Systems and methods for analyzing biological samples - Patents.com

Abstract

The present invention is directed to methods and systems for creating a library of cDNA on a surface and for analyzing nucleic acid molecules from multiple cells on the surface of a support. In some embodiments, the present invention includes a method for analyzing the transcriptomes of multiple single cells on separate areas of the same plane. The present invention is directed to methods and systems for creating a library of cDNA on a surface. In some embodiments, the present invention is directed to methods and systems for analyzing nucleic acid molecules, such as messenger RNA, from multiple cells on the surface of a solid support.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 209,544, filed June 11, 2021, which is incorporated by reference herein in its entirety. Incorporation by Reference

[0002] All publications, patents, and patent applications mentioned herein are 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 herein. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Background technology]

[0003] background Analysis of biological samples is one of the cornerstones of modern medicine. Although there have been recent developments that advance the analysis of specific deoxynucleic acid (DNA) molecules, analysis of nucleic acid molecules (e.g., DNA, ribonucleic nucleic acid (RNA)) created from specific cell or tissue samples remains a hurdle to overcome for the industry. For example, analysis of gene expression profiles (e.g., transcriptomes) in cells based on sequence and the abundance of nucleic acids to be sequenced in a sample remains inefficient and labor intensive. Currently available sequencing techniques have drawbacks. For example, sequencing of RNA samples requires sample preparation methods that first convert the RNA into double-stranded cDNA format prior to sequencing. Thus, preparation of biological samples containing RNA for sequencing is often labor intensive. Furthermore, current sequencing techniques are not the best at preserving the strand-specific information of the original single-stranded RNA molecules after they have been converted to double-stranded cDNA. Preserving strand-specific information can be important for annotation to determine gene expression levels.

[0004] The field of cell analysis will be advanced by the availability of multiplexed single-cell analysis methods, particularly RNA-seq methods, which provide sensitive and simple measurements of cellular gene expression. Summary of the Invention [Means for solving the problem]

[0005] overview The present invention is directed to a method and system for creating a library of cDNA on a surface. In some embodiments, the present invention is directed to a method and system for analyzing nucleic acid molecules, such as messenger RNA, from multiple cells on the surface of a solid support. In some embodiments, the present invention is directed to a method for synthesizing cDNA from nucleic acid molecules from multiple cells arranged on the surface of a solid support. In some embodiments, the method of the present invention is directed to determining the transcriptome of cells arranged on the surface of a solid support. In some embodiments, such a method may include the steps of: (a) providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes on the surface; (b) contacting separate areas of the surface with one or more nucleic acid molecules of different cells to generate one or more captured nucleic acid molecules in each of the separate areas, the captured nucleic acid molecules in the different separate areas being from different cells; and (c) synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives thereof, each of the cDNA molecules being bound to the surface of the solid support, and the cDNA molecules bound to the different separate areas being from different cells. In some embodiments, the surface-bound cDNA molecules comprise cDNA molecules covalently bound to the surface. In some embodiments, such methods further comprise amplifying the cDNA molecules or derivatives thereof to create a plurality of sets of amplicons of the cDNA molecules or derivatives thereof, each set of amplicons being from a different cell. In some embodiments, the transcriptomes of the plurality of cells are determined by sequencing the cDNA molecules of the amplicons. In some embodiments, the discrete regions of the surface of the solid support are determined by hydrogel chambers surrounding each of the plurality of cells. In some embodiments, the contacting step comprises treating the cells with a lysis agent that releases one or more nucleic acid molecules. In some embodiments, the one or more nucleic acid molecules are ribonucleic acid (RNA).In some embodiments, the surface comprises a diffusivity modifier that reduces or blocks diffusion of one or more nucleic acids away from the cell. In some embodiments, the diffusivity modifier comprises a gel barrier. In some embodiments, the gel barrier that modulates diffusivity comprises a hydrogel chamber. In some embodiments, the surface of the solid support comprises cells disposed thereon. In some embodiments, the surface of the solid support comprises a diffusivity modifier. In a variation of the above embodiments, the surface of the solid support is a planar surface. In some embodiments, the systems and methods of the invention employ an optical system to collect optical signals from labeled cells and / or molecules on the planar surface.

[0006] An aspect provided herein is a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, comprising the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives, the cDNA molecules being attached to the solid support; inserting an adaptor into the 3' region of the cDNA molecules or derivatives; and amplifying the cDNA molecules or derivatives to create a set of cDNA molecules or derivatives, the set of cDNA molecules or derivatives being attached to the solid support. In some embodiments, the synthesizing comprises performing reverse transcription and one or more second strand synthesis reactions. In some embodiments, the set of cDNA molecules or derivatives are attached to a plurality of surface primer probes. In some embodiments, the adaptor comprises a sequence configured to allow for the initiation of a sequencing reaction on the cDNA molecules of the set of cDNA molecules or derivatives. In some embodiments, the set of cDNA molecules or derivatives comprises an adaptor. In some embodiments, the method includes contacting the solid support with a moiety configured to inactivate at least a subset of the one or more nucleic acid molecule capture probes after contacting the solid support with the one or more nucleic acid molecules. In some embodiments, the subset of the one or more nucleic acid molecule capture probes includes one or more nucleic acid molecule capture probes that did not capture the nucleic acid molecule. In some embodiments, the moiety configured to inactivate at least a subset of the one or more nucleic acid molecule capture probes includes an exonuclease. In some embodiments, the one or more second strand synthesis reactions include template switch extension, random priming, or both. In some embodiments, prior to inserting an adaptor into the 3' region of the cDNA molecule, the method includes amplifying the cDNA molecule or a derivative thereof.In some embodiments, prior to inserting an adaptor into the 3' region of the cDNA molecule, the method includes a primer sequence in solution. In some embodiments, the cDNA molecule is amplified using the primer sequence in solution. In some embodiments, prior to inserting an adaptor into the 3' region of the cDNA molecule, the method includes fragmenting the cDNA molecule or a derivative thereof. In some embodiments, inserting an adaptor into the 3' region of the cDNA molecule includes single-stranded ligation. In some embodiments, inserting an adaptor into the 3' region of the cDNA molecule includes tagmentation. In some embodiments, inserting an adaptor into the 3' region of the cDNA molecule includes double-stranded ligation. In some embodiments, at least a subset of the plurality of surface primer probes includes a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, prior to amplifying the cDNA molecule, the plurality of surface primer probes is subjected to a reaction in which a blocking agent unblocks at least a subset of the plurality of surface primer probes to allow an extension reaction. In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule comprising a sequence complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule comprising a sequence partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. In some embodiments, the method comprises cleaving or linearizing at least a subset of the set of cDNA molecules or derivatives thereof. In some embodiments, the method comprises blocking the 3' end of a subset of the set of DNA molecules or derivatives thereof. In some embodiments, blocking the 3' end of a subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with terminal deoxynucleotidyl transferase (TdT).In some embodiments, blocking the 3' ends of the subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with an oligonucleotide comprising a sequence complementary to the 3' ends of the subset of the set of DNA molecules or derivatives thereof. In some embodiments, blocking the 3' ends of the subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with a cationic-neutral diblock polypeptide copolymer. In some embodiments, the method comprises sequencing the subset of the cDNA molecules or derivatives thereof in situ on a solid support. In some embodiments, the method comprises eluting at least the subset of the set of cDNA molecules or derivatives thereof from the solid support. In some embodiments, the one or more nucleic acid molecules comprise DNA or ribonucleic acid (RNA) molecules. In some embodiments, the DNA is fragmented single-stranded DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the RNA molecules comprise messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the RNA molecules comprise mRNA. In some embodiments, the one or more nucleic acid molecule capture probes include a sequence configured to bind to one or more nucleic acid molecules. In some embodiments, the sequence configured to bind to one or more nucleic acid molecules includes a poly-T sequence, a randomer, a sequence complementary to at least a subset of one or more nucleic acid molecules, or any combination thereof. In some embodiments, the solid support may include a well, a bead, a gel matrix, or a fluidic channel. In some embodiments, the one or more nucleic acid molecule capture probes and the plurality of surface primer probes are bound to a planar surface of the solid support. In some embodiments, the fluidic channel includes a flow cell. In some embodiments, the one or more nucleic acid molecule capture probes include one or more tags, the tags including a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the amplifying step includes solid-supported amplification.In some embodiments, the solid support amplification is bridge amplification. In some embodiments, the one or more nucleic acid molecules are derived from a single cell or a biological tissue. In some embodiments, the method occurs in a gel matrix, and the gel matrix is ​​adjacent to the solid support.

[0007] An aspect provided herein is a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, comprising the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing a cDNA molecule from the captured nucleic acid molecule or derivative; first amplifying the cDNA molecule or derivative to create an amplified cDNA population; inserting an adaptor into the 3' region of the amplified cDNA molecule or derivative, thereby creating a tagged amplified cDNA population; and performing solid support amplification on the tagged amplified cDNA population to create a set of cDNA molecules or derivatives thereof. In some embodiments, the synthesizing step comprises reverse transcription of the captured RNA template. In some embodiments, the solid support comprises a plurality of surface primer probes. In some embodiments, the cDNA molecule or derivative, the amplified cDNA population, the tagged amplified cDNA population, the set of cDNA molecules or derivatives thereof, or any combination thereof, is bound to a plurality of surface primer probes. In some embodiments, the adaptor comprises a sequence configured to allow initiation of a sequencing reaction on the cDNA molecules of the set of cDNA molecules or derivatives thereof. In some embodiments, the set of cDNA molecules or derivatives thereof comprises an adaptor. In some embodiments, the method comprises contacting the solid support with a moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes. In some embodiments, the subset of one or more nucleic acid molecule capture probes comprises one or more nucleic acid molecule capture probes that did not capture a nucleic acid molecule. In some embodiments, the moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes comprises an exonuclease. In some embodiments, the synthesizing comprises performing one or more second strand synthesis reactions comprising the cDNA molecules or derivatives thereof.In some embodiments, the one or more second strand synthesis reactions comprise template switch extension. In some embodiments, the one or more second strand synthesis reactions comprise random priming. In some embodiments, the method comprises fragmenting the amplified cDNA molecules. In some embodiments, the inserting of adapters comprises single stranded ligation. In some embodiments, the inserting of adapters comprises tagging fragmentation. In some embodiments, the inserting of adapters comprises double stranded ligation. In some embodiments, at least a subset of the plurality of surface primer probes comprises a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, the method comprises subjecting the blocking agent to a reaction that unblocks at least a subset of the plurality of surface primer probes to allow an extension reaction. In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule that comprises a sequence complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule comprising a sequence partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. In some embodiments, the method comprises cleaving or linearizing at least a subset of the set of cDNA molecules or derivatives thereof. In some embodiments, the method comprises blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof. In some embodiments, blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with terminal deoxynucleotidyl transferase (TdT).In some embodiments, blocking the 3' ends of the subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with an oligonucleotide comprising a sequence complementary to the 3' ends of the subset of the set of DNA molecules or derivatives thereof. In some embodiments, blocking the 3' ends of the subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with a cationic-neutral diblock polypeptide copolymer. In some embodiments, the method comprises sequencing at least a subset of the cDNA molecules or derivatives thereof in situ on a solid support. In some embodiments, the method comprises eluting at least a subset of the set of cDNA molecules or derivatives thereof from a solid support. In some embodiments, the one or more nucleic acid molecules comprise DNA or ribonucleic acid (RNA) molecules. In some embodiments, the DNA is fragmented single-stranded DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the RNA molecules comprise messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the RNA molecules comprise mRNA. In some embodiments, the one or more nucleic acid molecule capture probes include a sequence configured to bind to one or more nucleic acid molecules. In some embodiments, the sequence configured to bind to one or more nucleic acid molecules includes a poly-T sequence, a randomer, a sequence complementary to at least a subset of one or more nucleic acid molecules, or any combination thereof. In some embodiments, the solid support includes a well, a bead, a gel matrix, or a fluidic channel. In some embodiments, the fluidic channel is a flow cell. In some embodiments, the solid support is not a bead. In some embodiments, the one or more nucleic acid molecule capture probes include one or more tags, and the tag includes a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the first amplifying step includes solid-supported amplification. In some embodiments, the first amplifying step includes a primer sequence in solution.In some embodiments, the solid support amplification is bridge amplification. In some embodiments, the one or more nucleic acid molecules are derived from a single cell or a biological tissue. In some embodiments, the method occurs in a gel matrix, and the gel matrix is ​​adjacent to the solid support.

[0008] An aspect provided herein is a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, the method comprising the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes, at least a subset of the plurality of surface primer probes comprising a template switch moiety; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives, the synthesizing comprising performing reverse transcription; inserting an adaptor into the 3' end of the cDNA molecules or derivatives; and amplifying the cDNA molecules or derivatives to generate a set of cDNA molecules or derivatives thereof. In some embodiments, the synthesizing comprises performing one or more second strand synthesis reactions comprising the cDNA molecules or derivatives thereof. In some embodiments, the one or more second strand synthesis reactions are mediated by a subset of the plurality of surface primer probes comprising a template switch moiety. In some embodiments, the one or more second strand synthesis reactions comprise template switch extension. In some embodiments, the cDNA molecule or its derivatives, the set of cDNA molecules or its derivatives, or both are bound to a plurality of surface primer probes. In some embodiments, the adaptor comprises a sequence configured to allow initiation of a sequencing reaction on the cDNA molecules of the set of cDNA molecules or its derivatives. In some embodiments, the set of cDNA molecules or its derivatives comprises an adaptor. In some embodiments, the method comprises contacting the solid support with a moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes. In some embodiments, the subset of one or more nucleic acid molecule capture probes comprises one or more nucleic acid molecule capture probes that did not capture a nucleic acid molecule. In some embodiments, the moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes comprises an exonuclease.In some embodiments, the method comprises amplifying a cDNA molecule or a derivative thereof. In some embodiments, the amplifying comprises a primer sequence in solution. In some embodiments, the inserting of an adaptor comprises fragmenting a cDNA molecule. In some embodiments, the inserting of an adaptor comprises single-stranded ligation. In some embodiments, the inserting of an adaptor comprises tagging fragmentation. In some embodiments, the inserting of an adaptor comprises ligation. In some embodiments, at least a subset of the plurality of surface primer probes comprises a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, the method comprises subjecting the blocking agent to a reaction that unblocks at least a subset of the plurality of surface primer probes to allow an extension reaction. In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule that comprises a sequence complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents include a nucleic acid molecule comprising a sequence partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. In some embodiments, the method includes cleaving or linearizing at least a subset of the set of cDNA molecules or derivatives thereof. In some embodiments, the method includes blocking the 3' end of a subset of the set of DNA molecules or derivatives thereof. In some embodiments, blocking the 3' end of a subset of the set of DNA molecules or derivatives thereof includes contacting the subset of the set of DNA molecules or derivatives thereof with terminal deoxynucleotidyl transferase (TdT). In some embodiments, blocking the 3' end of a subset of the set of DNA molecules or derivatives thereof includes contacting the subset of the set of DNA molecules or derivatives thereof with an oligonucleotide comprising a sequence complementary to the 3' end of the subset of the set of DNA molecules.In some embodiments, blocking the 3' ends of the subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with a cationic-neutral diblock polypeptide copolymer. In some embodiments, the method comprises sequencing at least a subset of the set of cDNA molecules or derivatives thereof in situ on a solid support. In some embodiments, the method comprises eluting at least a subset of the set of cDNA molecules or derivatives thereof from a solid support. In some embodiments, the one or more nucleic acid molecules comprise DNA or ribonucleic acid (RNA) molecules. In some embodiments, the DNA is fragmented single-stranded DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the RNA molecule comprises messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the RNA molecule comprises mRNA. In some embodiments, the one or more nucleic acid molecule capture probes comprise a sequence configured to bind to the one or more nucleic acid molecules. In some embodiments, the sequence configured to bind to the one or more nucleic acid molecules comprises a poly-T sequence, a randomer, a sequence complementary to at least a subset of the one or more nucleic acid molecules, or any combination thereof. In some embodiments, the solid support comprises a well, a bead, a gel matrix, or a fluidic channel. In some embodiments, the fluidic channel is a flow cell. In some embodiments, the one or more nucleic acid molecule capture probes comprise one or more tags, the tags comprising a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the amplifying step comprises solid-supported amplification. In some embodiments, the solid-supported amplification is bridge amplification. In some embodiments, the one or more nucleic acid molecules are derived from a single cell or biological tissue. In some embodiments, the method occurs in or adjacent to a gel matrix, and the gel matrix is ​​adjacent to the solid support.

[0009] An aspect provided herein is a solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes, wherein at least a subset of the plurality of surface primer probes comprises a template switch portion. In some embodiments, the one or more nucleic acid molecule capture probes comprise a sequence configured to bind to one or more nucleic acid molecules. In some embodiments, the sequence configured to bind to one or more nucleic acid molecules comprises a poly-T sequence, a randomer, a sequence complementary to at least a subset of one or more nucleic acid molecules, or any combination thereof. In some embodiments, the solid support comprises a well, a bead, a gel matrix, or a fluidic channel. In some embodiments, the fluidic channel is a flow cell. In some embodiments, the one or more nucleic acid molecule capture probes comprise one or more tags, the tags comprising a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the one or more nucleic acid molecules comprise DNA or ribonucleic acid (RNA) molecules. In some embodiments, the DNA is fragmented. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the RNA molecule comprises messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the RNA molecule comprises mRNA, hi some embodiments, the solid support comprises a gel matrix, wherein the gel matrix is ​​adjacent to the solid support.

[0010] An aspect provided herein is a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, comprising the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing a cDNA molecule from the captured nucleic acid molecule or derivative, the synthesizing comprising reverse transcription, the cDNA molecule being bound to the solid support; inserting an adaptor into the 3' end of the cDNA molecule or derivative; and amplifying the cDNA molecule or derivative to create a set of cDNA molecules or derivatives thereof, the set of cDNA molecules or derivatives being bound to the solid support. In some embodiments, the set of cDNA molecules or derivatives thereof is bound to a plurality of surface primer probes. In some embodiments, the adaptor comprises a sequence configured to allow initiation of a sequencing reaction on the cDNA molecules of the set of cDNA molecules or derivatives thereof. In some embodiments, the set of cDNA molecules or derivatives thereof comprises an adaptor. In some embodiments, the method includes contacting the solid support with a moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes. In some embodiments, the subset of one or more nucleic acid molecule capture probes includes one or more nucleic acid molecule capture probes that did not capture a nucleic acid molecule. In some embodiments, the moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes includes an exonuclease. In some embodiments, the synthesizing step includes performing one or more second strand synthesis reactions including a cDNA molecule or a derivative thereof. In some embodiments, the one or more second strand synthesis reactions include template switch extension. In some embodiments, the one or more second strand synthesis reactions include random priming. In some embodiments, the method includes amplifying the cDNA molecule or a derivative thereof.In some embodiments, the amplifying step comprises a primer sequence in solution. In some embodiments, the inserting step of adaptors comprises fragmentation of cDNA molecules. In some embodiments, the inserting step of adaptors comprises single stranded ligation. In some embodiments, the inserting step of adaptors comprises tagging fragmentation. In some embodiments, the inserting step of adaptors comprises ligation. In some embodiments, at least a subset of the plurality of surface primer probes comprises a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, the method comprises subjecting the blocking agent to a reaction that unblocks at least a subset of the plurality of surface primer probes to allow an extension reaction. In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule that comprises a sequence that is complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule that comprises a sequence that is partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. In some embodiments, the method comprises cleaving or linearizing at least a subset of the set of cDNA molecules or derivatives thereof. In some embodiments, the method comprises blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof. In some embodiments, blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with terminal deoxynucleotidyl transferase (TdT). In some embodiments, blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with an oligonucleotide comprising a sequence complementary to the 3' ends of the subset of the set of DNA molecules.In some embodiments, blocking the 3' ends of the subset of the set of DNA molecules or derivatives thereof comprises contacting the subset of the set of DNA molecules or derivatives thereof with a cationic-neutral diblock polypeptide copolymer. In some embodiments, the method comprises sequencing at least a subset of the set of cDNA molecules or derivatives thereof in situ on a solid support. In some embodiments, the method comprises eluting at least a subset of the set of cDNA molecules or derivatives thereof from a solid support. In some embodiments, the one or more nucleic acid molecules comprise DNA or ribonucleic acid (RNA) molecules. In some embodiments, the DNA is fragmented single-stranded DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the RNA molecule comprises messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the RNA molecule comprises mRNA. In some embodiments, the one or more nucleic acid molecule capture probes comprise a sequence configured to bind to the one or more nucleic acid molecules. In some embodiments, the sequence configured to bind to the one or more nucleic acid molecules comprises a poly-T sequence, a randomer, a sequence complementary to at least a subset of the one or more nucleic acid molecules, or any combination thereof. In some embodiments, the solid support comprises a well, a bead, a gel matrix, or a fluidic channel. In some embodiments, the fluidic channel is a flow cell. In some embodiments, the solid support is not a bead. In some embodiments, the one or more nucleic acid molecule capture probes comprise one or more tags, the tags comprising a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the amplifying step comprises solid-supported amplification. In some embodiments, the solid-supported amplification is bridge amplification. In some embodiments, the one or more nucleic acid molecules are derived from a single cell or biological tissue. In some embodiments, the method occurs in a gel matrix, the gel matrix being adjacent to the solid support.

[0011] Aspects provided herein include a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules from a plurality of cells, comprising the steps of: providing a solid support comprising a surface comprising a plurality of cells disposed thereon, comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes bound thereto; contacting distinct areas of the surface with one or more nucleic acid molecules of different cells of the plurality of cells to generate one or more captured nucleic acid molecules in each of the distinct areas, wherein the nucleic acid molecules in the different distinct areas are from different cells of the plurality of cells; and synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives thereof, wherein each of the cDNA molecules is bound to the surface primer probes of the plurality of surface primer probes, and wherein the cDNAs bound to the different distinct areas are from different cells of the plurality of cells. In some embodiments, the contacting step comprises treating the different cells of the plurality of cells with a lysis reagent to release one or more nucleic acid molecules from the different cells of the plurality of cells. In some embodiments, the method further comprises amplifying the cDNA molecules or derivatives thereof to generate a plurality of sets of amplicons of the cDNA molecules or derivatives thereof. In some embodiments, the transcriptomes of the plurality of cells are determined by sequencing the cDNA molecules of the amplicons. In some embodiments, the cDNA molecules bound to the surface include spatial barcodes that code for locations on the surface, and the method further includes eluting the cDNA molecules from the surface prior to sequencing. In some embodiments, the contacting step of the one or more nucleic acid molecules is performed in the presence of a diffusivity modifier that reduces the diffusivity of the one or more nucleic acid molecules. In some embodiments, the surface includes a gel layer that encapsulates the cells disposed thereon, or each of the cells disposed on the surface is encapsulated by a separate gel body. In some embodiments, each of the cells disposed on the surface is surrounded by a hydrogel chamber.In some embodiments, the hydrogel chamber comprises an interior area, and the contacting step further comprises incubating the released nucleic acid molecule or molecules at a predetermined temperature such that the captured nucleic acid molecule or molecules are released and recaptured by the capture probe within the interior area. In some embodiments, the interior area of ​​the hydrogel chamber is selected such that the bound cDNA molecules have an expected nearest neighbor distance of at least 0.25 μm. In some embodiments, the interior area of ​​the hydrogel chamber is selected such that the bound cDNA molecules have an expected nearest neighbor distance of at least 1 μm. In some embodiments, the interior area of ​​the hydrogel chamber is selected such that the bound cDNA molecules have an expected nearest neighbor distance of at least 2 μm. In some embodiments, the bound cDNA molecules have an expected nearest neighbor distance in the range of 0.5 μm and 5 μm.

[0012] Aspects provided herein include a hydrogel chamber disposed on a surface, the hydrogel chamber comprising an internal area comprising a substantially uniform distribution of nucleic acid molecules from a single cell. In some embodiments, the nucleic acid molecules are mRNA molecules. In some embodiments, the substantially uniform distribution is a Poisson distribution with an expected nearest neighbor distance between nucleic acid molecules of 1 μm or greater. In some embodiments, the uniform distribution of nucleic acid molecules is substantially a Poisson distribution.

[0013]

[0013] Aspects provided herein include methods for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, the method comprising the steps of: providing a solid support comprising a surface, wherein the surface is unpartitioned, and wherein the solid support comprises one or more nucleic acid molecule capture probes and a plurality of surface primer probes; creating distinct regions on the solid support, wherein the distinct regions comprise one or more cells unique to the distinct regions; extracting one or more ribonucleic acid ("RNA") molecules from the one or more cells, wherein the one or more RNA molecules are captured by the one or more nucleic acid molecule capture probes located in the distinct regions, thereby creating one or more captured RNA molecules unique to the distinct regions; synthesizing a cDNA molecule or derivatives thereof from the one or more captured RNA molecules or derivatives thereof, wherein the cDNA molecule is bound to the surface primer probes of the plurality of surface primer probes located in the distinct regions; inserting an adaptor into the 3' region of the cDNA molecule or derivative; and amplifying the cDNA molecule or derivative to create a set of cDNA molecules or derivatives thereof, wherein the set of cDNA molecules or derivatives thereof is bound to the solid support. In some embodiments, the distinct regions are surrounded by a polymer matrix. In some embodiments, each distinct region comprises a single cell. In some embodiments, the method occurs within a polymer matrix. In some embodiments, the polymer matrix forms a hydrogel. In some embodiments, the polymer matrix is ​​formed from one or more polymer precursors. In some embodiments, the polymer matrix comprises pores sized to allow diffusion of reagents through the polymer matrix, and RNA molecules cannot diffuse through the pores of the polymer matrix. In some embodiments, the solid support comprises one or more distinct regions, and one or more distinct regions are not in fluid communication with another distinct region. In some embodiments, the solid support is not a bead.In some embodiments, the undivided surface comprises a plane, with each point or location on the plane in fluid communication with every other point or location on the plane.

[0014] This application for patent contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0015] [Figure 1A] FIG. 1A shows a diagram of current techniques for generating transcriptomes, according to some embodiments.

[0016] [Figure 1B] FIG. 1B shows a workflow for current techniques for generating transcriptomes, according to some embodiments.

[0017] [Figure 2A] Figure 2A shows a workflow for obtaining improved transcriptomes from in situ and direct creation of sequenceable libraries from captured mRNA. A library is created directly on the surface using surface coating of first and second primers (such as P5 and P7 primers). The amplification step can potentially be done using bridge amplification, but with primers in solution. The surface clusters thus created can be sequenced directly in situ, or the library can be eluted from the surface and sequenced separately.

[0018] [Figure 2B] FIG. 2B illustrates a problem addressed by an embodiment of the present invention related to the analysis of nucleic acid molecules released by neighboring cells on a flat surface.

[0019] [Figure 2C]FIG. 2C illustrates an embodiment for addressing the problem of FIG. 2B using a diffusible modifier.

[0020] [Figure 2D] FIG. 2D illustrates an embodiment in which the diffusible modifier is a gel layer that encapsulates cells disposed on a flat surface.

[0021] [Figure 2E] 2E-2F show an embodiment in which the diffusible modifier is a hydrogel chamber that surrounds a single cell. [Figure 2F] 2E-2F show an embodiment in which the diffusible modifier is a hydrogel chamber that surrounds a single cell.

[0022] [Figure 2G] FIG. 2G shows an embodiment in which the diffusive modifier is a hydrogel layer, excluding the cavities or wells around the cells.

[0023] [Figure 2H] FIG. 2H illustrates a problem associated with the distribution of captured nucleic acid molecules adjacent to the cells from which they are released.

[0024] [Figure 2I] FIG. 2I shows an embodiment of the invention that addresses the distribution problem of FIG. 2H by using a hydrogel chamber and a method for destabilizing and recapturing cellular nucleic acid molecules to produce a uniform distribution of captured molecules on the inner surface of the hydrogel chamber.

[0025] [Diagram 3]Figure 3 shows the use of 3' phosphate nucleotides to block and unblock primers as needed in the workflow of obtaining a transcriptome by utilizing the methods and systems described herein. This use is similar to that seen in Figure 2A. However, first, the P5 and P7 primer probes on the surface are blocked (shown as P5* and P7*). If they are to be used for amplification or library construction, they can then be unblocked using chemical or enzymatic processes. This reduces unwanted surface hybridization and background.

[0026] [Figure 4] Figure 4 shows the use of a complementary sequence that renders the primer unavailable by hybridizing to the primer. The primer can be activated or made available by dehybridization and washing away the complementary sequence. Similar to Figure 3, instead of blocking the P5 and P7 primer probes on the surface, the P5 and P7 primer probes can be hybridized to complementary P5' and P7' probes on the surface to reduce unwanted hybridization. The P5 and P7 primer probes can be dehybridized prior to surface activation.

[0027] [Diagram 5]FIG. 5 shows the use of terminal deoxynucleotidyl transferase (TdT, top panel) or complementary oligonucleotides (bottom panel) to reduce nucleic acid degradation due to secondary structures. The TdT enzyme can block the 3' ends of cDNA clusters, so that if this end unwinds on the cDNA molecule (more specifically on the polyT capture probe) and forms a secondary structure, it cannot extend and create noise signals during sequencing, resulting in a decrease in the quality of the sequencing signal. This step can be performed after the clustering and cleavage or linearization steps. Alternatively, complementary oligonucleotides can be used instead of TdT (bottom panel) to prevent self-folding.

[0028] [Figure 6] FIG. 6 shows in situ amplification of mRNA prior to fragmentation to improve the workflow to obtain and increase the quality of single-cell transcriptomes in an off-flow cell workflow (upper panel) and on-flow cell workflow (lower panel).

[0029] [Figure 7] Figure 7 shows the use of an exonuclease to digest unused and unbound primers on a surface or in solution, for example, unbound capture probes (e.g., probes with polyT) that have not captured any RNA molecules.

[0030] [Figure 8A] Figure 8A shows the use of primers for template switching. Instead of using free in-solution template switching in oligonucleotides, surface primers can be used for template switching. This improvement can increase the efficiency of the template switching process and can also avoid the formation of concatemers during the template switching process, thus increasing the efficiency of mRNA capture for the entire workflow.

[0031] [Figure 8B] FIG. 8B shows an embodiment in which the barcode and capture probes are grouped on different surface primers.

[0032] [Figure 9] FIG. 9 shows the combined use of the improvements described in FIG. 2 (library construction on a surface), FIG. 3 (3′ blocking of surface primers to avoid unwanted hybridization and extension), FIG. 5 (use of TdT to block the 3′ end of cDNA), and FIG. 7 (use of exonuclease treatment to remove unwanted capture probes) to improve currently available sequencing techniques.

[0033] [Figure 10] FIG. 10 shows a schematic diagram of a portion of a channel disposed in a fluidic device, according to some embodiments.

[0034] [Figure 11A] FIG. 11A shows a portion of a system provided herein including an energy source, according to some embodiments.

[0035] [Figure 11B] FIG. 11B shows a polymer matrix formed around a biological component in a portion of the systems provided herein, according to some embodiments.

[0036] [Figure 11C] FIG. 11C illustrates a method of forming a polymer matrix around a biological component in the systems provided herein, according to some embodiments.

[0037] [Figure 12] FIG. 12 is a flow chart depicting an embodiment of forming a polymer matrix.

[0038] [Figure 13A]FIG. 13A shows a portion of a channel including a capture element in a fluidic device, according to some embodiments.

[0039] [Figure 13B] FIG. 13B shows a biological component bound to a capture element on a surface of a portion of a system that includes a channel in a fluidic device, according to some embodiments.

[0040] [Figure 13C] FIG. 13C shows a polymer matrix disposed around a biological component in a system that includes a portion of a channel of a fluidic device, according to some embodiments.

[0041] [Figure 14] FIG. 14 is a flow chart depicting an embodiment of forming a polymer matrix around a surface-bound biocomponent.

[0042] [Figure 15A] FIG. 15A shows a portion of another embodiment of a system that includes a fluidic device that includes a sealable opening.

[0043] [Figure 15B] FIG. 15B illustrates a method of trapping a biological component in a portion of a system that includes a fluidic device, according to some embodiments.

[0044] [Figure 16A] FIG. 16A shows a top view of a schematic diagram of a portion of a system including a fluidic device, according to some embodiments.

[0045] [Figure 16B] FIG. 16B shows a top view of a schematic of a system including a portion of a fluidic device that includes a polymer matrix, according to some embodiments.

[0046] [Figure 17]FIG. 17 shows a top view of a schematic of a portion of a system including a fluidic device containing multiple different reagents, according to some embodiments.

[0047] [Figure 18A] FIG. 18A shows a portion of a spatial energy modulation element and a cylindrical polymer matrix according to some embodiments.

[0048] [Figure 18B] FIG. 18B shows a portion of a spatial energy modulation element and a polymer matrix in the shape of a hollow cylinder, according to some embodiments.

[0049] [Figure 19] FIG. 19 shows a micrograph of a polymer matrix compartment encapsulating one or more adult components, according to some embodiments.

[0050] [Figure 20A] FIG. 20A shows an open compartment formed in a multi-step polymer matrix formation process, according to some embodiments.

[0051] [Figure 20B] FIG. 20B shows a closed compartment formed in a multi-step polymer matrix formation process, according to some embodiments.

[0052] [Figure 21A] FIG. 21A is a schematic illustration of a portion of a surface of a fluidic device coated with repulsive elements, according to some embodiments.

[0053] [Figure 21B] FIG. 21B shows a micrograph of a biological component captured on a surface using a repulsive element, according to some embodiments.

[0054] [Figure 21C]FIG. 21C shows a high magnification micrograph of a biological component captured on a surface using a repulsive element, according to some embodiments.

[0055] [Figure 22A] 22A and 22B show a system for synthesizing a hydrogel chamber for use with the present invention. [Figure 22B] 22A and 22B show a system for synthesizing a hydrogel chamber for use with the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments. Detailed Description Overview

[0057] Provided herein are systems and methods, including functionalized solid supports, for creating nucleic acid molecules (e.g., sets of DNA molecules) that are ready for sequencing. The systems and methods described herein provide sets of nucleic acid molecules that can be sequenced in situ on the solid support on which they are created, or eluted from the solid support and sequenced off-site, where the systems and methods improve upon currently available sequencing techniques that focus on second strand synthesis (FIGS. 1A and 1B). In some embodiments, the systems and methods can sequence the first strand nucleic acid, the second strand nucleic acid, or both the first and second strands of the nucleic acid of the nucleic acid molecule. In some embodiments, the method includes preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules by the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing a cDNA molecule from the captured nucleic acid molecule or derivative, the synthesizing comprising reverse transcription and one or more second strand synthesis reactions, the cDNA molecule being bound to the solid support; inserting an adaptor into the 3' region of the cDNA molecule or derivative; and amplifying the cDNA molecule or derivative to create a set of cDNA molecules or derivatives, the set of cDNA molecules or derivatives being bound to the solid support. In some embodiments, the method includes contacting the solid support with a moiety configured to inactivate at least a subset of the one or more nucleic acid molecule capture probes.

[0058] In some embodiments, the method includes inserting an adaptor into the 3' region of the amplified cDNA molecule or derivative thereof, thereby creating a tagged amplified cDNA population; and performing solid-supported amplification on the tagged amplified cDNA population to create a set of cDNA molecules or derivatives thereof. In some instances, the subset of one or more nucleic acid molecule capture probes includes one or more nucleic acid molecule capture probes that did not capture a nucleic acid molecule. In some aspects, the moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes includes an exonuclease. In some cases, at least a subset of the plurality of surface primer probes includes a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, the method includes blocking the 3' end of a subset of the set of DNA molecules or derivatives thereof. In some embodiments, described herein is a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, the method comprising the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes, at least a subset of the plurality of surface primer probes comprising a template switch moiety; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives, the synthesizing comprising performing reverse transcription; inserting an adaptor at the 3' end of the cDNA molecule or derivative; and amplifying the cDNA molecule or derivative to generate the set of cDNA molecules or derivatives thereof.

[0059] In some embodiments, described herein is a method for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, the method comprising the steps of: providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes, at least a subset of the plurality of surface primer probes comprising a template switch moiety; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives, the synthesizing comprising performing reverse transcription; inserting an adaptor at the 3' end of the cDNA molecule or derivative; and amplifying the cDNA molecule or derivative to generate the set of cDNA molecules or derivatives thereof.

[0060] In some embodiments, a system is described herein that includes a solid support that includes one or more nucleic acid molecule capture probes and a plurality of surface primer probes, where at least a subset of the plurality of surface primer probes includes a template switch portion. In some embodiments, a method is described herein that utilizes a system for preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, comprising the steps of: providing a solid support, where the solid support includes one or more nucleic acid molecule capture probes and a plurality of surface primer probes; contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing a cDNA molecule from the captured nucleic acid molecule or derivative, where the synthesizing step includes a step of performing reverse transcription, where the cDNA molecule is bound to the solid support; inserting an adaptor into the 3' end of the cDNA molecule or derivative; and amplifying the cDNA molecule or derivative to generate a set of cDNA molecules or derivatives thereof, where the set of cDNA molecules or derivatives thereof is bound to the solid support. In some embodiments, the method of utilizing the system includes contacting a solid support with a moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes. In some examples, the subset of one or more nucleic acid molecule capture probes includes one or more nucleic acid molecule capture probes that did not capture a nucleic acid molecule. In some embodiments, the moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes includes an exonuclease. In some embodiments, the method includes amplifying a cDNA molecule or a derivative thereof. In some embodiments, at least a subset of the plurality of surface primer probes includes a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes.

[0061] In some embodiments, the systems or methods described herein include using a polymer matrix (e.g., a hydrogel matrix) formed adjacent to or around at least a portion of the individual components in the fluidic devices described herein. The hydrogel matrix can be selectively created to surround the components after the system detects the components, or the hydrogel matrix can be created according to a predefined pattern in the fluidic device. The hydrogel matrix can hold the individual components of the biological sample in place while allowing reagents and smaller entities to pass through. Since one or more individual components can be localized (e.g., encapsulated) in the fluidic device and the localized components can be exposed to one or more reagents and / or wash solutions during and / or between analyses, multiple assays can be performed in a compartment (e.g., simultaneously, substantially simultaneously, sequentially, etc.). Different assays can be performed in different locations of the fluidic device, for example, to test the effects of different processing conditions. In addition, since the components are generally not mixed and combined, low concentrations of the components (e.g., due to dilution) can be prevented. For example, when analyzing genomic material, an amplification step can be avoided due to the preservation of genetic material in each compartment. By having two or more components in a compartment, the interactions between the components can be studied as well. The polymer matrix can be degradable "on demand", allowing for control of localization and release mechanisms. The solutions provided herein can preserve spatial information of the components and generate data at the cellular, proteomic, transcriptomic, or genomic level. Because spatial information is preserved, the data can be associated (e.g., related) with phenotypic data. Furthermore, the solutions provided herein can preserve spatial information of the components and correlate data (e.g., phenotypic data) at the cellular, proteomic, transcriptomic, or genomic level.

[0062] In some embodiments, the present invention is directed to the synthesis of surface-bound cDNA molecules in separate regions of a surface set. Such surface-bound cDNA molecules are derived from captured nucleic acids, and the cDNA molecules in different separate regions are derived from substantially different cells. Whenever cells are randomly arranged on a surface, e.g., with a Poisson distribution, there is a trade-off between the concentration (or density) of cells to minimize the overlap of captured molecules and to maximize throughput, e.g., when measuring single-cell transcriptomes. That is, a higher density of cells on a surface means that the captured nucleic acids from adjacent cells are more likely to overlap, and a lower density means that fewer transcriptomes are measured. According to an aspect of the present invention, this trade-off can be influenced by providing a diffusibility modifier, i.e., an agent that reduces or blocks the diffusion of nucleic acid molecules released from cells, thereby promoting capture by capture probes closer to the original cell (than in the absence of the diffusibility modifier). The diffusion modifier may be a component of the reaction mixture or medium containing the cells, e.g., a soluble polymer such as agarose, poly(ethylene glycol) (PEG), dextran, poly(vinyl) alcohol, poly(vinyl) acetate, polyamide, polysaccharide, poly(lysine), polyacrylamide, poly(ethylene oxide), poly(acrylic acid), etc. In some embodiments, the diffusion modifier may be a viscosity modifier, e.g., glycerol, hydroxyethyl cellulose, carboxymethyl cellulose, etc. In some embodiments, the diffusion modifier may be a gel barrier that surrounds some or all of the cells on the surface, e.g., a gel layer on the surface. In other embodiments, the diffusion modifier may be an assembly of discontinuous gel barriers that encapsulate or surround individual cells. In some embodiments, such discontinuous gel barriers may comprise a single mass or body of gel material that each encapsulates a single cell, or such discontinuous gel barriers may be an assembly of gel chambers or cages that include polymer matrix walls that surround single cells but may or may not be in contact with the cells.In some embodiments, such gel chambers are hydrogel chambers (as further described herein). In some embodiments, contacting the nucleic acid molecules with the discrete regions of the surface is facilitated by providing a diffusivity modifier to the reaction mixture containing the cells. In other embodiments, such contacting is facilitated by providing a hydrogel chamber for each cell of the plurality of cells.

[0063] Aspects of the above embodiment are shown in Figures 2B-2H. The problem addressed by the above embodiment is shown in Figure 2B. In the top panel, cell (252) and cell (253) are positioned on a surface (250) with a distance D1 (256) between them. The surface (250) includes one or more nucleic acid molecule capture probes and a plurality of surface primer probes. For example, such capture probes may be designed to capture polyA mRNA, and such surface primer probes may include conventional primers, e.g., P5 and P7 primers (or their complements), for surface amplification of captured and reverse transcribed mRNA. In some embodiments, both sets of capture probes and surface primer probes are covalently attached to the surface (250) and uniformly coat the surface (250) at a predetermined density. After lysis (258) and release of the cell's nucleic acid molecules, e.g., messenger RNA (mRNA), a portion of the released nucleic acid molecules contact the surface (250) and are captured by the capture probes. The rate of capture depends on the concentration of the nucleic acid molecules of a cell, which is determined by their diffusion from their original cell after lysis. Therefore, at a given time after lysis, the most likely to be captured are those closest to the original cell, and then such likelihood decreases monotonically as the radial distance from the original cell increases. This phenomenon is shown for two pairs of cells in Figure 2B at different distances from each other. The density distribution of captured nucleic acid molecules ((254) and (255)) from lysed cells (252) and (253), respectively, is topologically shown in grayscale with the highest density being the darkest and the lowest density being the lightest. At a given time after lysis, nucleic acid molecules from a particular cell are preferentially captured by the capture probe adjacent to the lysed cell. For cells separated by a distance D1 at such a time, there is no (or there is minimal) overlap of captured molecules (256). As shown in the bottom panel, for cells close to each other separated by a distance D2, there is a significant overlap of captured nucleic acid molecules (264).According to some embodiments of the invention, the trade-off between density of cells disposed on a surface and, for example, number of transcriptome measurements of single cells may be improved by performing the lysis step in the presence of a diffusion modifier that reduces the rate of diffusion of nucleic acid molecules.

[0064] As noted above, a wide variety of agents are available to reduce the diffusivity of nucleic acid molecules, so that (as shown in FIG. 2C) pairs of cells at the same distance D1 and D2 ((266) and (267); (268) and (269)) do not produce overlapping captured nucleic acid molecules (265) because the rate of diffusion of released nucleic acid molecules is reduced. This allows a higher density of cells to be placed on the surface (250) for analysis of released cellular molecules, e.g., cellular nucleic acid molecules.

[0065] FIG. 2D illustrates an embodiment using a diffusivity modifier comprising a gel layer (272) surrounding cells disposed on a surface (270). Assay reagents, e.g., lysis reagents, polymerase, nucleoside triphosphates, primers, etc., can be delivered to the cells by flowing them (273) over the gel layer (272) in a liquid phase (277) and allowing them to diffuse to the cells, e.g., (274) and (275), disposed on the surface (270). The gel layer (272) can be formed by disposing the cells on the surface (270) in a reaction mixture that includes one or more polymer precursors. After disposing, the gel layer (272) can be formed by photopolymerizing one or more polymer precursors using conventional techniques, as described further below. The polymer precursors and reaction conditions can be selected such that the assay reagents can easily diffuse through the gel layer (272) while at the same time reducing the diffusivity of the nucleic acids of the cells of interest (e.g., mRNAs containing more than 200-300 nucleotides). After delivery of lysis reagent (278), the cells are incubated for a period of time to allow (i) the lysis reagent to reach the cells, (ii) cell lysis to occur, (iii) the cells' nucleic acid molecules to be released, and (iv) the released nucleic acid molecules to diffuse away from the original cells and / or be captured by capture probes. Illustratively, by distribution of captured nucleic acid molecules (280) and (282) from cells (274) and (275), respectively, gel layer (272) ensures that a greater proportion (on average) of captured nucleic acid molecules from different cells are in separate areas of surface (270). In some embodiments, gel layer (272) is a hydrogel layer formed by photocrosslinking polymer precursors.

[0066] Figures 2E-2G show a diffusive modifier comprising a hydrogel chamber. As shown in Figure 2E, cells and polymer precursors are loaded onto a surface (2902) that may be part of a channel of a fluidic device (described more fully below). Cells (e.g., 2901) are placed on the surface (2902), e.g., the position of the cells is determined by a detector (2904) that is used by a control system to create instructions for a spatial energy modulation element (2906) that generates a light beam to synthesize (2908) a hydrogel chamber in the channel (2900) around the single cell, as shown by hydrogel chambers (2912, 2913, and 2914). Enlargement (2910) shows that the solid-looking structures (2912, 2913, and 2914) have an interior (2911) and a wall (2921) with a predetermined thickness (2916). Similarly, the hydrogel chamber has a predetermined shape (e.g., circular with a diameter (2917)) and encloses a predetermined area. In the figures, the chambers are shown for convenience as being separate and unconnected with adjacent chambers and having a cylindrical or ring-like shape, however, the spatial energy modulation element may synthesize chambers of different shapes and sizes as may be useful for a particular application. In some embodiments, the surface (2902) may be part of a flow cell and / or channel in a fluidic device, and the hydrogel chamber may be synthesized between the surface (2902) and a parallel second surface (not shown in FIG. 2E). As used herein, a "channel" refers to a vessel that can hold a fluid (which may be static or flowing) and have at least one surface on which a cellular assay (such as a transcriptome measurement) may be performed. In some embodiments, the channel may have a first surface and / or a second surface on which the chamber may be synthesized and / or on which cells or assay components may be bound. In addition, in some embodiments, cells or assay components may be bound on the polymer matrix walls or captured therein by a capture element.As used herein, the attribute of a "first surface" (e.g., as the surface containing the capture elements) may also apply to a second surface, or, where appropriate, to a polymer matrix wall (all of which may be the interior surface of the hydrogel chamber). As used herein, a "channel" includes a surface, particularly a solid support, including a planar surface. In some embodiments, a channel may include a first surface and a second surface, which may be, for example, parallel to one another. In some embodiments, a channel may inhibit the flow of fluid therethrough from an inlet to an outlet. In other embodiments, a channel may include a non-flowing volume of fluid that may be removed, replaced or added by an opening or inlet, i.e., in some embodiments, a channel of the invention may be a well or well-like structure. The vertical distance between the first and second surfaces may range from 10 μm to 500 μm, or from 50 μm to 250 μm. In some embodiments, the vertical distance between the first and second surfaces may range from 2 times the average size of the cells to be analyzed to 5 times the average size of the cells to be analyzed.

[0067] In some embodiments, each hydrogel chamber synthesized on a surface has a depth of 0.001 to 0.1 mm. 2 or 0.001~1.0mm 2 In some embodiments, each hydrogel chamber synthesized has the same shape and area, e.g., ring-like, with an internal area selected from the range of 100 to 100,000. In some embodiments, each hydrogel chamber synthesized has the same shape and area for each different type of cell being assayed. In some embodiments in which mammalian cells are assayed, the number of hydrogel chambers synthesized around a single cell may be greater than 100, or greater than 1000, or greater than 10,000, or the number may range from 100 to 100,000, or from 1000 to 100,000, or from 1000 to 100,000. 6 may be in the range.

[0068] After the hydrogel chambers are synthesized, the cells are treated with a lysis reagent (2913) such that the cells' nucleic acids, e.g., mRNA, are released into the interior of the hydrogel chamber (e.g., 2924) where a portion is captured by the capture probe. In some embodiments, after capture, the hydrogel chamber may be depolymerized (2925) prior to introduction of extension and / or amplification reagents, e.g., polymerase, dNTPs, primers, etc., for cDNA synthesis and / or amplification.

[0069] 2G shows an embodiment of a diffusive modifier comprising a gel layer (2981) as described above, except that cavities or wells (2980) around cells (2982) are disposed on a surface (2984). So, for example, if cells (2982) are randomly disposed on a surface (2984), a random well array is formed around them, if so by selectively polymerizing polymer precursors beyond a given radius from each cell, for example, by photopolymerization. As in the case of a solid gel layer, assay reagents can be delivered to the cells by flowing them over the gel layer so that they can enter the wells.

[0070] As shown in FIG. 2H, after treating cells (2932) on a surface (2930) with a lysis reagent, the nucleic acid molecules of the cells, e.g., mRNA, are captured by adjacent capture probes as such molecules diffuse away from their original cell (2932) (lightly colored cells (e.g., 2933) with dashed outlines represent lysed cells). In addition, the initially captured molecules dissociate, diffuse a distance, and are recaptured, after which the process is repeated. In some embodiments, the captured mRNA is converted to cDNA, which is then surface amplified (e.g., by bridge PCR) to form clusters or clonal populations of cDNAs that are sequenced, e.g., using conventional sequencing-by-synthesis techniques. For successful sequencing by such an approach, the number of clonal cDNAs per cluster must be large enough to produce a detectable signal, and the spacing of the clusters must be large enough to avoid overlaps between clusters that would disrupt the signal due to different cDNAs in the overlapping regions. With regard to the latter parameter, for conventional sequencing-by-synthesis techniques, randomly distributed cDNA for surface amplification may have an expected nearest neighbor distance in the range of 0.25-5.0 μm, or in the range of 1.0-5.0 μm. In some embodiments, randomly distributed cDNA for surface amplification, e.g., bridge amplification, may have an expected nearest neighbor distance of 0.5 μm or greater, or an expected nearest neighbor distance of 1.0 μm or greater, or an expected nearest neighbor distance of 2.0 μm or greater. In some embodiments, the randomly distributed cDNA (or captured mRNA) on the surface is substantially distributed as a Poisson distribution. Returning to FIG. 2H, as the nucleic acid molecules of the cells are spread out on the surface (2930), the regions adjacent to the original cells with captured mRNA within the desired range can be qualitatively plotted, as shown in the bottom panel. At the time of lysis (2934), due to high concentration, the captured mRNA adjacent to the cells has an expected nearest neighbor distance that is too low for acceptable cluster formation.As mRNA diffusion progresses and the initially captured mRNA de-hybridizes and is recaptured (2936), the expected nearest neighbor distance increases in the region adjacent to the original cell. This process continues until equilibrium is reached (2938). In some embodiments where a hydrogel chamber is synthesized that prevents diffusion of mRNA molecules through its polymer matrix walls, the size of the hydrogel chamber (and thus its interior area) can be selected such that the distribution of captured mRNA approaches an equilibrium random distribution within the hydrogel chamber, where the expected nearest neighbor distance between the captured RNAs (or cDNAs) approaches a value within a desired range. Thus, as shown in FIG. 2I, cells (e.g., 2944) are placed on a surface (2946) and a hydrogel chamber (2948) is synthesized after the cells (2944) are lysed to release the mRNAs (e.g., ) that are captured by the capture probes on the surface (2946). After reaching an equilibrium distribution of captured mRNA, cDNA is synthesized with predicted nearest neighbor distances within the desired range. For example, approximately 2×10 cells (2944) are fully released into the hydrogel chamber (2948). 5 For a mammalian cell with 1000 mRNA molecules, the captured mRNA will be at least 1000 times larger than the internal area (2950) of the hydrogel chamber (2948) of approximately 2.8×10 5 μm 2 (e.g., the area of ​​a circle with a radius of 300 μm), has an expected nearest neighbor distance of about 1 μm, e.g., Pielou, Introduction to Mathematical Ecology (Wiley-Interscience, 1969). In some embodiments, the number of mRNAs remaining inside the hydrogel chamber may be controlled by controlling the permeability (or porosity) of the polymer matrix wall of the hydrogel chamber. For example, the porosity of the polymer matrix wall may be selected such that the number of retained mRNAs may have a molecular weight above a predetermined size. In some embodiments, the rate at which the nucleic acids of the cells approach equilibrium distribution at the inner surface of the hydrogel chamber may be increased by introducing agents that destabilize duplex formation, e.g., heat, low salt buffers, chaotropic agents, etc.

[0071] In some embodiments, the above method may be carried out by the following steps: (a) providing a solid support comprising a surface comprising a plurality of cells arranged thereon, comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes bound thereto; (b) contacting separate areas of the surface with one or more nucleic acid molecules of different cells in the presence of a diffusivity modifier to generate one or more captured nucleic acid molecules in each of the separate areas, wherein the nucleic acid molecules in the different separate areas are from different cells; and (c) synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives thereof, wherein each of the cDNA molecules is bound to the surface of the solid support, and wherein the cDNA bound to the different separate areas is from different cells. In some embodiments, the cDNA is "bound" to the surface by extending the capture probe in a polymerase reaction (such as a reverse transcriptase extension reaction) with the nucleic acid molecule of the captured cell as a template. In some embodiments, the contacting step comprises treating the cells with a lysis reagent to release one or more nucleic acid molecules, e.g., mRNA, from the cells. In some embodiments, the method further comprises amplifying the cDNA molecules or derivatives thereof to generate a plurality of sets of amplicons of the cDNA molecules or derivatives thereof. In some embodiments, the transcriptomes of the plurality of cells are determined by sequencing the cDNA molecules of the amplicons. In some embodiments, the cDNA molecules bound to the surface comprise spatial barcodes that code for locations on the surface, and the method further comprises eluting the cDNA molecules from the surface prior to sequencing. In some embodiments, the surface comprises one or more gel layers, e.g., one or more hydrogel layers, that encapsulate the cells disposed thereon. In some embodiments, a single hydrogel layer encapsulates substantially all of the cells disposed on the surface. In some embodiments, the cells disposed on the surface are encapsulated by separate gel layers or bodies.

[0072] In some embodiments, the diffusivity modifier comprises a hydrogel chamber surrounding the cells disposed on the surface. In some embodiments, each cell disposed on the surface is surrounded by a separate hydrogel chamber. In some embodiments, the porosity of the polymer matrix wall of the hydrogel chamber is selected to effectively prevent cellular nucleic acid molecules having a molecular weight within a predetermined size range from diffusing through such polymer matrix wall. In some embodiments in which mRNA is captured, such size ranges include mRNA molecules with more than 100 ribonucleotides, or more than 200 ribonucleotides, or more than 300 ribonucleotides, or more than 400 ribonucleotides, or more than 500 ribonucleotides. In some embodiments, after the cells are lysed, the hydrogel chamber is incubated at an elevated temperature for an interval of time to destabilize duplexes formed between the released cellular nucleic acid molecules and their capture probes, such that the released cellular nucleic acid molecules become randomly distributed among the capture probes on the interior surface of the hydrogel chamber. Such elevated temperature may be within the range of 25°C to 95°C, or such elevated temperature may be 10°C to 60°C above room temperature. In some embodiments, such time intervals may range from 30 seconds to 20 minutes, or from 30 seconds to 5 minutes, or from 30 seconds to 2 minutes. In some embodiments, after lysing the cells, the hydrogel chamber is treated with a duplex destabilizing reagent, e.g., a low salt buffer, for a time interval to destabilize duplexes formed between the nucleic acid molecules of the released cells and the capture probes. After the time interval, such low salt buffer is replaced with a buffer that forms stable duplexes. In some embodiments, the interior area of ​​the hydrogel chamber is selected such that the bound cDNA molecules (from which the clusters are created) have an expected nearest neighbor distance of greater than 1 μm, or in the range of 0.25 μm and 5 μm, or in the range of 1 μm and 3 μm.

[0073] In some embodiments, the invention includes a composition of matter comprising a hydrogel chamber disposed on a surface, where the hydrogel chamber comprises an internal area comprising a random distribution of nucleic acid molecules from a single cell. In some embodiments, the random distribution of nucleic acid molecules is a uniform distribution in that the probability of a given number of nucleic acid molecules binding within a given sub-area of ​​the internal area depends only on the size of the sub-area. In some embodiments, such a uniform distribution is substantially Poisson distributed. "Substantially Poisson distributed" as used herein means that the probability that the actual distribution was created by a Poisson process is greater than 50 percent, or greater than 70 percent, or greater than 90 percent. In some embodiments, the uniformly distributed nucleic acid molecules are mRNA. In some embodiments, the uniformly distributed nucleic acid molecules are cDNA. In some embodiments, such a uniform distribution of mRNA or cDNA molecules has an expected nearest neighbor distance between mRNA or cDNA molecules of 1 μm or greater.

[0074] In some embodiments, hydrogel chambers for use in the present invention may be synthesized by the following steps: (a) providing a fluidic device comprising (i) one or more channels each comprising a first surface, (ii) a spatial energy modulation element in optical communication with each of the first surfaces, and (iii) a detector for locating cells in each channel based on one or more optical signals therefrom; (b) loading cells and one or more polymer precursors into each channel such that the cells are disposed on the first surface; and (c) synthesizing one or more chambers in each channel, each chamber surrounding one or more cells by projecting light into each channel using the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, wherein the position of the synthesized chamber is determined in each channel by the position of the cells surrounded thereby as identified by the detector. In some embodiments, the method further includes the steps of (i) loading a lysis reagent into the channel such that the cellular messenger RNA is released and captured by a capture probe inside the hydrogel chamber, (ii) loading a reverse transcription reagent into the channel to copy the captured messenger RNA to generate complementary DNA, and (iii) sequencing the complementary DNA.

[0075] In some embodiments, the polymer matrix wall of the hydrogel chamber is 3×10 6 Permeates molecules with molecular weights less than 3 x 10 Daltons 6 In some embodiments, the polymer matrix wall of the hydrogel chamber is impermeable to molecules having a molecular weight greater than 3×10 Daltons. 5 Permeates molecules with molecular weights less than 3 x 10 Daltons 5 In some embodiments, the polymer matrix wall of the hydrogel chamber is impermeable to molecules having a molecular weight greater than 3×10 Daltons.4 Permeates molecules with molecular weights less than 3 x 10 Daltons 4 In some embodiments, the polymer matrix wall of the hydrogel chamber is impermeable to molecules having a molecular weight greater than 3×10 Daltons. 3 Permeates molecules with molecular weights less than 3 x 10 Daltons 3 It is impermeable to molecules with a molecular weight greater than a Dalton. Creating tagged sets of nucleic acid molecules for sequencing

[0076] A method for making tagged nucleic acids for sequencing is described herein. In some embodiments, the tagged nucleic acids are synthesized from one or more nucleic acid molecules captured on a solid support. Non-limiting examples of solid supports include gel matrices or fluidic channels. In some embodiments, the fluidic channel is a flow cell. In some embodiments, the solid support is not a bead. In some embodiments, the solid support is in contact with or encapsulated in a gel matrix, such as a hydrogel. In some embodiments, the solid support is a gel matrix. In some embodiments, the solid support is an encapsulated gel matrix. In some embodiments, the captured nucleic acid molecule is RNA. In some embodiments, the captured nucleic acid molecule (e.g., mRNA) is washed or degraded away from the solid support. In some embodiments, the first strand of nucleic acid is tagged and synthesized from the captured nucleic acid, while the second strand of tagged nucleic acid is synthesized from the first strand of nucleic acid after the captured nucleic acid molecule is washed or degraded away from the solid support. In some embodiments, the method includes preparing a set of complementary deoxynucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules. In some embodiments, the one or more nucleic acid molecules are contacted with a solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes. In some embodiments, the one or more nucleic acid molecules are captured by the capture probes. In some embodiments, a first strand nucleic acid comprising a cDNA molecule is synthesized from the captured nucleic acid molecule or derivative, where the synthesizing comprises performing reverse transcription, and the cDNA molecule is bound to a solid support. In some embodiments, the cDNA is tagged (e.g., with a barcode, e.g., a unique molecular index or UMI). In some embodiments, the cDNA is tagged with a tag comprising a cell-specific or spatial location-specific identifier sequence, and optionally a UMI sequence. In some embodiments, an adapter can be inserted or added to the 3' end of the cDNA molecule or derivative.In some embodiments, the cDNA molecule or derivatives thereof can be further amplified or sequenced. In some embodiments, an adaptor inserted at the 3' end of the cDNA molecule can serve as an initiation for sequencing. In some embodiments, the method includes synthesizing one or more second strands of the cDNA molecule or derivatives thereof. In some embodiments, the one or more second strand synthesis reactions include template switch extension. In some embodiments, the one or more second strand synthesis reactions include random priming. In some embodiments, the method includes synthesizing a cDNA library by single strand ligation. In some embodiments, the cDNA library synthesis includes tagging fragmentation. In some embodiments, the cDNA library synthesis includes fragmentation followed by adaptor ligation. In some embodiments, the cDNA can be cleaved or linearized. In some embodiments, the cDNA is cleaved or linearized after amplification. In some embodiments, the cDNA is synthesized by solid support amplification. In some embodiments, the solid support amplification is bridge amplification.

[0077] In some embodiments, the one or more nucleic acids that contact the solid support and serve as templates for cDNA synthesis can be DNA or RNA. In some cases, the DNA is single-stranded. In some embodiments, the DNA is genomic DNA. In some embodiments, the DNA is cell-free DNA. In some embodiments, the RNA is ncRNA, nmRNA, sRNA, smnRNA, tRNA, sRNA, mRNA, pcRNA, rRNA, 5S rRNA, 5.8S rRNA, SSU rRNA, LSU rRNA, NoRC RNA, pRNA, 6S RNA, SsrS RNA, aRNA, asRNA, asmiRNA, cis-NAT, crRNA, tracrRNA, CRISPR RNA, DD RNA, diRNA, dsRNA, endo-siRNA, exRNA, gRNA, hc-siRNA, hcsiRNA, hnRNA, RNAi, lincRNA, lncRNA, miRNA, mrpRNA, nat-siRNA, natsiRNA, OxyS RNA, piRNA, qiRNA, rasiRNA, RNase MRP, RNase P, scaRNA, scnRNA, scRNA, scRNA, SgrS RNA, shRNA, siRNA, SL RNA, SmY RNA, snoRNA, snRNA, snRNP, SRP In some embodiments, the RNA is an mRNA. In some embodiments, the RNA is an miRNA. In some embodiments, the RNA is an miRNA. In some embodiments, the RNA is an siRNA ... Probe inactivation

[0078] In some embodiments, the method includes inactivating at least a subset of the one or more nucleic acid molecule capture probes. In some embodiments, at least a subset of the one or more nucleic acid molecule capture probes can be inactivated (FIG. 7). In some embodiments, at least a subset of the one or more nucleic acid molecule capture probes can be inactivated before contacting with one or more nucleic acid molecules. In some embodiments, at least a subset of the one or more nucleic acid molecule capture probes can be inactivated after contacting with one or more nucleic acid molecules. In some embodiments, at least a subset of the one or more nucleic acid molecule capture probes can be inactivated by treating the nucleic acid molecule capture probes with a nuclease. In some embodiments, at least a subset of the one or more primer probes can be inactivated before contacting with one or more nucleic acid molecules. In some embodiments, at least a subset of the one or more primer probes can be inactivated after contacting with one or more nucleic acid molecules. In some embodiments, at least a subset of the one or more primer probes can be inactivated by treating the primer probes with a nuclease. In some cases, the nuclease can be an endonuclease. In some embodiments, nuclease is exonuclease.Non-limiting examples of nuclease for inactivating one or more nucleic acid molecule capture probes include S1 nuclease, P1 nuclease, N.crassa nuclease, Mycelia, Conidia, BAL 31 nuclease, U.Maydis nuclease, nuclease Bh1, Aspergillus nuclease, Physarum nuclease, SP nuclease, mung bean nuclease, wheat chloroplast nuclease, nuclease I, bean seed nuclease, tobacco nuclease I, alfalfa seedling nuclease, SK nuclease, Hen liver nuclease, rat liver nuclear nuclease or mouse mitochondrial nuclease.

[0079] In some embodiments, one or more of the nucleic acid molecule capture probes or primer probes can be inactivated by a non-nuclease moiety (FIG. 5). In some embodiments, one or more of the nucleic acid molecule capture probes or primer probes can be inactivated by a moiety that includes a TdT enzyme. In some embodiments, one or more of the nucleic acid molecule capture probes or primer probes can be inactivated by hybridizing with a complementary oligonucleotide. In some embodiments, one or more of the nucleic acid molecule capture probes or primer probes can be inactivated by hybridizing the one or more of the nucleic acid molecule capture probes or primer probes with at least a partially complementary oligonucleotide and incorporating a reversible terminator nucleotide with a polymerase. In some embodiments, one or more of the nucleic acid molecule capture probes or primer probes can be inactivated by contacting with a moiety that binds a phosphate to the 3' end of the oligonucleotide. In some embodiments, one or more of the nucleic acid molecule capture probes or primer probes can be inactivated by contacting with a moiety that includes a cationic-neutral diblock polypeptide copolymer. In some embodiments, inactivation of the probe reduces the self-folding of the probe. In some embodiments, inactivation of a probe reduces background signal associated with a probe that is not in contact with one or more nucleic acids or cDNA synthesized from one or more nucleic acids. Preparation of cDNA in solution

[0080] In some embodiments, methods for synthesizing or sequencing cDNA in solution are described herein. In some embodiments, the cDNA molecule or derivatives thereof are synthesized while one or more nucleic acid molecules are bound to a solid support (e.g., bound by contact with one or more nucleic acid molecule capture probes). In some embodiments, the first strand of the cDNA is synthesized while one or more nucleic acid molecules are bound to a solid support. In some embodiments, the second strand of the cDNA is synthesized while one or more nucleic acid molecules are bound to a solid support. In some embodiments, the synthesized cDNA (both the first and second strands) is eluted from the solid support prior to sequencing via contact with a primer in solution. In some embodiments, the cDNA molecule is contacted or encapsulated by a gel matrix (e.g., a hydrogel) prior to elution from the solid support. In some embodiments, the cDNA molecule is contacted or encapsulated by a gel matrix (e.g., a hydrogel) after elution from the solid support. In some embodiments, the cDNA molecule is suspended in an aqueous buffer after elution from the solid support.

[0081] In some embodiments, one or more nucleic acids are contacted and captured by a nucleic acid molecule capture probe. When contacted with the nucleic acid molecule capture probe, the first strand of tagged cDNA sequence can be synthesized. In some embodiments, the captured nucleic acid molecule (e.g., mRNA) is washed or degraded away from the solid support. In some embodiments, the first strand can be eluted from the solid support before the synthesis of the second strand of tagged cDNA sequence. In some embodiments, the first and second strands can be eluted from the solid support before cDNA sequencing.

[0082] In some embodiments, the cDNA molecule or derivative thereof is synthesized while the one or more nucleic acid molecules are not bound to a solid support. In some embodiments, the cDNA molecule or derivative thereof is synthesized while the one or more nucleic acid molecules are encapsulated in a gel matrix, such as a hydrogel as described herein. In some embodiments, the cDNA molecule or derivative thereof is synthesized after the one or more nucleic acid molecules are eluted from the solid support. In some embodiments, the first strand of the cDNA is synthesized after the one or more nucleic acid molecules are eluted from the solid support. In some embodiments, the second strand of the cDNA is synthesized after the one or more nucleic acid molecules are eluted from the solid support. In some embodiments, the cDNA is synthesized by contacting with a primer sequence in solution. In some embodiments, the cDNA synthesized through the use of a primer sequence in solution is fragmented. In some embodiments, the cDNA synthesized through the use of a primer sequence in solution includes tagging fragmentation. Primer-probe blocking

[0083] In some embodiments, methods are described herein for blocking surface primer probes by contacting them with blocking moieties described herein. In some embodiments, surface primer probes can be blocked by contacting and hybridizing with an oligonucleotide comprising a nucleic acid sequence complementary to the surface primer probe. In some embodiments, surface primer probes can be blocked by contacting with a blocking agent comprising one or more 3' phosphate nucleotides. Figure 3 shows a non-limiting example of the use of 3' phosphate nucleotides to block and unblock surface primers as needed in the workflow for obtaining a transcriptome described herein. This use is similar to that shown in Figure 2. However, first, the surface P5 and P7 primer probes are blocked (P5 * and P7 *(shown as ). If they are to be used for amplification or library construction, they can then be unblocked using chemical or enzymatic processes. This reduces unwanted surface hybridization and background signal. Figure 4 shows another example of blocking and unblocking surface primers, where an oligonucleotide containing a sequence complementary to the surface primer can make the surface primer unavailable by hybridizing to the surface primer. The surface primer can be activated or made available by dehybridization and washing away of the complementary sequence. Similar to Figure 3, instead of blocking the surface P5 and P7 primer probes, the P5 and P7 primer probes can be hybridized to complementary P5' and P7' probes on the surface to reduce unwanted hybridization. The P5 and P7 primer probes can be dehybridized prior to surface activation.

[0084] In some embodiments, the method includes preparing a set of cDNA molecules or derivatives thereof from one or more nucleic acid molecules. In some embodiments, the method includes contacting a solid support described herein with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules. In some embodiments, a first strand of the cDNA molecule is synthesized from the captured nucleic acid molecule. In some cases, a second strand of the cDNA molecule is synthesized from the first strand. In some embodiments, an adaptor is inserted at the 3' end of the cDNA molecule (first or second strand) or derivatives thereof. In some embodiments, the cDNA molecule is contacted with at least a subset of the plurality of surface primer probes for initiation of a sequencing reaction to sequence the cDNA molecule. In some aspects, the cDNA molecule or derivatives thereof is bound to the solid support prior to amplification. In some aspects, the cDNA molecule or derivatives thereof is eluted from the solid support prior to amplification. In some embodiments, the surface primer probe includes a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, prior to amplification of the cDNA molecules, the blocking agent is removed to unblock the plurality of surface primer probes and allow the extension reaction.

[0085] In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the blocking agent comprises an oligonucleotide comprising a sequence complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule comprising a sequence partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. In some embodiments, the plurality of surface primer probes are blocked by treating the plurality of surface primer probes with TdT. In some embodiments, blocking the 3' ends of the subset of the plurality of surface primer probes comprises contacting the plurality of surface primer probes with a cationic-neutral diblock polypeptide copolymer. Creating tagged second strands of nucleic acids for sequencing

[0086] Methods for generating second strands of nucleic acids (e.g., second strands of cDNA molecules) for sequencing are described herein. Figure 2 shows a non-limiting example for obtaining improved transcriptomes from in situ and direct generation of sequenceable libraries from captured mRNA and tagged second strands of nucleic acids (e.g., second strands of cDNA molecules). Surface coating of P5 and P7 primer probes can be used to generate libraries directly on the surface of a solid support. Amplification can be performed using bridge amplification, but also with primers in solution. Bridge amplification can include amplification using primer probes coated on the surface of a solid support. The primer probes can be attached to the 5' end by a flexible linker. At the end of the amplification, each clonal cluster contains several copies of a single member of the cDNA or one or more nucleic acid molecules. In some embodiments, amplification performed with primers in solution can include the use of emulsion PCR. In one embodiment, one of the PCR primers can be tethered to the surface of the solid support (5' attachment) and the other primer can be in solution. In some cases, the solid support comprises two or more primers, where the primers can target or hybridize to one or more of the nucleic acid molecules or cDNA molecules.

[0087] The created surface clusters can be sequenced directly in situ or the library can be eluted from the surface separately for sequencing. In some embodiments, the method includes preparing a set of cDNA molecules or derivatives thereof from one or more nucleic acid molecules. In some instances, the method includes providing a solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes. In some cases, the method includes contacting the solid support with one or more nucleic acid molecules to produce one or more captured nucleic acid molecules. In some embodiments, the cDNA molecules are synthesized from the captured nucleic acid molecules, where synthesizing the cDNA can occur when the cDNA or one or more nucleic acid molecules are bound to the solid support. In some embodiments, synthesizing the cDNA can occur when the cDNA or one or more nucleic acid molecules are eluted from the solid support. In some embodiments, an adaptor can be inserted into the 3' region of the cDNA molecule or derivatives thereof. In some embodiments, amplification of the cDNA occurs when the set of cDNA molecules or derivatives thereof is bound to the solid support.

[0088] In some embodiments, the set of cDNA molecules or derivatives thereof is bound to a plurality of surface primer probes. In some embodiments, the adapter inserted into the 3' region of the cDNA molecule comprises a sequence configured to allow the initiation of a sequencing reaction on the cDNA molecules of the set of cDNA molecules or derivatives thereof. In some embodiments, the one or more nucleic acid molecule capture probes can be inactivated or blocked by any one of the moieties described herein. In some embodiments, the one or more nucleic acid molecule capture probes can be inactivated by any one of the nucleases described herein. In some embodiments, the nuclease is an exonuclease. Figure 7 shows the use of an exonuclease to digest unused and unbound primers on a surface or in solution. For example, unbound capture probes (e.g., polyT) that have not captured any RNA molecules can be digested.

[0089] In some embodiments, the one or more second strand synthesis reactions include template switch extension, random priming, or both. In some embodiments, the amplification of the cDNA molecule or derivatives thereof occurs when the cDNA is bound to the solid support. In some embodiments, the amplification of the cDNA molecule or derivatives thereof occurs when the cDNA is eluted from the solid support. In some embodiments, the amplification of the cDNA molecule includes contacting the cDNA molecule with a primer sequence in solution. In some embodiments, the cDNA includes fragmentation. In some embodiments, the adapter inserted into the cDNA includes a sequence for tagging fragmentation. In some embodiments, the adapter is inserted into the cDNA by single strand ligation. In some embodiments, the adapter is inserted into the cDNA by double strand ligation.

[0090] In some embodiments, at least a subset of the plurality of surface primer probes comprises a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, the method comprises subjecting the blocking agent to a reaction that unblocks at least a subset of the plurality of surface primer probes to allow the extension reaction. In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule that comprises a sequence complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule that comprises a sequence partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. Figure 3 shows a non-limiting example of the use of 3' phosphate nucleotides to block and unblock surface primers as needed in the workflow for obtaining a transcriptome described herein. This use is similar to that shown in Figure 2. However, first, the surface P5 and P7 primer probes are blocked (P5 *and P7 * (shown as ). If they are to be used for amplification or library construction, they can then be unblocked using chemical or enzymatic processes. This reduces unwanted surface hybridization and background. Figure 4 shows another example of blocking and unblocking surface primers, where an oligonucleotide containing a sequence complementary to the surface primer can render the surface primer unavailable by hybridizing to the surface primer. The surface primer can be activated or made available by dehybridization and washing away of the complementary sequence. Similar to Figure 3, instead of blocking the surface P5 and P7 primer probes, the P5 and P7 primer probes can be hybridized to complementary P5' and P7' probes on the surface to reduce unwanted hybridization. The P5 and P7 primer probes can be dehybridized prior to surface activation.

[0091] In some embodiments, the method includes blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof by contacting the subset of the set of DNA molecules or derivatives thereof with terminal deoxynucleotidyl transferase (TdT). In other cases, the method includes blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof by contacting the subset of the set of DNA molecules or derivatives thereof with an oligonucleotide that includes a sequence complementary to the 3' ends of the subset of the set of DNA molecules. FIG. 5 shows the use of terminal deoxynucleotidyl transferase (TdT, top panel) or complementary oligonucleotides (bottom panel) to reduce nucleic acid degradation due to secondary structures. The TdT enzyme can block the 3' ends of cDNA clusters, so that if this end unwinds on the cDNA molecule (more specifically, on the polyT capture probe) and forms a secondary structure, it cannot be extended and cannot create a noise signal during sequencing, resulting in a decrease in the quality of the sequencing signal. This step can be performed after the clustering and cleavage or linearization steps. Alternatively, a complementary oligonucleotide can be used in place of TdT to prevent self-folding. In some embodiments, the method includes blocking the 3' ends of a subset of the set of DNA molecules or derivatives thereof by contacting the subset of the set of DNA molecules or derivatives thereof with a cationic-neutral diblock polypeptide copolymer.

[0092] In some embodiments, the one or more nucleic acid molecule capture probes comprise a sequence configured to bind to one or more nucleic acid molecules. In some aspects, the sequence configured to bind to one or more nucleic acid molecules comprises a poly-T sequence, a randomer, a sequence complementary to at least a subset of one or more nucleic acid molecules, or any combination thereof. The method of any one of the preceding claims, wherein the one or more nucleic acid molecule capture probes comprise one or more tags, the tags comprising a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the cDNA synthesis or amplification occurs when the one or more nucleic acid molecules or cDNA molecules are bound to the solid support. In some embodiments, the cDNA synthesis or amplification occurs when the one or more nucleic acid molecules or cDNA molecules are eluted from the solid support. In some embodiments, the cDNA synthesis or amplification occurs when the one or more nucleic acid molecules or cDNA molecules are contacted with or encapsulated in a gel matrix as described herein. In some embodiments, cDNA synthesis or amplification occurs when one or more nucleic acid molecules or cDNA molecules are eluted from the solid support and contacted with or encapsulated in a gel matrix. Template switching oligonucleotides

[0093] A method for synthesizing cDNA by utilizing template switching oligonucleotides is described herein. FIG. 8A shows an example of using a template switching primer. Instead of using a template switch in free solution in oligonucleotides, a surface primer can be used as a template switching primer. This improvement can increase the efficiency of the template switching process and can also avoid the formation of concatemers during the template switching process, thus increasing the efficiency of mRNA capture for the entire workflow. In some embodiments, the method includes preparing a set of cDNA molecules or derivatives thereof from one or more nucleic acid molecules. In some embodiments, the method includes providing a solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes, where at least a subset of the plurality of surface primer probes comprises a template switch portion (e.g., a template switch primer). In some embodiments, the method includes contacting the solid support with one or more nucleic acid molecules to produce one or more captured nucleic acid molecules, and synthesizing a cDNA molecule from the captured nucleic acid molecule or derivative. In some embodiments, an adaptor can be inserted at the 3' end of the cDNA molecule or derivative. In some embodiments, the cDNA molecule or derivative can be amplified for sequencing.

[0094] In some embodiments, the method includes a step of synthesizing comprising performing one or more second strand synthesis reactions comprising the cDNA molecule or a derivative thereof. In some embodiments, the one or more second strand synthesis reactions are mediated by a subset of a plurality of surface primer probes comprising a template switch moiety. In some embodiments, the second strand is synthesized by template switch extension. In some embodiments, the cDNA molecule or a derivative thereof is bound to a plurality of surface primer probes or template switching moieties. In some embodiments, the adapter inserted in the 3' region of the cDNA comprises a sequence configured to allow initiation of a sequencing reaction on the cDNA molecule or a derivative thereof. In some embodiments, the one or more nucleic acid molecule capture probes or surface primer probes can be inactivated or blocked by any one of the moieties described herein.

[0095] In some embodiments, cDNA synthesis or amplification occurs when one or more nucleic acid molecules or cDNA molecules are bound to solid support. In some embodiments, cDNA synthesis or amplification occurs when one or more nucleic acid molecules or cDNA molecules are eluted from solid support. In some embodiments, cDNA synthesis or amplification occurs when one or more nucleic acid molecules or cDNA molecules contact with primer sequences in solution as described herein. In some embodiments, cDNA synthesis or amplification occurs when one or more nucleic acid molecules or cDNA molecules contact with or are encapsulated in a gel matrix as described herein. In some embodiments, cDNA synthesis or amplification occurs when one or more nucleic acid molecules or cDNA molecules are eluted from solid support and contact with or are encapsulated in a gel matrix.

[0096] In some embodiments, the methods described herein can be utilized to obtain transcriptome sequencing. In some embodiments, first strand synthesis can be extended using a primer probe that includes a randomer. The randomer can include the same combination of sample barcode and unique molecular identifier, in addition to other adapter and primer binding sites, such as transposome adapter sequences or template switching (TS) primers. Template switching and second strand synthesis can be performed for oligo-dT primed cDNA synthesis. The resulting double-stranded cDNA can then be subjected to tagging fragmentation.

[0097] Figure 8B shows an embodiment in which the capture sequence and the barcode sequence (which may or may not contain a UMI) are grouped or located on separate surface primers. This results in shorter surface primers, which is advantageous both from a synthesis point of view and also because there are fewer issues with surface tagging fragmentation (if used). Also, the system is modular such that multiple different capture probes can be used without changing the design of the rest of the system. For example, a polyDT oligo for mRNA capture, and a handle sequence for capturing a specific nucleic acid sequence, e.g., a complementary oligo barcode.

[0098] In one embodiment, the solid support (850) has a surface (852) that includes (i) a capture element or probe (851) that includes a surface primer (854) (which may be, for example, a P7 primer) and a capture probe (856), and (ii) an oligonucleotide sequence (861) that includes a surface primer (862) (which may be, for example, a P5 surface primer), a complement (R1') of primer binding site (R1) (864) (which may be used in a sequencing step, e.g., to identify a barcode or UMI), a barcode sequence (866), a UMI (868), and a complement (R2') of primer binding site, R2 (which may be used in a sequencing step, e.g., to identify a target template). Also shown is a complement (860) to the capture probe (856) (e.g., a polyA region of an mRNA, or a nucleic acid molecule of another cell or an artificial sequence, e.g., a so-called "handle" sequence for an antibody label), and a captured sequence (863) that includes a template region (858) that is copied to form part of a cDNA. After reverse transcription, tagging fragmentation (or a similar procedure) and denaturation (871), a preliminary cDNA (873) is generated. In an alternative embodiment, R2 (872) may be attached via template switching when tagging fragmentation is not used. Under hybridization conditions and in the presence of polymerase and dNTPs, R2 (872) anneals to its complement R2' (870) and copies and extends the surface primer (861) (875). This results in a final cDNA product (878) that contains primer binding site R2 (884), UMI (888), barcode (886), primer binding site R1 (885), and primer binding site P5 (881) (877). This final cDNA product may be, for example, surface amplified to form clusters for sequencing, or it may be copied and the copies eluted for external sequencing.

[0099] In some embodiments, the above method for synthesizing barcoded cDNA can be carried out by the following steps: (a) providing a solid support, the solid support comprising one or more nucleic acid molecule capture probes and a plurality of surface primer probes, at least one of the surface primer probes comprising a barcode sequence; (b) contacting the solid support with the one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; (c) synthesizing an initial cDNA molecule from the captured nucleic acid molecule or derivative, the cDNA molecule being bound to the solid support; (d) ligating an adaptor to the free end of the cDNA molecule, the adaptor comprising a 3' segment complementary to the 3' end of the surface primer probe comprising the barcode sequence; providing conditions under which the 3' segment of the adaptor is annealed to the 3' end of the surface primer and extended, thereby generating a second cDNA molecule comprising the barcode sequence. In some embodiments, the adaptor is ligated to the initial cDNA by tagging fragmentation. In some embodiments, the second cDNA molecule is surface amplified to form a cluster. Amplification of nucleic acid molecules prior to tagging

[0100] Methods for amplifying nucleic acid molecules before tagging or fragmentation are described herein. Figure 6 shows a non-limiting example of amplifying cDNA before fragmentation to improve workflows to obtain single-cell transcriptomes and increase their quality in off-flow cell workflow (top panel) and on-flow cell workflow (bottom panel). In some embodiments, the method includes preparing a set of cDNA molecules or derivatives thereof from one or more nucleic acid molecules by providing a solid support, the solid support including one or more nucleic acid molecule capture probes. In some aspects, the method includes contacting the solid support with one or more nucleic acid molecules to generate one or more captured nucleic acid molecules. In some cases, the method includes synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives, and amplifying the cDNA molecules or derivatives to create an amplified cDNA population. As mentioned before, an adaptor can be inserted into the 3' region of the amplified cDNA molecules or derivatives to create a tagged amplified cDNA population. In some embodiments, the method includes performing solid support amplification on the tagged amplified cDNA population to create a set of cDNA molecules or derivatives thereof. In some embodiments, the method comprises performing amplification after the cDNA molecules or derivatives thereof are eluted from the solid support. In some embodiments, the cDNA molecules or derivatives thereof, the amplified cDNA population, the tagged amplified cDNA population, the set of cDNA molecules or derivatives thereof, or any combination thereof, are bound to a plurality of surface primer probes.

[0101] In some embodiments, the adaptor comprises a sequence configured to allow the initiation of a sequencing reaction on the cDNA molecules of the set of cDNA molecules or derivatives thereof. In some embodiments, the method comprises contacting the solid support with a moiety configured to inactivate at least a subset of one or more nucleic acid molecule capture probes. In some embodiments, the subset of one or more nucleic acid molecule capture probes comprises one or more nucleic acid molecule capture probes that did not capture a nucleic acid molecule. In some embodiments, one or more of the nucleic acid molecule capture probes or surface primer probes can be inactivated or blocked by any one of the moieties described herein. Polymer Matrix Embodiments

[0102] The present disclosure provides a system for compartmentalizing or isolating one or more biological components. The system can include a fluidic device that contains or includes one or more biological components. The fluidic device can contain or include one or more polymer precursors. In some cases, the fluidic device can include a first surface configured to bind or receive at least one of the one or more biological components to form a bound biological component. The system can also include at least one energy source, where the energy source is in communication with the fluidic device. In various embodiments, the at least one energy source can form a polymer matrix on or adjacent to at least a portion of the one or more biological components.

[0103] In some cases, a sample may be introduced or provided of the system. In certain cases, the sample may include one or more biological components. The system may be used to separate one or more biological components from each other. In various cases, the biological components may be physically separated. In some cases, the biological components may be in fluid communication with each other. In certain cases, the biological components may be in chemical communication with each other. The system may be used for single cell analysis. In some embodiments, the system may be used for single cell analysis at the 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. The system may be used for DNA sequencing, whole genome sequencing, whole exome sequencing, targeted sequencing, or 16S sequencing of cell-free DNA. The system may be used to study DNA tags attached to a biological molecule of interest. The biological molecule may include proteins, metabolites, etc. In some cases, the DNA may be nuclear DNA or mitochondrial DNA. The system may be used for single cell analysis or bulk analysis at the 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, cellular immune repertoire studies, or full-length mRNA analysis. In some embodiments, the system may be used for single cell analysis at the proteome level. The system may be used for functional assays of biological components. The system may be used to study surface proteins, secreted proteins, or metabolic products of biological components. In some cases, the system may be used to study epigenomics, DNA methylation, or chromatin accessibility in biological components. The system may be used for other suitable assays, experiments, and processes.

[0104] In certain embodiments, the system may be used for single cell analysis at the indirect cell-cell interaction level. For example, the effect of one or more molecules produced from a first cell on a second cell can be analyzed using the system provided herein. In various embodiments, the system may be used to analyze 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 one or more effects of the first cell on the second cell, or vice versa, can be analyzed using the system 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 to analyze the response of a biological component to various physiological conditions (e.g., various media, temperatures, mechanical stimuli, etc.).

[0105] In some cases, the sample comprises a biological sample. The biological sample may comprise biological components. In some embodiments, the biological sample comprises 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, 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 20 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 10000, 15000, 25000, 30000, 40000, 50000, 60000, 70000, 800 20The biological components may include more than 10 base pairs (bp) in length. The biological components may include cells. In some embodiments, the cells may include eukaryotic cells, prokaryotic cells, fungal cells, protozoa, algal cells, plant cells, animal cells (e.g., human cells), or any other suitable cells. The biological components may include cells, viruses, bacteria, nucleic acids (e.g., DNA, or RNA), proteins, or combinations thereof. The combinations may include DNA-protein complexes, RNA-protein complexes, or combinations thereof. In certain embodiments, the nucleic acids may include DNA. The DNA may be at least 10 base pairs (bp) in length. In some embodiments, the DNA is at least 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp in length, or longer than 800 bp.

[0106] In certain embodiments, the one or more polymer precursors may be added to or included with the biological sample. The one or more biological samples and the one or more polymer precursors may be introduced into the system (e.g., into a 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 before the polymer precursor(s), the polymer precursor(s) may be introduced before the biological sample(s), the biological sample(s) and the polymer precursor(s) may be introduced simultaneously (or substantially simultaneously), or in any other suitable manner or order. In some embodiments, the polymer precursor may include one or more hydrogel precursors. 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 one or more biological components prior to introduction into the system. In various cases, the one or more polymer precursors may be mixed with one or more biological components after introduction into the system.

[0107] The system may include a fluidic device. In some embodiments, the fluidic device may include one or more polymer precursors. In other words, the 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 the channel of the fluidic device). In some embodiments, the fluidic device may include 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 two numbers listed herein. In some embodiments, the fluidic device includes 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 this disclosure unless otherwise indicated. For example, a channel or chamber of a fluidic device may include a first surface, a second surface, or more surfaces.

[0108] A channel or chamber of a fluidic device may receive or be configured to receive a biological sample. FIG. 10 shows a schematic diagram of a portion of a channel 100 that may be disposed in at least a portion of a fluidic device of a system provided herein. The fluidic device may include a channel 100. The channel 100 may include a first surface 101. Additionally, the channel 100 may include a second surface 102. In some embodiments, the first surface 101 and the second surface 102 are disposed, placed, or positioned opposite each other (e.g., as depicted in FIG. 10). In some embodiments, the first surface 101 may be a lower surface. In certain embodiments, the second surface 102 may be an upper surface. The terms "lower" and "upper" are used herein for convenience and not intended to be limiting when referring to the figures. The channel 100 may receive a biological sample including one or more biological components 50, 51. The channel 100 may receive one or more polymer precursors. As shown in FIG. 10, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, and the like. In some embodiments, the first surface 101, the second surface 102, or both surfaces may bind or receive, or be configured to bind or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 101 may bind or receive, or be configured to bind or receive, a biological component (e.g., biological component 50, 51). In certain cases, the second surface 102 may bind or receive, or be configured to bind or receive, a biological component (e.g., biological component 50, 51).

[0109] In certain cases, the channel may have a rectangular, circular, semicircular, oval, or other suitably shaped cross section. Thus, the channel may have a single internal surface. In some cases, the channel may have a triangular, square, rectangular, polygonal, or other cross section. Thus, the channel may have three or more internal surfaces. One or more internal surfaces may bind or receive, or be configured to bind or receive, one or more biological components.

[0110] In some cases, the first surface 101, the second surface 102, or both surfaces 101 and 102 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 for capturing one or more moieties (e.g., chemical moieties), acrylamide, agarose, biotin, streptavidin, strep-tag II, linker, aldehyde, phosphate, silicate, ester, acid, amide, alkyne, azide, aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group for capturing one or more moieties. For example, acrylamide, agarose, etc. may include such functional groups. In certain embodiments, the surface polymer may include polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or a combination 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.

[0111] In some cases, the first surface 101, the second surface 102, or both surfaces 101 and 102 may include one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the first surface 101, the second surface 102, or both surfaces 101 and 102 may include 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 between any of the two numbers listed herein. 2 ~about 500nm 2In some embodiments, the first surface 101, the second surface 102, or both surfaces 101 and 102 may include a total number of barcodes of at most about 10,000,000. The barcodes may be different from each other (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of barcodes may be different from a second portion or subset of barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of barcodes, or any number of portions or subsets of barcodes between any of the two numbers listed herein. In some cases, the barcode (or portion / subset of barcode) may be associated with the location of the barcode on the surface (coordinates (e.g., x, y coordinates) of the location on the surface of the channel). The barcode may be bound or linked to the captured biocomponent. In some embodiments, the barcode may be a unique identifier that distinguishes the biocomponent from other biocomponents (e.g., identifies a first biocomponent to a second biocomponent). In some embodiments, the barcode may include a nucleic acid sequence (e.g., a consensus sequence) for capturing the biocomponent or may be used in amplification. In some embodiments, the barcode may include a unique identifier that includes a unique nucleic acid sequence (e.g., a DNA sequence, an RNA sequence, etc.), a protein tag, an antibody, or an aptamer. In some embodiments, the barcode may include a fluorescent molecule. In some embodiments, the location of the captured biocomponent may be associated with the unique identifier, e.g., that holds spatial information of the biocomponent.

[0112] 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.

[0113] The systems disclosed herein may include one or more energy sources. The energy source may be in communication with the fluidic device. In some cases, the energy source may be used to form one or more polymer matrices in the fluidic device (e.g., on or adjacent to the surface of a channel or chamber of the fluidic device). In some embodiments, the energy source may include a light-generating device, a heat-generating device, an electrochemical reaction-generating device, an electrode, or a microwave device. The polymer matrix may be formed in the channel of the fluidic device. The energy source may direct or transfer energy to a predetermined location of the fluidic device. The energy may cause or activate one or more polymer precursors to form (e.g., polymerize) a polymer matrix at the predetermined location.

[0114] In some embodiments, the polymer matrix may comprise a hydrogel. In some embodiments, the hydrogel may be sufficiently porous or have pores of a suitable size to allow the movement or translocation of reagents (e.g., enzymes, compounds, small molecules, antibodies, etc.) through the polymer matrix, while the hydrogel may not allow the movement or translocation of biological components (e.g., DNA, RNA, proteins, cells, etc.) through the polymer matrix. In some embodiments, the pores may have a diameter of 5 nm to 100 nm. In some embodiments, the pores may have a diameter of 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 greater than 100 nm. In some embodiments, the pores may have a diameter less than 5 nm. The reagent may comprise an enzyme or a primer having a size less than 50 base pairs (bp). The primer may comprise single stranded DNA (ssDNA). In some embodiments, the primer may have a size of 5 bp to 50 bp. In some embodiments, the primer may have a size of 5 bp to 10 bp, 10 bp to 20 bp, 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, the primer may have a size greater than 50 bp. In certain cases, the primer may have a size less than 5 bp. The reagent may comprise lysozyme, proteinase K, hexamers (e.g., random hexamers), polymerase, transposase, ligase, catalytic enzyme, deoxyribonuclease, deoxyribonuclease inhibitor, ribonuclease, ribonuclease inhibitor, DNA oligo, deoxynucleotide triphosphate, buffer, detergent, salt, divalent cation, or any other suitable reagent.

[0115] FIG. 11A shows a portion of a system presented herein that includes an energy source 1103. The embodiment of FIG. 11A may include components that resemble those of FIG. 10 in some respects. For example, the embodiment of FIG. 11A includes a channel 1100 that may resemble the channel 100 of FIG. 10. It is recognized that the illustrated embodiments may have similar features. Accordingly, similar features are designated with similar reference numbers, with the leading digit incremented to "2". As such, the relevant disclosures set forth above regarding similarly identified features may not be repeated hereinafter in this specification. Also, certain features of the systems provided herein and related components shown in FIG. 11A may not be indicated or identified by reference numbers in the drawings or specifically discussed in the writing that follows. However, such features may clearly be the same or substantially the same as features depicted in and / or described with respect to other embodiments. As such, the relevant descriptions of such features apply equally to the features and related components of the system of FIG. 11A. Any suitable combination of features and variations thereof described with respect to the system and components shown in Figure 10 can be used with the system and components of Figure 11A, and vice versa. This pattern of disclosure applies equally to further embodiments depicted in subsequent figures and described herein below.

[0116] With continued reference to FIG. 11A, the channel 1100 of the system may include a first surface 1101 and a second surface 1102. In some embodiments, the energy source 1103 may include one or more energy emitting portions (e.g., energy emitting portion 1105). In some embodiments, the energy source 1103 may include one or more non-emitting portions (e.g., non-emitting portion 1104). The non-emitting portion 1104 may not emit energy or may not be configured to emit energy. In some embodiments, the emitting portion 1105 may 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 1105 may emit energy to the fluidic device. In some embodiments, the fluidic channel may be coupled to a movable stage. In other embodiments, light may be projected onto or at least a portion of the fluidic channel to create one or more polymer matrices. The light may be directed to different portions of the fluidic channel. In some embodiments, the emitting portion 1105 may be coupled to an objective lens (e.g., a microscope objective lens or lens), where the objective lens may be moved to different portions of the fluidic device. The objective lens may provide a shape (e.g., a virtual mask or a physical mask) that allows the light to form a pattern on the fluidic device to form a polymer matrix similar or complementary to the pattern. In various embodiments, one or more polymer precursors in the fluidic device or mixed with the biological sample may absorb the emitted energy 1106. In some embodiments, the emitted energy 1106 may 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 in the channel 1100 of the fluidic device may be activated by the emitted energy and initiate a polymerization reaction to form a polymer matrix.

[0117] In some embodiments, the energy source may emit energy into a majority of the fluidic channel or into substantially the entire surface of the fluidic channel. A physical mask may be used to block the energy emitted into one or more portions of the fluidic channel. The energy source (e.g., a light source) may be coupled to the fluidic device via an objective lens (e.g., a microscope objective lens or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective lens). In some cases, the light source, the objective lens, and / or the fluidic channel are movable to allow the emission of energy into the fluidic channel to generate a pattern on at least a portion of the surface of the fluidic device. The polymer matrix may be formed similarly or complementary to the pattern of energy emission.

[0118] The polymer precursors may include activating molecules capable of absorbing the radiated energy 1106 to initiate polymerization of one or more polymer precursors in the fluidic device. Non-limiting examples of activating molecules may include photocatalysts, photoactivators, photoacid generators, or photobase generators. In some embodiments, the first polymer matrix 1108 and / or the second polymer matrix 1109 may be formed on or adjacent to the biocomponent 50. In certain embodiments, the first polymer matrix 1108 and the second polymer matrix 1109 may form an analysis chamber or compartment 1120 that separates (e.g., physically separates) the biocomponent 50 from other biocomponents (e.g., biocomponents 51, 52, or 53) in the fluidic device. In other words, the polymer matrix may compartmentalize a channel (e.g., channel 1100). In various embodiments, the polymer matrix may partially surround the biocomponent. For example, the polymeric structure surrounding the biological component may form a closed structure (e.g., a hollow cylindrical shaped polymeric structure) or a partially open structure (e.g., a crescent shaped polymeric structure). In some embodiments, two or more polymeric matrices may be formed adjacent to a biological component forming a compartment that separates the biological component from other biological components. In certain embodiments, the polymeric matrix may include or form a wall (e.g., a polymeric matrix wall).

[0119] With continued reference to FIG. 11A, the polymer matrix 1108, 1109, or at least a portion of the polymer matrix 1108, 1109, may be attached to the first surface 1101, the second surface 1102, or both surfaces 1101 and 1102. In certain embodiments, the polymer matrix, or at least a portion of the polymer matrix, may be attached to a third surface, a fourth surface, a fifth surface, etc., as appropriate. In various embodiments, the polymer matrix 1108, 1109 may extend from the first surface 1101 to the second surface 1102 (e.g., through at least a portion of the lumen of the channel 200 or the cavity of the chamber) such that the polymer matrix surrounds or substantially surrounds the biocomponent 50. In some embodiments, two or more biocomponents that are in physical proximity (e.g., biocomponents 50, 51 in FIG. 11C) may be separated (e.g., by stirring or shaking the fluidic device). The fluidic device may be agitated or shaken by physical movement, by using acoustic pulses, by altering the flow in the channel, or by any other suitable method of agitation. A polymer matrix may then be formed that surrounds (or partially surrounds) the separated biocomponents. FIG. 11B shows a polymer matrix 1108, 1109 formed surrounding the biocomponent 50 after it has been separated from the biocomponent 51. FIG. 11C shows a process of separating two adjacent biocomponents 50, 51 according to various embodiments. That is, the biocomponents 50, 51 can be separated by agitating or shaking the fluidic device. In some embodiments, the separation of the biocomponents is achieved by fluid pressure, pulsatile flow, dielectrophoresis, photothermal flow, or some combination thereof. In some cases, the separation of the biocomponents is achieved by acoustic vibration. FIG. 11C also shows a polymer matrix formed to create a compartment 1122 surrounding the biocomponent 50 after separation of the biocomponents 50, 51.

[0120] With continued reference to FIG. 11A, in some cases, the energy source 1103 may form or produce, or may be configured to form or produce, one or more emitting portions 1105 and one or more non-emitting portions 204. The systems disclosed herein may further include a spatial energy modulation element that directs energy from the energy source to one or more target portions of the fluidic device. For example, the spatial energy modulation element may be configured to selectively direct energy from the energy source to form a polymer matrix at or adjacent to at least a portion of the biocomponent. The spatial energy modulation element may be configured to selectively direct energy, for example, by inhibiting or preventing the energy from being directed to one or more portions of the fluidic device other than the one or more target portions. In some embodiments, the spatial energy modulation element may include a physical mask. In some cases, the spatial energy modulation element may include a virtual mask. In certain cases, the spatial energy modulation element may be configured to control one or more electrodes that can selectively provide energy to one or more target portions of the fluidic device. The concept of electrodes may also be used to provide spatially modulated energy to form a hydrogel structure. In some implementations, one or more electrodes can be aligned at predetermined locations in the fluidic channel, thus allowing the formation of hydrogel at these locations. In an alternative implementation, the electrodes can be in the form of an array. The electrodes of the array can be turned on or off as required to create a desired spatial pattern of energy to form a desired shape of the hydrogel. For example, one or more electrodes (e.g., an array of electrodes) can be disposed within one or more portions of a fluidic device. For another example, one or more electrodes (e.g., an array of electrodes) can be in communication (e.g., electrical communication) with one or more portions of a fluidic device.

[0121] In some embodiments, the mask may prevent or be configured to prevent one or more portions of the energy emitting surface 1110 of the energy source 1103 from emitting energy (e.g., non-emitting portions 1104). In some embodiments, the mask may be a virtual mask (e.g., computer code or digital system). In certain embodiments, the mask may prevent energy from being emitted to locations where the biological component is present. This may allow or permit a polymer matrix to form adjacent to, on, or encapsulating the biological component (e.g., to hold cells, proteins, DNA molecules, RNA molecules, or other target molecules in place on a fluidic channel). In other embodiments, the mask may promote polymerization such that the polymer matrix is ​​on the biological component. In various embodiments, the mask may be a physical mask (e.g., an opaque material, a heat shield, or an electromagnetic shield). In some embodiments, the mask (e.g., a virtual mask or a physical mask) may be created using or in combination with a detector that detects or identifies the location of the biological component. In some embodiments, the detector includes a camera. In some embodiments, the detector comprises a light detector, a conductivity detector, an ultrasonic detector, an ultrasonic sensor, a piezoelectric sensor, a combination thereof, or another suitable detection device.

[0122] In some embodiments, the first surface 1101 or the second surface 1102 may include a detector configured to detect or detect one or more locations of one or more biological components in the fluidic device (e.g., in the channel 1100). In certain embodiments, the energy source 1103 may include, be coupled to, or be in communication with a detector configured to detect or detect the location of the biological components in the fluidic device. In various embodiments, a mask may be created using an image obtained from at least a portion of the fluidic device. The mask may enable or allow the energy source 1103 to emit energy at or toward one or more locations or positions where one or more biological components are present on or adjacent to the first surface 1101. The mask may inhibit or prevent the energy source 1103 from emitting energy at or toward one or more locations or positions where one or more biological components are present on or adjacent to the first surface 1101. In some embodiments, the image may be obtained from a camera (e.g., a digital camera, a microscope imaging camera, etc.). In some embodiments, the camera may be coupled, connected, or in communication with the energy source 1103. For example, a camera (not shown) may be in electrical communication with the energy source 1103. In some embodiments, the energy source 1103 may include a camera. In various embodiments, the energy source 203 may include a microscope (e.g., a fluorescent microscope, a confocal microscope, a lens-free imaging system, a transmission electron microscope (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used (e.g., in combination with a detector) to detect one or more locations of one or more biological components.

[0123] FIG. 18A shows an example of a mask including an energy masking region 1810 and an energy transparent region 1815. Energy from an energy source can be blocked by the energy masking region 1810 to prevent the energy from forming any polymer matrix in a portion of the fluidic device (e.g., portion 1820). The energy transparent region 1815 can allow the energy to communicate with the fluidic device to form a polymer matrix 1825. FIG. 18B shows another example of a mask, where the energy transparent region 1835 is in the shape of a hollow cylinder (e.g., donut-shaped). Energy masked by the masking region 1830 can prevent energy communication with a portion of the fluidic device (e.g., portion 1840). The energy transparent region 1835 can deliver energy to the fluidic device to form a polymer matrix 1845. The polymer matrix 1845 can be in the shape of a hollow cylinder.

[0124] FIG. 19 shows examples of biological components encapsulated and / or localized (i.e., shown as white spots) using a polymer matrix. In some cases, biological components 1901 may be localized within a hollow region of a polymer matrix compartment 1902. In some other cases, a polymer matrix 1903 may be formed on a biological component 1904. In some alternative cases, a polymer matrix 1905 may localize two or more biological components. A polymer matrix 1906 of a biological compartment may encapsulate one or more biological components.

[0125] FIG. 12 is a flow chart of forming a polymer matrix on or adjacent to one or more biological components according to some embodiments of the present disclosure. Process 1200 may be performed manually or automatically (e.g., by a suitably programmed computer system). In step 1210, a biological sample may be deposited, introduced, or provided to at least a portion of a fluidic device. In some embodiments, a mask may then be formed or created such that one or more portions of the energy source directed toward the biological components are non-emitting (step 1220). In step 1230, an energy source may apply or provide energy to at least a portion of the fluidic device. In some embodiments, the energy source may activate or initiate the polymer precursors (e.g., via the energy provided by the energy source) such that the polymer precursors form the polymer matrix. In some embodiments, imaging of the fluidic device may be performed after step 1210 and before step 1220 to determine or identify the location of the biological components to create the mask. In some embodiments, the mask is a virtual mask. In some embodiments, the polymer matrix may form a compartment that partially or completely surrounds the biological component.

[0126] In certain cases, the energy source may be manipulated so that the polymer matrix is ​​formed in different steps. For example, the energy source may initiate multiple polymer precursors so that the polymer precursors form open compartments (e.g., crescent-shaped or semi-cylindrical polymer matrices). The open compartments may be manipulated to capture and / or contain biological components (e.g., cells) or portions of the sample in a portion of the fluidic device. The orientation of the energy source or the fluidic device may be adjusted and an additional portion of the polymer matrix may be formed. This additional portion may be used to form one or more compartments in conjunction with the pre-formed semi-cylindrical polymer matrix. In other embodiments, the polymer matrix compartments may be formed in at least 2, 3, 4, 5, or more matrix formation steps.

[0127] 20A and 20B show an example of multi-step polymer matrix compartment creation. FIG. 20A shows a first step of multi-step creation, where an open compartment (e.g., open compartment 2001 made from a polymer matrix) can be created to capture and / or contain a biological component (e.g., biological component 2002). A sample containing biological component 2002 can have a flow direction 2003 in a fluidic device (e.g., a portion of fluidic device 2000). The open compartment 2001 can be formed by creating a polymer matrix using an energy source and an energy modulation unit described herein. The open compartment can intersect a portion of the flow direction 2003 of the sample in the fluidic device. The open compartment 2001 of the polymer matrix can be oblique or perpendicular to the flow direction 2003 of the sample in the fluidic device. FIG. 11B shows a second step of the multi-step fabrication, where an open compartment (e.g., open compartment 2001) is sealed or closed by forming a polymer matrix adjacent to, around, or on a biological component (e.g., biological component 2012). In some cases, in the second step, the biological component may be completely or substantially completely encapsulated by the polymer matrix (e.g., to form closed compartment 2011). In some cases, the polymer matrix, which may form adjacent to, around, or on the biological component, localizes the biological component to a location on the fluidic device 2000. Genomic and / or proteomic material may be extracted from the localized biological component. The polymer matrix may further localize the extracted material. The fluidic device may then provide a surface on which the extracted material may be sequenced. In some embodiments, the extracted material may be eluted and transferred to another device or surface for sequencing.In other embodiments, sequencing may be performed by short read sequencing, nanopore sequencing, sequencing by synthesis, sequencing by in situ hybridization, any optical reading using a microscope, or any other suitable sequencing method.

[0128] One or more surfaces of the fluidic device may include optical (e.g., fluorescent), mechanical, electrical, or biochemical sensing elements or sensors. Sensing elements may include fluorescent tags, enzymes, primers, oligonucleotides, or sensor molecules (e.g., biochemical sensor molecules). Sensing elements may be used to detect and / or measure pH, oxygen concentration, CO2 concentration, or any other suitable variable. Sensing elements may detect and / or measure parameters locally. For example, sensing elements may detect and / or measure pH, oxygen concentration, or CO2 concentration within a compartment (e.g., a polymer matrix shell cylinder) that surrounds a biological component. System with Capture Element

[0129] The present disclosure also provides a system including one or more capture elements (e.g., nucleic acid capture probes described herein) for immobilizing and / or compartmentalizing one or more biological components. The system can include a fluidic device. The fluidic device can include or contain one or more biological components. Additionally, the fluidic device can include or contain one or more polymer precursors. In some embodiments, the fluidic device can include a first surface (e.g., in a channel and / or chamber of the fluidic device). The fluidic device can include one or more capture elements. The capture element can immobilize or be configured to immobilize at least one of the one or more biological components on or adjacent to the first surface (or any suitable surface). Immobilization or binding of the biological component to the capture element can form an immobilized biological component. The system can further include at least one energy source in communication with the fluidic device. In certain embodiments, the at least one energy source can provide or supply energy to, or can be configured to provide or supply energy to, at least a portion of the fluidic device. Thus, the energy source can activate or cause one or more polymer precursors (e.g., disposed in the fluidic device) to form at least one polymer matrix on or adjacent to the immobilized biocomponents. In various embodiments, the fluidic device may further include a platform or stage that holds the fluidic device. In some embodiments, the system may also include a sequencing device (e.g., a next-generation sequencing device) to obtain sequencing data. The polymer matrix formed in the fluidic device may be used to capture and localize the biocomponents. Genomic and / or proteomic material may be extracted using the fluidic device. The fluidic device may then provide a surface on which the extracted material can be sequenced.In some embodiments, the extracted material may be eluted and transferred to another device or surface for sequencing, in other embodiments, sequencing may be performed by short read sequencing, nanopore sequencing, sequencing by synthesis, sequencing by in situ hybridization, or any optical reading using a microscope.

[0130] To immobilize a biological component, the fluidic device may include one or more capture sites. The capture site may include a capture element. In some embodiments, the one or more capture elements or sites may include or be arranged in a pattern. The fluidic device may include 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 ... 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 20 or any number between any two numbers recited herein. In some embodiments, the fluidic device may include 10 20 The substrate may contain more capture elements than the substrate.

[0131] The fluidic device may include a channel. The fluidic device may include a chamber. FIG. 13A shows an example of at least a portion of a channel 1300 in a fluidic device. One or more capture elements 1311 may be disposed or located on a first surface 1301 of the fluidic device. In some cases, the second surface 1302 may include one or more capture elements. The capture elements may be disposed on both surfaces or any other suitable surface. The capture elements may include or be at least partially formed by a functional group. Some non-limiting examples of functional groups include a functional group that can react with a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), biotin, streptavidin, strep-tag II, a linker, or a molecule (e.g., an aldehyde, phosphate, silicate, ester, acid, amide, alkyne, azide, or aldehyde dithiolane). The functional group may be specifically attached to the N-terminus or C-terminus of a peptide. The functional group may be specifically attached to an amino acid side chain. The functional group may be attached to the side chain of an amino acid (e.g., an acid of glutamate or aspartate, a thiol of cysteine, an amine of lysine, or an amide of glutamine or asparagine). The functional group may be specifically attached to a reactive group on a particular species, for example, a membrane-bound molecule on a cell (e.g., a glycoprotein on a eukaryotic cell, or a pilus on the protoplasm of a prokaryotic organism). In some examples, the capture element may include fibronectin. In another example, the capture element may include an RGD peptide. In some cases, the capture element may include an antibody. In some cases, the functional motif may be reversibly attached and cleaved (e.g., by using an enzyme). Figure 13B shows an example of a biological component 51 in contact with or attached to the capture element 1311. In some cases, a repelling surface coating (e.g., PEG) may be used to prevent the polymer matrix from covering or trapping the biological component.

[0132] In various examples, the capture element may include a physical trap, a hydrodynamic trap, a geometric trap, a well, an electrochemical trap (e.g., a trap for a charged molecule), a streptavidin, an antibody, an aptamer, an affinity bond (e.g., a peptide that can bind to a surface protein of a cell), one or more magnetic materials (e.g., a magnetic disk, a magnetic array, or a magnetic particle), a dielectrophoretic trap (e.g., an electrode array), or a combination thereof. The trap may include a polymer matrix or a hydrogel. The polymer matrix or hydrogel trap may be constructed or dismantled as required using an energy source and / or disassembly similar to the polymer matrix compartments described herein. For example, the capture element may include a well. The well may have a diameter of 1 μm to 50 μm. In some embodiments, the well may have a diameter of 1 μm to 20 μm, 20 μm to 30 μm, 30 μm to 40 μm, or 40 μm to 50 μm. The well may be larger than 50 μm in diameter. The wells may be less than 1 μm in diameter. In some embodiments, the wells may be between 0.1 μm and 100 μm deep. In certain embodiments, the wells may be greater than 100 μm deep. The wells may be less than 0.1 μm deep. The well depths are: 0.1μm~0.5μm, 0.1μm~1μm, 0.1μm~5μm, 0.1μm~10μm, 0.1μm~20μm, 0.1μm~30μm, 0.1μm~50μm, 0.1μm~100μm, 0.5μm~1μm, 0.5μm~5μm, 0.5μm~10μm, 0.5μm~20μm, 0.5μm~30μm, 0.5μm~50μm, 0.5μm~100μm, 1μm~5μm, 1μm~10μm, 1μm~ It may be 20 μm, 1 μm to 30 μm, 1 μm to 50 μm, 1 μm to 100 μm, 5 μm to 10 μm, 5 μm to 20 μm, 5 μm to 30 μm, 5 μm to 50 μm, 5 μm to 100 μm, 10 μm to 20 μm, 10 μm to 30 μm, 10 μm to 50 μm, 10 μm to 100 μm, 20 μm to 30 μm, 20 μm to 50 μm, 20 μm to 100 μm, 30 μm to 50 μm, 30 μm to 100 μm, or 50 μm to 100 μm.The depth of the wells may be about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 50 μm, or about 100 μm. The depth of the wells may be at least 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, or 50 μm. The depth of the wells may be at most 0.5 μm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, or 100 μm.

[0133] In some embodiments, the fluidic device may include a repulsive surface coating that may be used to prevent capture of a biological component at a predefined location. FIG. 21A shows a portion of a surface 2101 of a fluidic device, where the surface 2101 may include capture sites 2102 and repulsive sites 2103. The surface 2101 may be functionalized using a surface coating (e.g., PEG) to create the repulsive sites 2103. The repulsive sites 2103 may prevent the biological component from binding to the surface 2101 at the locations of the repulsive sites 2103 and may transport the biological component to the capture sites 2102. In some cases, the surface 2101 may include only repulsive sites without capture sites. The repulsive sites may initially localize the biological component. The polymer matrix may be formed by directing an energy source to the repulsive sites to form a compartment adjacent to the biological component that may be located between the repulsive sites. FIG. 21B shows an example of a biological component contained at a predefined location in a fluidic device. FIG. 21C shows a high magnification example of a biocomponent contained in a predefined location in a fluidic device.

[0134] An energy source may be used to form a polymer matrix on, around, or adjacent to at least a portion of the captured biocomponents. In some embodiments, a mask may be used to enable or allow the energy source to direct energy toward the location or position of the captured biocomponents. In certain embodiments, a mask may be used to inhibit or prevent the energy source from directing energy toward the location or position of the captured biocomponents. The mask may be configured to direct energy to predetermined or selected locations to form a polymer matrix that surrounds or at least partially surrounds one or more biocomponents. The mask may be created based at least in part on a pattern of capture sites (e.g., a pattern of capture sites / capture elements on a surface of a fluidic device). In some embodiments, the mask may be configured to prevent energy from being directed to locations surrounding capture sites or capture elements that have not captured or are not associated with a biocomponent. In certain cases, for analyzing single cells, the mask may be configured to prevent energy from being emitted adjacent to locations of capture elements that have captured or are associated with two or more biocomponents. In some embodiments, the mask may be configured to allow or permit energy to be emitted adjacent to the location of the capture element that has captured two or more biological components, for example, to allow analysis of cell-cell interactions. In certain embodiments, the mask may be a photolithographic mask or another suitable mask, as described herein. In some embodiments, the system may further include a detector, for example, to detect the location of the biological components, as described herein. The mask may be created at least in part based on the detected location of the biological components. In addition, the mask may selectively direct or provide energy from an energy source to the fluidic device, as described herein.

[0135] FIG. 13C shows an example of a method of forming a polymer matrix adjacent (e.g., surrounding) a biological component. The polymer matrix 1308 may be formed adjacent to a capture element 1311. The polymer matrix 1308 may be configured to hold the biological component 51 in place or within an analysis chamber or compartment 1320. The compartment 1320 may be formed, at least in part, by the polymer matrix 1308, the first surface 1301, and the second surface 1302 to form a chamber or at least a partially enclosed space within the fluidic device (e.g., around the biological component 51). In some embodiments, the polymer matrix 1308 may form a compartment 1320 that surrounds the biological component 51. The compartment 1320 may hold the biological component 51 in place. The polymer matrix 1308 and / or the compartment 420 may inhibit or prevent a compound associated with the biological component 51 from leaving the compartment. In some embodiments, the compound associated with the biological component may include a nucleic acid (e.g., DNA or RNA), a protein, a metabolite, an enzyme, an antibody, a combination thereof, or any other suitable compound or material. In some embodiments, the surface of the polymer matrix or hydrogel may be functionalized by binding a functional group to the polymer matrix or hydrogel. The functionalized surface of the polymer matrix inside the compartment may be bound to a capture element (e.g., an antibody) to capture a molecule secreted by the biological component (e.g., a secreted protein). The capture element or the captured molecule may then be read by a sensing molecule or by a labeling method, for example, by fluorescent labeling. In some embodiments, the polymer matrix may be configured to allow the passage of one or more compounds associated with the biological component. In some embodiments, the polymer matrix may be configured to allow the passage of a reagent.The reagents may include, for example, one or more enzymes, chemicals, oligonucleotides (e.g., one or more primers having a size of less than 50 base pairs), lysozyme, proteinase K, random hexamers, polymerases, transposases, ligases, catalytic enzymes, deoxynucleotide triphosphates, buffers, cell culture media, divalent cations, combinations thereof, or any other suitable reagents.

[0136] In certain embodiments, the first surface, the second surface, or both surfaces of the channel in the fluidic device may be functionalized as described herein. The surfaces of the fluidic device (e.g., the first surface, the second surface, the third surface, etc.) may include compounds configured to bind to biocomponents (e.g., captured biocomponents). In some embodiments, the surfaces of the fluidic device (e.g., the first surface, the second surface, the third surface, etc.) may include one or more barcodes. The one or more surfaces may include oligos to from DNA clusters for sequencing. In some cases, the one or more surfaces may include one or more nanopore readers for direct DNA and / or RNA reading. The one or more surfaces may include nanowells that capture single RNA and / or single DNA molecules or contain DNA / RNA libraries. In some alternative cases, the one or more surfaces may include patterned hydrophobic / hydrophilic features for selective deposition of DNA nanoballs. Nanoballs may be created from DNA / RNA molecules by circularization and amplification of DNA libraries.

[0137] One or more surfaces of the fluidic device may include optical (e.g., fluorescent), mechanical, electrical, or biochemical sensing elements or sensors. Sensing elements may include fluorescent tags, enzymes, primers, oligonucleotides, or sensor molecules (e.g., biochemical sensor molecules). Sensing elements may be used to detect and / or measure pH, oxygen concentration, CO2 concentration, or any other suitable variable. Sensing elements may detect and / or measure parameters locally. For example, sensing elements may detect and / or measure pH, oxygen concentration, or CO2 concentration within a compartment (e.g., a polymer matrix shell cylinder) that surrounds or encapsulates a biological component.

[0138] In some embodiments, the fluidic device described herein comprises a nucleic acid molecule capture probe and a plurality of surface primer probes. In some embodiments, the nucleic acid molecule capture probe is located within one or more compartments described herein (e.g., well, polymer matrix). In some embodiments, the nucleic acid molecule capture probe is located adjacent to one or more compartments. In some embodiments, the surface primer probe is located within one or more compartments described herein (e.g., well, polymer matrix). In some embodiments, the surface primer probe is located adjacent to one or more compartments. In some embodiments, the nucleic acid molecule capture probe and / or the surface primer probe comprises an adapter sequence. In some embodiments, the nucleic acid molecule capture probe and / or the surface primer probe comprises an amplification primer sequence. In some embodiments, the adapter comprises a sequence configured to allow initiation of a sequencing reaction on a nucleic acid molecule or a derivative thereof (e.g., cDNA). In some embodiments, the microfluidic device comprises a moiety configured to inactivate at least a subset of the one or more nucleic acid molecule capture probes. In some embodiments, the subset of the one or more nucleic acid molecule capture probes comprises one or more nucleic acid molecule capture probes that are not occupied by a nucleic acid molecule. In some embodiments, the moiety configured to inactivate at least a subset of the one or more nucleic acid molecule capture probes comprises an exonuclease. In some embodiments, the nucleic acid molecule capture probe and / or the surface primer probe comprises a template switch oligo. In some embodiments, the microfluidic device comprises one or more compartments for the insertion of primer sequences in solution. In some embodiments, at least a subset of the plurality of surface primer probes comprises a blocking agent that blocks an extension reaction in at least a subset of the plurality of surface primer probes. In some embodiments, the blocking agent can be removed by a reaction that unblocks at least a subset of the plurality of surface primer probes to allow an extension reaction.In some embodiments, the one or more blocking agents comprise one or more 3' phosphate nucleotides. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule comprising a sequence complementary to at least a subset of the plurality of surface primer probes. In some embodiments, the one or more blocking agents comprise a nucleic acid molecule comprising a sequence partially complementary to at least a subset of the plurality of surface primer probes, a reversible terminator nucleotide, and a polymerase, or any derivative thereof. In some embodiments, the microfluidic device comprises sufficient reagents to sequence at least a subset of the nucleic acid molecules or derivatives thereof in situ in the microfluidic device. In some embodiments, the one or more nucleic acid molecules comprise DNA or ribonucleic acid (RNA) molecules. In some embodiments, the DNA is fragmented single-stranded DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the RNA molecule comprises messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the RNA molecule comprises mRNA. In some embodiments, the one or more nucleic acid molecule capture probes comprise a sequence configured to bind to one or more nucleic acid molecules. In some embodiments, the sequence configured to bind to the one or more nucleic acid molecules comprises a poly-T sequence, a randomer, a sequence complementary to at least a subset of the one or more nucleic acid molecules, or any combination thereof. In some embodiments, the microfluidic device is a well, a bead, or a fluidic channel. In some embodiments, the fluidic channel is a flow cell. In some embodiments, the microfluidic device is not a bead. In some embodiments, the one or more nucleic acid molecule capture probes comprise one or more tags, the tags comprising a cell-specific or spatial location-specific identifier sequence, and optionally a unique molecular identifier (UMI) sequence. In some embodiments, the amplifying step comprises solid-supported amplification. In some embodiments, the solid-supported amplification is bridge amplification. In some embodiments, the one or more nucleic acid molecules are derived from a single cell or biological tissue. In some embodiments, the method occurs in a gel matrix, the gel matrix being adjacent to a solid support. Multi-layered system

[0139] Also provided herein is a system for analyzing a biological component including at least a flow channel (e.g., a first layer or top layer) and an analysis channel (e.g., a second layer or bottom layer). The system may include a fluidic device including a flow channel, an analysis channel, and a layer or wall disposed between the flow channel and the analysis channel. The system may include at least one energy source in communication with the fluidic device as described herein. The analysis channel may be disposed adjacent to the flow channel, where at least one flow impeding element may be disposed within the flow channel to impede or stop the flow of the biological component in the flow channel. The layer disposed between the flow channel and the analysis channel may include at least one sealable opening disposed at or adjacent to the at least one flow impeding element. One or more biological components may be stopped or trapped adjacent to the sealable opening. The at least one sealable opening may be configured to allow the passage of one or more biological components. For example, the sealable opening may be configured to allow the passage of one or more biological components from the flow channel to the analysis channel. The at least one energy source may be in communication with the analysis channel. Furthermore, the at least one energy source may form or be configured to form a polymer matrix within the analysis channel.

[0140] As described herein, in some embodiments, the fluidic device may include a microfluidic device or a nanofluidic device. In certain embodiments, the fluidic device may be used for nucleic acid sequencing. In some cases, the fluidic device may include a nucleic acid sequencing flow cell. In other cases, the sequencing may include short-read sequencing, nanopore sequencing, sequencing by synthesis, sequencing by in situ hybridization, sequencing by collecting any optical read, or any other suitable method of sequencing.

[0141] As described herein, the biological components may include cells, cell lysates, nucleic acids, microbiomes, proteins, mixtures of cells, spatially linked biological components, metabolites, combinations thereof, or any other suitable biological components. In some cases, the mixtures of cells may include two or more different cell types. For example, the mixtures of cells may include a first cell type and a second cell type. In some cases, the mixtures of cells may include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cell types. The cells may be mammalian cells (e.g., human cells), fungal cells, bacterial cells, tumor spheroids, combinations thereof, or any other suitable cells. In some cases, the biological components may include tumor spheroids or spatially linked biological components (or samples).

[0142] In some cases, the nucleic acid may comprise at least 100 bases or base pairs. In certain embodiments, the nucleic acid comprises DNA or RNA. The DNA may be at least 100 bp in length. In some embodiments, the DNA may comprise at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1,000 bp, 10 kilobase pairs (kbp), 100 kbp, 1 megabase pair (Mbp), 100 Mbp, 1 gigabase pair (Gbp), 10 Gbp, 100 Gbp, or more base pairs. The biological component may comprise a DNA molecule comprising any number of base pairs between the numbers listed herein. For example, the DNA may comprise 50 bp to 1,000 bp, 300 bp to 10 kbp, or 1,000 bp to 10 Gbp. The RNA may be dsRNA. The dsRNA may comprise at least 50bp, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1,000bp, 10kbp, or 100kbp. The biological component may comprise a dsRNA molecule comprising any number of base pairs between the numbers listed herein. For example, the dsRNA may comprise 50bp to 1,000bp, 300bp to 10kbp, or 1,000bp to 100kbp. The RNA may be ssRNA. The ssRNA may comprise at least 50 nucleotides to 100,000nt. The ssRNA may comprise 50nt to 100nt, 50nt to 1,000nt, 50nt to 10,000nt, 50nt to 100,000nt, 100nt to 1,000nt, 100nt to 10,000nt, 100nt to 100,000nt, 1,000nt to 10,000nt, 1,000nt to 100,000nt, or 10,000nt to 100,000nt. In some cases, the ssRNA may be less than 50 nucleotides in length. The ssRNA may be greater than 100,000 nucleotides in length.

[0143] In some embodiments, the flow channel or a portion thereof may be parallel or substantially parallel to the analysis channel or at least a portion thereof. In some embodiments, the flow channel may be removably coupleable to the analysis channel. For example, a user may remove the flow channel from the analysis channel. Thus, a portion of the fluidic device including the analysis channel may be used to perform various analyses or experiments. Removal of the portion of the fluidic device including the flow channel may make the portion of the fluidic device including the analysis channel more accessible, for example, to a detector, camera, or other device for analyzing biological components in the analysis channel.

[0144] In some cases, the analysis channel may include a polymer matrix structure for capturing or trapping the biological component, or a molecule or compound produced by the biological component (e.g., prior to the introduction of the biological component into the fluidic device). For example, the user may obtain an analysis channel that includes a polymer matrix structure. That is, the user may not form a polymer matrix structure. In various cases, the analysis channel may be configured to include a polymer matrix structure for capturing or trapping the biological component, or a molecule or compound produced by the biological component. For example, in such an embodiment, after the introduction of the biological component into the fluidic device and the analysis channel, one or more polymer matrix structures may be formed in the analysis channel. The analysis channel may be configured for a screening process, library preparation, or another suitable process. In some embodiments, the screening process may be for drug screening, antibiotic screening, culture condition screening, or CRISPR screening. In certain cases, multiple samples may be placed in multiple channels. Multiple samples may be screened against various conditions in other signal-containing channels.

[0145] The sealable opening may be configured to transition from a sealed state to an open state. For example, the sealable opening may include a thermosensitive polymer that may melt, for example, when subjected to heat, to open the sealable opening. In some cases, the passage of biological components through the sealable opening may be inhibited in the sealed state. In certain cases, the passage of biological components through the sealable opening may be permitted in the open state. In some cases, the sealable opening may be sealed with agarose gel, a temperature-soluble polymer, an N-isopropylacrylamide (NIPAAm) polymer, a wax compound, an alginate, or any other suitable compound or material.

[0146] 15A and 15B show a portion of a fluidic device configured to trap a biological component 50. The fluidic device may include a flow channel or chamber 1551, an analysis channel or chamber 1552, and a layer or wall 1553 disposed between at least a portion of the flow channel 1551 and the analysis channel 1552. The layer 1553 may include one or more sealable openings or apertures 1554. Additionally, one or more flow impediment elements 1555 may inhibit or prevent or be configured to inhibit or prevent the biological component 50 from flowing along the flow channel 1511. The flow impediment elements 1555 may be configured to stop or trap the biological component 50 adjacent the sealable openings 1554. As described herein, the sealable openings 1514 may be configured to transition from a sealed state (e.g., a closed state) or configuration to an open state or configuration. FIG. 15A shows an example of a sealable opening 1514 in a sealed state. FIG. 15B shows an example of a sealable opening 1554 in an open state. Upon transition from a sealed state to an open state, the sealable opening 1554 may enable or permit passage of a biological component 50 from at least a portion of the flow channel 1551 to at least a portion of the analysis channel 1552. In certain instances, the analysis channel 1552 may be placed or configured to be placed under the flow channel 1551 to allow the biological component 50 to move from the flow channel 1551 to the analysis channel 1552 by an provided force (e.g., via gravity, high pressure pulses by pressurizing the flow in the flow channel, and generating negative pressure in the analysis channel). In some embodiments, the fluidic device may be spun or centrifuged to place one or more biological components from the flow channel into the analysis channel. Reagents may be placed or passed through at least a portion of the analysis channel 1552, for example, to conduct analyses or experiments are provided herein.

[0147] 15A , the flow impediment element 1555 can be disposed within at least a portion of the flow channel 1551 to impede or prevent flow of a biological component (e.g., biological component 50) in the flow channel 1551. The flow impediment element 1555 can be configured to capture or trap the biological component 50 in at least a portion of the flow channel 1551. In some cases, the flow impediment element 1555 can extend from a surface (e.g., surface 1569) of the flow channel 1551. In some cases, the surface 1569 can be disposed opposite the flow channel surface 1561 that is adjacent to the layer 1553.

[0148] In various cases, the analysis channel 1552 may include a surface 1559 disposed opposite the analysis channel surface 1563 adjacent or at the surface of the layer 1553. The analysis channel 1552 may include one or more polymer matrices 1556. The analysis channel 1552 may include one or more polymer precursors. For example, the one or more polymer precursors may be disposed in at least a portion of the analysis channel 1552. The one or more polymer matrices 1556 may be formed using an energy source that provides energy to the one or more polymer precursors in the analysis channel 1502. The energy source may be in optical, electrochemical, electromagnetic, thermal, or microwave communication with the fluidic device or the analysis channel 1552. In some cases, the energy source may be a light generating device, a heat generating device, an electrochemical generating device, an electrode, a microwave device, or a combination thereof. The energy source may selectively provide energy to the analysis channel 1552 to form the polymer matrix at a predefined location. A spatial energy modulating element may be used to selectively provide energy to the analysis channels 1552 .

[0149] In some cases, the spatial energy modulation element may include a photolithographic mask, a DMD system, or other suitable mask. The one or more polymer matrices 1556 may be formed before the sealable opening 1554 transitions to an open state (e.g., as shown in FIG. 15A). For example, the polymer matrix may be formed or associated with the sealable opening such that when the sealable opening 1554 is opened, the inhibiting element 1555 may be directed (e.g., by gravity or by fluid pressure) into the compartment 1520, thereby retaining the biological component 1550. The one or more polymer matrices 1556 may be formed after the sealable opening 1554 transitions to an open state (e.g., as shown in FIG. 15B). The one or more polymer matrices 1556 may form the analysis chamber or compartment 1520, as described herein.

[0150] 16A and 16B show top views of the fluidic device. The flow inhibition channel 1675 can be configured to inhibit the biological component 20 from flowing along the flow channel 1651. The flow of fluid (e.g., a fluid containing the biological component) through the flow channel 651 and the flow inhibition channel 1651 can cause the biological component 20 to be trapped or stopped upon opening of the flow inhibition channel 1675, as depicted in FIG. 16A. As shown, the dimensions (e.g., width) of the flow inhibition channel 1675 can be too small or narrow to allow or permit the biological component 20 to pass through the flow inhibition channel 1675. As shown in FIG. 16B, a polymer matrix 1676 can be formed on or adjacent to (e.g., surrounding) the biological component 20. In some cases, the polymer matrix can surround at least a portion of the biological component. The fluidic device of FIG. 16A and 16B can be a single-layer fluidic device. That is, the polymer matrix may be formed in flow channel 1651. As shown, the path of flow channel 1651 may be circuitous. For example, flow channel 1651 may include one or more bends. In some embodiments, the path of the flow channel may be straight, substantially straight, a zigzag pattern, or any other suitable shape.

[0151] In certain embodiments, the fluidic device of Figures 16A and 16B may include two or more layers. For example, the fluidic device may include a flow channel and an analysis channel (similar to the system shown in Figures 15A and 15B). Additionally, a sealable opening may be disposed in or adjacent to a portion of the flow inhibition channel. In such embodiments, the biological component may migrate through the sealable opening to the analysis channel (e.g., disposed adjacent to or below the flow channel) as described herein. In some cases, the analysis channel may receive two or more biological components. For example, the analysis channel may receive 2, 3, 4, 5, 6, 7, 8, 9, 10, or more biological components.

[0152] FIG. 17 shows an example of a fluidic device including or configured for multiple reagents and / or analytes (R1, R2, R3, and R4). The fluidic device may include a first flow channel 1751a to receive one or more biological components from a first sample. The first flow channel 1751a may enable or allow the flow or passage of one or more biological components from the first sample. Additionally, the first flow channel 1751a may enable or allow the flow or passage of one or more polymer precursors. The fluidic device may include a second flow channel 1751b to receive one or more biological components from a second sample. The second flow channel 1751b may enable or allow the flow or passage of one or more biological components from the second sample. Additionally, the second flow channel 1751b may enable or allow the flow or passage of one or more biological components from the second sample.

[0153] The first flow channel 1751a and / or the second flow channel 1751b may include a plurality of inhibiting elements (e.g., inhibiting elements 1755). The biological component (e.g., biological component 50) may be trapped or localized by the inhibiting elements 1755. As described herein, the first flow channel 1751a and / or the second flow channel 1751b may include one or more sealable openings disposed at or adjacent to one or more inhibiting elements 1755 that may be opened (e.g., transition from a sealed state to an open state) to allow the biological component to move to the first analysis channel 1752a or the second analysis channel 1752b. The first and second flow channels 1751a and 1751b may be disposed above the first and second analysis channels 1752a and 1752b (e.g., in upper and lower layers similar to the fluidic device shown in FIG. 15A and FIG. 15B). The polymer matrix 1756 can be formed surrounding the biological component 50. The polymer matrix 1756 may partially surround the biological component 50. The polymer matrix 1756 can form a compartment or analysis chamber 1720 to localize the biological component 50 within at least a portion of the analysis channel (e.g., analysis channels 1751a and 1751b).

[0154] The first analysis channel 1752a may include one or more reagents and / or analytes that are different from the one or more reagents and / or analytes in the second analysis channel 1752b. The first analysis channel 1752a may include one or more reagents and / or analytes that are the same as the one or more reagents and / or analytes in the second analysis channel 1752b. In some cases, the fluidic device may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, or more flow channels, and in certain cases, the fluidic device may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, or more analysis channels. The fluidic device may analyze multiple biological components in parallel. The multiple biological components may be exposed to one or more different reagents and / or analytes as provided herein. Such a configuration (e.g., as shown in FIG. 17) may allow multiple biological components in one or more samples to be analyzed under various conditions provided by different reagents and / or analytes. The fluidic device shown in FIG. 17 may be used for a screening process. The screening process may be for drug screening, antibiotic screening, culture condition screening, or CRISPR screening. The screening process may be performed in a combinatorial manner. For example, multiple samples may be loaded into multiple flow channels (e.g., in parallel) that may be screened against multiple conditions in multiple analytical channels.

[0155] The first and / or second sample may be homogenous or heterogenous. For example, one or more biocomponents in the first sample may be the same or different. The first sample may be different from the second sample. In some cases, the biocomponents may be released from the compartment or analysis chamber 1720 by selectively degrading the polymer matrix as described herein. In other words, the polymer matrix may be degradable "on demand" (e.g., by a user or as directed by a computer). In various embodiments, the degradation may be achieved by the use of a localized stimulus. In certain embodiments, the degradation may be achieved by the use of heat, light, electrochemical reactions, or some combination thereof. The released biocomponents may be collected using an outlet channel (e.g., outlet channel 1781a or 1781b).

[0156] 15A and 15B, the layer may be disposed between flow channels 1751a and 1751b and analytical channels 1752a and 1752b. The analytical channel surface adjacent to the layer (e.g., similar to surface 1561 shown in FIG. 15A), the analytical channel surface opposite the layer (e.g., similar to surface 1559 shown in FIG. 15A), or both, may include one or more barcodes, as described herein.

[0157] In some cases, the channels and / or analyses may include molecules in addition to or instead of one or more barcodes. For example, one or more channels and / or any of the surfaces of the analysis channels may include optical (e.g., fluorescent), mechanical, electrical, or biochemical sensing elements or sensors. The sensing elements may include fluorescent tags, enzymes, primers, oligonucleotides, or sensor molecules (e.g., biochemical sensor molecules). The sensing elements may be used to detect and / or measure pH, oxygen concentration, CO2 concentration, or any other suitable variable. The sensing elements may detect and / or measure parameters locally. For example, the sensing elements may detect and / or measure pH, oxygen concentration, or CO2 concentration in a compartment (e.g., a polymer matrix shell cylinder) that surrounds the biological component. Hydrogel Chamber

[0158] In some embodiments, the hydrogel chamber may be used as a diffusion regulator to limit the distance that the nucleic acid molecules of a given cell may travel away from the lysed cell. A wide variety of photosynthesizable gels may be used in connection with the present invention. In some embodiments, hydrogels are used in the present invention, particularly because of their compatibility with living cells and their versatility to form gels with desired properties, including but not limited to porosity (which in large part determines what is contained in and passes through the gel (or polymer matrix) wall, degradability, mechanical strength, ease and speed of synthesis, etc.).

[0159] Porosity. In some embodiments, the porosity of the hydrogel is selected to allow the passage of selected reagents while preventing the passage of other reagents at the same time. In some embodiments, the porosity of the hydrogel is selected to prevent the passage of biological cells but allow the passage of reagents, including proteins such as polymerases. In some embodiments, the reagents that are permeable through the polymer matrix walls include lysozyme, proteinase K, random hexamers, polymerases, transposases, ligases, deoxynucleotide triphosphates, buffers, cell culture media, or divalent cations. In some embodiments, the at least one polymer matrix includes pores that allow diffusion of reagents through the at least one polymer matrix but are too small in size to allow DNA or RNA for analysis to traverse the pores (having a size greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs). In some embodiments, crosslinking of the polymer chains of the hydrogel structure forms a hydrogel matrix with pores (i.e., a porous hydrogel matrix). In some versions, the size of the pores in the hydrogel structure may be adjusted or tailored to encapsulate sufficiently large genetic material (e.g., greater than about 300 base pairs) but allow smaller materials, such as reagents, or smaller sized nucleic acids (e.g., less than about 50 base pairs), such as primers, to pass through the pores and thereby into and out of the hydrogel structure. In some embodiments, the hydrogel may include pore sizes with diameters sufficient to allow the reagents listed above to diffuse through its structure while retaining nucleic acid molecules of lengths greater than 500 nucleotides or base pairs. In some embodiments, the pores have diameters of about 10 nm to about 100 nm. In some embodiments, the pore size of the hydrogel structure may be determined by routine experimentation, by varying the ratio of the concentration of polymer precursors to the concentration of crosslinker, varying pH, salt concentration, temperature, light intensity, etc.In some embodiments, the average diameter of the pores in the polymer matrix wall prevents the passage of molecules having a molecular weight of 25 kilodaltons (kDa) or greater, or having a molecular weight of 50 kDa or greater, or having a molecular weight of 75 kDa or greater, or having a molecular weight of 100 kDa or greater, or having a molecular weight of 150 kDa or greater.

[0160] In some embodiments, the retained DNA or RNA has a length that allows sequence determination using conventional sequencing-by-synthesis techniques. For example, such DNA or RNA comprises at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the pore may have an average diameter of 5 nm to 100 nm. In some embodiments, the pore may have an average diameter of 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 pore may have an average diameter greater than 100 nm. In some embodiments, the pore may have an average diameter smaller than 5 nm. The reagent may include an enzyme or primer having a size of less than 50 base pairs (bp). The primer may include single-stranded DNA (ssDNA). In some embodiments, the primer may have a size of 5 bp to 50 bp. In some embodiments, the primers may have a size of 5 bp to 10 bp, 10 bp to 20 bp, 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, the primers may have a size greater than 50 bp. In certain cases, the primers may have a size less than 5 bp. In some embodiments, the pores may have a diameter of 5 nm to 100 nm. In some embodiments, the pores may have a diameter of 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 greater than 100 nm. In some embodiments, the pores may have an average diameter less than 5 nm. The polymer matrix may have a pore size of about 5 nanometers (nm) to about 100 nm.The polymer matrix may be about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10nm to approx. 80nm, approx. 10nm to approx. 90nm, approx. 10nm to approx. I00nm, approx. 10nm to approx. 110nm, approx. 20nm to approx. 30nm, approx. 70nm, approximately 20nm to approximately 80nm, approximately 20nm to approximately 90nm, approximately 20nm to approximately 100nm, approximately 20nm to approximately 110nm, approximately 30nm to approximately 40nm, approximately 30nm to approximately 50nm, approximately 30nm to approximately 60nm, approximately 30nm to approximately 70nm, approximately 30nm to approx. 80nm, approx. 30nm to approx. 90nm, approx. 30nm to approx. I00nm, approx. 30nm to approx. 110nm, approx. 40nm to approx. 50nm, approx. 90nm, approx. 40nm ~ approx. I00nm, approx. 40nm ~ approx. 110nm, approx. 50nm ~ approx. 60nm, approx. 50nm ~ approx. 70nm, approx. The polymer matrix may have a pore size of about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 90 nm, about 110 nm, about 100 nm, about 1 ...The polymer matrix may have a pore size of at least about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, or less. The polymer matrix may have a pore size of at most about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or more.

[0161] Porosity modulation. The pore size in the polymer matrix may be modulated using chemical reagents or by applying heat, electric field, light, or another suitable stimulus. In other words, the polymer matrix may include a tunable property (e.g., pore size). In some cases, the polymer matrix may include a thermoresponsive polymer or a temperature-responsive polymer. A thermoresponsive polymer (e.g., poly(N-isopropylacrylamide) (NIPAAM)) may phase separate from a solution upon heating or cooling (e.g., a polymer that exhibits a lower critical solution temperature (LCST) or an upper critical solution temperature (UCST)). The polymer matrix may include a polymer that may collapse at high temperatures, for example, to control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that may be used to form hydrogel / polymer matrices with tunable properties may include poly(N-vinylcaprolactam), poly(N-ethyloxazoline), poly(methylvinylether), poly(acrylic acid-coacrylamide), or combinations thereof. A change in temperature can expand or contract the average pore size in the polymer matrix, allowing selected molecules, such as nucleic acid molecules, proteins, or any biomolecule or molecule smaller than the tailored pore size, to be released from the hydrogel chamber.

[0162] Size and shape of the hydrogel chamber. In some embodiments, the polymer matrix wall of the chamber inhibits passage of certain components, such as mammalian cells, genomic DNA, larger polynucleotides (e.g., mRNAs of greater than 200 ribonucleotides, or greater than 300 ribonucleotides, or 500 ribonucleotides). In some embodiments, the polymer matrix wall extends from a first surface to a second surface (parallel to the first surface) to form a chamber within the channel. In some embodiments, the chamber has an interior having an interior area that is the area of ​​the surface enclosed by the polymer matrix wall and the chamber. In some embodiments, the interior of the chamber is sized to enclose a cell. For example, such a chamber may include a cylindrical or polygonal shell that includes an interior space, or an interior and a polymer matrix wall. In some embodiments, such a chamber has an annulus-like cross section. As used herein, the term "annulus-like cross section" refers to a cross section that is topologically equivalent to an annulus. In some embodiments, the interior space or interior of the chamber has a diameter in the range of 1 μm to 500 μm and a volume in the range of 1 picoliter to 200 nanoliters, or 100 picoliters to 100 nanoliters, or 100 picoliters to 10 nanoliters. 2 ~1×10 6 μm 2 range, or 400 μm 2 ~7×10 5 μm 2In some embodiments, the polymer matrix walls have a thickness of at least 1 μm (micrometer). In some embodiments, the height of the chamber having a ring-like cross section has a value in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm. In some embodiments, the polymer matrix walls having a ring-like cross section have an aspect ratio (i.e., height / width) of 1 or less. In some embodiments, the aspect ratio and the thickness of the polymer matrix wall are selected to maximize the stability of the chamber against forces, e.g., flow of reagents through the channel, washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix is ​​degradable. In some embodiments, the degradation of at least one polymer matrix is ​​"on demand". In some embodiments, the chambers in the channel are discontinuous. In some embodiments, the chambers in the channel may be continuous with adjacent chambers. In some embodiments, the chambers may share polymer matrix walls with each other. In some embodiments, chambers may be synthesized with slits or other orifaces that are large enough to allow the passage of certain components, e.g., beads, but small enough to prevent the passage of other components, e.g., cells.

[0163] Hydrogel Compositions. In some embodiments, the channels of the fluidic devices of the systems of the invention comprise one or more polymer precursors for forming the chambers. In some embodiments, the one or more polymer precursors comprise hydrogel precursors. Such precursors include, but are 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, heptane, glycerol ... The hydrogel may be selected from a wide variety of compounds including parin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylopropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof. In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEG thiol / PEG-acrylate, acrylamide / N,N'-bis(acryloyl)cystamine (BACy), or PEG / PPO. In some embodiments, the following precursors and crosslinkers may be used to form chambers having degradable polymer matrix (hydrogel) walls. The polymer precursor may be formed by using any of the hydrogel precursors and crosslinkers in Table IA (columns 1 and 3, respectively).The resulting polymer matrix can be degraded by the degrading agents shown in Table 1A (column 4). [Table 1A] [Table 1B]

[0164] Hydrogel Degradation. In some embodiments, the hydrogel chambers of the present invention are generally degradable or depolymerizable "on demand" within the channel or within the channel. Generally degradable hydrogel chambers are degraded by treatment with a degrading agent, or equivalently, a depolymerizing agent that is exposed to all chambers within the channel. Depolymerizing agents may include, but are not limited to, heat, light, and / or chemical depolymerizing agents (sometimes referred to as cleaving or decomposing agents). In some embodiments, on-demand degradation may be performed using polymer precursors that allow for photocrosslinking and photodecomposition, for example, using different wavelengths for crosslinking and for decomposition. For example, Eosin Y may be used for radical polymerization in defined regions using a wavelength of 500 nm, and then irradiation at 380 nm may be used to cleave the crosslinker. In other embodiments, a photocaged hydrogel cleaving reagent may be included in the formation of the polymer matrix wall. For example, an acid-labile crosslinker (e.g., esters, etc.) may be used to create a hydrogel, and then UV light may be used to generate localized acidic conditions that then degrade the hydrogel. In some embodiments, the at least one polymer matrix is ​​degradable by at least one of the following steps: (i) contacting the at least one polymer matrix with a cleavage 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 the photocleavable crosslinkers that crosslink the polymers of the at least one polymer matrix. In some embodiments, the at least one polymer matrix comprises a hydrogel. In some embodiments, the cleavage reagent degrades the hydrogel. In some embodiments, the cleavage reagent comprises a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage reagent, or a combination thereof. In some embodiments, the cleavage 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 hydrogel may be functionalized by attaching a functional group to the polymer matrix or hydrogel. Some non-limiting examples of functional groups may include functional groups including capture reagents (e.g., pyridinecarboxaldehyde (PCA)), acrylamide, agarose, biotin, streptavidin, strep-tag II, linkers, aldehydes, phosphates, silicates, esters, acids, amides, aldehyde dithiolanes, PEG, thiols, alkenes, alkynes, azides, or combinations 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) a biocomponent. The biomolecules may be produced by the biocomponent (e.g., cell-derived secretome). The functionalized surface of the polymer matrix inside the compartment may be used to capture reagents or molecules from outside the compartment. A functionalized surface can increase the surface area covered by a reagent, a molecular sensor, or any molecule of interest (eg, an antibody).

[0165] Partial Degradation. In some embodiments, an existing polymer matrix wall may be partially degraded, for example, to change the porosity. In some embodiments, the polymer precursor may include degradable beads that when synthesized form part of the polymer matrix wall and are embedded therein and can then be degraded on demand or generally, thereby creating an increase in porosity.

[0166] Photosynthesis. In some embodiments, the creation of a polymer matrix in the fluidic device comprises exposing 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 between 350 nm and 800 nm. In some embodiments, the light generating device generates light between 350 nm and 600 nm. In some embodiments, the light generating device generates light between 350 nm and 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the creation of a polymer matrix in the fluidic device is performed using a spatial light modulator (SLM) (i.e., a spatial energy modulating element capable of spatially generating a desired light intensity pattern). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam directed using a galvanometer. In some embodiments, the SLM is liquid crystal based. Sequencing, barcoding, genomic fragments and transcriptomes

[0167] RNA or nucleic acid molecules, including oligonucleotide labels, barcodes, genome fragments, messenger RNA and other polynucleotide targets, can be sequenced by the methods and systems of the present invention. In some embodiments, a capture element, or capture probe, for this purpose comprises an oligonucleotide bound to a surface, where such an oligonucleotide comprises a sequence segment that is complementary to that of the nucleic acid to be captured, which may be (for example) an mRNA or a polyA segment of any sequence, or a "handle" segment adjacent to the barcode or oligonucleotide label. When the sequencing operation is performed, the surface is provided with such a capture element (such as an oligonucleotide), and optionally a surface primer for surface amplification of the captured nucleic acid or its derivatives. Such captured oligonucleotides may be attached to the first surface by many chemistries known in the art, such as the Integrated DNA Technologies brochure entitled "Strategies for attaching oligonucleotides to solid supports," (2014). The sequencing step may be performed on the surface adjacent to the lysed cells ("in situ" sequencing), or the templates may be optionally amplified, released, and eluted from the surface and sequenced in an external sequencing instrument ("external" sequencing). In the latter approach, the capture element may contain spatial barcodes that provide surface location information, allowing sequences associated with specific locations on the surface, e.g., individual chamber sites, to be determined externally. In some embodiments, the spatial barcodes are present at a sufficiently high density that each chamber covers an area of ​​the surface that is uniquely associated with one or more spatial barcodes, and usually a single spatial barcode. In some embodiments, preparation of polynucleotides for sequencing operations is performed after the target templates (e.g., oligonucleotide labels, mRNA, genomic fragments) are released and captured by complementary sequences in the capture element. The releasing step depends on the nature of the target templates.For example, oligonucleotide labels attached to antibodies by disulfide bonds may be released by a reducing agent (which may be the same as the lysis reagent). mRNA may be released by treating cells with a conventional lysis reagent. Release of genomic fragments may require a lysis and pre-amplification step. Lysis conditions may vary widely and may be based on the action of heat, detergents, proteases, alkali, or combinations of such factors. The following references provide guidance for the selection of lysis reagents, or lysis buffers, for single cell lysis conditions for mRNA and / or genomic DNA: Thronhill et al, Prenatal Diagnosis, 21: 490-497 (2001), Kim et al, Fertility and Sterility, 92: 814-818 (2009), Spencer et al, ISME Journal, 10: 427-436 (2016), Tamminen et al, Frontiers Microbiol. Methods, 6: article 195 (2015), etc. Lysis conditions may include: 1) cells in HO at 96° C. for 15 min followed by 10° C. for 15 min; 2) 200 mM KOH, 50 mM dithiotheitol, heated at 65° C. for 10 min; 3) for 4 μL protease-based lysis buffer: 1 μL 17 μM SDS combined with 3 μL 125 μg / mL proteinase K, followed by incubation at 37° C. for 60 min and then at 95° C. for 15 min (to inactivate proteinase K); 4) for 10 μL detergent-based lysis buffer: 2 μL HO, 2 μL 10 mM EDTA, 2 μL 250 mM dithiothreitol, 2 μL 0.5% N-lauryl sarcosine salt solution; 5) for 200 mM Tris pH 7.5, 20 mM EDTA, 2% sarcoyl, 6% Ficoll.

[0168] In embodiments using spatial barcodes on a surface, the spatial barcodes may be created using a wide variety of methods, including but not limited to those described in the following references, which are incorporated herein by reference: Horgan et al., International Patent Application Publication No. WO2022 / 013094; Frisen et al., U.S. Patent No. 9,593,365; Fan et al., U.S. Patent Application Publication No. US2019 / 0360121; Chen et al, bioRxiv (https: / / doi.org / 10.1101 / 2021.01.17.427004); Cho et al, bioRxiv (https: / / doi.org / 10.1101 / 2021.01.25.427807); Quan et al, Nature Biotechnology,29(5): 449-453 (2011); Singh-Gasson et al, Nature Biotechnology, 17: 974- (1999); and the like. Systems and Equipment

[0169] A system for carrying out an embodiment using a gel barrier as a diffusivity modifier (e.g., as shown in Figures 2E-2F) is shown in Figure 22A. A flow cell (2200) is a component of a fluidic device that provides one or more channels under programmable control and liquid handling components for delivering beads and reagents to the channels. In this example, four channels (2202, 2204, 2206, and 2208) are shown, and a close-up (2212) of a segment (2210) of channel 2 (2204) is shown below. In the conceptualized representation of the flow cell (2200) in Figure 22A, the inlets, outlets, and other features of the channels are not shown. At a first surface (2214) of channel 2 (2204), a plurality of beads, e.g., (2218), are each surrounded by a hydrogel chamber, e.g., (2216). In some embodiments, the porosity of the polymer matrix walls of the hydrogel chamber is selected to be impermeable to the beads but permeable to the reagents to form the spatial barcode. In this way, reagents can be introduced to and removed from the interior of the hydrogel chamber by flowing them through the channel (2220) and retaining the beads inside. The enlargement (2212) of the channel segment (2210) below shows an optical system (2221) for photosynthesizing the hydrogel chamber at the location of the beads in the channel. Those skilled in the art will recognize that optical systems having configurations different from those of Figures 22A and 22B may be used to perform these functions. In some embodiments, the speed of synthesis may be increased by synthesizing multiple structures simultaneously using one or more DMD-objective lens subsystems for synthesizing hydrogel structures.

[0170] Returning to FIG. 22A, to photosynthesize the hydrogel chambers, a light source (2222) generates a beam of light (e.g., UV light) of an appropriate wavelength (2223) that passes through an appropriate photomask or beam shaping or beam steering (Galvo) system to shape the beam and synthesize the desired structure or structures in the channels. In some embodiments, a digital micromirror device (DMD) (2224) is used, while in other embodiments, a physical photomask is used. The position, shape and thickness of the polymer matrix wall of the chamber are determined, at least in part, from the positional information of the beads determined from the image collected by the detector (2232). The reflected light from the DMD (2224) is shaped using conventional optics, e.g., collimating optics (2228), and directed through an objective lens system (2234) to the channel 2 segment (2210). The objective lens (2234) and flow cell (2200) move relative to each other in the xy directions (2236) to photosynthesize a chamber at any position in any of the channels. In some embodiments, the flow cell (2200) moves and the optical system (2221) is stationary. In some embodiments, the objective lens (2234) can also direct the light beam (2227) from the light source (2229) to targets, e.g., cells, at the first surface (2214) and collect optical signals, e.g., fluorescent signals, from the assay performed at the first surface (2214). Alternatively, the collection of optical signals can be performed using separate objective lenses, as shown (if) in FIG. 22B. The information collected by the detector (2232) or its counterpart in the embodiment of FIG. 22B, particularly the position of the cells in their respective channels, is used by the computer (2238) and / or auxiliary controller to instruct the DMD (2224) and translation device, which control the relative position of the objective lens (2234) and the flow cell (2200), to synthesize hydrogel chambers of the appropriate shape and size at the appropriate location.

[0171] FIG. 22B shows an alternative optical system in which the detection portion (2250) of the optical system moves (2272) independently of the movement (2268) of the combination portion (2252) of the optical system. The detection portion (2250) of the optical system includes a detector (2256), an objective lens (2258), a light source (2260), and interconnecting optical elements, such as a dichroic mirror (2262). As in the embodiment of FIG. 22A, the detector (2256) is operatively associated with a computer (2264) and the combination portion (2252) of the optical system to provide bead position information to the combination portion (2252). The computers (2264) and (2238) are also operatively associated with stages and / or motors that control the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the combination portion (2252) of the optical system is located on the opposite side of the first surface (2264) from the detection portion (2250). As in the embodiment of FIG. 22A, this includes conventional components objective lens (2274), mirror (2276), collimating optics (2280), DMD (2282), and light source (2278).

[0172] In some embodiments, the beads in Figure 22A, e.g., (2218), are randomly arranged on the first surface (2214). In alternative embodiments, the first surface (2214) may include regularly spaced sites or features for capturing beads, such that they are substantially the only such sites or features arranged on the first surface. For example, in some embodiments, such sites or features may be linear or hexagonal arrays of spots.

[0173] In some embodiments, the system of the present invention includes: (a) a channel including a first surface, a plurality of cells disposed on the first surface, and one or more polymer precursors; (b) a spatial energy modulation element in optical communication with the first surface; (c) a detector in optical communication with the first surface and operably associated with the spatial energy modulation element, the detector detecting each of the plurality of cells and determining its location on the first surface; and (d) a plurality of gel chambers, each gel chamber surrounding a single cell of the plurality of cells, where the gel chambers are synthesized by projecting light into the channel using the spatial energy modulation element such that the projected light causes crosslinking of one or more polymer precursors to form a polymer matrix wall of the chamber, and the location of the synthesized chamber is determined by the location of the enclosed cell, thereby identified by the detector. It is understood that the term "detector" as used herein may include, but is not limited to, a microscope element that collects and optionally magnifies an image of a portion of the channel, as well as an image analysis element including software for identifying cells, cell features, chambers, and other objects, to store such information as well as associated location information. The computing element uses such information generated by the detectors together with user input to generate commands to other elements, e.g., spatial energy modulation elements, to perform various functions including, but not limited to, synthesis of chambers, disassembly of chambers "on demand", photolysis of cells, etc. Configurations of such embodiments are shown in Figures 22A-22B described above. In some embodiments, the channels of the fluidic device further include a second surface, where the first surface and the second surface are disposed opposite each other across the channel, and the polymer matrix walls of the chambers extend from the first surface to the second surface to form chambers each having an interior. In some embodiments, the chambers in the channels each enclose a single cell.In some embodiments, both the first and second walls are made of an optically transparent material, e.g., glass, plastic, etc., and are positioned such that the first and second surfaces are substantially parallel to each other. The vertical distance between the first and second surfaces may be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm. In some embodiments, the vertical distance between the first and second surfaces may be 2 times the average size of the cells to be analyzed to 5 times the average size of the cells to be analyzed.

[0174] In some embodiments, the first surface may include a capture element to capture cells at a predetermined location. For example, the capture element may include, but is not limited to, a capture antibody specific to all or a subpopulation of cells. The capture element may also include, but is not limited to, a non-specific capture material, such as, but not limited to, polylysine, fibronectin, treated plastic (e.g., Maxysorb™ plastic, ThermoFisher), and the like. In some embodiments, the capture moieties (e.g., antibodies) of such cells may be restricted to spots or reaction sites arranged in a regular pattern on the first surface, so that the cells captured at such reaction sites may be arranged on the first surface in a regular pattern that may be more efficient for chamber synthesis and / or optical signal detection than a random arrangement. Guidance for providing cell capture antibodies on surfaces can be found in the following references: Zhu et al, Analytica Chemica Acta, 608: 186-196 (2008); Sekine et al, J. Immunol. Methods, 313(1-2): 96-109 (2006); etc. In some embodiments, such reaction sites or spots have diameters in the range of 5-500 μm, or in the range of 10-1000 μm. In some embodiments, such spots or reaction sites are arranged in a linear array, or in a hexagonal array. In some embodiments, such arrays of such spots or reaction sites have diameters in the range of 10-2500 sites / mm. 2, or 10 to 1000 sites / mm 2 , or 10-500 sites / mm 2 , or 10-100 sites / mm 2 It has a density in the range of

[0175] The spatial energy modulation element that uses light energy for polymerization may include a physical photomask or a virtual photomask, such as a digital micromirror device (DMD).The following references, which are incorporated herein by reference, provide guidance in the selection and operation of a DMD for photopolymerizing gels: Chung et al., U.S. Pat. No. 10,464,307; Hribar et al., U.S. Pat. No. 10,351,819; Das et al., U.S. Pat. No. 9,561,622; Huang et al., Biomicrofluidics, 5: 034109 (2011); and the like.

[0176] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and explanation of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions shown herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is therefore contemplated that the present invention should encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby. EXAMPLES

[0177] The following illustrative examples are representative of embodiments of the stimuli, systems, and methods described herein and are not meant to be limiting in any way. Example 1 RNA Sequencing Using the Methods Described Herein

[0178] Experiments are performed with P7 or P5 adapted libraries derived from RNA transcripts. RNA transcripts are made from a plasmid containing green fluorescent protein (GFP). The pMA-T based plasmid contains P5, T7 polymerase promoter, start codon, His tag, FLAG tag, GFP sequence, TAA stop codon, T7 terminator, and P7. RNA transcripts can extend from the promoter sequence to the T7 termination sequence. However, the T7 terminator does not completely stop transcription, so some of the resulting RNA transcripts are His_FLAG_GFP_P7'. RNA transcripts are treated with DNase to remove DNA that would otherwise form clusters. To check that the DNase treatment is effective, reactions are run on gel and analyzed to prove that the DNase treatment is effective in removing DNA.

[0179] The PhiX DNA library and DNase-treated GFP-P7'RNA transcripts are hybridized on different lanes of a flow cell following standard cluster protocols for template hybridization. For example, lanes 1-4 can contain PhiX DNA, while lanes 5-8 contain GFP RNA. Lanes 5 and 6 can contain RNA that has been pretreated with DNase to remove DNA. Lanes 7 and 8 can be RNA that has been pretreated with DNase and treated with RNase on the flow cell as an additional control. The PhiX DNA library can be hybridized via P5 or P7 as both sequences, their complements are present in the template. In contrast, the GFP-P7'RNA template hybridizes only with the P7 surface primer due to their complementarity and the lack of a P5 sequence. The first extension can be performed by using any commercially available reverse transcriptase, such as Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. Some lanes can be transposed using a transposon complex that contains the P5 adapter sequence of the transposon sequence. Gaps in the DNA sequence left after the transposition event can be filled using a strand displacement extension reaction. A transposition event is necessary in the lane containing the GFP_P7'RNA to add a P5 adaptor to create a template from which clusters can be made. Isothermal cluster amplification can be performed as standard.

[0180] The nucleic acid strand synthesized from the first extension can be sequenced for the analysis of biological samples as described herein.In some cases, the strand synthesized from the first extension can be sequenced to obtain transcriptome data.In other examples, the strand synthesized from the first extension can serve as a template for second extension or second strand synthesis to create a second strand of nucleic acid.The second strand can be sequenced to obtain transcriptome data.The first strand, second strand, or amplification of cDNA molecules can also be obtained by a combinatorial approach, which can utilize one or more of the methods described herein in combination to obtain transcriptome data. FIG. 9 provides a non-limiting example of such a combination, where the combined use of the improvements described in FIG. 2 (library construction on a surface), FIG. 3 (3' blocking of surface primers to avoid unwanted hybridization and extension), FIG. 5 (use of TdT to block the 3' end of cDNA), and FIG. 7 (use of exonuclease treatment to remove unwanted capture probes) to improve currently available sequencing techniques. Example 2 RNA sequencing of human biological samples

[0181] A flow cell with multiple lanes can be prepared containing primers capable of hybridizing to RNA molecules containing poly-A tails. For example, lane 1 can be grafted with a standard oligonucleotide (oligo) mix containing only P5 and P7 oligonucleotides, while lanes 2-8 are grafted with the standard mix (P5 and P7 oligos) and capture oligos (i.e., primers containing poly-T sequences for binding to RNA molecules containing poly-A tails). After primer grafting, the flow cell can be stored at 4°C until use. In this example, 5 pM of PhiX control library sample can be prepared and added to the flow cell for hybridization in lanes 1 and 2. For each of lanes 3-8, 400 ng of RNA sample can be prepared and added to the flow cell for hybridization. Lanes 3-6 can contain human RNA, such as a biological sample obtained from a subject. Lanes 7 and 8 can contain Universal Human Reference (UHR) RNA. After template hybridization, a wash buffer can be administered through the flow cell for removal of unhybridized template. Un-hybridized templates in all lanes could be extended using AMV-RT (NEB, Ipswich, Mass.), which generated DNA:RNA complexes.

[0182] Lanes 3-8 can be contacted with the transposome complex, while lanes 1 and 2 are only contacted with an equal volume of wash buffer. Two different concentrations of transposome complex mixes are prepared. The mix for lanes 3, 5 and 7 can be prepared with 1.25 μl of transposome complex, 100 μl of buffer, and 400 μl of water. The mix for lanes 4, 6 and 8 can be prepared with 0.625 μl of transposome complex, 100 μl of buffer, and 400 μl of water. 95 μl of transposome complex mix is ​​added to lanes 3-8 of the flow cell for tagging fragmentation. To remove the transposase after tagging fragmentation, a chaotropic buffer is added to lanes 3-8 of the flow cell and incubated for 2 min. The lanes of the flow cell are then washed twice. After cleaning, Bst enzyme is used for strand displacement extension of the tagged fragmented DNA:RNA complex to remove the non-mobile strand of the transposon and make the DNA strand of the DNA:RNA complex full length for clustering. The RNA strand is removed and then the cluster is created using isothermal amplification. The cluster is then sequenced.

[0183] The sequencing results are compared with those obtained for standard RNA sequencing of universal human reference RNA, which is performed according to standard sequencing methods using standard sequencing reagents. The results may show normal alignment distribution for RNA samples sequenced using the methods provided herein. The results may show more masked clusters of repeats, possibly due to the higher number of polyA sequences and more repeats in the 3'UTR region of the RNA samples analyzed by the tagging fragmentation method. The usable reads may be about 10% lower than for standard RNA sequencing protocols, possibly due to the higher number of repeats in the RNA that can be analyzed. The amount of ribosomal RNA may be lower as expected, since mRNA is isolated and sequenced in the tagging fragmentation method provided herein. Mitochondrial RNA is within normal limits.

[0184] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to one skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the disclosure. For example, all of the techniques and devices described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated herein by reference for all purposes. Terms and Definitions

[0185] "Amplicon" refers to the product of a polynucleotide amplification reaction, i.e., a clonal population of polynucleotides that may be single-stranded or double-stranded, replicated from one or more starting sequences. "Amplifying" refers to generating an amplicon by performing an amplification reaction. The one or more starting sequences may be one or more copies of the same sequence, or they may be a mixture of different sequences. In some embodiments, an amplicon is formed by the amplification of a single starting sequence, such that the amplicon is a clonal population of starting sequences. An amplicon may be generated by a variety of amplification reactions whose products include replicating one or more starting or target nucleic acids. In one aspect, an amplification reaction that generates an amplicon is "template-driven" in that the base pairing of either the nucleotide or oligonucleotide reactants has a complement in the template polynucleotide that is required for the creation of the reaction product. In one aspect, the template-driven reaction is primer extension by a nucleic acid polymerase, or oligonucleotide ligation by a nucleic acid ligase. Such reactions include, but are not limited to, polymerase chain reaction (PCR), linear polymerase reaction, nucleic acid sequence-based amplification (NASBA), rolling circle amplification, and the like, as disclosed in the following documents, which are incorporated herein by reference: Mullis et al., U.S. Pat. Nos. 4,683,195; 4,965,188; 4,683,202; and 4,800,159 (PCR); Gelfand et al., U.S. Pat. No. 5,210,015 (real-time PCR using "taqman" probes); Wittwer et al., U.S. Pat. No. 6,174,670; Kacian et al., U.S. Pat. No. 5,399,491 ("NASBA"); Lizardi, U.S. Pat. No. 5,854,033; Aono et al., Japanese Patent Application Publication No. JP4-262799 (rolling circle amplification); and the like. With respect to amplification reactions, "reaction mixture" means a solution containing all the necessary reactants to carry out the reaction, which may include, but is not limited to, buffers, salts, cofactors, scavengers, etc. to maintain the pH at a selected level during the reaction.Of particular interest are solid-phase amplification techniques in which a starting sequence is amplified to generate surface-bound copies, such as bridge amplification, e.g., U.S. Pat. Nos. 6,090,592, 6,060,288, 6,787,308, 9,057,097, 9,169,513, 9,476,080, 9,476,080; Adessi et al, Nucleic Acids Research, 28(20): e87 (2000), and the like, which are incorporated herein by reference.

[0186] "Barcode" refers to a molecular label or identifier. In some embodiments, the barcode is a molecule bound to an analyte or a segment of an analyte that can be used to identify the analyte (e.g., in the case of polynucleotide barcodes and polynucleotide analytes). In some embodiments, the barcode (referred to herein as a "spatial barcode") can be bound to a surface to identify a location on the surface. In some embodiments, a population of identical spatial barcodes may be located within a specific area on the surface. In some embodiments, there may be a one-to-one correspondence between different spatial barcodes and different areas on the surface, i.e., each different area has a different unique barcode. In some embodiments, the identity of the spatial barcode is determinable, for example, by sequencing, whenever the spatial barcode is a polynucleotide. In some embodiments, the spatial barcode is an oligonucleotide. In some embodiments, the barcode comprises a random sequence oligonucleotide. Random sequence oligonucleotides are typically synthesized by "split and mix" synthesis techniques, for example, as described in the following references, which are incorporated herein by reference: Church, U.S. Pat. No. 4,942,124; Godron et al., International Patent Publication No. WO2020 / 120442; Seelig et al., U.S. Patent Application Publication No. 2016 / 0138086; etc. Random oligonucleotides are sometimes represented as "NNN...N". In some embodiments, the term "barcode" includes composite barcodes, i.e., oligonucleotide segments that include sub-segments that identify different objects. For example, a first segment of a composite barcode can identify a specific area of ​​a surface, and a second segment of the composite barcode can identify a specific molecule (a so-called "unique molecular identifier" or UMI).

[0187] "Cell" refers to a biological cell that may be assayed by the methods and systems of the present invention, including, but not limited to, vertebrate, invertebrate, eukaryotic, mammalian, microbial, protozoan, prokaryotic, bacterial, insect, or fungal cells. In some embodiments, mammalian cells are assayed by the methods and systems of the present invention. In particular, any mammalian cell that may be or has been genetically modified for use in medical, industrial, environmental, or therapeutic processes may be analyzed by the methods and systems of the present invention. In some embodiments, "cell" as used herein includes genetically modified mammalian cells. In some embodiments, "cell" includes stem cells. In some embodiments, "cell" refers to cells modified by CRISPR Cas9 technology. In some embodiments, "cell" refers to cells of the immune system, including, but not limited to, cytotoxic T lymphocytes, regulatory T cells, CD4+ T cells, CD8+ T cells, natural killer cells, antigen presenting cells, or dendritic cells. Of particular interest are cytotoxic T lymphocytes engineered for therapeutic applications, such as cancer therapy.

[0188] "Cluster" refers to an amplicon or clonal population of a single polynucleotide amplified by a surface amplification technique such as bridge PCR. In some embodiments, the term "cluster" includes amplicons generated by rolling circle amplification.

[0189] "Hydrogel" means a gel comprising a crosslinked hydrophilic polymer network that has the ability to absorb and retain large amounts of water (e.g., 60-90 percent water, or 70-80 percent water) without dissolution due to the establishment of physical or chemical bonds between the polymer chains, which may be covalent, ionic, or hydrogen bonds. Hydrogels exhibit high permeability for oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications. Hydrogels may comprise natural or synthetic polymers and may be reversible (i.e., degradable or depolymerizable) or irreversible. Synthetic hydrogel polymers may include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and poly(vinyl alcohol). Natural hydrogel polymers include alginate, hyaluronic acid, and collagen. The following references describe hydrogels and their biomedical uses: Drury et al, Biomaterials, 24: 4337-4351 (2003); Garagorri et al, Acta Biomatter, 4(5): 1139-1147 (2008); Caliari et al, Nature Methods, 13(5): 405-414 (2016); Bowman et al., U.S. Pat. No. 9,631,092; Koh et al, Langmuir, 18(7): 2459-2462 (2002).

[0190] "On demand" means that the operation can be directed to individual, separate, selected locations (e.g., spatial locations of the polymer precursor solution, or selected polymer matrix chambers). Such selection can be based on manual observation of the optical signal or data collected by the detector, or such selection can be based on a computer algorithm operating on the optical signal or data collected by the detector. Manual observation of the optical signal or data collected by the detector can include either real-time detection, or detection for a period of time before modulating the unit of energy to polymerize the polymer precursor or decomposing the chamber. For example, a subset of the chambers (all formed with photodegradable polymer matrix walls) can be preselected to release and remove their contents based on the position information and the value of the optical signal from the analytical assay performed in the chambers. The preselected chambers can be photolyzed by selectively projecting a beam of light of appropriate wavelength characteristics (e.g., using a spatial energy modulation element) to decompose the polymer matrix walls of the preselected chambers. In another example, the multiple chambers may be observed in real time (e.g., via fluorescence microscopy) for detection of an analyte of interest, and one or more chambers of the multiple chambers are selected for degradation in real time upon detection of the analyte of interest.

[0191] A "physical photomask" generally refers to a physical structure having a plurality of openings or holes through which light can be projected. A physical photomask can be used to create the hydrogel matrix described herein by polymerizing a polymer precursor solution and forming a three-dimensional structure corresponding to the pattern on the photomask. The physical photomask can be patterned with a specific layout or geometric pattern. The physical photomask can be attached to the top surface of the flow cell.

[0192] "Polymerase chain reaction" or "PCR" refers to a reaction for the in vitro amplification of a specific DNA sequence by sequential primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicas of a target nucleic acid flanked by primer binding sites, such a reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing the primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled in a thermal cycler instrument through different temperatures optimized for each step. The specific temperatures, the duration of each step, and the rate of change between steps depend on many factors well known to those skilled in the art, as exemplified, for example, by the literature: McPherson et al, editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in conventional PCR using Taq DNA polymerase, double-stranded target nucleic acid may be denatured at a temperature >90°C, primers may be annealed at a temperature ranging from 50-75°C, and primers may be extended at a temperature ranging from 72-78°C. The term "PCR" encompasses derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, bridge PCR, and the like. Reaction volumes range from hundreds of nanoliters, e.g., 200 nL, to hundreds of μL, e.g., 200 μL. "Reverse transcription PCR" or "RT-PCR" refers to PCR that proceeds by a reverse transcription reaction that converts target RNA into complementary single-stranded DNA that is then amplified. See, for example, Tecott et al., U.S. Patent No. 5,168,038, which is incorporated herein by reference. "Real-time PCR" or "quantitative PCR" refers to PCR in which the amount of reaction product, i.e., amplicon, is monitored as the reaction progresses.There are many forms of real-time PCR, which mainly differ in the detection chemistry used to monitor reaction products.For example, Gelfand et al., U.S. Patent No. 5,210,015 ("taqman"); Wittwer et al., U.S. Patent No. 6,174,670 and U.S. Patent No. 6,569,627 (intercalating dye); Tyagi et al., U.S. Patent No. 5,925,517 (molecular beacon), which are incorporated herein by reference.Detection chemistry for real-time PCR is reviewed in Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporated herein by reference.

[0193] "Polymer matrix" generally refers to a phase material (e.g., a continuous phase material) that includes at least one polymer. In some embodiments, the polymer matrix refers to at least one polymer and the interstitial space not occupied by the polymer. The polymer matrix may be composed of one or more types of polymers. The polymer matrix may include linear, branched, and crosslinked polymer units. The polymer matrix may also contain non-polymeric species intercalated within the interstitial space not occupied by the polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term "polymer matrix" may encompass dry hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. The polymer matrix may include polymer precursors, which generally refer to one or more molecules that, upon activation, can trigger or initiate a polymerization reaction. The polymer precursors may be activated by electrochemical energy, photochemical energy, photons, magnetic energy, or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which polymerize to produce a polymer matrix) and crosslinking compounds, which may include photoinitiators and other compounds necessary or useful for creating polymer matrices, particularly polymer matrices that are hydrogels.

[0194] "Polynucleotide" and "oligonucleotide" are used interchangeably and refer to a linear polymer of nucleotide monomers, respectively. The monomers that make up polynucleotides and oligonucleotides can specifically bind to natural polynucleotides by regular patterns of monomer-monomer interactions, such as Watson-Crick base pairing, base stacking, Hoogsteen or reverse Hoogsteen base pairing, etc. Such monomers and their internucleoside linkages can be naturally occurring or can be analogs thereof, such as naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs can include PNA, phosphorothioate internucleoside linkages, bases that contain linking groups that allow the attachment of labels, such as fluorophores or haptens, etc. Whenever the use of an oligonucleotide or polynucleotide requires enzymatic processing, such as elongation by polymerase, ligation by ligase, etc., those skilled in the art will understand that the oligonucleotide or polynucleotide in these cases does not contain certain analogs of internucleoside linkages, sugar moieties, or bases at any or some positions. Polynucleotides typically range in size from a few monomeric units, e.g., 5-40 to several thousand monomeric units when they are usually referred to as "oligonucleotides". Whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (upper or lower case), such as "ATGCCTG", it is understood that the nucleotides are in 5'→3' order from left to right, and that, unless otherwise indicated or obvious from the context, "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, "I" denotes deoxyinosine, and "U" denotes uridine. Unless otherwise noted, the terminology and atom numbering conventions are as disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999).Polynucleotides usually contain the four natural nucleosides linked by phosphodiester bonds (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA, or their ribose counterparts for RNA), however, they can also contain non-natural nucleotide analogs, including, for example, modified bases, sugars, or internucleoside linkages. It will be apparent to one of skill in the art that if an enzyme has a requirement for a particular oligonucleotide or polynucleotide substrate for activity, such as, for example, single-stranded DNA, RNA / DNA duplexes, the selection of the appropriate composition for the oligonucleotide or polynucleotide substrate is well within the knowledge of the skilled artisan, especially with guidance from treatises such as Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) and similar references.

[0195] "Primer" refers to an oligonucleotide, either natural or synthetic, that can act as an initiation point for nucleic acid synthesis when it forms a duplex with a polynucleotide template and can be extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually performed with a nucleic acid polymerase, e.g., a DNA polymerase or an RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Typically, a primer is extended by a DNA polymerase. A primer usually has a length in the range of 14-40 nucleotides, or in the range of 18-36 nucleotides. Primers are used in various types of nucleic amplification reactions, e.g., linear amplification reactions using a single primer, or polymerase chain reactions using two or more primers. Guidance for selecting primer length and sequence for a particular application is well known to those of skill in the art, as evidenced by the following reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003), which is incorporated herein by reference.

[0196] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "subsequently," "before," "after," "lastly," and "finally," is not meant to limit the scope of the embodiments disclosed herein, but is by way of example only.

[0197] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0198] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that function both conjunctively and disjunctively. For example, each of the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0199] As used herein, "or" may refer to "and," "or," or "and / or," and may be used both exclusively and inclusively. For example, the term "A or B" may refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0200] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0201] The term "about," when referring to a number or numerical range, means that the number or numerical range referred to is approximate within experimental variation (or within statistical experimental error), and that the number or numerical range may vary, for example, by 1% to 15% of the stated number or numerical range. In an example, the term "about" refers to ±10% of the stated number or value.

[0202] The terms "increased", "increasing" or "increase" are used herein generally to mean an increase of a statically significant amount. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a reference level, for example, an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% increase, or any increase between 10-100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0203] The terms "decreased", "decreasing" or "decrease" are used herein to generally mean a statistically significant amount of decrease. In some embodiments, "decreased" or "decrease" means at least a 10% decrease compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% decrease (e.g., nonexistent or undetectable levels compared to a reference level), including a 100% decrease compared to a reference level, or any decrease between 10-100%. In the context of a marker or symptom, these terms mean a statistically significant decrease in such levels. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, preferably to a level accepted as within the normal range for an individual without a given disease.

[0204] A DNA:RNA duplex may refer to the complex that is formed when RNA is captured and complementary DNA (cDNA) is reverse transcribed from the captured RNA. In some cases, the RNA of the DNA:RNA duplex can be denatured or washed away, and the cDNA can be subjected to a second strand synthesis reaction to create double-stranded DNA (dsDNA).

[0205] A transposon or transposase can refer to a small sequence of nucleotides that has the ability to move (translocate) from place to place almost arbitrarily.

[0206] The term "sample" as used herein generally refers to a chemical sample or biological sample that contains biological components. The biological components may include cells, nucleic acids, microbiomes, proteins, combinations of cells, metabolites, combinations thereof, or any other suitable components of a biological sample. For example, a sample may be a biological sample that includes one or more cells. For another example, a sample may be a biological sample that includes one or more nucleic acids. A biological sample may be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal discharge, sputum, feces, and tears. A biological sample may be a fluid or tissue sample (e.g., a skin sample). In some instances, a sample may be derived from a homogenized tissue sample (e.g., a brain homogenate, a liver homogenate, or a kidney homogenate). In certain embodiments, a sample may include a particular type of cell (e.g., a nerve cell, a muscle cell, a liver cell, or a kidney cell). The sample may include or be obtained from diseased cells or tissues (e.g., tumor cells or necrotic cells). In some embodiments, the sample may include or be derived from disease-related inclusions (e.g., plaque, biofilm, tumor, or non-cancerous mass). In certain embodiments, the sample may include or be obtained from acellular bodily fluids, such as whole blood, saliva, or urine. In various embodiments, the sample may include circulating tumor cells. In some cases, the sample may include or be an environmental sample (e.g., soil, waste, or ambient air), an industrial sample (e.g., a sample from any industrial process), or a food sample (e.g., dairy, vegetable, or meat products). The sample may have been processed prior to loading into the microfluidic device. For example, the sample may have been processed to purify certain cell types or nucleic acids and / or to include reagents.

[0207] As used herein, "tagging fragmentation" refers to modification of DNA by a transposome complex that includes a transposase enzyme complexed with an adaptor that includes a transposon end sequence. Tagging fragmentation results in simultaneous DNA fragmentation and adaptor ligation to the 5'-end of both strands of the double-stranded fragment. After a purification step to remove the transposase enzyme, additional sequences can be added to the ends of the adapted fragments, for example, by PCR, ligation, or any other suitable methodology. A "transposome" is composed of at least a transposase enzyme and a transposase recognition site. In some such systems, referred to as "transposomes," the transposase can form a functional complex with the transposon recognition site that can catalyze a transposition reaction. A transposase or integrase can bind to the transposase recognition site and insert the transposase recognition site into a target nucleic acid in a process that may be referred to as "tagging fragmentation." In some such insertion events, one strand of the transposase recognition site can be transferred to the target nucleic acid. In standard sample preparation methods, each template contains an adaptor at either end of the insert, and often several steps are required to modify both the DNA or RNA and purify the desired products of the modification reaction. These steps are performed in solution prior to addition of the adapted fragments to a flow cell where they are bound to a surface by a primer extension reaction that copies the hybridized fragments onto the ends of primers covalently attached to the surface.

[0208] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and explanation of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions shown herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is therefore contemplated that the present invention should encompass any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A method for preparing a set of complementary deoxyribonucleic acid (cDNA) molecules or derivatives thereof from one or more nucleic acid molecules, comprising: contacting one or more nucleic acid molecule capture probes on a solid support with the one or more nucleic acid molecules to generate one or more captured nucleic acid molecules; synthesizing cDNA molecules from the captured nucleic acid molecules or derivatives thereof and inserting an adapter into the 3' region of the cDNA molecules or derivatives thereof, wherein the cDNA molecules are bound to surface primer probes of the plurality of surface primer probes; amplifying at least a portion of the cDNA molecules or derivatives thereof to generate the set of cDNA molecules or derivatives thereof, wherein the set of cDNA molecules or derivatives thereof is bound to surface primer probes of the plurality of surface primer probes; A method comprising the steps above.

2. The method according to claim 1, wherein the adapter comprises a sequence configured to enable initiation of a sequencing reaction in the cDNA molecules of the set of cDNA molecules or derivatives thereof.

3. The method according to claim 1, further comprising, after the contacting step, inactivating at least a subset of the one or more nucleic acid molecule capture probes, wherein the subset of the one or more nucleic acid molecule capture probes comprises one or more nucleic acid molecule capture probes that did not capture nucleic acid molecules.

4. The method according to claim 3, wherein the inactivating step comprises contacting the one or more nucleic acid molecule capture probes or the subset of the one or more nucleic acid molecule capture probes with an exonuclease.

5. The method according to claim 1, wherein the synthesizing step comprises performing one or more second strand synthesis reactions comprising the cDNA molecules or derivatives thereof. **Claim 6**: The method according to claim 1, further comprising, prior to the step of inserting, a step of amplifying the cDNA molecule or its derivative. **Claim 7**: The method according to claim 1, further comprising, prior to the step of inserting, fragmentation of the cDNA molecule or its derivative. **Claim 8**: The method according to claim 1, wherein the step of inserting the adapter into the 3'-region of the cDNA molecule or its derivative comprises one of single-strand ligation, tagged fragmentation, and double-strand ligation. **Claim 9** At least a subset of the plurality of surface primer probes comprises a blocking agent that blocks the extension reaction in the at least subset of the plurality of surface primer probes, and the method further comprises, prior to the step of inserting, subjecting the blocking agent to a reaction that unblocks the at least subset of the plurality of surface primer probes or the subset of the plurality of surface primer probes to enable the extension reaction. The method according to claim 1. **Claim 10**: The method according to claim 1, further comprising, after the step of amplifying, a step of cleaving or linearizing at least a subset of the set of cDNA molecules or their derivatives. **Claim 11**: The method according to claim 1, wherein after the step of amplifying, the 3'-end of the subset of the set of DNA molecules or their derivatives is blocked. **Claim 12** The method according to claim 1, further comprising a step of sequencing at least a subset of the cDNA molecule or its derivative in situ on the solid support. **Claim 13** The method according to claim 1, further comprising a step of eluting at least a subset of the set of cDNA molecules or their derivatives from the solid support. **Claim 14**: The method according to claim 1, wherein the sequence of one nucleic acid molecule capture probe among the one or more nucleic acid molecule capture probes is configured to bind to the nucleic acid molecule among the one or more nucleic acid molecules, and the sequence of the nucleic acid molecule capture probe includes a poly-T sequence, a randomer, a sequence complementary to at least a subset of the one or more nucleic acid molecules, or any combination thereof. **Claim 15** The method according to claim 1, wherein the solid support is the surface of a fluid channel. **Claim 16** The method according to claim 15, wherein the fluid channel is a flow cell. **Claim 17** The method according to claim 1, wherein the solid support is not beads. **Claim 18** The method according to claim 1, wherein the one or more nucleic acid molecule capture probes include one or more tags, and one of the one or more tags includes a cell-specific or spatially location-specific identifier sequence and, optionally, a unique molecular identifier (UMI) sequence. **Claim 19** The method according to claim 1, wherein the amplifying step includes solid support amplification. **Claim 20** The method according to claim 1, wherein the one or more nucleic acid molecules are derived from a single cell or biological tissue. **Claim 21**: The method according to claim 1, wherein the solid support includes a hydrogel chamber disposed thereon, and the hydrogel chamber includes one or more polymer matrix walls. **Claim 22**: The method according to claim 21, wherein the one or more polymer matrix walls extend from the solid support to the upper surface on the opposite side of the solid support, thereby forming the interior of the hydrogel chamber. **Claim 23**: The method according to claim 1, wherein the inserting step is after the cDNA molecule is bound to the surface primer probe.

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