WO2025264702A1

WO2025264702A1 - 3d hydrogels for nucleic acid sequencing

Info

Publication number: WO2025264702A1
Application number: PCT/US2025/034017
Authority: WO
Inventors: Alexander GORYAYNOV; Hongbo Feng; Jeremy Lackey; David Hoffman; Patrick J. Marks; Benjamin PRUITT
Original assignee: 10X Genomics Inc
Current assignee: 10X Genomics Inc
Priority date: 2024-06-18
Filing date: 2025-06-17
Publication date: 2025-12-26
Anticipated expiration: 2026-12-18

Abstract

The present disclosure relates in some aspects to methods and compositions for sequencing nucleic acids in a 3D hydrogel. In some aspects, the methods comprise generating 3D hydrogels comprising immobilized RCPs that are generated from isolated nucleic acids, and sequencing the RCPs to determine the sequences of the isolated nucleic acids. In some embodiments, the isolated nucleic acids comprise sequencing libraries.

Description

3D HYDROGELS FOR NUCLEIC ACID SEQUENCING

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63/661,499, filed on June 18, 2024, entitled “3D HYDROGELS FOR NUCLEIC ACID SEQUENCING”, which is herein incorporated by reference in its entirety for all purposes.

FIELD

[0002] The present disclosure relates in some aspects to methods for sequencing amplification products generated from isolated nucleic acid molecules that are immobilized and/or processed in a 3-dimensional (3D) hydrogel.

BACKGROUND

[0003] Next-generation sequencing methods allow researchers to rapidly determine the sequences of vast numbers of nucleic acids, providing valuable insight into biological systems and disease. Improved methods of high-throughput nucleic acid sequencing approaches are needed. Provided herein are methods and compositions that address these and other needs.

SUMMARY

[0004] In some aspects, provided herein are methods and compositions for nucleic acid sequencing, in particular for sequencing isolated nucleic acids using a 3-dimensional (3D) hydrogel. In some aspects, the isolated nucleic acids are used as templates in a rolling circle amplification (RCA) reaction to generate rolling circle amplification products (RCPs) in the 3D hydrogel. In some embodiments, the nucleic acids are hybridized to capture probes that are immobilized in the 3D hydrogel, and the capture probes are extended in the rolling circle amplification reaction to generate the RCPs. In some embodiments, the RCPs are sequenced in the 3D hydrogel in at least a first RCP layer and second RCP layer of the 3D hydrogel. In some aspects, sequencing of RCPs arranged within a 3D hydrogel allows for an increased sequencing capacity, for example in comparison to RCPs arranged on a 2-dimensional (2D) substrate. In some embodiments, the RCPs are distributed at defined densities and/or patterns (e.g. layers) within the 3D hydrogel, which are beneficial for the quality and efficiency of sequencing.

[0005] In some aspects, provided herein is a method, comprising: providing a 3- dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and sequencing RCP molecules of the library of RCPs that are immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel. In some embodiments, the method comprises generating the library of RCPs. In some embodiments, the method comprises immobilizing the RCP molecules of the library of RCPs in the first RCP layer and the second RCP layer.

[0006] In some embodiments, the method comprises: distributing the library of isolated nucleic acids within the 3D hydrogel, wherein the 3D hydrogel comprises capture probes that are immobilized in the 3D hydrogel; allowing nucleic acid molecules of the library of isolated nucleic acids to hybridize to the immobilized capture probes; and performing a rolling circle amplification (RCA) reaction to extend the immobilized capture probes using as template the nucleic acid molecules hybridized to the immobilized capture probes, thereby generating the library of RCPs immobilized in the 3D hydrogel. In some embodiments, the capture probes are immobilized in the first RCP layer and the second RCP layer. In some embodiments, the library of isolated nucleic acids comprises circular or circularizable nucleic acids. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circular or circularizable. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circularized prior to the RCA reaction. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circularized by ligation using the capture probes as template. In some embodiments, gap filling using the capture probes as template is performed prior to the ligation. In some embodiments, the gap filling comprises extending 3’ ends of the nucleic acid molecules hybridized to the capture probes in a nucleic acid extension reaction. In some embodiments, gap filling using the capture probes as template is not performed prior to the ligation. In some embodiments, the capture probes comprise a capture sequence, nucleic acids of the library of isolated nucleic acids comprise a target region, and the capture sequence hybridizes to the target region. In some embodiments, the target region is a contiguous sequence. In some embodiments, the target region is not a contiguous sequence. In some embodiments, the target region is the same among the nucleic acids of the library of isolated nucleic acids. In some embodiments, subsets of the nucleic acids of the library of isolated nucleic acids comprise different subset-specific target regions. In some embodiments, a target region of a given nucleic acid of the library of isolated nucleic acids comprises a first target sequence at a 3’ end of the given nucleic acid and a second target sequence at a 5’ end of the given nucleic acid. In some embodiments, the capture sequence is the same among the capture probes. In some embodiments, subsets of the capture probes comprise different subset-specific capture sequences. In some embodiments, the different subset-specific capture sequences hybridize to the different subset-specific target regions. In some embodiments, the capture sequence is a contiguous capture sequence. In some embodiments, the capture sequence is a non-contiguous capture sequence. In some embodiments, the non-contiguous capture sequence comprises a first portion and a second portion, and the first portion and the second portion of the capture sequence are separated by an intervening sequence. In some embodiments, the first portion is at a 3’ end of the capture probe. In some embodiments, a capture sequence of a given capture probe comprises a first portion at the 3’ end of the capture probe and a second portion that is not contiguous with the first portion, and the first and second portion are separated by an intervening sequence. In some embodiments, a first subset of capture probes comprises a first intervening sequence and a second subset of capture probes comprises a second intervening sequence; the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are circularized using either the first intervening sequence or the second intervening sequence of the capture probes as templates; and the RCA generates first RCPs comprising multiple copies of the first intervening sequence and second RCPs comprising multiple copies of the second intervening sequence. In some embodiments, the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are extended in a gap-filling reaction using either the first intervening sequence or the second intervening sequence of the capture probes as templates prior to ligation. In some embodiments, the nucleic acids of the library of isolated nucleic acids comprise the same noncontiguous target region. In some embodiments, the first and second intervening sequences serve as sequencing primer binding sites for sequencing the first and second RCPs, respectively. In some embodiments, the first RCPs are sequenced using a first sequencing primer comprising a sequence complementary to the first intervening sequence. In some embodiments, the second RCPs are sequenced using a second sequencing primer comprising a sequence complementary to the second intervening sequence. In some embodiments, the first and second RCPs are sequenced in parallel and/or simultaneously. In some embodiments, the method comprises sequencing the first and second RCPs simultaneously. In some embodiments, the method comprises sequencing the first and second RCPs in the same sequencing reactions. In some embodiments, the first and second RCPs are not sequenced simultaneously. In some embodiments, the first RCPs are sequenced in first sequencing reactions and the second RCPs are sequenced in second sequencing reactions which occur after the first sequencing reactions and/or do not occur simultaneously with the first sequencing reactions. In some embodiments, the first sequencing reactions comprise using the first sequencing primer to sequence one or more nucleotides of the first RCPs and the second sequencing reactions comprise using the second sequencing primer to sequence one or more nucleotides of the second RCPs. In some embodiments, nucleic acids of the library of isolated nucleic acids that do not hybridize to the immobilized capture probes are removed from the hydrogel and/or are not amplified in the RCA reaction. In some embodiments, nucleic acids of the library of isolated nucleic acids that do not hybridize to the immobilized capture probes are not sequenced.

[0007] In some embodiments, the capture probes and/or the RCP molecules of the library of RCPs are immobilized in the first RCP layer and second RCP layer. In some embodiments, the first RCP layer and second RCP layer are separated by a spacer layer. In some embodiments, the spacer layer is positioned between the first and second RCP layers. In some embodiments, the spacer layer does not comprise immobilized capture probes or immobilized RCPs. In some embodiments, the spacer layer is substantially free of immobilized capture probes and/or immobilized RCPs. In some embodiments, the spacer layer comprises a lower concentration of RCPs than the RCP layer. In some embodiments, the concentration of RCPs in the RCP layer is at least 2-fold, at least 5-fold, at least 10-fold, or at least 100-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the first RCP layer, second RCP layer, and/or spacer layer are flat, planar, and/or 2-dimensional. In some embodiments, an upper boundary and lower boundary of any of the first RCP layer, second RCP layer, and/or spacer layer are planar. In some embodiments, an upper boundary and lower boundary of any of the first RCP layer, second RCP layer, and/or spacer layer are 2-dimensional. In some embodiments, the first RCP layer and second RCP layer do not overlap. In some embodiments, the 3D hydrogel further comprises a third RCP layer. In some embodiments, the spacer layer is a first spacer layer, and the 3D hydrogel further comprises a second spacer layer positioned between the second RCP layer and the third RCP layer. In some embodiments, the first spacer layer and second spacer layer are positioned on opposite sides of the second RCP layer. In some embodiments, the 3D hydrogel further comprises a fourth RCP layer. In some embodiments, the 3D hydrogel further comprises a third spacer layer positioned between the third RCP layer and the fourth RCP layer. In some embodiments, the third spacer layer and second spacer layer are positioned on opposite sides of the third RCP layer. In some embodiments, the 3D hydrogel further comprises one or more further RCP layers. In some embodiments, the 3D hydrogel comprises one or more further spacer layers positioned between the one or more further RCP layers.

[0008] In some embodiments, the method comprises arranging the capture probes and/or the RCP molecules within the 3D hydrogel to generate the RCP layers and/or spacer layers. In some embodiments, the arranging comprises applying an electrical current to the 3D hydrogel. In some embodiments, the electrical current comprises a direct current and/or an alternating current. In some embodiments, the capture probes and/or the RCP molecules are immobilized after the arranging. In some embodiments, the 3D hydrogel is provided on a solid support. In some embodiments, the solid support comprises a substantially flat, horizontal, and/or 2-dimensional surface. In some embodiments, the solid support comprises a slide. In some embodiments, the immobilized capture probes and/or RCPs are uniformly distributed throughout the 3D hydrogel. In some embodiments, the immobilized capture probes and/or RCPs are randomly distributed throughout the 3D hydrogel. In some embodiments, the immobilized capture probes and/or RCPs are distributed at a defined density or density range within the first RCP layer and second RCP layer and/or throughout the 3D hydrogel. In some embodiments, the defined density or density range comprises at or about, or comprises at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the defined density is at or about, or is at least 0.02, 0.07, or 0.1 RCPs per cubic micron. In some embodiments, the defined density or density range comprises at or about, or comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 immobilized capture probes and/or RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of equal to or greater than 0.005, 0.01, 0.05, or 0.1 cubic millimeters. In some embodiments, the method comprises sequencing at least or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within the first RCP layer and second RCP layer. In some embodiments, the method comprises sequencing at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron within the first RCP layer and second RCP layer. In some embodiments, the method comprises sequencing at least or at or about 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron of one or more areas of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 1, 2, 5, or 10 RCPs per square micron of one or more areas of the 3D hydrogel. In some embodiments, the one or more areas of the 3D hydrogel comprise a contiguous area of at least or at or about 0.1 square millimeters of the 3D hydrogel or at least or at or about 1 square millimeter of the 3D hydrogel. In some embodiments, the 3D hydrogel is at least or at or about 5, 10, 50, 100, 200, 300, 400, or 500 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 50 microns thick. In some embodiments, the 3D hydrogel has an area of at least or at or about 1, 5, 10, 50, 100, 200, 300, 400, 500, or 1000 square millimeters. In some embodiments, the 3D hydrogel has an area of at least or at or about 500 square millimeters. In some embodiments, the method comprises sequencing at least or at or about lxlO^A6, lxlO^A7, lxl0^A8, lxlO^A9, or lxl0^A10 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about lxlO^A9 RCPs in the 3D hydrogel. In some embodiments, sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 10. In some embodiments, sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 20. In some embodiments, sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence is determined that has a minimum phred- scaled quality value (q-score) of at least 10, at least 20, or at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred-scaled quality value (q-score) of at least 10, at least 20, or at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred-scaled quality value (q-score) of at least 20.

[0009] In some embodiments, sequencing the RCP molecules comprises sequencing one or more nucleotides of the RCP molecules. In some embodiments, sequencing the RCP molecules comprises sequencing the entire RCP molecules or portions thereof. In some embodiments, sequencing the one or more nucleotides comprises detecting one or more signals in the 3D hydrogel corresponding to the one or more nucleotides. In some embodiments, the one or more signals corresponding to the one or more nucleotides are detected in one or more sequential imaging cycles. In some embodiments, the method further comprises analyzing the one or more signals corresponding to the one or more nucleotides to determine sequences of one or more RCPs in the 3D hydrogel. In some embodiments, sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding. In some embodiments, sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis.

[0010] In some embodiments, sequencing RCP molecules of the library of RCPs comprises imaging the 3D hydrogel. In some embodiments, the imaging comprises fluorescence microscopy. In some embodiments, the fluorescence microscopy comprises epifluorescence imaging. In some embodiments, the fluorescence microscopy comprises wide-field epifluorescence imaging. In some embodiments, imaging the 3D hydrogel comprises imaging a first focal plane of the 3D hydrogel and imaging a second focal plane of the 3D hydrogel. In some embodiments, the first focal plane coincides with the first RCP layer and the second focal plane coincides with the second RCP layer. In some embodiments, imaging the 3D hydrogel comprises imaging a plurality of focal planes coinciding with a plurality of RCP layers in the 3D hydrogel. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 1.0. In some embodiments, the imaging is performed using an objective having a field of view (FOV) having a diagonal of equal to or greater than 0.5 millimeters. In some embodiments, the imaging is performed using an objective having a field of view (FOV) of equal to or greater than 1.0 millimeters. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9 and a field of view having a diagonal of equal to or greater than 0.5 millimeters. In some embodiments, imaging the 3D hydrogel comprises optically sectioning the hydrogel to image a first optical section of the first RCP layer and a second optical section of the second RCP layer. In some embodiments, sequencing RCP molecules of the library of RCPs comprises optically sectioning the hydrogel to image a first optical section of the first RCP layer and a second optical section of the second RCP layer.

[0011] In some embodiments, the library of RCPs comprises a sequencing library. In some embodiments, the library of RCPs comprises a single-cell sequencing library. In some embodiments, the library of RCPs comprises an RNA sequencing library. In some embodiments, the library of RCPs comprises a DNA sequencing library. In some embodiments, the library of RCPs comprises a single-cell gene expression sequencing library.

[0012] In some aspects, provided herein is a system, comprising: a 3-dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and an imaging system for imaging the 3D hydrogel. In some embodiments, the first RCP layer and second RCP layer are separated by a spacer layer In some embodiments, the library of RCPs comprises a sequencing library. In some embodiments, the sequencing library is a single-cell sequencing library. In some embodiments, the imaging system is configured to image a first focal plane coinciding with the first RCP layer and a second focal plane coinciding with to the second RCP layer. In some embodiments, the imaging system is configured to image the 3D hydrogel by wide-field epifluorescence microscopy. In some embodiments, the system further comprises one or more reagents for sequencing the library of RCPs in the 3D hydrogel. In some embodiments, the system is configured for sequencing the library of RCPs. In some embodiments, the system is configured for sequencing the library of RCPs by sequencing by synthesis, sequencing by ligation, or sequencing by binding. In some embodiments, the system comprises one or more computers. In some embodiments, the one or more computers are configured to determine and/or analyze one or more sequences present in the library of RCPs.

[0013] In some aspects, provided herein is a kit, comprising: a 3-dimensional (3D) hydrogel comprising capture oligonucleotides immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the first RCP layer and second RCP layer are separated by a spacer layer; wherein the capture oligonucleotides comprise a capture sequence that is complementary to a target region present in circular or circularizable isolated nucleic acids of a sequencing library. In some embodiments, the capture oligonucleotides are configured to hybridize to the circular or circularizable isolated nucleic acids of the sequencing library via hybridization between the capture sequence and the target region. In some embodiments, the circular or circularizable isolated nucleic acids of the sequencing library are circular, and the capture oligonucleotides are configured to be extended in an RCA reaction using the circular isolated nucleic acids as template to generate RCPs immobilized in the first RCP layer and second RCP layer. In some embodiments, the circular or circularizable isolated nucleic acids of the sequencing library are circularizable, wherein the capture oligonucleotides are configured to serve as ligation templates for the circularizable isolated nucleic acids to generate circularized isolated nucleic acids, and wherein the capture oligonucleotides are configured to be extended in an RCA reaction using the circularized isolated nucleic acids as template to generate RCPs immobilized in the first RCP layer and second RCP layer. In some embodiments, the kit further comprises the sequencing library. In some embodiments, the sequencing library is a single-cell sequencing library. In some embodiments, the kit further comprises one or more reagents for sequencing the RCPs immobilized in the 3D hydrogel. In some embodiments, the sequencing comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding.

[0014] In some aspects, provided herein is a composition, comprising: a 3- dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the first RCP layer and second RCP layer are separated by a spacer layer, and wherein the library of RCPs is generated from a library of isolated nucleic acids. In some embodiments, the library of isolated nucleic acids and/or the library of RCPs comprises a single-cell sequencing library. In some embodiments, the composition further comprises a sequencing primer that is hybridized to the RCPs immobilized in the first RCP layer and to the RCPs immobilized in the second RCP layer. In some embodiments, the composition further comprises a modified nucleotide configured to be incorporated into the 3’ end of the sequencing primer and detected. In some embodiments, the modified nucleotide comprises a reversible terminator and/or a fluorescent moiety. In some embodiments, the composition further comprises a polymerase capable of extending the sequencing primer with the modified nucleotide. [0015] In some aspects, provided herein is a method, comprising: providing a 3- dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and sequencing RCP molecules of the library of RCPs that are immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel. In some aspects, provided herein is a method for nucleic acid sequencing comprising: providing a 3- dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and sequencing RCP molecules of the library of RCPs that are immobilized in at least a first layer and a second layer of the 3D hydrogel; thereby determining sequences of molecules of the library of isolated nucleic acids. In some embodiments, providing the library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel comprises: stochastically distributing the library of isolated nucleic acids within the 3D hydrogel, wherein the 3D hydrogel comprises immobilized capture probes; allowing nucleic acid molecules of the library of isolated nucleic acids to hybridize to the immobilized capture probes; and performing a rolling circle amplification (RCA) reaction to extend the immobilized capture probes using as template the nucleic acid molecules hybridized to the immobilized capture probes, thereby generating the library of RCPs that is immobilized in the 3D hydrogel.

[0016] In some embodiments, the library of isolated nucleic acids comprises circular or circularizable nucleic acids. In some embodiments, the nucleic acid molecules hybridized to the immobilized capture probes are circular or circularizable. In some embodiments, the nucleic acid molecules hybridized to the immobilized capture probes are circularized prior to the RCA reaction. In some embodiments, the nucleic acid molecules hybridized to the immobilized capture probes are circularized using the immobilized capture probes as template. In some embodiments, gap filling using the capture probes as template is performed prior to ligation. In some embodiments, gap filling using the capture probes as template is not performed prior to ligation. In some embodiments, the immobilized capture probes comprise a capture sequence, the nucleic acids of the library of nucleic acids comprise a target region, and the capture sequence hybridizes to the target region. In some embodiments, the target region is a contiguous sequence. In some embodiments, the target region is not a contiguous sequence. In some embodiments, the target region is the same among all nucleic acids of the library of isolated nucleic acids. In some embodiments, subsets of the nucleic acids of the library of isolated nucleic acids comprise different subset-specific target regions. In some embodiments, a target region of a given nucleic acid of the library of isolated nucleic acids comprises a first target sequence at a 3’ end of the given nucleic acid and a second target sequence at a 5’ end of the given nucleic acid. In some embodiments, the capture sequence is the same among all immobilized capture probes. In some embodiments, subsets of the immobilized capture probes comprise different subset-specific capture sequences. In some embodiments, the capture sequence is a contiguous sequence. In some embodiments, the capture sequence is not a contiguous sequence. In some embodiments, the non-contiguous capture sequence comprises a first portion and second portion, and the first portion and second portion of the capture sequence are separated by an intervening sequence. In some embodiments, a capture sequence of a given capture probe comprises a first portion at the 3’ end of the capture probe and a second portion that is not contiguous with the first portion, and wherein the first and second portion are separated by an intervening sequence. In some embodiments, a first subset of immobilized capture probes comprises a first intervening sequence and a second subset of immobilized capture probes comprises a second intervening sequence; the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are circularized using as template either the first intervening sequence or second intervening sequence of the capture probes; and the RCA generates first RCPs comprising multiple copies of the complement of the first intervening sequence and second RCPs comprising multiple copies of the complement of the second intervening sequence. In some embodiments, the nucleic acids of the library of isolated nucleic acids comprise the same noncontiguous target region. In some embodiments, the first and second intervening sequences serve as sequencing primers for sequencing the first and second RCPs, respectively. In some embodiments, the first and second RCPs are sequenced in parallel. In some embodiments, the first and second RCPs are sequenced separately and/or not in parallel. In some embodiments, nucleic acids of the library of nucleic acids that do not hybridize to the immobilized capture probes are removed from the hydrogel and/or are not amplified in the RCA reaction.

[0017] In some embodiments, the immobilized capture probes and/or the RCPs are immobilized in the first layer and second layer. In some embodiments, the first layer and second layer are separated by a spacer layer. In some embodiments, the spacer layer does not comprise immobilized capture probes or immobilized RCPs. In some embodiments, the spacer layer is substantially free of immobilized capture probes and immobilized RCPs. In some embodiments, the first layer, second layer, and/or spacer layer are flat, planar, and/or 2-dimensional. In some embodiments, an upper boundary and lower boundary of the first layer, second layer, and/or spacer layer are substantially planar. In some embodiments, an upper boundary and lower boundary of the first layer, second layer, and/or spacer layer are substantially 2-dimensional. In some embodiments, the first layer and second layer do not overlap. In some embodiments, the 3D hydrogel is provided on a solid support. In some embodiments, the 3D hydrogel is provided on a substantially flat, horizontal, and/or 2-dimensional surface. In some embodiments, the 3D hydrogel is provided on a slide. In some embodiments, the immobilized capture probes and/or RCPs are uniformly distributed throughout the 3D hydrogel.

[0018] In some embodiments, the immobilized capture probes and/or RCPs are distributed at a defined density or density range within the first layer and second layer and/or throughout the hydrogel. In some embodiments, the defined density or density range is at least or at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 capture probes and/or RCPs per cubic micron. In some embodiments, the density of immobilized capture probes and/or RCPs is at least 0.5 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the method comprises sequencing at least or at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 0.5 RCPs per cubic micron of the 3D hydrogel. In some embodiments, the defined density or density range of immobilized capture probes and/or RCPs is at least or at or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 immobilized capture probes and/or RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron of the 3D hydrogel.

[0019] In some embodiments, sequencing RCP molecules of the library of RCPs comprises performing sequencing reactions in the 3D hydrogel. In some embodiments, sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding. In some embodiments, sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis. In some embodiments, sequencing RCP molecules of the library of RCPs comprises imaging the 3D hydrogel. In some embodiments, imaging the 3D hydrogel comprises fluorescence microscopy. In some embodiments, the fluorescence microscopy comprises wide-field epifluorescence imaging. In some embodiments, sequencing RCP molecules of the library of RCPs comprises optically sectioning the hydrogel to image a first optical section of the first layer and a second optical section of the second layer. In some embodiments, sequencing RCP molecules of the library of RCPs comprises performing iterative rounds of sequencing reactions and imaging. In some embodiments, sequencing RCP molecules of the library of RCPs comprises performing iterative rounds of fluorescently labeled nucleotide incorporation and imaging.

[0020] In some aspects, provided herein is a composition or kit comprising any 3D hydrogel described herein or produced by any of the methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings illustrate certain features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

[0022] FIG. 1 shows a schematic illustrating an exemplary 3D hydrogel comprising immobilized capture probes and generation of RCPs.

[0023] FIG. 2 shows a schematic illustrating a cross-section of exemplary RCP layers and spacer layers in a 3D hydrogel.

[0024] FIGS. 3A-3C show schematics illustrating exemplary capture probe / isolated nucleic acid configurations and corresponding exemplary downstream workflows for regenerating RCPs.

[0025] FIG. 4 shows a plot illustrating an exemplary relationship between RCP density (x-axis) and sequencing output (y-axis).

[0026] FIG. 5 shows schematics illustrating exemplary RCP images resulting from an optical section or focal plane taken of RCPs that are arranged in a layer (left) or randomly distributed (right) in a 3D hydrogel.

[0027] FIG. 6 shows representative images from 10 imaging cycles from sequencing- by-synthesis of a single RCP species immobilized in a 3D hydrogel.

[0028] FIG. 7 shows representative images from 10 imaging cycles from sequencing- by-synthesis of multiple different RCP species immobilized in a 3D hydrogel. [0029] FIG. 8 shows representative images of RCPs in individual layers of a 3D hydrogel imaged at different focal planes.

[0030] FIG. 9 shows a representative cross-section view of 3 different RCPs decoded in a first and second layer of a 3D hydrogel.

[0031] FIG. 10 shows a plot of the 3D locations of different RCPs sequenced within a 3D hydrogel volume.

[0032] FIGS. 11A-11C show results of RCP sequencing in 3D hydrogels with varying concentrations of RCPs. FIG. 11A shows the proportion of sequenced RCPs having a q- score of greater than or equal to 20. FIG. 11B shows sequenced RCPs having a q-score of greater than or equal to 30 per 100 square micron of a 3D hydrogel. FIG. 11C shows the density of detected RCPs at different quality thresholds.

[0033] FIG. 12 shows images of individual focal planes from a single fluorescent channel in 3D hydrogels having different RCP input concentrations.

[0034] FIGS. 13A-13B show schematics illustrating exemplary methods for arranging RCPs in a 3D hydrogel using electrical currents. FIG. 13A illustrates an exemplary method for arranging RCPs in a 3D hydrogel using a direct current (DC). FIG. 13B illustrates an exemplary method for arranging RCPs in a 3D hydrogel using an alternating current (AC).

[0035] FIG. 14 shows a schematic illustrating an exemplary workflow for generating a molecule of a gene expression sequencing library as described herein.

DETAILED DESCRIPTION

[0036] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0037] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Overview

[0038] The ability to sequence vast numbers of isolated nucleic acids using nextgeneration sequencing methods is an essential tool that allows researchers to gain insight into biological systems and disease states. New methods for sequencing nucleic acid libraries can provide opportunities for new insights, improved workflows, and increased sequencing throughput.

[0039] Various nucleic acid sequencing methods involve amplification of isolated nucleic acids which are arranged on a substrate for iterative rounds of sequencing reactions and fluorescent imaging (e.g. methods comprising sequencing by synthesis). In some aspects the flexibility and capacity of such methods can be limited by the 2-dimensional nature of the substrate on which nucleic acids are arranged and sequenced. Methods are needed to increase the flexibility, efficiency, and capacity of nucleic acid sequencing.

[0040] In some aspects, provided herein are methods and compositions for nucleic acid sequencing, in particular for sequencing isolated nucleic acids using a 3-dimensional (3D) hydrogel. In some aspects, the isolated nucleic acids are used as templates in a rolling circle amplification (RCA) reaction to generate rolling circle amplification products (RCPs) in the 3D hydrogel. In some embodiments, the isolated nucleic acids are hybridized to capture probes that are immobilized in the 3D hydrogel, and the capture probes are extended in the rolling circle amplification reaction to generate the RCPs. In some embodiments, the RCPs are sequenced in the 3D hydrogel in at least a first layer (also referred to herein as a first RCP layer) and second layer (also referred to herein as a second RCP layer) of the 3D hydrogel. In some aspects, sequencing of RCPs arranged in a 3D hydrogel allows for an increased sequencing capacity, for example in comparison to RCPs arranged on a 2-dimensional (2D) substrate. In some embodiments, the RCPs are distributed at defined densities and/or in specific patterns (e.g. layers) within the 3D hydrogel, which are beneficial for the quality and efficiency of sequencing.

[0041] The methods provided herein provide several advantages in comparison to certain other compositions and methods for nucleic acid sequencing. For example, by providing RCPs in a 3D hydrogel, the sequencing capacity of the workflow can be increased in comparison to methods in which RCPs are arranged on a 2-dimensional surface. In addition, by providing RCPs arranged in defined densities and patterns (e.g. layers) within the 3D hydrogel, improvements in the efficiency and quality of RCP imaging and/or sequencing can be achieved. For example, a particular 3D density of RCPs can maximize sequencing output while avoiding optical crowding during imaging steps. A particular pattern of RCPs, such as RCPs arranged in 2D layers within the hydrogel, can increase the quality of images captured from the RCPs (e.g. in a single focal plane or optical section), promote uniformity of RCP signals, and reduce computational burden in downstream analysis steps.

[0042] In some aspects, provided herein is a method for nucleic acid sequencing comprising providing a 3-dimensional (3D) hydrogel. In some embodiments, the 3D hydrogel comprises a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel. In some embodiments, the library of RCPs is generated from a library of isolated nucleic acids. In some embodiments, the method comprises sequencing RCP molecules of the library of RCPs that are immobilized in the 3D hydrogel. In some embodiments, the method comprises sequencing RCP molecules of the library of RCPs that are immobilized in at least a first layer (e.g. first RCP layer) and a second layer (e.g. second RCP layer) of the 3D hydrogel. In some embodiments, the method comprises determining sequences of molecules of the library of isolated nucleic acids. In some embodiments, the method comprises distributing the library of isolated nucleic acids within the 3D hydrogel. In some embodiments, the method comprises stochastically distributing the library of isolated nucleic acids within the 3D hydrogel. In some aspects, the stochastically distributing comprises introducing the isolated nucleic acids into the 3D hydrogel in an arrangement that does not reflect the spatial arrangement of the isolated nucleic acids in their native context (e.g. in the sample from which they are derived). In some aspects, the distributing comprises contacting the 3D hydrogel with the isolated nucleic acids.

[0043] In some embodiments, the 3D hydrogel comprises immobilized capture probes. In some embodiments, the method comprises allowing nucleic acid molecules of the library of isolated nucleic acids to hybridize to the immobilized capture probes. In some embodiments, the method comprises performing a rolling circle amplification (RCA) reaction to extend the immobilized capture probes using as template the nucleic acid molecules hybridized to the immobilized capture probes, thereby generating the library of RCPs that is immobilized in the 3D hydrogel.

[0044] Various additional details and advantages of the methods and compositions are described in more detail in the sections herein. II. 3D Hydrogels for Nucleic Acid Sequencing and Related Methods and Compositions

[0045] In some aspects, provided herein is a method, comprising: providing a 3- dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and sequencing RCP molecules of the library of RCPs that are immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel. In some aspects, provided herein are methods, such as methods for sequencing RCPs in 3D hydrogels. In some embodiments, provided herein are methods for generating any of the 3D hydrogels provided herein. In some embodiments, provided herein is a method comprising providing a 3- dimensional (3D) hydrogel. In some embodiments, the 3D hydrogel comprises a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel. In some embodiments, the library of RCPs is generated from a library of isolated nucleic acids. For example, generating the library of RCPs from the library of isolated nucleic acids can comprise performing a rolling circle amplification (RCA) reaction using as templates nucleic acid molecules of the library of isolated nucleic acids or products thereof. In some embodiments, generating the library of RCPs from the library of isolated nucleic acids comprises using the library of isolated nucleic acids to generate the library of RCPs, for example via one or more processing steps such as any described herein, including hybridization, extension, ligation, and/or RCA. In some embodiments, the method comprises sequencing RCP molecules of the library of RCPs that are immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel. In some embodiments, the method comprises generating the library of RCPs. In some embodiments, the method comprises immobilizing the RCP molecules of the library of RCPs in the first RCP layer and the second RCP layer.

[0046] In some embodiments, the method comprises: distributing the library of isolated nucleic acids within the 3D hydrogel, wherein the 3D hydrogel comprises capture probes that are immobilized in the 3D hydrogel; allowing nucleic acid molecules of the library of isolated nucleic acids to hybridize to the immobilized capture probes; and performing a rolling circle amplification (RCA) reaction to extend the immobilized capture probes using as template the nucleic acid molecules hybridized to the immobilized capture probes, thereby generating the library of RCPs immobilized in the 3D hydrogel. In some embodiments, the method comprises distributing the library of isolated nucleic acids within the 3D hydrogel. In some embodiments, the 3D hydrogel comprises capture probes that are immobilized in the 3D hydrogel. In some embodiments, the method comprises allowing nucleic acid molecules of the library of isolated nucleic acids to hybridize to the immobilized capture probes. In some embodiments, the method comprises performing a rolling circle amplification (RCA) reaction to extend the immobilized capture probes using as template the nucleic acid molecules hybridized to the immobilized capture probes. In some embodiments, the RCA reaction generates the library of RCPs immobilized in the 3D hydrogel.

[0047] In some embodiments, the method comprises distributing the library of isolated nucleic acids within the 3D hydrogel, wherein the 3D hydrogel comprises capture probes that are immobilized in the 3D hydrogel. However, processing, distribution and immobilization of molecules can happen in any suitable alternative order for any of the methods provided herein. For example, in some embodiments, the nucleic acid molecules of the library of isolated nucleic acids are hybridized to the capture probes before the capture probes are immobilized in the 3D hydrogel, and/or before the nucleic acid molecules of the library of isolated nucleic acids are distributed within the 3D hydrogel. In some embodiments, the nucleic acid molecules of the library of isolated nucleic acids are first hybridized to the capture probes, and then the nucleic acid molecules of the library of isolated nucleic acids and the capture probes hybridized thereto are distributed and immobilized within the 3D hydrogel prior to RCA. In some embodiments, the nucleic acid molecules of the library of isolated nucleic acids are first hybridized to the capture probes, RCA is performed to generate RCPs comprising the capture probes, and then the RCPs are distributed and immobilized within the 3D hydrogel prior to sequencing. In some embodiments, one or more processing steps, such as circularization steps (e.g. gap-filling and/or ligation reactions) can also be carried out to circularize the nucleic acid molecules of the library of isolated nucleic acids prior to distribution in the hydrogel. In some embodiments, steps of any of the methods provided herein can be performed in the order in which they are described. In some embodiments, steps of any of the methods provided herein can also be performed in any other suitable order.

[0048] In some embodiments, the capture probes are immobilized in the first RCP layer and the second RCP layer. In some embodiments, the library of isolated nucleic acids comprises circular or circularizable nucleic acids. In some embodiments, the library of isolated nucleic acids comprises circular nucleic acids. In some embodiments, the library of isolated nucleic acids comprises circularizable nucleic acids. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circular or circularizable. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circular. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circularizable. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circularized prior to the RCA reaction. In some embodiments, the nucleic acid molecules hybridized to the capture probes are circularized by ligation using the capture probes as template. In some embodiments, gap filling using the capture probes as template is performed prior to the ligation. In some embodiments, the gap filling comprises extending 3’ ends of the nucleic acid molecules hybridized to the capture probes in a nucleic acid extension reaction (e.g. using a polymerase), for example according to gap-filling as described elsewhere herein. In some embodiments, gap filling using the capture probes as template is not performed prior to the ligation.

[0049] In some embodiments, the capture probes comprise a capture sequence. In some embodiments, nucleic acids of the library of isolated nucleic acids comprise a target region. In some embodiments, the capture sequence hybridizes to the target region. In some embodiments, the capture probes comprise a capture sequence, nucleic acids of the library of isolated nucleic acids comprise a target region, and the capture sequence hybridizes to the target region. In some embodiments, the target region is a contiguous sequence. In some embodiments, the target region is not a contiguous sequence. In some embodiments, the target region is the same among the nucleic acids of the library of isolated nucleic acids. For example, in some embodiments, all nucleic acids of the library of nucleic acids comprise the same target region. In some embodiments, subsets of the nucleic acids of the library of isolated nucleic acids comprise different subset-specific target regions. In some embodiments, a target region of a given nucleic acid of the library of isolated nucleic acids comprises a first target sequence at a 3’ end of the given nucleic acid and a second target sequence at a 5’ end of the given nucleic acid. In some embodiments, the capture sequence is the same among the capture probes. In some embodiments, subsets of the capture probes comprise different subset-specific capture sequences. In some embodiments, different subset-specific capture sequences hybridize to the different subset-specific target regions. In some embodiments, the capture sequence is a contiguous capture sequence. In some embodiments, the capture sequence is a non-contiguous capture sequence. In some embodiments, the non-contiguous capture sequence comprises a first portion and a second portion, and the first portion and the second portion of the capture sequence are separated by an intervening sequence. In some embodiments, the first portion is at a 3’ end of the capture probe. In some embodiments, a capture sequence of a given capture probe comprises a first portion at the 3’ end of the capture probe and a second portion that is not contiguous with the first portion. In some embodiments, the first and second portion are separated by an intervening sequence. In some embodiments, a capture sequence of a given capture probe comprises a first portion at the 3’ end of the capture probe and a second portion that is not contiguous with the first portion, and wherein the first and second portion are separated by an intervening sequence. In some embodiments, a first subset of capture probes comprises a first intervening sequence and a second subset of capture probes comprises a second intervening sequence. In some embodiments, the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are circularized using either the first intervening sequence or the second intervening sequence of the capture probes as templates. In some embodiments, the RCA generates first RCPs comprising multiple copies of the first intervening sequence and second RCPs comprising multiple copies of the second intervening sequence. In some embodiments, a first subset of capture probes comprises a first intervening sequence and a second subset of capture probes comprises a second intervening sequence; the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are circularized using either the first intervening sequence or the second intervening sequence of the capture probes as templates; and the RCA generates first RCPs comprising multiple copies of the first intervening sequence and second RCPs comprising multiple copies of the second intervening sequence. In some embodiments, the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are extended in a gap-filling reaction using either the first intervening sequence or the second intervening sequence of the capture probes as templates prior to ligation. In some embodiments, the nucleic acids of the library of isolated nucleic acids comprise the same noncontiguous target region. In some embodiments, the first and second intervening sequences serve as sequencing primer binding sites for sequencing the first and second RCPs, respectively. In some embodiments, the first RCPs are sequenced using a first sequencing primer comprising a sequence complementary to the first intervening sequence. In some embodiments, the second RCPs are sequenced using a second sequencing primer comprising a sequence complementary to the second intervening sequence. [0050] In some embodiments, the first and second RCPs are sequenced in parallel and/or simultaneously. In some embodiments, the method comprises sequencing the first and second RCPs simultaneously. In some embodiments, the method comprises sequencing the first and second RCPs in the same sequencing reactions. In some embodiments, the first and second RCPs are not sequenced simultaneously. In some embodiments, the first RCPs are sequenced in first sequencing reactions and the second RCPs are sequenced in second sequencing reactions which occur after the first sequencing reactions and/or do not occur simultaneously with the first sequencing reactions. In some embodiments, the first RCPs are sequenced in first sequencing reactions and the second RCPs are sequenced in second sequencing reactions which occur after the first sequencing reactions. In some embodiments, the first sequencing reactions comprise using the first sequencing primer to sequence one or more nucleotides of the first RCPs and the second sequencing reactions comprise using the second sequencing primer to sequence one or more nucleotides of the second RCPs. In some embodiments, the method comprises comprise using the first sequencing primer to sequence the first RCPs and using the second sequencing primer to sequence the second RCPs. In some embodiments, the method comprises comprise using the first sequencing primer to sequence one or more nucleotides of the first RCPs and using the second sequencing primer to sequence one or more nucleotides of the second RCPs. In some embodiments, nucleic acids of the library of isolated nucleic acids that do not hybridize to the immobilized capture probes are removed from the hydrogel and/or are not amplified in the RCA reaction. In some embodiments, nucleic acids of the library of isolated nucleic acids that do not hybridize to the immobilized capture probes are not sequenced.

[0051] In some embodiments, the capture probes and/or the RCP molecules of the library of RCPs are immobilized in the first RCP layer and second RCP layer. In some embodiments, the capture probes are immobilized in the first RCP layer and second RCP layer. In some embodiments, the RCP molecules of the library of RCPs are immobilized in the first RCP layer and second RCP layer. In some embodiments, the first RCP layer and second RCP layer are separated by a spacer layer. In some embodiments, the spacer layer is positioned between the first and second RCP layers. In some embodiments, the spacer layer does not comprise immobilized capture probes or immobilized RCPs. In some embodiments, the spacer layer is substantially free of immobilized capture probes and/or immobilized RCPs. In some embodiments, the spacer layer is substantially free of immobilized capture probes. In some embodiments, the spacer layer is substantially free of immobilized RCPs. In some embodiments, the spacer layer comprises a lower concentration of RCPs than the RCP layer. In some embodiments, the concentration of RCPs in the RCP layer is at least 2-fold, at least 5-fold, at least 10-fold, or at least 100-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the concentration of RCPs in the RCP layer is at least 2-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the concentration of RCPs in the RCP layer is at least 5-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the concentration of RCPs in the RCP layer is at least 10-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the concentration of RCPs in the RCP layer is at least 100-fold greater than the concentration of RCPs in the spacer layer.

[0052] In some embodiments, the first RCP layer, second RCP layer, and/or spacer layer are flat, planar, and/or 2-dimensional. In some embodiments, an upper boundary and lower boundary of any of the first RCP layer, second RCP layer, and/or spacer layer are planar. In some embodiments, an upper boundary and lower boundary of any of the first RCP layer, second RCP layer, and/or spacer layer are 2-dimensional. In some embodiments, an upper boundary and lower boundary of each of the first RCP layer, second RCP layer, and/or spacer layer are planar. In some embodiments, an upper boundary and lower boundary of each of the first RCP layer, second RCP layer, and/or spacer layer are 2-dimensional. In some embodiments, the first RCP layer and second RCP layer do not overlap.

[0053] In some embodiments, the 3D hydrogel further comprises a third RCP layer. In some embodiments, the spacer layer is a first spacer layer, and the 3D hydrogel further comprises a second spacer layer positioned between the second RCP layer and the third RCP layer. In some embodiments, the first spacer layer and second spacer layer are positioned on opposite sides of the second RCP layer. In some embodiments, the 3D hydrogel further comprises a fourth RCP layer. In some embodiments, the 3D hydrogel further comprises a third spacer layer positioned between the third RCP layer and the fourth RCP layer. In some embodiments, the third spacer layer and second spacer layer are positioned on opposite sides of the third RCP layer. In some embodiments, the 3D hydrogel further comprises one or more further RCP layers. In some embodiments, the 3D hydrogel comprises one or more further spacer layers positioned between the one or more further RCP layers. [0054] In some embodiments, the method comprises arranging the capture probes and/or the RCP molecules within the 3D hydrogel to generate the RCP layers and/or spacer layers. In some embodiments, the arranging comprises applying an electrical current to the 3D hydrogel. In some embodiments, the electrical current comprises a direct current and/or an alternating current. In some embodiments, the capture probes and/or the RCP molecules are immobilized after the arranging. In some embodiments, the immobilization can be initiated by a stimulus, such as light or the introduction of a reagent.

[0055] In some embodiments, the 3D hydrogel is provided on a solid support. In some embodiments, the solid support comprises a substantially flat, horizontal, and/or 2- dimensional surface. In some embodiments, the solid support comprises a slide. In some embodiments, the immobilized capture probes and/or RCPs are uniformly distributed throughout the 3D hydrogel. In some embodiments, the immobilized capture probes and/or RCPs are randomly distributed throughout the 3D hydrogel.

[0056] In some embodiments, the immobilized capture probes and/or RCPs are distributed at a defined density or density range within the first RCP layer and second RCP layer and/or throughout the 3D hydrogel. In some embodiments, the immobilized capture probes are distributed at a defined density. In some embodiments, the immobilized RCPs are distributed at a defined density. In some embodiments, the immobilized capture probes are distributed within the first RCP layer and second RCP layer. In some embodiments, the immobilized RCPs are distributed within the first RCP layer and second RCP layer. In some embodiments, the immobilized capture probes are distributed throughout the 3D hydrogel. In some embodiments, the immobilized RCPs are distributed throughout the 3D hydrogel.

[0057] In some embodiments, the defined density or density range comprises at or about, or comprises at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the defined density is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the defined density is at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the defined density is between 0.01 and 0.1, between 0.1 and 1.0, between 1.0 and 2.0, or between 1.0 and 5.0 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the defined density is at most 10.0, 5.0, or 2.0 immobilized capture probes and/or RCPs per cubic micron. In some embodiments, the defined density is at or about, or is at least 0.02, 0.07, or 0.1 RCPs per cubic micron. In some embodiments, the defined density is at or about 0.02, 0.07, or 0.1 RCPs per cubic micron. In some embodiments, the defined density is at least 0.02, 0.07, or 0.1 RCPs per cubic micron.

[0058] In some embodiments, the defined density or density range comprises at or about, or comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 immobilized capture probes and/or RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 immobilized capture probes and/or RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 immobilized capture probes and/or RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 1 RCP per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 2 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 3 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 4 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 5 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at least 10 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is about 1 RCP per square micron of the 3D hydrogel. In some embodiments, the defined density is about 2 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is about 3 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is about 4 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is about 5 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is about 10 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at most 100 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at most 50 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at most 20 RCPs per square micron of the 3D hydrogel. In some embodiments, the defined density is at most 10 RCPs per square micron of the 3D hydrogel. [0059] In some embodiments, the method comprises sequencing at least or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the method comprises sequencing about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 0.02, 0.07, or 0.1 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the method comprises sequencing about 0.02, 0.07, or 0.1 RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of equal to or greater than 0.005, 0.01, 0.05, or 0.1 cubic millimeters. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of about or at least 0.005, 0.01, 0.05, or 0.1 cubic millimeters. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of at least 0.005 cubic millimeters. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of at least 0.01 cubic millimeters. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of at least 0.05 cubic millimeters. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of at least 0.1 cubic millimeters. In some embodiments, the one or more regions of the 3D hydrogel comprise a contiguous volume of at least 1 cubic millimeter.

[0060] In some embodiments, the method comprises sequencing at least or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within the first RCP layer and second RCP layer. In some embodiments, the method comprises sequencing at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within the first RCP layer and second RCP layer. In some embodiments, the method comprises sequencing about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within the first RCP layer and second RCP layer. In some embodiments, the method comprises sequencing at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron within the first RCP layer and second RCP layer.

[0061] In some embodiments, the method comprises sequencing at least or at or about 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron of one or more areas of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron of one or more areas of the 3D hydrogel. In some embodiments, the method comprises sequencing about 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron of one or more areas of the 3D hydrogel. In some embodiments, the method comprises sequencing at least or at or about 1, 2, 5, or 10 RCPs per square micron of one or more areas of the 3D hydrogel. In some embodiments, the one or more areas of the 3D hydrogel comprise a contiguous area of at least or at or about 0.1 square millimeters of the 3D hydrogel or at least or at or about 1 square millimeter of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 1 RCP per square micron of the 3D hydrogel (e.g. of one or more areas of the 3D hydrogel). In some embodiments, the method comprises sequencing at least 2 RCPs per square micron of the 3D hydrogel. In some embodiments, method comprises sequencing at least 3 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 4 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 5 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at least 10 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing about 1 RCP per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing about 2 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing about 3 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing about 4 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing about 5 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing about 10 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at most 100 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at most 50 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at most 20 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing at most 10 RCPs per square micron of the 3D hydrogel. In some embodiments, the method comprises sequencing between about 1 and about 10 RCPs per square micron of the 3D hydrogel. In any of the foregoing embodiments, the RCPs sequenced per square micron of the 3D hydrogel can be per square micron of the entire 3D hydrogel or a portion (e.g. one or more areas) thereof.

[0062] In some embodiments, the 3D hydrogel is at least or at or about 5, 10, 50, 100, 200, 300, 400, or 500 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 5 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 10 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 20 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 30 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 40 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 50 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 100 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 200 microns thick. In some embodiments, the 3D hydrogel is at least or at or about 300 microns thick. In some embodiments, the 3D hydrogel is between about 5 and about 100 microns thick. In some embodiments, the 3D hydrogel is at most 500 microns thick. In some embodiments, the 3D hydrogel is at most 300 microns thick. In some embodiments, the 3D hydrogel is at most 100 microns thick.

[0063] In some embodiments, the 3D hydrogel has an area of at least or at or about 1, 5, 10, 50, 100, 200, 300, 400, 500, or 1000 square millimeters. In some embodiments, the 3D hydrogel has an area of at least 1 square millimeters. In some embodiments, the 3D hydrogel has an area of at least 10 square millimeters. In some embodiments, the 3D hydrogel has an area of at least 100 square millimeters. In some embodiments, the 3D hydrogel has an area of at least 500 square millimeters. In some embodiments, the 3D hydrogel has an area of at least 1000 square millimeters. In some embodiments, the 3D hydrogel has an area of between about 100 square millimeters and about 1000 square millimeters. In some embodiments, the 3D hydrogel has an area of between about 100 square millimeters and about 10,000 square millimeters.

[0064] In some embodiments, the method comprises sequencing at least or at or about lx!0^A6, lx!0^A7, lxl0^A8, lxlO^A9, or lxl0^A10 RCPs in the 3D hydrogel. In some 1 embodiments, the method comprises sequencing at least lxlO^A6, lxlO^A7, lxlO^A8, lxlO^A9, or lxl0^A10 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing at least lxlO^A6 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing at least lxlO^A7 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing at least lxlO^A8 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing at least lxlO^A9 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing at least lxl0^A10 RCPs in the 3D hydrogel. In some embodiments, the method comprises sequencing between about lxlO^A6 RCPs and about lxl0^A10 RCPs in the 3D hydrogel. In some embodiments, method of any of claims 1-85, wherein the method comprises sequencing at least or at or about lxlO^A9 RCPs in the 3D hydrogel.

[0065] In some embodiments, sequencing a given density or number of RCPs (e.g. any of the densities or numbers of RCPs provided above or herein) comprises sequencing the given density or number of RCPs at a minimum phred- scaled quality value (q- score) threshold. For example, in some embodiments, sequencing a given RCP comprises determining a sequence of the RCP, and determining that the sequence of the RCP was determined with a minimum q- score. In some embodiments, sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 10. In some embodiments, sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 20. In some embodiments, sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence is determined that has a minimum phred-scaled quality value (q-score) of at least 10, at least 20, or at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence is determined that has a minimum phred-scaled quality value (q-score) of at least 20. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence is determined that has a minimum phred-scaled quality value (q-score) of at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred-scaled quality value (q-score) of at least 10, at least 20, or at least 30. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred-scaled quality value (q-score) of at least 20. In some embodiments, the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred- scaled quality value (q-score) of at least 30. In some embodiments, any other suitable metric for sequencing quality and/or confidence in base pair calling can be used as a threshold for determining the number of sequenced RCPs.

[0066] In some embodiments, sequencing the RCP molecules comprises sequencing one or more nucleotides of the RCP molecules. In some embodiments, sequencing the RCP molecules comprises sequencing the entire RCP molecules or portions thereof. In some embodiments, sequencing the one or more nucleotides comprises detecting one or more signals in the 3D hydrogel corresponding to the one or more nucleotides. In some embodiments, the one or more signals corresponding to the one or more nucleotides are detected in one or more sequential imaging cycles. In some embodiments, the method further comprises analyzing the one or more signals corresponding to the one or more nucleotides to determine sequences of one or more RCPs in the 3D hydrogel. In some embodiments, sequencing RCP molecules of the library of RCPs comprises performing any suitable sequencing method provided herein. In some embodiments, sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding. In some embodiments, sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis.

[0067] In some embodiments, sequencing RCP molecules of the library of RCPs comprises imaging the 3D hydrogel. In some embodiments, the imaging comprises fluorescence microscopy. In some embodiments, the fluorescence microscopy comprises epifluorescence imaging. In some embodiments, the fluorescence microscopy comprises wide-field epifluorescence imaging. In some embodiments, imaging the 3D hydrogel comprises imaging a first focal plane of the 3D hydrogel and imaging a second focal plane of the 3D hydrogel. In some embodiments, the first focal plane coincides with the first RCP layer and the second focal plane coincides with the second RCP layer. In some embodiments, imaging the 3D hydrogel comprises imaging a plurality of focal planes coinciding with a plurality of RCP layers in the 3D hydrogel. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 1.0. In some embodiments, the imaging is performed using an objective having a field of view (FOV) having a diagonal of equal to or greater than 0.5 millimeters. In some embodiments, the imaging is performed using an objective having a field of view (FOV) of equal to or greater than 1.0 millimeters. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9 and a field of view having a diagonal of equal to or greater than 0.5 millimeters. In some embodiments, imaging the 3D hydrogel comprises optically sectioning the hydrogel to image a first optical section of the first RCP layer and a second optical section of the second RCP layer. In some embodiments, sequencing RCP molecules of the library of RCPs comprises optically sectioning the hydrogel to image a first optical section of the first RCP layer and a second optical section of the second RCP layer.

[0068] In some embodiments, the library of RCPs comprises a sequencing library. In some embodiments, the library of RCPs comprises a single-cell sequencing library. In some embodiments, the library of RCPs comprises an RNA sequencing library. In some embodiments, the library of RCPs comprises a DNA sequencing library. In some embodiments, the library of RCPs comprises a single-cell gene expression sequencing library.

A. 3D Hydrogels

[0069] In some aspects, provided herein are methods and compositions for sequencing isolated nucleic acids in a 3-dimensional (3D) hydrogel. In some aspects, provided herein are 3D hydrogels, including any of the 3D hydrogels described herein or 3D hydrogels produced by any of the methods described herein. In some aspects, provided herein are kits and/or compositions comprising any of the 3D hydrogels described herein or 3D hydrogels produced by any of the methods described herein.

[0070] Any suitable 3D hydrogel can be used in connection with the methods provided herein. In general, the term hydrogel refers to a composition comprising a porous, permeable solid component (such as a matrix) and a fluid component (such as water or a composition comprising water). The 3D hydrogel may include a polymer matrix (e.g., a matrix formed by polymerization or cross -linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and/or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductive interactions, or physical entanglement. In some embodiments, the 3D hydrogel comprises a polyacrylamide matrix. The matrix may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic, and may include nucleic acid and/or non-nucleic acid molecules. The 3D hydrogel may be rigid. The 3D hydrogel may be flexible and/or compressible. The gross physical characteristics of the 3D hydrogel can be adjusted to accommodate any of the methods provided herein.

[0071] In some embodiments, a hydrogel can include hydrogel subunits, such as, but not limited to, acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly (hydroxy ethyl acrylate), and poly (hydroxy ethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof.

[0072] In some embodiments, a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Patent Application Publication Nos. 2017/0253918, 2018/0052081 and 2010/0055733, the entire contents of each of which are incorporated herein by reference.

[0073] In some embodiments, additional reagents are added to the hydrogel subunits before, contemporaneously with, and/or after polymerization. For example, additional reagents can include but are not limited to oligonucleotides (e.g., probes), DNA polymerase enzymes, and dNTPs used to amplify nucleic acids. Other enzymes can be used, including without limitation, RNA polymerase, ligase, proteinase K, and DNAse. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers, and oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, contemporaneously with, and/or after polymerization.

[0074] In some embodiments, the 3D hydrogel has a defined shape. In some embodiments, the 3D hydrogel has a shape resembling a sheet with a thickness. In some embodiments, the 3D hydrogel comprises a sheet that is elongated in 2 dimensions (e.g. x and y dimensions) and that has a thickness in a third dimension (e.g. z dimension). The 3D hydrogel can comprise a plurality of layers (e.g. layers comprising capture probes and/or RCPs, and spacer layers) that are elongated in the x and y dimensions and stacked on top of one another in the z dimension. In some embodiments, an area of the 3D hydrogel is defined according to the x and y dimensions. In some embodiments, a thickness of the 3D hydrogel is defined according to the z dimension.

[0075] In some embodiments, the 3D hydrogel is provided on a solid support. In some embodiments, the 3D hydrogel is provided on a surface of the solid support. In some embodiments, the surface is a flat, horizontal, and/or 2-dimensional surface. In some embodiments, the surface is a substantially flat, horizontal, and/or 2-dimensional surface. In some embodiments, the 3D hydrogel is provided on a slide. In some embodiments, the solid support is a slide. In some embodiments, the solid support is a glass slide. In some embodiments, the solid support is compatible with imaging and sequencing applications, such as fluorescence imaging of the 3D hydrogel on the solid support. In some embodiments, the solid support is part of and/or can be provided in a device for sequencing and/or imaging nucleic acids within the 3D hydrogel.

[0076] A wide variety of different solid supports can be used. In some embodiments, the solid support is compatible with the methods provided herein, including hybridization, RCA, sequencing, and/or imaging steps (e.g., optical imaging such as fluorescence microscopy). The solid support can comprise any suitable support material. In some embodiments, the solid support is transparent or comprises transparent components. In some embodiments, a glass slide is used. In some embodiments, a cover slip is used. The solid support can include, but is not limited to including, glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics, nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene and polycarbonate. In some embodiments, the solid support is between about 0.01 mm and about 5 mm, e.g., between about 0.05 mm and about 3 mm, between about 0.1 mm and about 2.5 mm, between about 0.2 mm and about 2 mm, between about 0.5 mm and about 1.5 mm, or about 1 mm in thickness. In some embodiments, the 3D hydrogel is less than, greater than, or at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm in thickness, or of a thickness in between any one of the aforementioned values.

[0077] In some embodiments, the sequencing is not performed in a flow cell. In some embodiments, the detection (e.g. imaging) steps performed herein are not performed in a flow cell. For example, in some embodiments, the detection (e.g. imaging) steps performed herein are performed in an open system. In some embodiments, the 3D hydrogel is not fully enclosed and/or contacted by a solid structure on all sides (e.g. the 3D hydrogel is not encased in a flow cell). For example, in some embodiments, the 3D hydrogel is provided on a slide and/or in an open well. In some embodiments, the 3D hydrogel is provided on a slide. In some embodiments, the 3D hydrogel is provided in an open well. In some embodiments, the 3D hydrogel is provided on a slide in an open well. In some embodiments, providing a 3D hydrogel in an open format (e.g. not in a flow cell) provides various advantages. For example, in some embodiments, the 3D hydrogel is directly accessible from above, and can be imaged directly without a solid structure (such as the top of a flow cell, which may comprise glass or other solid materials) between the imaging objective and the 3D hydrogel. This arrangement can improve the quality of imaging. In some embodiments, this arrangement can also simplify reagent exchange, such as to carry out various sequencing reaction steps as described herein. In some embodiments, the solid support can correspond to a flow cell. In some embodiments, the solid support is not a flow cell.

[0078] In some embodiments, the 3D hydrogel is stationary. In some embodiments, the 3D hydrogel is stationary during one or more of the signal detection (e.g. imaging) steps performed in any of the sequencing methods provided herein. In some embodiments, the 3D hydrogel remains stationary between acquisition of one or more images. In some embodiments, the 3D hydrogel remains stationary in one or more of the x-, y-, and z-dimensions during and/or between one or more of the imaging steps. In some embodiments, the 3D hydrogel is translated (e.g. moved) in one or more of the x-, y-, and z-dimensions between one or more of the imaging steps. In some embodiments, the z-dimension refers to movement along the axis defining the depth or thickness of the hydrogel, and x- and y-dimensions refer to lateral movement. In some embodiments, the 3D hydrogel is translated (e.g. moved) along the Z-dimension. For example, in some embodiments, the 3D hydrogel is moved along the Z-dimension between acquisition of individual images, such as when performing z- stepping to acquire multiple images in a z- stack (e.g. images of different focal planes or optical sections). However, in some embodiments, the 3D hydrogel is not moved along the z-dimension during acquisition of multiple images (e.g. the objective may move instead of the 3D hydrogel to acquire z-stacks). In some embodiments, the 3D hydrogel is translated (e.g. moved) along the x- and/or y-dimension between acquisition of one or more images or z-stacks. In some embodiments, the 3D hydrogel is not translated (e.g. moved) along the x- and/or y-dimension between acquisition of one or more images or z-stacks, such as to image different areas of the 3D hydrogel. In some embodiments, the 3D hydrogel is moved in the Z-dimension but not the x- and y- dimensions. In some embodiments, the 3D hydrogel is not moved in the Z-dimension but is moved in the x- and/or y- dimensions. In some embodiments, the 3D hydrogel is not translated rotationally during the imaging (e.g. signal detection) steps. In some embodiments, the 3D hydrogel is not translated rotationally between one or more of the imaging (e.g. signal detection) steps. In some embodiments, the methods provided herein involve sequential rounds of sequencing reactions (and/or hybridization) interspersed between rounds of 3D hydrogel imaging (e.g. as in SBS). In some embodiments, the 3D hydrogel can be moved in any suitable dimension(s) to facilitate the various stages of sequencing reactions or hybridization events between rounds of imaging, for example to perform reagent exchange.

[0079] In some aspects, provided herein are methods and compositions for sequencing nucleic acids. In some aspects, the nucleic acids are isolated nucleic acids. In some embodiments, the isolated nucleic acids can comprise nucleic acids obtained from any biological sample. In some embodiments, the isolated nucleic acids can comprise products of nucleic acids obtained from any biological sample (e.g. nucleic acid products resulting from reactions such as amplification, reverse transcription, ligation, digestion, polymerization, or any combination thereof). In some aspects, the methods comprise sequencing (e.g. determining the sequences of) rolling circle amplification products (RCPs) that are generated using (e.g. from) the isolated nucleic acids and that are immobilized in a 3-dimensional (3D) hydrogel.

[0080] In some embodiments, the isolated nucleic acids are not provided in their native context. For example, the isolated nucleic acids can be removed (e.g. isolated) from a native context (e.g. from a biological sample) from which they were obtained or derived. In some aspects, the isolated nucleic acids do not retain a spatial relationship to one another that is representative of the spatial relationship of the nucleic acids within a biological sample from which they were derived. For example, isolation of the nucleic acids from a biological sample can comprise suspending the nucleic acids in a medium (e.g. aqueous solution) in which the nucleic acids are not fixed in place and/or move about freely. In some embodiments, the method comprises stochastically distributing the isolated nucleic acids in a 3D hydrogel which does not comprise the biological sample from with the isolated nucleic acids were obtained or derived. By extension, in some embodiments, the products (e.g. RCPs) generated from the isolated nucleic acids are also not provided, sequenced, and/or analyzed in the native and/or spatial context from which the isolated nucleic acids were obtained or derived. Thus, in some aspects, the methods provided herein do not comprise analysis or sequencing of nucleic acids in situ in a biological sample. Instead, the methods provided herein relate to sequencing of nucleic acids that have been removed from their native spatial context.

[0081] In some embodiments, the isolated nucleic acids are provided as a library of isolated nucleic acids. The library of isolated nucleic acids can be any plurality of isolated nucleic acids suitable for use in connection with the methods provided herein. In some embodiments, the library of isolated nucleic acids is a next-generation sequencing library. In some embodiments, the isolated nucleic acids comprise sequences that are identical or complimentary to sequences of nucleic acids present in a biological sample. Thus, in some embodiments, sequencing the isolated nucleic acids comprises determining sequences of one or more nucleic acids that are present in the biological sample from which the isolated nucleic acids were obtained or derived. In some embodiments, the isolated nucleic acids comprise one or more barcode sequences that alone or in combination correspond to and/or identify an analyte (e.g. nucleic acid) present in a biological sample. Thus, in some embodiments, sequencing the isolated nucleic acids comprises determining sequences of one or more nucleic acids that are not present in the biological sample but which correspond to analytes in the biological sample.

[0082] In some embodiments, the isolated nucleic acids comprise additional sequences (e.g. functional sequences), such as sequences that are added to the isolated nucleic acids and/or that are not present in the native context from which the isolated nucleic acids were obtained or derived. In some embodiments, the additional sequences can facilitate processing and/or sequencing of the isolated nucleic acids. In some embodiments, the isolated nucleic acids comprise functional sequences that can be used for processing and/or sequencing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a primer or primer binding sequence, or a sequencing primer or primer binding sequence. In some embodiments, the isolated nucleic acids comprise a sequence or region that is configured to hybridize to another nucleic acid. For example, the isolated nucleic acids can comprise a target region that hybridizes to a capture sequence present on a capture probe, as described herein. In some embodiments, the isolated nucleic acids comprise barcode regions which can encode features of individual isolated nucleic acid molecules or subsets thereof. For example, barcode regions can be associated with the biological sample from which the isolated nucleic acid was obtained or derived, the location within the biological sample from which the isolated nucleic acid was obtained or derived, and/or any other desirable feature. The isolated nucleic acids can comprise one or more sequencing primer binding sequences that facilitate sequencing of the isolated nucleic acids or products thereof in the 3D hydrogel as described herein. In some embodiments, subsets of the isolated nucleic acids can comprise different sequencing primer binding sequences, which can facilitate sequencing of subsets of RCPs within the 3D hydrogel that are generated from the isolated nucleic acids.

[0083] In some embodiments, the isolated nucleic acids comprise one or more barcodes. In some embodiments, the one or more barcodes correspond to and/or are indicative of an analyte. In some embodiments, the one or more barcodes correspond to and/or are indicative of one or more characteristics of an analyte, such as its cell and/or sample of origin. In some embodiments, the one or more barcodes correspond to and/or are indicative of an analyte in a biological sample from which the isolated nucleic acids are obtained or derived (e.g. an endogenous analyte such as an mRNA). In some embodiments, the one or more barcodes are sufficient to identify (e.g. uniquely correspond to) the analyte, either alone or in combination. In some embodiments, an isolated nucleic acid comprising one or more barcodes is used to generate an RCP that is immobilized in the 3D hydrogel. In some embodiments, the RCP comprises the one or more barcodes or complements thereof. In some embodiments, the one or more barcodes identify (e.g. uniquely correspond to) the analyte, either alone or in combination. In some embodiments, the method comprises sequencing the one or more barcodes or complements thereof (e.g. by sequencing the RCP). In some embodiments, an isolated nucleic acid comprises a sequence of an analyte from a biological sample (e.g. an endogenous analyte such as an mRNA), or a complement thereof. In some embodiments, an RCP comprises a sequence of an analyte from a biological sample (e.g. an endogenous analyte such as an mRNA), or a complement thereof. In some embodiments, the method comprises sequencing the sequence of the analyte or the complement thereof (e.g. by sequencing the RCP).

[0084] In some embodiments, the isolated nucleic acids are circular or circularizable, and thus can serve as suitable templates for rolling circle amplification (RCA). In some embodiments, the isolated nucleic acids can be circularized at any suitable processing step prior to RCA. In some embodiments, the isolated nucleic acids are circularized prior to being distributed in the 3D hydrogel. In some embodiments, the isolated nucleic acids are circularized after being distributed in the 3D hydrogel. In some embodiments, the isolated nucleic acids are circularized after hybridizing to the capture probes. In some embodiments, the isolated nucleic acids are circularized by ligation using the capture probes as template, with or without gap filling prior to ligation. In some embodiments, the isolated nucleic acids are circularized by ligation using as template a nucleic acid molecule that is not a capture probe. For example, the isolated nucleic acids can be circularized by ligation using as template a separate splint nucleic acid. In some embodiments, the isolated nucleic acids serve as templates for rolling circle amplification (RCA) to generate rolling circle amplification products (RCPs) in the 3D hydrogel. In some embodiments, the RCPs in the 3D hydrogel are sequenced to determine the sequences of the isolated nucleic acids.

[0085] In some embodiments, the RCPs generated from the isolated nucleic acids are provided in defined patterns and/or densities within the 3D hydrogel. In some aspects, the defined patterns and/or densities provide advantages for detecting, imaging, and/or sequencing the RCPs.

[0086] In some embodiments, the RCPs generated from the isolated nucleic acids are provided in a defined density within the 3D hydrogel. The defined density can be a density or density range. In some embodiments, a defined density of RCPs within the 3D hydrogel can maximize and/or have a beneficial effect on an imaging-based sequencing output.

[0087] FIG. 4 shows an example of a defined density of RCPs immobilized in a 3D hydrogel that maximizes and/or is beneficial for an imaging-based sequencing output. At the lowest densities of RCPs shown in the figure, there are few available RCPs to sequence, and the number of sequenced RCPs will consequently be low (e.g. limited by the low density of RCPs). At the highest densities of RCPs, overcrowding of RCPs leads to a high incidence of overlapping optical signals (i.e optical crowding) generated from neighboring RCPs that are too close to distinguish based on the imaging method (e.g. fluorescence microscopy, such as epifluorescence microscopy). As optical crowding increases, the number of sequenced RCPs declines. At a certain defined density of RCPs, the number of successfully sequenced RCPs is maximized by providing RCPs at a sufficiently high density for high-throughput sequencing without a high rate of optical crowding. Put another way, as the density of RCPs increases from 0, the number of successfully sequenced RCPs also increases, until an inflection point is reached at which the negative effect of optical crowding is greater than the positive effect of increasing RCP density. The inflection point represents the density at which sequencing output is maximized in this example. While certain primary exemplary factors affecting the optimal RCP density for imaging-based sequencing are highlighted in FIG. 4, other factors may also affect the optimal defined density for RCPs in a 3D hydrogel for sequencing. In some embodiments, the defined density is determined empirically.

[0088] In some embodiments, the number of sequenced RCPs is defined as the number of RCPs sequenced with a minimum quality metric. In some embodiments, the number of sequenced RCPs is defined as the number of RCPs sequenced with a minimum phred- scaled quality value (also known as q-score). In some embodiments, the minimum q-score is 20. In some embodiments, the minimum q-score is 30. In some embodiments, sequencing a given number of RCPs comprises sequencing the given number of RCPs with a a q-score of greater than or equal to 20 (q>=20). In some embodiments, sequencing a given number of RCPs comprises sequencing the given number of RCPs with a a q-score of greater than or equal to 30 (q>=30).

[0089] In some embodiments, the RCP density is homogenous throughout the 3D hydrogel. In some embodiments, the RCP density is not homogenous throughout the 3D hydrogel. In some embodiments, the RCP density is homogenous within one or more layers (e.g. RCP layers) of the 3D hydrogel. In some embodiments, the density of RCPs (such as any of the densities described below) represents the density of RCPs that can be sequenced within the 3D hydrogel simultaneously and/or in parallel (e.g. not in sequential sequencing reactions for subsets of RCPs as described elsewhere herein).

[0090] In some embodiments, the density of RCPs can be defined per unit volume (e.g. cubic micron (pm³)). In some embodiments, the density of RCPs can be defined per unit area. In some embodiments, the density of RCPs can be defined per square micron (pm²) of the 3D hydrogel and/or solid support. In some embodiments, because the hydrogel is a 3D hydrogel, the density expressed as a function of area (e.g. in square microns) represents the number of RCPs within a 3D space defined by a square micron extending throughout the thickness of the hydrogel. For example, in some embodiments, the density of RCPs in a square micron is the number of RCPs present in the total volume of the 3D hydrogel that is positioned directly above a square micron of a flat solid support on which the 3D hydrogel rests. In some embodiments, the density of RCPs in a square micron is the number of RCPs present across the entire z- dimension (e.g. thickness) of the hydrogel within a square micron defined in the x-dimension and y-dimension.

[0091] In some embodiments, the density of RCPs is at least, at most, or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron. In some embodiments, the density of RCPs is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron. In some embodiments, the density of RCPs is at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron. In some embodiments, the density of RCPs is at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron. In some embodiments, the density of RCPs is at least or at or about 0.02 RCPs per cubic micron. In some embodiments, the density of RCPs is at least or at or about 0.07 RCPs per cubic micron. In some embodiments, the density of RCPs is at least or at or about 0.1 RCPs per cubic micron. In some embodiments, the density of RCPs is at least or at or about 0.5 RCPs per cubic micron. In some embodiments, the density of RCPs is at least or at or about 1 RCPs per cubic micron.

[0092] In some embodiments, the density of RCPs in the 3D hydrogel or in a layer (e.g. RCP layer) of the hydrogel is at most, at least, or at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron. In some embodiments, the density of RCPs is at least 0.5 RCPs per cubic micron. In some embodiments, the density of RCPs is at least 0.6 RCPs per cubic micron. In some embodiments, the density of RCPs is at least 0.7 RCPs per cubic micron. In some embodiments, the density of RCPs is at least 0.8 RCPs per cubic micron.

[0093] In some embodiments, the density of RCPs (such as any of the densities defined above or herein) is the density of RCPs per cubic micron within one or more regions of the 3D hydrogel. In some embodiments, the one or more regions of the 3D hydrogel comprise a volume of at least 0.005, 0.01, 0.05, or 0.1 cubic millimeters. In some embodiments, the volume is contiguous. In some embodiments, the density of RCPs is the density of RCPs per cubic micron within a contiguous region of the 3D hydrogel having a volume of at least 0.005, 0.01, 0.05, or 0.1 cubic millimeters. In some embodiments, the density of RCPs is the density of RCPs per cubic micron within one or more layers (e.g. RCP layers). In some embodiments, the density of RCPs is the density of RCPs per cubic micron distributed throughout the 3D hydrogel.

[0094] In some embodiments, the density of RCPs is at least, at most, or at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron. In some embodiments, the density of RCPs is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron. In some embodiments, the density of RCPs is at or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron. In some embodiments, the density of RCPs is at least 1 RCP per square micron. In some embodiments, the density of RCPs is at least 2 RCPs per square micron. In some embodiments, the density of RCPs is at least 3 RCPs per square micron. In some embodiments, the density of RCPs is at least 4 RCPs per square micron. In some embodiments, the density of RCPs is at least 5 RCPs per square micron. In some embodiments, the density of RCPs is at least 10 RCPs per square micron. In some embodiments, the density of RCPs is at least 20 RCPs per square micron. In some embodiments, the density of RCPs is at least 50 RCPs per square micron.

[0095] In some embodiments, the density of RCPs in a 5 pm thick hydrogel is at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, or at least 5 RCPs per square micron. In some embodiments, the density of RCPs in an RCP layer (e.g. the first layer and/or second layer) is at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, or at least 5 RCPs per square micron.

[0096] In some embodiments, the density of RCPs in a 10 pm thick hydrogel is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 RCPs per square micron. In some embodiments, the density of RCPs in an RCP layer (e.g. the first layer and/or second layer) is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 RCPs per square micron. [0097] In some embodiments, the density of RCPs (such as any of the densities defined above or herein) is the density of RCPs per square micron within one or more areas of the 3D hydrogel. In some embodiments, the one or more areas of the 3D hydrogel comprise at least 0.1 square millimeters of the 3D hydrogel. In some embodiments, the one or more areas of the 3D hydrogel comprise at least 1 square millimeter of the 3D hydrogel. In some embodiments, the density of RCPs (such as any of the densities defined above or herein) is the density of RCPs per square micron within a contiguous area of at least 0.1 square millimeters of the 3D hydrogel. In some embodiments, the density of RCPs is the density of RCPs per square micron within a contiguous area of at least 1 square millimeter of the 3D hydrogel.

[0098] In some embodiments, the hydrogel is at least 5 microns thick. In some embodiments, the hydrogel is at or about 5 microns thick. In some embodiments, the hydrogel is at least or at or about 10 microns thick. In some embodiments, the hydrogel is greater than 10 microns thick. In some embodiments, the hydrogel is at least or at or about 20 microns thick. In some embodiments, the hydrogel is at least or at or about 30 microns thick. In some embodiments, the hydrogel is at least or at or about 40 microns thick. In some embodiments, the hydrogel is at least or at or about 50 microns thick. In some embodiments, the hydrogel is at least or at or about 100 microns thick. In some embodiments, the hydrogel is at least or at or about 200 microns thick. In some embodiments, the hydrogel is at least or at or about 300 microns thick. In some embodiments, the hydrogel is at least or at or about 400 microns thick. In some embodiments, the hydrogel is at least or at or about 500 microns thick. In some embodiments, the thickness of the hydrogel is defined in the z-dimension (e.g. perpendicular to the plane defined by a solid support on which a substantially flat, or sheet-like hydrogel rests). In some embodiments, for a hydrogel having a thickness higher than a threshold value (e.g. greater than 10 microns thick, or greater than 50 microns thick), the hydrogel refractive index can be matched to a refractive index of an immersion medium used to image the hydrogel. In some aspects, the number of RCPs that can be sequenced in parallel in the hydrogel can scale proportionally with the thickness of the hydrogel. In some aspects, the number of RCPs that can be sequenced in parallel in the hydrogel scales proportionally with the number of RCP layers provided in the hydrogel.

[0099] In an exemplary illustrative embodiment, the 3D hydrogel is 10 pm thick, has an imageable area of 6.75xl0^A8 pm2 (e.g. an imageable area of 45 millimeters (mm) length by 15 mm width), and comprises a density of 7 RCPs per pm². In this example, the imageable area can comprise 4.7xlO^A9 (4.7 billion) RCPs. In some embodiments, the imageable area can accommodate the parallel sequencing of 4.7 billion RCPs. In a similar exemplary embodiment, increasing the thickness of the hydrogel to 20 pm could result in an imageable area that can accommodate parallel sequencing of 9.4 billion RCPs.

[0100] In some embodiments, the RCPs generated from the isolated nucleic acids are provided in a defined pattern within the 3D hydrogel. For example, the RCPs can be arranged in a pattern comprising regions of the 3D hydrogel that comprise RCPs and regions that do not comprise RCPs (or that comprise significantly lower concentrations of RCPs). Any suitable arrangement or pattern of RCPs can be used. In some embodiments, the pattern of RCPs comprises layers, rows, columns, any other suitable 2D or 3D configuration of RCPs within the 3D hydrogel, or a combination thereof. In some embodiments, the RCPs are arranged in discrete layers, rows, columns, any other suitable 2D or 3D configuration of RCPs within the 3D hydrogel, or a combination thereof. In some embodiments, the pattern of RCPs comprises layers. In some embodiments, the RCPs are arranged in layers (e.g. RCP layers as described herein). In some embodiments, the pattern of RCPs in the 3D hydrogel is beneficial and provides various advantages for downstream sequencing applications (e.g. any of the methods provided herein). For example, RCPs arranged in defined, planar layers (e.g. a first RCP layer and second RCP layer) separated by spacer layers, e.g. as described herein, can improve resolution of RCPs when imaging, including along the Z-axis. In some embodiments, arranging RCPs in defined layers allows each layer to be imaged in a single plane (e.g. focal plane) or optical section, which can in turn improve the efficiency of imaging (e.g. during sequencing steps), for example by reducing imaging time. Arranging RCPs in defined layers can improve, simplify, and increase the efficiency of both imaging and image analysis. For example, by arranging RCPs in a defined layer, a single focal plane or optical section can be imaged that captures maximum diameters and intensities from all or most of the RCPs in the layer, resulting in a single image (e.g. focal plane image or optical section) with a high degree of uniformity in the sizes and/or intensities of the imaged optical signals generated from the RCPs (e.g. as shown in FIG. 5; left). Uniformity of RCP signals in turn can greatly simplify and increase the efficiency of downstream image analysis, for example by decreasing the number of images that need to be acquired and analyzed, and reducing computational burden in analyzing signals generated from RCPs. In contrast, an image of a focal plane or optical section of RCPs randomly distributed in 3 dimensions will capture partial RCPs, resulting in non-uniform RCP signals that vary in both size and intensity (e.g. as shown in FIG. 5; right). In some aspects, providing RCPs within discrete RCP layers can allow image acquisition to be performed only for certain depths (e.g. focal planes) of the 3D hydrogel and not for others. Thus, in some embodiments, the method comprises imaging first portions (e.g. focal planes) of the 3D hydrogel comprising RCPs (such as RCP layers) and not imaging second portions of the 3D hydrogel (e.g. spacer layers). In some embodiments, the images acquired in a given cycle of imaging (e.g. a cycle of imaging performed in any of the sequencing methods provided herein, such as SBS) do not represent the entire depth of the 3D hydrogel. In some embodiments, the images acquired in a given cycle of imaging (e.g. a cycle of imaging performed in any of the sequencing methods provided herein, such as SBS) do not represent the entire volume of the 3D hydrogel. In some embodiments, the images acquired in a given cycle of imaging (e.g. a cycle of imaging performed in any of the sequencing methods provided herein, such as SBS) represent a subset of the depths (e.g. layers) of the 3D hydrogel. In some embodiments, the method comprises imaging RCP layers and not imaging one or more spacer layers in the 3D hydrogel. In some embodiments, the method comprises imaging RCP layers and not imaging one or more spacer layers in the 3D hydrogel in at least one cycle of imaging.

[0101] It can be seen that providing a plurality of defined layers of RCPs in a 3D hydrogel can provide the benefits (e.g. simplicity and efficiency) of imaging RCPs in 2D (in individual layers), while also taking advantage of the third dimension of the 3D hydrogel to increase the capacity of the 3D hydrogel to hold a large number of RCPs for sequencing (e.g. by providing multiple 2D layers instead of a single 2D layer of RCPs).

[0102] In some embodiments, the 3D hydrogel comprises a spacer layer. In some embodiments, the 3D hydrogel comprises one or more spacer layers. In some embodiments, the 3D hydrogel comprises a plurality of spacer layers that separate a plurality of layers comprising RCPs (i.e. RCP layers) from one another. In some aspects, spacer layers in the 3D hydrogel can provide space between RCP layers. In some embodiments, spacer layers can provide space between layers of RCPs such that the layers of RCPs are separated, discrete, and/or well-defined. In some aspects, spacer layers contribute to providing defined RCP layers. Thus, in some aspects, spacer layers provide the same advantages as defined RCP layers, since the spacer layers themselves contribute to defining the RCP layers. In some embodiments, complete exclusion of all RCPs (and/or immobilized capture probes) from spacer layers may not be achieved. Thus, in some embodiments, the spacer layers are substantially free of RCPs. In some embodiments, a spacer layer comprises a lower concentration of RCPs than an RCP layer. In some embodiments, the concentration of RCPs in the RCP layers of the 3D hydrogel is at least 2-fold, at least 5-fold, at least 10-fold, or at least 100-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the concentration of RCPs in the RCP layers of the 3D hydrogel is at least 10-fold greater than the concentration of RCPs in the spacer layer. In some embodiments, the concentration of RCPs in the RCP layers of the 3D hydrogel is at least 100-fold greater than the concentration of RCPs in the spacer layer.

[0103] In some embodiments, the RCPs generated from the isolated nucleic acids are provided in both a defined pattern and a defined density within the 3D hydrogel. For example, the RCPs can be arranged in a particular pattern comprising regions of the 3D hydrogel that comprise RCPs and regions that do not comprise RCPs (or are substantially free of RCPs or have lower concentrations of RCPs as described above), and within the regions that comprise RCPs (e.g. layers), the RCPs can be provided at a defined density. In some embodiments, the RCPs provided in a layer (e.g. the first RCP layer and/or second RCP layer) are provided at a defined density, such as any density described above. In some embodiments, different layers comprise the same RCP densities. In some embodiments, different layers comprise different RCP densities.

[0104] In some embodiments, the distribution of RCPs in the 3D hydrogel (e.g. in a defined density and/or pattern) can be controlled based on the distribution of immobilized capture probes, which prime the RCA reactions and are extended to generate the RCPs. In some embodiments, the capture probes are immobilized in defined layers, such as any of the defined layers described above for RCPs. In some embodiments, the capture probes are immobilized in defined densities, such as any of the defined densities described above for RCPs. In some embodiments, the capture probes are immobilized in defined patterns and densities. For example, the capture probes can be immobilized in defined layers, with defined densities in each layer. In some embodiments, the capture probes are immobilized in layers that are separated by spacer layers. [0105] In some embodiments, the capture probes are immobilized prior to hybridization with the isolated nucleic acids. For example, following immobilization of the capture probes within the 3D hydrogel, the isolated nucleic acids can be distributed within the 3D hydrogel and allowed to hybridize to the immobilized capture probes. Optionally, isolated nucleic acids that do not hybridize to the capture probes can then be washed out of the 3D hydrogel (e.g. excess isolated nucleic acids or nucleic acids lacking sequences that are complementary to the capture probes). In some embodiments, the capture probes are immobilized in the 3D hydrogel after hybridization with the isolated nucleic acids.

[0106] Immobilization of the capture probes or RCPs can be performed by any suitable method. Various methods for immobilizing oligonucleotides within hydrogels have been described and can be readily performed by a person having skill in the art. In some embodiments, the capture probes are immobilized via a 5’ end of the capture probes. In some embodiments, the 5’ ends of the capture probes are immobilized and the 3’ ends of the capture probes serve to hybridize to the isolated nucleic acids and prime the RCA reactions. In some embodiments, the capture probes comprise a 5’ moiety to facilitate covalent or non-covalent attachment to the 3D hydrogel. In some embodiments, the capture probes comprise a 5’ acrydite modification, which may also be referred to as a 5’ acrydite moiety, to facilitate covalent attachment to the 3D hydrogel. In some embodiments, a 5’ acrydite moiety can be covalently attached to the polyacrylamide matrix of the 3D hydrogel. In some embodiments, the capture probes are immobilized (e.g. attached to the 3D hydrogel) before RCA is performed. In some embodiments, the capture probes are immobilized (e.g. attached to the 3D hydrogel) after RCA is performed and the capture probes have been extended to form RCA products. Thus, in some embodiments, the RCA products (RCPs) are immobilized.

[0107] In some embodiments, immobilization of the capture probes results in immobilized RCPs, which are generated by extending the immobilized capture probes, and are thus immobilized themselves (e.g. via the 5’ acrydite moiety). In some embodiments, immobilization of the capture probes and RCPs facilitates the maintenance of a stable distribution of RCPs in the 3D hydrogel, which is maintained during subsequent processing, sequencing, and imaging steps. In some embodiments, the RCPs are not further immobilized after the RCA. In some embodiments, the RCPs are further immobilized (e.g. via crosslinking to the gel) after the RCA. In some embodiments, the RCPs are immobilized (e.g. via crosslinking to the gel) as an alternative to capture probe immobilization. In some embodiments, RCPs are generated prior to being contacted with the 3D hydrogel and are immobilized in the 3D hydrogel.

[0108] In some cases, an acrydite moiety can refer to an acrydite analogue generated from the reaction of acrydite with one or more species, such as, the reaction of acrydite with other monomers and cross-linkers during a polymerization reaction. Acrydite moieties may be modified to form chemical bonds with a species to be attached, such as a nucleic acid molecule (e.g., an oligonucleotide). Acrydite moieties may be modified with thiol groups capable of forming a disulfide bond or may be modified with groups already comprising a disulfide bond. The thiol or disulfide (via disulfide exchange) may be used as an anchor point for a species to be attached or another part of the acrydite moiety may be used for attachment. In some cases, attachment is reversible, such that when the disulfide bond is broken (e.g., in the presence of a reducing agent), the attached species is released from the bead. In other cases, an acrydite moiety can comprise a reactive hydroxyl group that may be used for attachment.

[0109] In some aspects, the capture probes and/or RCPs are anchored to a polymer matrix, such as a polymer matrix of the 3D hydrogel. In some embodiments, one or more of the capture probes and/or RCPs is modified to contain functional groups that can be used as an anchoring site for attachment to the polymer matrix. Examples of modification and polymer matrix that can be employed in accordance with the provided embodiments comprise those described in, for example, US 2018/0051332, US 2019/0241950, US 2016/0024555, US 2018/0251833 and US 2017/0219465, which are herein incorporated by reference in their entireties. In some examples, the polymer matrix also contains modifications or functional groups that can react with or incorporate the modifications or functional groups of the capture probes or RCPs. In some examples, the polymer matrix of the 3D hydrogel can comprise oligonucleotides, polymers, and/or chemical groups, to provide a matrix and/or support structures.

[0110] Amplification products (e.g. RCPs) may be immobilized within the matrix of the 3D hydrogel generally at the location of the nucleic acid being amplified, thereby creating a localized colony of amplicons. The amplification products may be immobilized within the matrix by steric factors. The amplification products may also be immobilized within the matrix by covalent or noncovalent bonding. In this manner (e.g. by covalent or noncovalent bonding), the amplification products may be considered to be attached to the matrix. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the size and spatial relationship the amplicons is maintained. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the amplification products are resistant to movement or unraveling under mechanical stress, various processing steps, and/or sequencing steps.

[0111] In some embodiments, the capture probes or RCPs are copolymerized with the matrix. In some embodiments, the capture probes or RCPs are covalently attached to the surrounding matrix. In some embodiments, the provided methods involve embedding the capture probes or RCPs in the presence of hydrogel subunits to form one or more hydrogel-embedded capture probes or RCPs. In some embodiments, the described hydrogel chemistry comprises covalently attaching nucleic acids (e.g. capture probes or RCPs) to the hydrogel for reagent (e.g. nucleic acid or enzyme) diffusion, and multiple-cycle sequencing workflows. In some embodiments, to enable embedding in the hydrogel setting, one or more amine-modified nucleotides are included in the capture probes or RCPs, functionalized with an acrylamide moiety using acrylic acid N-hydroxysuccinimide esters, and copolymerized with acrylamide monomers to form a hydrogel.

[0112] In some embodiments, one or more functional moieties can be included in a capture probe or RCP to facilitate attachment to the 3D hydrogel (e.g. immoibilization). In some embodiments, one or more functional moieties can be included (e.g. incorporated during RCA) in an RCP. In some embodiments, the functional moiety is incorporated via a modified dNTP during RCA. In some embodiments, the functional moiety is attached to the hydrogel covalently or non-covalently. In some embodiments, the functional moiety is attached to the hydrogel via crosslinking. The functional moiety can be attached to the hydrogel via click chemistry, biotin/streptavidin binding, or other suitable interactions, such as any provided herein. In some embodiments, the functional moiety is attached to the hydrogel during hydrogel polymerization (e.g. is co-polymerized with the hydrogel). In some embodiments, the functional moiety is attached to the hydrogel after the hydrogel is formed. In some embodiments, the functional moiety is attached to hydrogel monomers prior to polymerization of the hydrogel monomers to form the 3D hydrogel.

[0113] In some embodiments, the capture probes capture the isolated nucleic acids to be sequenced within the 3D hydrogel. In some embodiments, the capture probes capture the isolated nucleic acids via hybridization. In some embodiments, the capture probes comprise a capture sequence that hybridizes to a target region of the isolated nucleic acids. In some embodiments, the capture sequence is complementary to the target region. In some embodiments, the capture sequence is at the 3’ end of the capture probe. In some embodiments, the capture sequence or at least a portion thereof is at the 3’ end of the capture probe.

[0114] In some embodiments, the capture sequence is a common capture sequence (e.g. common to all capture probes). In some embodiments, the capture sequence hybridizes to a common target region of the isolated nucleic acids. In some embodiments, the capture sequence is a common capture sequence (e.g. common to all capture probes), and the capture sequence hybridizes to a common target region of the isolated nucleic acids (e.g. a target region that is common to all of the isolated nucleic acids). In some embodiments, the target region is a functional sequence that is added to the isolated nucleic acids, such as an adapter region (e.g. a common adapter region) that is added to a sequencing library of isolated nucleic acids. In some embodiments, subsets of capture probes can comprise different capture sequences. For example, the capture probes can be provided in a first subset of capture probes having a first capture sequence and a second subset of capture probes having a second capture sequence. Similarly, subsets of isolated nucleic acids can comprise different target regions. For example, the isolated nucleic acids can be provided in a first subset of isolated nucleic acids having a first target region and a second subset of isolated nucleic acids having a second target region. Any suitable capture sequence(s) and target region(s) can be used in accordance with the described methods, and could be readily designed by one having ordinary skill in the art.

[0115] In some embodiments, the target region is a contiguous sequence. In some embodiments, the target region is not a contiguous sequence. For example, the target region can be a split sequence having a first portion at the 3’ end and a second portion at the 5’ end of a given nucleic acid of the isolated nucleic acids. In some embodiments, the first and second portion of the split sequence are ligated, thereby circularizing the isolated nucleic acid. In some embodiments, the first and second portion of the split sequence are ligated using the capture sequence of the capture probe as template, with or without gap filling prior to ligation. Thus, in some embodiments, the capture probes can serve as template to ligate/circularize the isolated nucleic acids.

[0116] In some embodiments, the capture sequence is a contiguous sequence. In some embodiments, the capture sequence is not a contiguous sequence. For example, the capture sequence can be a split capture sequence comprising a first portion at the 3’ end of the capture probe and a second portion that is not contiguous with the first portion. The first portion and second portion of the split capture sequence can be separated by any suitable intervening sequence. In some embodiments, the intervening sequence serves as template for gap filling prior to ligation and circularization of the isolated nucleic acids. Thus, in some embodiments, the intervening sequence can be used to incorporate any desired sequences into the isolated nucleic acids and/or RCPs.

[0117] In some embodiments, the intervening sequences can be used for downstream detection and/or sequencing of subsets of the generated RCPs. In an exemplary embodiment, a first subset and second subset of capture probes comprise a common split capture sequence that hybridizes to a common split target region of circularizable isolated nucleic acids. The first and second subset of capture probes comprise a first intervening sequence and second intervening sequence, respectively, which are incorporated by gap filling into the hybridized isolated nucleic acids, which are then ligated and circularized, and amplified by RCA. The resulting RCPs comprise a first RCP subset (e.g. first RCPs) comprising multiple copies of the first intervening sequence and a second RCP subset (e.g. second RCPs) comprising multiple copies of the second intervening sequence. The first and second intervening sequences can be used to detect and/or sequence the two subsets of RCPs within the 3D hydrogel in different (e.g. sequential) steps, such as in separate sequencing reactions that are performed one after another. For example, in some embodiments, the first RCPs are sequenced using a first sequencing primer corresponding to the first intervening sequence, and the second RCPs are sequenced using a second sequencing primer corresponding to the second intervening sequence. In some embodiments, the first RCPs are sequenced using a first sequencing primer comprising a sequence complementary to the first intervening sequence, and the second RCPs are sequenced using a second sequencing primer comprising a sequence complementary to the second intervening sequence. Detection and/or sequencing of subsets of RCPs in different steps can be advantageous, for example for overcoming limitations associated with optical crowding at high densities of RCPs. In some embodiments, by detecting and/or sequencing subsets of RCPs within the 3D hydrogel, optical crowding can be reduced and/or the density of RCPs provided in the 3D hydrogel can be increased. [0118] In some aspects, it can be seen that the capture probes can facilitate multiple steps of the method, including: a) capturing the isolated nucleic acids, b) optionally serving as template to ligate/circularize the isolated nucleic acids, and c) serving as primers for the RCA reaction using the isolated nucleic acids as template to extend the capture probes, thereby generating the RCPs.

[0119] In some embodiments, the isolated nucleic acids are amplified by rolling circle amplification (RCA) to generate rolling circle amplification products (RCPs). In some embodiments, the RCA is performed in the 3D hydrogel. In some embodiments, the RCA is not performed in the 3D hydrogel. Any suitable method of RCA can be used in connection with the methods described herein. A variety of methods for performing RCA have been described herein and elsewhere and can be readily performed by those having skill in the art.

[0120] In some embodiments, an RCP is generated using an RCA primer (e.g. the capture probe) that is or comprises a single-stranded nucleic acid sequence having a 3’ end that is used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction (e.g. the RCA reaction). In some embodiments, the RCA primer is the capture probe. In some embodiments, the RCA primer is a separately provided oligonucleotide that is not the capture probe (e.g. in certain instances where the RCPs are directly immobilized in the 3D hydrogel). In some embodiments, RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. In some examples, DNA primers are used to prime RNA synthesis and vice versa (e.g., RNA primers are used to prime DNA synthesis). Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. Primers can vary in length. For example, primers are about 6 bases to about 120 bases. In some embodiments, primers can include up to about 25 bases. A primer, in some cases, refers to a primer binding sequence. A primer extension reaction generally refers to any method where two nucleic acid sequences are hybridized, and one or both 3’ termini of the hybridized nucleic acids are extended using the other nucleic acid sequence as a template for extension. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and/or a reverse transcriptase. [0121] In some embodiments, the method comprises performing amplification of circular or circularizable nucleic acids (e.g., following circularization of the circularizable nucleic acids). In some embodiments, the amplification is performed at a temperature between or between about 20°C and about 60°C. In some embodiments, the amplification is performed at a temperature between or between about 30°C and about 40°C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, or 49°C.

[0122] In some embodiments, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, a primer (e.g. capture probe) is elongated to produce multiple copies of the circular template (e.g. the circular or circularized isolated nucleic acid). This amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, the isolated nucleic acid is rolling-circle amplified to generate an amplification product (RCP, sometimes referred to as a DNA nanoball) containing multiple copies of the complement of the sequence of the isolated nucleic acid. Techniques for the rolling circle amplification (RCA) can include linear RCA, branched RCA, dendritic RCA, or any combination thereof (see, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 November 15; 49(11): 2540-2550; Schweitzer et al. Proc. Natl Acad. Sci. USA 97:101 13- 1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el l8, 2001; Dean et al. Genome Res. 11:1095-1099, 2001; Schweitzer et al, Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). In some embodiments, the RCA is linear RCA. Examples of polymerases for use in RCA include DNA polymerases such as phi29 (cp29) polymerase, Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some embodiments, DNA polymerases that have been engineered or mutated to have desirable characteristics are employed. In some embodiments, the polymerase is phi29 DNA polymerase.

[0123] In some aspects, provided herein are 3D hydrogels with specific arrangements and/or densities of RCPs. The specific arrangements and/or densities of RCPs can be provided by any suitable method. In some embodiments, the RCPs are provided in discrete RCP layers separated by spacer layers, as described elsewhere herein. [0124] In some embodiments, nucleic acids such as the RCPs or capture probes provided herein can be arranged in the 3D hydrogel using various techniques available to those skilled in the art. Various methods for generating multi-layered hydrogels may be employed in accordance with the methods provided herein, for example to provide 3D hydrogels comprising RCP layers and intervening spacer layers (e.g. as illustrated in FIG. 2). Methods have been described for generating hydrogels comprising multiple discrete regions and/or layers having different geometries, compositions, and densities of biomolecules, components of which can be readily adapted to generate the 3D hydrogels provided herein. Suitable methods for generating multi-layered hydrogels can include, for example, methods involving layer-by-layer assembly, layer stacking, step-wise assembly, photo-polymerization, photo-crosslinking, 3D printing, and/or other processes (see, e.g. Liu et al., “Multi-Layered Hydrogels for Biomedical Applications” Front Chem. 2018 Sep 25:6:439; Ko et al., “A simple layer- stacking technique to generate biomolecular and mechanical gradients in photocrosslinkable hydrogels” Biofabrication. 2019 Mar 28;l l(2):025014; and US20110212501A1, each of which is incorporated in its entirety herein). In some embodiments, alternating RCP and spacer layers are generated and/or assembled one on top of another in a step-wise fashion. In some embodiments, the layers (e.g. RCP layers and spacer layers) are generated by sequential loading, for example by polymerizing a first hydrogel layer comprising RCPs (or capture probes or moieties for attachment thereto), then polymerizing a second hydrogel layer not comprising RCPs, and repeating this process any suitable number of times to generate alternating RCP and spacer layers. In some embodiments, the RCP layers are generated comprising RCPs, and the RCPs are immobilized within the RCP layers. In some embodiments, the RCP layers are initially generated comprising immobilized capture probes, and isolated nucleic acids are subsequently contacted with the RCP layers and/or the assembled hydrogel, captured, and amplified to generate RCPs in the RCP layers. In some embodiments, the RCP layers are initially generated comprising a moiety that is capable of being attached to an RCP or capture probe (e.g. a 5’ acrydite moiety of a capture probe), and the RCP or capture probe is subsequently attached to the moiety in the RCP layers.

B. Electric Field Manipulation of 3D Hydrogels

[0125] In some embodiments, the RCPs and/or capture probes are arranged in the 3D hydrogel using an electric field. In some embodiments, the method comprises applying an electric field to the RCPs or capture probes. In some embodiments, the method comprises applying a current, such as a direct current (DC) or alternating current (AC) to the 3D hydrogel and/or RCPs or capture probes therein. In some embodiments, application of the electric field drives the RCPs and/or capture probes to the desired locations within the 3D hydrogel (e.g. RCP layers). In some aspects, capture probes and/or RCPs can be loaded into and/or arranged within the 3D hydrogel in defined patterns using a direct current and/or an alternating current. In some embodiments, provided herein is a method for arranging nucleic acids in a 3D hydrogel. In some embodiments, provided herein is a method for arranging the capture probes and/or RCPs provided herein in a 3D hydrogel.

[0126] In some embodiments, a direct current is applied to drive the RCPs and/or capture probes into the 3D hydrogel. For example, in some embodiments, RCPs or capture probes are deposited into a loading zone of the hydrogel, which may be an edge (e.g. top) of the 3D hydrogel, and a direct current electric field is applied to electrophoretically drive the negatively charged RCPs or capture probes into the 3D hydrogel. In some embodiments, the direct current is generated by applying voltage across electrodes on opposite sides of the 3D hydrogel. In some embodiments, the RCPs or capture probes are generally evenly distributed across the loading zone (e.g. top of the hydrogel) and pulled into the hydrogel via the electric field to a defined depth, thereby being arranged in a layer. In some embodiments, different batches of RCPs or capture probes can be added to the loading zone at timed intervals during the application of the current, thereby creating multiple concentrated layers of RCPs or capture probes at different depths within the 3D hydrogel, separated by spacer layers. In some embodiments, the RCPs or capture probes are driven into the 3D hydrogel using the direct current but are not yet arranged in discrete or well-defined RCP layers. In some embodiments, further manipulation of the RCPs or capture probes is performed after application of the direct current.

[0127] In some embodiments, the direct current (DC) is applied with any suitable voltage strength and duration. In some embodiments, the direct current (DC) can be tuned in terms of voltage strength and duration. A higher DC voltage will inject RCPs faster and deeper into the gel, but if too high could cause RCPs to overshoot or even produce undesired heating/electrolysis. The timing (how long the DC field is applied) can be adjusted to load a desired number or distribution of RCPs. DC may also be applied in pulses or stages (e.g., a short high-voltage pulse to quickly introduce RCPs, then a lower voltage to homogeneously distribute them). The polarity of the DC field may be reversed (e.g. to move RCPs or capture probes in the opposite direction).

[0128] In some embodiments, an alternating current is applied to arrange and/or evenly space the RCPs and/or capture probes within the 3D hydrogel. In some embodiments, prior to application of the alternating current, the RCPs or capture probes are randomly distributed or do not form discrete or well-defined RCP layers. In some embodiments, prior to application of the alternating current, the RCPs or capture probes are arranged in layers within the 3D hydrogel, but the layers do not have the ultimate desired spacing and/or arrangement. In some embodiments, the alternating current is applied to form and/or further refine discrete layers of RCPs or capture probes within the 3D hydrogel.

[0129] In some embodiments, an alternating current (AC) electric field is applied in order to align the RCPs or capture probes in a layer-by-layer manner, such as along the vertical (z) axis of the 3D hydrogel, e.g. using electrodes placed at the top and bottom of the 3D hydrogel. In some embodiments, the AC field can produce any suitable waveform, such as a sine wave or square wave. In some embodiments, the AC field is a sinusoidal voltage waveform. In some embodiments, the waveform creates an oscillating electric field through the hydrogel. In some embodiments, by applying specific frequencies, a standing-wave electric field having periodic electrical force nodes can be established within the 3D hydrogel. Consequently, the RCPs or capture probes will be driven to and accumulate at electric field nodes (or anti-nodes) along the standing wave, thereby forming distinct layers. As a result, the RCPs or capture probes distributed in the 3D hydrogel are ordered or refined into a series of discrete RCP layers within the 3D hydrogel, with each RCP layer being separated from other RCP layers by a depleted region (e.g. spacer layer). In some embodiments, the spacing of layers is controlled by the AC frequency. For example, a higher frequency will generate more closely spaced layers, whereas a lower frequency waveform will generate more widely spaced layers. In some embodiments, the field amplitude and waveform, and duration of time of AC application can be adjusted to control the distribution of layers and how tightly RCPs or capture probes are confined within a given layer (e.g. the distribution of RCPs or capture probes along the z-axis within a given RCP layer). In some embodiments, the AC alignment phase is maintained for a sufficient time to allow the system to reach equilibrium (e.g. RCPs or capture probes positioned into layers). [0130] In some embodiments, the method comprises first applying a DC to the 3D hydrogel (e.g. for introduction of RCPs or capture probes into the 3D hydrogel and/or formation of layers) and then applying an AC to the 3D hydrogel (e.g. for formation or refinement of layers). In some embodiments, the method does not comprise applying a DC current to the hydrogel. For example, in some embodiments, the RCPs or capture probes can initially be randomly dispersed within the 3D hydrogel or a monomer precursor solution and an AC current is applied to generate the layers without first applying a DC. In some embodiments, the method does not comprise applying an AC to the hydrogel. For example, in some embodiments, periodic introduction of capture probes or RCPs into the 3D hydrogel via application of DC and loading of capture probes or RCPs at regular intervals can establish the RCP layers and spacer layers without the need for further refinement.

[0131] FIG. 13A illustrates an exemplary embodiment of how a direct current (DC) can be used to move RCPs (or, interchangeably, capture probes) into a 3D hydrogel in accordance with the methods provided herein. A cathode and anode are positioned at the top and bottom of the 3D hydrogel to establish a DC that is constantly applied and configured to move negatively charged molecules (e.g. RCPs or capture probes) downwards into the 3D hydrogel. First RCPs are loaded on top of the 3D hydrogel and migrate downwards into the hydrogel to form a first RCP layer. After a first time interval, second RCPs are loaded on top of the 3D hydrogel and migrate downwards into the hydrogel to establish a second RCP layer. After a second time interval, third RCPs are loaded on top of the 3D hydrogel and will migrate downwards into the hydrogel to establish a third RCP layer. The process can be repeated any number of times to establish a sufficient number of RCPs and/or layers thereof within the 3D hydrogel. In some embodiments, following migration of RCPs or capture probes into the 3D hydrogel, the RCPs or capture probes may not be arranged in well-defined layers, and/or may require further refinement. FIG. 13B illustrates an exemplary embodiment of how an alternating current can be used to generate a standing wave electric field having nodes and anti-nodes that are arranged along the z-axis (e.g. depth) of the 3D hydrogel. Application of the standing wave electric field causes movement of RCPs or capture probes such that they become arranged according to the nodes or antinodes present in the standing wave electric field. In some embodiments, the RCPs or capture probes aggregate at the antinodes of the standing wave electric field. In some embodiments, the RCPs or capture probes aggregate at the nodes of the standing wave electric field. In some embodiments, the nodes and antinodes are regularly spaced along the z-axis (e.g. vertical direction or thickness) of the 3D hydrogel. In some embodiments, aggregation of the RCPs or capture probes at the nodes or antinodes generates regions of concentrated RCPs or capture probes (e.g. RCP layers) and regions of relatively sparse RCPs or capture probes (e.g. spacer layers).

[0132] In some embodiments, the DC and/or AC electric field can be adjusted as needed to reconfigure the RCPs or capture probes after an initial electric field application and/or layer alignment. In some embodiments, electrodes or arrays thereof can be positioned in any suitable configuration. In some embodiments, electrodes are positioned above and below the 3D hydrogel to control distribution of RCPs or capture probes along the z-axis (e.g. for formation of layers). In some embodiments, electrodes or arrays thereof can be positioned on the sides of the 3D hydrogel to create more complex electric fields and consequently distributions of RCPs or capture probes.

[0133] Any suitable apparatus for controlling the electric fields and currents described herein can be used. For example, in some embodiments, a programmable waveform generator is used. In some embodiments, the waveform generator produces any desirable current (e.g. DC or AC) combination, or sequence thereof (e.g. a DC bias plus an AC modulation, frequency sweeps, etc.) In some embodiments, the sequence of DC-to-AC switching can be automated.

[0134] In some embodiments, the 3D hydrogel can be placed within a device (e.g. microfluidic device) for implementing the various manipulations provided in this section, including RCP or capture probe loading, and electric field application. The 3D hydrogel may be removed from the device for subsequent reactions and/or imaging steps. The 3D hydrogel may be remain in the device for subsequent reactions and/or imaging steps.

[0135] In some aspects, the AC field inherently can act via dielectrophoresis (DEP), inducing dipoles in the RCPs (or capture probes) and moving them in non-uniform fields. In some embodiments, this effect can be enhanced by using shaped or multiple electrodes to create field gradients. For example, interdigitated electrodes or patterned electrodes within the hydrogel could generate localized high-field regions. In some embodiments, this would allow fine positioning of RCPs or capture probes in layers, and/or into particular zones or micro-patterns within each layer. In some embodiments, DEP forces keep RCPs tightly clustered at the nodes of the standing wave. In some embodiments, by adjusting frequency, RCPs or capture probes can be switched between experiencing positive DEP (attraction to field maxima) and negative DEP (repulsion to field maxima). In some aspects, DEP tuning provides another degree of control to move RCPs or capture probes to desired locations (for instance, concentrating them at either the nodes or antinodes of the standing wave field, whichever yields optimal alignment).

[0136] In some embodiments, attachment and/or immobilization of components such as RCPs or capture probes in the hydrogel comprises photopolymerization. For example, once RCPs or capture probes are aligned in the desired layers or other configurations, the method can comprise photopolymerizing the hydrogel to lock the RCPs or capture probes in place. In some embodiments, the hydrogel or hydrogel monomers comprise photo-crosslinkable components. In some embodiments, exposure to light (e.g. UV or visible light) can further polymerize the hydrogel to stabilize the positions of the RCPs or capture probes. In some embodiments, this can allow RCPs or capture probes to remain in place in the absence of an electric field to maintain their positions. In some embodiments, electric field alignment is performed prior to hydrogel polymerization (e.g. while the hydrogel is still in a liquid or semi-cured state), and the hydrogel is polymerized (e.g. cured) once the RCPs or capture probes have been aligned.

[0137] Any suitable configuration of electrodes can be used for electric field alignment. In some aspects, the geometry and placement of electrodes can vary. In some embodiments, parallel plate electrodes can be used. In some embodiments, two parallel plate electrodes are positioned above and below the 3D hydrogel, respectively, to generate an electric field along the z-axis of the 3D hydrogel (e.g. vertically). In some embodiments, multiple planar electrodes are pattenered around the sides of the gel. In some embodiments, this can create more complex field distributions. In some embodiments, interdigitated electrode arrays are used to produce non-uniform fields for enhanced DEP effects. In some embodiments, the electrode material is transparent (e.g. for simultaneous viewing and/or imaging of the 3D hydrogel). In some embodiments, the electrode material is opaque. In some embodiments, the spacing between electrodes (e.g. based on the thickness of the hydrogel) can be adjusted. In some embodiments, the spacing of electrodes relative to the AC field wavelength can determine how many RCP layers are formed. In some embodiments, electrode surface coatings (e.g. passivation layers) are employed to prevent electrochemical reactions or to favor certain electrical field profiles. [0138] In some embodiments, the type of hydrogel and/or its viscosity or stiffness can be adjusted. The viscosity of a pre-cured solution comprising hydrogel monomers (or mesh size of the cured gel) will affect how easily RCPs move under the electric field. For example, a more viscous or tightly crosslinked gel will slow RCP movement (which might be useful for stability, but requires stronger fields or longer times to achieve alignment), whereas a looser gel permits faster movement. The dielectric properties of the hydrogel may also influence the DEP behavior of RCPs. In some embodiments, charged groups incorporated into the hydrogel provide a baseline uniform distribution (e.g., a uniformly charged gel could counteract some electrophoretic drift and make the layering crisper, depending on interactions). In some embodiments, the thickness of the hydrogel layer can be any suitable thickness. In some embodiments, a thicker gel can accommodate more layers, but may require more careful field tuning to ensure even alignment across depth.

[0139] In some embodiments, the RCPs or capture probes are modified for improved or enhanced manipulation within the hydrogel, such as by electric fields. In some embodiments, the RCPs or capture probes comprise or are attached to one or more tags (e.g. molecules) that enhance manipulation by electric fields. In some embodiments, the tag is a dieletric bead. In some embodiments, the tag is a nanoparticle. In some embodiments, the nanoparticle is a metal nanoparticle. In some embodiments, the tag is covalently attached. In some embodiments, the tag is non-covalently attached (e.g. via hybridization to a sequence in the RCP or capture probe). In some embodiments, the tag increases the polarizability and/or size of the RCPs or capture probes, allowing them to respond more strongly to an electric field (e.g. to the DC and/or AC), and/or improving DEP and alignment.

[0140] In some embodiments, the RCPs, capture probes, or moieties for attaching thereto, comprise or are attached to a magnetic tag (e.g. paramagnetic bead). In some embodiments, the magnetic tag facilitates manipulation of RCPs and capture probe arrangements by application of magnets or magnetic fields.

[0141] Any other suitable means for manipulating the positions of RCPs or capture probes within the 3D hydrogel, or moieties for attaching thereto, may be performed in accordance with the methods provided herein. III. Nucleic Acid Sequencing in 3D Hydrogels

[0142] In some aspects, once RCPs are generated and immobilized in the 3D hydrogel, the RCPs can be sequenced by any suitable sequencing method. Various non-limiting sequencing methods that may be adapted to the 3D hydrogel method provided herein can include, for example, sequencing by synthesis, sequencing by ligation, sequencing by binding, or sequencing by avidity.

[0143] In some embodiments, “sequencing” as used herein can comprise any of the sequencing methods provided herein. In some embodiments, “sequencing” comprises generating and/or detecting a plurality of signals that correspond to and/or are indicative of a plurality of nucleotides in a sequence of a nucleic acid, such as an RCP, for example as is performed in a sequencing-by- synthesis (SBS) method. In some embodiments, the plurality of signals are generated and detected in a plurality of sequential cycles of nucleic acid extension and imaging (e.g. as in SBS). In some embodiments, “sequencing” further comprises using (e.g. analyzing) the plurality of signals (e.g. fluorescent signals detected in a sequencing-by-synthesis method) to deduce sequences of nucleic acids in the 3D hydrogel (e.g. sequences of RCPs). However, in some embodiments, “sequencing” as used herein does not further comprise analyzing the signals.

[0144] In some embodiments, the sequencing is performed using a base-by-base sequencing method, e.g., sequencing-by-synthesis (SBS), sequencing-by-avidity (SBA) or sequencing-by-binding (SBB). In some embodiments, the 3D hydrogel is contacted with a sequencing primer and base-by-base sequencing is performed using a cyclic series of nucleotide incorporation or binding, respectively, thereby generating extension products of the sequencing primer, followed by removing, cleaving, or blocking the extension products of the sequencing primer.

[0145] In some embodiments, the sequencing is performed using sequencing by synthesis (SBS). In general, the sequencing-by-synthesis (SBS) method for sequencing nucleic acid molecules is based on incorporation of a fluorescent, reversibly terminated nucleotide into an extended priming strand, where the incorporated nucleotide is complementary to a nucleotide at the position of the template nucleic acid molecule that is being probed. Following imaging to detect incorporation of the nucleotide at the position, the reversible terminator and the fluorescent moiety are cleaved off the newly incorporated nucleotide before progressing to the next cycle of incorporation and imaging. Iterative rounds of incorporation and imaging facilitate base-by-base sequencing. Exemplary SBS methods comprise those described for example, but not limited to, US 2007/0166705, US 2006/0188901, US 7,057,026, US 2006/0240439, US 2006/0281109, US 2011/005986, US 2005/0100900, US 9,217,178, US 2009/0118128, US 2012/0270305, US 2013/0260372, and US 2013/0079232.

[0146] Generally in sequencing-by- synthesis methods, a first population of detectably labeled nucleotides (e.g., dNTPs) are introduced to contact a template nucleotide in a template nucleic acid hybridized to a sequencing primer, and a first detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by a polymerase to extend the sequencing primer in the 5’ to 3’ direction using a complementary nucleotide (a first nucleotide residue) in the template nucleic acid as template. A signal from the first detectably labeled nucleotide can then be detected. The first population of nucleotides may be continuously introduced, but in order for a second detectably labeled nucleotide to incorporate into the extended sequencing primer, nucleotides in the first population of nucleotides that have not incorporated into a sequencing primer are generally removed (e.g., by washing), and a second population of detectably labeled nucleotides are introduced into the reaction. Then, a second detectably labeled nucleotide (e.g., A, T, C, or G nucleotide) is incorporated by the same or a different polymerase to extend the already extended sequencing primer in the 5’ to 3’ direction using a complementary nucleotide (a second nucleotide residue) in the template nucleic acid as template. Thus, in some embodiments, cycles of introducing and removing detectably labeled nucleotides are performed in sequencing- by-synthesis.

[0147] In some embodiments, the base-by-base sequencing comprises using a polymerase that is fluorescently labeled. In some embodiments, the base-by-base sequencing comprises using a polymerase-nucleotide conjugate comprising a fluorescently labeled polymerase linked to a nucleotide moiety that is not fluorescently labeled. In some embodiments, the base-by-base sequencing comprises using a multivalent polymer-nucleotide conjugate comprising a polymer core, multiple nucleotide moieties, and one or more fluorescent labels.

[0148] In some embodiments, the sequencing is performed using sequencing by ligation (e.g. single molecule sequencing by ligation). In general, such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. Aspects and features involved in sequencing by ligation are described, for example, in Shendure et al. Science (2005), 309: 1728-1732, and in US 5,599,675; US 5,750,341; US 6,969,488; US 6,172,218; US and 6,306,597.

[0149] In some embodiments, the sequencing is performed by sequencing-by -binding (SBB). Various aspects of SBB are described in U.S. Pat. No. 10,655,176 B2, the content of which is herein incorporated by reference in its entirety. In some embodiments, SBB comprises performing repetitive cycles of detecting a stabilized complex that forms at each position along the template nucleic acid to be sequenced (e.g. a ternary complex that includes the primed template nucleic acid, a polymerase, and a cognate nucleotide for the position), under conditions that prevent covalent incorporation of the cognate nucleotide into the primer, and then extending the primer to allow detection of the next position along the template nucleic acid. In the sequencing-by-binding approach, detection of the nucleotide at each position of the template occurs prior to extension of the primer to the next position. Generally, the methodology is used to distinguish the four different nucleotide types that can be present at positions along a nucleic acid template by uniquely labelling each type of ternary complex (e.g., different types of ternary complexes differing in the type of nucleotide it contains) or by separately delivering the reagents needed to form each type of ternary complex. In some instances, the labeling may comprise fluorescence labeling of, e.g., the cognate nucleotide or the polymerase that participate in the ternary complex.

[0150] In some embodiments, the sequencing is performed by sequencing-by-avidity (SBA). Some aspects of SBA approaches are described in U.S. Pat. No. 10,768,173 B2, the content of which is herein incorporated by reference in its entirety. In some embodiments, SBA comprises detecting a multivalent binding complex formed between a fluorescently-labeled polymer-nucleotide conjugate, and a one or more primed target nucleic acid sequences (e.g., barcode sequences). Fluorescence imaging is used to detect the bound complex and thereby determine the identity of the N+l nucleotide in the target nucleic acid sequence (where the primer extension strand is N nucleotides in length). Following the imaging step, the multivalent binding complex is disrupted and washed away, the correct blocked nucleotide is incorporated into the primer extension strand, and the sequencing cycle is repeated.

[0151] In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET), as described for example in Levene et al., Science (2003), 299, 682-686, Lundquist et al., Opt. Lett. (2008), 33, 1026-1028, and Korlach et al., Proc. Natl. Acad. Sci. USA (2008), 105, 1176-1181.

[0152] In some embodiments, the analysis and/or sequence determination involves washing to remove unbound polynucleotides, thereafter revealing a fluorescent product for imaging.

[0153] In some embodiments, the sequencing is performed in 3 dimensions. For example, RCPs can be sequenced in layers at different depths of the 3D hydrogel (such as any of the layers described herein, including the first RCP layer, second RCP layer, and other RCP layers). In some embodiments, the RCP layers are defined and separated by spacer layers. In some embodiments, the layers in which sequencing of RCPs is performed are defined in 3D space within the 3D hydrogel, but the hydrogel comprises a homogenous distribution of RCPs.

[0154] In some embodiments, the RCPs can be sequenced in parallel. In some embodiments, subsets of RCPs can be sequenced in sequential rounds of sequencing. Sequencing different subsets of RCPs in sequential rounds of sequencing can be advantageous for reducing optical crowding and/or increasing the total density of RCPs that can be sequenced within the hydrogel. In some embodiments, an RCP detection step can be performed to determine the locations and/or density of RCPs and/or subsets of RCPs in the 3D hydrogel, for example prior to sequencing. The detection step may inform subsequent sequencing steps, such as whether to sequence all RCPs in parallel, or to sequence subsets of RCPs in sequential rounds of sequencing. The detection step may also be used for image registration purposes (e.g. to align images acquired in sequential imaging rounds of sequencing). In some embodiments, the RCP detection comprises any suitable method for visualizing the RCPs.

[0155] In some embodiments, different subsets of RCPs can be generated with different functional sequences that facilitate the sequential rounds of sequencing. For example, different subsets of the isolated nucleic acids can comprise different functional sequences that allow hybridization of different sequencing primers. The different functional sequences can be present in the isolated nucleic acids prior to circularization and/or being contacted with the 3D hydrogel. In another example, a first subset of immobilized capture probes comprises a first intervening sequence and a second subset of immobilized capture probes comprises a second intervening sequence; the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are circularized using as template either the first intervening sequence or second intervening sequence of the capture probes; and the RCA generates first RCPs comprising multiple copies of the first intervening sequence and second RCPs comprising multiple copies of the second intervening sequence. The first and second RCPs can be sequenced using different sequencing primers that hybridize to the copies of the first or second intervening sequence present in the first and second RCPs. The different sequencing primers can be used to sequence the first and second RCPs in separate (e.g. sequentially performed) rounds of sequencing (e.g. SBS). Alternatively, the different sequencing primers can be used to sequence the first and second RCPs simultaneously, for example if optical crowding is determined to not present a challenge in certain contexts.

[0156] In some embodiments, images acquired in sequential imaging rounds of sequencing (e.g. iterative rounds of base calling) are aligned, for example by image registration. In some embodiments, alignment and/or registration of images allows for a sequence of signals corresponding to a sequence of a nucleic acid (e.g. RCP) at a particular location in the 3D hydrogel to be associated with one another, and thereby for the sequence of the nucleic acid to be determined. In some embodiments, image alignment and/or registration can be facilitated by landmarks within the 3D hydrogel which remain consistent and can be included in sequential images. The landmarks can include RCPs themselves, or additional landmarks within the 3D hydrogel. In some embodiments, the landmarks include fiduciary markers on the solid support. In some embodiments, the landmarks include fiduciary markers within the 3D hydrogel. In some embodiments, the landmarks include beads, such as fluorescent beads within the 3D hydrogel. The landmarks, such as beads, can be distributed in a defined pattern or randomly within the 3D hydrogel. In some embodiments, the landmarks retain a consistent physical position within the 3D hydrogel and with relation to the RCPs being imaged and sequenced.

[0157] In some embodiments, sequencing the RCPs comprises determining sequences in the RCPs that correspond to sequences in the isolated nucleic acids from which the RCPs were generated. Thus, in some aspects, sequencing the RCPs comprises sequencing the isolated nucleic acids. In some aspects, determining the sequences of RCPs allows for the determination of sequences of the isolated nucleic acids. In some embodiments, the isolated nucleic acids comprise sequences corresponding to the native sequences of nucleic acids that are present in a biological sample from which the isolated nucleic acids were obtained or generated. In some embodiments, the isolated nucleic acids can further comprise additional sequences that are associated with the native sequences but that are not present in the sample, such as barcode sequences, unique molecular identifiers (UMIs), or other functional sequences. In some embodiments, the additional sequences are also sequenced. Thus, in some embodiments, sequencing the RCPs can allow for the identification of specific nucleic acids (e.g. transcripts) from a biological sample as well as information associated therewith.

[0158] In some aspects, the sequencing methods provided herein rely on imaging of the 3D hydrogel. In some embodiments, the imaging comprises 3D imaging, and/or imaging of multiple substantially flat layers arranged in 3 dimensions within the 3D hydrogel. In some embodiments, the imaging comprises fluorescent imaging. In some embodiments, the fluorescent imaging comprises epifluorescent imaging. In some embodiments, the fluorescent imaging comprises wide-field epifluorescent imaging. Any suitable imaging approach can be used. In some aspects, the imaging is carried out using any one of a number of different types of microscopy, e.g., confocal microscopy, two-photon microscopy, light- field microscopy, or light sheet microscopy. In some embodiments, the imaging comprises acquiring images representative of different depths within the 3D hydrogels, such as images of focal planes or optical sections. In some embodiments, the imaging comprises acquiring images of one or more focal planes. In some embodiments, the one or more focal planes correspond to one or more RCP layers. In some embodiments, the imaging comprises acquiring images of one or more optical sections. In some embodiments, the one or more optical sections correspond to one or more RCP layers. As used herein, optical section may refer to an image acquired via confocal microscopy, or may refer to an image of a focal plane acquired by a different mode of imaging, such as wide-field epifluorescence imaging.

[0159] In some embodiments, fluorescence microscopy is used. In some aspects, a fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. In fluorescence microscopy, a sample (e.g. the 3D hydrogel) is illuminated with light of a wavelength which excites fluorescence in the sample. The fluoresced light, which is usually at a longer wavelength than the illumination, is then imaged through a microscope objective. Two filters may be used in this technique; an illumination (or excitation) filter which ensures the illumination is near monochromatic and at the correct wavelength, and a second emission (or barrier) filter which ensures none of the excitation light source reaches the detector. Alternatively, these functions may both be accomplished by a single dichroic filter. The fluorescence microscope can be any microscope that uses fluorescence to generate an image, whether it is a more simple set up like an epifluorescence microscope, or a more complicated design such as a confocal microscope, which uses optical sectioning to obtain higher resolution of the fluorescent image.

[0160] In some embodiments, confocal microscopy is used. Confocal microscopy uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal. As only light produced by fluorescence very close to the focal plane can be detected, the image's optical resolution, particularly in the sample depth direction, is much better than that of wide-field microscopes. However, as much of the light from sample fluorescence is blocked at the pinhole, this increased resolution is at the cost of decreased signal intensity - so long exposures can be required. As only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (e.g., a rectangular pattern of parallel scanning lines). The achievable thickness of the focal plane is defined mostly by the wavelength of the used light divided by the numerical aperture of the objective lens, but also by the optical properties of the specimen. The thin optical sectioning possible makes these types of microscopes particularly well-suited for 3D imaging and surface profiling of samples.

[0161] Any suitable detectable labels can be used in connection with the methods provided herein. A detectable label can comprise a directly or indirectly detectable moiety, e.g., any fluorophores, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.

[0162] A fluorophore can comprise a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range. Particular examples of labels that may be used in accordance with the provided embodiments comprise, but are not limited to phycoerythrin, Alexa dyes, fluorescein, YPet, CyPet, Cascade blue, allophycocyanin, Cy3, Cy5, Cy7, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, biotin, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase, chloramphenical acetyl transferase, and urease.

[0163] Examples of detectable labels comprise but are not limited to various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, protein-protein binding pairs and protein- antibody binding pairs. Examples of fluorescent proteins comprise, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.

[0164] Examples of bioluminescent markers comprise, but are not limited to, luciferase (e.g., bacterial, firefly and click beetle), luciferin, aequorin and the like. Examples of enzyme systems having visually detectable signals comprise, but are not limited to, galactosidases, glucorimidases, phosphatases, peroxidases and cholinesterases. Identifiable markers also comprise radioactive compounds such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H. Identifiable markers are commercially available from a variety of sources.

[0165] Examples of fluorescent labels and nucleotides and/or polynucleotides conjugated to such fluorescent labels comprise those described in, for example, Hoagland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227- 259 (1991). In some embodiments, exemplary techniques and methods methodologies applicable to the provided embodiments comprise those described in, for example, US 4,757,141, US 5,151,507 and US 5,091,519, all of which are herein incorporated by reference in their entireties. In some embodiments, one or more fluorescent dyes are used as labels for labeled target sequences, for example, as described in US 5,188,934 (4,7-dichlorofluorescein dyes); US 5,366,860 (spectrally resolvable rhodamine dyes); US 5,847,162 (4,7- dichlororhodamine dyes); US 4,318,846 (ether-substituted fluorescein dyes); US 5,800,996 (energy transfer dyes); US 5,066,580 (xanthine dyes); and US 5,688,648 (energy transfer dyes), all of which are herein incorporated by reference in their entireties. Labeling can also be carried out with quantum dots, as described in US 6,322,901, US 6,576,291, US 6,423,551, US 6,251,303, US 6,319,426, US 6,426,513, US 6,444,143, US 5,990,479, US 6,207,392, US 2002/0045045 and US 2003/0017264, all of which are herein incorporated by reference in their entireties. In some embodiments, a fluorescent label comprises a signaling moiety that conveys information through the fluorescent absorption and/or emission properties of one or more molecules. Exemplary fluorescent properties comprise fluorescence intensity, fluorescence lifetime, emission spectrum characteristics and energy transfer.

[0166] Examples of commercially available fluorescent nucleotide analogues readily incorporated into nucleotide and/or polynucleotide sequences comprise, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, N.J.), fluorescein- 12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY TMTR-14- dUTP, RHOD AMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™ 630/650- 14-dUTP, BODIPY™ 650/665- 14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546- 14-dUTP, fluorescein- 12-UTP, tetramethylrhodamine-6-UTP, TEXAS RED™-5- UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR- 14-UTP, BODIPY™ TR-14-UTP, RHOD AMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, and ALEXA FLUOR™ 546- 14-UTP (Molecular Probes, Inc. Eugene, Oreg.). Methods are known for custom synthesis of nucleotides having other fluorophores (See, Henegariu et al. (2000) Nature Biotechnol. 18:345).

[0167] Other fluorophores available for post-synthetic attachment comprise, but are not limited to, ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg.), Cy2, Cy3.5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, N.J.). FRET tandem fluorophores may also be used, comprising, but not limited to, PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE- Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), and APC-Alexa dyes. [0168] In some cases, metallic silver or gold particles may be used to enhance signal from fluorescently labeled nucleotide and/or polynucleotide sequences (Lakowicz et al. (2003) Bio Techniques 34:62).

[0169] Biotin, or a derivative thereof, may also be used as a label on a nucleotide and/or a polynucleotide sequence, and subsequently bound by a detectably labeled avidin/streptavidin derivative (e.g., phycoerythrin-conjugated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently bound by a detectably labeled anti-digoxigenin antibody (e.g., fluoresceinated anti-digoxigenin). An aminoallyl-dUTP residue may be incorporated into a polynucleotide sequence and subsequently coupled to an N-hydroxy succinimide (NHS) derivatized fluorescent dye. In general, any member of a conjugate pair may be incorporated into a detection polynucleotide provided that a detectably labeled conjugate partner can be bound to permit detection. Other suitable labels for a polynucleotide sequence may comprise fluorescein (FAM), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), and phosphor- amino acids (e.g., P-tyr, P-ser, P-thr). In some embodiments the following hapten/antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin/a-biotin, digoxigenin/a- digoxigenin, dinitrophenol (DNP)/a-DNP, 5-Carboxyfluorescein (FAM)/a-FAM.

[0170] In some embodiments, a nucleotide and/or a oligonucleotide sequence is indirectly labeled, especially with a hapten that is then bound by a capture agent, e.g., as disclosed in US 5,344,757, US 5,702,888, US 5,354,657, US 5,198,537 and US 4,849,336, and US 5,073,562, all of which are herein incorporated by reference in their entireties. Many different hapten-capture agent pairs are available for use. Exemplary haptens comprise, but are not limited to, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, Cy5, and digoxigenin. For biotin, a capture agent may be avidin, streptavidin, or antibodies. Antibodies may be used as capture agents for the other haptens (many dye-antibody pairs being commercially available, e.g., Molecular Probes, Eugene, Oreg.).

IV. Isolated Nucleic Acids and Sequencing Libraries

[0171] In some aspects, the isolated nucleic acids are provided as isolated nucleic acid libraries. In some aspects, isolated nucleic acid libraries can be provided and/or generated in any suitable format. In some aspects, isolated nucleic acid libraries can be considered as “sequencing libraries” that can be prepared and sequenced according to any of the methods provided herein. The methods provided herein are widely compatible with virtually any sequencing library that can be used to generate a rolling circle amplification product. This includes sequencing libraries that are not traditionally provided in the form of circular nucleic acid molecules, since one skilled in the art of molecular biology and cloning can readily circularize a library of linear nucleic acids and/or add adapters that provide compatibility with the methods provided herein (e.g. a target sequence for hybridizing to a capture probe). In some embodiments, the sequencing library is a next-generation sequencing library. Isolated nucleic acid sequencing libraries can be generated for interrogation of various types of biological information. For example, isolated nucleic acid sequencing libraries may be used for genomic or transcriptomic sequencing, either at the bulk or single-cell level. In some embodiments, the isolated nucleic acids comprise a single-cell sequencing library. In some embodiments, a singlecell sequencing library is a library that can be sequenced to determine the presence and/or abundance of analytes in each of a plurality of different cells, at the individual cell level. In some aspects, a molecule of a single-cell sequencing library contains information regarding both an analyte and its cell of origin. In some embodiments, the single-cell sequencing library can be generated from (e.g. contain barcoded nucleic acid molecules generated from) any suitable number of cells. In some embodiments, the single-cell sequencing library is generated from at least 100 cells. In some embodiments, the single-cell sequencing library is generated from at least 1,000 cells. In some embodiments, the single-cell sequencing library is generated from at least 10,000 cells. In some embodiments, the single-cell sequencing library is generated from at least 100,000 cells. In some embodiments, the single-cell sequencing library is generated from at least 1,000,000 cells. In some embodiments, both the isolated nucleic acids and RCPs generated therefrom can be considered as sequencing libraries. In some embodiments, the isolated nucleic acids comprise a sequencing library. In some embodiments, the RCPs comprise a sequencing library. In some embodiments, a sequencing library of RCPs is generated from a sequencing library of isolated nucleic acids. Provided herein are exemplary embodiments of how such libraries may be generated and/or sequenced.

[0172] In some aspects, the sequencing libraries can be generated according to methods described in and/or adapted from any of the following publications: US Patent Application Publication No. US20240002914A1; US Patent No. 9,701,998; US Patent No. 10,400,280; US Patent No. 10,344,329; US Patent Application Publication No. US20180105808A1; and US Patent Application Publication No. US20220025438A1, each of which is incorporated by reference in its entirety herein.

A. DNA sequencing libraries

[0173] In some embodiments, the library of RCPs comprises a DNA sequencing library. In some embodiments, the library of RCPs comprises a single-cell DNA sequencing library. In some embodiments, the isolated nucleic acids comprise a DNA sequencing library. In some embodiments, the isolated nucleic acids comprise a single-cell DNA sequencing library. In some embodiments, provided herein are sequencing libraries for sequencing DNA. In some embodiments, the DNA is genomic DNA. In some embodiments, the DNA is cell-free DNA or synthetic DNA. In some embodiments, provided herein are sequencing libraries for sequencing genomic DNA. In some embodiments, DNA (e.g. genomic DNA) can be isolated, fragmented, and incorporated into circular or circularizable molecules, which may serve as templates for the generation of RCPs as provided herein. In some embodiments, the RCPs are immobilized in 3D hydrogels and sequenced according to any of the methods provided herein. In some embodiments, the genomic DNA fragments are barcoded (e.g. with partition-specific and/or single-cell barcodes) by any suitable means. In some embodiments, the DNA fragments are generated in a tagmentation reaction (e.g. in an assay for analyzing chromatin accessibility).

[0174] In some aspects, provided below is an exemplary, non-limiting workflow for generating a genomic DNA sequencing library.

[0175] Genomic DNA is isolated from a sample and digested with one or more restriction enzymes to generate gDNA fragments. In some embodiments, the digestion occurs in 2 parallel reactions, each with a different restriction enzyme. In some embodiments, different restriction enzymes can have different densities of target sequences at different portions of the genome. Thus, using two different restriction enzymes can improve the generation of appropriately sized gDNA fragments across the entire genome and improve coverage in downstream sequencing steps. For example, the restriction enzyme MluCI cuts double- stranded DNA at the palindromic sequence AATT, providing improved coverage at AT-rich regions. The restriction enzyme Sau3AI cuts double-stranded DNA at the palindromic sequence GATC, providing improved coverage at GC-rich regions, for example in comparison to MluCI. In some embodiments, the two different restriction enzymes are MluCI and Sau3AI. Any suitable restriction enzymes or combinations thereof can be used. [0176] Following digestion, the gDNA fragments are ligated to adapters to form adapter-ligated gDNA fragments, for example utilizing the overhangs generated by digestion with the one or more restriction enzymes to facilitate adapter hybridization and ligation. In some embodiments, the adapters comprise one or more features to facilitate downstream processing, as described below. In some embodiments, the adapters are “Y-adapters”. In some embodiments, a Y-adapter comprises two nucleic acid (e.g. DNA) molecules, which form: 1) a double-stranded region formed by hybridization between the two nucleic acid molecules, and 2) non- complementary arms. In some embodiments, a Y-adapter further comprises an overhang adjacent to the double-stranded region, which hybridizes to the gDNA fragments via the gDNA fragment overhangs generated as a result of enzymatic digestion.

[0177] In some embodiments, after adapter ligation, the adapter-ligated gDNA fragments are amplified, such as in a limited cycle PCR (e.g. 4 cycles), to generate doublestranded amplification products.

[0178] In some embodiments, the double-stranded amplification products are digested with a restriction enzyme to generate complementary overhangs at opposite ends of each double-stranded amplification product. In some embodiments, the complementary overhangs are then used to circularize the double- stranded amplification products (e.g. via hybridization and ligation), thereby generating double-stranded circularized molecules. In some embodiments, the Y-adapters comprise sequences that are used as template in the amplification, and which encode targets for the restriction enzyme to generate the complementary overhangs.

[0179] In some embodiments, a purification step can be performed to remove unwanted small digested fragments and/or larger unintended products of the circularization reaction.

[0180] In some embodiments, the double-stranded circularized molecules are used to generate single-stranded circular molecules that can serve as templates for RCA. In some embodiments, the double-stranded circularized molecules are contacted with a nicking endonuclease, which facilitates single- stranded cleavage to generate nicked DNA. Following nicking, the nicked strand can be removed. In some embodiments, the nicked strand is removed by enzymatic digestion, for example with T5 exonuclease, yielding a single- stranded circular molecules. [0181] In some embodiments, the double-stranded circularized molecules can be cleaved on a specific strand of the double strand. In some embodiments, this can be facilitated by different nicking endonucleases that target different single- stranded nicking sites. The different single- stranded sites can be encoded by the Y-adapters and generated upon the amplification reaction to generate the double- stranded amplification product. The different single- stranded nicking sites can be arranged such that they yield nicking events on different strands of the double-stranded circularized molecules. Exemplary nicking endonucleases that can be used include Nt.BspQI (which nicks at GCTCTTCN sequences), and Nb.BbvCI (which nicks at GCTGAGG). In some embodiments, nicking and removal of different strands in separate reactions allows for downstream amplification and sequencing of both strands of a given gDNA fragment, for example using RCA primers that are complementary to the different strands.

[0182] In some embodiments, the method further comprises using the single- stranded circular molecules as template in an RCA reaction to generate RCPs. In some embodiments, the RCPs are immobilized in a 3D hydrogel and sequenced according to any of the methods provided herein.

B. RNA sequencing libraries

[0183] In some embodiments, the library of RCPs comprises an RNA sequencing library. In some embodiments, the isolated nucleic acids comprise an RNA sequencing library. In some embodiments, provided herein are sequencing libraries for RNA sequencing. In some embodiments, the RNA comprises mRNA. In some embodiments, the RNA comprises noncoding RNA. In some embodiments, the RNA comprises CRISPR guide RNA.

[0184] In some embodiments, any suitable RNA (e.g. mRNA) sequencing library can be adapted to the methods provided herein. In some embodiments, the RNA sequencing library is an mRNA sequencing library. In some embodiments, the RNA sequencing library is a singlecell RNA sequencing library. In some embodiments, the RNA sequencing library is a single-cell mRNA sequencing library. For example, a given molecule of the RNA sequencing library can comprise information (e.g. in the form of barcode sequences) that identifies the cell of origin of a given RNA sequence or complement thereof. In some embodiments, the RNA sequencing library is generated according to any suitable method. Various methods available to those skilled in the art may be used to generate such single-cell sequencing libraries, for example as described in the various references cited above in this section.

[0185] In an exemplary embodiment, cells are co-partitioned with beads comprising a plurality of nucleic acid barcode molecules, each comprising a partition- specific barcode. mRNAs are released from the cells, and the mRNAs and nucleic acid barcode molecules are used to generate barcoded nucleic acid molecules, each barcoded nucleic acid molecule comprising: 1) a sequence of an mRNA molecule or complement thereof, and 2) a partition-specific barcode or complement thereof. In some embodiments, a poly A sequence of an mRNA molecule hybridizes to a poly-T sequence of a barcoded nucleic acid molecule and an extension reaction is performed to generate a barcoded nucleic acid molecule. In some embodiments, template switching is performed to generate the barcoded nucleic acid molecule. For example, in some embodiments, a primer comprising a poly-T sequence is hybridized to an mRNA molecule in the partition and is extended using a reverse transcriptase capable of adding non-templated 3’ nucleotides, the non-templated 3’ nucleotides hybridize to a nucleic acid barcode molecule, and a further extension incorporates a complement of the nucleic acid barcode molecule.

[0186] In some embodiments, the molecules of the RNA sequencing library are circularized to generate circular templates. In some embodiments, the circular templates are amplified by RCA to generate RCPs. In some embodiments, the RCPs are immobilized in 3D hydrogels and sequenced according to any of the methods provided herein.

C. Expression Profiling

[0187] In some embodiments, the library of RCPs comprises a gene expression sequencing library. In some embodiments, the library of RCPs comprises a single-cell gene expression sequencing library. In some embodiments, the isolated nucleic acids can comprise a sequencing library for analysis of gene expression. In some embodiments, the isolated nucleic acids can comprise a sequencing library for analysis of expression of one or more genes, products thereof (e.g. mRNA), and/or other molecules (e.g. proteins) from a biological sample, such as a cell or tissue.

[0188] In some embodiments, the sequencing library is generated by a method involving ligatable probes or probe pairs that are ligated in the presence of specific analytes (e.g. mRNA molecules) in a biological sample, such as a cell, and thereby detected in downstream steps. Thus, in some embodiments, the ligatable probe pairs are analyte- specific ligatable probe pairs. In some embodiments, the ligatable probe pairs can comprise or can be configured according to any of the probes or probe sets described, for example, in US Patent Application Publication No. US20240002914A1, which is herein incorporated herein in its entirety. In some embodiments, the ligatable probe pairs are used to generate a single-cell sequencing library.

[0189] In some embodiments, a ligatable probe pair comprises a first probe molecule and a second probe molecule. In some embodiments, the first probe molecule comprises a first hybridization region that hybridizes to a first sequence of a target analyte (e.g. mRNA) and the second probe molecule comprises a second hybridization region that hybridizes to a second sequence of the target analyte. In some embodiments, the first and second probe molecules hybridized to the target analyte are ligated. In some embodiments, the ligated ligatable probe pair can be referred to as a ligated probe pair. In some embodiments, the ligation is templated by the target analyte. In some embodiments, the ligation is RNA-templated DNA ligation (e.g. when the target analyte is mRNA and the probe molecules comprise DNA). In some embodiments, the first sequence of the target analyte and second sequence of the target analyte are adjacent, and the first and second probe molecules are ligated without performing a gap-filling step prior to ligation. In some embodiments, the first and second sequence of the target analyte are not adjacent (e.g. are separated by one or more intervening nucleotides), and a gap-filling step (e.g. an extension reaction) is performed prior to the ligation. In some embodiments, a plurality (e.g. library) of ligatable probe pairs are contacted with the sample, allowed to hybridize to a plurality of different target analytes (e.g. mRNAs), and ligated using the target analytes as template. In some embodiments, unligated probe molecules of the ligatable probe pairs are removed (e.g. by one or more wash steps).

[0190] In some embodiments, the ligatable probe pairs comprise one or more overhangs. In some embodiments, the one or more overhangs of a ligatable probe pair comprise one or more barcodes, such as a sample- specific barcode and/or one or more barcodes that alone or in combination correspond to and/or identify the analyte. In some embodiments, the one or more overhangs can be used to capture, extend, and/or further ligate the ligatable probe pair to append additional sequences, which may include a cell-specific and/or partition-specific barcode, and/or unique molecular identifier (UMI) sequence. In some embodiments, the one or more overhangs comprise one or more additional functional sequences, such as a unique molecular identifier (UMI), a capture sequence, a primer sequence or primer binding site (e.g., a R1/R2 sequence), and/or a target region (e.g. a target region complementary to a capture sequence, as described in detail elsewhere herein).

[0191] In some embodiments, the ligatable probe pairs are used to generate a singlecell sequencing library. In some embodiments, the ligatable probe pairs are ligated in cells (e.g. fixed cells), and are subsequently partitioned and barcoded with a partition- specific barcode. For example, in some embodiments, after the ligation, a cell is co-partitioned (e.g. in microwells or droplets) along with a plurality of nucleic acid barcode molecules. The plurality of nucleic acid barcode molecules comprise a partition- specific barcode, for example a barcode sequence that is common to the nucleic acid barcode molecules in the partition, and/or that is different from partition- specific barcodes present in other partitions. In some embodiments, the ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic acid barcode molecules are used to generate a barcoded nucleic acid molecule (e.g. a barcoded ligated probe pair) comprising: 1) the partition- specific barcode or a complement thereof, and 2) a sequence of the ligated probe pair or a complement thereof. In some embodiments, the barcoded nucleic acid molecule comprises a sequence that identifies the analyte (e.g. the ligated hybridization regions), and a sequence that identifies the partition and/or cell of origin (e.g. the partition- specific barcode). Thus, in some embodiments, a plurality of barcoded nucleic acid molecules comprises a single-cell sequencing library that can be sequenced to assess gene expression at the level of single cells for a plurality of cells. In some embodiments, generation of the barcoded nucleic acid molecule comprises ligation. In some embodiments, generation of the barcoded nucleic acid molecule comprises hybridization and extension.

[0192] In an exemplary embodiment, cells comprising ligated probe pairs are copartitioned with beads comprising a plurality of nucleic acid barcode molecules, each nucleic acid barcode molecule comprising a partition-specific barcode (which may also be referred to as a bead-specific or cell-specific barcode). The beads can be any suitable bead, such as a gel bead (e.g. hydrogel bead). In some embodiments, the nucleic acid barcode molecules are released from the bead upon partitioning or after partitioning and upon provision of a stimulus that releases the nucleic acid barcode molecules from the bead. In some embodiments, the nucleic acid barcode molecules are not released from the bead in the partition. The ligated probe pairs are released from the cells. The ligated probe pairs and nucleic acid barcode molecules are used to generate barcoded nucleic acid molecules, each barcoded nucleic acid molecule comprising: 1) a sequence of a ligated probe pair or complement thereof, and 2) a partition-specific barcode or complement thereof. In some embodiments, a ligated probe pair hybridizes to a nucleic acid barcode molecule and an extension reaction is performed to generate a barcoded nucleic acid molecule. In some embodiments, the extension reaction extends the nucleic acid barcode molecule using the ligated probe pair as template, and/or the extension reaction extends the ligated probe pair using the nucleic acid barcode molecule as template. In some embodiments, a ligated probe pair is ligated to a nucleic acid barcode molecule to generate a barcoded nucleic acid molecule. In some embodiments, the ligation is templated by a splint nucleic acid.

[0193] In some embodiments, the nucleic acid barcode molecule comprises a partition- specific barcode, a unique molecular identifier (UMI), and/or one or more functional sequences for downstream processing. In some embodiments, the nucleic acid barcode molecule comprises a primer sequence or primer binding site (e.g., a R1/R2 sequence), and/or a target region (e.g. a target region complementary to a capture sequence of a capture probe, as described in detail elsewhere herein). In some embodiments, any and/or all of the sequences of the nucleic acid barcode molecule are incorporated into the barcoded nucleic acid molecule (e.g. barcoded ligated probe pair).

[0194] In some embodiments, the ligatable probe pairs or products thereof comprise sample- specific barcode regions that correspond to different samples, which may be assayed in parallel. In some embodiments, this can allow for ligatable probe pairs from different samples to be pooled for downstream processing steps while retaining information about the sample of origin.

[0195] In some embodiments, a plurality (e.g. library) of ligatable probe pairs is contacted with the sample. In some embodiments, the plurality of ligatable probe pairs target a plurality of different analytes (e.g. mRNA molecules). In some embodiments, the plurality of ligatable probe pairs target at least 10, at least 100, at least 1,000, at least 10,000, at least 20,000, or at least 30,000 analytes. In some embodiments, at least 10, at least 100, at least 1,000, at least 10,000, at least 20,000, or at least 30,000 ligatable probe pairs are contacted with the biological sample. In some embodiments, the ligatable probe pairs collectively target a transcriptome. In some embodiments, the ligatable probe pairs target mRNA molecules and are suitable for gene expression profiling (e.g. transcriptomic profiling). In some embodiments, the plurality of ligatable probe pairs is a library of ligatable probe pairs. In some embodiment, the plurality of ligatable probe pairs is used to generate a library of corresponding RCPs, which is sequenced to identify one or more of the analytes targeted by the plurality of ligatable probe pairs.

[0196] In some embodiments, the ligated probe pair or a product thereof (e.g. the barcoded nucleic acid molecule) is circularized to generate a circular template. Any suitable method can be performed for the circularization. In some embodiments, the ligatable probe pair or product thereof is circularized using a splint molecule. In some embodiments, the splint molecule hybridizes to sequences at the 3’ end and 5’ end of the ligated probe pair or product thereof (e.g. barcoded nucleic acid molecule) and acts as a template for circularization (e.g. by ligation with or without gap-fill prior to ligation). In some embodiments, the circular template generated from the ligated probe pair or product thereof then serves as a template for RCA to generate RCPs in accordance with any of the methods provided herein. In some embodiments, the RCPs are distributed and/or arranged in a 3D hydrogel according to any of the embodiments provided herein.

[0197] In some embodiments, the ligated probe pair or product thereof (e.g. barcoded nucleic acid molecule) comprises a target region that is complementary to and/or hybridizes to a capture sequence of a capture probe, for example as described in detail elsewhere herein. In some embodiments, the target region is a split target region. In such embodiments, the capture probe can serve as a ligation template and act as an RCA primer to generate the RCP, for example within the 3D hydrogel.

[0198] In some embodiments, ligatable probe pairs described above can alternatively be designed comprising more than 2 probes. In some embodiments, the components of the ligatable probe pairs described above can be provided as a single circularizable molecule that is circularized upon the ligation, for example in the form of a padlock probe comprising hybridization regions that are ligated using the target analyte as template and a backbone comprising the one or more barcode sequences corresponding to the analyte. In such embodiments, ligation forms the circular template that is used in the downstream RCA.

[0199] In some embodiments, the RCPs are immobilized. In some embodiments, the RCPs are immobilized in a 3D hydrogel, such as any provided herein. In some embodiments, the RCPs are immobilized in a 3D hydrogel in a particular density and/or arrangement, for example according to any of the methods provided herein. In some embodiments, the RCPs are sequenced. In some embodiments, the sequencing comprises detecting one or more signals from the RCPs corresponding to one or more nucleotides in a sequence of the RCPs, such as in a sequencing-by- synthesis reaction.

[0200] FIG. 14 illustrates an exemplary embodiment of utilizing a ligatable probe pair to generate a ligated probe pair corresponding to a target analyte (shown as a target mRNA), for example as provided herein, and generating a barcoded nucleic acid molecule. The ligatable probe pair comprises a first probe molecule and a second probe molecule. The first probe molecule comprises a first hybridization region that is complementary to a first sequence in the target mRNA (these complementary sequences are shown with diagonal lines), and the second probe molecule comprises a second hybridization region that is complementary to a second sequence in the target mRNA (these complementary sequences are shown with vertical lines). The ligatable probe pair further comprises overhang regions, which may comprise barcodes (e.g. sample- specific barcodes) and/or other functional sequences as described herein. The ligatable probe pair is contacted with a biological sample (e.g. cell) comprising the target mRNA. The ligatable probe pair hybridizes to the target mRNA and is ligated to generate a ligated probe pair. The cell comprising the ligated probe pair is partitioned with a plurality of nucleic acid barcode molecules, and the ligated probe pair and a nucleic acid barcode molecule of the plurality of nucleic barcode molecules are used to generate a barcoded nucleic acid molecule comprising: 1) a sequence of the ligated probe pair or complement thereof, and 2) a sequence of the nucleic acid barcode molecule or complement thereof. The barcoded nucleic acid molecule can be generated by ligation, or by hybridization and extension. The barcoded nucleic acid molecule is circularized, and used as a template in an RCA reaction to generate an RCP. The RCP is immobilized in a 3D hydrogel and sequenced according to any of the methods provided herein. V. Samples, Analytes, and Target Sequences

A. Samples

[0201] In some aspects, the isolated nucleic acids provided herein are from a sample, such as a biological sample. In some aspects, analysis of the sample comprises isolation and/or sequencing of nucleic acids from the sample. In some aspects, analysis of the sample comprises sequencing of nucleic acids obtained and/or generated from the biological sample. The sample can be or be derived from any biological sample. Methods and compositions disclosed herein may be used for analyzing a biological sample, which may be obtained from a subject using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject. In addition to the subjects described above, a biological sample can be obtained from a prokaryote such as a bacterium, an archaea, a virus, or a viroid. A biological sample can also be obtained from non-mammalian organisms (e.g., a plant, an insect, an arachnid, a nematode, a fungus, or an amphibian). A biological sample can also be obtained from a eukaryote, such as a tissue sample, a patient derived organoid (PDO) or patient derived xenograft (PDX). A biological sample from an organism may comprise one or more other organisms or components therefrom. For example, a mammalian tissue section may comprise a prion, a viroid, a virus, a bacterium, a fungus, or components from other organisms, in addition to mammalian cells and non-cellular tissue components. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a pre-disposition to a disease, and/or individuals in need of therapy or suspected of needing therapy.

[0202] The biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei). The biological sample can include nucleic acids (such as DNA or RNA), proteins/polypeptides, carbohydrates, and/or lipids. In some embodiments, the biological sample is obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate. In some embodiments, the biological sample is or comprise a cell pellet or a section of a cell pellet. In some embodiments, the biological sample is or comprise a cell block or a section of a cell block. The sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions. In some embodiments, the biological sample may comprise cells which are deposited on a surface.

[0203] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms. Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells. Biological samples can also include fetal cells and immune cells.

[0204] A variety of steps can be performed to prepare or process a biological sample for and/or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for analysis.

[0205] A biological sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro on a growth substrate or culture dish as a population of cells, and prepared for analysis. Grown samples, and samples obtained via biopsy or sectioning, can be prepared as tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome. Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade.

[0206] In some embodiments, the biological sample is prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (e.g., the physical characteristics) of the tissue structure and/or components thereof (e.g. nucleic acids). In some embodiments, the biological sample can be prepared using formalin-fixation and paraffin-embedding (FFPE), which are established methods. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formalin-fixation and paraffin-embedding. As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives prior to analysis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0207] In some embodiments, the methods provided herein comprises one or more post-fixing (also referred to as postfixation) steps.

[0208] In some embodiments, a biological sample is permeabilized to facilitate transfer of species, for example to facilitate transfer of nucleic acids out of the sample. In general, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100™ or Tween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample is incubated with a cellular permeabilizing agent to facilitate permeabilization of the sample. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in connection with the samples described herein.

[0209] In some embodiments, the biological sample can be permeabilized by any suitable methods. For example, one or more lysis reagents can be added to the sample. Examples of suitable lysis agents include, but are not limited to, bioactive reagents such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be added to the biological sample to facilitate permeabilization. For example, surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chao tropic agents.

[0210] Additional reagents can be added to a biological sample to perform various functions prior to analysis of the sample (e.g. isolation and sequencing of nucleic acids). In some embodiments, DNase and RNase inactivating agents or inhibitors such as proteinase K, and/or chelating agents such as EDTA, is added to the sample.

B. Analytes

[0211] A biological sample may comprise one or a plurality of analytes of interest. Methods for performing multiplexed assays to analyze two or more different analytes in a single biological sample are provided. The methods and compositions disclosed herein can be used to detect and analyze a wide variety of different analytes, for example so long as the analytes are associated with a nucleic acid that can be sequenced in accordance with the methods provided herein. In some aspects, an analyte can include any biological substance, structure, moiety, or component to be analyzed. In some aspects, a target disclosed herein may similarly include any analyte of interest. In some embodiments, an analyte is an isolated nucleic acid molecule as provided herein. In some embodiments, an analyte is used to generate an isolated nucleic acid molecule as provided herein. In some embodiments, a plurality of analytes is a library of isolated nucleic acids as provided herein. In some embodiments, a plurality of analytes is used to generate a library of isolated nucleic acids as provided herein. In some embodiments, a plurality of analytes is used to generate a library of RCPs as provided herein.

[0212] Analytes can be derived from a specific type of cell and/or a specific sub- cellular region. For example, analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell. Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis, and/or allow access of one or more reagents to the analytes in the cell or cell compartment or organelle.

[0213] The analyte may include any biomolecule or chemical compound, including a macromolecule such as a protein or peptide, a lipid or a nucleic acid molecule, or a small molecule, including organic or inorganic molecules. The analyte may be a cell or a microorganism, including a virus, or a fragment or product thereof. An analyte can be any substance or entity for which a specific binding partner (e.g. an affinity binding partner) can be developed, and which results in a product that can be detected via the sequencing methods provided herein.

[0214] Analytes of particular interest may include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g. mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and/or modified nucleic acid molecules, (e.g. including nucleic acid domains comprising or consisting of synthetic or modified nucleotides such as LNA, PNA, morpholino, etc.), proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof, or a lipid or carbohydrate molecule, or any molecule which comprise a lipid or carbohydrate component. The analyte may be a single molecule or a complex that contains two or more molecular subunits, e.g. including but not limited to protein-DNA complexes, which may or may not be covalently bound to one another, and which may be the same or different. Thus in addition to cells or microorganisms, such a complex analyte may also be a protein complex or protein interaction. Such a complex or interaction may thus be a homo- or hetero-multimer. Aggregates of molecules, e.g. proteins may also be target analytes, for example aggregates of the same protein or different proteins. The analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNA or RNA, e.g. interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNA or RNA.

(i) Endogenous Analytes

[0215] In some embodiments, an analyte herein is endogenous to a biological sample and can include nucleic acid analytes and non-nucleic acid analytes. Methods and compositions disclosed herein can be used to analyze nucleic acid analytes and/or non-nucleic acid analytes (e.g., using a labeling agent that comprises a reporter oligonucleotide and binds directly or indirectly to a non-nucleic acid analyte) in any suitable combination.

[0216] Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte is inside a cell or on a cell surface, such as a transmembrane analyte or one that is attached to the cell membrane. In some embodiments, the analyte is an organelle (e.g., nuclei or mitochondria). In some embodiments, the analyte is an extracellular analyte, such as a secreted analyte. Exemplary analytes include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.

[0217] Examples of nucleic acid analytes include DNA analytes such as singlestranded DNA (ssDNA), double- stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA/DNA hybrids. The DNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as mRNA) present in a tissue sample. [0218] Examples of nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA. Examples of the different types of RNA analytes include messenger RNA (mRNA), including a nascent RNA, a pre-mRNA, a primary-transcript RNA, and a processed RNA, such as a capped mRNA (e.g., with a 5’ 7-methyl guanosine cap), a polyadenylated mRNA (poly- A tail at the 3’ end), and a spliced mRNA in which one or more introns have been removed. Also included in the analytes disclosed herein are non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. The RNA analyte can be a transcript of another nucleic acid molecule (e.g., DNA or RNA such as viral RNA) present in a tissue sample. Examples of a non-coding RNAs (ncRNA) that is not translated into a protein include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), as well as small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), small Cajal body-specific RNAs (scaRNAs), and the long ncRNAs such as Xist and HOTAIR. The RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include 5.8S ribosomal RNA (rRNA), 5S rRNA, tRNA, miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). The RNA can be double-stranded RNA or single- stranded RNA. In some embodiments, the RNA comprises circular RNA. In some embodiments, the RNA is a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).

[0219] In some embodiments described herein, an analyte may be a denatured nucleic acid, wherein the resulting denatured nucleic acid is single-stranded. The nucleic acid may be denatured, for example, optionally using formamide, heat, or both formamide and heat. In some embodiments, the nucleic acid is not denatured for use in a method disclosed herein.

[0220] Methods and compositions disclosed herein can be used to analyze any number of analytes. For example, the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes. (ii) Labeling Agents

[0221] In some embodiments, provided herein are methods and compositions for analyzing endogenous analytes (e.g., RNA, ssDNA, cell surface or intracellular proteins, and/or metabolites) from a sample using one or more labeling agents. In some embodiments, an analyte labeling agent may include an agent that interacts with an analyte (e.g., an endogenous analyte in a sample). In some embodiments, the labeling agents can comprise a reporter oligonucleotide that is indicative of the analyte or portion thereof interacting with the labeling agent. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labeling agent. In some embodiments, the labeling agent can be identified as having been within the biological sample (and optionally having bound to the analyte in the biological sample) by sequencing the reporter oligonucleotide (or any other oligonucleotide sequence associated with the labeling agent) in a downstream sequencing step, such as any provided herein. For example, in some embodiments, the labeling agent can be contacted with the biological sample and allowed to bind analyte. Unbound labeling agent can be removed from the sample (e.g. by washing). The remaining bound labeling agent can then be isolated, and the reporter oligonucleotide (or a product thereof) can be detected (e.g. by sequencing using the methods provided herein). In some embodiments, the reporter oligonucleotide is an isolated nucleic acid as described herein. In some embodiments, a product of the reporter oligonucleotide is an isolated nucleic acid as described herein. In some embodiments, the reporter oligonucleotide is a nucleic acid of the library of isolated nucleic acids. In some embodiments, a product of the reporter oligonucleotide is a nucleic acid of the library of isolated nucleic acids.

[0222] In some embodiments, the analyte labeling agent comprises an analyte binding moiety and a labeling agent barcode domain comprising one or more barcode sequences, e.g., a barcode sequence that corresponds to the analyte binding moiety, the analyte, and/or the sample of interest. An analyte binding moiety barcode can include a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode (according to any of the sequencing methods provided herein), the analyte to which the analyte binding moiety binds can also be identified. An analyte binding moiety barcode can be a nucleic acid sequence of a given length and/or sequence that is associated with the analyte binding moiety. In some embodiments, the barcode domain is part of a reporter oligonucleotide. In some embodiments, the labeling agent is a polynucleotide probe or probe set. Thus, in some embodiments, the binding domain and the reporter oligonucleotide are part of the same nucleic acid molecule. In some embodiments, the binding domain is a split (e.g. ligatable) binding domain, such as two hybridization regions of a circularizable (e.g. padlock) probe, or ligatable probe pair. In some embodiments, the reporter oligonucleotide comprises one or more barcode sequences. In some embodiments, the labeling agent is a circularizable polynucleotide probe (e.g. a padlock probe) or probe set. In some embodiments, the labeling agent is a ligatable probe or probe set. In some embodiments, the labeling agent comprises a probe or probe set that is ligated using an analyte of a biological sample as template.

[0223] In some embodiments, the method comprises one or more post-fixing (also referred to as post- fixation) steps after contacting the sample with one or more labeling agents.

[0224] In the methods and systems described herein, one or more labeling agents capable of binding to or otherwise coupling to one or more features may be used to characterize analytes, cells and/or cell features. In some instances, cell features include cell surface features. In some embodiments, analytes are nucleic acid analytes. In some embodiments, analytes are endogenous analytes. In some embodiments, the analytes are endogenous nucleic acid analytes (e.g. RNA such as mRNA, or DNA). Analytes may include, but are not limited to, a protein, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features may include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post-translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof.

[0225] In some embodiments, an analyte binding moiety may include any molecule or moiety capable of binding to an analyte (e.g., a biological analyte, e.g., a macromolecular constituent). A labeling agent may include, but is not limited to, a nucleic acid, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bispecific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. The labeling agents can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature (or other analyte) to which the binding group binds. For example, the reporter oligonucleotide may comprise a barcode sequence that permits identification of the labeling agent via sequencing, as described herein. For example, a labeling agent that is specific to one type of cell feature (e.g., a first cell surface feature) may have coupled thereto a first reporter oligonucleotide, while a labeling agent that is specific to a different cell feature (e.g., a second cell surface feature) may have a different reporter oligonucleotide coupled thereto. For a description of exemplary labeling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub. 20190177800; and U.S. Pat. Pub. 20190367969, which are each incorporated by reference herein in their entirety.

[0226] In some embodiments, an analyte binding moiety includes one or more antibodies or epitope-binding fragments thereof. The antibodies or epitope-binding fragments including the analyte binding moiety can specifically bind to a target analyte. In some embodiments, the analyte is a protein (e.g., a protein on a surface of the biological sample (e.g., a cell) or an intracellular protein). In some embodiments, a plurality of analyte labeling agents comprising a plurality of analyte binding moieties bind a plurality of analytes present in a biological sample. In some embodiments, the plurality of analytes includes a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labeling agents are the same. In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte labeling agents are the different (e.g., members of the plurality of analyte labeling agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites). In some embodiments, the plurality of analytes includes multiple different species of analyte (e.g., multiple different species of polypeptides or multiple different species of nucleic acids (such as different mRNAs)).

[0227] In other instances, e.g., to facilitate sample multiplexing, a labeling agent that is specific to a particular cell feature may have a first plurality of the labeling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labeling agent coupled to a second reporter oligonucleotide.

[0228] In some aspects, these reporter oligonucleotides may comprise nucleic acid barcode sequences that permit identification of the labeling agent which the reporter oligonucleotide is coupled to. The selection of oligonucleotides as the reporter may provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using the sequencing methods provided herein.

[0229] Attachment (e.g. coupling) of the reporter oligonucleotides to the labeling agents may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides may be covalently attached to a portion of a labeling agent (such a protein, e.g., an antibody or antibody fragment) using chemical conjugation techniques (e.g., Lightning-Link® antibody labeling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which is entirely incorporated herein by reference for all purposes. Furthermore, click reaction chemistry may be used to couple reporter oligonucleotides to labeling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art may be used to couple reporter oligonucleotides to labeling agents as appropriate. In another example, a labeling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide comprising a barcode sequence that identifies the label agent. For instance, the labeling agent may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that comprises a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labeling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labeling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide may be attached to the labeling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein.

[0230] In some cases, the labeling agent can comprise a reporter oligonucleotide and a label. A label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. In some embodiments, the label is conjugated to a labeling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labeling agent or reporter oligonucleotide). In some cases, a label is conjugated to a first oligonucleotide that is complementary (e.g., hybridizes) to a sequence of the reporter oligonucleotide. In some embodiments, the label can be used to isolate the reporter oligonucleotide for sequencing, (iii) Generation of Products

[0231] In some embodiments, provided herein are methods and compositions for sequencing nucleic acids, such as endogenous nucleic acids or products thereof, which are isolated from and/or derived from a biological sample. The biological sample can be any suitable biological sample. In some embodiments, the biological sample is a cell, population of cells, or a tissue sample. In some embodiments, the methods comprise generating a product from an analyte by any suitable method. In some embodiments, an endogenous analyte (e.g., a viral or cellular DNA or RNA) or a product (e.g., a hybridization product, a ligation product, an extension product (e.g., by a DNA or RNA polymerase), a replication product, a transcription/reverse transcription product, and/or an amplification product such as a rolling circle amplification (RCA) product) thereof is sequenced and/or analyzed. In some embodiments, the isolated nucleic acid is an endogenous analyte. In some embodiments, the isolated nucleic acid is a product of an endogenous analyte. In some embodiments, a library of isolated nucleic acids (such as any provided herein) is a library comprising endogenous analytes, or products of endogenous analytes. The products of endogenous analytes can be products of hybridization, ligation, extension, amplification, other biochemical reactions, or combinations thereof. In some embodiments, the products of endogenous analytes comprise sequences of endogenous analytes or complements thereof. In some embodiments, the products of endogenous analytes comprise one or more sequences (e.g. one or more barcode sequences) that are indicative of and/or correspond to endogenous analytes.

[0232] In some embodiments, hybridization is performed in the methods provided herein. In some embodiments, the hybridization comprises the pairing of substantially complementary or complementary nucleic acid sequences, for example between two different nucleic acid molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another.

[0233] In some embodiments, ligation is performed in the methods provided herein. In some embodiments, the ligation involves chemical ligation (e.g., click chemistry ligation). In some embodiments, the chemical ligation involves template dependent ligation. In some embodiments, the chemical ligation involves template independent ligation. In some embodiments, the click reaction is a template-independent reaction (see, e.g., Xiong and Seela (2011), J. Org. Chem. 76(14): 5584-5597, incorporated by reference herein in its entirety). In some embodiments, the click reaction is a template-dependent reaction or template-directed reaction. In some embodiments, the template-dependent reaction is sensitive to base pair mismatches such that reaction rate is significantly higher for matched versus unmatched templates. In some embodiments, the click reaction is a nucleophilic addition templatedependent reaction. In some embodiments, the click reaction is a cyclopropane-tetrazine template-dependent reaction.

[0234] In some embodiments, the ligation involves enzymatic ligation. In some embodiments, the enzymatic ligation involves use of a ligase. In some aspects, the ligase used herein comprises an enzyme that is commonly used to join polynucleotides together or to join the ends of a single polynucleotide. An RNA ligase, a DNA ligase, or another variety of ligase can be used to ligate two nucleotide sequences together. Ligases comprise ATP-dependent doublestrand polynucleotide ligases, NAD-i-dependent double-strand DNA or RNA ligases and singlestrand polynucleotide ligases, for example any of the ligases described in EC 6.5.1.1 (ATP- dependent ligases), EC 6.5.1.2 (NAD+-dependent ligases), EC 6.5.1.3 (RNA ligases). Specific examples of ligases comprise bacterial ligases such as E. coli DNA ligase, Tth DNA ligase, Thermococcus sp. (strain 9° N) DNA ligase (9°N™ DNA ligase, New England Biolabs), Taq DNA ligase, Ampligase™ (Epicentre Biotechnologies) and phage ligases such as T3 DNA ligase, T4 DNA ligase and T7 DNA ligase and mutants thereof. In some embodiments, the ligase is a T4 RNA ligase. In some embodiments, the ligase is a splintR ligase. In some embodiments, the ligase is a single stranded DNA ligase. In some embodiments, the ligase is a T4 DNA ligase. In some embodiments, the ligase is a ligase that has an DNA-splinted DNA ligase activity. In some embodiments, the ligase is a ligase that has an RNA-splinted DNA ligase activity.

[0235] In some embodiments, the ligation herein is a direct ligation. In some embodiments, the ligation herein is an indirect ligation. In some aspects, "direct ligation" refers to ligation in which the ends of the polynucleotides hybridize immediately adjacently to one another to form a substrate for a ligase enzyme resulting in their ligation to each other (e.g. intramolecular ligation). In some embodiments, "indirect ligation" refers to ligation in which the ends of the polynucleotides hybridize non- adjacently to one another, i.e., are separated by one or more intervening nucleotides or "gaps". In some embodiments, said ends are not ligated directly to each other, but instead the ligation occurs either via the intermediacy of one or more intervening (so-called "gap" or "gap-filling" (oligo)nucleotides) or by the extension of the 3' end of a probe (e.g. one of the hybridized polynucleotides) to "fill" the "gap" corresponding to said intervening nucleotides (e.g. intermolecular ligation). In some cases, the gap of one or more nucleotides between the hybridized ends of the polynucleotides may be "filled" by one or more "gap" (oligo)nucleotide(s) which are complementary to a splint, padlock probe, or target nucleic acid. The gap may be a gap of 1 to 60 nucleotides or a gap of 1 to 40 nucleotides or a gap of 3 to 40 nucleotides. In specific embodiments, the gap may be a gap of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides, of any integer (or range of integers) of nucleotides in between the indicated values. In some embodiments, the gap between said terminal regions may be filled by a gap oligonucleotide or by extending the 3' end of a polynucleotide. In some cases, ligation involves ligating the ends of the probe to at least one gap (oligo)nucleotide, such that the gap (oligo)nucleotide becomes incorporated into the resulting polynucleotide. In some embodiments, the ligation herein is preceded by gap filling. In other embodiments, the ligation herein does not require gap filling.

[0236] In some embodiments, ligation of the polynucleotides produces polynucleotides with melting temperature higher than that of unligated polynucleotides. Thus, in some aspects, ligation stabilizes the hybridization complex containing the ligated polynucleotides prior to subsequent steps, such as steps comprising amplification and sequencing.

[0237] In some aspects, a high fidelity ligase, such as a thermostable DNA ligase (e.g., a Taq DNA ligase), is used. Thermostable DNA ligases are active at elevated temperatures, allowing further discrimination by incubating the ligation at a temperature near the melting temperature (T_m) of the DNA strands. This selectively reduces the concentration of annealed mismatched substrates (expected to have a slightly lower T_m around the mismatch) over annealed fully base-paired substrates. Thus, high-fidelity ligation can be achieved through a combination of the intrinsic selectivity of the ligase active site and balanced conditions to reduce the incidence of annealed mismatched dsDNA.

[0238] In some embodiments, the ligation herein is a proximity ligation of ligating two (or more) nucleic acid sequences that are in proximity with each other, e.g., through enzymatic means (e.g., a ligase). In some embodiments, proximity ligation can include a “gapfilling” step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic acid molecules of interest (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference). A wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt- end” ligations. Additionally, single- stranded ligation can be used to perform proximity ligation on a single- stranded nucleic acid molecule. Sticky-end proximity ligations involve the hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be joined, prior to the ligation event itself. Blunt-end proximity ligations generally do not include hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack a single- stranded overhang at the site of ligation.

[0239] In some embodiments, primer extension and/or amplification (e.g. RCA) is performed. A primer is generally a single-stranded nucleic acid sequence having a 3’ end that, in some embodiments, is used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. In some aspects, RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis. Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality. In some examples, DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis). Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases. A primer, may in some cases, refer to a primer binding sequence. A primer extension reaction generally refers to any method where two nucleic acid sequences become linked (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (e.g., 3’ termini). Such linking can be followed by nucleic acid extension (e.g., an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for extension. In some embodiments, enzymatic extension is performed by an enzyme including, but not limited to, a polymerase and/or a reverse transcriptase.

[0240] In some embodiments, the methods provided herein comprise performing rolling circle amplification (RCA). In some embodiments, the amplification (e.g. RCA) is performed at a temperature between or between about 20°C and about 60°C. In some embodiments, the amplification is performed at a temperature between or between about 30°C and about 40°C. In some aspects, the amplification step, such as the rolling circle amplification (RCA) is performed at a temperature between at or about 25°C and at or about 50°C, such as at or about 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, or 49°C.

[0241] In some embodiments, upon addition of a DNA polymerase in the presence of appropriate dNTP precursors and other cofactors, a primer is elongated to produce multiple copies of the circular template. This amplification step can utilize isothermal amplification or non-isothermal amplification. In some embodiments, after the formation of the hybridization complex and association of the amplification probe, the hybridization complex is rolling-circle amplified to generate a cDNA nanoball (i.e., amplicon) containing multiple copies of the cDNA. Techniques for rolling circle amplification (RCA) include linear RCA, a branched RCA, a dendritic RCA, or any combination thereof. (See, e.g., Baner et al, Nucleic Acids Research, 26:5073-5078, 1998; Lizardi et al, Nature Genetics 19:226, 1998; Mohsen et al., Acc Chem Res. 2016 November 15; 49(11): 2540-2550; Schweitzer et al. Proc. Natl Acad. Sci. USA 97:101 13- 1 19, 2000; Faruqi et al, BMC Genomics 2:4, 2000; Nallur et al, Nucl. Acids Res. 29:el 18, 2001; Dean et al. Genome Res. 1 1 :1095- 1099, 2001; Schweitzer et al, Nature Biotech. 20:359-365, 2002; U.S. Patent Nos. 6,054,274, 6,291,187, 6,323,009, 6,344,329 and 6,368,801). Exemplary polymerases for use in RCA comprise DNA polymerase such phi29 (cp29) polymerase, Klenow fragment, Bacillus stearothermophilus DNA polymerase (BST), T4 DNA polymerase, T7 DNA polymerase, or DNA polymerase I. In some aspects, DNA polymerases that have been engineered or mutated to have desirable characteristics can be employed. In some embodiments, the polymerase is phi29 DNA polymerase.

[0242] In some aspects, during the amplification step, modified nucleotides are added to the reaction to incorporate the modified nucleotides in the amplification product (e.g., RCP, sometimes referred to as a nanoball). Exemplary of the modified nucleotides comprise amine- modified nucleotides. In some aspects of the methods, for example, for anchoring or crosslinking of the generated amplification product (e.g., nanoball) to a scaffold (e.g. the polymer matrix). In some aspects, the amplification products comprises a modified nucleotide, such as an amine-modified nucleotide. In some embodiments, the amine-modified nucleotide comprises an acrylic acid N- hydroxysuccinimide moiety modification. Examples of other amine-modified nucleotides comprise, but are not limited to, a 5-Aminoallyl-dUTP moiety modification, a 5- Propargylamino-dCTP moiety modification, a N6-6-Aminohexyl-dATP moiety modification, or a 7-Deaza-7-Propargylamino-dATP moiety modification.

[0243] In some aspects, the polynucleotides and/or amplification product (e.g., amplicon such as RCP) are anchored to a polymer matrix (e.g. of the 3D hydrogel). For example, the polymer matrix can be a hydrogel. In some embodiments, one or more of the polynucleotide probe(s) is modified to contain functional groups that can be used as an anchoring site to attach the polynucleotide probes and/or amplification product (e.g. capture probes or RCPs) to a polymer matrix. Exemplary modification and polymer matrix that can be employed in accordance with the provided embodiments comprise those described in, for example, WO 2014/163886, US20160024555A1, WO 2017/079406, US20180251833 Al, US 2016/0024555, US 2018/0251833 and US 2017/0219465, which are herein incorporated by reference in their entireties. In some examples, the scaffold also contains modifications or functional groups that can react with or incorporate the modifications or functional groups of the probe set or amplification product. In some examples, the scaffold can comprise oligonucleotides, polymers or chemical groups, to provide a matrix and/or support structures. [0244] The amplification products (e.g. RCPs) may be immobilized within the matrix generally at the location of the nucleic acid being amplified, thereby creating a localized colony of amplicons. The amplification products may be immobilized within the matrix by steric factors. The amplification products may also be immobilized within the matrix by covalent or noncovalent bonding. In this manner, the amplification products may be considered to be attached to the matrix. By being immobilized to the matrix, such as by covalent bonding or cross-linking, the size and spatial relationship of the original amplicons is maintained (e.g. within the 3D hydrogel). By being immobilized to the matrix, such as by covalent bonding or cross-linking, the amplification products are resistant to movement or unraveling under mechanical stress, allowing for consistent positioning, e.g. between iterative imaging steps of a sequencing method.

[0245] In some aspects, the amplification products (e.g. RCPs) are copolymerized and/or covalently attached to the surrounding matrix. For example, if the amplification products are those generated from DNA or RNA, the amplification products can also be functionalized to form covalent attachment to the matrix preserving their position. In some embodiments, the provided methods involve embedding the one or more polynucleotide probe sets (e.g. capture probes) and/or the amplification products (e.g. RCPs) in the presence of hydrogel subunits to form one or more hydrogel-embedded amplification products. In some embodiments, amine- modified nucleotides are comprised in the amplification step (e.g., RCA), functionalized with an acrylamide moiety using acrylic acid N-hydroxy succinimide esters, and copolymerized with acrylamide monomers to form a hydrogel.

[0246] In some embodiments, the RCA template may comprise a target analyte, or a part thereof, or a sequence thereof, or a complement of a sequence thereof, where the target analyte is a nucleic acid, or it may be provided or generated as a proxy, or a marker, for the analyte.

[0247] In some embodiments, a product herein (e.g. an isolated nucleic acid or RCP) includes a molecule or a complex generated in a series of reactions, e.g., hybridization, ligation, extension, replication, transcription/reverse transcription, and/or amplification (e.g., rolling circle amplification), in any suitable combination.

[0248] In some embodiments, the methods provided herein comprise sequencing barcodes comprised by the isolated nucleic acids and/or RCPs. In some embodiments, an analyte described herein is associated with one or more barcode(s), e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more barcodes. In some embodiments, barcodes can provide information regarding the spatial distribution or compartmentalization of molecular components found in biological samples, for example, within a cell or a tissue sample. For example, a barcode can be present in an isolated nucleic acid that is no longer in its native context or position within the biological sample, wherein the barcode encodes information regarding the original location of the isolated nucleic acid within the biological sample, and/or wherein the barcode encodes the cell and/or sample of origin of the isolated nucleic acid. A barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner. In some aspects, a barcode comprises about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides.

[0249] In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more nonbarcode sequences. In some embodiments, the one or more barcode(s) can also provide a platform for targeting functionalities that may be useful in processing steps to facilitate the sequencing methods provided herein.

VI. Systems, Kits and Compositions

[0250] In some aspects, provided herein are compositions and kits comprising any of the 3D hydrogels described herein, or 3D hydrogels generated according to any of the methods provided herein. The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container. In some embodiments, the kits further contain instructions for using the components of the kit to practice the provided methods. In some embodiments, the kit comprises a 3D hydrogel comprising immobilized capture probes. The immobilized capture probes are arranged in layers separated by spacer layers within the 3D hydrogel.

[0251] In some embodiments, the kits can contain reagents and/or consumables required for performing one or more steps of the provided methods. In some embodiments, the kits contain reagents, such as enzymes and buffers for ligation and/or amplification, such as ligases and/or polymerases. In some aspects, the kit can also comprise any of the reagents described herein, e.g., wash buffer and ligation buffer. In some embodiments, the kits contain reagents for detection and/or sequencing, such as detectable labels. In some embodiments, the kits optionally contain other components, for example nucleic acid primers.

[0252] In some aspects, provided herein are kits, systems, and compositions. In some aspects, provided herein are kits, systems, and compositions for sequencing RCPs within 3D hydrogels, for example in accordance with any of the methods provided herein. In some aspects, any of the kits, systems, or compositions described herein can comprise any component described in connection with another one of the kits, systems, or compositions provided herein. In some aspects, any of the kits, systems, or compositions described herein can comprise any component described in connection with the methods provided herein. Similarly, any of the methods provided herein can comprise the use of any component described in the kits, compositions, or systems provided herein. Such components include but are not limited to any of the isolated nucleic acids or products thereof, RCPs, reagents (e.g. primers, enzymes, polymerases, detectably labeled nucleotides, chemicals), instruments, imaging systems, apparatuses, portions or sub-components of any of the foregoing, or combinations of any of the foregoing. In some aspects, any of the compositions provided herein can comprise a composition that is generated in the course of performing any of the methods provided herein.

[0253] In some aspects, provided herein are systems. In some aspects, provided herein is a system comprising a 3D hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel. In some embodiments, the library of RCPs is generated from a library of isolated nucleic acids. In some embodiments, the system further comprises an imaging system for imaging the 3D hydrogel. In some aspects, provided herein is a system comprising: a 3-dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and an imaging system for imaging the 3D hydrogel.

[0254] In some embodiments, the first RCP layer and second RCP layer are separated by a spacer layer. In some embodiments, the 3D hydrogel can comprise any suitable number of additional RCP layers and spacer layers, such as any provided herein. In some embodiments, the library of RCPs comprises a sequencing library, such as any suitable sequencing library provided herein. In some embodiments, the sequencing library is a single-cell sequencing library, such as a single-cell RNA sequencing library.

[0255] In some embodiments, the imaging system is configured to image a first focal plane coinciding with the first RCP layer and a second focal plane coinciding with to the second RCP layer. In some embodiments, the imaging system is configured to image any suitable number of focal planes coinciding with RCP layers. In some embodiments, the imaging system is any imaging system described herein (e.g. in section VII). In some embodiments, the imaging system is configured to image the 3D hydrogel by wide-field epifluorescence microscopy.

[0256] In some embodiments, the system further comprises one or more reagents for sequencing the library of RCPs in the 3D hydrogel. In some embodiments, the system is configured for sequencing the library of RCPs, such as by any of the sequencing methods provided herein. In some embodiments, the system is configured for sequencing the library of RCPs by sequencing by synthesis, sequencing by ligation, or sequencing by binding. In some embodiments, the system comprises one or more computers. In some embodiments, the one or more computers are configured to determine and/or analyze one or more sequences present in the library of RCPs.

[0257] In some aspects, provided herein are kits. In some aspects, provided herein is a kit comprising a 3-dimensional (3D) hydrogel comprising capture oligonucleotides immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel. In some embodiments, the first RCP layer and second RCP layer are separated by a spacer layer. In some embodiments, the capture oligonucleotides comprise a capture sequence that is complementary to a target region present in circular or circularizable isolated nucleic acids of a sequencing library. In some aspects, provided herein is a kit, comprising: a 3-dimensional (3D) hydrogel comprising capture oligonucleotides immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the first RCP layer and second RCP layer are separated by a spacer layer; wherein the capture oligonucleotides comprise a capture sequence that is complementary to a target region present in circular or circularizable isolated nucleic acids of a sequencing library.

[0258] In some embodiments, the capture oligonucleotides are any of the capture oligonucleotides provided herein. In some embodiments, the capture oligonucleotides are configured to hybridize to the circular or circularizable isolated nucleic acids of the sequencing library via hybridization between the capture sequence and the target region. In some embodiments, the circular or circularizable isolated nucleic acids of the sequencing library are circular, and wherein the capture oligonucleotides are configured to be extended in an RCA reaction using the circular isolated nucleic acids as template to generate RCPs immobilized in the first RCP layer and second RCP layer. In some embodiments, the circular or circularizable isolated nucleic acids of the sequencing library are circularizable, wherein the capture oligonucleotides are configured to serve as ligation templates for the circularizable isolated nucleic acids to generate circularized isolated nucleic acids, and wherein the capture oligonucleotides are configured to be extended in an RCA reaction using the circularized isolated nucleic acids as template to generate RCPs immobilized in the first RCP layer and second RCP layer. In some embodiments, the kit further comprises the sequencing library, such as any sequencing library described herein. In some embodiments, the sequencing library is a singlecell sequencing library. In some embodiments, the kit further comprises one or more reagents for sequencing the RCPs immobilized in the 3D hydrogel. In some embodiments, the sequencing comprises any of the methods for sequencing provided herein. In some embodiments, the sequencing comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding.

[0259] In some aspects, provided herein are compositions. In some aspects, provided herein is a composition comprising a 3-dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel. In some embodiments, the first RCP layer and second RCP layer are separated by a spacer layer. In some embodiments, the library of RCPs is generated from a library of isolated nucleic acids. In some embodiments, the library of isolated nucleic acids and/or the library of RCPs comprises a single-cell sequencing library. In some embodiments, the composition further comprises a sequencing primer that is hybridized to the RCPs immobilized in the first RCP layer and to the RCPs immobilized in the second RCP layer. In some embodiments, the composition further comprises a modified nucleotide configured to be incorporated into the 3’ end of the sequencing primer and detected. In some embodiments, the modified nucleotide comprises a reversible terminator and/or a fluorescent moiety. In some embodiments, the composition further comprises a polymerase capable of extending the sequencing primer with the modified nucleotide. VII. Opto-Fluidic Instruments for Analysis of Biological Samples and 3D Hydrogels

[0260] In some embodiments, the 3D hydrogels comprising RCPs provided herein can be imaged, sequenced, and/or analyzed using an instrument, such as any instrument described below.

[0261] In some aspects, provided herein is an instrument having integrated optics and fluidics modules (an “opto-fluidic instrument” or “opto-fluidic system”) for imaging and/or sequencing target molecules (e.g. RCPs or isolated nucleic acid molecules) as described herein. In some embodiments, in an opto-fluidic instrument, the fluidics module is configured to deliver one or more reagents to the 3D hydrogel and/or remove spent reagents therefrom. Additionally, in some embodiments, the optics module is configured to illuminate the biological sample, which may be a 3D hydrogel, with light having one or more spectral emission curves (over a range of wavelengths) and subsequently capture one or more images of emitted light signals from the biological sample (e.g. corresponding to an RCP or sequence thereof) during one or more cycles. In various embodiments, the captured images may be processed in real time and/or at a later time to determine the presence of the one or more target molecules (e.g. RCPs) in the 3D hydrogel, as well as three-dimensional position information associated with each detected target molecule within the 3D hydrogel. Additionally, in some embodiments, the opto-fluidics instrument includes a sample module configured to receive (and, optionally, secure) one or more 3D hydrogels. In some instances, the sample module includes an X-Y stage configured to move the biological sample (or 3D hydrogel) along an X-Y plane (e.g., perpendicular to an objective lens of the optics module).

[0262] In various embodiments, the opto-fluidic instrument is configured to image, sequence, and/or analyze one or more target molecules (e.g. RCPs) in the 3D hydrogel.

[0263] The opto-fluidic instrument may include a sample module configured to receive the sample (e.g. 3D hydrogel), and an optics module including an imaging system for illuminating (e.g., exciting one or more fluorescent probes within the sample) and/or imaging light signals received from the probed sample (e.g. 3D hydrogel). The analysis system may also include other ancillary modules configured to facilitate the operation of the opto-fluidic instrument, such as, but not limited to, cooling systems, motion calibration systems, etc.

[0264] In various embodiments, the 3D hydrogel may be placed in the opto-fluidic instrument for sequencing of the target molecules (e.g. RCPs). In various embodiments, the opto- fluidic instrument can be a system configured to facilitate the conditions conducive for the detection and/or sequencing of the target molecules. For example, the opto-fluidic instrument can include a fluidics module, an optics module, a sample module (e.g. 3D hydrogel module), and an ancillary module, and these modules may be operated by a system controller to create the experimental conditions for detecting, sequencing, and/or imaging nucleic acids in the 3D hydrogel (e.g. RCPs). In various embodiments, the various modules of the opto-fluidic instrument may be separate components in communication with each other, or at least some of them may be integrated together.

[0265] In various embodiments, the sample module may be configured to receive the sample (e.g. 3D hydrogel) into the opto-fluidic instrument. For instance, the sample module may include a sample interface module (SIM) that is configured to receive a sample device (e.g., cassette) onto which the sample can be deposited. That is, the sample may be placed in the opto- fluidic instrument by depositing the sample (e.g., the 3D hydrogel) on a sample device that is then inserted into the SIM of the sample module. In some instances, the sample module may also include an X-Y stage onto which the SIM is mounted. The X-Y stage may be configured to move the SIM mounted thereon (e.g., and as such the sample device containing the sample inserted therein) in perpendicular directions along the two-dimensional (2D) plane of the opto- fluidic instrument. Additional discussion related the SIM can be found in US Provisional Application No.: 63/348,879, filed June 3, 2022, titled “Methods, Systems, and Devices for Sample Interface,” and in US 2024/0044754 Al, each of which is incorporated herein by reference in its entirety.

[0266] The experimental conditions that are conducive for the detection of the molecules in the sample (e.g. 3D hydrogel) may depend on the target molecule detection technique that is employed by the opto-fluidic instrument. For example, in various embodiments, the opto-fluidic instrument can be a system that is configured to detect molecules in the sample (e.g. 3D hydrogel) via sequencing. In such cases, the experimental conditions can include molecule hybridization conditions that result in the intensity of hybridization of the target molecule (e.g., nucleic acid) to a probe (e.g., oligonucleotide) being significantly higher when the probe sequence is complementary to the target molecule than when there is a single-base mismatch. The hybridization conditions include the preparation of the sample using reagents such as washing/stripping reagents, hybridizing reagents, etc., and such reagents may be provided by the fluidics module.

[0267] In various embodiments, the fluidics module may include one or more components that may be used for storing the reagents, as well as for transporting said reagents to and from the sample device containing the sample (e.g. 3D hydrogel). For example, the fluidics module may include reservoirs configured to store the reagents, as well as a waste container configured for collecting the reagents (e.g., and other waste) after use by the opto-fluidic instrument to sequence, image, process, analyze, and/or detect the molecules of the sample (e.g. 3D hydrogel). Further, the fluidics module may also include pumps, tubes, pipettes, etc., that are configured to facilitate the transport of the reagent to the sample device (e.g., and as such the sample). For instance, the fluidics module may include pumps (“reagent pumps”) that are configured to pump washing/stripping reagents to the sample device for use in washing/stripping the sample (e.g., as well as other washing functions such as washing an objective lens of the imaging system of the optics module).

[0268] In various embodiments, an ancillary module can be a cooling system of the opto-fluidic instrument. The cooling system may include a network of coolant-carrying tubes that are configured to transport coolants to various modules of the opto-fluidic instrument for regulating the temperatures thereof. In such cases, the fluidics module may include coolant reservoirs for storing the coolants and pumps (e.g., “coolant pumps”) for generating a pressure differential, thereby forcing the coolants to flow from the reservoirs to the various modules of the opto-fluidic instrument via the coolant-carrying tubes. In some instances, the fluidics module may include returning coolant reservoirs that may be configured to receive and store returning coolants, i.e., heated coolants flowing back into the returning coolant reservoirs after absorbing heat discharged by the various modules of the opto-fluidic instrument. In such cases, the fluidics module may also include cooling fans that are configured to force air (e.g., cool and/or ambient air) into the returning coolant reservoirs to cool the heated coolants stored therein. In some instance, the fluidics module may also include cooling fans that are configured to force air directly into a component of the opto-fluidic instrument so as to cool said component. For example, the fluidics module may include cooling fans that are configured to direct cool or ambient air into the system controller to cool the same. [0269] As discussed above, the opto-fluidic instrument may include an optics module which includes various optical components of the opto-fluidic instrument, such as but not limited to a camera, an illumination module (e.g., LEDs), an objective lens, and/or the like. The optics module may include a fluorescence imaging system that is configured to image signals generated from target molecules (e.g. RCPs), for example during iterative sequencing/imaging cycles, following excitation by light from the illumination module of the optics module.

[0270] In some instances, the optics module may also include an optical frame onto which the camera, the illumination module, and/or the X-Y stage of the sample module may be mounted.

[0271] In various embodiments, the system controller may be configured to control the operations of the opto-fluidic instrument (e.g., and the operations of one or more modules thereof). In some instances, the system controller may take various forms, including a processor, a single computer (or computer system), or multiple computers in communication with each other. In various embodiments, the system controller may be communicatively coupled with data storage, set of input devices, display system, or a combination thereof. In some cases, some or all of these components may be considered to be part of or otherwise integrated with the system controller, may be separate components in communication with each other, or may be integrated together. In other examples, the system controller can be, or may be in communication with, a cloud computing platform.

[0272] In various embodiments, the opto-fluidic instrument may analyze the sample (e.g. 3D hydrogel) and may generate an output that is indicative of the sequences of target molecules (e.g. RCPs) within the sample. In some embodiments, the opto-fluidics instrument can be used as a sequencer for the sequencing methods provided herein involving the use of a 3D hydrogel.

[0273] In some aspects, provided herein is an instrument suitable for performing sequencing of target analytes (e.g. RCPs) within a three-dimensional sample (e.g. 3D hydrogel), which includes an optical subsystem that is capable of imaging optical signals (e.g., fluorescent emissions) from the target analytes in one or more color channels. For example, the optical signals may be fluorescent emissions from one or more nucleotides tagged (e.g. labeled) with a fluorescent dye of a particular color (e.g., red, yellow, green, blue, nUV, etc.) for multicolor volumetric imaging. In various embodiments, the fluorescent dyes also include a reversible terminator that blocks further nucleotide addition until the terminator is removed (e.g., via cleavage). In various embodiments, the three-dimensional sample is a 3D hydrogel, such as any provided herein. In various embodiments, 3D hydrogel is suitable for epifluorescent imaging and is sufficiently permeable to allow for reagents to contact the target analytes (e.g. RCPs) therein. In various embodiments, the three-dimensional sample is a hydrogel having a plurality of RCPs disposed (e.g., deposited and/or immobilized) therein.

[0274] In some embodiments, the sequencing performed herein involves detecting a sequence of signals (e.g. fluorescent signals) at a given location within a 3D hydrogel across sequential imaging cycles in a sequencing workflow such as SBS. Thus, in some embodiments, the optical subsystem is configured for high spatial resolution imaging of target analytes (e.g. RCPs) in X, Y, and Z axes. In various embodiments, the optical subsystem for high-resolution 3D sequencing, particularly adapted for three-dimensional samples such as tissue sections or 3D hydrogels containing target analytes, includes at least one objective lens. In some embodiments, the objective lens is an infinity-corrected objective lens. In embodiments where an infinity- corrected objective lens is used, the optical subsystem includes at least one tube lens configured to receive parallel rays from the infinity-corrected objective lens and focus the rays to a focal point, where an image sensor (e.g., a CMOS sensor) is positioned. In various embodiments, the optical subsystem is configured for epifluorescence microscopy (e.g. where excitation light provided to the sample in the excitation channel is filtered out from any emission light provided to the image sensor in the emission channel). An infinity-corrected objective lens may be particularly suited for epifluorescence microscopy because the parallel rays in the infinity space (i.e., the space between the objective and the tube lens in which rays from the objective travel in a parallel, collimated beam to the tube lens) allow for the insertion of additional optical components, such as beamsplitters and filters, without introducing significant optical aberrations. To achieve the high resolution necessary for imaging individual, small clusters, or amorphous/diffuse regions of target analytes (e.g. RCPs), the objective lens ideally possesses a high numerical aperture (NA). For example, objectives with NAs greater than or equal to 0.9, and more preferably, greater than or equal to 1.0, are contemplated to maximize resolution and light collection efficiency from fluorescently tagged analytes. In various embodiments, to achieve a higher NA, an objective capable of immersion in a liquid having a higher refractive index than air (e.g., water with a refractive index of about 1.33 or oil with a refractive index of about 1.51) may be needed. Examples of such objective lenses include water immersion objectives (e.g., for NAs as high as -1.27) or oil immersion (e.g., for NAs as high as -1.4). However, it is understood that objectives with lower NAs may also be utilized depending on the specific resolution requirements and/or sample characteristics. For example, the NA of the objective lens may be at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, from 0.6 to 1.4, from 0.7 to 1.4, from 0.8 to 1.4, from 0.9 to 1.4, from 1.0 to 1.4, from 0.9 to 1.1, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, or about 1.4. In various embodiments, the tube lens is selected to further optimize the imaging performance, ensuring that the combined optical system delivers sharp, high-contrast images of the target analytes (e.g. RCPs) throughout the field of view (FOV) in all imaging color channels (e.g., red, yellow, green, blue, nUV). In various embodiments, the objective lens includes a large FOV to maximize the image volume of a single z-stack of images (thereby reducing the number of z- stacks required to image an entire sample). For example, the FOV may have a diagonal of at least 0.50 mm, at least 0.75 mm, at least 0.80 mm, at least 0.90 mm, at least 1.00 mm, at least 1.10 mm, at least 1.20 mm, at least 1.30 mm, at least 1.40 mm, at least 1.50 mm, at least 1.60 mm, at least 1.70 mm, at least 1.80 mm, at least 1.90 mm, at least 2.00 mm, at least 2.25mm, at least 2.50mm, at least 2.75mm, at least 3.00 mm, from 0.50 mm to 5.00 mm, from 0.75 to 4.00 mm, from 0.75 mm to 3.00 mm, from 0.75 mm to 2.00 mm, from 1.00 mm to 4.00 mm, from 1.00 mm to 3.00 mm, from 1.00 mm to 2.00 mm, about 1.00 mm, about 1.25 mm, about 1.5 mm, about 1.75 mm, about 2.00 mm, about 2.5 mm, or about 3.00 mm. In some embodiments, the FOV may have a diagonal of at most 0.50 mm, at most 0.75 mm, at most 0.80 mm, at most 0.90 mm, at most 1.00 mm, at most 1.10 mm, at most 1.20 mm, at most 1.30 mm, at most 1.40 mm, at most 1.50 mm, at most 1.60 mm, at most 1.70 mm, at most 1.80 mm, at most 1.90 mm, at most 2.00 mm, at most 2.25mm, at most 2.50mm, at most 2.75mm, at most 3.00 mm, at most 4.0mm, or at most 5.0mm. In some embodiments, a diagonal of a FOV is the maximum distance from one corner of the field of view to the opposite corner of the field of view. In some embodiments, a diagonal of a FOV is the maximum distance from one point on the border of the field of view to another point on the border of the field of view.

[0275] In various embodiments, the optical subsystem is designed to facilitate multicolor volumetric (e.g., z-stack) imaging at a plurality of FOVs of the sample (e.g. 3D hydrogel), enabling the capture of high-resolution volumetric data from the sample in a plurality of color channels. In various embodiments, the instrument and/or optical subsystem is designed such that z-repeatability of relative z-motion of the objective lens and sample is less than the depth of focus of the objective lens. In various embodiments, the objective lens moves in Z and the stage is stationary. In various embodiments, the objective lens is stationary and the stage moves in Z. In various embodiments, both the objective lens and the stage have Z-motion capability. In various embodiments, the optical subsystem is designed such that the wavefront error, chromatic shift, and/or field curvature is less than the depth of focus of the objective lens and/or less than the step size between z-slices in the z-stack. In various embodiments, the z-step size is about 0.25 pm to about 2.00 pm, about 0.50 pm to about 1.50 pm, about 0.50 pm to about 1.00 pm, about 1.00 pm, about 0.90 pm, about 0.80 pm, about 0.75 pm, about 0.70 pm, about 0.60 pm, about 0.50 pm, or about 0.25 pm.

[0276] In various embodiments, the optical subsystem is designed to minimize various optical aberrations to maximize image quality across the entire z-stack of images. Specifically, the objective lens and tube lens are designed such that wavefront error, chromatic shift, and field curvature are very small. In various embodiments, the objective lens is designed such that substantially all of the illuminated FOV (which may be a smaller area than the full area of the circular FOV) is usable for decoding target analytes. In various embodiments, wavefront error, chromatic shift, and field curvature are significantly less than the depth of focus of the objective lens. By designing an optical subsystem with minimal wavefront aberration, light collected from the sample is accurately focused, preserving spatial resolution. Moreover, designing an optical subsystem with minimal chromatic shift is particularly useful for multicolor fluorescence imaging as misregistration of the different color channels is reduced (e.g., minimized). Lastly, designing an optical subsystem with corrected (minimal) field curvature ensures that the entire field of view remains in focus across each z-plane, allowing for greater spatial resolution in the Z-axis and potentially increasing the effective imaging area and throughput. In various embodiments, tight control of optical aberration(s) contributes to consistent and high image quality throughout the entire acquired z-stack in multicolor volumetric imaging, ultimately resulting in higher quality and reliable decoding and spatial localization of target analytes (e.g. RCPs).

[0277] In various embodiments, the optical subsystem is designed for high- throughput imaging, allowing for rapid sequencing workflows, such as in 3D hydrogels as described herein. In various embodiments, this optimization is achieved through various design considerations. Firstly, in some embodiments, the optical subsystem is configured to image fluorescent dyes that require short exposure times to emit strong optical signals, thereby minimizing photobleaching and maximizing imaging speed. Secondly, in some embodiments, the optical subsystem provides a large FOV, enabling the imaging of large areas of the sample and reducing the number of z-stack acquisitions (e.g. focal plane acquisitions) required to cover a given sample volume. Thirdly, in some embodiments, the subsystem is engineered for rapid z- stack imaging, allowing for quick stepping between discrete z-slices in each z-stack. In various embodiments, quick z-stepping can be achieved through the integration of fast axial scanning mechanisms, which may integrate voice coil actuators, piezoelectric actuators, or other actuators to enable precise and rapid adjustment of the focal plane as well as high precision, high speed linear XY or XYZ stages (belt, screw, or electromagnetic driven), and tight feedback control loops and/or vibration control, which may integrate proportional control, proportional-integral control, or proportional-integral-derivative control, for precise and rapid switching between z- slices and/or FOVs. In some embodiments, the optical subsystem uses epifluorescence imaging to image the 3D hydrogel.

[0278] In some embodiments, epifluorescence imaging with design characteristics provided herein for high-throughput imaging provide the advantage of rapid acquisition of images from the volume of the 3D hydrogel. For example, the acquisition of images from the volume of the 3D hydrogel by epifluorescence imaging can provide advantages over other forms of imaging, such as confocal microscopy, which typically requires more time to acquire an optical section of a given area (e.g. RCP layer) than the time required to acquire a focal plane of the same area via epifluorescence imaging. In some embodiments, the imaging comprises epifluorescence imaging. In some embodiments, the imaging comprises wide-field epifluorescence imaging. In some embodiments, imaging the 3D hydrogel comprises imaging a plurality of focal planes coinciding with a plurality of RCP layers in the 3D hydrogel. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9 In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 1.0. In some embodiments, the imaging is performed using an objective having a field of view (FOV) having a diagonal of equal to or greater than 0.5 millimeters. In some embodiments, the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9 and a field of view having a diagonal of equal to or greater than 0.5 millimeters.

[0279] Examples of suitable optical subsystems for sequencing are described in detail in U.S. Patent Application Pub. Nos. 2024-0171833, 2024-0167956 and U.S. Provisional Application Nos. 63/701,676, 63/739,889, US 63/723,065, and US 63/723,065, each of which is hereby incorporated by reference in its entirety.

VIII. Terminology

[0280] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0281] The terms "polynucleotide," "polynucleotide," and "nucleic acid molecule", used interchangeably herein, can refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term comprises, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.

[0282] A “primer” as used herein, in some embodiments, is an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers usually are extended by a DNA polymerase.

[0283] In some instances, “ligation” refers to the formation of a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides, in a template-driven reaction. The nature of the bond or linkage may vary widely and the ligation, in some embodiments, is carried out enzymatically or chemically. As used herein, ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon terminal nucleotide of one oligonucleotide with a 3' carbon of another nucleotide.

[0284] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein comprises (and describes) embodiments that are directed to that value or parameter per se. In some embodiments, the term “about” refers to a value within 20% of an indicated value. In some embodiments, the term “about” refers to a value within 10% of an indicated value.

[0285] As used herein, the singular forms "a," "an," and "the" comprise plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more."

[0286] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be comprised in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range comprises one or both of the limits, ranges excluding either or both of those comprised limits are also comprised in the claimed subject matter. This applies regardless of the breadth of the range.

[0287] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

EXAMPLES

[0288] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: Nucleic acid sequencing using a 3D hydrogel

[0289] A 3D hydrogel provided on a solid support (e.g. as shown in FIG. 1) is provided. The 3D hydrogel comprises immobilized capture probes that are immobilized in a polymer matrix of the 3D hydrogel. The capture probes are immobilized at a 5’ end (e.g. via a 5’ acrydite), and comprise a capture sequence at a 3’ end. The 3D hydrogel is contacted with a library of isolated nucleic acids (e.g. a sequencing library) comprising a target region. Nucleic acids of the library of isolated nucleic acids are hybridized to the immobilized capture probes via hybridization between the capture sequence and target region. The nucleic acids hybridized to the immobilized capture probes serve as template for an RCA reaction that extends the immobilized capture probes, thereby generating a library of RCPs that are immobilized in the 3D hydrogel.

[0290] The resulting 3D hydrogel comprises RCPs arranged in a specific density and/or pattern, such as any described herein, which may be suitable or advantageous for sequencing of the RCPs as described herein. It can be seen that the specific density and/or pattern of RCPs in the resulting hydrogel can be determined by the specific density and/or pattern of the immobilized capture probes, which in turn can be provided in any suitable density and/or pattern during the formation of the 3D hydrogel. In some examples, the RCPs are arranged in RCP layers (e.g. a first layer and second layer) separated by one or more spacer layers, for example as shown in FIG. 2.

[0291] The nucleic acids hybridized to the capture probes can be pre-circularized (e.g. as shown in FIG. 3A), or can be circularized using the capture probes as template. In some examples, the nucleic acids hybridized to the capture probes can be circularized without gap filling prior to ligation (e.g. as shown in FIG. 3B). In some examples, the nucleic acids hybridized to the capture probes are circularized with gap filling prior to ligation, using as template an intervening sequence present between two portions the capture sequence on the capture probes (e.g. as shown in FIG. 3C). In some examples, different intervening sequences can be included in subsets of capture probes to facilitate the non-parallel and/or separate (e.g. sequential) sequencing of subsets of RCPs within the 3D hydrogel, for example to reduce optical crowding or increase sequencing capacity, as described in detail herein.

[0292] RCPs in the 3D hydrogel within at least a first and second layer (e.g. RCP layers) are sequenced using any suitable method for sequencing (e.g. sequencing by synthesis, sequencing by binding, sequencing by ligation, etc.). Sequencing of RCPs in the 3D hydrogel with a defined density and/or pattern of RCPs provides advantages for sequencing output, quality, analysis, and efficiency, as described herein.

Example 2: Nucleic acid sequencing of rolling circle amplification products (RCPs) in a 3D hydrogel

[0293] A single circular nucleic acid having a known sequence was synthesized in solution via splint-templated ligation of a circularizable probe, followed by exonuclease treatment to remove any remaining linear nucleic acid molecules. Molecules of the circular nucleic acid were hybridized to capture probes that were immobilized in a 3-dimensional (3D) hydrogel via a 5’ acrydite moiety. The capture probes were extended in a rolling circle amplification (RCA) reaction within the 3D hydrogel using the circular nucleic acid as template, thereby forming RCA products (RCPs) distributed throughout the 3D hydrogel.

[0294] A sequencing primer was hybridized to the RCPs generated from the circular nucleic acid in the 3D hydrogel, and the RCPs were sequenced within the 3D hydrogel using sequential cycles of fluorescent nucleotide incorporation and imaging in a sequencing-by- synthesis (SBS) reaction. Fluorescently labeled A, C, T, and G nucleotides were used for the SBS reaction, each labeled with a different fluorophore corresponding to base identity. In each cycle, a single base of the fluorescent nucleotides was incorporated onto the 3’ end of each sequencing primer and imaged. Iterative rounds of fluorescent nucleotide incorporation and imaging were performed to sequence 10 base pairs downstream of the sequencing primer binding site on the RCP.

[0295] The experiments for 3D hydrogel based sequencing described below were performed using an automated imaging system having a wide-field epifluorescence microscope with a large field of view acquiring images of focal planes along the z-dimension. This imaging approach allowed for rapid image acquisition across a large 3D volume of hydrogel, facilitating high-throughput sequencing.

I l l [0296] FIG. 6 shows representative images from 10 SBS imaging cycles. The representative images show maximum intensity projections from the 3D volume of the imaged hydrogel, in 4 different fluorescent channels corresponding to T, C, A, and G. As expected, since all RCPs were generated using the same circular nucleic as template, fluorescent signals corresponding only to a single base pair identity were observed in each cycle across the hydrogel. Across 10 SBS imaging cycles, the identified base pair corresponded to the expected signal based on the known sequence of the circular nucleic acid and the sequence of the RCP downstream of the sequencing primer. The sequence of each individual RCP in the hydrogel was determined based on the sequence of fluorescent signals identified at a single 3D location across the 10 SBS imaging cycles, with different 3D locations corresponding to different RCPs in the hydrogel. >95% of RCPs were decoded with a phred-scaled quality value (q-score) of greater than or equal to 20 (q>=20), and >99.9% of RCPs were decoded with a q-score of >=10. The density of sequenced RCPs with a q-score of >=20 was approximately 0.02 RCPs per cubic micron (RCP/pm³), corresponding to approximately 1 RCP per square micron (RCP/pm²) of the approximately 50 pm-thick 3D hydrogel. The results show that sequencing was achieved with both high density and high quality in the exemplary 3D hydrogel SBS workflow.

[0297] Next, the ability to sequence multiple different nucleic acids in a single 3D hydrogel was investigated. An experiment was performed using the workflow as described above, this time using 9 different circular nucleic acids, each containing a different known sequence to be sequenced by SBS. The 9 different circular nucleic acids were hybridized to capture probes immobilized in a single 3D hydrogel, and used as templates to generate 9 different corresponding RCPs across the 3D hydrogel via extension of the capture probes using the circular nucleic acids as templates in an RCA reaction. A sequencing primer was hybridized to a common sequence in the 9 different RCPs adjacent to the different known sequences, and the different sequences of the RCPs were sequenced within the 3D hydrogel in 10 sequential SBS imaging cycles. The RCPs were analyzed in a 3D context.

[0298] FIG. 7 shows representative images of maximum intensity projections from the 3D volume of the imaged hydrogel across 10 SBS imaging cycles. As expected, signals were observed in multiple channels for each cycle at various locations in the hydrogel, since multiple sequences corresponding to different RCPs were present. [0299] FIG. 8 shows representative images of individual layers imaged in separate focal planes from the 3D volume of the imaged hydrogel across the 10 SBS imaging cycles. The first layer (top) and second layer (bottom) contain different RCPs that can be clearly distinguished along the z-axis of the 3D hydrogel. RCPs detected in the first layer are not detected in the second layer, and vice versa.

[0300] FIG. 9 shows a representative reconstructed cross-section view of 3 different RCPs decoded in a first and second z-layer (e.g. RCP layers and/or focal planes) of the 3D hydrogel across the 10 SBS imaging cycles. The z-dimension corresponding to the plane of imaging focus (e.g. hydrogel depth) is shown on the vertical axis, as indicated in the inset (right). The top right RCP in each image has a different sequence from the top left RCP and bottom right RCP. In the figure, the RCPs are overlayed with consistent shapes (circle, triangle, or rectangle, as shown in inset) across the imaging cycles for ease of identification. The figure demonstrates fine resolution of RCPs along the z-axis in the 3D hydrogel SBS workflow.

[0301] FIG. 10 shows a plot of the 3D locations of the 9 different sequenced RCPs within a representative imaged 3D hydrogel volume (left), or a zoomed in portion of the imaged volume (right). Each dot represents a sequenced RCP, with different dot colors representing different RCP sequences.

[0302] The sequence of each individual RCP in the hydrogel was determined based on the sequence of fluorescent signals identified at a single 3D location across the 10 SBS imaging cycles, with different 3D locations corresponding to different RCPs in the hydrogel. >96.7% of RCPs were decoded with a q-score of >=20, and >99.9% of RCPs were decoded with a q-score of >=10. The density of sequenced RCPs with a q-score of >=20 was approximately 0.07 RCPs per cubic micron (RCP/pm3), corresponding to approximately 3.5 RCPs per square micron (RCP/pm2) of an approximately 50 pm-thick 3D hydrogel.

[0303] The results show that sequencing of multiple different sequences in the same 3D hydrogel was achieved with both high density and high quality across multiple distinct layers of the hydrogel. The results show that imaging-based sequencing in a 3D hydrogel increases sequencing capacity while facilitating rapid sequencing with high density and quality.

Example 3: Sequencing in 3D hydrogels with controlled densities

[0304] Sequencing of RCPs generated from the 9 different circular nucleic acids in a 3D hydrogel was performed to assess sequencing performance at different RCP densities. [0305] Hydrogels were formed containing immobilized capture probes hybridized to a mixture of the 9 different circular nucleic acids, and the capture probes were extended using the circular nucleic acids as templates in an RCA reaction to generate RCPs within the 3D hydrogel, which were sequenced via SBS, generally as described above. To generate hydrogels with different RCP densities, the capture probes hybridized to the circular nucleic acids were immobilized at different input densities in different 3D hydrogels, with the input concentration of circular nucleic acids hybridized to immobilized capture probes varying from 30 pM to 10,000 pM.

[0306] FIG. 11A shows the proportion of sequenced RCPs that had a q-score of >=20 (squares) and the proportion of sequenced RCPs that: 1) had a q-score of >=20, and 2) had a sequence corresponding to one of the 9 known sequences of the circular nucleic acids (circles), as a function of circular nucleic acid input concentration range (input concentrations of 30 pM, 100 pM, 500 pM, and 1,000 pM). As expected, decoding quality remained constant over lower concentrations and began to drop at a certain density threshold, in this experiment between the 500 pM to 1,000 pM input concentrations, e.g. as predicted in FIG. 4. The drop in sequencing quality likely reflects optical tradeoffs such as optical crowding or out-of-focus background fluorescence, for example as discussed below.

[0307] FIG. 11B shows sequenced RCPs having a q-score of >=30 per 100 square microns. The maximum density of RCP sequences at q>=30 in the current workflow was achieved at 500 pM input concentration, resulting in approximately 1.5 high-quality RCP reads per square micron. In this experiment, an approximately 20 micron-thick volume of a 3D hydrogel was imaged. Accordingly, increasing the thickness of the 3D hydrogel and/or the imaged 3D volume of the hydrogel is expected to proportionally increase the density of sequenced RCPs per square micron.

[0308] FIG. 11C shows the density of detected RCPs (squares), and the density of RCPs sequenced with different q-score thresholds (circles and triangles as shown in inset), as a function of circular nucleic acid input concentration. As expected, the overall density of detected RCPs (squares) increased across the entire input concentration range. Similarly, the density of sequenced RCPs with a q-score of >=10 also increased across the entire input concentration range, indicating that the ability to sequence RCPs with this quality was not maximized in this concentration range. The density of sequenced RCPs with a q-score of >=20 plateaued, reaching a maximum of about 0.1 RCPs per cubic micron at both 500 pM and 1,000 pM input concentrations. This suggests that a flexible window of input concentrations (in this case -500 pM to at least -1000 pM) may facilitate maximum sequencing output density while maintaining high sequencing quality (e.g. q-scores of >=20), within the exemplified workflow. The density of sequenced RCPs with a q-score of >=30 reached a maximum of 0.1 RCPs per cubic micron at 500 pM input concentration and decreased at higher input concentrations. Overall, the results show that high sequencing output in a 3D hydrogel-based sequencing workflow can be achieved by providing an RCP density that achieves high throughput while avoiding decreased optical performance associated with optical crowding or other optical tradeoffs at higher densities, e.g. as predicted in FIG. 4. The results also show that the exemplary 3D hydrogel sequencing workflow facilitates high-quality nucleic acid sequencing at a density of at least 0.1 sequenced analytes (e.g. RCPs) per cubic micron. This translates to an area-based sequencing density of approximately 5 RCPs per square micron in a 50 micron-thick 3D hydrogel.

[0309] FIG. 12 shows individual focal planes from a single fluorescent channel generated with different input concentrations. Observation of the focal planes revealed that out- of-focus background fluorescence may contribute to decreased signal-to-noise ratio for detected RCP puncta at higher concentrations, and thus decreased sequencing quality. This out-of-focus background fluorescence may be an additional factor contributing to decreased sequencing quality, independently of overlapping signals from RCPs that are too close to be resolved (e.g. optical crowding). This is supported by the images which show non-overlapping puncta that are still discernible within an overall increased background fluorescence signal. These results suggest that the density of sequencing in 3D hydrogels may be further increased by addressing out-of-focus fluorescence from neighboring RCPs. For example, generating discrete RCP layers separated by spacer layers (e.g. as shown in FIG. 2) would be expected to decrease background from neighboring planes within acquired focal planes while maintaining high density within the imaged sections. Thus, the results further support the utility of patterned 3D hydrogels as described in the current application.

[0310] Other approaches could additionally or alternatively be used to address the observed out-of-focus background fluorescence. For example, fluorescence intensity of individual RCPs could be decreased based on biochemical or optical adjustments. Improvements in the employed signal extraction algorithm to account for the higher background fluorescence could also be made. Finally, a different imaging method could be used to eliminate out-of-focus light (e.g. confocal microscopy instead of wide-field epifluorescence), although such an approach could sacrifice imaging speed for the increased sequencing density.

[0311] Together, the results demonstrate the utility of 3D hydrogels for nucleic acid sequencing as described in the current application. The results support that approaches for nucleic acid sequencing using both patterned and non-pattemed 3D hydrogels have the potential to markedly increase both the quality and throughput of nucleic acid sequencing workflows.

Claims

1. A method, comprising: providing a 3-dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and sequencing RCP molecules of the library of RCPs that are immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel.

2. The method of claim 1, wherein the method comprises generating the library of RCPs.

3. The method of claim 1 or 2, wherein the method comprises immobilizing the RCP molecules of the library of RCPs in the first RCP layer and the second RCP layer.

4. The method of any of claims 1-3, wherein the method comprises: distributing the library of isolated nucleic acids within the 3D hydrogel, wherein the 3D hydrogel comprises capture probes that are immobilized in the 3D hydrogel; allowing nucleic acid molecules of the library of isolated nucleic acids to hybridize to the immobilized capture probes; and performing a rolling circle amplification (RCA) reaction to extend the immobilized capture probes using as template the nucleic acid molecules hybridized to the immobilized capture probes, thereby generating the library of RCPs immobilized in the 3D hydrogel.

5. The method of claim 4, wherein the capture probes are immobilized in the first RCP layer and the second RCP layer.

6. The method of any of claims 1-5, wherein the library of isolated nucleic acids comprises circular or circularizable nucleic acids.

7. The method of any of claims 4-6, wherein the nucleic acid molecules hybridized to the capture probes are circular or circularizable.

8. The method of any of claims 4-7, wherein the nucleic acid molecules hybridized to the capture probes are circularized prior to the RCA reaction.

9. The method of any of claims 4-8, wherein the nucleic acid molecules hybridized to the capture probes are circularized by ligation using the capture probes as template.

10. The method of claim 9, wherein gap filling using the capture probes as template is performed prior to the ligation.

11. The method of claim 10, wherein the gap filling comprises extending 3’ ends of the nucleic acid molecules hybridized to the capture probes in a nucleic acid extension reaction.

12. The method of claim 9, wherein gap filling using the capture probes as template is not performed prior to the ligation.

13. The method of any of claims 4-12, wherein the capture probes comprise a capture sequence, nucleic acids of the library of isolated nucleic acids comprise a target region, and the capture sequence hybridizes to the target region.

14. The method of claim 13, wherein the target region is a contiguous sequence.

15. The method of claim 13, wherein the target region is not a contiguous sequence.

16. The method of any of claims 13-15, wherein the target region is the same among the nucleic acids of the library of isolated nucleic acids.

17. The method of any of claims 13-15, wherein subsets of the nucleic acids of the library of isolated nucleic acids comprise different subset-specific target regions.

18. The method of any of claims 15-17, wherein a target region of a given nucleic acid of the library of isolated nucleic acids comprises a first target sequence at a 3’ end of the given nucleic acid and a second target sequence at a 5’ end of the given nucleic acid.

19. The method of any of claims 13-18, wherein the capture sequence is the same among the capture probes.

20. The method of any of claims 13-18, wherein subsets of the capture probes comprise different subset-specific capture sequences.

21. The method of claim 20, wherein the different subset-specific capture sequences hybridize to the different subset-specific target regions.

22. The method of any of claims 13-21, wherein the capture sequence is a contiguous capture sequence.

23. The method of any of claims 31-21, wherein the capture sequence is a non-contiguous capture sequence.

24. The method of claim 23, wherein the non-contiguous capture sequence comprises a first portion and a second portion, and the first portion and the second portion of the capture sequence are separated by an intervening sequence.

25. The method of claim 24, wherein the first portion is at a 3’ end of the capture probe.

26. The method of claim 24 or 25, wherein a capture sequence of a given capture probe comprises a first portion at the 3’ end of the capture probe and a second portion that is not contiguous with the first portion, and wherein the first and second portion are separated by an intervening sequence.

27. The method of claim 26, wherein: a first subset of capture probes comprises a first intervening sequence and a second subset of capture probes comprises a second intervening sequence; the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are circularized using either the first intervening sequence or the second intervening sequence of the capture probes as templates; and the RCA generates first RCPs comprising multiple copies of the first intervening sequence and second RCPs comprising multiple copies of the second intervening sequence.

28. The method of claim 27, wherein the nucleic acids of the library of isolated nucleic acids that hybridize to the capture probes are extended in a gap-filling reaction using either the first intervening sequence or the second intervening sequence of the capture probes as templates prior to ligation.

29. The method of claim 27 or 28, wherein the nucleic acids of the library of isolated nucleic acids comprise the same non-contiguous target region.

30. The method of any of claims 27-29, wherein the first and second intervening sequences serve as sequencing primer binding sites for sequencing the first and second RCPs, respectively.

31. The method of any of claims 27-30, wherein the first RCPs are sequenced using a first sequencing primer comprising a sequence complementary to the first intervening sequence.

32. The method of any of claims 27-31, wherein the second RCPs are sequenced using a second sequencing primer comprising a sequence complementary to the second intervening sequence.

33. The method of any of claims 27-32, wherein the first and second RCPs are sequenced in parallel and/or simultaneously.

34. The method of any of claims 27-33, wherein the method comprises sequencing the first and second RCPs simultaneously.

35. The method of any of claims 27-34, wherein the method comprises sequencing the first and second RCPs in the same sequencing reactions.

36. The method of any of claims 27-32, wherein the first and second RCPs are not sequenced simultaneously.

37. The method of any of claims 27-32, wherein the first RCPs are sequenced in first sequencing reactions and the second RCPs are sequenced in second sequencing reactions which occur after the first sequencing reactions and/or do not occur simultaneously with the first sequencing reactions.

38. The method of claim 37, wherein the first sequencing reactions comprise using the first sequencing primer to sequence one or more nucleotides of the first RCPs and the second sequencing reactions comprise using the second sequencing primer to sequence one or more nucleotides of the second RCPs.

39. The method of any of claims 4-38, wherein nucleic acids of the library of isolated nucleic acids that do not hybridize to the immobilized capture probes are removed from the hydrogel and/or are not amplified in the RCA reaction.

40. The method of any of claims 4-39, wherein nucleic acids of the library of isolated nucleic acids that do not hybridize to the immobilized capture probes are not sequenced.

41. The method of any of claims 1-40, wherein the capture probes and/or the RCP molecules of the library of RCPs are immobilized in the first RCP layer and second RCP layer.

42. The method of any of claims 1-41, wherein the first RCP layer and second RCP layer are separated by a spacer layer.

43. The method of claim 42, wherein the spacer layer is positioned between the first and second RCP layers.

44. The method of claim 42 or 43, wherein the spacer layer does not comprise immobilized capture probes or immobilized RCPs.

45. The method of any of claims 42-44, wherein the spacer layer is substantially free of immobilized capture probes and/or immobilized RCPs.

46. The method of any of claims 42-44, wherein the spacer layer comprises a lower concentration of RCPs than the RCP layer.

47. The method of claim 46, wherein the concentration of RCPs in the RCP layer is at least 2-fold, at least 5-fold, at least 10-fold, or at least 100-fold greater than the concentration of RCPs in the spacer layer.

48. The method of any of claims 42-47, wherein the first RCP layer, second RCP layer, and/or spacer layer are flat, planar, and/or 2-dimensional.

49. The method of any of claims 42-48, wherein an upper boundary and lower boundary of any of the first RCP layer, second RCP layer, and/or spacer layer are planar.

50. The method of any of claims 42-49, wherein an upper boundary and lower boundary of any of the first RCP layer, second RCP layer, and/or spacer layer are 2-dimensional.

51. The method of any of claims 1-50, wherein the first RCP layer and second RCP layer do not overlap.

52. The method of any of claims 1-51, wherein the 3D hydrogel further comprises a third RCP layer.

53. The method of any of claims 42-52, wherein the spacer layer is a first spacer layer, and the 3D hydrogel further comprises a second spacer layer positioned between the second RCP layer and the third RCP layer.

54. The method of claim 53, wherein the first spacer layer and second spacer layer are positioned on opposite sides of the second RCP layer.

55. The method of any of claims 52-54, wherein the 3D hydrogel further comprises a fourth RCP layer.

56. The method of any of claims 53-55, wherein the 3D hydrogel further comprises a third spacer layer positioned between the third RCP layer and the fourth RCP layer.

57. The method of claim 56, wherein the third spacer layer and second spacer layer are positioned on opposite sides of the third RCP layer.

58. The method of claim 57, wherein the 3D hydrogel further comprises one or more further RCP layers.

59. The method of claim 58, wherein the 3D hydrogel comprises one or more further spacer layers positioned between the one or more further RCP layers.

60. The method of any of claims 1-59, wherein the method comprises arranging the capture probes and/or the RCP molecules within the 3D hydrogel to generate the RCP layers and/or spacer layers.

61. The method of claim 60, wherein the arranging comprises applying an electrical current to the 3D hydrogel.

62. The method of claim 61, wherein the electrical current comprises a direct current and/or an alternating current.

63. The method of any of claims 60-62, wherein the capture probes and/or the RCP molecules are immobilized after the arranging.

64. The method of any of claims 1-63, wherein the 3D hydrogel is provided on a solid support.

65. The method of claim 64, wherein the solid support comprises a substantially flat, horizontal, and/or 2-dimensional surface.

66. The method of claim 64 or 65, wherein the solid support comprises a slide.

67. The method of any of claims 1-66, wherein the immobilized capture probes and/or RCPs are uniformly distributed throughout the 3D hydrogel.

68. The method of any of claims 1-67, wherein the immobilized capture probes and/or RCPs are randomly distributed throughout the 3D hydrogel.

69. The method of any of claims 1-68, wherein the immobilized capture probes and/or RCPs are distributed at a defined density or density range within the first RCP layer and second RCP layer and/or throughout the 3D hydrogel.

70. The method of claim 69, wherein the defined density or density range comprises at or about, or comprises at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 immobilized capture probes and/or RCPs per cubic micron.

71. The method of 69 or 70, wherein the defined density is at or about, or is at least 0.02, 0.07, or 0.1 RCPs per cubic micron.

72. The method of any of claims 69-71, wherein the defined density or density range comprises at or about, or comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 immobilized capture probes and/or RCPs per square micron of the 3D hydrogel.

73. The method of any of claims 1-72, wherein the method comprises sequencing at least or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within one or more regions of the 3D hydrogel.

74. The method of any of claims 1-73, wherein the method comprises sequencing at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron within one or more regions of the 3D hydrogel.

75. The method of claim 73 or 74, wherein the one or more regions of the 3D hydrogel comprise a contiguous volume of equal to or greater than 0.005, 0.01, 0.05, or 0.1 cubic millimeters.

76. The method of any of claims 1-75, wherein the method comprises sequencing at least or at or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 RCPs per cubic micron within the first RCP layer and second RCP layer.

77. The method of any of claims 1-76, wherein the method comprises sequencing at least or at or about 0.02, 0.07, or 0.1 RCPs per cubic micron within the first RCP layer and second RCP layer.

78. The method of any of claims 1-77, wherein the method comprises sequencing at least or at or about 0.2, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 RCPs per square micron of one or more areas of the 3D hydrogel.

79. The method of any of claims 1-78, wherein the method comprises sequencing at least or at or about 1, 2, 5, or 10 RCPs per square micron of one or more areas of the 3D hydrogel.

80. The method of claim 78 or 79, wherein the one or more areas of the 3D hydrogel comprise a contiguous area of at least or at or about 0.1 square millimeters of the 3D hydrogel or at least or at or about 1 square millimeter of the 3D hydrogel.

81. The method of any of claims 1-80, wherein the 3D hydrogel is at least or at or about 5, 10, 50, 100, 200, 300, 400, or 500 microns thick.

82. The method of any of claims 1-81, wherein the 3D hydrogel is at least or at or about 50 microns thick.

83. The method of any of claims 1-82, wherein the 3D hydrogel has an area of at least or at or about 1, 5, 10, 50, 100, 200, 300, 400, 500, or 1000 square millimeters.

84. The method of any of claims 1-83, wherein the 3D hydrogel has an area of at least or at or about 500 square millimeters.

85. The method of any of claims 1-84, wherein the method comprises sequencing at least or at or about lxlO^A6, lxlO^A7, lxl0^A8, lxlO^A9, or lxl0^A10 RCPs in the 3D hydrogel.

86. The method of any of claims 1-85, wherein the method comprises sequencing at least or at or about lxlO^A9 RCPs in the 3D hydrogel.

87. The method of any of claims 1-86, wherein sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 10.

88. The method of any of claims 1-86, wherein sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 20.

89. The method of any of claims 1-86, wherein sequencing a density or number of RCPs comprises sequencing the density or number of RCPs at a minimum phred-scaled quality value (q-score) of at least 30.

90. The method of any of claims 1-86, wherein the density or number of sequenced RCPs is the density or number of RCPs from which a sequence is determined that has a minimum phred- scaled quality value (q-score) of at least 10, at least 20, or at least 30.

91. The method of any of claims 1-86, wherein the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred-scaled quality value (q-score) of at least 10, at least 20, or at least 30.

92. The method of any of claims 1-86, wherein the density or number of sequenced RCPs is the density or number of RCPs from which a sequence of at least 10 base pairs is determined that has a minimum phred-scaled quality value (q-score) of at least 20.

93. The method of any of claims 1-92, wherein sequencing the RCP molecules comprises sequencing one or more nucleotides of the RCP molecules.

94. The method of any of claims 1-93, wherein sequencing the RCP molecules comprises sequencing the entire RCP molecules or portions thereof.

95. The method of claim 93 or 94, wherein sequencing the one or more nucleotides comprises detecting one or more signals in the 3D hydrogel corresponding to the one or more nucleotides.

96. The method of claim 95, wherein the one or more signals corresponding to the one or more nucleotides are detected in one or more sequential imaging cycles.

97. The method of claim 95 or 96, wherein the method further comprises analyzing the one or more signals corresponding to the one or more nucleotides to determine sequences of one or more RCPs in the 3D hydrogel.

98. The method of any of claims 1-97, wherein sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding.

99. The method of claim 98, wherein sequencing RCP molecules of the library of RCPs comprises sequencing by synthesis.

100. The method of any of claims 1-99, wherein sequencing RCP molecules of the library of RCPs comprises imaging the 3D hydrogel.

101. The method of claim 100, wherein the imaging comprises fluorescence microscopy.

102. The method of claim 101, wherein the fluorescence microscopy comprises epifluorescence imaging.

103. The method of claim 101, wherein the fluorescence microscopy comprises wide-field epifluorescence imaging.

104. The method of any of claims 100-103, wherein imaging the 3D hydrogel comprises imaging a first focal plane of the 3D hydrogel and imaging a second focal plane of the 3D hydrogel.

105. The method of claim 104, wherein the first focal plane coincides with the first RCP layer and the second focal plane coincides with the second RCP layer.

106. The method of any of claims 100-105, wherein imaging the 3D hydrogel comprises imaging a plurality of focal planes coinciding with a plurality of RCP layers in the 3D hydrogel.

107. The method of any of claims 100-106, wherein the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9.

108. The method of any of claims 100-106, wherein the imaging is performed using an objective having a numerical aperture of equal to or greater than 1.0.

109. The method of any of claims 100-108, wherein the imaging is performed using an objective having a field of view (FOV) having a diagonal of equal to or greater than 0.5 millimeters.

110. The method of any of claims 100-109, wherein the imaging is performed using an objective having a field of view (FOV) of equal to or greater than 1.0 millimeters.

111. The method of any of claims 100-110, wherein the imaging is performed using an objective having a numerical aperture of equal to or greater than 0.9 and a field of view having a diagonal of equal to or greater than 0.5 millimeters.

112. The method of any of claims 100-111, wherein imaging the 3D hydrogel comprises optically sectioning the hydrogel to image a first optical section of the first RCP layer and a second optical section of the second RCP layer.

113. The method of any of claims 1-112, wherein sequencing RCP molecules of the library of RCPs comprises optically sectioning the hydrogel to image a first optical section of the first RCP layer and a second optical section of the second RCP layer.

114. The method of any of claims 1-113, wherein the library of RCPs comprises a sequencing library.

115. The method of claim 114, wherein the library of RCPs comprises a single-cell sequencing library.

116. The method of claim 114 or 115, wherein the library of RCPs comprises an RNA sequencing library.

117. The method of claim 114 or 115, wherein the library of RCPs comprises a DNA sequencing library.

118. The method of claim 114, wherein the library of RCPs comprises a single-cell gene expression sequencing library.

119. A system, comprising: a 3-dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the library of RCPs is generated from a library of isolated nucleic acids; and an imaging system for imaging the 3D hydrogel.

120. The system of claim 119, wherein the first RCP layer and second RCP layer are separated by a spacer layer

121. The system of claim 119 or 120, wherein the library of RCPs comprises a sequencing library.

122. The system of claim 121, wherein the sequencing library is a single-cell sequencing library.

123. The system of any of claims 119-122, wherein the imaging system is configured to image a first focal plane coinciding with the first RCP layer and a second focal plane coinciding with to the second RCP layer.

124. The system of any of claims 119-123, wherein the imaging system is configured to image the 3D hydrogel by wide-field epifluorescence microscopy.

125. The system of any of claims 119-124, wherein the system further comprises one or more reagents for sequencing the library of RCPs in the 3D hydrogel.

126. The system of any of claims 119-125, wherein the system is configured for sequencing the library of RCPs.

127. The system of any of claims 119-126, wherein the system is configured for sequencing the library of RCPs by sequencing by synthesis, sequencing by ligation, or sequencing by binding.

128. The system of any of claims 119-127, wherein the system comprises one or more computers.

129. The system of claim 128, wherein the one or more computers are configured to determine and/or analyze one or more sequences present in the library of RCPs.

130. A kit, comprising: a 3-dimensional (3D) hydrogel comprising capture oligonucleotides immobilized in at least a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the first RCP layer and second RCP layer are separated by a spacer layer; wherein the capture oligonucleotides comprise a capture sequence that is complementary to a target region present in circular or circularizable isolated nucleic acids of a sequencing library.

131. The kit of claim 130, wherein the capture oligonucleotides are configured to hybridize to the circular or circularizable isolated nucleic acids of the sequencing library via hybridization between the capture sequence and the target region.

132. The kit of claim 130 or 131, wherein the circular or circularizable isolated nucleic acids of the sequencing library are circular, and wherein the capture oligonucleotides are configured to be extended in an RCA reaction using the circular isolated nucleic acids as template to generate RCPs immobilized in the first RCP layer and second RCP layer.

133. The kit of any of claims 130 or 131, wherein the circular or circularizable isolated nucleic acids of the sequencing library are circularizable, wherein the capture oligonucleotides are configured to serve as ligation templates for the circularizable isolated nucleic acids to generate circularized isolated nucleic acids, and wherein the capture oligonucleotides are configured to be extended in an RCA reaction using the circularized isolated nucleic acids as template to generate RCPs immobilized in the first RCP layer and second RCP layer.

134. The kit of any of claims 130-133, wherein the kit further comprises the sequencing library.

135. The kit of any of claims 130-134, wherein the sequencing library is a single-cell sequencing library.

136. The kit of any of claims 132-135, wherein the kit further comprises one or more reagents for sequencing the RCPs immobilized in the 3D hydrogel.

137. The kit of claim 136, wherein the sequencing comprises sequencing by synthesis, sequencing by ligation, or sequencing by binding.

138. A composition, comprising: a 3-dimensional (3D) hydrogel comprising a library of rolling circle amplification products (RCPs) immobilized in a first RCP layer and a second RCP layer of the 3D hydrogel, wherein the first RCP layer and second RCP layer are separated by a spacer layer, and wherein the library of RCPs is generated from a library of isolated nucleic acids.

139. The composition of claim 138, wherein the library of isolated nucleic acids and/or the library of RCPs comprises a single-cell sequencing library.

140. The composition of claim 138 or 139, wherein the composition further comprises a sequencing primer that is hybridized to the RCPs immobilized in the first RCP layer and to the RCPs immobilized in the second RCP layer.

141. The composition of claim 140, wherein the composition further comprises a modified nucleotide configured to be incorporated into the 3’ end of the sequencing primer and detected.

142. The composition of claim 141, wherein the modified nucleotide comprises a reversible terminator and/or a fluorescent moiety.

143. The composition of claim 141 or 142, wherein the composition further comprises a polymerase capable of extending the sequencing primer with the modified nucleotide.