one or more knock-out or knock-in genes or gene edits.

[0190] FIGs. 10A, 10B, 11 A, and 1 IB show a top view of an example of a barcoded surface. The surface may comprise one or more lanes (also sometimes referred to as a “flow channel”). In some cases, the fluidic device comprises 5 lanes to 50 lanes. In some cases, the fluidic device comprises 5 lanes to 10 lanes, 5 lanes to 20 lanes, 5 lanes to 30 lanes, 5 lanes to 50 lanes, 10 lanes to 20 lanes, 10 lanes to 30 lanes, 10 lanes to 50 lanes, 20 lanes to 30 lanes, 20 lanes to 50 lanes, or 30 lanes to 50 lanes. In some cases, the fluidic device comprises 5 lanes, 10 lanes, 20 lanes, 30 lanes, or 50 lanes. In some cases, the fluidic device comprises at least 5 lanes, 10 lanes, 20 lanes, or 30 lanes. In some cases, the fluidic device comprises at most 10 lanes, 20 lanes, 30 lanes, or 50 lanes. Each lane may be associated with a spatial barcode unique to the lane in which it is located. Similarly, a single spatial barcode may uniquely identify the location of a barcode (e.g., a barcoded primer) within a lane and the lane in which the barcode is located. FIG. 10A shows a portion of a surface of a fluidic device with eight lanes.

[0191] Each lane may include one or more discrete locations referred to as a “field” or “array” with oligonucleotides disposed thereon. The oligonucleotides disposed thereon can include the “barcode primers” as described herein (for example, as shown in FIGs. 2B and 3B). The oligonucleotides disposed thereon can include the “capture probes” as described herein (for example, as shown in FIG. 1).

[0192] In some cases, each lane comprises 5 fields to 30 fields. In some cases, each lane comprises 5 fields to 10 fields, 5 fields to 15 fields, 5 fields to 20 fields, 5 fields to 30 fields, 10 fields to 15 fields, 10 fields to 20 fields, 10 fields to 30 fields, 15 fields to 20 fields, 15 fields to 30 fields, or 20 fields to 30 fields. In some cases, each lane comprises 5 fields, 10 fields, 15 fields, 20 fields, or 30 fields. In some cases, each lane comprises at least 5 fields, 10 fields, 15 fields, or 20 fields. In some cases, each lane comprises at most 10 fields, 15 fields, 20 fields, or 30 fields. Each field may be associated with a spatial barcode unique to the field in which it is located. In FIG.10A, each lane comprises 15 fields.

[0193] FIGs. 10B and 11 A show an exploded view of one of the fields of FIG.10A. Each field may include one or more discrete locations referred to as a “reaction site” or “dot” or “spot” with oligonucleotides disposed thereon. The oligonucleotides disposed thereon can include the “barcode primers” as described herein (for example,Attorney Docket No. 59528-731601as shown in FIGs. 2B and 3B). The oligonucleotides disposed thereon can include the “capture probes” as described herein (for example, as shown in FIG. 1). In some cases, each field comprises 100 dots to 3,000 dots. In some cases, each field comprises 100 dots to 500 dots, 100 dots to 1,000 dots, 100 dots to 1,500 dots, 100 dots to 2,000 dots, 100 dots to 3,000 dots, 500 dots to 1,000 dots, 500 dots to 1,500 dots, 500 dots to 2,000 dots, 500 dots to 3,000 dots, 1,000 dots to 1,500 dots, 1,000 dots to 2,000 dots, 1,000 dots to 3,000 dots, 1,500 dots to 2,000 dots, 1,500 dots to 3,000 dots, or 2,000 dots to 3,000 dots. In some cases, each field comprises 100 dots, 500 dots, 1,000 dots, 1,500 dots, 2,000 dots, or 3,000 dots. In some cases, each field comprises at least 100 dots, 500 dots, 1,000 dots, 1,500 dots, or 2,000 dots. In some cases, each field comprises at most 500 dots, 1,000 dots, 1,500 dots, 2,000 dots, or 3,000 dots. Each reaction site may be associated with a spatial barcode unique to the dot or spot in which it is located. In FIGs. 10B and 11 A, the field includes 1536 dots. FIG. 1 IB shows a further zoomed in view of the field shown in FIGs. 10B and 11 A. FIG. 1 IB shows a field with 80 pm diameter dots and a 130 pm hexagonal pitch. As shown in FIG. 1 IB, a hydrogel chamber can be formed on the fluidic device that is at least partially vertically aligned with a dot. Based on the spatial barcoding scheme described above, the unique spatial location (lane, field, and spot) of an mRNA can be determined upon sequencing of the mRNA and oligonucleotide associated therewith.

[0194] The capture probes and barcode primers disclosed herein may also be used for spatial analysis on collections of cells such as tissues, tumors, solid biopsies, spheroids, neurospheres, and the like. As used herein, the term “spatial analysis” denotes a method that generates location-specific information. When performed on collections of cells, a spatial analysis method can identify the locations of particular cell types (e.g., cancerous cells or tumor infiltrating T cells) or features (e.g., hypoxia or necrosis) within the collection of cells. Spatial analysis can also identify the locations of cells that express a particular transcript or analyte, such as the location of activation factor-expressing (e.g., a-SMA-expressing) cells within a tissue sample.

[0195] FIGS. 13A-C illustrate a spatial analysis method that utilizes capture probes and spatially-tagged nucleic acid barcode primers to determine the 2-dimensional locations of nucleic acids in a tissue sample. As depicted in FIG. 13 A, the tissue sample 1301 can be disposed on a substrate 1300 such as a glass slide or flow cell surface that includes capture probes and barcoded primers. The capture probes and barcoded primers can be disposed in discrete regions 1302, such as spots.Attorney Docket No. 59528-731601The capture probes and barcode primers can be coupled to the substrate 1300, for example by nuclease-cleavable linkers. Alternatively, the capture probes and barcode primers can be coupled to beads or other removable structures that are disposed within the discrete regions 1302 and can be collected following nucleic acid capture or cDNA synthesis. The tissue sample may optionally be a living or fixed tissue sample.

[0196] Cells within the tissue sample can be permeabilized or lysed to release nucleic acids. The capture probes can be configured to bind a particular type of nucleic acid, such as mRNA. cDNA can be generated from the nucleic acids using the capture probes and barcode primers. Barcoded primers within each region 1302 can share a uniquely identifiable spatial barcode such that each cDNA product can be mapped to a particular location on the substrate 1300. For example, as depicted in FIG. 13B, nucleic acid sequences ‘SEQUENCE 1’ through ‘SEQUENCE 5’ from the tissue sample 1301 can be mapped to specific regions 1302 on the substrate 1300. This spatial sequence information can be correlated to other forms of spatial data, such as an image of the tissue sample 1301. For example, as depicted in FIG. 13C, a particular sequence (e.g., ‘SEQUENCE 1’) can be mapped to a feature 1301A of the sample, such as a necrotic region identified through tissue staining.Hydrogel Compositions

[0197] In some embodiments, a channel of a fluidic device of a system of the invention comprises one or more polymer precursors for forming chambers. In some embodiments, the one or more polymer precursors comprise hydrogel precursors. Such precursors may be selected from a wide variety of compounds including, but not limited to, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoaneAttorney Docket No. 59528-731601trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof. In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEGthiol / PEG-acrylate, acrylamide / N,N'-bis(acryloyl)cystamine (BACy), or PEG / PPO. In some embodiments, the following precursors and crosslinker may be used to form chambers with degradable polymer matrix (hydrogel) walls. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel. In some embodiments, the degrading comprises exposing the hydrogel polymer wall to UV light. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are made of different materials. In some embodiments, the degrading in (b) does not degrade the hydrogel chamber. In some embodiments, the method further comprises degrading the hydrogel chamber. In some embodiments, the method further comprises imaging the analyte, the first biological material, the hydrogel chamber, the fluidic device, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a cPEG monomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure listed in Table 1.Table 1Attorney Docket No. 59528-731601< " < " "<"Attorney Docket No. 59528-731601Attorney Docket No. 59528-731601" <<>>""<""<<Attorney Docket No. 59528-731601Attorney Docket No. 59528-731601<<""Attorney Docket No. 59528-731601Attorney Docket No. 59528-731601<Attorney Docket No. 59528-731601<<"Attorney Docket No. 59528-731601<"<<Attorney Docket No. 59528-731601

[0198] A polymer precursor can further comprise additional reagents that affect polymerization and polymer matrix properties. As examples, a polymer precursor can include a crosslinker, a porogen, a viscosity -modifying agent, an acid, a base, a catalyst, a salt, a photoinitiator, or a combination thereof.

[0199] As used herein, the term “crosslinker” denotes a species with two or more polymerizable groups. For example, where the polymerizable group is an ethylenically unsaturated group, a crosslinker would contain two or more ethylenically unsaturated groups. In another example, a crosslinker can contain three or more reactive centers for bifunctional polymer synthesis (e.g., the three methoxy groups of trimethoxybenzene in the context of polyester synthesis).

[0200] As used herein, the term “porogen” can denote a species that modulates the porosity of a polymer matrix. A porogen can be dispersed with the reactants before the polymerization process of forming the polymer matrix. Porogens typically diffuse out of polymer matrices following polymerization, leaving pores in the regions that they occupied. Porogen size, concentration, hydrophobicity, and hydrophilicity can thus influence pore density and pore size in polymer matrices. Examples of porogensAttorney Docket No. 59528-731601consistent with the present disclosure include particles (e.g., polymeric, ceramic, metal, metal oxide, or hydrogel particles), polymers such as polyethylene glycol and alginate, and vesicles such as liposomes or micelles.

[0201] As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In many cases, a photoinitiator included in a polymer precursor formulation is a type I photoinitiator, that is a molecule that generates radicals through intramolecular cleavage (e.g., homolysis) upon photoexcitation, or a type II photoinitiator, that is a molecule that abstract an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of photoinitiators utilizable in the present methods include acetophenone, anisoin, anthraquinone, anthraquinone-2-sulfonic acid, benzil, benzoin, benzophenone, 3,3’,4,4’-benzophenonetetracarboxylic dianydride, 4-benzoylbiphenyl, 2-benzyl-2-(dimethylamino)-4’-morpholinobutyrophenone, dibenzosuberenone, 2,2-diethoxyacetophenone, 2-ethylanthraquinone, ferrocene, 2-isopropylthioxanthone, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, methyl-2-benzoylbenzoate, and thiooxanthen-9-one.

[0202] In some embodiments, the generation of a polymer matrix within said fluidic device comprises exposing the one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light at 350 nm to 800 nm. In some embodiments, the light generating device generates light at 350 nm to 600 nm. In some embodiments, the light generating device generates light at 350 nm to 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of the polymer matrix comprises between about 1 and 3 seconds of illumination, between about 1 and 5 seconds of illumination, between about 1 and 10 seconds of illumination, between about 1 and 15 seconds of illumination, between about 1 and 20 seconds of illumination, between about 1 and 30 seconds of illumination, between about 1 and 50 seconds of illumination, between about 3 and 5 seconds of illumination, between about 3 and 10 seconds of illumination, between about 3 and 15 seconds of illumination, between about 3 and 20 seconds of illumination, between about 3 and 30 seconds of illumination, between about 3 and 50 seconds of illumination, between about 5 and 10 seconds of illumination, between about 5 and 15 seconds of illumination, between about 5 and 20 seconds of illumination, between about 5 and 30 seconds of illumination, between about 5 and 50 seconds of illumination, between about 10 and 20 seconds of illumination, between about 10 and 30 seconds of illumination, between about 10 and 50Attorney Docket No. 59528-731601seconds of illumination, between about 20 and 30 seconds of illumination, or between about 20 and 50 seconds of illumination. In some embodiments, the generation of a polymer matrix within said fluidic device is performed using a spatial light modulator (SLM) (i.e. a spatial energy modulation element that is capable of generating desired light intensity pattern spatially). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid crystal based.

[0203] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer, the monomer comprising: an oligomeric domain comprising three or more arms, wherein each arm of said oligomeric domain comprises a degradable unit and a crosslinkable unit, wherein the crosslinkable unit of an arm of the three or more arms is configured to crosslink with another crosslinkable unit of another polymer precursor in response to a first stimulus, thereby obtaining the polymerized form of the monomer, and wherein the degradable unit is configured to be cleaved in response to a second stimulus, thereby solubilizing the polymerized form of the monomer. In some embodiments, the oligomeric domain comprises four or more arms. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a degradable functional group. In some embodiments, said degradable function group comprises disulfide, beta-thioether ester, amidomethylol and vicinal diol, vicinal diol, alginate backbone, dextran backbone, chitosan backbone, hyaluronic acid backbone, chondroitin sulfate backbone, or carboxy methyl cellulose backbone, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a hydrogel macromonomer. In some embodiments, the hydrogel macromonomer comprises cPEG, cSEL-BTEEC, cSEL-DHEBA, cSEL-diol, cSEL-alginate, cSEL-dextran, cSEL-chitosan, cSEL-hyaluronic acid, cSEL-chondroitin sulfate, or cSEL-cellulose, or a combination thereof. In some embodiments, the degradation unit is degraded by inputting a degradation reagent into the fluidic device. In some embodiments, the degradation reagent comprises DTT, TCEP, BME, GSH, DMEM, RPMI, PBS buffer, DMEM, RPMI, PBS buffer, sodium (meta)periodate, Alginate lyase (enzyme), Dextranase, Lysozyme and chitinase, Hyaluronidase, Chondroitinase, or Cellulases, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises at least one beta-thioether ester. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a PEG-macromonomer containing beta-thioether esters. InAttorney Docket No. 59528-731601some embodiments, the beta-thioether ester is formed by reacting an acrylate with a thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Michael donor. In some embodiments, the Michael donor is PEG-thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a cSEL beta-thioether ester with one beta-thioether ester per arm. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises are formed from any material that comprises a PEG with a Michael acceptor chain. In some embodiments, the Michael acceptor chain comprises PEG-acrylamide, PEG-vinyl sulfone, PEG-maleimide, or PEG-carbonyl acrylic, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by cleavage of disulfide bonds. In some embodiments, the disulfide bonds are cleavable by one or more reducing agents. In some embodiments, the one or more reducing agents comprise DTT, TCEP, BME, or GSH, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more arms each comprising one or more amides.

[0204] In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by oxidative cleavage of vicinal diol by sodium (meta)periodate. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photocleavable 4-arm PEG-macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is photodegradable via an ortho-nitrobenzyl moiety. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Coumarin-based photodegradable macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a 4-arm PEG-acrylamide comprising one or more disulfides. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more cage disulfide bonds in a hydrogel cage formation. In some embodiment, the hydrogel cages degrade using light and a photoinitiator. In some embodiments, the hydrogel chamber or the hydrogel polymer wall enables hydrogel formation. In some embodiments, the hydrogel enables spatiotemporal control of hydrogel cage degradation, therefore enabling selective retention of cells with a single hydrogel formulation. In some embodiments, upon exposure to light, photogenerated radicals’ initial multiple fragmentation and disulfide exchange reactions, thereby permitting and promoting photodeformation, photowelding and photodegradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, one or more polymer precursors enable formation of theAttorney Docket No. 59528-731601hydrogel chamber or the hydrogel polymer wall. In some embodiments, the hydrogel exhibits a chemical or physical change in response to an external stimulus. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photolabile nitrobenxyl ester which lyses upon photon absorption, thereby allowing a user to exogenously control degradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the method further comprises controlling a network degradation of the hydrogel chamber or the hydrogel polymer wall by concentration of a photoinitaitor infused into the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the first hydrogel polymer wall comprises a shape configured to contain the first biological material. In some embodiments, the fluidic device comprises a top layer, a bottom layer, and a spacer layer.

[0205] In some embodiments, the spacer layer includes a cut-out region, where the spacer layer is sandwiched in between the bottom layer and the top layer to form a channel in the cut-out region. In some embodiments, the hydrogel chamber is at least partly formed by the top layer and the bottom layer. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are the same material. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are different materials.

[0206] In some embodiments, hydrogel chambers of the invention are degradable or depolymerizable either generally within a channel or “on demand” within a channel. Hydrogel chambers that are generally degradable are degraded by treatment with a degradation agent, or equivalently, a depolymerization agent that is exposed to all chambers within channel. Depolymerization agents include, but are not limited to, heat, light, and / or chemical depolymerization reagents (also sometimes referred to a cleaving reagents or degradation reagents). In some embodiments, on demand degradation may be implemented using polymer precursors that permit photocrosslinking and photo-degradation, for example, using different wavelengths for crosslinking and for degradation. For example, Eosin Y may be used for radical polymerization at defined regions using 500 nm wavelength, after which illumination at 380 nm can be used to cleave the cross linker. In other embodiments, photo-caged hydrogel cleaving reagents may be included in the formation of polymer matrix walls. For example, acid labile crosslinkers (such as esters, or the like) can be used to create the hydrogel and then UV light can be used to generate local acidic conditions which, in turn, degrades the hydrogel. In some embodiments, the at least one polymer matrix is degradable by at least one of: (i) contacting the at least one polymer matrix with aAttorney Docket No. 59528-731601cleaving reagent; (ii) heating the at least one polymer matrix to at least 90 °C; or (iii) exposing the at least one polymer matrix to a wavelength of light that cleaves a photo-cleavable cross linker that cross links the polymer of the at least one polymer matrix. In some embodiments, the at least one polymer matrix comprises a hydrogel. In some embodiments, the cleaving reagent degrades the hydrogel. In some embodiments, the cleaving reagent comprises a reducing agent, an oxidative agent, an enzyme, a pH based cleaving reagent, or a combination thereof. In some embodiments, the cleaving reagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3 -hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments, the surface of the polymer matrix or hydro gel may be functionalized by coupling a functional group to the polymer matrix or hydrogel. Some nonlimiting examples of functional group may include a capture reagent ( e.g., pyridinecarboxaldehyde (PC A)), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an aldehyde dithiolane, PEG, a thiol, an alkene, an alkyne, an azide, or a combination thereof. In some cases, the functionalized polymer matrix may be used to capture biomolecules inside a polymer matrix compartment formed adjacent to (e.g., around or on) the biological component. The biomolecule may be produced by the biological component ( e.g., secretome from a cell). The functionalized surface of the polymer matrix inside the compartment may be used to capture reagents or molecules from outside the compartment. The functionalized surface may increase surface area covered by a reagent, a molecular sensor, or any molecule of interest (e.g., an antibody).

[0207] In some embodiments, existing polymer matrix walls may be partially degraded, e.g. to change porosity. In some embodiments, polymer precursors may include degradable beads that form part of, and are embedded in, the polymer matrix walls when synthesized, after which either on-demand or generally, may be degraded, thereby creating an increase in porosity.

[0208] In some embodiments, the hydrogel chamber is made of a first material that degrades upon exposure to a first stimulus. In some cases, a hydrogel polymer wall is made of a second material that degrades upon exposure to a second stimulus. The first stimulus and second stimulus can be different. In some cases, the first stimulus comprises light, and the second stimulus comprises a degradation reagent. In some cases, the first stimulus comprises a degradation reagent, and the second stimulusAttorney Docket No. 59528-731601comprises light. In some cases, the first stimulus comprises a first degradation reagent, and the second stimulus comprises a second degradation reagent different from the first degradation reagent. In some cases, the first stimulus comprises light in a first wavelength range, and the second stimulus comprises a light in a second wavelength range different from the first wavelength range.

[0209] The pore size in the polymer matrix may be modulated using a chemical reagent, or by applying heat, electrical field, light, or another suitable stimulus. In other words, the polymer matrix may comprise tunable properties (e.g., the pore size) In some cases, the polymer matrix may comprise a thermoresponsive or temperature-responsive polymer. A thermoresponsive polymer (e.g., poly(N-isopropylacrylamide) (NIPAAM)) may phase separate from a solution upon heating or upon cooling (e.g., polymer showing lower critical solution temperature (LCST) or upper critical solution temperature (UCST). The polymer matrix may comprise polymer which may collapse at high temperature in order to, for example, control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that may be used to form hydrogel / polymer matrix with tunable properties may include Poly(N-vinyl caprolactam), Poly(N-ethyl oxazoline), Poly(methyl vinyl ether), Poly(acrylic acid-coacrylamide), or a combination thereof. A change in temperature may enlarge or contract average pore size in the polymer matrix to allow selected molecules, such as a nucleic acid molecule, a protein, or any biomolecule or molecule smaller than the adjusted pore size to be released from a hydrogel chamber.Computer Systems

[0210] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 12 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameter using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic deviceAttorney Docket No. 59528-731601of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0211] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and / or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1530 in some cases may be a telecommunication and / or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server.

[0212] The CPU 1505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions can be directed to the CPU 1505, which can subsequently program or otherwise configure the CPU 1505 to implement methods of the present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0213] The CPU 1505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).

[0214] The storage unit 1515 can store files, such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, e.g., user preferences and user programs. The computer system 1501 in some cases can include one or more additionalAttorney Docket No. 59528-731601data storage units that are external to the computer system 1501, such as located on a remote server that may be in communication with the computer system 1501 through an intranet or the Internet.

[0215] The computer system 1501 can communicate with one or more remote computer systems through the network 1530. For instance, the computer system 1501 can communicate with a remote computer system of a user (e.g., a laptop, a personal computer, a tablet, or a mobile phone). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1501 via the network 1530.

[0216] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations, the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.

[0217] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0218] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, randomaccess memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the softwareAttorney Docket No. 59528-731601programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0219] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0220] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, forAttorney Docket No. 59528-731601example, an image of a biological component, a barcode, a signal or measurement of a local parameter. Examples of UI’ s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0221] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1505. The algorithm can, for example, identify a biological component, detect a barcode, generate a spatial modulating element (e.g., a mask), provide energy from an energy source, detect or measure a local parameter using a sensor, etc.

[0222] While the present invention has been described with reference to several particular example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present invention. The present invention is applicable to a variety of sensor implementations and other subject matter, in addition to those discussed above.EXAMPLES EXAMPLE 15’-Nucleic Acid Barcoding

[0223] This example covers a method for generating 5’-barcoded cDNA in a flow cell channel. Four channels of an eight-channel flow cell were used for these experiments. The bottom surface of each channel of the flow cell included 8 fields of spots functionalized with equal amounts of capture probes and barcode primers. 1 ng / uL Jurkat cell RNA was loaded into each channel. Channels 2 and 4 were additionally loaded with reverse transcription reagents that included a templateswitching oligonucleotide (TSO) containing a 5’ primer sequence that enabled amplification of TSO-extended cDNA. Channels 1 and 3 were loaded with reverse transcription reagents that included a template-switching oligonucleotide (TSO) that included a 5’ sequence identical to the 3’ sequence of the barcode primer present in the channels. Accordingly, cDNA generated in these channels (and extended over the TSO) hybridized to the barcode primer to facilitate extension of the cDNA molecule over the barcode primer and extension of the barcode primer over the cDNA.Channels 1 and 2 were subjected to a single turnaround step performed by melting double stranded nucleic acids with formamide and then inputting a strand displacing DNA polymerase. Channels 3 and 4 were not subjected to turnaround steps. DNAAttorney Docket No. 59528-731601products were then eluted from the channels, amplified, quantitated, and sequenced from the barcode primer.

[0224] As shown in FIG. 14 A, DNA product was observed in all four channels. The greatest amount of product was observed in channels 2 and 4, which included a TSO that enabled amplification of the TSO-extended product. Higher DNA product elution was observed in channel 1 than in channel 3, indicating that 5’ barcoding (i.e., barcode primer extension over cDNA and cDNA extension over the barcode primer) occurred following turnaround.

[0225] DNA from channel 2 was collected and sequenced. FIG. 14B is a plot of positional nucleobase frequencies beginning 6 nucleotides upstream from the TSOs. The highly conserved (> 90%) nucleobase frequencies from positions 7-36 indicate that the TSO sequence was successfully incorporated into the DNA products.FIG. 14C is a plot of nucleobase frequencies at internal positions within the DNA products corresponding to reverse transcribed mRNA sequences. The nucleobase distributions in these reads were consistent with the Jurkat transcriptome, indicating that the queried mRNA profiling method was independent of target sequence.Consistent with this finding, 86.67% of the sequences mapped to exonic sequences, 7.7% of the sequences mapped to intronic sequences, and 5.63% of the sequences mapped to intergenic sequences, reasonable distributions for a Jurkat cell mRNA profile. As shown in the gene coverage profile plot in FIG. 14D, which shows read counts (y-axis) as a function of transcript position (x-axis, % of distance from 5’ to 3’ end), the sequences reads were biased towards the 5’ ends of genes from the Jurkat cells.EXAMPLE 25’-Barcoding with Spatially-Barcoded Primers

[0226] This example covers 5 ’-barcoding using an array of nucleic acid barcode primers that include spatial location tags. Four channels of an eight-channel flow cell were used for these experiments. The bottom surface of each channel of the flow cell included about 1500 spots that each contained a unique barcode primer that included, from 5’ to 3’, a PCR handle, a first barcode sequence, a spacer sequence, a second barcode sequence, a random sequence, and a sequence present in TSOs used for cDNA 3’ tagging (and therefore configured to hybridize to TSO extension products). Channels 1 and 3 were additionally loaded with reverse transcriptionAttorney Docket No. 59528-731601reagents that included a template-switching oligonucleotide (TSO) containing a 5’ primer sequence that enabled amplification of TSO-extended cDNA. Channels 2 and 4 were loaded with reverse transcription reagents that included a template-switching oligonucleotide (TSO) that included a 5’ sequence identical to the 3’ sequence of the barcode primer present in the channels. Channels 1 and 2 were subjected to a single turnaround step performed by melting double stranded nucleic acids with formamide and then inputting further reverse transcription reagents. Channels 3 and 4 were not subjected to turnaround steps. DNA products were then eluted from the channels, amplified, quantitated, and sequenced.

[0227] As shown in FIG. 15 A, DNA product was observed in channels 1 and 3, which included a TSO that enabled amplification of the TSO-extended product. Higher DNA product elution was observed in channel 1 than in channel 3, indicating that 5’ barcoding (i.e., barcode primer extension over cDNA and cDNA extension over the barcode primer) occurred following melting and rehybridization to a barcode primer (i.e., turnaround). Nucleobase frequence per sequenced position of the cDNA products are shown in FIG. 15B. This figure provides conserved nucleotide frequencies within the cDNA products that are indicative of successful and consistent 5’ barcoding.EXAMPLE 3Variable Turnaround Steps in 5’ cDNA Barcoding

[0228] This example covers the effects of turnaround number in 5 ’-barcoding assays. This assay utilized reverse transcription to generate cDNA from mRNA, a first turnaround step to simultaneously extend the cDNA over a first barcode primer and to extend the first barcode primer over the cDNA, and optional subsequent turnaround steps to extend additional barcode primers over the cDNA. The assay utilized a flow cell with a bottom surface that included about 1500 spots. Each spot included a capture probe and a unique barcode primer that contained, from 5’ to 3’, a PCR handle, a first barcode sequence, a spacer sequence, a second barcode sequence, a random “unique molecular identifier” (UMI) sequence, and a sequence present in TSOs used for cDNA 3’ tagging (and therefore configured to hybridize to TSO extension products).

[0229] In a first assay, two channels were loaded with 1 ng / pL Jurkat mRNA, reverse transcription reagents, and a TSO with a unique molecular identifier and aAttorney Docket No. 59528-731601sequence identical to the 3’ sequence on the barcode primers to facilitate semisuppressive PCR. The unique molecular identifier on the TSO prevented inflation of barcode primer UMI counts generated from multiple turnaround steps involving multiple rounds of barcode primer extension over a single cDNA molecule. The first channel was subjected to a single turnaround step. The second channel was subjected to two turnaround steps. FIG. 9A shows the total DNA product generated from the 1 and 2 turnaround lanes. As can be seen from this figure, a greater amount of DNA product was generated in the lane that utilized 2 turnaround steps (8.6 ng / pL) than the lane that utilized 1 turnaround step (6.5 ng / pL). FIG. 9B shows the number of counts for each gene read from the 1 and 2 turnaround channels. While the total counts were higher in the 2 turnaround channel, no bias was generated or propagated by introducing a second turnaround step. This lack of bias is further reflected by the linearity of the plot in FIG. 9C, which provides total gene expression counts from the 1 turnaround (x-axis) and 2 turnaround (y-axis) channels.

[0230] A second set of variable turnaround step assays were performed as described above. In this assay, four channels were subjected to a single turnaround step, 3 turnaround steps, 6 turnaround steps, or 9 turnaround steps, respectively. The results of these assays are shown in FIGS. 9D-I. FIG. 9D is a plot of raw gene counts from each of the four channels. Total counts increased in going from 1 to 3 to 6 turnaround steps. However, minimal difference in gene counts were observed in going from 6 to 9 turnaround steps, indicating that the barcode primers were largely exhausted after 6 turnaround steps. FIG. 9E provides total counts of unique molecular identifier sequences in the reads from each channel. FIG. 9F is a set of plot that compares gene counts between the four channels. The linearity of these plots indicates that sequence bias was not introduced by increasing the number of turnaround steps.FIGS. 9G and 9H are, respectively, plots of read lengths and GC content in reads from each channel. These figures collectively show that length and GC bias were not introduced by increasing the number of turnaround steps. FIG. 91 is a plot of DNA product generated in each of the four lanes, and showing that turnaround number correlated with DNA product yield. As shown in this figure, the product yields from the 1, 3, 6, and 9 turnaround lanes were 0.8, 1.5, 2.8, and 4.1 ng / pL, respectively.Attorney Docket No. 59528-731601EXAMPLE 4Fluidic Device Fabrication

[0231] This example covers capture probe and barcode primer spotting on a fluidic device surface to configure the fluidic device for 5 ’-barcoding assays. The method outlined in this example is illustrated in FIGS. 18A-C.

[0232] As shown in FIG. 16A, a surface 1801 of a fluidic device is coated with a thin layer of polymer 1802 that contains NHS ester reactive sites 1803 (i.e., N-hydroxysuccinimide). A ratio of capture probes 1804 and barcode primers 1818 are spotted on multiple discrete locations (“spots”) along the surface 1801. The capture probes 1804 and barcode primers 1818 have 5’ amines 1806A that are reactive towards the NHS ester reactive sites 1803 along the fluidic device surface 1801. The amines 1806A of the capture probes 1804 and barcode primer 1818 react with the NHS ester reactive sites 1803 to couple the capture probes 1804 and barcode primers 1818 to the polymer through amide bonds 1806B.

[0233] As shown in FIG. 16B, following the coupling step, the capture probes 1804 include, from 5’ to 3’, an amide linkage 1806B to the polymer coating 1802 on the fluidic device surface 1801, an optional restriction enzyme recognition site 1807, a first primer binding site 1808, and a nucleic acid capture sequence 1809. The barcode primers 1818 include, from 5’ to 3’, an amide linkage to the polymer coating 1806B, an optional restriction enzyme recognition site 1810, a second primer binding site 1811, a first barcode sequence 1812, second barcode sequences 1816, and known sequence 1817. Known sequence 1817 may be a 15 to 40 nucleotide sequence corresponding to a template switch oligonucleotide (TSO) or a complement to the TSO that can be utilized during a 5 ’-barcoding assay. In various embodiments, an optional spacer sequence may be used in between first barcode sequence 1812 and second barcode sequence 1816. The barcode sequence 1812 and the second barcode sequence 1816 may be incorporated into the barcode primer 1818 that is attached to the surface using an extension reaction or a ligation reaction.

[0234] Multiple arrays of spots are generated along the fluidic device surface 1801. Each array contains about 100 spots. However, the number of spots per array can be adjusted based on desired assay multiplex. Within each array, each spot includes a unique barcode primer 1818 with a unique first barcode sequence 1812. All barcode primers include identical second primer binding sites 1811. Within the fluidicAttorney Docket No. 59528-731601device, each array includes a unique barcode primer 1818 with a unique second barcode sequence 1816. The ratio of capture probe 1804 to barcode primer 1818 may be uniform among all spots.

[0235] The second barcode sequence 1816 varies between arrays so that arrays can be distinguished based on their second barcode sequences. The first barcode sequence 1812 varies between spots within each array. Accordingly, each barcode primer can be mapped to a particular array using the second barcode sequence 1816 and can be mapped to a particular spot within the array using the first barcode sequence 1812. The first primer binding site 1808 and second primer binding site 1811 have high degrees of homology, such that 5’-barcoded cDNA generated with the capture probe 1804 and barcode primer 1818 are configured to self-hybridize for semi-suppressive amplification.

[0236] Within each spot, the capture probes and barcode primers are present at densities of about 5xl013oligonucleotides (capture probes and barcode primers) per square centimeter. At these densities, the capture probes and barcode primers are sufficiently closely packed to support multiple turnaround steps during nucleic acid analysis (for example as described in Example 3).

Claims

1. Attorney Docket No. 59528-731601CLAIMSWhat is claimed is:

1. A method for processing a biological material, comprising:(a) introducing the biological material into a fluidic device, wherein the biological material comprises a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, wherein a surface of the fluidic device comprises (i) one or more capture probes, and (ii) one or more barcode primers comprising one or more barcode sequences;(b) hybridizing a 3’ end of the RNA molecule to a capture probe of the one or more capture probes;(c) extending the capture probe to generate a complementary deoxyribonucleic acid (cDNA) molecule comprising a sequence complementary to at least a portion of the nucleic acid sequence of the RNA molecule;(d) hybridizing at least a portion of the cDNA molecule generated in (c) to a barcode primer of the one or more barcode primers; and(e) extending:the barcode primer using at least a portion of the cDNA molecule generated in (c) as a template, thereby obtaining a tagged molecule complementary to the cDNA molecule generated in (c),the cDNA molecule generated in (c) using at least a portion of the barcode primer as a template, ora combination thereof.

2. The method of claim 1, further comprising releasing the RNA molecule from the biological material prior to (b).

3. The method of claim 1, wherein in (d), the cDNA molecule is simultaneously coupled to the capture probe and hybridized to the barcode primer of the one or more barcode primers.

4. The method of claim 1, further comprising incubating the biological material in the fluidic device prior to (b).

5. The method of claim 1, wherein the method comprises quantitating an amount of the RNA molecule in the biological material.

6. The method of claim 5, wherein the quantitating comprises comparing a relative amount of the RNA molecule to an amount of a second RNA molecule.Attorney Docket No. 59528-7316017. The method of claim 1, wherein the capture probe comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% homologous to a sequence of the one or more barcode primers.

8. The method of claim 1, wherein the tagged molecule is configured to selfhybridize.

9. The method of claim 1, further comprising cleaving the cDNA, the tagged molecule, or the cDNA and the tagged molecule from the surface of the fluidic device.

10. The method of claim 9, further comprising pooling the cDNA, the tagged molecule, or the cDNA and the tagged molecule subsequent to the cleaving.

11. The method of claim 1, wherein the surface comprises a planar surface.

12. The method of claim 11, wherein the planar surface is a surface of a flow cell.

13. The method of claim 12, wherein the planar surface is a top surface of the flow cell.

14. The method of claim 12, wherein the planar surface is a bottom surface of the flow cell.

15. The method of claim 1, wherein the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material.

16. The method of claim 15, wherein the capture element comprises a physical trap.

17. The method of claim 15, wherein the capture element comprises one or more functional groups capable of interacting with the biological material.

18. The method of claim 17, wherein the one or more functional groups comprise an integrin-binding group.

19. The method of claim 18, wherein the integrin-binding group comprises fibronectin.

20. The method of claim 18, wherein the integrin-binding group comprises an arginylglycylaspartic acid (RGD) peptide.

21. The method of claim 17, wherein the one or more functional groups comprise antibodies.

22. The method of claim 14, wherein the fluidic device further comprises a top surface opposite of the bottom surface, and wherein the top surface comprises a capture element configured to capture the biological material.

23. The method of claim 1, wherein the surface is a top surface of the fluidic device.Attorney Docket No. 59528-73160124. The method of claim 23, wherein the fluidic device further comprises a bottom surface opposite of the top surface, and wherein the bottom surface comprises a capture element configured to capture the biological material.

25. The method of claim 1, wherein the capture probe comprises a poly(T) tail.

26. The method of claim 25, wherein the poly(T) tail is located at a 3’ end of the capture probe.

27. The method of claim 25, wherein the poly(T) tail comprises a sequence of three or more thymine bases.

28. The method of claim 25, wherein in (c), the 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe.

29. The method of claim 25, wherein the poly(T) tail comprises a reverse transcriptase primer.

30. The method of claim 29, wherein the capture probe comprises a T30VN reverse transcriptase primer.

31. The method of claim 30, wherein the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe.

32. The method of claim 1, wherein the one or more capture probes do not include barcode sequences.

33. The method of claim 1, wherein in (e), the extending is performed using an enzyme.

34. The method of claim 33, wherein the enzyme incorporates, at the 3’ end of the cDNA molecule, a sequence that is complementary to the at least a portion of the barcode primer.

35. The method of claim 1, further comprising, subsequent to (c), incorporating a sequence complementary to a template switch oligonucleotide (TSO) at a 3’ end of the cDNA molecule.

36. The method of claim 35, wherein the incorporating the sequence complementary to the template switch oligonucleotide at the 3’ end of the cDNA molecule comprises hybridizing the template switch oligonucleotide to the cDNA molecule and extending the cDNA molecule over at least a portion of the template switch oligonucleotide.

37. The method of claim 36, wherein the sequence complementary to the TSO comprises a portion that is complementary to at least a portion of the barcode primer.Attorney Docket No. 59528-73160138. The method of claim 36, wherein the TSO comprises a complement of a unique molecular identifier or a unique molecular identifier.

39. The method of claim 1, wherein the method comprises extending the cDNA molecule using the barcode primer or the portion of the barcode primer as a template.

40. The method of claim 1, wherein the method comprises extending the barcode primer using the cDNA molecule or the portion of the cDNA molecule as a template.

41. The method of claim 1, wherein the one or more barcode primers comprise a unique molecular identifier (UMI) or a complement thereof.

42. The method of claim 41, wherein the extending in (e) comprises incorporating the unique molecular identifier (UMI) or a complement thereof into the cDNA molecule.

43. The method of claim 1, further comprising sequencing the tagged molecule, the cDNA molecule, an amplicon generated therefrom, or a combination thereof.

44. The method of claim 1, further comprising sequencing a subset of the nucleic acid sequence of the RNA molecule or a sequence complementary thereto.

45. The method of claim 44, wherein the subset of the nucleic acid sequence of the RNA molecule comprises a 5’ end of the nucleic acid sequence of the RNA molecule.

46. The method of claim 44, wherein the subset of the nucleic acid sequence of the RNA molecule does not comprise a 3’ end of the nucleic acid sequence of the RNA molecule.

47. The method of claim 1, further comprising generating an amplicon of the tagged molecule.

48. The method of claim 47, wherein the generating the amplicon uses a gene-specific primer.

49. The method of claim 48, wherein the gene-specific primer binds to an internal position of the cDNA or the tagged molecule.

50. The method of claim 47, wherein the generating the amplicon uses exponential amplification.

51. The method of claim 47, wherein the generating the amplicon uses nestedamplification.Attorney Docket No. 59528-73160152. The method of claim 47, wherein the generating the amplicon uses any combination of i) a gene-specific primer, (ii) exponential amplification, and (iii) nested amplification.

53. The method of claim 47, wherein the amplicon comprises a nucleic acid sequence that corresponds to at least a portion of a sequence of the RNA molecule or to at least a portion of a complementary sequence of the RNA molecule.

54. The method of claim 53, wherein the nucleic acid sequence corresponds to at least a portion of a 5’ end of the nucleic acid sequence of the RNA molecule.

55. The method of claim 53, wherein the nucleic acid sequence does not correspond to a 3 ’ end of the nucleic acid sequence of the RNA molecule.

56. The method of claim 53, wherein the amplicon does not comprise a 3’ end of the nucleic acid sequence of the RNA molecule.

57. The method of claim 53, wherein the amplicon comprises (i) the one or more barcode sequences or (ii) complements to the barcode sequences.

58. The method of claim 57, wherein the amplicon comprises a double stranded deoxyribonucleic acid molecule that corresponds to a subset of the nucleic acid sequence of the RNA molecule.

59. The method of claim 1, wherein the tagged molecule comprises, from a 5’ end to a 3’ end, (i) the one or more barcode sequences and (ii) a modified nucleic acid sequence, wherein the modified nucleic acid sequence corresponds to at least a portion of the nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

60. The method of claim 59, wherein the tagged molecule further comprises an additional sequence that is 3’ to the modified nucleic acid sequence and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to a barcode sequence of the one or more barcode sequences.

61. The method of claim 1, wherein the barcode primer further comprises one or more unique molecular identifiers (UMIs).

62. The method of claim 1, wherein the one or more barcode sequences comprise a barcode which indicates a position of the capture probe within the fluidic device.

63. The method of claim 1, further comprising, subsequent to (e), hybridizing the 3’ end of the cDNA molecule to an additional barcode primer of the one or more barcode primers.Attorney Docket No. 59528-73160164. The method of claim 63, further comprising extending the additional barcode primer using the cDNA molecule as a template, thereby obtaining an additional tagged molecule complementary to the cDNA molecule.

65. The method of claim 64, wherein the additional tagged molecule comprises, from a 5’ end to a 3’ end, (i) the one or more barcode sequences and (ii) an additional modified nucleic acid sequence, wherein the additional modified nucleic acid sequence corresponds to the nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

66. The method of claim 64, wherein the additional tagged molecule further comprises a complement to at least a portion of the capture probe.

67. The method of claim 1, wherein the nucleic acid sequence of the RNA molecule comprises, from a 5’ end to a 3’ end, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence.

68. The method of claim 67, wherein the tagged molecule comprises, from a 5’ end to a 3 ’ end of the tagged molecule, (i) the one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the modified first nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the modified second nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

69. The method of claim 68, further comprising sequencing the one or more barcode sequences and the modified first nucleic acid sequence of the tagged molecule.

70. The method of claim 69, wherein the modified second nucleic acid sequence of the tagged molecule is not sequenced.

71. The method of claim 68, wherein the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of an antigen binding molecule of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the antigen binding molecule.

72. The method of claim 68, wherein the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR.Attorney Docket No. 59528-73160173. The method of claim 68, wherein the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

74. The method of claim 1, wherein (a) comprises introducing a plurality of biological materials into the fluidic device, and wherein the plurality of biological materials comprises the biological material.

75. The method of claim 74, further comprising selectively encapsulating the biological material.

76. The method of claim 75, wherein the biological material is selectively encapsulated prior to (b).

77. The method of claim 76, further comprising, prior to the selective encapsulation of the biological material, determining a location of the biological material within the fluidic device.

78. The method of claim 1, wherein the biological material is encapsulated within a chamber comprising one or more polymer matrix walls.

79. The method of claim 78, wherein the one or more polymer matrix walls extend from the surface to an additional surface opposite of the surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material.

80. The method of claim 79, wherein the one or more polymer matrix walls extend partially from the surface towards an additional surface opposite of the surface.

81. The method of claim 80, wherein the chamber comprises a gap between the polymer matrix wall and the additional surface.

82. The method of claim 78, further comprising introducing one or more polymer precursors into the fluidic device.

83. The method of claim 82, wherein the one or more polymer precursors are polymerized, thereby forming the one or more polymer matrix walls.

84. The method of claim 82, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photo-initiator.

85. The method of claim 82, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen.

86. The method of claim 82, wherein the one or more polymer precursors are polymerized by light.Attorney Docket No. 59528-73160187. The method of claim 82, further comprising determining a location of the biological material within the fluidic device and selectively polymerizing the one or more polymer precursors to generate the one or more polymer matrix walls.

88. The method of claim 87, wherein the determining of the location of the biological material within the fluidic device comprises using a detector to image the fluidic device.

89. The method of claim 88, wherein the detector is coupled to an energy source configured to emit energy.

90. The method of claim 89, wherein the energy source is a light generating device, and wherein the energy comprises light.

91. The method of claim 90, further comprising generating a virtual mask based on the location of the biological material within the fluidic device and projecting the virtual mask using the light emitted from the light generating device.

92. The method of claim 91, wherein the virtual mask is generated from a spatial light modulator (SLM).

93. The method of claim 92, wherein the SLM is a digital micromirror device (DMD).

94. The method of claim 89, wherein the energy source is in optical communication with the fluidic device.

95. The method of claim 78, wherein the chamber has an annular-like cross section.

96. A method for biological analysis, comprising:(a) providing a fluidic device comprising a surface, wherein the surface comprises (i) one or more capture probes, and (ii) one or more barcode primers separate from said one or more capture probes;(b) introducing a biological material comprising a ribonucleic acid (RNA) molecule into the fluidic device, wherein the RNA molecule comprises, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence; and(c) on the surface, generating a tagged molecule from the RNA molecule, wherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the modified second nucleic acid sequence is identical to the second nucleicAttorney Docket No. 59528-731601acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

97. The method of claim 96, further comprising, prior to (c), hybridizing a 3’ end of the RNA molecule to a capture probe of the one or more capture probes.

98. The method of claim 97, further comprising extending the capture probe to generate a complementary deoxyribonucleic acid (cDNA) molecule comprising (i) a sequence complementary to the first nucleic acid sequence of the RNA molecule and (ii) a sequence complementary to the second nucleic acid sequence of the RNA molecule.

99. The method of claim 98, further comprising hybridizing the cDNA molecule generated to a barcode primer of the one or more barcode primers.

100. The method of claim 99, further comprising extending the barcode primer using the cDNA molecule generated in (c) as a template, thereby obtaining the tagged molecule, wherein the tagged molecule is complementary to the cDNA molecule.

101. The method of claim 96, wherein the surface comprises a planar surface.

102. The method of claim 101, wherein the planar surface is a top surface of a flow cell.

103. The method of claim 96, wherein the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material.

104. The method of claim 103, wherein the capture element comprises a physical trap.

105. The method of claim 103, wherein the capture element comprises one or more functional groups capable of interacting with the biological material.

106. The method of claim 105, wherein the one or more functional groups comprise an integrin-binding group.

107. The method of claim 106, wherein the integrin-binding group comprises fibronectin.

108. The method of claim 106, wherein the integrin-binding group comprises an Arginylglycylaspartic acid (RGD) peptide.

109. The method of claim 105, wherein the one or more functional groups comprise antibodies.Attorney Docket No. 59528-731601110. The method of claim 96, wherein the surface is a top surface of the fluidic device.

111. The method of claim 110, wherein the fluidic device further comprises a bottom surface opposite of the top surface, and wherein the bottom surface comprises a capture element configured to capture the biological material.

112. The method of claim 96, wherein the surface is a bottom surface of the fluidic device.

113. The method of claim 112, wherein the fluidic device further comprises a top surface opposite of the bottom surface, and wherein the top surface comprises a capture element configured to capture the biological material.

114. The method of claim 96, wherein the capture probe comprises a poly(T) tail.

115. The method of claim 114, wherein the poly(T) tail is located at a 3’ end of the capture probe.

116. The method of claim 114, wherein the poly(T) tail comprises a sequence of three or more thymine bases.

117. The method of claim 114, wherein in (c), the 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe.

118. The method of claim 114, wherein the poly(T) tail comprises a reverse transcriptase primer.

119. The method of claim 118, wherein the capture probe comprises a T30VN reverse transcriptase primer.

120. The method of claim 119, wherein the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe.

121. The method of claim 96, wherein the one or more capture probes do not include barcode sequences.

122. The method of claim 98, wherein the extending is performed using an enzyme.

123. The method of claim 122, wherein the enzyme incorporates, at the 3’ end of the cDNA molecule, a sequence that is complementary to the at least a portion of the barcode primer.

124. The method of claim 98, further comprising incorporating a sequence complementary to a template switch oligonucleotide (TSO) at a 3’ end of the cDNA molecule.Attorney Docket No. 59528-731601125. The method of claim 124, wherein the sequence complementary to the TSO comprises a portion that is complementary to at least a portion of the barcode primer.

126. The method of claim 98, further comprising incorporating, at a 3' end of the cDNA molecule, a sequence that is complementary to at least a portion of the barcode primer.

127. The method of claim 126, wherein the incorporating comprises ligating the sequence that is complementary to at least the portion of the barcode primer to the 3’ end of the cDNA molecule.

128. The method of claim 98, wherein the one or more barcode primers comprise a unique molecular identifier (UMI).

129. The method of claim 128, wherein the extending comprises incorporating the unique molecular identifier (UMI) or a complement thereof into the cDNA molecule.

130. The method of claim 96, further comprising sequencing the tagged molecule or an amplicon generated therefrom, or a combination thereof.

131. The method of claim 96, further comprising sequencing the one or more barcode primers and the modified first nucleic acid sequence.

132. The method of claim 131, wherein the modified second nucleic acid is not sequenced.

133. The method of claim 96, further comprising generating an amplicon of at least a portion of the tagged molecule.

134. The method of claim 133, wherein the generating the amplicon uses a genespecific primer.

135. The method of claim 134, wherein the gene-specific primer binds to an internal position of the tagged molecule.

136. The method of claim 133, wherein the generating the amplicon uses exponential amplification.

137. The method of claim 133, wherein the generating the amplicon uses nested amplification.

138. The method of claim 133, wherein the generating the amplicon uses any combination of (i) a gene-specific primer, (ii) exponential amplification, and (iii) nested amplification.Attorney Docket No. 59528-731601139. The method of claim 133, wherein the amplicon comprises the modified first nucleic acid sequence.

140. The method of claim 132, wherein the amplicon does not comprise the modified second nucleic acid sequence.

141. The method of claim 133, wherein the amplicon comprises the one or more barcode sequences.

142. The method of claim 96, wherein the barcode primer further comprises one or more unique molecular identifiers (UMIs).

143. The method of claim 96, wherein the one or more barcode sequences comprise a barcode which indicates a position of the capture probe within the fluidic device.

144. The method of claim 100, further comprising, hybridizing a 3’ end of the cDNA molecule to an additional barcode primer of the one or more barcode primers.

145. The method of claim 144, further comprising extending the additional barcode primer using the cDNA molecule as a template, thereby obtaining an additional tagged molecule complementary to the cDNA molecule.

146. The method of claim 145, wherein the additional tagged molecule comprises, from a 5’ end to a 3’ end, (i) the one or more barcode sequences, (ii) an additional modified first nucleic acid sequence, and (iii) an additional modified second nucleic acid sequence, wherein the additional modified first nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the additional modified second nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

147. The method of claim 146, wherein the additional tagged molecule further comprises a complement to at least a portion of the additional capture probe.

148. The method of claim 96, wherein the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of an antigen binding molecule of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the antigen binding molecule.

149. The method of claim 96, wherein the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR.Attorney Docket No. 59528-731601150. The method of claim 96, wherein the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

151. The method of claim 96, wherein (a) comprises introducing a plurality of biological materials into the fluidic device, and wherein the plurality of biological materials comprises the biological material.

152. The method of claim 151, further comprising selectively encapsulating the biological material.

153. The method of claim 152, wherein the biological material is selectively encapsulated prior to (c).

154. The method of claim 153, further comprising, prior to the selective encapsulation of the biological material, determining a location of the biological material within the fluidic device.

155. The method of claim 96, wherein the biological material is encapsulated within a chamber comprising one or more polymer matrix walls.

156. The method of claim 155, wherein the one or more polymer matrix walls extend from the surface to an additional surface opposite of the surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material.

157. The method of claim 155, wherein the one or more polymer matrix walls extend partially from the surface towards an additional surface opposite of the surface.

158. The method of claim 157, wherein the chamber comprises a gap between the polymer matrix wall and the additional surface.

159. The method of claim 155, further comprising introducing one or more polymer precursors into the fluidic device.

160. The method of claim 159, wherein the one or more polymer precursors are polymerized, thereby forming the one or more polymer matrix walls.

161. The method of claim 159, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photo-initiator.

162. The method of claim 159, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen.Attorney Docket No. 59528-731601163. The method of claim 159, wherein the one or more polymer precursors are polymerized by light.

164. The method of claim 159, further comprising determining a location of the biological material within the fluidic device and selectively polymerizing the one or more polymer precursors to generate the one or more polymer matrix walls.

165. The method of claim 164, wherein the determining of the location of the biological material within the fluidic device comprises using a detector to image the fluidic device.

166. The method of claim 165, wherein the detector is coupled to an energy source configured to emit energy.

167. The method of claim 166, wherein the energy source is a light generating device, and wherein the energy comprises light.

168. The method of claim 167, further comprising generating a virtual mask based on the location of the biological material within the fluidic device and projecting the virtual mask using the light emitted from the light generating device.

169. The method of claim 168, wherein the virtual mask is generated from a spatial light modulator (SLM).

170. The method of claim 169, wherein the SLM is a digital micromirror device (DMD).

171. The method of claim 166, wherein the energy source is in optical communication with the fluidic device.

172. The method of claim 155, wherein the chamber has an annular-like cross section.

173. A method for processing a biological sample, comprising:(a) introducing a biological material into a fluidic device, wherein the biological material comprises a ribonucleic acid (RNA) molecule comprising, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence;(b) introducing one or more polymer precursors into the fluidic device;(c) determining a location of the biological material within the fluidic device; (d) selectively polymerizing the polymer precursors to generate a polymer matrix from the polymer precursors within the fluidic device, wherein the polymer matrix at least partially encapsulates the biological material; andAttorney Docket No. 59528-731601(e) generating a tagged molecule from the RNA molecule, wherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) a modified first nucleic acid sequence and (ii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the second modified nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

174. A fluidic device, comprising:a planar surface comprising a tagged molecule generated from a ribonucleic acid (RNA) molecule,wherein the RNA molecule comprises, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence, andwherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the second modified nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

175. The fluidic device of claim 174, wherein the planar surface is a bottom surface of a flow cell.

176. The fluidic device of claim 174, wherein the RNA molecule is released from a biological material, wherein the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material.

177. The fluidic device of claim 176, wherein the capture element comprises a physical trap.

178. The fluidic device of claim 176, wherein the capture element comprises one or more functional groups capable of interacting with the biological material.

179. The fluidic device of claim 178, wherein the one or more functional groups comprise an integrin-binding group.Attorney Docket No. 59528-731601180. The fluidic device of claim 179, wherein the integrin-binding group comprises fibronectin.

181. The fluidic device of claim 179, wherein the integrin-binding group comprises an arginylglycylaspartic acid (RGD) peptide.

182. The fluidic device of claim 178, wherein the one or more functional groups comprise antibodies.

183. The fluidic device of claim 174, wherein the planar surface is a bottom surface of the fluidic device.

184. The fluidic device of claim 183, wherein the fluidic device further comprises a top surface opposite of the bottom surface, wherein the RNA molecule is released from a biological material, and wherein the top surface comprises a capture element configured to capture the biological material.

185. The fluidic device of claim 174, wherein the planar surface is a top surface of the fluidic device.

186. The fluidic device of claim 185, wherein the fluidic device further comprises a bottom surface opposite of the top surface, wherein the RNA molecule is released from a biological material, and wherein the bottom surface comprises a capture element configured to capture the biological material.

187. The fluidic device of claim 174, wherein the planar surface further comprises one or more capture probes.

188. The fluidic device of claim 187, wherein the capture probe comprises a poly(T) tail.

189. The fluidic device of claim 188, wherein the poly(T) tail is located at a 3’ end of the capture probe.

190. The fluidic device of claim 188, wherein the poly(T) tail comprises a sequence of three or more thymine bases.

191. The fluidic device of claim 188, wherein a 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe.

192. The fluidic device of claim 188, wherein the poly(T) tail comprises a reverse transcriptase primer.

193. The fluidic device of claim 192, wherein the capture probe comprises a T30VN reverse transcriptase primer.

194. The fluidic device of claim 193, wherein the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe.Attorney Docket No. 59528-731601195. The fluidic device of claim 187, wherein the one or more capture probes do not include barcode sequences.

196. The fluidic device of claim 174, wherein the planar surface further comprises one or more barcode primers, and wherein the one or more barcode primers comprise the one or more barcode sequences.

197. The fluidic device of claim 196, wherein the one or more barcode primers comprise a unique molecular identifier (UMI).

198. The fluidic device of claim 174, wherein the one or more barcode sequences comprise a barcode which indicates a position of the RNA molecule within the fluidic device.

199. The fluidic device of claim 174, wherein the RNA molecule is released from a biological material.

200. The fluidic device of claim 199, wherein the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the immune cell.

201. The fluidic device of claim 199, wherein the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR.

202. The fluidic device of claim 199, wherein the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

203. The fluidic device of claim 199, wherein the biological material is encapsulated within a chamber comprising one or more polymer matrix walls.

204. The fluidic device of claim 203, wherein the one or more polymer matrix walls extend from the planar surface to an additional surface opposite of the planar surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material.

205. The fluidic device of claim 203, wherein the one or more polymer matrix walls extend partially from the planar surface towards an additional surface opposite of the planar surface.Attorney Docket No. 59528-731601206. The fluidic device of claim 205, wherein the chamber comprises a gap between the polymer matrix wall and the additional surface.

207. The fluidic device of claim 203, wherein the one or more polymer matrix walls are formed by polymerization of one of more polymer precursors.

208. The fluidic device of claim 207, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photo-initiator.

209. The fluidic device of claim 207, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen.

210. The fluidic device of claim 207, wherein the one or more polymer precursors are polymerized by light.

211. The fluidic device of claim 203, wherein the chamber has an annular-like cross section.

212. A fluidic device, comprising:a hydrogel chamber comprising a tagged molecule generated from a ribonucleic acid (RNA) molecule,wherein the RNA molecule comprises, from a 5’ end to a 3’ end of the RNA molecule, (i) a first nucleic acid sequence, and (ii) a second nucleic acid sequence, andwherein the tagged molecule comprises, from a 5’ end to a 3’ end of the tagged molecule, (i) one or more barcode sequences, (ii) a modified first nucleic acid sequence, and (iii) a modified second nucleic acid sequence, wherein the first modified nucleic acid sequence is identical to the first nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases, and wherein the second modified nucleic acid sequence is identical to the second nucleic acid sequence of the RNA molecule except that all uracil bases are replaced with thymine bases.

213. The fluidic device of claim 212, wherein the hydrogel chamber is located on a planar surface of the fluidic device.

214. The fluidic device of claim 212, wherein the RNA molecule is released from a biological material, wherein the fluidic device further comprises one or more capture elements, and wherein a capture element of the one or more capture elements is configured to capture the biological material.Attorney Docket No. 59528-731601215. The fluidic device of claim 214, wherein the capture element comprises a physical trap.

216. The fluidic device of claim 214, wherein the capture element comprises one or more functional groups capable of interacting with the biological material.

217. The fluidic device of claim 216, wherein the one or more functional groups comprise an integrin-binding group.

218. The fluidic device of claim 217, wherein the integrin-binding group comprises fibronectin.

219. The fluidic device of claim 217, wherein the integrin-binding group comprises an Arginylglycylaspartic acid (RGD) peptide.

220. The fluidic device of claim 216, wherein the one or more functional groups comprise antibodies.

221. The fluidic device of claim 213, wherein the planar surface is a bottom surface of the fluidic device.

222. The fluidic device of claim 221, wherein the fluidic device further comprises a top surface opposite of the bottom surface, wherein the RNA molecule is released from a biological material, and wherein the bottom surface comprises a capture element configured to capture the biological material.

223. The fluidic device of claim 213, wherein the planar surface is a top surface of the fluidic device.

224. The fluidic device of claim 223, wherein the fluidic device further comprises a bottom surface opposite of the top surface, wherein the RNA molecule is released from a biological material, and wherein the bottom surface comprises a capture element configured to capture the biological material.

225. The fluidic device of claim 212, wherein the fluidic device further comprises one or more capture probes.

226. The fluidic device of claim 225, wherein a capture probe of the one or more capture probes comprises a poly(T) tail.

227. The fluidic device of claim 226, wherein the poly(T) tail is located at a 3’ end of the capture probe.

228. The fluidic device of claim 226, wherein the poly(T) tail comprises a sequence of three or more thymine bases.

229. The fluidic device of claim 226, wherein a 3’ end of the RNA molecule hybridizes to the poly(T) tail of the capture probe.Attorney Docket No. 59528-731601230. The fluidic device of claim 226, wherein the poly(T) tail comprises a reverse transcriptase primer.

231. The fluidic device of claim 230, wherein the capture probe comprises a T30VN reverse transcriptase primer.

232. The fluidic device of claim 231, wherein the T30VN reverse transcriptase primer is located at a 3’ end of the capture probe.

233. The fluidic device of claim 225, wherein the one or more capture probes do not include barcode sequences.

234. The fluidic device of claim 212, wherein the fluidic device further comprises one or more barcode primers, and wherein the one or more barcode primers comprise the one or more barcode sequences.

235. The fluidic device of claim 234, wherein the one or more barcode primers comprise a unique molecular identifier (UMI).

236. The fluidic device of claim 212, wherein the one or more barcode sequences comprise a barcode which indicates a position of the RNA molecule within the fluidic device.

237. The fluidic device of claim 212, wherein the RNA molecule is released from a biological material.

238. The fluidic device of claim 237, wherein the biological material comprises an immune cell, wherein the first nucleic acid sequence comprises a variable region of the immune cell, and wherein the second nucleic acid sequence comprises a constant region of the immune cell.

239. The fluidic device of claim 237, wherein the biological material comprises a T cell comprising a T-cell receptor (TCR), wherein the first nucleic acid sequence comprises a variable region of the TCR, and wherein the second nucleic acid sequence comprises a constant region of the TCR.

240. The fluidic device of claim 237, wherein the biological material comprises a B cell comprising a B-cell receptor (BCR), wherein the first nucleic acid sequence comprises a variable region of the BCR, and wherein the second nucleic acid sequence comprises a constant region of the BCR.

241. The fluidic device of claim 212, wherein the hydrogel chamber comprises one or more polymer matrix walls.

242. The fluidic device of claim 241, wherein the one or more polymer matrix walls extend from the planar surface to an additional surface opposite of theAttorney Docket No. 59528-731601surface, thereby forming an interior of the chamber, wherein the interior of the chamber comprises the biological material.

243. The fluidic device of claim 241, wherein the one or more polymer matrix walls extend partially from the planar surface towards an additional surface opposite of the surface.

244. The fluidic device of claim 241, wherein the hydrogel chamber comprises a gap between the polymer matrix wall and the additional surface.

245. The fluidic device of claim 241, wherein the one or more polymer matrix walls are formed by polymerization of one of more polymer precursors.

246. The fluidic device of claim 245, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers and (ii) a photo-initiator.

247. The fluidic device of claim 245, wherein the one or more polymer precursors comprise (i) one or more cleavable crosslinkers, (ii) a photo-initiator, and (iii) a porogen.

248. The fluidic device of claim 245, wherein the one or more polymer precursors are polymerized by light.

249. The fluidic device of claim 212, wherein the hydrogel chamber has an annular-like cross section.

250. A method for processing a ribonucleic acid (RNA) molecule, comprising:introducing the RNA molecule into a fluidic device, wherein the fluidic device comprises (i) a plurality of capture probes and (ii) a plurality of barcode primers comprising one or more barcode sequences;hybridizing a 3’ end of the RNA molecule to a capture probe of the plurality of capture probes;extending the capture probe using at least a portion of the RNA molecule as a template, thereby generating a complementary deoxyribonucleic acid (cDNA) molecule comprising a sequence complementary to the nucleic acid sequence of the RNA molecule;hybridizing the cDNA molecule to a first barcode primer of the plurality of barcode primers;extending the first barcode primer using at least a portion of the cDNA molecule as a template, thereby obtaining a first tagged molecule complementary to the cDNA molecule;Attorney Docket No. 59528-731601hybridizing the cDNA molecule to a second barcode primer of the plurality of barcode primers; andextending the second barcode primer using the cDNA molecule or the portion of the cDNA molecule as a template, thereby obtaining a second tagged molecule complementary to the cDNA molecule or the portion of the cDNA molecule.

251. The method of claim 250, further comprising hybridizing the cDNA molecule to additional barcode primers of the plurality of barcode primers, and extending the additional barcode primers using the cDNA molecule, the portion of the cDNA molecule, or an additional portion of the cDNA molecule as a template.

252. A method for processing a ribonucleic acid (RNA) molecule, comprising:introducing the RNA molecule into a fluidic device, wherein the fluidic device comprises (i) a capture probe coupled to a surface and (ii) a barcode primer comprising one or more barcode sequences;hybridizing a 3’ end of the RNA molecule to the capture probe; extending the capture probe using at least a portion of the RNA molecule as a template to generate a complementary deoxyribonucleic acid (cDNA) molecule coupled to the capture probe and comprising a sequence complementary to the nucleic acid sequence of the RNA molecule;hybridizing the cDNA molecule to the barcode primer; extending:the cDNA molecule using at least a portion of the barcode primer as a template, thereby incorporating a complement of a barcode sequence of the one or more barcode sequences to the cDNA molecule,the barcode primer using at least a portion of the cDNA molecule as a template, thereby incorporating a complement of the cDNA molecule or the portion of the cDNA molecule to the barcode primer, ora combination thereof; andcleaving:the capture probe from the surface,the cDNA molecule from the capture probe,Attorney Docket No. 59528-731601the barcode primer from the surface, ora combination thereof.

253. A method for processing ribonucleic acid (RNA) molecules, comprising:introducing the RNA molecules into a fluidic device, wherein the fluidic device comprises (i) a plurality of capture probes coupled to a surface and (ii) a plurality of barcode primers comprising one or more barcode sequences;hybridizing 3’ ends of the RNA molecules to capture probes of the plurality of capture probes;extending the capture probes using the RNA molecules or portions of the RNA molecules as templates to generate complementary deoxyribonucleic acid (cDNA) molecules coupled to the capture probes and comprising sequences complementary to nucleic acid sequences of the RNA molecules;hybridizing the cDNA molecules to barcode primers of the plurality of barcode primers;extending the cDNA molecules using at least portions of the barcode primers as templates, thereby incorporating complements of barcode sequences of the one or more barcode sequences to the cDNA molecules; cleaving the capture probes from the surface or cleaving the cDNA molecules from the capture probe, thereby releasing the cDNA molecules from the surface;amplifying a first portion of the cDNA molecules using a first amplification reaction;amplifying a second portion of the cDNA molecules using a second amplification reaction; andsequencing amplicons generated from the first and second amplification reaction.

254. The method of claim 253, wherein the first amplification reaction comprises a whole transcriptome amplification method.

255. The method of claim 254, wherein the whole transcriptome amplification method comprises tagmentation, fragmentation, adapter ligation, a random primer, or a combination thereof.Attorney Docket No. 59528-731601256. The method of claim 253, wherein the first amplification reaction comprises amplification with a gene-specific primer.

257. The method of claim 253, wherein the second amplification reaction comprises a whole transcriptome amplification method.

258. The method of claim 257, wherein the whole transcriptome amplification method comprises tagmentation, fragmentation, adapter ligation, a random primer, or a combination thereof.

259. The method of claim 253, wherein the second amplification reaction comprises amplification with a gene-specific primer.

260. The method of claim 253, wherein the first amplification reaction comprises amplification with a first gene-specific primer and the second amplification reaction comprises amplification with a second gene-specific primer.

261. The method of claim 253, wherein during the incorporating the complements of barcode sequences of the one or more barcode sequences to the cDNA molecules, the barcode primers are extended over the cDNA molecules, thereby generating tagged molecules, and wherein the tagged molecules are amplified with the cDNA molecules in the first amplification reaction and the second amplification reaction.

262. The method of claim 261, following the incorporating the complements of barcode sequences of the one or more barcode sequences to the cDNA molecules, the method further comprises melting the cDNA molecules from the barcode primers, hybridizing the cDNA molecules to additional barcode primers of the plurality of barcode primers; and extending the additional barcode primers over the cDNA molecules, thereby generating additional tagged molecules, wherein the additional tagged molecules are amplified with the cDNA molecules in the first amplification reaction and the second amplification reaction.

263. The method of claim 253, wherein the first amplification reaction and the second amplification reaction are different.

264. The method of claim 253, wherein the first amplification reaction and the second amplification reaction are the same.

265. A method for processing a ribonucleic acid (RNA) molecule, comprising:introducing the RNA molecule into a fluidic device, wherein the fluidic device comprises (i) a capture probe comprising a first primer bindingAttorney Docket No. 59528-731601site and (ii) a barcode primer comprising one or more barcode sequences and a second primer binding site;hybridizing a 3’ end of the RNA molecule to the capture probe; extending the capture probe using at least a portion of the RNA molecule as a template to generate a complementary deoxyribonucleic acid (cDNA) molecule coupled to the capture probe, wherein the cDNA comprises a sequence complementary to the RNA molecule or a portion of the RNA molecule;incorporating a sequence that is complementary to a template switch oligonucleotide (TSO) at a 3’ end of the cDNA molecule;hybridizing the cDNA molecule to the barcode primer; and extending the cDNA molecule using the barcode primer as a template, thereby incorporating complements of the one or more barcode sequences and the second primer binding site 3’ to the sequence that is complementary to the template switch oligonucleotide, thereby generating a product strand comprising the first primer binding site and a complement of the second primer binding site, wherein the first primer binding site is configured to hybridize to the complement of the second primer binding site.

266. A fluidic device comprising:i) a surfaceii) a capture probe coupled to the surface, wherein the capture probe comprises, in a direction from 5’ to 3’:a) a first primer binding site, andb) a nucleic acid capture sequence; andiii) a barcode primer coupled to the surface, wherein the barcode primer comprises, in a direction from 5’ to 3’:a) a second primer binding site,b) one or more barcode sequences, andc) a known sequence.

267. The fluidic device of claim 266, wherein the nucleic acid capture sequence comprises a poly(T) sequence.

268. The fluidic device of claim 266, wherein:Attorney Docket No. 59528-731601i) the capture probe comprises a cleavage domain at a 5’ end of the first primer binding site;ii) the barcode primer comprises a cleavage domain at a 5’ end of the second primer binding site; oriii) the capture probe comprises a cleavage domain at a 5’ end of the first primer binding site and the barcode primer comprises a cleavage domain at a 5’ end of the second primer binding site.

269. The fluidic device of claim 266, wherein the first primer binding site comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 99%, or 100% sequence homology with the second primer binding site.

270. The fluidic device of claim 266, wherein the known sequence comprises about 10 to 100 nucleotides or about 15 to 40 nucleotides.

271. The fluidic device of claim 266, wherein the one or more barcode sequences comprise a sequence associated with a location on the surface.

272. The fluidic device of claim 266, wherein a spot on the surface of the fluidic device comprises a plurality of instances of the capture probe and a plurality of instances of the barcode primer.

273. The fluidic device of claim 272, wherein the one or more barcode sequences in each instance of the barcode primer comprise a sequence associated with a location of the spot on the surface.

274. The fluidic device of claim 273, wherein the spot has a diameter ranging from about 80 microns to about 140 microns.

275. The fluidic device of claim 273, wherein the fluidic device comprises a plurality of spots each comprising a plurality of instances of the capture probe and a plurality of instances of the barcode primer, wherein the one or more barcode sequences in each instance of the barcode primer comprises a sequence uniquely associated with the spot of the plurality of spots in which the barcode primer is located.Attorney Docket No. 59528-731601276. The fluidic device of claim 266, wherein a 5’ end of the first primer binding site and a 5’ end of the second primer binding site are each coupled to the surface.

277. The fluidic device of claim 266, wherein the surface comprises a first surface, wherein the fluidic device further comprises a second surface, wherein the first surface is opposite of the second surface, and wherein the second surface comprises a cell adherent support.

278. The fluidic device of claim 277, wherein the cell adherent support comprises a material selected from the group consisting of actinin, collagen, fibrinogen, fibronectin, gelatin, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, poly-1 - lysine (PLL), poly-d-lysine (PDL), poly-l-omithine, talin, VCAM-1, vinculin, vitronectin, a cell adherent peptide, or a combination thereof.

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