Methods for Transposase-Mediated Spatial Tagging and Analysis of Genomic DNA in Biological Samples

Abstract

The present invention relates to methods and materials for the spatial analysis of nucleic acids that have been fragmented with a transposase, alone or in combination with other types of analytes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 724,483 filed on August 29, 2018, U.S. Provisional Patent Application No. 62 / 779,342 filed on December 13, 2018, U.S. Provisional Patent Application No. 62 / 723,950 filed on August 28, 2018, U.S. Provisional Patent Application No. 62 / 723,957 filed on August 28, 2018, U.S. Provisional Patent Application No. 62 / 723,960 filed on August 28, 2018, U.S. Provisional Patent Application No. 62 / 723,964 filed on August 28, 2018, and U.S. Provisional Patent Application No. 62 / 723,970 filed on August 28, 2018. No. 3,970, U.S. Provisional Patent Application No. 62 / 724,483, filed on August 29, 2018, U.S. Provisional Patent Application No. 62 / 723,972, filed on August 29, 2018, U.S. Provisional Patent Application No. 62 / 724,489, filed on August 28, 2018, U.S. Provisional Patent Application No. 62 / 724,561, filed on August 29, 2018, U.S. Provisional Patent Application No. 62 / 788,905, filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,867, filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,906, filed on January 6, 2019, 871, U.S. Provisional Patent Application No. 62 / 788,897 filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,885 filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 822,565 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 819,496 filed on March 15, 2019, U.S. Provisional Patent Application No. 62 / 819,486 filed on March 15, 2019, U.S. Provisional Patent Application No. 62 / 819,467 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 822,6 32, U.S. Provisional Patent Application No. 62 / 822,618 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 822,592 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 819,468 filed on March 15, 2019, U.S. Provisional Patent Application No. 62 / 822,627 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 819,448 filed on March 15, 2019, U.S. Provisional Patent Application No. 62 / 822,649 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 819,468 filed on March 15, 2019,456, U.S. Provisional Patent Application No. 62 / 819,478 filed on March 15, 2019, U.S. Provisional Patent Application No. 62 / 819,449 filed on March 15, 2019, U.S. Provisional Patent Application No. 62 / 822,554 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 822,575 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 822,605 filed on March 22, 2019, U.S. Provisional Patent Application No. 62 / 812,219 filed on February 28, 2019, U.S. Provisional Patent Application No. 62 / 819,458 filed on March 15, 2019 No. 62 / 839,223, filed April 26, 2019, U.S. Provisional Patent Application No. 62 / 839,320, filed April 26, 2019, U.S. Provisional Patent Application No. 62 / 839,346, filed April 26, 2019, U.S. Provisional Patent Application No. 62 / 842,463, filed May 2, 2019, U.S. Provisional Patent Application No. 62 / 860,993, filed June 13, 2019, U.S. Provisional Patent Application No. 62 / 839,526, filed April 26, 2019, and U.S. Provisional Patent Application No. 62 / 858,331, filed June 7, 2019. The contents of each of these applications are incorporated herein by reference in their entirety. Background Art

[0003] Cells within a subject's tissue differ in cell morphology and / or function due to different analyte levels (e.g., gene and / or protein expression) within different cells. The specific location of a cell within a tissue (e.g., the location of the cell relative to neighboring cells or the location of the cell relative to the tissue microenvironment) can affect, for example, the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and crosstalk with other cells in the tissue.

[0004] Spatial heterogeneity has been previously studied using techniques that provide data for only a few analytes in intact tissue or partial tissue contacts, or provide data for a large number of analytes in single cells, but do not provide information about the location of single cells in the maternal biological sample (e.g., tissue sample).

[0005] Chromatin structure can vary between cells within a biological sample or between biological samples from the same tissue. Analysis of differences in accessible chromatin can indicate transcriptionally active sequences, such as genes, within a particular cell. Further understanding of transcriptionally active regions within chromatin will help identify genes that contribute to a cell's function and / or phenotype. Summary of the Invention

[0006] The present invention generally describes a method for spatially analyzing genomic DNA present in a biological sample. In one aspect, the method comprises providing an array having a plurality of capture probes, such that the plurality of capture probes comprise a spatial barcode and a capture domain; permeabilizing the biological sample under conditions sufficient to render the genomic DNA in the biological sample insertable by a transposon; providing a transposon sequence and a transposase to the biological sample under conditions wherein the transposon sequence is inserted into the genomic DNA; allowing the transposase to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA; contacting the biological sample comprising the fragmented genomic DNA with the array under conditions wherein the capture probes interact with the fragmented genomic DNA; and correlating the positions of the capture probes on the array with positions in the biological sample, thereby spatially analyzing the fragmented genomic DNA.

[0007] In some embodiments, an array comprising a plurality of capture probes is provided on a substrate. In some embodiments, an array comprising a plurality of capture probes is provided on a feature. In some embodiments, the capture probes are attached directly or indirectly. In some embodiments, an array comprising a plurality of capture probes is provided on a feature on a substrate. In some embodiments, the substrate comprises a microfluidic channel. In some embodiments, the capture probe further comprises one or more of a cleavage domain, a functional domain, and a unique identifier, or a combination thereof.

[0008] In some embodiments, another migration step includes a step in which the fragmented genomic DNA is migrated to a substrate. In some embodiments, the migration step is an active migration step, comprising applying an electric field to the fragmented genomic DNA. In some embodiments, the migration step is a passive migration step comprising diffusion. In some embodiments, the migration of the fragmented genomic DNA from the biological sample comprises exposing the biological sample and the feature to heat. In some embodiments, the biological sample is fixed on a substrate.

[0009] In some embodiments, the transposase is a dimer comprising a first monomer that is complexed with a first adaptor (comprising a transposon end sequence and a sequence complementary to a capture domain), wherein a second monomer is complexed with a second adaptor (comprising a transposon end sequence and a second adaptor sequence), wherein the transposase ligates the first adaptor and the second adaptor to the fragmented genomic DNA. In some embodiments, the first adaptor and the second adaptor have a 5′ end and a 3′ end, wherein the 5′ end is phosphorylated in situ. In some embodiments, prior to fragmenting the DNA, the 5′ end of the first adaptor complexed with the first monomer and the 5′ end of the second adaptor complexed with the second monomer are phosphorylated. In some embodiments, the step of phosphorylating the 5′ end of the first adaptor complexed with the first monomer and the 5′ end of the second adaptor complexed with the second monomer comprises contacting the first monomer:first adaptor complex and the second monomer:second adaptor complex with a polynucleotide kinase in the presence of ATP.

[0010] In some embodiments, the capture domain of the capture probe comprises a sequence that hybridizes to a sequence that is complementary to the capture domain of the first adaptor. In some embodiments, the capture probe is a partially double-stranded molecule comprising a first strand comprising a capture domain that hybridizes to a second strand, and wherein the first strand serves as a template for connecting the first adaptor to the second strand. In some embodiments, the first adaptor sequence complementary to the capture domain or a portion thereof that hybridizes to the capture probe serves as a template for connecting and connecting the 5′ end of the first adaptor to the 3′ end of the capture probe. In some embodiments, the capture probe comprises a surface probe and a splint oligonucleotide, the splint oligonucleotide comprising a sequence that is complementary to the hybridization domain of the surface probe. In some embodiments, the splint oligonucleotide comprises a capture domain having a sequence that is complementary to the first adaptor or a portion thereof. In some embodiments, the splint oligonucleotide hybridizes to the first adaptor or a portion thereof and hybridizes to the hybridization domain or a portion thereof of the surface probe. In some embodiments, the connection is performed in the presence of the splint oligonucleotide, thereby connecting the surface probe and the first adaptor of the capture probe.

[0011] In some embodiments, the fragmented genomic DNA hybridized with the capture probe by the first adapter is an extension template for generating an extended capture probe, and the extended capture probe includes a sequence of a spatial barcode and a sequence complementary to the fragmented genomic DNA. In some embodiments, the capture probe hybridized with the fragmented genomic DNA is extended with a DNA polymerase. In some embodiments, the DNA polymerase has strand displacement activity. In some embodiments, the single-strand breaks in the fragmented genomic DNA are subjected to a further step of gap repair.

[0012] In some embodiments, the sequence complementary to the capture domain is a unique sequence. In some embodiments, the capture probe is ligated to the fragmented genomic DNA by DNA ligase. In some embodiments, the transposase is a Tn5 transposase or a functional derivative thereof. In some embodiments, the Tn5 transposase comprises a sequence having at least 80% identity to SEQ ID NO: 1. In some embodiments, the transposase is a Mu transposase or a functional derivative thereof. In some embodiments, the Mu transposase comprises a sequence having at least 80% identity to SEQ ID NO: 2. In some embodiments, the transposon end sequence comprises a sequence having at least 80% identity to SEQ ID NO: 8. In some embodiments, the transposon end sequence comprises a sequence having at least 80% identity to any one of SEQ ID NOs: 9 to 14.

[0013] In some embodiments, the biological sample is permeabilized under chemical permeabilization conditions, enzymatic permeabilization conditions, or both. In some embodiments, the chemical permeabilization conditions comprise contacting the biological sample with an alkaline solution. In some embodiments, the enzymatic permeabilization conditions comprise contacting the biological sample with an acidic solution comprising a protease. In some embodiments, the protease is an aspartyl protease, preferably pepsin, a pepsin-like enzyme, or a functional equivalent thereof. In some embodiments, the pepsin, pepsin-like enzyme, or a functional equivalent thereof comprises a sequence that is at least 80% identical to SEQ ID NO: 3 or 4.

[0014] In some embodiments, the enzymatic permeabilization conditions comprise contacting the biological sample with a zinc endopeptidase, a collagenase, a collagenase-like enzyme, or a functional equivalent thereof; with a serine protease, a proteinase K enzyme, a proteinase K-like enzyme, or a functional equivalent thereof; or both. In some embodiments, the collagenase, collagenase-like enzyme, or a functional equivalent thereof comprises a sequence that is at least 80% identical to SEQ ID NO: 5 or 6. In some embodiments, the proteinase K enzyme, proteinase K-like enzyme, or a functional equivalent thereof comprises a sequence that is at least 80% identical to SEQ ID NO: 7.

[0015] In some embodiments, the fragmented genomic DNA that hybridizes with the capture probe as an extension template produces a DNA molecule. In some embodiments, the fragmented genomic DNA that hybridizes with the capture probe is used as a connection template to produce a DNA molecule. In some embodiments, the step includes the step of analyzing the generated DNA molecule. In some embodiments, the step of analyzing the DNA molecule includes sequencing. In some embodiments, the step of associating the spatial barcode of the capture probe with the fragmented genomic DNA associated with the capture probe spatially analyzes the fragmented genomic DNA. In some embodiments, the biological sample is imaged before or after the biological sample contacts the substrate.

[0016] In another aspect, the invention generally describes a kit for use in a method for spatially detecting nucleic acids in a biological sample, wherein the kit comprises any two or more of an array having a plurality of capture probes; one or more biological sample permeabilization reagents; one or more transposases; one or more reverse transcriptases; and one or more lytic enzymes.

[0017] In various aspects, the present invention generally describes methods for spatially analyzing genomic DNA and RNA present in a biological sample, wherein an array is provided and the array comprises a plurality of capture probes, wherein a first capture probe of the plurality of capture probes comprises a spatial barcode and a first capture domain, and wherein a second capture probe of the plurality of capture probes comprises a spatial barcode and a second capture domain; permeabilizing the biological sample under conditions sufficient to cause transposon insertion into the genomic DNA in the biological sample; providing a transposon sequence and a transposase to the biological sample under conditions wherein the transposon sequence is inserted into the genomic DNA;

[0018] A transposase is allowed to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA; a biological sample containing fragmented genomic DNA and RNA is contacted with an array under conditions wherein the first capture domain interacts with the fragmented genomic DNA and the second capture domain interacts with the RNA; and the position of the first capture probe on the array is associated with the position in the biological sample, and the position of the second capture probe on the array is associated with the position in the biological sample, thereby spatially analyzing the fragmented genomic DNA and RNA at the position in the biological sample.

[0019] In some embodiments, the RNA is mRNA. In some embodiments, the first capture domain and the second capture domain are identical. In some embodiments, the first capture domain and the second capture domain comprise a homopolymeric poly(T) sequence. In some embodiments, the first capture domain and the second capture domain are different. In some embodiments, the first capture domain comprises a random sequence and the second capture domain comprises a poly(T) sequence. In some embodiments, an array comprising a plurality of capture probes is provided on a substrate. In some embodiments, an array comprising a plurality of capture probes is provided on a feature. In some embodiments, the feature comprises a first capture probe, a second capture probe, or both. In some embodiments, the first capture probe, the second capture probe, or both are attached directly or indirectly. In some embodiments, an array comprising a plurality of capture probes is provided on a feature on a substrate. In some embodiments, the substrate comprises a microfluidic channel. In some embodiments, the first capture probe, the second capture probe, or both comprise one or more of the following: a cleavage domain, a functional domain, and a unique identifier, or a combination thereof.

[0020] In some embodiments, there is a migration step in which the fragmented genomic DNA and RNA migrate to a substrate. In some embodiments, the migration step is an active migration step. In some embodiments, the migration step is a passive migration step. In some embodiments, the migration of the fragmented genomic DNA and RNA from the biological sample comprises exposing the biological sample to heat. In some embodiments, the biological sample is fixed to a substrate.

[0021] In some embodiments, the fragmented genomic DNA is repaired by connecting the breaks with a ligase. In some embodiments, the single-strand breaks in the fragmented genomic DNA undergo gap repair. In some embodiments, a sequence complementary to the first capture domain of the first capture probe is introduced into the fragmented genomic DNA. In some embodiments, the first capture domain of the first capture probe hybridizes with a sequence complementary to the capture domain introduced into the fragmented genomic DNA. In some embodiments, the random sequence of the first capture domain hybridizes with the fragmented genomic DNA. In some embodiments, the second capture domain of the second capture probe hybridizes with a complementary sequence in the mRNA. In some embodiments, the sequence complementary to the first capture domain and the complementary sequence in the mRNA are homopolymeric sequences. In some embodiments, the homopolymer sequence is a poly (A) sequence.

[0022] In some embodiments, the first capture probe is extended using fragmented genomic DNA as an extension template, and the second capture probe is extended using RNA as an extension template. In some embodiments, the first capture probe is extended using a DNA polymerase. In some embodiments, the second capture probe is extended using a reverse transcriptase.

[0023] In some embodiments, the transposase is a Tn5 transposase or a functional derivative thereof. In some embodiments, the Tn5 transposase comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the transposase is a Mu transposase or a functional derivative thereof. In some embodiments, the Mu transposase comprises a sequence that is at least 80% identical to SEQ ID NO: 2. In some embodiments, the transposase is complexed with an adaptor comprising a transposon end sequence. In some embodiments, the transposon end sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the transposon end sequence comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 9 to 14.

[0024] In some embodiments, a step of permeabilizing the biological sample is performed. In some embodiments, 7. The method of any one of claims 51 to 86, wherein the permeabilization of the biological sample is performed under chemical permeabilization conditions, enzymatic permeabilization conditions, or both. In some embodiments, the chemical permeabilization conditions comprise contacting the biological sample with an alkaline solution. In some embodiments, the enzymatic permeabilization conditions comprise contacting the biological sample with an acidic solution comprising a protease. In some embodiments, the protease is an aspartyl protease, preferably pepsin, a pepsin-like enzyme, or a functional equivalent thereof. In some embodiments, the pepsin, pepsin-like enzyme, or a functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 3 or 4. In some embodiments, the enzymatic permeabilization conditions comprise contacting the biological sample with a zinc endopeptidase, a collagenase, a collagenase-like enzyme, or a functional equivalent thereof; with a serine protease, a proteinase K enzyme, a proteinase K-like enzyme, or a functional equivalent thereof; or both. In some embodiments, the collagenase, collagenase-like enzyme, or a functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 5 or 6. In some embodiments, the Proteinase K enzyme, Proteinase K-like enzyme, or a functional equivalent thereof comprises a sequence that is at least 80% identical to SEQ ID NO:7.

[0025] In some embodiments, the step of analyzing DNA molecules includes sequencing. In some embodiments, the spatial barcode of the first capture probe is associated with the fragmented genomic DNA associated with the first capture probe to spatially analyze the fragmented genomic DNA. In some embodiments, the spatial barcode of the second capture probe is associated with the mRNA associated with the second capture probe to spatially analyze the mRNA. In some embodiments, the biological sample is imaged before or after the biological sample contacts the substrate.

[0026] All publications, patents, and patent applications available on the Internet mentioned in this specification are incorporated herein by reference as if each individual publication, patent, patent application, and information item was specifically and individually incorporated herein by reference. To the extent that publications, patents, patent applications, and information items incorporated by reference conflict with the disclosure contained in this specification, it is intended that this specification control and / or take precedence over any conflicting material.

[0027] Where values are described in a range, it should be understood that the description includes disclosure of all possible sub-ranges within that range as well as disclosure of specific values within that range, regardless of whether a specific value or specific sub-range is explicitly stated.

[0028] The term "each" when referring to a group of items is intended to identify an individual item in the group, but not necessarily every item in the group unless expressly stated otherwise or unless the context of usage clearly indicates otherwise.

[0029] Various embodiments of the features of the present invention are described herein. However, it should be understood that these embodiments are provided as examples only, and that many variations, changes, and substitutions may be made by those skilled in the art without departing from the scope of the present disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following drawings illustrate certain embodiments of the features and advantages of the present invention. These embodiments are not intended to limit the scope of the appended claims in any way. The same reference numerals in the drawings represent the same elements.

[0031] Figure 1 An exemplary spatial analysis workflow is shown.

[0032] Figure 2 An exemplary spatial analysis workflow is shown.

[0033] Figure 3 An exemplary spatial analysis workflow is shown.

[0034] Figure 4 An exemplary spatial analysis workflow is shown.

[0035] Figure 5 An exemplary spatial analysis workflow is shown.

[0036] Figure 6 is a schematic diagram showing an example of a barcoded capture probe as described herein.

[0037] Figure 7is a schematic diagram illustrating a cleavable capture probe, wherein the cleaved capture probe can enter a non-permeabilized cell and bind to a target analyte within a sample.

[0038] Figure 8 is a schematic diagram of an exemplary multi-space marking feature.

[0039] Figure 9 is a schematic diagram of an exemplary analyte-capture agent.

[0040] Figure 10 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1024 and an analyte-capture agent 1026.

[0041] Figure 11A 、 11B and 11C are schematic diagrams illustrating how streptavidin cell tagging can be used in an array-based system to generate spatially barcoded cells or cell contents.

[0042] Figure 12 is a schematic diagram showing the arrangement of barcoding features within an array.

[0043] Figure 13 is a schematic diagram showing a side view of an anti-diffusion medium (eg, a cover).

[0044] Figure 14A and 14B This diagram shows the development of the electrophoretic transfer system. Figure 14A and side view Figure 14B Schematic diagram of the electrophoretic transfer system configured to direct transcript analytes to a spatially barcoded capture probe array.

[0045] Figure 15 is a schematic diagram illustrating an exemplary workflow scheme utilizing an electrophoretic transfer system.

[0046] Figure 16 An example of a microfluidic channel structure 1600 for partitioning a dissociated sample, such as biological particles or individual cells from a sample, is shown.

[0047] Figure 17A An example of a microfluidic channel structure 1700 for delivering beads carrying spatial barcodes to droplets is shown.

[0048] Figure 17B A cross-sectional view of another example of a microfluidic channel structure 1750 having geometric features for controlled partitioning is shown.

[0049] Figure 17C A workflow diagram is shown.

[0050] Figure 18 Schematic diagram depicting cell labeling using covalent coupling of analyte-binding moieties to the cell surface or non-covalent interactions with cell membrane components.

[0051] Figure 19 is a schematic diagram depicting cell labeling using a cell-penetrating peptide or delivery system.

[0052] Figure 20A is a schematic diagram of a workflow illustrating exemplary, non-limiting, non-exhaustive steps for "pixelating" a sample, wherein the sample is cut, stamped, microdissected, or transferred by a hollow needle or microneedle to move small portions of the sample into individual partitions or wells.

[0053] Figure 20B A schematic depicting multi-needle pixelation, where a set of needles penetrates a sample on a scaffold and into a nanopore below containing gel beads and reagents. Once the needles are in the nanopore, the cell pops out.

[0054] Figure 21 A workflow diagram showing exemplary, non-limiting, non-exhaustive steps for dissociating spatially barcoded samples for analysis by droplet or flow cell analysis methods.

[0055] Figure 22A-D is a schematic diagram showing an example of spatial processing of DNA from a biological sample.

[0056] Figure 23A-C is a schematic diagram showing an example of the spatial ATAC-seq method.

[0057] Figure 24 AC are schematic diagrams showing examples of multiplexed detection of analytes in biological samples.

[0058] Figure 25 is a schematic diagram showing a representative workflow of the present invention.

[0059] Figure 26 is a schematic diagram showing a representative workflow of the procedures used to study Tn5 transposase / transposome efficiency.

[0060] Figure 27 is a schematic diagram showing a representative workflow of the procedure used to study labeling conditions in fixed tissue sections.

[0061] Figure 28 is a schematic diagram showing a representative workflow of the procedure used to study hybridization and ligation conditions for phosphorylated DNA tags.

[0062] Figure 29Shown are DNA fragment analysis of a reference tagmentation reaction performed in cell suspension as described (Corces, MR et al., Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution, Nat Genetic. Vol. 48(10): pp. 1193-1203 (2016)). Fragment distribution analysis was used to determine the success of open chromatin tagmentation, where successful tagmentation reactions of accessible chromatin revealed periodicity in the size of PCR-amplified nucleosome-protected DNA fragments (approximately 170-180 bp; nucleosome-wrapped DNA and PCR handles).

[0063] Figure 30 shows the Figure 27 DNA fragment analysis of a tagmentation reaction performed according to the workflow in [ 15 ], comparing different detergents during a 10-minute permeabilization step at 25°C: a) no detergent; b) 0.1% Triton-X-100; c) IGEPAL 0.1%; d) Tween 0.1%, digitonin 0.01%, and NP-40 0.1%. In e), insert size distribution analysis of tissue sections permeabilized with IGEBAL 0.1% and processed as described in (Chen 2016 Nat Meth) failed to reveal significant nucleosome periodicity.

[0064] Figure 31 shows the Figure 27 Figure 5. DNA fragmentation analysis of tagmentation reactions performed according to the workflow in Figure 5. Comparison of different protease treatments (3 minutes) on fixed tissue sections: a) pepsin (0.1 mg / ml) in the presence of 100 mM HCl; b) pepsin (0.5 mg / ml) in the presence of 0.5 M acetic acid; c) pepsin (0.1 mg / ml) in the presence of 0.5 M acetic acid; and d) proteinase K.

[0065] Figure 32 shows the Figure 27 DNA fragment analysis of tagmentation reactions performed according to the workflow in Figure 5 compares different permeabilization treatments on fixed tissue sections: a) pepsin (0.1 mg / ml) in the presence of 0.5% acetic acid; b) chemical permeabilization using 1X Exonuclease-I buffer (67 mM glycine KOH, 6.7 mM MgCl2, 10 mM β-ME); and c) collagenase.

[0066] Figure 33 shows the Figure 27We compared DNA fragment analysis of tagmentation reactions performed according to the workflow in

[15] , and compared different Tn5 assembly methods on fixed tissue sections: a) MEDS-Tn5 assembled on a column as in (Picelli, S. et al., Tn5 transposase and tagmentation procedures for massively scaled sequencing projects; Genome Res., Vol. 24, 2033-2040 (2014)); b) MEDS-Tn5 assembled in solution (Picelli et al., 2014, supra); and c) MEDS-Tn5 assembly of 5′ phosphorylated oligonucleotides assembled in solution.

[0067] Figure 34 is a schematic diagram showing assays to assess the effects of T4-PNK phosphorylation and reaction conditions on the MEDS-Tn5 complex after assembly.

[0068] Figure 35 Shows the Figure 26 Analysis of DNA fragments from tagmentation reactions performed according to the workflow in

[15] , investigating the compatibility of post-assembly 5′ phosphorylation with DNA tagmentation: a) MEDS-AB-Tn5 assembled on-column as in (Picelli et al., 2014, supra); b) as in a), but exposed to T4-PNK reaction conditions at 37°C for 30 min; c) as in b), but including T4-PNK enzyme; and d) bar graph showing quantification of the relative proportions of nucleosome-protected fragments recovered in a)-c).

[0069] Figure 36 shows the Figure 28 Photographs of arrays generated by the workflow in , depicting the ligation efficiency of DNA tagment to capture probe oligonucleotides (a) without and (b) with post-assembly phosphorylation.

[0070] Figure 37 is a schematic diagram depicting a representative embodiment of the present invention, in which the taggant is gap-filled using a polymerase with slippery activity (e.g., stuttering), a terminal transferase is used to generate a poly-A-sticky end (3' overhang) at the 3' end (mimicking the mRNA poly(A)-tail), and then hybridized to the capture domain of a capture probe (this embodiment will allow for simultaneous hybridization of mRNA transcripts). Alternatively, the taggant can be extended using a polymerase before capture.

[0071] Figure 38is a schematic diagram of a representative embodiment of the invention, in which a tagment is ligated to a partially double-stranded capture probe using the capture domain strand (eg, capture domain oligonucleotide) of the capture probe as a ligation template.

[0072] Figure 39 Figure 1 is a schematic diagram showing a representative workflow for studying phospho-DNA tag ligation and downstream qPCR analysis across the entire human genome.

[0073] Figure 40 Schematic diagram showing exemplary oligonucleotide capture strategies and corresponding sequences. Readout was performed using qPCR using oligonucleotides specific for tagments that were successfully attached to the surface (e.g., A-short and Nextera Reverse) or for all tagments (e.g., Nextera Forward and Nextera Reverse).

[0074] FIG41A shows the results of various experimental conditions. Figure 39 Schematic diagram of the basic overview of the workflow shown (ligation of phosphorylated DNA fragments from the entire human genome).

[0075] FIG. 41B shows the Figure 39 Figure 2 shows the DNA fragment analysis of the tagmentation reaction performed with the workflow shown. The PCR primer pair "Ashort Next" covers the surface probe and the tagment. This primer pair only produces PCR products when hybridization and ligation occur. Samples 1 and 2 represent tagment with phosphate groups added to facilitate ligation. Samples 3 and 4 contain tagment without phosphate groups, and as a negative control, samples 5 and 6 contain MQ water instead of the tag. In addition, a pair of Nextera primers ("NEXT only", samples 7-11) shows PCR products when both ligation and hybridization occur, resulting in signals from wells D and E.

[0076] Figure 41C Alignment of PCR products is shown. Ligation of the "Ashort-Next" primers is shown (ligated qPCR product), while minimal ligation occurred in all four negative controls.

[0077] Figure 42 Figure 1 is a schematic diagram showing a representative workflow for permeabilization and labeling conditions used to study DNA tagmentation in fixed tissue sections. The results of partial protein digestion with trypsin or proteinase K during pre-permeabilization are shown.

[0078] Figure 43 according to Figure 42 The workflow shown shows collagenase treatment followed by proteinase-K ( Figure 43 A) or trypsin ( Figure 43B) Graph showing the effect of pre-permeabilization on labeling efficiency. Experiments were performed in duplicate. Figure 43 C) Proteinase-K pre-permeabilization treatment resulted in uniformly high signals for the amplified tagged species compared to the negative control (phosphate-negative marker).

[0079] Figure 44 Figure 2 is a schematic diagram showing a representative workflow for studying the capture of DNA tagment from fixed tissue sections.

[0080] Figure 45 shows the Figure 44 The workflow shown is a diagram and photograph of the successful capture of DNA tagging from fixed tissue sections by pre-permeabilization with collagenase and proteinase-K. Each experiment was performed in duplicate: one for PCR downstream analysis and the other for hybridization using fluorescently labeled (Cy5) oligonucleotides (complementary to the connected tagging). Phosphate-positive samples produced detectable signals (Figures 45A and B), while phosphate-negative samples did not (Figure 45C). Figure 45D shows a hematoxylin-eosin image (left) and a corresponding spatial pattern of connected DNA tagging (right), showing the successful capture of DNA from tissue sections.

[0081] Figure 46A is a schematic diagram illustrating an exemplary sample processing device that can be used to implement the various steps and methods described herein.

[0082] Figure 46B is a schematic diagram illustrating an example imaging device that can be used to obtain images of biological samples, analytes, and feature arrays.

[0083] Figure 46C yes Figure 46A and Figure 46B Schematic diagram of an example of a control unit of a device. DETAILED DESCRIPTION

[0084] I. Introduction

[0085] The present invention describes devices, systems, methods and compositions for spatial analysis of biological samples. This section specifically describes certain general terms, analytes, sample types and preparation steps that will be referred to in later sections of the present invention.

[0086] (a) Spatial analysis

[0087] Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from unicellular or multicellular organisms (e.g., mammals). Tissues and cells obtained from mammals (e.g., humans) typically have different analyte levels (e.g., gene and / or protein expression), which can lead to differences in cell morphology and / or function. The location of cells in a tissue can affect the fate, behavior, morphology, and signal transduction and crosstalk of cells with other cells in the tissue. Information about the differences in analyte levels (gene and / or protein expression) in different cells of mammalian tissue can also help doctors select or give effective treatments for unicellular or multicellular organisms (e.g., mammals) based on the differences in analyte levels in different cells of the detected tissue. The differences in analyte levels in different cells of mammalian tissue can also provide information on how tissues (e.g., healthy and diseased tissues) function and / or develop. The differences in analyte levels in different cells of mammalian tissue can also provide different information on the pathogenesis of diseases in tissues and information on therapeutic mechanisms of action in tissues. The differences in analyte levels in different cells of mammalian tissue can also provide information on drug resistance mechanisms and the development of drug resistance mechanisms in mammalian tissues. The differences in the presence or absence of an analyte in different cells in a tissue of a multicellular organism (eg, a mammal) can provide information on the mechanisms of drug resistance and its development in the tissue of the multicellular organism.

[0088] Spatial analysis methods are used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells in a mammalian tissue or within a single cell of a mammal. For example, spatial analysis methods can be used to detect differences in analyte levels (e.g., gene and / or protein expression) within different cells in a tissue section sample, and data therefrom can be recombined to generate a three-dimensional map of analyte levels (e.g., gene and / or protein expression) in a tissue sample obtained from a mammal, for example, at a certain degree of spatial resolution (e.g., single-cell resolution).

[0089] Spatial heterogeneity in developmental systems is typically investigated using RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subpopulations, and subsequent genomic profiling (e.g., RNA-seq). However, such approaches rely on a relatively small set of predefined markers and therefore introduce selection bias that limits discovery. These previous methods also rely on a priori knowledge. Traditionally, spatial RNA analysis has relied on staining of a limited number of RNA species. In contrast, single-cell RNA sequencing allows for deep profiling of cellular gene expression, including noncoding RNAs, but established methods isolate cells from their native spatial context.

[0090] Current spatial analysis methods provide a large amount of analyte levels and / or expression data for a variety of analytes in a sample with high spatial resolution, for example, while retaining the natural spatial environment. Spatial analysis methods include, for example, using a capture probe that includes a spatial barcode (e.g., a nucleic acid sequence providing information about the position of the capture probe in a cell or tissue sample (e.g., a mammalian cell or mammalian tissue sample)) and a capture domain capable of binding to an analyte (e.g., protein and / or nucleic acid) produced by a cell or present in a cell. As described herein, a spatial barcode can be a nucleic acid with a unique sequence, a unique fluorophore or a unique combination of fluorophores, a unique amino acid sequence, a unique heavy metal or a unique combination of heavy metals, or any other unique detectable agent. The capture domain can be any reagent capable of binding to an analyte produced by a cell and / or present in a cell (e.g., a nucleic acid capable of hybridizing with nucleic acid from a cell (e.g., mRNA, genomic DNA, mitochondrial DNA or miRNA)), a substrate or binding partner of an analyte, or an antibody specifically bound to an analyte). The capture probe can also include a nucleic acid sequence complementary to a universal forward and / or universal reverse primer sequence. The capture probe can also include a cleavage site (eg, a cleavage recognition site for a restriction endonuclease), a photolabile bond, a heat-sensitive bond, or a chemical-sensitive bond.

[0091] Binding of the analyte to the capture probe can be detected using a variety of different methods, such as nucleic acid sequencing, fluorophore detection, nucleic acid amplification, nucleic acid ligation detection, and / or nucleic acid cleavage product detection. In some examples, detection is used to associate a specific spatial barcode with a specific analyte produced by and / or present in a cell (e.g., a mammalian cell).

[0092] The capture probe can be, for example, attached to a surface, such as a solid array, a bead, or a coverslip. In some examples, the capture probe is not attached to a surface. In some instances, the capture probe can be encapsulated in, embedded in, or layered on a surface of a permeable composition (e.g., any substrate described herein). For example, the capture probe can be encapsulated or arranged in permeable beads (e.g., gel beads). In some instances, the capture probe can be encapsulated in, embedded in, or layered on a surface of a substrate (e.g., any exemplary substrate described herein, such as a hydrogel or a porous membrane).

[0093] In some embodiments, cells or tissue samples comprising cells are contacted with capture probes attached to a substrate (e.g., a surface of a substrate), and the cells or tissue samples are permeabilized to release analytes from the cells and bind to the capture probes attached to the substrate. In some examples, analytes released from cells can be actively guided to the capture probes attached to the substrate using a variety of methods (e.g., electrophoresis, chemical gradients, pressure gradients, fluid flow, or magnetic fields).

[0094] In other examples, a variety of methods can be used to direct the capture probe to interact with the cell or tissue sample, including, for example, lipid anchors in the capture probe, reagents that specifically bind to or form covalent bonds with membrane proteins in the capture probe, fluid flow, pressure gradients, chemical gradients, or magnetic fields.

[0095] Non-limiting aspects of spatial analysis methods are described in WO 2011 / 127099, WO 2014 / 210233, WO 2014 / 210225, WO 2016 / 162309, WO 2018 / 091676, WO 2012 / 140224, WO 2014 / 060483, U.S. Patent No. 10,002,316, U.S. Patent No. 9,727,810, U.S. Patent Application Publication No. 2017 / 0016053, Rodriques et al., Science 363(6434):1463-1467, 2019; WO 2018 / 045186, Lee et al., Nat. Protoc. 10(3):442-458, 2015; WO 2016 / 007839, WO 2018 / 045181, WO 2014 / 163886, Trejo et al., PLoS ONE 14(2):e0212031, 2019, U.S. Patent Application Publication No. 2018 / 0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol. 15:50, 2017, WO 2017 / 144338, WO 2018 / 107054, WO 2017 / 222453, WO 2019 / 068880, WO 2011 / 094669, U.S. Patent No. 7,709,198, U.S. Patent No. 8,604,182, U.S. Patent No. 8,951,726, U.S. Patent No. 9,783,841, U.S. Patent No. 10,041,949, WO 2016 / 057552, WO 2017 / 147483, WO 2018 / 022809, WO 2016 / 166128, WO 2017 / 027367, WO 2017 / 027368, WO 2018 / 136856, WO 2019 / 075091, U.S. Patent No. 10,059,990, WO 2018 / 057999, WO 2015 / 161173, and Gupta et al., Nature Biotechnol. 36: 1197-1202, 2018, and may be used in any combination herein. Further non-limiting aspects of the spatial analysis method are described herein.

[0096] (b) General terms

[0097] Specific terminology is used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions. This section contains explanations of certain terms that appear in subsequent sections of this disclosure. If the descriptions in this section clearly conflict with usage in other sections of this disclosure, the definitions in this section will prevail.

[0098] (i) Barcode

[0099] A "barcode" is a label or identifier that conveys or is capable of conveying information (e.g., information about an analyte, bead, and / or capture probe in a sample). A barcode can be part of an analyte or can be independent of the analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes.

[0100] Barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. Barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. For example, a barcode can be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow identification and / or quantification of individual sequencing reads (e.g., a barcode can be or can include a unique molecular identifier or "UMI").

[0101] A barcode can spatially resolve molecular components found in a biological sample, e.g., at single-cell resolution (e.g., a barcode can be or can include a "spatial barcode"). In some embodiments, a barcode includes both a UMI and a spatial barcode. 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) separated by one or more non-barcode sequences.

[0102] (ii) Nucleic acids and nucleotides

[0103] The terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and include naturally occurring substances or functional analogs thereof. Particularly useful functional analogs of nucleic acids can hybridize with nucleic acids in a sequence-specific manner (e.g., can hybridize with two nucleic acids so that connection can occur between the two hybridized nucleic acids), or can be used as a template for replicating a specific nucleotide sequence. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures can have other backbone connections, including any of a variety of connections known in the art. Naturally occurring nucleic acids typically have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)).

[0104] Nucleic acids can include nucleotides having any of the various analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acids can have one or more bases selected from adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acids can have one or more bases selected from uracil (U), adenine (A), cytosine (C) or guanine (G). Useful non-natural bases that can be included in nucleic acids or nucleotides are known in the art.

[0105] (iii) Probes and targets

[0106] "Probe" or "target," when applied to a nucleic acid or nucleic acid sequence, is intended to serve as a semantic identifier for the nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the nucleic acid or sequence beyond that explicitly indicated.

[0107] (iv) Oligonucleotides and polynucleotides

[0108] The terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polymers of nucleotides having a length of about 2 to about 500 nucleotides. Oligonucleotides can be synthesized, enzymatically prepared (e.g., by polymerization), or prepared using a "split-pool" method. Oligonucleotides can include ribonucleotide monomers (i.e., oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some examples, an oligonucleotide can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in an oligonucleotide (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). For example, the length of the oligonucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400 to 500 nucleotides. The oligonucleotide may include (e.g., covalently or non-covalently) one or more functional moieties attached to the polymer structure. For example, the oligonucleotide may include one or more detectable labels (e.g., radioisotopes or fluorophores).

[0109] (v) Object

[0110] A "subject" is an animal, such as a mammal (eg, a human or non-human ape), or avian (eg, a bird), or other organism, such as a plant. Examples of subjects include, but are not limited to, mammals, such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates (i.e., humans or non-human primates); plants, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, rapeseed, or soybeans; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, or honey bees; arachnids, such as spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi, such as Pneumocystis carinii, Takifugu rubripes), yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe; or Plasmodium falciparum.

[0111] (vi) Genome

[0112] "Genome" generally refers to genomic information from a subject, for example, at least part or all of the genetic information encoded by the subject's genes. A genome can include coding regions (e.g., regions encoding proteins) as well as non-coding regions. A genome can include the sequences of some or all of the subject's chromosomes. For example, the human genome typically has a total of 46 chromosomes. The sequences of some or all of these chromosomes can constitute a genome.

[0113] (vii) Adapters, adapters, and tags

[0114] "Adapter," "joiner," and "tag" are terms used interchangeably in this disclosure and refer to substances that can be coupled to a polynucleotide sequence using any of a number of different techniques, including but not limited to ligation, hybridization, and tagging (in a process known as "tagmentation"). An adapter can also be a nucleic acid sequence that adds functionality, such as a spacer sequence, a primer sequence / site, a barcode sequence, or a unique molecular identifier sequence.

[0115] (viii) Hybridization, hybridization, annealing, and chain recombination

[0116] The terms "hybridization," "annealing," and "strand recombination" are used interchangeably in this disclosure and refer to the pairing of substantially complementary or fully complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence is joined by base pairing with a substantially complementary or fully complementary sequence to form a hybridization complex. For the 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 the individual bases in the two nucleic acid sequences are complementary to each other.

[0117] (ix) Primers

[0118] " Primer " is a single-stranded nucleic acid sequence with a 3 'end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed from RNA nucleotides and are used for RNA synthesis, while DNA primers are composed of DNA nucleotides and are used for DNA synthesis. Primers can also include RNA nucleotides and DNA nucleotides (for example, in a random or designed pattern). Primers can also include other natural or synthetic nucleotides with other functionalities as described herein. In some examples, a DNA primer can be used to initiate RNA synthesis, and vice versa (for example, an RNA primer can be used to initiate DNA synthesis). The length of the primer can be different. For example, a primer can be about 6 bases to about 120 bases. For example, a primer can include up to about 25 bases.

[0119] (x) Primer extension

[0120] "Primer extension" refers to any method in which two nucleic acid sequences (e.g., constant regions from each of two different capture probes) are linked (e.g., hybridized) by overlapping complementary nucleic acid sequences at their respective ends (e.g., 3' ends). Following such ligation, nucleic acid extension (e.g., enzymatic extension) can be performed at one or both ends using another nucleic acid sequence as an extension template. Enzymatic extension can be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0121] (xi) Proximity Connection

[0122] "Proximity ligation" is a method of joining two (or more) nucleic acid sequences that are adjacent to each other by enzymatic means (e.g., ligase). In some embodiments, proximity ligation may include a "gap filling" step, which involves the inclusion of one or more nucleic acids spanning the distance between two nucleic acid molecules of interest by a polymerase based on the nucleic acid sequence of the template nucleic acid molecule (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference).

[0123] A variety of methods can be used for proximity joining of nucleic acid molecules, including, but not limited to, "sticky-end" and "blunt-end" ligation. In addition, single-stranded ligation can be used to proximity join single-stranded nucleic acid molecules. Sticky-end proximity ligation involves hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be joined prior to the ligation event. Blunt-end proximity ligation generally does not involve hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack single-stranded overhangs at the site of ligation.

[0124] (xii) Nucleic acid extension

[0125] "Nucleic acid extension" generally involves incorporating one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, or derivatives thereof) into a molecule (e.g., but not limited to a nucleic acid sequence) in a template-dependent manner such that the contiguous nucleic acid is incorporated by an enzyme (e.g., a polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. For example, by using a complementary nucleic acid sequence as a template for nucleic acid synthesis, a primer that hybridizes to the complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule. Similarly, the 3' polyadenylated tail of an mRNA transcript that hybridizes to a poly (dT) sequence (e.g., a capture domain) can be used as a template for single-stranded synthesis of a corresponding cDNA molecule.

[0126] (xiii) PCR amplification

[0127] "PCR amplification" refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. For example, suitable reagents and conditions for implementing PCR are described in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers for a primer extension reaction, and reagents for the reaction. The oligonucleotide primers are of sufficient length to hybridize with the complementary genetic material under annealing conditions. The length of the primer generally depends on the length of the amplification domain, but is generally at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and can be up to 40 bp or longer, with the length of the primer generally being between 18 and 50 bp. The genetic material can be contacted with a single primer or a set of two primers (forward and reverse primers), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.

[0128] In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In certain embodiments, the DNA polymerase is from bacteria, for example, the DNA polymerase is a bacterial DNA polymerase. For example, the DNA polymerase can be from bacteria of the genus Escherichia coli, Bacillus, Thermobacterium, or Thermococcus.

[0129] Suitable examples of DNA polymerases that can be used include, but are not limited to, E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot Start Taq DNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase, DNA polymerase, Quick DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-fidelity DNA polymerases, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase, and T7 DNA polymerase.

[0130] The term "DNA polymerase" includes not only naturally occurring enzymes, but also all modified derivatives thereof, and also derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase can be modified to remove 5'-3' exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that can be used include, but are not limited to, mutants that retain at least some of their functionality, such as the DNA polymerase activity of the wild-type sequence. Under different reaction conditions, such as temperature, template concentration, primer concentration, etc., mutations can affect the activity profile of the enzyme, such as increasing or decreasing the polymerization rate. Mutations or sequence modifications may also affect the exonuclease activity and / or thermal stability of the enzyme.

[0131] In some embodiments, PCR amplification can include reactions such as, but not limited to, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, isothermal amplification, and / or loop-mediated amplification.

[0132] In some embodiments, PCR amplification uses a single primer that is complementary to the 3' tag of the target DNA fragment. In some embodiments, PCR amplification uses a first and a second primer, wherein at least the 3' terminal portion of the first primer is complementary to at least a portion of the 3' tag of the target nucleic acid fragment, and wherein at least the 3' terminal portion of the second primer displays at least a portion of the sequence of the 5' tag of the target nucleic acid fragment. In some embodiments, the 5' terminal portion of the first primer is not complementary to the 3' tag of the target nucleic acid fragment, and the 5' terminal portion of the second primer does not display at least a portion of the sequence of the 5' tag of the target nucleic acid fragment. In some embodiments, the first primer includes a first universal sequence and / or the second primer includes a second universal sequence.

[0133] In some embodiments (e.g., when PCR amplification is performed on the captured DNA), DNA ligase can be used to connect the PCR amplification product to other sequences. DNA ligase activity can be provided by one or more different DNA ligases. In some embodiments, the DNA ligase is from bacteria, for example, the DNA ligase is a bacterial DNA ligase. In some embodiments, the DNA ligase is from a virus (e.g., a bacteriophage). For example, the DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Thermococcus (strain 9oN) DNA ligase (9oN™ DNA ligase, available from New England Biolabs, Ipswich, Massachusetts) and Ampligase TM (Available from Epicentre Biotechnologies, Madison, Wisconsin.) Derivatives, such as sequence-modified derivatives and / or mutants thereof, may also be used.

[0134] In some embodiments, the genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The desired reverse transcriptase activity can be provided by one or more different reverse transcriptases, suitable examples of which include but are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScript TM 、MultiScribe TM 、ThermoScript TM ,and I, II, III and IV enzymes. "Reverse transcriptase" includes not only the naturally occurring enzyme but also all such modified derivatives thereof, and also includes derivatives of the naturally occurring reverse transcriptase.

[0135] In addition, sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases can be used for reverse transcription, including mutants that retain at least some functional (e.g., reverse transcriptase) activity of the wild-type sequence. The reverse transcriptase can be provided as part of a composition comprising other components, such as a stabilizing component that enhances or improves the activity of the reverse transcriptase, such as an RNase inhibitor, an inhibitor of DNA-dependent DNA synthesis, such as actinomycin D. Many sequence-modified derivatives or mutants of reverse transcriptases, such as M-MLV, and compositions comprising unmodified and modified enzymes, such as ArrayScript TM 、MultiScribe TM 、ThermoScript TM ,and I, II, III and IV enzymes are commercially available.

[0136] Certain reverse transcriptases (e.g., avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase) can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that can use both RNA and ssDNA as templates for the extension reaction, such as AMV or MMLV reverse transcriptase.

[0137] In some embodiments, quantification of RNA and / or DNA is performed by real-time PCR (also known as quantitative PCR or qPCR) using techniques well known in the art, such as, but not limited to, "TAQMAN TM "or or on a capillary (" Capillary". In some embodiments, quantification of genetic material is determined by light absorbance and real-time PCR. In some embodiments, quantification of genetic material is determined by digital PCR. In some embodiments, the analyzed gene can be compared to a reference nucleic acid extract (DNA and RNA) for both expression (mRNA) and quantity (DNA) to compare expression levels of the target nucleic acid.

[0138] (xiv) Antibodies

[0139] An "antibody" is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a molecular structural shape that resembles a Y-shape. Naturally occurring antibodies, known as immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can also be synthesized artificially. For example, recombinant antibodies known as monoclonal antibodies can be synthesized using synthetic genes by recovering the antibody gene from a source cell, amplifying it into an appropriate vector, and introducing the vector into a host so that the host expresses the recombinant antibody. In general, recombinant antibodies can be cloned from any antibody-producing animal species using appropriate oligonucleotide primers and / or hybridization probes. Recombinant technology can be used to generate antibodies and antibody fragments, including non-endogenous substances.

[0140] Synthetic antibodies can be obtained from non-immunoglobulin sources. For example, antibodies can be produced from nucleic acids (e.g., aptamers) and non-immunoglobulin scaffolds (e.g., peptide aptamers), into which hypervariable loops are inserted to form antigen-binding sites. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue permeabilization.

[0141] Antibodies can also include affimer proteins, which are affinity reagents typically having a molecular weight of about 12-14 kDa. Affimer proteins typically bind to targets (e.g., target proteins) with high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive proteins. In some embodiments, affimer proteins are derived from cysteine protease inhibitors and include a peptide loop and a variable N-terminal sequence that provides a binding site.

[0142] Antibodies may also include single domain antibodies (V H H domain and VNAR domain), scFv and Fab fragments.

[0143] (xv) Affinity group

[0144] An "affinity group" is a molecule or portion of a molecule that has a high affinity or preference for associating or binding with another specific or particular molecule or portion. The association or binding with another specific or particular molecule or portion can be through non-covalent interactions, such as hydrogen bonds, ionic forces, and van der Waals interactions. For example, an affinity group can be biotin, which has a high affinity or preference for associating or binding with the proteins avidin or streptavidin. For example, an affinity group can also refer to avidin or streptavidin that has affinity for biotin. Other examples of affinity groups and specific or particular molecules or portions that they bind or associate with include, but are not limited to, antibodies or antibody fragments and their respective antigens, such as digoxin and anti-digoxin antibodies, lectins, and carbohydrates (e.g., sugars, monosaccharides, disaccharides, or polysaccharides), as well as receptors and receptor ligands.

[0145] Any pair of an affinity group and the specific or particular molecule or moiety to which it is bound or associated can have its effects reversed, for example, between a first molecule and a second molecule, where in a first instance the first molecule is characterized as an affinity group for the second molecule and in a second instance the second molecule is characterized as an affinity group for the first molecule.

[0146] (xvi) Labels, detectable labels and optical labels

[0147] The terms "detectable marker," "optical marker," and "label" are used interchangeably herein to refer to a directly or indirectly detectable portion that is associated (e.g., coupled) with a molecule to be detected (e.g., a capture probe or analyte). Detectable labels can be directly detectable by themselves (e.g., radioisotope labels or fluorescent labels), or, in the case of enzyme labels, can be indirectly detectable, e.g., by catalyzing the chemical alteration of a substrate compound or composition that is directly detectable. Detectable labels can be suitable for small-scale detection and / or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.

[0148] Detectable markers can be detected qualitatively (e.g., optical or spectral), or can be quantitative. Qualitative detection generally includes a detection method that confirms the presence or occurrence of a detectable marker, while quantitative detection generally includes a detection method with a quantifiable (e.g., digitally reportable) value, such as intensity, duration, polarization and / or other properties. In some embodiments, detectable markers are bound to a feature or a capture probe associated with a feature. For example, a feature that is detectably labeled can include fluorescent, colorimetric or chemiluminescent markers attached to a bead (see, e.g., Rajeswari et al., J.Microbiol Methods 139: 22-28, 2017, and Forcucci et al., J. Biomed Opt. 10: 105010, 2015, the entire contents of which are incorporated herein by reference).

[0149] In some embodiments, multiple detectable labels can be attached to a feature, capture probe, or composition to be detected. For example, a detectable label can be incorporated during nucleic acid polymerization or amplification (e.g., -labeled nucleotides, e.g. Any suitable detectable label can be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be from the following group: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), Allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, aniline blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Feulgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBO TM -1 / BO-PROTM -1. BOBO TM -3 / BO-PRO TM -3. BTC, Calcium Crimson, Calcein Blue TM , Calcium Green-1 TM , Calcium Orange TM , White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade CascadeYellow TM , CCF2 (GeneBLAzer TM ), CFP (cyan fluorescent protein), CFP / YFP FRET, chromomycin A3, C1-NERF (low pH), CPM, 6-CR 6G, CTC-formazan Cychrome (PE-Cy5), dansylamide, dansylcadaverine, dansyl chloride, DAPI, dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18 (5)), DIDS, Di1 (DilC18 (3)), DiO (DiOC18 (3)), DiR (DilC18 (7)), Di-4ANEPPS, Di-8ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, Alcohol, eosin, erythromycin, ethidium bromide, ethidium homodimer-1 (EthD-1), europium (III) chloride, 5-FAM (5-carboxyfluorescein), fast blue, fluorescein dT phosphoamidite, FITC, Fluo-3, Fluo-4, Fluoro-Gold TM (High pH), Fluoro-Gold TM (low pH), Fluoro-Jade, Fura-2 (high calcium), Fura-2 / BCECF, FuraRed TM (High Calcium), Fura Red TM / Fluo-3, GeneBLAzer TM(CCF2), red-shifted GFP (rsGFP), wild-type GFP, GFP / BFP-FRET, GFP / DsRed-FRET, Hoechst 33342 & 33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5-indoleacetic acid, Indo-1 (high calcium), Indo-1 (low calcium), indole dicarbocyanine, indole tricarbocyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS 751(+DNA), LDS 751(+RNA), LOLO TM -1 / LO-PRO TM -1, Lucifer Yellow, LysoSensor TM Blue (pH 5), LysoSensor TM Green (pH 5), LysoSensor TM Yellow / blue (pH 4.2), green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM , Marina 4-Methylumbelliferone, Mitramycin, green, orange, Red, NBD(amine), Nile Red, Oregon Oregon Oregon Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinyl Chlorophyll Protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1, POPO TM -3 / PO-PRO TM -3, propidium iodide (PI), PyMPO, pyrene, pyrroline Y, Quantum Red (PE-Cy5), quinacridone mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), red fluorescent protein (DsRed), resorufin, RH 414, Rhod-2, rhodamine B, Rhodamine Green TM , Rhodamine Red TM, Rhodamine phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, (high pH), (high pH), (low pH), Sodium Green TM , blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), Texas Texas (NHS ester), thiadicarbocyanine, thiazole orange, Tricolor (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima Yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO647N, TYE 563, TYE 665, TYE 705, (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, (NHS ester) and Dy 750 (NHS ester).

[0150] As described above, in some embodiments, the detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases, such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze a chemiluminescent reaction given an appropriate substrate (e.g., an oxidizing agent plus a chemiluminescent compound). Many families of compounds are known to provide chemiluminescence under various conditions. Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-1,4-phthalazinedione luminal, 5-amino-6,7,8-trimethoxy- and dimethylamino[ca]benzene analogs. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of chemiluminescent compound families include, for example, 2,4,5-triphenylimidazole, p-dimethylamino and -methoxy substituents, oxalates (e.g., oxalyl active esters), p-nitrophenyl, N-alkyl acridinium esters, fluorescein, lucigenin, or acridinium esters.

[0151] (xvii) Template switching oligonucleotides

[0152] A "template switching oligonucleotide" is an oligonucleotide that hybridizes to non-templated nucleotides added by a reverse transcriptase (e.g., an enzyme with terminal transferase activity) during reverse transcription. In some embodiments, the template switching oligonucleotide hybridizes to non-templated poly(C) nucleotides added by the reverse transcriptase. In some embodiments, the template switching oligonucleotide adds a common 5' sequence to the full-length cDNA used for cDNA amplification.

[0153] In some embodiments, a template-switching oligonucleotide adds a consensus sequence to the 5' end of the RNA being reverse transcribed. For example, the template-switching oligonucleotide can hybridize to an untemplated poly(C) nucleotide added to the end of a cDNA molecule and provide a template for the reverse transcriptase to continue replication to the 5' end of the template-switching oligonucleotide, thereby generating a full-length cDNA capable of further amplification. In some embodiments, once the full-length cDNA molecule is generated, the template-switching oligonucleotide can be used as a primer in a cDNA amplification reaction.

[0154] In some embodiments, a template-switching oligonucleotide is added before, simultaneously with, or after reverse transcription or other terminal transferase-based reactions. In some embodiments, a template-switching oligonucleotide is included in a capture probe. In certain embodiments, a sample analysis method using a template-switching oligonucleotide can involve generating a nucleic acid product from an analyte in a tissue sample, followed by further processing of the nucleic acid product using a template-switching oligonucleotide.

[0155] The template switching oligonucleotide may include a hybridization region and a template region. The hybridization region may include any sequence capable of hybridizing with a target. In some embodiments, the hybridization region may, for example, include continuous G bases to complement the overhanging C bases at the 3' end of the eDNA molecule. Continuous G bases may include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The template sequence may include any sequence to be incorporated into the eDNA. In other embodiments, the hybridization region may include at least one base in addition to at least one G base. In other embodiments, hybridization may include bases other than G bases. In some embodiments, the template region includes at least one (e.g., at least 2, 3, 4, 5 or more) tag sequence and / or functional sequence. In some embodiments, the template region and the hybridization region are separated by a spacer.

[0156] In some embodiments, the template region includes a barcode sequence. The barcode sequence can function as a spatial barcode and / or as a unique molecular identifier. The template switching oligonucleotide can include deoxyribonucleic acid; RNA; modified nucleic acid, including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reverse dT, 5-methyl dC, 2′-deoxyinosine, super T (5-hydroxybutyl-2′-deoxyuridine), super G (8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, e.g., UNA-A, UNA-U, UNA-C, UNA-g), Iso-dG, Iso-dC, 2′ fluoro bases (e.g., fluoro C, fluoro U, fluoro A, and fluoro G), or any combination thereof.

[0157] In some embodiments, the template-switching oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides in length or longer. In some embodiments, the template-switching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides in length or longer.

[0158] (xviii) Splint oligonucleotides

[0159] A "splint oligonucleotide" is an oligonucleotide that, when hybridized with other polynucleotides, acts as a "splint," bringing the polynucleotides adjacent to each other so that they can be linked together. In some embodiments, the splint oligonucleotide is DNA or RNA. The splint oligonucleotide may include a nucleotide sequence that is partially complementary to a nucleotide sequence from two or more different oligonucleotides. In some embodiments, the splint oligonucleotide assists in joining a "donor" oligonucleotide and an "acceptor" oligonucleotide. Generally, RNA ligase, DNA ligase, or other types of ligases are used to join the two nucleotide sequences together.

[0160] In some embodiments, the splint oligonucleotide is between 10 and 50 oligonucleotides in length, for example, between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides in length. In some embodiments, the splint oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25 nucleotides in length.

[0161] (c) Analyte

[0162] The devices, systems, methods, and compositions described in this disclosure can be used to detect and analyze a variety of different analytes. For purposes of the present invention, an "analyte" can include any biological substance, structure, part, or component to be analyzed. The term "target" can similarly refer to an analyte of interest.

[0163] Analytes can be broadly divided into two categories: nucleic acid analytes and non-nucleic acid analytes. 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 phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitination variants of proteins, sulfated variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte can be an organelle (e.g., a nucleus or mitochondria).

[0164] Cell surface features corresponding to the analyte may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, clusters of differentiation proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) status of cell surface proteins, gap junctions, and adherens junctions.

[0165] Analytes can be derived from specific cell types and / or specific subcellular regions. For example, analytes can be derived from the cytoplasm, nucleus, mitochondria, microsomes, or more generally, any other compartment, organelle, or part of a cell. Permeabilizing agents specifically targeted to certain cellular compartments and organelles can be used to selectively release analytes from cells for analysis.

[0166] Examples of nucleic acid analytes include DNA analytes, such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids.

[0167] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (miRNA) and viral RNA. RNA can be a transcript (e.g., present in a tissue section). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA with a length greater than 200 nucleic acid bases). Small RNA mainly includes 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snRNA), Piwi interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16srRNA or 23s rRNA).

[0168] Other examples of analytes include mRNA and cell surface features (e.g., using markers described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cellular methylation status, mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq and / or Micrococcal enzyme-seq), mRNA and metabolites (e.g., using markers described herein), barcoded markers (e.g., oligonucleotide-tagged antibodies described herein), and V(D)J sequences of immune cell receptors (e.g., T cell receptors), mRNA and perturbators (e.g., CRISPR crRNA / sgRNA, TALENs, zinc finger nucleases, and / or antisense oligonucleotides as described herein).

[0169] The analyte may include a nucleic acid molecule having a nucleic acid sequence encoding at least a portion of the V(D)J sequence of an immune cell receptor (e.g., a TCR or BCR). In some embodiments, the nucleic acid molecule is first generated from reverse transcription of the corresponding mRNA using a poly(T)-containing primer. The generated cDNA can then be barcoded using a capture probe having a barcode sequence (and optionally a UMI sequence) that hybridizes to at least a portion of the generated cDNA. In some embodiments, the template-switching oligonucleotide hybridizes to the poly(C) tail added to the 3′ end of the cDNA by a reverse transcriptase. The original mRNA template and the template-switching oligonucleotide can be denatured from the cDNA, and the barcoded capture probe can then hybridize to the cDNA and generate a complement of the cDNA. Other methods and compositions suitable for barcoding cDNA generated from mRNA transcripts, including those encoding V(D)J regions of immune cell receptors, and / or compositions and barcoding methods comprising template-switching oligonucleotides are described in PCT patent application PCT / US2017 / 057269, filed on October 18, 2017, and U.S. patent application serial number 15 / 825,740, filed on November 29, 2017, both of which are incorporated herein by reference in their entirety. V(D)J analysis can also be accomplished by using one or more markers that bind to specific surface features of immune cells and are associated with a barcode sequence. The one or more markers can include MHC or MHC multimers.

[0170] As described above, the analyte may include a nucleic acid capable of functioning as a component of a gene editing reaction, such as, for example, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing. Thus, the capture probe may include a nucleic acid sequence complementary to the analyte (e.g., a sequence hybridizable to a CRISPR RNA (crRNA), a single guide RNA (sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).

[0171] In certain embodiments, analytes can be extracted from living cells. Processing conditions can be adjusted to ensure that the biological sample remains viable during analysis and that the analyte is extracted (or released) from the living cells of the sample. Analytes derived from living cells can be obtained only once from the sample, or at intervals while the sample remains viable.

[0172] In general, the systems, devices, methods, and compositions can be used to analyze any number of analytes. For example, the number of analytes 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 1000, at least about 10,000, at least about 100,000, or more different analytes present in a sample region or within an individual feature of a substrate. Methods for performing multiplex analysis to analyze two or more different analytes will be discussed in subsequent sections of the present invention.

[0173] (d) Biological samples

[0174] (i) Type of biological sample

[0175] A "biological sample" is obtained from a subject for analysis using any of a variety of techniques, including but not limited to biopsy, surgery, and laser capture microscopy (LCM), and typically includes cells and / or other biological material from the subject. In addition to the above-mentioned subjects, biological samples can also be obtained from prokaryotes, such as bacteria, such as Escherichia coli, Staphylococcus aureus, or Mycoplasma pneumoniae; Archaea; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Biological samples can be obtained from non-mammalian organisms (such as plants, insects, arachnids, nematodes, fungi, or amphibians). Biological samples can also be obtained from eukaryotic organisms, such as patient-derived organoids (PDOs) or patient-derived xenografts (PDXs). The subject from whom the biological sample can be obtained can be a healthy or asymptomatic individual, an individual having or suspected of having a disease (e.g., a patient having a disease such as cancer) or an individual with a pre-treatment for a disease, and / or an individual in need of treatment or suspected of needing treatment.

[0176] The biological sample can include any number of macromolecules, such as cellular macromolecules and organelles (such as mitochondria and nuclei). The biological sample can be a nucleic acid sample and / or a protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, a biopsy, a core biopsy, a needle aspiration or a fine needle aspiration. The sample can be a liquid sample, such as a blood sample, a urine sample or a saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histological sample, a histopathological sample, a plasma or serum sample, a tumor sample, a living cell, a cultured cell, a clinical sample, such as whole blood or a blood-derived product, a blood cell or a cultured tissue or cell, including a cell suspension.

[0177] Cell-free biological samples may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from body samples such as blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0178] A biological sample may be from a homogenous culture or population of a subject or organism as described herein, or may alternatively be from a collection of several different organisms, for example, in a community or ecosystem.

[0179] A biological sample may include one or more diseased cells. Diseased cells may exhibit altered metabolic properties, gene expression, protein expression, and / or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, neurological disorders, and cancer. Cancer cells may be derived from solid tumors, hematologic malignancies, cell lines, or obtained as circulating tumor cells.

[0180] Biological samples can also include fetal cells. For example, a procedure such as amniocentesis can be performed to obtain a fetal cell sample from the maternal circulation. Fetal cell sequencing can be used to identify any of a number of genetic diseases, including, for example, aneuploidy, such as Down syndrome, Edwards syndrome, and Patau syndrome. In addition, cell surface characteristics of fetal cells can be used to identify any of a variety of diseases or conditions.

[0181] Biological samples can also include immune cells. Sequence analysis of the immune function of such cells, including genomics, proteomics, and cell surface characteristics, can provide rich information to help understand the state and function of the immune system. For example, determining the status (e.g., negative or positive) of minimal residual disease (MRD) in multiple myeloma (MM) patients after autologous stem cell transplantation is considered to be a predictor of MRD in MM patients (see, e.g., U.S. Patent Application Publication No. 2018 / 0156784, which is incorporated herein by reference in its entirety).

[0182] Examples of immune cells in a biological sample include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and helper T cells), natural killer cells, cytokine-induced killer (CIK) cells, myeloid cells, such as granulocytes (basophils, eosinophils, neutrophils / multinuclear neutrophils), monocytes / macrophages, mast cells, platelets / megakaryocytes, and dendritic cells.

[0183] As described above, a biological sample may include a single analyte of interest or multiple analytes of interest. Methods for performing multiplexing to analyze two or more different analytes in a single biological sample will be discussed in subsequent sections of the present invention.

[0184] (ii) Preparation of biological samples

[0185] Various steps can be performed to prepare a biological sample for analysis. Unless otherwise indicated, the preparation steps described below can generally be combined in any manner to appropriately prepare a particular sample for analysis.

[0186] (1) Tissue sections

[0187] Biological samples can be obtained from a subject (e.g., by surgical biopsy, whole subject sectioning) or grown in vitro as a cell population on a growth substrate or culture dish and prepared as tissue sections or tissue slices for analysis. The grown sample can be thin enough to be analyzed without further processing steps. Alternatively, the grown sample and the sample obtained by biopsy or sectioning can be prepared as thin tissue slices using a mechanical cutting device (e.g., a vibrating blade microtome). As another alternative, in some embodiments, thin tissue slices can be prepared by applying a biological sample print to a suitable substrate material.

[0188] The thickness of the tissue section can be a fraction of the maximum cross-sectional dimension of the cells (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections having a thickness greater than the maximum cross-sectional dimension of the cells can also be used. For example, cryostat sections having a thickness of, for example, 10-20 microns can be used.

[0189] In some embodiments, the thickness of tissue section is 1-100 micron, 1-50 micron, 1-30 micron, 1-25 micron, 1-20 micron, 1-15 micron, 1-10 micron, 2-8 micron, 3-7 micron or 4-6 micron, but as mentioned above, thickness can also be analyzed to be greater than or less than the section of these scopes.

[0190] It is also possible to obtain multiple slices from a single biological sample. For example, multiple tissue slices can be obtained from a surgical biopsy sample by serially sectioning the biopsy sample using a microtome blade. In this way, spatial information between serial slices can be preserved, and the slices can be analyzed sequentially to obtain three-dimensional information about the biological sample.

[0191] (2) Freezing

[0192] In some embodiments, biological sample (for example, tissue section as described above) can be prepared by deep freezing at a temperature suitable for maintaining or keeping the integrity (for example, physical properties) of tissue structure. For example, the temperature can be lower than -20 ° C, or lower than -25 ° C, -30 ° C, -40 ° C, -50 ° C, -60 ° C, -70 ° C, -80 ° C, -90 ° C, -100 ° C, -110 ° C, -120 ° C, -130 ° C, -140 ° C, -150 ° C, -160 ° C, -170 ° C, -180 ° C, -190 ° C or -200 ° C. Frozen tissue sample can be cut (for example, thin slice) onto the substrate surface using any number of suitable methods. For example, a freezing microtome (for example, cryostat) can be used to prepare a tissue sample, and the freezing microtome is arranged on a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of nucleic acid in the sample. For example, this temperature can be lower than -15 ° C, lower than -20 ° C or lower than -25 ° C.

[0193] (3) Formaldehyde fixation and paraffin embedding

[0194] In some embodiments, biological samples can be prepared using formaldehyde fixation and paraffin embedding (FFPE), which is an established method. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formaldehyde fixation and paraffin embedding. After the sample is fixed and embedded in paraffin or resin blocks, the sample can be sectioned as described above. Prior to analysis, paraffin-embedded material can be removed from the tissue sections (e.g., deparaffinized) by incubating the tissue sections in an appropriate solvent (e.g., xylene) and then rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

[0195] (4) Fixed

[0196] As an alternative to formaldehyde fixation described above, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, the sample can be fixed by immersing it in ethanol, methanol, acetone, paraformaldehyde-triton, and combinations thereof.

[0197] In some embodiments, acetone fixation is used with fresh frozen samples, which may include, but are not limited to, cortical tissue, mouse olfactory bulbs, human brain tumors, human postmortem brain, and breast cancer samples. When acetone fixation is performed, a pre-permeabilization step (described below) may not be performed. Alternatively, acetone fixation may be performed in conjunction with a permeabilization step.

[0198] (5) Embedding

[0199] As an alternative to paraffin embedding, biological samples can be embedded in any of a variety of other embedding materials to provide a structural substrate for the sample prior to sectioning and other processing steps. Generally, the embedding material is removed prior to analyzing the tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylates), epoxies, and agar.

[0200] (6) Dyeing

[0201] For ease of visualization, biological samples can be stained using a variety of stains and staining techniques. In some embodiments, for example, any number of stains can be used to stain the sample, including but not limited to acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hodgkin's stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranine.

[0202] The sample can be stained using hematoxylin-eosin (H&E) staining, Papanicolaou staining, Masson's trichrome staining, silver staining, Sudan red staining, and / or periodic acid-Schiff (PAS) staining. PAS staining is typically performed after formaldehyde or acetone fixation. In some embodiments, the sample can be stained using Romanowski staining, including Wright staining, Jenner staining, Kangrunwald staining, Leishman staining, and Giemsa staining.

[0203] In some embodiments, the biological sample can be decolorized. Methods for decontamination or decolorization of biological samples are known in the art and generally depend on the nature of the stain applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. These stains can be removed using techniques such as cleavage of disulfide bonds by washing with reducing agents and detergents, treatment with hygroscopic salts, treatment with antigen retrieval solutions, and treatment with acidic glycine buffers. For example, methods for multiple staining and decolorization are described in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6: 8390, Pirici et al., J. Histochem. Cytochem. 2009; 57: 567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57: 899-905, the entire contents of which are incorporated herein by reference.

[0204] (7) Hydrogel embedding

[0205] In some embodiments, a biological sample can be embedded in a hydrogel matrix. Embedding a sample in this manner generally involves contacting the biological sample with the hydrogel, such that the biological sample is surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymeric material and activating the polymeric material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.

[0206] In some embodiments, the biological sample is immobilized in the hydrogel by crosslinking of the hydrogel-forming polymeric material. Crosslinking can be performed chemically and / or photochemically, or by any other hydrogel-forming method known in the art.

[0207] The composition and application of the hydrogel substrate to a biological sample generally depends on the nature and preparation of the biological sample (e.g., sliced, non-sliced, fixed type). As an example, where the biological sample is a tissue slice, the hydrogel substrate may include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, when the biological sample consists of cells (e.g., cultured cells or cells isolated from a tissue sample), the cells may be incubated with a monomer solution and an APS / TEMED solution. For cells, the hydrogel substrate gel is formed in a compartment, including but not limited to a device for culturing, maintaining, or transporting cells. For example, the hydrogel substrate can be formed using a monomer solution plus APS / TEMED, which is added to a compartment to a depth of between about 0.1 μm and about 2 mm.

[0208] Other methods and aspects of hydrogel embedding of biological samples are described, for example, in Chen et al., Science 347(6221): 543-548, 2015, the entire contents of which are incorporated herein by reference.

[0209] (8) Isometric Expansion

[0210] In some embodiments, biological samples embedded in a hydrogel can be expanded isometrically. An isometric expansion method that can be used includes hydration, which is a preparatory step in expansion microscopy, as described by Chen et al., Science 347(6221):543-5482015.

[0211] Isometric expansion can be achieved by anchoring one or more components of the biological sample to the gel, followed by gel formation, protein hydrolysis, and swelling. Isometric expansion of the biological sample can occur before the biological sample is fixed on the substrate, or after the biological sample is fixed on the substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate before contacting the substrate with the capture probe, which will be discussed in more detail in subsequent sections.

[0212] In general, the steps used to perform isometric spreading of biological samples may depend on the characteristics of the sample (e.g., thickness of tissue sections, fixation, cross-linking) and / or the analyte of interest (e.g., different conditions for anchoring RNA, DNA, and proteins to gels).

[0213] In some embodiments, proteins in a biological sample are anchored to a swellable gel (e.g., a polyelectrolyte gel). Antibodies can be directed to the protein before, after, or in conjunction with anchoring to the swellable gel. DNA and / or RNA in a biological sample can also be anchored to the swellable gel via a suitable linker. Examples of such linkers include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X-SE) (available from ThermoFisher, Waltham, Massachusetts), Label IT amine (available from MirusBio, Madison, Wisconsin), and Label X (described, for example, in Chen et al., Nat. Methods 13: 679-684, 2016, the entire contents of which are incorporated herein by reference).

[0214] The equidistant spreading of the sample can improve the spatial resolution of subsequent analysis of the sample. The increased resolution in the spatial profile can be determined by comparing the equidistantly spread sample with a sample that was not equidistantly spread.

[0215] In some embodiments, the biological sample is isometrically expanded to at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non-expanded size. In some embodiments, the sample is isometrically expanded to at least 2 times and less than 20 times its non-expanded size.

[0216] (9) Substrate attachment

[0217] In some embodiments, a biological sample can be attached to a hydrogel substrate. Examples of substrates suitable for this purpose are described in detail below. Attachment of the biological sample can be irreversible or reversible, depending on the nature of the sample and the subsequent steps in the analytical method.

[0218] In certain embodiments, a sample can be reversibly attached to a substrate by applying a suitable polymer coating to the substrate and contacting the sample with the polymer coating. The sample can then be separated from the substrate using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers suitable for this purpose.

[0219] More generally, in some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate sample attachment to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.

[0220] (10) Cell disaggregation

[0221] In some embodiments, the biological sample corresponds to a cell (e.g., from a cell culture or tissue sample). In a cell sample having a plurality of cells, the plurality of individual cells may not naturally aggregate. For example, the cells may be from a cell suspension and / or from cells separated or disaggregated from a tissue or tissue section.

[0222] Alternatively, the cells in the sample can aggregate and can be depolymerized into multiple individual cells using, for example, enzymes or mechanical techniques. Examples of enzymes for enzymatic depolymerization include, but are not limited to, dispase, collagenase, trypsin, and combinations thereof. For example, a tissue homogenizer can be used for mechanical depolymerization.

[0223] (11) Suspension and adherent cells

[0224] In some embodiments, the biological sample can be obtained from a cell culture grown in vitro. A sample from a cell culture can include one or more suspended cells that are anchorage-independent within the cell culture. Examples of such cells include, but are not limited to, cell lines derived from hematopoietic cells and cell lines derived from the following cell lines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI-H322M, and MALME-3M.

[0225] The sample from the cell culture may include one or more adherent cells grown on the surface of a container containing culture medium. Non-limiting examples of adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical carcinoma) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myeloid leukemia) cells, LNCaP (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells, SH-SY5Y (neuroblastoma, cloned from myeloma) cells, and SC-17Y (bone cancer) cells. cells, T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, National Cancer Institute Cancer Cell Line Panel 60 (NCI60), Vero (African green monkey kidney epithelial cell line), MC3T3 (embryonic skull) cells, GH3 (pituitary tumor) cells, PC12 (pheochromocytoma) cells, dog MDCK kidney epithelial cells, Xenopus laevis A6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insect epithelial cells.

[0226] Other examples of adherent cells are shown in Table 1 and are categorized in, for example, “A Catalog of in Vitro Cell Lines, Transplantable Animal and Human Tumors and Yeast,” Division of Cancer Treatment and Diagnostics (DCTD), National Cancer Institute (2013) and Abaan et al., “The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology,” Cancer Research 73(14): 4372-82, 2013, the entire contents of which are incorporated herein by reference.

[0227] Table 1: Examples of adherent cells

[0228]

[0229]

[0230]

[0231] In some embodiments, the adherent cells are cells corresponding to one or more of the following cell lines: BT549, HS578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31, A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES, DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1, U87, vero, MC3T3, GH3, PC12, dog MDCK kidney epithelial, Xenopus A6 kidney epithelial, zebrafish AB9, and Sf9 insect epithelial cell lines.

[0232] (12) Tissue permeabilization

[0233] In some embodiments, the biological sample can be permeabilized to facilitate the transfer of analytes from the sample and / or to facilitate the transfer of substances (e.g., capture probes) into the sample. If the sample is not permeabilized enough, the amount of analyte captured from the sample may be too low to be fully analyzed. Conversely, if the tissue sample is too permeabilized, the relative spatial relationships of the analytes within the tissue sample may be lost. Therefore, it is ideal to fully permeabilize the tissue sample to obtain good signal intensity while still maintaining a balance between the spatial resolution of the analyte distribution in the sample.

[0234] Generally, biological samples 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, TM or Tween-20 TM) and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample can be incubated with a cell permeabilizing agent to facilitate permeabilization of the sample. For example, other methods for sample permeabilization are described 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 conjunction with the samples described herein.

[0235] In some embodiments, when an anti-diffusion medium is used to limit the migration of an analyte or other substance during an analytical procedure, the anti-diffusion medium may include at least one permeabilization agent. For example, the anti-diffusion medium may include holes (e.g., micropores, nanopores, or picopores) containing a permeabilization buffer or reagent. In some embodiments, wherein the anti-diffusion medium is a hydrogel, the hydrogel may include a permeabilization buffer. In some embodiments, before the hydrogel contacts the sample, the hydrogel is soaked in a permeabilization buffer. In some embodiments, when the anti-diffusion medium is applied to a biological sample, the hydrogel or other anti-diffusion medium may include a dry reagent or a monomer to deliver the permeabilization agent. In some embodiments, the anti-diffusion medium (i.e., a hydrogel) is covalently attached to a solid substrate (i.e., an acrylic glass slide). In some embodiments, the hydrogel may be modified to include both capture probes and permeabilization agents. For example, the hydrogel membrane may be modified to include a spatial barcoded capture probe. Then, before the spatial barcoded hydrogel membrane is contacted with the sample, the spatial barcoded hydrogel membrane is soaked in a permeabilization buffer. Therefore, the spatial barcoded hydrogel membrane transports the permeabilization reagent to the sample surface in contact with the spatial barcoded hydrogel, enhancing analyte migration and capture. In some embodiments, the spatial barcoded hydrogel is applied to the sample and placed in a large amount of permeabilization solution. In some embodiments, the hydrogel membrane soaked in the permeabilization reagent is sandwiched between the sample and the spatial barcoded array. In some embodiments, the target analyte can diffuse through the hydrogel soaked with the permeabilization reagent and hybridize or bind to the capture probe on the other side of the hydrogel. In some embodiments, the thickness of the hydrogel is proportional to the resolution loss. In some embodiments, the hole (e.g., micropore, nanopore or picometer hole) may contain spatial barcoded capture probes and permeabilization reagent and / or buffer. In some embodiments, the spatial barcoded capture probe and permeabilization reagent are maintained between the spacer. In some embodiments, the sample is punched, cut or transferred to the hole, wherein the target analyte diffuses to the spatial barcoded capture probe by the permeabilization reagent / buffer. In some embodiments, the resolution loss may be proportional to the gap thickness (e.g., the amount of permeabilization buffer between the sample and the capture probe). In some embodiments, the anti-diffusion medium (e.g., hydrogel) has a thickness between about 50-500 microns, including 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 microns, or any thickness between 50 and 500 microns.

[0236] In some embodiments, the permeabilizing solution can be delivered to the sample through a porous membrane. In some embodiments, the porous membrane is used to limit the loss of the analyte by diffusion while allowing the permeabilizing agent to reach the sample. The membrane chemistry and pore size can be controlled to minimize the loss of the analyte. In some embodiments, the porous membrane can be made of glass, silicon, paper, hydrogel, polymer monolith or other materials. In some embodiments, the material can be naturally porous. In some embodiments, the material can have pores or holes etched into the solid material. In some embodiments, the permeabilizing agent flows through a microfluidic chamber or channel on the porous membrane. In some embodiments, the flow controls the accessibility of the sample to the permeabilizing agent. In some embodiments, the porous membrane is sandwiched between the spatial barcoding array and the sample, wherein the permeabilizing solution is applied to the porous membrane. The permeabilizing agent diffuses into the tissue through the membrane pores.

[0237] In some embodiments, the biological sample can be permeabilized by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive agents, such as lysing enzymes for cracking different cell types, such as gram-positive or gram-negative bacteria, plants, yeasts, mammals, such as lysozyme, colorless peptidase, lysostaphin, Labiase enzyme, kitalase, lytic enzyme, and a variety of other commercially available lysing enzymes.

[0238] Other lysing agents can be added to the biological sample in addition or alternatively to promote permeabilization. For example, a lysis solution based on a surfactant can be used for lysis sample cells. The lysis solution can include an ionic surfactant, such as sarkosyl and sodium lauryl sulfate (SDS). More generally, chemical lysing agents can include but are not limited to organic solvents, chelating agents, detergents, surfactants and chaotropic agents.

[0239] In some embodiments, the biological sample can be permeabilized by a non-chemical permeabilization method. Non-chemical permeabilization methods are known in the art. For example, usable non-chemical permeabilization methods include but are not limited to physical dissolution techniques, such as electroporation, mechanical permeabilization methods (e.g., bead milling using a homogenizer and grinding balls to mechanically disrupt the sample's tissue structure), acoustic permeabilization (e.g., ultrasound), and thermal decomposition techniques, such as heating to induce thermal permeabilization of the sample.

[0240] (13) Selective enrichment of RNA substances

[0241] In some embodiments in which RNA is an analyte, one or more RNA analytes of interest can be selectively enriched. For example, one or more RNAs of interest can be selected by adding one or more oligonucleotides to the sample. In some embodiments, other oligonucleotides are sequences for initiating reactions by polymerase. For example, one or more primer sequences with sequence complementarity to one or more RNAs of interest can be used to amplify one or more RNAs of interest, thereby selectively enriching these RNAs. In some embodiments, oligonucleotides with sequence complementarity to the complementary strand of the RNA (e.g., cDNA) captured can be bound to the cDNA. For example, biotinylated oligonucleotides with sequences complementary to one or more cDNAs of interest can be bound to the cDNA and can utilize biotinylation-streptavidin affinity, using any one of various methods known in the art (e.g., streptavidin beads) to select.

[0242] Alternatively, any one of a variety of methods can be used to downwardly select (e.g., remove) one or more RNAs. For example, a probe can be given to the sample that selectively hybridizes with ribosomal RNA (rRNA), thereby reducing the collection and concentration of rRNA in the sample. Subsequently, the capture probe is applied to the sample to improve the capture of other types of RNA resulting from the reduction of the non-specific RNA present in the sample. Alternatively, double-stranded specific nuclease (DSN) can be used to remove rRNA (see, e.g., Archer et al., Selective and flexible depletion of problematic sequencesw from RNA-seq libraries at the cDNA stage at the cDNA stage, BMC Genomics, 15401, (2014), the entire contents of which are incorporated herein by reference). Additionally, hydroxyapatite chromatography can remove abundant species (e.g., rRNA) (see, e.g., Vandernoot, VA, cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6)373-80, (2012), the entire contents of which are incorporated herein by reference).

[0243] (14) Other reagents

[0244] Prior to analyzing the sample, other reagents may be added to the biological sample to perform various functions. In some embodiments, DNase and RNase inactivators or inhibitors (e.g., proteinase K) and / or chelating agents (e.g., EDTA) may be added to the sample.

[0245] In some embodiments, the sample can be treated with one or more enzymes. For example, one or more endonucleases for fragmenting DNA, DNA polymerases, and dNTPs for amplifying nucleic acids can be added. Other enzymes that can also be added to the sample include, but are not limited to, polymerases, transposases, ligases, DNA enzymes, and RNA enzymes.

[0246] In some embodiments, a reverse transcriptase enzyme can be added to the sample, including an enzyme with terminal transferase activity, primers, and a conversion oligonucleotide. Template switching can be used to increase the length of a cDNA, for example, by appending a predefined nucleic acid sequence to the cDNA.

[0247] (15) Pretreatment for capture probe interaction

[0248] In some embodiments, the analyte in the biological sample can be pre-treated before interacting with the capture probe. For example, before interacting with the capture probe, a polymerization reaction catalyzed by a polymerase (such as DNA polymerase or reverse transcriptase) is carried out in the biological sample. In some embodiments, the primer for polymerization reaction includes a functional group that enhances hybridization with the capture probe. The capture probe can include an appropriate capture domain to capture the biological analyte of interest (for example, a poly (dT) sequence to capture poly (A) mRNA).

[0249] In some embodiments, the biological analyte is pre-processed to generate a library by next generation sequencing. For example, the analyte can be pre-processed by adding a modifier (e.g., attaching a sequence that interacts with the capture probe). In some embodiments, the analyte (e.g., DNA or RNA) is fragmented using a fragmentation technique (e.g., using a transposase and / or a fragmentation buffer).

[0250] After fragmentation, the analyte can be modified. For example, the modification can be added by connecting an adapter sequence that allows hybridization with the capture probe. In some embodiments, where the analyte of interest is RNA, poly (A) tailing is performed. Adding a poly (A) tail to an RNA that does not contain a poly (A) tail can promote hybridization with a capture probe that contains a capture domain with a functional amount of a poly (dT) sequence.

[0251] In some embodiments, before interacting with the capture probe, a ligation reaction catalyzed by a ligase is carried out in a biological sample. In some embodiments, connection can be carried out by chemical connection. In some embodiments, click chemistry can be used to connect, as described below. In some embodiments, the capture domain includes a DNA sequence with complementarity to an RNA molecule, wherein the RNA molecule has complementarity with the second DNA sequence, and wherein the RNA-DNA sequence complementarity is used to connect the second DNA sequence to the DNA sequence in the capture domain. In these embodiments, the RNA molecule can be directly detected.

[0252] In some embodiments, a target-specific reaction is performed in a biological sample prior to interacting with a capture probe. Examples of target-specific reactions include, but are not limited to, connection of target-specific adapters, probes, and / or other oligonucleotides, target-specific amplification using one or more analyte-specific primers, and target-specific detection using in situ hybridization, DNA microscopy, and / or antibody detection. In some embodiments, a capture probe includes a capture domain (e.g., amplification or connection) that is targeted to a target-specific product.

[0253] II. General spatial array-based analysis methods

[0254] This section of the invention describes methods, devices, systems, and compositions for spatial array-based analysis of biological samples.

[0255] (a) Spatial analysis methods

[0256] Array-based spatial analysis methods involve transferring one or more analytes from a biological sample to an array of features on a substrate, each associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes involves determining the identity of the analytes and the spatial location of each analyte in the sample. The spatial location of each analyte in the sample is determined based on the features bound by each analyte in the array and the relative spatial locations of the features in the array.

[0257] There are at least two general approaches to associating a spatial barcode with one or more adjacent cells such that the spatial barcode identifies the one or more cells and / or the contents of the one or more cells as being associated with a specific spatial location. One general approach is to drive the target analyte out of the cell and toward the spatially barcoded array. Figure 1 An exemplary embodiment of this general method is depicted. Figure 1In the present invention, a spatially barcoded array having a collection of capture probes (as further described herein) is contacted with a sample 101 and the sample is permeabilized to allow target analytes to migrate from the sample to the array. The target analytes interact with the capture probes on the spatially barcoded array 102. Once the target analytes hybridize / bind to the capture probes, the sample is optionally removed from the array and the capture probes are analyzed to obtain spatially resolved analyte information 103.

[0258] Another general approach is to cleave the spatially barcoded capture probes from the array and drive the spatially barcoded capture probes toward and / or into or onto the sample. Figure 2 An exemplary embodiment of the general method is depicted, whereby a spatially barcoded array aggregated with capture probes (as further described herein) can be contacted with a sample 201. The spatially barcoded capture probes are cleaved and then interact with cells within a provided sample 202. This interaction can be a covalent or non-covalent cell surface interaction. The interaction can be an intracellular interaction promoted by a delivery system or a cell penetrating peptide. Once the spatially barcoded capture probes are associated with specific cells, the sample can be optionally removed for analysis. The sample can be selectively separated before analysis. Once the labeled cells are associated with the spatially barcoded capture probes, the capture probes can be analyzed to obtain spatially resolved information about the labeled cells 203.

[0259] Figure 3 An exemplary workflow including preparing samples on a spatial barcoding array 301 is shown. Sample preparation can include placing the sample on a slide, fixing the sample, and / or staining the sample for imaging. The stained sample is then imaged on an array 302 using bright field (for imaging of sample hematoxylin and eosin staining) and fluorescence (for imaging of features) modes. In some embodiments, the target analyte is then released from the sample, and the capture probes forming the spatial barcoding array hybridize or bind to the released target analyte 303. The sample is then removed from the array 304, and the capture probes are cleaved from the array 305. The sample and array are then optionally imaged a second time in two modes 305B, while the analyte is reverse transcribed into cDNA, and an amplicon library 306 is prepared and sequenced 307. The two sets of images are then spatially overlapped to associate the sample information 308 identified in the space. When the sample and array do not have a second imaging 305B, a point coordinate file is provided by the manufacturer. The point coordinate file replaces the second imaging step 305B. Additionally, amplicon library preparation 306 can be performed using unique PCR adapters and sequencing 307 .

[0260] Figure 4Another exemplary workflow is shown, which utilizes a spatial marker array on a substrate, wherein capture probes labeled with spatial bar codes are gathered in areas called features. The spatially labeled capture probes can include a cleavage domain, one or more functional sequences, a spatial bar code, a unique molecular identifier, and a capture domain. The spatially labeled capture probes can also include a 5' end modification for reversibly attaching to the substrate. The spatial bar code array is contacted with the sample 401 and the sample is permeabilized by applying a permeabilization reagent 402. The permeabilization reagent can be given by placing the array / sample assembly in a large amount of solution. Alternatively, the permeabilization reagent can be given to the sample via an anti-diffusion medium and / or a physical barrier (e.g., a cover), wherein the sample is sandwiched between the anti-diffusion medium and / or barrier and the substrate comprising the array. Analytes are migrated to the spatial barcoded capture array using any number of techniques disclosed herein. For example, analyte migration can be performed using an anti-diffusion medium cover and passive migration. As another example, for example, using an electrophoretic transfer system, analyte migration can be active migration. Once the analyte approaches the spatial barcoded capture probe, the capture probe can hybridize or otherwise bind to the target analyte 403. The sample can optionally be removed from the array 404 .

[0261] The capture probes can be optionally cleaved from the array 405, and the captured analytes can be spatially labeled by performing a reverse transcriptase first-chain cDNA reaction. The first-chain cDNA reaction can optionally be performed using a template conversion oligonucleotide. For example, the template conversion oligonucleotide can hybridize with a poly (C) tail added to the 3' end of the cDNA by reverse transcriptase. The original mRNA template and template conversion oligonucleotide can be denatured from the cDNA, and then the barcoded capture probe can hybridize with the cDNA and can generate a complement of the cDNA. The first-chain cDNA can then be purified and collected for downstream amplification steps. The first-chain cDNA can be amplified using PCR 406, in which the forward and reverse primers flank the spatial barcode of interest and the target analyte region, generating a library associated with a specific spatial barcode. In some embodiments, the cDNA comprises a sequencing-by-synthesis (SBS) primer sequence. The library amplicons are sequenced and analyzed to decode spatial information 407.

[0262] Figure 5An exemplary workflow is described in which samples are removed from a spatially barcoded array and spatially barcoded capture probes are removed from the array for barcoded analyte amplification and library preparation. Another embodiment includes performing first-strand synthesis on a spatially barcoded array using template switching oligonucleotides without cutting the capture probes. In this embodiment, sample preparation 501 and permeabilization 502 are performed as described elsewhere herein. Once the capture probe captures the target analyte, the first-strand cDNA produced by template switching and reverse transcriptase 503 is subsequently denatured, and the second strand is subsequently extended 504. The second-strand cDNA is then denatured, neutralized, and transferred to a tube 505 from the first-strand cDNA. cDNA quantification and amplification can be performed using standard techniques discussed herein. The cDNA can then undergo library preparation 506 and indexing 507, including fragmentation, end repair, A-tailing, and indexing PCR steps.

[0263] In some non-limiting examples of the above workflow, the sample can be immersed in 100% frozen methanol and incubated at -20°C for 30 minutes. After 20 minutes, the sample can be removed and rinsed in ultrapure water. After rinsing the sample, prepare a fresh eosin solution and cover the sample with isopropyl alcohol. After the sample is incubated in isopropyl alcohol for 1 minute, the reagent can be removed by holding the slide at a certain angle (the bottom edge of the slide can be in contact with a laboratory wipe and air-dried). The sample can be evenly covered in hematoxylin solution and incubated at room temperature for 7 minutes. After the sample is incubated in hematoxylin for 7 minutes, the reagent can be removed by holding the slide at a certain angle (the bottom edge of the slide can be in contact with a laboratory wipe). The slide containing the sample can be immersed in water and the excess liquid can be removed. The sample is then covered with bluing buffer and incubated at room temperature for 2 minutes. The slide containing the sample is immersed in water again, evenly covered with eosin solution, and incubated at room temperature for 1 minute. The slide can be air-dried and incubated at 37°C for 5 minutes. Samples can be imaged using the methods disclosed herein.

[0264] The following are non-limiting exemplary steps for sample permeabilization and cDNA generation. The sample can be exposed to a permeabilizing enzyme and incubated at 37°C for 6 minutes. Other permeabilization methods are described herein. The permeabilizing enzyme can be removed by adding SSC buffer, and the sample can be prepared for analyte capture. The sample is then subjected to a pre-equilibrated thermal cycling protocol, and the SSC buffer is removed. A master mix containing nuclease-free water, reverse transcriptase reagent, template-switching oligonucleotide, reducing agent, and reverse transcriptase can be added, and the sample with the master mix can be subjected to a thermal cycling protocol. The reagents can be removed from the sample, and NaOH can be applied and incubated at room temperature for 5 minutes. The sodium hydroxide can be removed, and an elution buffer can be added and removed from the sample. A second-strand mix, including second-strand reagent, second-strand primer, and second-strand enzyme, can be added to the sample, and the sample can be sealed and incubated. After the incubation is complete, the reagents are removed, and an elution buffer is added and removed from the sample. NaOH is then added to the sample, and the sample is incubated at room temperature for 10 minutes. Tris-HCl can be added and the reagents mixed.

[0265] The following steps are non-limiting exemplary steps for cDNA amplification and quality control. A qPCR mixture can be prepared, including nuclease-free water, a qPCR master mix, and cDNA primers, and a NaOH / Tris-HCl mixture can be mixed with the qPCR mixture and sample and thermally cycled according to a predetermined thermal cycling protocol. After completing the thermal cycle, a cDNA amplification mixture can be prepared and merged and mixed with the sample. The sample is then incubated and thermally cycled. The sample can then be resuspended in SPRIselect reagent and pipetted to ensure proper mixing. The sample can then be incubated for 5 minutes at room temperature and placed on a magnet (e.g., with the magnet in the high position) for cleaning. The supernatant is removed, and 80% ethanol is added to the precipitate and incubated for 30 seconds. The ethanol can be removed and the precipitate can be washed again. The sample is then centrifuged and placed on a magnet (e.g., with the magnet in the low position). Any residual ethanol can be removed and the sample can be air-dried. The magnet can be removed, elution buffer added to the sample, mixed, and incubated at room temperature for 2 minutes. The sample is then placed on a magnet (e.g., on high) until the solution is clear. A portion of the sample can be run on an Agilent Bioanalyzer High Sensitivity Chip, where a region can be selected and the cDNA concentration measured to calculate the total cDNA yield. Alternatively, quantification can be performed on an Agilent Bioanalyzer or Agilent TapeStation.

[0266] The following steps are non-limiting exemplary steps for constructing a spatial gene expression library. A fragmentation mixture can be prepared on ice, including a fragmentation buffer and a fragmentation enzyme. Elution buffer and a fragmentation mixture can be added to each sample, mixed and centrifuged. The sample mixture can then be placed in a thermal cycler and cycled according to a predetermined scheme. SPRIselect reagent can be added to the sample and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (e.g., at a high position) until the solution is clear and the supernatant can be transferred to a new tube strip. SPRIselect reagent can be added to the sample, mixed and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (e.g., at a high position) until the solution is clear. The supernatant is removed, 80% ethanol is added to the precipitate, the precipitate is incubated for 30 seconds, and the ethanol can be removed. The ethanol wash can be repeated, and the sample can be placed on a magnet (e.g., at a low position) until the solution is clear. Residual ethanol can be removed, elution buffer is added to the sample, mixed, and incubated at room temperature for 2 minutes. The sample can be placed on a magnet (e.g., in the high position) until the solution is clear and a portion of the sample can be transferred to a new tube strip. An adapter ligation mix comprising ligation buffer, DNA ligase, and adapter oligonucleotides can be prepared and centrifuged. The adapter ligation mix can be added to the sample, pipetted to mix, and briefly centrifuged. The sample can then be thermally cycled according to a predetermined protocol. SPRIsleect reagent can be added to the sample, incubated at room temperature for 5 minutes, and placed on a magnet (e.g., in the high position) until the solution is clear. The supernatant can be removed, the precipitate washed with 80% ethanol, incubated for 30 seconds, and the ethanol can be removed. The ethanol wash can be repeated, and the sample can be briefly centrifuged before placing the sample on the magnet (e.g., in the low position). Any residual ethanol can be removed and the sample can be air-dried. Elution buffer can be added to the sample, the sample removed from the magnet, and then the sample can be mixed with a pipette, incubated at room temperature for 2 minutes, and placed on a magnet (e.g., in the low position) until the solution is clear. A portion of the sample can be transferred to a new tube strip. A sample index PCR mixture, including the amplification mixture and SI primers, can be prepared and combined with the sample. The sample / sample index PCR mixture can be loaded into individual Chrome i7 sample index wells and a thermal cycling protocol can be used. SPRIselect reagent can be added to each sample, mixed and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (e.g., in the high position) until the solution is clear and the supernatant can be transferred to a new tube strip. SPRIselect reagent can be added to each sample, pipetted to mix and incubated at room temperature for 5 minutes. The sample can then be placed on a magnet (e.g., in the high position) until the solution is clear. The supernatant is removed and the precipitate is washed with 80% ethanol, incubated for 30 seconds, and then the ethanol can be removed. The ethanol wash can be repeated, the sample centrifuged and placed on a magnet (e.g., in the low position) to remove any remaining ethanol.The sample can be removed from the magnet, elution buffer can be added to the sample, pipetted to mix, and incubated at room temperature for 2 minutes. The sample can be placed on the magnet (e.g., on high) until the solution clears, and a portion of the sample can be transferred to a new tube strip. Average fragment size can be determined using a bioanalyzer tracer or Agilent TapeStation.

[0267] In some embodiments, correlation analysis of data generated by this workflow and other workflows described herein can produce a 95% or greater correlation (e.g., 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater) of genes expressed across two capture regions. When the workflow is performed using single-cell RNA sequencing of nuclei, in some embodiments, correlation analysis of the data can produce a correlation of greater than 90% (e.g., greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of genes expressed across two capture regions.

[0268] (b) Capture probe

[0269] "Capture probe" refers to any molecule capable of capturing (directly or indirectly) and / or labeling an analyte of interest in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe is a conjugate (e.g., an oligonucleotide-antibody conjugate). In some embodiments, the capture probe comprises a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI)) and a capture domain.

[0270] Figure 6It is a schematic diagram illustrating an example of a capture probe as described herein. As shown in the figure, capture probe 602 is optionally coupled to feature 601 by a cleavage domain 603 such as a disulfide bond joint. Capture probe may include a functional sequence useful to subsequent processing, such as a functional sequence 604, which may include a sequencer-specific flow cell attachment sequence, such as a P5 sequence, and a functional sequence 606, which may include a sequencing primer sequence, such as an R1 primer binding site. In some embodiments, sequence 604 is a P7 sequence, and sequence 606 is an R2 primer binding site. Spatial barcode 605 may be included in the capture probe for barcoding target analytes. Typically, functional sequences may be selected to be compatible with any of a variety of sequencing systems, such as 454 sequencing, ion torrent proton (Ion Torrent Proton) or PGM, Illumina X10, PacBio, nanopore (Nanopore) etc. and requirements thereof. In some embodiments, functional sequences may be selected to be compatible with non-commercial sequencing systems. Examples of such sequencing systems and techniques for which suitable functional sequences can be used include, but are not limited to, Roche 454 sequencing, Ion Torrent proton or PGM sequencing, Illumina X10 sequencing, PacBio SMRT sequencing, and Oxford nanopore sequencing. In addition, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercial sequencing systems.

[0271] In some embodiments, the spatial barcode 605, functional sequence 604 (e.g., flow cell attachment sequence) and 606 (e.g., sequencing primer sequence) can be common to all probes attached to a given feature. The spatial barcode can also include a capture domain 607 to facilitate capture of target analytes.

[0272] Capture Domain

[0273] As described above, each capture probe comprises at least one capture domain. A "capture domain" is an oligonucleotide, polypeptide, small molecule, or any combination thereof, that specifically binds to a desired analyte. In some embodiments, a capture domain can be used to capture or detect a desired analyte.

[0274] In some embodiments, the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes, such as one or more different types of nucleic acids (e.g., RNA molecules and DNA molecules). In some embodiments, the functional nucleic acid sequence may include an N-polymer sequence (e.g., a random N-polymer sequence), wherein the N-polymer sequence is configured to interact with multiple DNA molecules. In some embodiments, the functional sequence may include a poly (T) sequence, which is configured to interact with a messenger RNA (mRNA) molecule via a poly (A) tail of an mRNA transcript. In some embodiments, the functional nucleic acid sequence is a binding target of a protein (e.g., a transcription factor, a DNA binding protein, or an RNA binding protein), wherein the analyte of interest is a protein.

[0275] The capture probe can include ribonucleotides and / or deoxyribonucleotides and synthetic nucleotide residues that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the capture domain can trigger a reverse transcription reaction to produce cDNA complementary to the captured RNA molecule. In some embodiments, the capture domain of the capture probe can trigger a DNA extension (polymerase) reaction to produce DNA complementary to the captured DNA molecule. In some embodiments, the capture domain can serve as a template for the ligation reaction between the captured DNA molecule and the surface probe directly or indirectly fixed on the substrate. In some embodiments, the capture domain can be connected to one chain of the captured DNA molecule. For example, SplintR ligase together with RNA or DNA sequence (e.g., degenerate RNA) can be used to connect single-stranded DNA or RNA to the capture domain. In some embodiments, a ligase with RNA templated ligase activity (e.g., SplintR ligase, T4 RNA ligase 2 or KOD ligase) can be used to connect single-stranded DNA or RNA to the capture domain. In some embodiments, the capture domain includes a splint oligonucleotide. In some embodiments, the capture domain captures the splint oligonucleotide.

[0276] In some embodiments, the capture domain is located at the 3' end of the capture probe and includes a free 3' end, which can be extended, for example, by template-dependent polymerization to form an extended capture probe as described herein. In some embodiments, the capture domain includes a nucleotide sequence capable of hybridizing with nucleic acids (e.g., RNA or other analytes) present in cells of a tissue sample in contact with the array. In some embodiments, the capture domain can be selected or designed to selectively or specifically bind to the target nucleic acid. For example, the capture domain can be selected or designed to capture mRNA by hybridizing with the mRNA poly (A) tail. Therefore, in some embodiments, the capture domain includes a poly (T) DNA oligonucleotide, i.e., a series of consecutive deoxythymidine residues connected by a phosphodiester bond, which can hybridize with the poly (a) tail of the mRNA. In some embodiments, the capture domain may include nucleotides that are functionally or structurally similar to the poly (T) tail. For example, a poly (U) oligonucleotide or an oligonucleotide comprising a deoxythymidine analog. In some embodiments, the capture domain comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capture domain comprises at least 25, 30, or 35 nucleotides.

[0277] In some embodiments, a random sequence (e.g., a random hexamer or similar sequence) can be used to form all or part of a capture domain. For example, a random sequence can be used together with a poly(T) (or poly(T) analog) sequence. Thus, where the capture domain includes a poly(T) (or "poly(T)-like") oligonucleotide, it can also include a random oligonucleotide sequence (e.g., a "poly(T)-random sequence" probe). For example, this can be located 5' or 3' of the poly(T) sequence, such as at the 3' end of the capture domain. The poly(T)-random sequence probe can facilitate the capture of the mRNA poly(A) tail. In some embodiments, the capture domain can be a completely random sequence. In some embodiments, a degenerate capture domain can be used.

[0278] In some embodiments, the collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly (T) sequence, and the capture domain of one or more capture probes includes a random sequence. In some embodiments, the collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly (T) sample sequence, and the capture domain of one or more capture probes includes a random sequence. In some embodiments, the collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly (T) random sequence, and the capture domain of one or more capture probes includes a random sequence. In some embodiments, probes with degenerate capture domains can be added to any of the aforementioned combinations listed herein. In some embodiments, probes with degenerate capture domains can replace one of the probes in each pair described herein.

[0279] The capture domain can be based on the specific gene sequence or specific motif sequence or common / conserved sequence (i.e., sequence-specific capture domain) that it is designed to capture. Thus, in some embodiments, the capture domain can selectively bind to a desired nucleic acid subtype or subset, such as a specific type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tRNA, snRNA, snRNA, SmY RNA, sARNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of RNA. In a non-limiting example, the capture domain can selectively bind to a desired subset of ribonucleic acids, such as microbiome RNA, such as 16SrRNA.

[0280] In some embodiments, the capture domain includes an "anchor" or "anchor sequence", which is a nucleotide sequence designed to ensure that the capture domain hybridizes with the expected biological analyte. In some embodiments, the anchor sequence includes a nucleotide sequence, including a 1-mer, a 2-mer, a 3-mer or a longer sequence. In some embodiments, the short sequence is random. For example, a capture domain including a poly (T) sequence can be designed to capture mRNA. In such embodiments, the anchor sequence can include a random 3-mer (e.g., GGG) that helps to ensure that the poly (T) capture domain hybridizes with the mRNA. In some embodiments, the anchor sequence can be VN, N or NN. Alternatively, a specific nucleotide sequence can be used to design the sequence. In some embodiments, the anchor sequence is located at the 3' end of the capture domain. In some embodiments, the anchor sequence is located at the 5' end of the capture domain.

[0281] In some embodiments, the capture domain of the capture probe is blocked before the biological sample is contacted with the array, and a blocking probe is used when the nucleic acid in the biological sample is modified before it is captured on the array. In some embodiments, the blocking probe is used to block or modify the free 3' end of the capture domain. In some embodiments, the blocking probe can hybridize with the capture probe to shield the free 3' end of the capture domain, such as a hairpin probe or a partially double-stranded probe. In some embodiments, the free 3' end of the capture domain can be blocked by chemical modification, for example, adding an azidomethyl group as a chemically reversible capping portion so that the capture probe does not include a free 3' end. Before the biological sample is contacted with the array, the capture probe is blocked or modified, particularly at the free 3' end of the capture domain, to prevent modification of the capture probe, for example, to prevent the addition of a poly (A) tail to the free 3' end of the capture probe.

[0282] Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC), 3′ inverted dT, 3′C3 spacer, 3′ amino group, and 3′ phosphorylation. In some embodiments, nucleic acids in biological samples can be modified so that they can be captured by the capture domain. For example, an adapter sequence (including a binding domain capable of binding to the capture domain of a capture probe) can be added to the end of a nucleic acid (e.g., fragmented genomic DNA). In some embodiments, this is achieved by connecting adapter sequences or extending nucleic acids. In some embodiments, enzymes are used to incorporate additional nucleotides, such as poly (A) tails, at the ends of nucleic acid sequences. In some embodiments, the capture probe can be reversibly shielded or modified so that the capture domain of the capture probe does not include a free 3′ end. In some embodiments, the 3′ end is removed, modified, or made inaccessible so that the capture domain is not susceptible to the process (e.g., connection or extension) used to modify the nucleic acid of the biological sample.

[0283] In some embodiments, the capture domain of the capture probe is modified to remove any modifications of the capture probe that occurred during modification of the nucleic acid molecules of the biological sample.In some embodiments, the capture probe can include additional sequences downstream of the capture domain, i.e., to 3' of the capture domain, i.e., a blocking domain.

[0284] In some embodiments, the capture domain of the capture probe can be a non-nucleic acid domain. Examples of suitable capture domains that are not entirely nucleic acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any capture domain described herein.

[0285] cleavage domain

[0286] Each capture probe may optionally include at least one cleavage domain. A cleavage domain represents the portion of the probe that is used to reversibly attach the probe to an array feature, as described below. In addition, one or more segments or regions of the capture probe may optionally be released from the array feature by cleaving the cleavage domain. For example, a spatial barcode and / or a universal molecular identifier (UMI) may be released by cleaving the cleavage domain.

[0287] Figure 7 Figure 7 is a schematic diagram illustrating a cleavable capture probe that can enter non-permeabilized cells and bind to target analytes within a sample. Capture probe 701 comprises a cleavage domain 702, a cell-penetrating peptide 703, a reporter molecule 704, and a disulfide bond (-SS-). 705 represents all other parts of the capture probe, such as the spatial barcode and capture domain.

[0288] In some embodiments, the cleavage domain that connects the capture probe to the feature is a disulfide bond. A reducing agent can be added to break the disulfide bond, thereby releasing the capture probe from the feature. As another example, heating can also cause degradation of the cleavage domain and release the attached capture probe from the array feature. In some embodiments, laser radiation is used to heat and degrade the cleavage domain of the capture probe at a specific location. In some embodiments, the cleavage domain is a photosensitive chemical bond (i.e., a chemical bond that dissociates when exposed to light such as ultraviolet light).

[0289] Other examples of cleavage domains include labile chemical bonds, such as, but not limited to, an ester bond (e.g., cleavable with acid, base, or hydroxylamine), a vicinal diol bond (e.g., cleavable by sodium periodate), a Diels-Alder bond (e.g., cleavable by heat), a sulfone bond (e.g., cleavable by base), a silyl ether bond (e.g., cleavable by acid), a glycosidic bond (e.g., cleavable by amylase), a peptide bond (e.g., cleavable by a protease), or a phosphodiester bond (e.g., cleavable by a nuclease (e.g., DNase)).

[0290] In some embodiments, the cleavage domain includes a sequence recognized by one or more enzymes capable of cutting nucleic acid molecules, for example, capable of destroying the phosphodiester bond between two or more nucleotides. The bond can be cut by other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases). For example, the cleavage domain can include a restriction endonuclease (restriction enzyme) recognition sequence. Restriction endonucleases cut double-stranded or single-stranded DNA at specific recognition nucleotide sequences known as restriction sites. In some embodiments, rare-cutting restriction enzymes are used, i.e., enzymes with long recognition sites (at least 8 base pairs in length) to reduce the possibility of cutting at other positions in the capture probe.

[0291] In some embodiments, the cleavage domain includes a poly (U) sequence that can be cleaved by uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER TM Once released, the releasable capture probe can be used for reaction. Thus, for example, the activatable capture probe can be activated by releasing the capture probe from the feature.

[0292] In some embodiments, when the capture probe is indirectly attached to the substrate, for example, via a surface probe, the cleavage domain includes one or more mismatched nucleotides such that the complementary portions of the surface probe and the capture probe are not 100% complementary (e.g., the number of mismatched base pairs can be one, two, or three base pairs). For example, such mismatches are recognized by MutY and T7 endonuclease I enzymes, which result in cleavage of the nucleic acid molecule at the position of the mismatch.

[0293] In some embodiments, when the capture probe is indirectly attached to the feature, for example, via a surface probe, the cleavage domain includes a nickase recognition site or sequence. Nickase is a single-stranded endonuclease that can only cut a DNA duplex. Therefore, the cleavage domain can include a nickase recognition site near the 5' end of the surface probe (and / or the 5' end of the capture probe) so that the cracking of the surface probe or the capture probe destabilizes the duplex between the surface probe and the capture probe, thereby releasing the capture probe from the feature.

[0294] Nickase can also be used for capturing probe and is directly attached to some embodiments of feature.For example, substrate can be contacted with the nucleic acid molecule that hybridizes to the cleavage domain of capture probe to provide or reorganize nickase recognition site, such as cracking auxiliary probe.Therefore, contacting with nickase will cause the cracking of cleavage domain, thereby discharges capture probe from feature.This cracking auxiliary probe can also be used for providing or reorganizing cleavage recognition site for other cleavage enzymes (such as restriction endonucleases).

[0295] Some nickases, by binding and recognizing specific nucleotide recognition sequences, only introduce single-stranded nicks at specific sites of the DNA molecule. Many natural nickases have been found, and the sequence recognition characteristics of at least four nickases have been determined. Nickases are described in U.S. Patent No. 6,867,028, which is incorporated herein by reference in its entirety. In general, any suitable nickase can be used to bind to the complementary nickase recognition site of the cleavage domain. After use, the nickase can be removed from the analysis, or inactivated after releasing the capture probe to prevent unnecessary cutting of the capture probe.

[0296] Examples of suitable capture domains that are not solely nucleic acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any of the capture domains described herein.

[0297] In some embodiments, the cleavage domain is absent from the capture probe. Examples of substrates with attached capture probes lacking a cleavage domain are described, for example, in Macosko et al., (2015) Cell 161, 1202-1214, the entire contents of which are incorporated herein by reference.

[0298] In some embodiments, the capture probe region corresponding to the cleavage domain can be used for certain other functions. For example, other regions for nucleic acid extension or amplification can be included in the position where the cleavage domain would normally be located. In such embodiments, this region can supplement the functional domain or even exist as another functional domain. In some embodiments, there is a cleavage domain, but its use is optional.

[0299] Functional domain

[0300] Each capture probe can optionally include at least one functional domain. Each functional domain typically includes a functional nucleotide sequence for use in a downstream analysis step in the overall analysis procedure.

[0301] In some embodiments, the capture probe may include a functional domain for attachment to a sequencing flow cell, e.g., In some embodiments, the capture probe or its derivative may include another functional domain, such as a domain for attachment to a target region. The functional domains can be selected to be compatible with any of a variety of sequencing systems, such as 454 sequencing, Ion Torrent Proton or PGM, Illumina X10, etc. and their requirements.

[0302] In some embodiments, the functional domain comprises a primer. The primer may comprise a primer for The deleted R1 primer sequence, and in some embodiments, includes Examples of such capture probes and their uses are described in U.S. Patent Publication Nos. 2014 / 0378345 and 2015 / 0376609, the entire contents of which are incorporated herein by reference.

[0303] Spatial Barcode

[0304] As described above, the capture probe may include one or more spatial barcodes (e.g., two or more, three or more, four or more, five or more) spatial barcodes. A "spatial barcode" is a continuous nucleic acid segment or two or more non-continuous nucleic acid segments that serve as a label or identifier that transmits or is capable of transmitting spatial information. In some embodiments, the capture probe includes a spatial barcode with a spatial aspect, wherein the barcode is associated with a specific position within the array or a specific position on the substrate.

[0305] The spatial barcode can be part of the analyte or independent of the analyte (i.e., part of the capture probe). The spatial barcode can be a label attached to the analyte (e.g., a nucleic acid molecule), or a combination of labels other than the endogenous characteristics of the analyte (e.g., the size or terminal sequence of the analyte). The spatial barcode can be unique. In some embodiments where the spatial barcode is unique, the spatial barcode acts both as a spatial barcode and as a unique molecular identifier (UMI) associated with a specific capture probe.

[0306] Spatial barcodes can have a variety of different formats. For example, spatial barcodes can include polynucleotide spatial barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. In some embodiments, the spatial barcode is attached to the analyte in a reversible or irreversible manner. In some embodiments, the spatial barcode is added to a fragment of, for example, a DNA or RNA sample before, during, and / or after sample sequencing. In some embodiments, the spatial barcode enables identification and / or quantification of individual sequence readings. In some embodiments, the spatial barcode is used as a fluorescent barcode, wherein a fluorescently labeled oligonucleotide probe hybridizes to the spatial barcode.

[0307] In some embodiments, the spatial barcode is a nucleic acid sequence that does not substantially hybridize to an analyte nucleic acid molecule in a biological sample. In some embodiments, the spatial barcode has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequence over a substantial portion (e.g., 80% or more) of the nucleic acid molecules in the biological sample.

[0308] The spatial barcode sequence may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the spatial barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0309] In some embodiments, the spatial barcode subsequences can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0310] For multiple capture probes attached to a common array feature, the one or more spatial barcode sequences of the multiple capture probes can include a sequence that is the same for all capture probes coupled to the feature and / or a sequence that differs between all capture probes coupled to the feature.

[0311] Figure 8 is a schematic diagram of an exemplary multi-space marking feature. Figure 8 , feature 801 can be coupled to spatially barcoded capture probes, wherein the spatial barcoded probes of a particular feature can have the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with multiple target analytes. For example, a feature can be coupled to four different types of spatially barcoded capture probes, each type of spatially barcoded capture probe having a spatial barcode 802. One type of capture probe associated with the feature comprises a combination of a spatial barcode 802 and a poly (T) capture domain 803, which is designed to capture mRNA target analytes. A second type of capture probe associated with the feature comprises a combination of a spatial barcode 802 and a random N-mer capture domain 804 for gDNA analysis. A third type of capture probe associated with the feature comprises a spatial barcode 802 and a capture domain combination that is complementary to a capture domain on an analyte capture agent capture agent barcode domain 805. The fourth type of capture probe associated with this feature includes a spatial barcode 802 in combination with a capture probe that can specifically bind to a nucleic acid molecule 806 that can function in a CRISPR assay (e.g., CRISPR / Cas9). Figure 8 Only four different capture probe barcoding constructs are shown, but capture probe barcoding constructs can be customized for analysis of any given analyte associated with nucleic acids and can be combined with such constructs. For example, Figure 8The scheme shown in can also be used for the simultaneous analysis of other analytes disclosed herein, including but not limited to: (a) mRNA, lineage tracing constructs, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or MNAzyme-seq) cell surface or intracellular proteins and metabolites, and perturbators (e.g., CRISPR-crRNA / sgRNA, TALEN, zinc finger nucleases and / or antisense oligonucleotides as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, barcoding markers (e.g., MHC multimers described herein) and V(D)J sequences of immune cell receptors (e.g., T cell receptors).

[0312] The capture probes attached to a single array feature can include the same (or common) spatial barcode sequence, different spatial barcode sequences, or a combination of the two. The capture probes attached to a feature can include multiple groups of capture probes. The capture probes of a given group can include the same spatial barcode sequence. The same spatial barcode sequence can be different from the spatial barcode sequence of another group of capture probes.

[0313] A plurality of capture probes may include a spatial barcode sequence (e.g., a nucleic acid barcode sequence) associated with a specific position on the spatial array. For example, a first plurality of capture probes may be associated with a first region based on a spatial barcode sequence common to the capture probes within the first region, and a second plurality of capture probes may be associated with a second region based on a spatial barcode sequence common to the capture probes within the second region. The second region may or may not be associated with the first region. Other multiple capture probes may be associated with a spatial barcode sequence shared by capture probes within other regions. In some embodiments, the spatial barcode sequence may be the same between multiple capture probe molecules.

[0314] In some embodiments, multiple different spatial barcodes are incorporated into a single array capture probe. For example, a mixed but known set of spatial barcode sequences can provide stronger addressing or attributes of the spatial barcode to a given point or position by providing repeated or independent confirmation of positional characteristics. In some embodiments, multiple spatial barcodes represent increased specificity of the position of a particular array point.

[0315] Unique molecular identifier

[0316] The capture probe can include one or more (e.g., two or more, three or more, four or more, five or more) unique molecular identifiers (UMIs). A unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that serves as a tag or identifier for a specific analyte or a capture probe that binds a specific analyte (e.g., via a capture domain).

[0317] A UMI can be unique. A UMI can include one or more specific polynucleotide sequences, one or more random nucleic acid and / or amino acid sequences, and / or one or more synthetic nucleic acid and / or amino acid sequences.

[0318] In some embodiments, a UMI is a nucleic acid sequence that does not substantially hybridize to an analyte nucleic acid molecule in a biological sample. In some embodiments, a UMI has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to a nucleic acid sequence over a substantial portion (e.g., 80% or more) of the nucleic acid molecules in a biological sample.

[0319] A UMI can include about 6 to about 20 or more nucleotides within a capture probe sequence. In some embodiments, a UMI can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or longer. In some embodiments, a UMI can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or longer. In some embodiments, a UMI can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or shorter.

[0320] These nucleotides can be completely contiguous, i.e., in a single sequence stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences separated by one or more nucleotides. The length of the isolated UMI subsequences is about 4 to 16 nucleotides. In some embodiments, the UMI subsequences can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequences can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0321] In some embodiments, a UMI is attached to an analyte in a reversible or irreversible manner. In some embodiments, a UMI is added to fragments of, for example, a DNA or RNA sample before, during, and / or after sequencing of the analyte. In some embodiments, a UMI enables identification and / or quantification of individual sequencing reads. In some embodiments, a UMI is used as a fluorescent barcode, where a fluorescently labeled oligonucleotide probe hybridizes to the UMI.

[0322] Other aspects of capture probes

[0323] For the capture probes attached to array features, individual array features can include one or more capture probes. In some embodiments, individual array features include hundreds or thousands of capture probes. In some embodiments, the capture probes are associated with specific individual features, wherein the individual features include capture probes that include a spatial barcode unique to a region or position defined on the array.

[0324] In some embodiments, a specific feature may include capture probes that include more than one spatial barcode (e.g., one capture probe at a specific feature may include a spatial barcode that is different from the spatial barcode included in another capture probe at the same specific feature, while both capture probes include a second common spatial barcode), wherein each spatial barcode corresponds to a specific defined area or position on the array. For example, multiple spatial barcode sequences associated with a specific feature on the array can provide a stronger address or attribute to a given position by providing repetition or independent confirmation of the position. In some embodiments, multiple spatial barcodes represent increased specificity of the position of a specific array point. In a non-limiting example, a specific array point can be encoded using two different spatial barcodes, wherein each spatial barcode identifies a specific defined area within the array, and an array point having two spatial barcodes identifies a sub-area where the two defined areas overlap, for example, such as the overlapping portion of a Venn diagram.

[0325] In another non-limiting example, a particular array spot can be encoded with three different spatial barcodes, where a first spatial barcode identifies a first region within the array, a second spatial barcode identifies a second region, where the second region is a subregion completely within the first region, and a third spatial barcode identifies a third region, where the third region is a subregion completely within the first and second subregions.

[0326] In some embodiments, the capture probe attached to the array features is released from the array features to perform sequencing. Or, in some embodiments, the capture probe remains attached to the array features, and the probe is sequenced while remaining attached to the array features (e.g., by in situ sequencing). Other aspects of the sequencing of the capture probe are described in subsequent parts of the present invention.

[0327] In some embodiments, array features can include different types of capture probes attached to features. For example, an array feature can include a first class capture probe and a second class capture probe, wherein the first class capture probe has a capture domain designed to be bound to a class of analytes, and the second class capture probe has a capture domain designed to be bound to a second class of analytes. Typically, an array feature can include one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more) different types of capture probes attached to a single array feature.

[0328] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is attached to the array feature via its 5′ end. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a barcode (e.g., spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs) and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI and a capture domain. In some embodiments, the capture probe does not include a spatial barcode. In some embodiments, the capture probe does not include a UMI. In some embodiments, the capture probe includes a sequence for initiating a sequencing reaction.

[0329] In some embodiments, the capture probe is fixed to the feature via its 3' end. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: a barcode (e.g., spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.

[0330] In some embodiments, capture probe comprises the oligonucleotide synthesized in situ.In some embodiments, the oligonucleotide synthesized in situ comprises one or more constant sequences, wherein one or more are used as initiator sequences (for example, primers for amplifying target nucleic acid).In some embodiments, constant sequence is a cleavable sequence.In some embodiments, the oligonucleotide synthesized in situ comprises a barcode sequence, for example, a variable barcode sequence.In some embodiments, the oligonucleotide synthesized in situ is attached to the feature of the array.

[0331] In some embodiments, the capture probe is the product of two or more oligonucleotide sequences, eg, two or more oligonucleotide sequences ligated together. In some embodiments, one of the oligonucleotide sequences is an oligonucleotide synthesized in situ.

[0332] In some embodiments, the capture probe comprises a splint oligonucleotide.Two or more oligonucleotides can be ligated together using a splint oligonucleotide and any type of ligase known in the art or described herein (eg, a splint ligase).

[0333] In some embodiments, one of the oligonucleotides comprises: a constant sequence (e.g., a sequence complementary to a portion of the splint oligonucleotide), a degenerate sequence, and a capture domain (e.g., as described herein). In some embodiments, the capture probe is generated by enzymatically adding a polynucleotide to the end of the oligonucleotide sequence. The capture probe can comprise a degenerate sequence that can serve as a unique molecular identifier.

[0334] Capture probes can include degenerate sequences, which are sequences that contain many possible bases at certain positions in a nucleotide sequence. A degenerate sequence can be a degenerate nucleotide sequence comprising about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides. In some embodiments, a nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 15, 20, 25, or more degenerate positions within a nucleotide sequence. In some embodiments, a degenerate sequence is used as a UMI.

[0335] In some embodiments, the capture probe includes a restriction endonuclease recognition sequence or a nucleotide sequence that can be cut by a specific enzymatic activity. For example, a uracil sequence can be cut by a specific enzymatic activity. As another example, other modified bases (for example, modified by methylation) can be recognized and cut by a specific endonuclease. The capture probe can be subjected to enzyme digestion, which removes the blocking domain and any other nucleotides added to the capture probe 3 ' end during the modification process. Removing the blocking domain will display and / or restore the free 3 ' end of the capture domain of the capture probe. In some embodiments, other nucleotides can be removed to display and / or restore the 3 ' end of the capture domain of the capture probe.

[0336] In some embodiments, the blocking domain can be incorporated into the capture probe during or after its synthesis. The terminal nucleotide of the capture domain is a reversible terminator nucleotide (e.g., a 3′-O-blocked reversible terminator and a 3′-unblocked reversible terminator) and can be included in the capture probe during or after probe synthesis.

[0337] Extended capture probe

[0338] An "extended capture probe" is a capture probe having an expanded nucleic acid sequence. For example, where the capture probe comprises a nucleic acid, an "extended 3' end" means that additional nucleotides are added to the very terminal 3' nucleotide of the capture probe to extend the length of the capture probe, e.g., by extending the nucleic acid molecule via a standard polymerization reaction, including template polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase).

[0339] In some embodiments, extending the capture probe includes generating cDNA from the captured (hybridized) RNA. This process involves synthesizing a complementary strand of a hybrid nucleic acid, for example, generating cDNA based on a captured RNA template (RNA hybridized to the capture domain of the capture probe). Thus, in the initial step of extending the capture probe (e.g., cDNA generation), the captured (hybridized) nucleic acid (e.g., RNA) serves as a template for extension (e.g., a reverse transcription step).

[0340] In some embodiments, the capture probe is extended using reverse transcription. For example, reverse transcription includes using reverse transcriptase to synthesize cDNA (complementary or copy DNA) from RNA (such as messenger RNA). In some embodiments, reverse transcription is carried out when the tissue is still in situ, to produce an analyte library, wherein the analyte library includes a spatial barcode from a neighboring capture probe. In some embodiments, the capture probe is extended using one or more DNA polymerases.

[0341] In some embodiments, the capture domain of the capture probe includes a primer for generating a complementary strand of nucleic acid that hybridizes to the capture probe, for example, a primer for DNA polymerase and / or reverse transcription. The nucleic acid (e.g., DNA and / or cDNA) molecule produced by the extension reaction comprises the sequence of the capture probe. The extension of the capture probe, such as DNA polymerase and / or reverse transcription reaction, can be performed using various suitable enzymes and protocols.

[0342] In some embodiments, full-length DNA (e.g., cDNA) molecules are produced. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if the nucleic acid (e.g., RNA) is partially degraded in the tissue sample, the captured nucleic acid molecule will be different from the initial RNA length in the tissue sample. In some embodiments, the 3' end of the extended probe (e.g., the first-strand cDNA molecule) is modified. For example, a linker or adapter can be connected to the 3' end of the extended probe. This can be accomplished by using a single-stranded ligase such as T4 RNA ligase or CircleGase TM (available from Epicentre Biotechnologies, Madison, Wisconsin). In some embodiments, a template switching oligonucleotide is used to extend the cDNA to produce full-length cDNA (or as close to full-length cDNA as possible). In some embodiments, a second-strand synthesis auxiliary probe (a partially double-stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe) can be ligated to the 3′ end of the extended probe, such as a first-strand cDNA molecule, using a double-stranded ligase (e.g., T4 DNA ligase). Other enzymes suitable for the ligation step are known in the art and include, for example, Tth DNA ligase, Taq DNA ligase, Thermococcus (strain 9°N) DNA ligase (9°N TM DNA ligase, New England Biolabs), Ampligase TM(available from Epicentre Biotechnologies, Madison, Wisconsin) and SplintR (available from New England Biolabs, Ipswich, Massachusetts). In some embodiments, a polynucleotide tail (e.g., a poly(A) tail) is incorporated into the 3′ end of the extended probe molecule. In some embodiments, the polynucleotide tail is enzymatically incorporated using terminal transferase activity.

[0343] In some embodiments, the double-stranded extended capture probe is treated to remove any unextended capture probe prior to amplification and / or analysis (e.g., sequence analysis). This can be achieved by a variety of methods, for example, using an enzyme to degrade the unextended probe, such as an exonuclease, or using a purification column.

[0344] In some embodiments, the extended capture probe is amplified to produce a quantity sufficient for analysis, such as by DNA sequencing. In some embodiments, the first strand (e.g., a DNA and / or cDNA molecule) of the extended capture probe is used as a template for an amplification reaction (e.g., a polymerase chain reaction).

[0345] In some embodiments, the amplification reaction incorporates an affinity group onto an extended capture probe (e.g., RNA-cDNA hybrid) using a primer comprising an affinity group. In some embodiments, the primer comprises an affinity group and the extended capture probe comprises an affinity group. The affinity group can correspond to any of the affinity groups described above.

[0346] In some embodiments, the capture probe comprising an extension of an affinity group can be coupled to an array feature specific for the affinity group. In some embodiments, the substrate can include an antibody or antibody fragment. In some embodiments, the array feature includes avidin or streptavidin, and the affinity group includes biotin. In some embodiments, the array feature includes maltose, and the affinity group includes maltose binding protein. In some embodiments, the array feature includes maltose binding protein, and the affinity group includes maltose. In some embodiments, as long as a copy of the extended probe is not attached to the array feature, the capture probe that has been amplified can serve to release the amplified probe from the array feature.

[0347] In some embodiments, the capture probe or its complement or amplicon that extends is released from the array characteristics.The step of releasing the capture probe or its complement or amplicon that extends from the array characteristics can be achieved in many ways.In some embodiments, the capture probe or its complement that extends is released from the feature by nucleic acid cleavage and / or denaturation (for example, by heating to denature double-stranded molecules).

[0348] In some embodiments, the extended capture probe or its complement or amplicon is released from the array feature by physical means. For example, the method of inducing physical release includes denaturing double-stranded nucleic acid molecules. Another method of releasing the extended capture probe is to use a solution that interferes with the hydrogen bonds of the double-stranded molecules. In some embodiments, the extended capture probe is released by applying heated water (e.g., water or buffer at least 85°C, such as water at least 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C or 99°C). In some embodiments, a solution comprising salts, surfactants, etc. is added to further destabilize the interactions between nucleic acid molecules to release the extended capture probe from the array feature. In some embodiments, formamide solution can be used to destabilize the interactions between nucleic acid molecules to release the extended capture probe from the array feature.

[0349] Analyte capture agent

[0350] The present invention also provides methods and materials for spatial profiling of biological analytes (e.g., mRNA, genomic DNA, accessible chromatin and cell surface or intracellular proteins and / or metabolites) using analyte capture agents. As used herein, an "analyte capture agent" (sometimes also referred to previously as a "cell marker") refers to a substance that interacts with an analyte (e.g., an analyte in a sample) and a capture probe (e.g., a capture probe attached to a substrate) to identify the analyte. In some embodiments, the analyte capture agent comprises an analyte binding portion and a capture agent barcode domain.

[0351] Figure 9 Schematic diagram of an exemplary analyte capture agent 902 consisting of an analyte binding portion 904 and a capture agent barcode domain 908. The analyte binding portion 904 is a molecule capable of binding to an analyte 906 and interacting with a spatially barcoded capture probe. The analyte binding portion can bind to the analyte 906 with high affinity and / or high specificity. The analyte capture agent may include a capture agent barcode domain 908, a nucleotide sequence (e.g., an oligonucleotide) that can hybridize with at least a portion or all of the capture domain of the capture probe. The analyte binding portion 904 may include a polypeptide and / or an aptamer (e.g., an oligonucleotide or peptide molecule that binds to a specific target analyte). The analyte binding portion 904 may include an antibody or antibody fragment (e.g., an antigen binding fragment).

[0352] As used herein, the term "analyte binding moiety" refers to a molecule or portion that is capable of binding to a macromolecular component (e.g., an analyte, e.g., a biological analyte). In some embodiments of any of the spatial profiling methods described herein, the analyte binding moiety of the analyte capture agent that binds to a biological analyte may include, but is not limited to, an antibody or epitope binding fragment thereof, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bispecific antibody, a bispecific T cell engager, a T cell receptor engager, a B cell receptor engager, a precursor, an aptamer, a monomer, an adhesin, a DARPin, and a protein scaffold, or any combination thereof. The analyte binding moiety can bind to a macromolecular component (e.g., an analyte) with high affinity and / or high specificity. The analyte binding moiety may include a nucleotide sequence (e.g., an oligonucleotide) that may correspond to at least part or all of the analyte binding moiety. The analyte binding moiety may include a polypeptide and / or an aptamer (e.g., a polypeptide and / or an aptamer that binds to a specific target molecule, e.g., an analyte). The analyte binding moiety may include an antibody or antibody fragment (e.g., an antigen binding fragment) that binds to a specific analyte (e.g., a polypeptide).

[0353] In some embodiments, the analyte capture agent can bind to an analyte present in the cell. In some embodiments, the analyte capture agent can bind to a cell surface analyte, which may include but is not limited to receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation or lipidation) state, gap junctions and adherens junctions. In some embodiments, the analyte capture agent can be bound to a post-translationally modified cell surface analyte. In such embodiments, the analyte capture agent can be specific to a cell surface analyte based on a given state of post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation or lipidation) such that a cell surface analyte profile may include post-translational modification information of one or more analytes.

[0354] In some embodiments, the analyte capture agent comprises a capture agent barcode domain coupled to or otherwise attached to an analyte binding moiety. In some embodiments, the capture agent barcode domain is covalently linked to the analyte binding moiety. In some embodiments, the capture agent barcode domain is a nucleic acid sequence. In some embodiments, the capture agent barcode domain comprises an analyte binding moiety barcode and an analyte capture sequence.

[0355] As used herein, the term "analyte-binding moiety barcode" refers to a barcode that is associated with or otherwise identifies an analyte-binding moiety. In some embodiments, by identifying an analyte-binding moiety (by identifying its associated analyte-binding moiety barcode), the analyte to which the analyte-binding moiety binds can also be identified. The analyte-binding moiety barcode can be a nucleic acid sequence of a given length and / or a sequence associated with the analyte-binding moiety. The analyte-binding moiety barcode can generally include any of the various aspects of barcodes described herein. For example, an analyte capture agent specific for one type of analyte can have a first capture agent barcode domain coupled thereto (e.g., comprising a first analyte-binding moiety barcode), while an analyte capture agent specific for a different analyte can have a different capture agent barcode domain coupled thereto (e.g., comprising a second barcode analyte-binding moiety barcode). In some aspects, such a capture agent barcode domain can include an analyte binding moiety barcode that identifies the analyte binding moiety to which the capture agent barcode domain is coupled. The selection of the capture agent barcode domain can yield significant diversity in sequence while also being easily attached to most analyte binding moieties (e.g., antibodies) and easily detected (e.g., using sequencing or array technology). In some embodiments, the analyte capture agent can include an analyte binding moiety having a capture agent barcode domain attached thereto. For example, the analyte capture agent can include a first analyte binding moiety (e.g., an antibody that binds to an analyte, e.g., a first cell surface feature) having associated therewith a capture agent barcode domain that includes the first analyte binding moiety barcode.

[0356] In some embodiments, the capture agent barcode domain of the analyte capture agent includes an analyte capture sequence. As used herein, the term "analyte capture sequence" refers to a region or portion that is configured to hybridize to, bind to, couple to, or otherwise interact with the capture domain of a capture probe. In some embodiments, the analyte capture sequence includes a nucleic acid sequence that is complementary or substantially complementary to the capture domain of the capture probe, such that the analyte capture sequence hybridizes with the capture domain of the capture probe. In some embodiments, the analyte capture sequence comprises a poly(A) nucleic acid sequence that hybridizes to a capture domain comprising a poly(T) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a poly(T) nucleic acid sequence that hybridizes to a capture domain comprising a poly(A) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a non-homogeneous nucleic acid sequence that hybridizes to a capture domain, the capture domain comprising a non-homogeneous nucleic acid sequence that is complementary (or substantially complementary) to the non-homogeneous nucleic acid sequence of the analyte capture region.

[0357] In some embodiments of any of the spatial analysis methods described herein using analyte capture agents, the capture agent barcode domains can be directly coupled to the analyte binding moiety, or they can be attached to beads, molecular lattices, such as linear, globular, cross-linked or other polymers, or other frameworks attached to or otherwise associated with the analyte binding moiety that enable attachment of multiple capture agent barcode domains to a single analyte binding moiety. Attachment (coupling) of the capture agent barcode domains to the analyte binding moiety can be achieved by any of a variety of direct or indirect, covalent or non-covalent binding or attachments. For example, where the capture agent barcode domains are coupled to an analyte binding moiety comprising an antibody or antigen binding fragment, chemical coupling techniques (e.g., Lightning Biopharmaceuticals available from Innova Biosciences) can be used. Antibody labeling kit) these capture agent barcode domains are covalently attached to portions of the antibody or antigen binding fragment. In some embodiments, the capture agent barcode domain can be coupled to the antibody or antigen binding fragment using a non-covalent attachment mechanism (e.g., using a biotinylated antibody and an oligonucleotide or bead comprising one or more biotinylated linkers, coupled to the oligonucleotide with an avidin or streptavidin linker). Antibody and oligonucleotide biotinylation techniques can be used and are described, for example, in Fang et al., Nucleic Acids Res. (2003), 31(2):708-715, the entire contents of which are incorporated herein by reference. Similarly, protein and peptide biotinylation techniques have been developed and can be used and are described, for example, in U.S. Patent No. 6,265,552, the entire contents of which are incorporated herein by reference. In addition, click reaction chemistry (e.g., methyl tetrazine-PEG5-NHS ester reaction, TCO-PEG4-NHS ester reaction, etc.) can be used to couple the capture agent barcode domain to the analyte binding portion. The reactive moieties on the analyte binding portion may also include amines for targeting aldehydes, amines for targeting maleimides (e.g., free sulfhydryls), azides for targeting click chemistry compounds (e.g., alkynes), biotin for targeting streptavidin, phosphates for targeting EDC, which in turn target active esters (e.g., NH2). The reactive moieties on the analyte binding portion may be compounds or groups that bind to the reactive moieties on the analyte binding portion. Exemplary strategies for coupling the analyte binding portion to the capture agent barcode domain include the use of commercial kits (e.g., Solulink, Thunder link), slight reduction of the hinge region and binding of maleimide labels, click chemistry reactions promoted by dyeing of labeled amides (e.g., copper-free), coupling of periodate oxidation of sugar chains, and coupling of amines. In the case where the analyte binding portion is an antibody, the antibody can be modified prior to or simultaneously with oligonucleotide coupling. For example, antibodies can be glycosylated with the substrate-permissive mutant GalT (Y289L) of β-1,4-galactosyltransferase and the azide-containing uridine diphosphate-N-acetylgalactosamine analog uridine diphosphate-GalNAz. The modified antibodies can be conjugated to oligonucleotides bearing dibenzocyclooctyne-PEG4-NHS groups. In some embodiments, certain steps (e.g., COOH activation (e.g., EDC) and homobifunctional crosslinkers) can be avoided to prevent the analyte-binding moiety from conjugating to itself.In some embodiments of any of the spatial profiling methods described herein, an analyte capture agent (e.g., an analyte binding portion coupled to an oligonucleotide) can be delivered to the cell by transfection (e.g., using transfectamine, a cationic polymer, calcium phosphate, or electroporation), transduction (e.g., using a phage or recombinant viral vector), by mechanical delivery (e.g., magnetic beads), by lipids (e.g., 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporters. The analyte capture agent can be delivered to the cell via an exosome. For example, a first cell can be generated that releases an exosome containing an analyte capture agent. The analyte capture agent can be attached to an exosome membrane. The analyte capture agent can be contained in the cytoplasm of the exosome. The released exosome can be harvested and provided to a second cell, thereby delivering the analyte capture agent to the second cell. The analyte capture agent can be released from the exosome membrane before, during, or after delivery to the cell. In some embodiments, cells are permeabilized to allow coupling of the analyte capture agent to intracellular cellular components (such as, but not limited to, intracellular proteins, metabolites, and nuclear membrane proteins). Following intracellular delivery, the analyte capture agent can be used to analyze the intracellular components as described herein.

[0358] In some embodiments of any of the spatial profiling methods described herein, the capture agent barcode domain coupled to the analyte capture agent may include modifications that render it non-amplifiable by polymerases. In some embodiments, the capture agent barcode domain can be used as a template rather than a primer when bound to a nucleic acid in a sample or a capture domain of a capture probe for a primer extension reaction. When the capture agent barcode domain also includes a barcode (e.g., an analyte binding moiety barcode), such a design can improve the efficiency of molecular barcoding by increasing the affinity between the capture agent barcode domain and the unencoded sample nucleic acid and eliminate the potential formation of adapter artifacts. In some embodiments, the capture agent barcode domain may include a random N-mer sequence that is capped by modifications that render it non-extensible by polymerases. In some cases, the composition of the random N-mer sequence can be designed to maximize binding efficiency to free, non-encoded ssDNA molecules. Designs may include random sequence compositions with higher GC content, partially random sequences with fixed G or C at specific positions, the use of guanosine, the use of locked nucleic acids, or any combination thereof.

[0359] The modification of primer extension blocked by polymerase can be carbon spacer groups or dideoxynucleotides of different lengths. In some embodiments, the modification can be an analog of a baseless site with no purine or no pyrimidine structure, a base analog or a phosphate backbone, such as N-(2-aminoethyl)-glycine backbone, tetrahydrofuran or 1 ', 2'-dideoxyribose connected by an amide bond. The modification can also be uracil base, 2'OMe modified RNA, C3-18 spacers (e.g., structures with 3-18 continuous carbon atoms, such as C3 spacers), ethylene glycol polymer spacers (e.g., spacer 18 (hexaethylene glycol spacers)), biotin, dideoxynucleotide triphosphates, ethylene glycol, amines or phosphates.

[0360] In some embodiments of any spatial profile analysis method described herein, the capture agent barcode domain coupled to the analyte binding portion includes a cleavable domain. For example, after the analyte capture agent is bound to the analyte (e.g., cell surface analyte), the capture agent barcode domain can be cut and collected according to the methods described herein for downstream analysis. In some embodiments, the cleavable domain of the capture agent barcode domain includes a U-excision element that releases the substance from the bead. In some embodiments, the U-excision element may include a single-stranded DNA (ssDNA) sequence comprising at least one uracil. The substance can be attached to the bead by the ssDNA sequence. The substance can be released by a combination of uracil-DNA glycosylase (e.g., removing uracil) and an endonuclease (e.g., inducing ssDNA breakage). If the endonuclease produces a 5' phosphate group from the cleavage, other enzymes can be included in the downstream processing to eliminate the phosphate group, for example, before connecting other sequencing handle elements (e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence and / or partial R1 sequence).

[0361] In some embodiments, the analyte-binding portion of an analyte-capture agent comprises one or more antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment comprising the analyte-binding portion can specifically bind to the target analyte. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, multiple analyte-capture agents comprising multiple analyte-binding portions bind to multiple analytes present in a biological sample. In some embodiments, the multiple analytes comprise a single type of analyte (e.g., a single type of polypeptide). In some embodiments where the multiple analytes comprise a single type of analyte, the analyte-binding portions of the multiple analyte-capture agents are identical. In some embodiments where the multiple analytes comprise a single type of analyte, the analyte-binding portions of the multiple analyte-capture agents are different (e.g., members of the multiple analyte-capture agents can have two or more analyte-binding portions, wherein each of the two or more analyte-binding portions binds to a single type of analyte (e.g., at different binding sites)). In some embodiments, the multiple analytes comprise multiple different types of analytes (e.g., multiple different types of polypeptides).

[0362] In some embodiments, a variety of different types of analytes (e.g., polypeptides) from a biological sample can then be associated with one or more physical properties of the biological sample. For example, a variety of different types of analytes can be associated with the location of the analyte in the biological sample. Such information (e.g., proteomic information when the analyte binding moiety recognizes the polypeptide) can be used in conjunction with other spatial information (e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e., transcript sequence), or both). For example, a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., the shape, size, activity, or type of the cell). One or more physical properties can be characterized by imaging the cell. The cell can be bound by an analyte capture agent comprising an analyte binding moiety that binds to a cell surface protein and an analyte binding moiety barcode that recognizes the analyte binding moiety, and the cell can be spatially analyzed (e.g., any of the various spatial analysis methods described herein). For example, an analyte capture agent that is bound to a cell surface protein can be bound to a capture probe (e.g., a capture probe on an array) that includes a capture domain that interacts with an analyte capture sequence present on a capture agent barcode domain of the analyte capture agent. All or part of the capture agent barcode domain (including the analyte binding portion barcode) can be replicated using a polymerase, using the 3' end of the capture domain as a priming site to generate an extended capture probe that includes all or part of the capture probe (including the spatial barcode present on the capture probe) and a copy of the analyte binding portion barcode. In some embodiments, a spatial array with extended capture probes can be contacted with a sample, wherein the analyte capture agent associated with the spatial array captures the target analyte. The analyte capture agent containing an extended capture probe (which includes the spatial barcode of the capture probe and the analyte binding portion barcode) can then be denatured from the capture probe of the spatial array. In this way, the spatial array can be reused. The sample can be separated into non-aggregated cells (e.g., single cells) and analyzed by the single cell / droplet method described herein. The extended capture probe can be sequenced to obtain a nucleic acid sequence, wherein the spatial barcode of the capture probe is associated with the analyte binding portion barcode of the analyte capture agent. Thus, the nucleic acid sequence of the extended capture probe can be associated with an analyte (e.g., a cell surface protein) and, in turn, associated with one or more physical properties of the cell (e.g., shape or cell type). In some embodiments, the nucleic acid sequence of the extended capture probe can be associated with an intracellular analyte of a nearby cell, wherein the intracellular analyte is released using any cell permeabilization or analyte migration technique described herein.

[0363] In some embodiments of any of the spatial profiling methods described herein, the capture agent barcode domains released from the analyte capture agents can then be sequenced to identify which analyte capture agents bind to the analyte. Based on the presence of capture agent barcode domains and analyte binding portion barcode sequences associated with features on the spatial array (e.g., features at a specific location), an analyte file can be created for the biological sample. Files for individual cells or cell populations can be compared with files for other cells (e.g., "normal" cells) to determine changes in the analyte, thereby providing diagnostic-related information. In some embodiments, these files can be used to diagnose various diseases characterized by changes in cell surface receptors, such as cancer and other diseases.

[0364] Figure 10 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1024 and an analyte capture agent 1026. The feature-immobilized capture probe 1024 can include a spatial barcode 1008 and one or more functional sequences 1006 and 1010, as described elsewhere herein. The capture probe can also include a capture domain 1012 capable of binding to the analyte capture agent 1026. The analyte capture agent 1026 can include a functional sequence 1018, a capture agent barcode domain 1016, and an analyte capture sequence 1014 capable of binding to the capture domain 1012 of the capture probe 1024. The analyte capture agent can also include a linker 1020 that couples the capture agent barcode domain 1016 to the analyte binding moiety 1022.

[0365] In some embodiments of any of the spatial profiling methods described herein, the method is used to identify immune cell profiles. Immune cells express various adaptive immune receptors associated with immune function, such as T cell receptors (TCRs) and B cell receptors (BCRs). T cell receptors and B cell receptors play a role in immune responses by specifically recognizing and binding antigens and assisting in their destruction.

[0366] The T cell receptor (TCR) is a molecule present on the surface of T cells that is typically responsible for recognizing antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. TCRs are typically heterodimers of two chains, each a member of the immunoglobulin superfamily, with an N-terminal variable (V) domain and a C-terminal constant domain. In humans, 95% of T cells have TCRs composed of α and β chains, while 5% of T cells have TCRs composed of γ and δ (γ / δ) chains. This ratio varies during individual development, in disease states, and across species. When the TCR binds to an antigenic peptide and MHC (peptide / MHC or pMHC), T lymphocytes are activated through signal transduction.

[0367] Each of the two chains of a TCR contains multiple copies of gene segments—a variable "V" gene segment, a diversity "D" gene segment, and a connecting "J" gene segment. The TCR α chain (TCRα) is generated by the recombination of the V and J segments, while the β chain (TCRβ) is generated by the recombination of the V, D, and J segments. Similarly, the generation of the TCR γ chain involves the recombination of the V and J gene segments, while the generation of the TCR δ chain is achieved by the recombination of the V, D, and J gene segments. The intersection of these specific regions (V and J for the α or γ chain, V, D, and J for the β or δ chain) corresponds to the CDR3 region, which is important for antigen MHC recognition. The complementarity determining regions (e.g., CDR1, CDR2, and CDR3), or hypervariable regions, are the sequences in the variable domain of antigen receptors (e.g., T cell receptors and immunoglobulins) that complement the antigen. The majority of the CDR diversity is found in the CDR3, which is generated by somatic recombination events during T lymphocyte development. The unique nucleotide sequences generated during the gene sequencing process can be called clonotypes.

[0368] The B cell receptor (BCR) is a molecule present on the surface of B cells. The antigen-binding portion of the BCR consists of membrane-bound antibodies, which, like most antibodies (e.g., immunoglobulins), have unique and randomly determined antigen-binding sites. The antigen-binding portion of the BCR includes membrane-bound immunoglobulin molecules of one isotype (e.g., IgD, IgM, IgA, IgG, or IgE). When a B cell is activated by its first encounter with a cognate antigen, the cell proliferates and differentiates to produce populations of antibody-secreting plasma B cells and memory B cells. Different immunoglobulin isotypes differ in their biological properties, structure, target specificity, and distribution. There are multiple molecular mechanisms to generate initial diversity, including genetic recombination at multiple sites.

[0369] The BCR is composed of two genes, IgH and IgK (or IgL), which encode the heavy and light chains of antibodies. Immunoglobulins are formed through recombination between gene segments, sequence diversification at the junctions of these segments, and point mutations throughout the gene. Each heavy chain gene contains multiple copies of three different gene segments - the variable "V" gene segment, the diversity "D" gene segment, and the connecting "J" gene segment. Each light chain gene contains multiple copies of two different gene segments of the protein variable region - the variable "V" gene segment and the connecting "J" gene segment.

[0370] Recombination can produce a molecule with one V, D, and J segment each. In addition, several bases can be deleted at each junction of the two junctions, and other bases (called N and P nucleotides) can be added, thereby generating further diversity. After B cell activation, affinity maturation occurs through somatic hypermutation. In this process, the daughter cells of the activated B cell accumulate different somatic mutations throughout the gene, with a higher concentration of mutations in the CDR region, thereby producing antibodies with higher affinity for the antigen.

[0371] In addition to somatic hypermutation, activated B cells also undergo a process of isotype switching. Antibodies with the same variable segment can have different forms (isotypes) depending on the constant segment. While all naive B cells express IgM (or IgD), activated B cells mostly express IgG, but also express IgM, IgA, and IgE. This expression switch from IgM (and / or IgD) to IgG, IgA, or IgE occurs through a recombination event, which causes a cell to exclusively produce a specific isotype. The unique nucleotide sequence generated during the gene arrangement process can be similarly referred to as a clonotype.

[0372] Some methods described herein are used to analyze various sequences of TCR and BCR from immune cells, such as various clonal types. In some embodiments, the method is used to analyze the sequence of TCR α chain, TCR β chain, TCR δ chain, TCR γ chain or any fragment thereof (for example, including the variable region, constant region, transmembrane region, its fragment, its combination and its fragment combination of V (D) J or VJ area). In some embodiments, the methods described herein can be used to analyze the sequence of B cell receptor heavy chain, B cell receptor light chain or any fragment thereof (for example, including the variable region, constant region, transmembrane region, its fragment, its combination and its fragment combination of V (D) J or VJ area).

[0373] In the case of immune cells to be analyzed, primer sequences for any of the various operations for attaching barcode sequences and / or amplification reactions may include gene-specific sequences targeting genes or gene regions of immune cell proteins (e.g., immune receptors). These gene sequences include, but are not limited to, sequences of various T cell receptor alpha variable genes (TRAV genes), T cell receptor alpha joining genes (TRAJ genes), T cell receptor alpha constant genes (TRAC genes), T cell receptor beta variable genes (TRBV genes), T cell receptor beta diversity genes (TRBD genes), T cell receptor beta joining genes (TRBJ genes), T cell receptor beta constant genes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), T cell receptor gamma joining genes (TRGJ genes), T cell receptor gamma constant genes (TRGC genes), T cell receptor delta variable genes (TRDV genes), T cell receptor delta diversity genes (TRDD genes), T cell receptor delta joining genes (TRDJ genes), and T cell receptor delta constant genes (TRDC genes).

[0374] In some embodiments, the analyte binding portion is based on a class I or class II major histocompatibility complex (MHC). In some embodiments, the analyte binding portion is an MHC multimer, including but not limited to MHC dextrorotatory bodies, MHC tetramers, and MHC pentamers (e.g., see U.S. Patent Application Publication Nos. US 2018 / 0180601 and US 2017 / 0343545, the entire contents of which are incorporated herein by reference). An MHC comprising all or part of an MHC peptide (e.g., a soluble MHC monomer molecule) can be used as an analyte binding portion of an analyte capture agent, which is coupled to a capture agent barcode domain that includes an analyte binding portion barcode that recognizes its associated MHC (thus, for example, a TCR binding partner of an MHC). In some embodiments, MHC is used to analyze one or more cell surface features of a T cell, such as a TCR. In some cases, multiple MHCs are combined together in a larger complex (MHC multimer), and the binding affinity of MHC to TCR is increased through multi-ligand binding synergy.

[0375] Figure 11A 、 11B11C is a schematic diagram illustrating how streptavidin cell tags can be used in array-based systems to generate spatially barcoded cells or cell contents. For example, as shown in FIG11 , peptide-bound major histocompatibility complex (pMHC) can be individually bound to biotin and bound to a streptavidin portion such that the streptavidin portion comprises multiple pMHC portions. Each of these portions can be bound to a TCR such that streptavidin binds to a target T cell through multiple MCH / TCR binding interactions. Multiple interactions act synergistically to greatly increase binding affinity. This improved affinity can improve labeling of T cells and also reduce the likelihood of label dissociation from the T cell surface. As Figure 11B As shown, the capture agent barcode domain 1101 can be modified with streptavidin 1102 and contacted with multiple molecules of biotinylated MHC 1103 (e.g., pMHC), such that the biotinylated MHC 1103 molecules are coupled to the streptavidin-conjugated capture agent barcode domain 1101. The result is a barcoded MHC multimer complex 1105. Figure 11B As shown, the capture agent barcode domain sequence 1101 can recognize the MHC as its associated tag and also includes optional functional sequences, such as sequences for hybridization with other oligonucleotides. Figure 11CAs shown, an example oligonucleotide is a capture probe 1106 that includes a complementary sequence (e.g., rGrGrG corresponding to C CC), a barcode sequence, and other functional sequences, such as a UMI, an adapter sequence (e.g., including a sequencing primer sequence (e.g., R1 or a portion of R1 ("pR1")), a flow cell attachment sequence (e.g., P5 or P7 or a portion thereof), etc. In some cases, the capture probe 1106 can be first associated with a feature (e.g., a gel bead) and released from the feature. In other embodiments, the capture probe 1106 can be hybridized to the capture agent barcode domain 1101 of the MHC-oligonucleotide complex 1105. The hybridized oligonucleotide (spacer CCC and spacer rGrGrG) can then be extended in a primer extension reaction to generate a barcode sequence corresponding to two spatial barcode sequences (the spatial barcode associated with the capture probe and the spacer associated with the MHC-oligonucleotide complex). In some cases, one or both of these corresponding sequences can be the complement of the original sequence in the capture probe 1106 or the capture agent barcode domain 1101. In other embodiments, the capture probe and the capture agent barcode domain are connected together. The resulting construct can be optionally further processed (e.g., to add any other sequences and / or for cleaning) and sequenced. As described elsewhere herein, sequences derived from the spatial barcode sequence of the capture probe 1106 can be used to identify features, and sequences derived from the spatial barcode sequence on the capture agent barcode domain 1101 can be used to identify specific peptide MHC complexes 1104 bound to the cell surface (e.g., when using an MHC peptide library for screening immune cells or immune cell populations).

[0376] (c) Base material

[0377] For the analytical method based on spatial array described in this section, substrate has played the effect of supporting capture probe to be directly or indirectly attached to array feature.In addition, in some embodiments, substrate (for example, identical substrate or different substrates) can be used for biological sample, particularly, for example thin tissue section provides support.Therefore, " substrate " is the support that is insoluble in aqueous solution, and it makes biological sample, analyte, feature and / or capture probe be positioned on substrate.

[0378] A variety of different substrates can be used for the above purposes. Generally, the substrate can be any suitable support material. Exemplary substrates include, but are not limited to, glass, modified and / or functionalized glass, hydrogels, membranes, films, plastics (including, for example, acrylic acid, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM, cyclic olefins, polyimides, etc.), 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 copolymer (COC), cyclic olefin polymer (COP), polypropylene, polyethylene and polycarbonate.

[0379] The substrate may also correspond to a flow cell. A flow cell may be formed from any of the materials described above and may include channels that allow reagents, solvents, features, and molecules to pass through the flow cell.

[0380] In the example of substrate materials discussed above, polystyrene is a hydrophobic material suitable for binding negatively charged macromolecules because it generally contains few hydrophilic groups. For nucleic acids immobilized on glass slides, the immobilization of nucleic acids can be increased by increasing the hydrophobicity of the glass surface. This enhancement can allow for relatively denser stacking (e.g., providing improved specificity and resolution).

[0381] In some embodiments, the substrate is coated with a surface treatment agent (e.g., poly (L) -lysine). Additionally or alternatively, the substrate can be treated by silanization (e.g., with epoxy silanes, amino silanes) and / or by polyacrylamide treatment.

[0382] The substrate can generally have any suitable form or morphology. For example, the substrate can be flat, curved, such as convex or concave toward the region where the interaction between the biological sample (e.g., tissue sample) and the substrate occurs. In some embodiments, the substrate is flat, such as a plane, a chip, or a glass slide. The substrate can include one or more patterned surfaces (e.g., channels, holes, protrusions, ridges, pits, etc.) within the substrate.

[0383] The substrate can be any desired shape. For example, the substrate can be generally a thin flat shape (for example, a square or a rectangle). In some embodiments, the substrate structure has rounded corners (for example, to increase safety or robustness). In some embodiments, the substrate structure has one or more cutoff angles (for example, for sliding clamps or cross-tables). In some embodiments, when the substrate structure is flat, the substrate structure can be a support (for example, a chip or a slide, such as a microscope slide) of any appropriate type with a flat surface.

[0384] The substrate can optionally include various structures, such as but not limited to projections, ridges and channels. The substrate can be micropatterned to limit lateral diffusion (e.g., to prevent the overlapping of spatial barcodes). The substrate modified with this structure can be modified to allow analyte, feature (e.g., pearl) or probe to be combined at various positions. For example, the site of the substrate modified with various structures can be continuous or discontinuous with other sites.

[0385] In some embodiments, the surface of the substrate can be modified to form discrete locations that can only have or accommodate a single feature. In some embodiments, the surface of the substrate can be modified so that features are attached to random sites.

[0386] In some embodiments, the surface of the substrate is modified to include one or more holes using techniques such as, but not limited to, stamping techniques, microetching techniques, and molding techniques. In some embodiments where the substrate includes one or more holes, the substrate can be a concave glass slide or a recessed glass slide. For example, the holes can be formed by one or more shallow depressions on the surface of the substrate. In some embodiments where the substrate includes one or more holes, the holes can be formed by attaching a box (e.g., a box comprising one or more chambers) to the surface of the substrate structure.

[0387] In some embodiments, the structures of the substrate (e.g., pores) can each carry a different capture probe. The different capture probes attached to each structure can be identified based on the location of the structure in or on the substrate surface. Exemplary substrates include arrays in which separate structures are located on a substrate, including, for example, arrays of pores that accommodate features.

[0388] In some embodiments, the substrate includes one or more markings on the surface of the substrate, for example, to provide guidance for associating spatial information with the characterization of the analyte of interest. For example, the substrate can be marked with a grid of lines (e.g., to easily estimate the size of an object seen under magnification and / or to provide a reference area for counting objects). In some embodiments, fiducial markings can be included on the substrate. Such markings can be made using techniques including, but not limited to, printing, sandblasting, and surface deposition.

[0389] In some embodiments, where a substrate is modified to include one or more structures, including but not limited to pores, protrusions, ridges, or markings, the structures may include sites of physical alteration. For example, a substrate modified with various structures may include physical properties including but not limited to physical configuration, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and / or electrostatically altered sites.

[0390] In some embodiments, where the substrate is modified to contain various structures, including but not limited to holes, protrusions, ridges, or logos, the structures are applied in a pattern. Alternatively, the structures can be randomly distributed.

[0391] In some embodiments, the substrate is treated to minimize or reduce non-specific analyte hybridization within or between features. For example, the treatment may include coating the substrate with a hydrogel, a membrane, and / or a film that forms a physical barrier to non-specific hybridization. Any suitable hydrogel can be used. For example, a hydrogel substrate prepared according to the methods described in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422 and U.S. Patent Application Publication Nos. US2017 / 0253918 and US2018 / 0052081 can be used. The entire contents of each of the above documents are incorporated herein by reference.

[0392] Treatment can include adding functional groups that are reactive or capable of being activated so that they become reactive upon receipt of a stimulus (e.g., photoactivation). Treatment can include treatment with a polymer having one or more physical properties (e.g., mechanical, electrical, magnetic, and / or thermal) to minimize nonspecific binding (e.g., activating the substrate at certain locations to allow analyte hybridization at those locations).

[0393] A substrate (e.g., a bead or feature on an array) can include tens of thousands to hundreds of thousands or millions of individual oligonucleotide molecules (e.g., at least about 10,000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 100,000,000, 100,000,000, 100,000,000, or 1,000,000,000 oligonucleotide molecules).

[0394] In some embodiments, the surface of the substrate is coated with a cell-permissive coating to allow living cells to adhere. A "cell-permissive coating" is a coating that allows or helps cells maintain cell viability (e.g., maintain viability) on a substrate. For example, a cell-permissive coating can enhance cell attachment, cell growth, and / or cell differentiation, e.g., a cell-permissive coating can provide nutrients to living cells. Cell-permissive coatings can include biomaterials and / or synthetic materials. Non-limiting examples of cell-permissive coatings include those characterized by having one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrous proteins, such as collagen, elastin, fibronectin, and laminin), polylysine, poly(L)-ornithine, and / or biocompatible silicones (e.g., For example, a cell-permissive coating comprising one or more extracellular matrix components may include type I collagen, type II collagen, type IV collagen, elastin, fibronectin, laminin, and / or vitronectin. In some embodiments, the cell-permissive coating comprises cells isolated from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., ). In some embodiments, the cell-permissive coating comprises collagen. The cell-permissive coating can be used to culture adherent cells on a spatially barcoded array or to maintain cell viability in a tissue sample or section while in contact with a spatially barcoded array.

[0395] When the substrate comprises a gel (e.g., a hydrogel or gel substrate), the oligonucleotides within the gel can be attached to the substrate. The terms "hydrogel" and "hydrogel substrate" are used interchangeably herein to refer to a macromolecular polymer gel comprising a network. In the network, some polymer chains can be selectively cross-linked, although cross-linking does not always occur.

[0396] In some embodiments, hydrogel may include hydrogel subunits." hydrogel subunits " are hydrophilic monomers, molecular precursors or polymerizable (e.g., cross-linked) polymers to form three-dimensional (3D) hydrogel networks. Hydrogel subunits may include any convenient hydrogel subunits, such as but not limited to acrylamide, bisacrylamide, polyacrylamide and derivatives thereof, polyethylene glycol and derivatives thereof (e.g., PEG-acrylates (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohol, polypropylene glycol, polyoxybutylene, polyvinyl pyrrolidone, polyacrylamide, polyhydroxyethyl acrylate and polyhydroxyethyl methacrylate, collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, etc. and combinations thereof.

[0397] In some embodiments, the hydrogel comprises a hybrid material, for example, a hydrogel material comprising elements of a synthetic polymer and a natural polymer. Examples of suitable hydrogels are described, for example, in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Patent Application Publication Nos. 2017 / 0253918, 2018 / 0052081, and 2010 / 0055733, the entire contents of which are incorporated herein by reference.

[0398] In some embodiments, a crosslinker and / or initiator is added to the hydrogel subunits. Examples of crosslinkers include, but are not limited to, bisacrylamide and bisaziridine. Examples of initiators include, but are not limited to, azobisisobutyronitrile (AIBN), riboflavin, and L-arginine. Inclusion of a crosslinker and / or initiator can result in increased covalent bonding between interacting biomacromolecules during later polymerization steps.

[0399] In some embodiments, the hydrogel can have a colloidal structure (eg, agarose) or a polymer network structure (eg, gelatin).

[0400] In some embodiments, some of the hydrogel subunits are covalently or physically cross-linked and polymerized (e.g., undergo "forming") to form a hydrogel network. For example, the hydrogel subunits can be polymerized by any method, including but not limited to thermal cross-linking, chemical cross-linking, physical cross-linking, ionic cross-linking, photocross-linking, radiation cross-linking (e.g., x-ray, electron beam), and combinations thereof. Techniques such as photolithographic polymerization can also be used to form hydrogels.

[0401] The polymerization method of the hydrogel subunits can be selected to form hydrogels with different properties (e.g., pore size, swelling properties, biodegradability, conductivity, transparency and / or permeability of the hydrogel). For example, the hydrogel can include pores of sufficient size to allow macromolecules (e.g., nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies, carbohydrates, peptides, metabolites and / or small molecules) to enter the sample (e.g., tissue section). It is well known that pore size generally decreases with increasing concentration of hydrogel subunits and generally increases with increasing ratio of hydrogel subunits to crosslinking agent. Therefore, a fixative / hydrogel composition can be prepared that includes a concentration of hydrogel subunits that allows such biomacromolecules to pass through.

[0402] In some embodiments, the hydrogel can form a substrate. In some embodiments, the substrate comprises a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on top of the one or more second materials. For example, the hydrogel can be pre-formed and then placed with the one or more second materials on top, on the bottom, or in any other configuration. In some embodiments, hydrogel formation occurs after contact with the one or more second materials during substrate formation. Hydrogel formation can also occur within structures (e.g., holes, ridges, protrusions, and / or markings) located on the substrate.

[0403] In some embodiments, hydrogel formation on the substrate occurs before, simultaneously with, or after features (e.g., beads) are attached to the substrate. For example, when capture probes are attached (e.g., directly or indirectly) to the substrate, hydrogel formation can be performed on a substrate that already contains capture probes.

[0404] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue section) is embedded in a hydrogel. In some embodiments, hydrogel subunits are injected into a biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

[0405] In embodiments where a hydrogel is formed within a biological sample, functionalized chemistry can be used. In some embodiments, the functionalized chemistry comprises hydrogel histochemistry (HTC). Any hydrogel tissue skeleton suitable for HTC (e.g., synthetic or natural) can be used to anchor biomacromolecules and regulate functionalization. Non-limiting examples of methods using HTC skeleton variants include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, the hydrogel formation within the biological sample is permanent. For example, biomacromolecules can be permanently adhered to the hydrogel, thereby allowing multiple rounds of interrogation. In some embodiments, the hydrogel formation within the biological sample is reversible.

[0406] In some embodiments, other reagents are added to the hydrogel subunits before, simultaneously and / or after polymerization. For example, other reagents may include but are not limited to oligonucleotides (e.g., capture probes), endonucleases for fragmented DNA, fragmentation buffers for DNA, DNA polymerases, dNTPs for amplifying nucleic acids and attaching barcodes to the amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, transposases, ligases, proteinase K and DNA enzymes. Other reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers and conversion oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, simultaneously and / or after polymerization.

[0407] In some embodiments, an HTC agent is added to the hydrogel before, simultaneously with, and / or after polymerization. In some embodiments, a cell labeling agent is added to the hydrogel before, simultaneously with, and / or after polymerization. In some embodiments, a cell permeabilizing agent is added to the hydrogel before, simultaneously with, and / or after polymerization.

[0408] The hydrogel embedded in the biological sample can be cleared using any suitable method. For example, electrophoretic tissue clearing methods can be used to remove biomacromolecules from the hydrogel-embedded sample. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixing medium, methylcellulose, or other semisolid medium) before or after the hydrogel is cleared.

[0409] A "conditionally removable coating" refers to a coating that can be removed from the surface of a substrate after application of a release agent. In some embodiments, the conditionally removable coating comprises a hydrogel as described herein, for example, a hydrogel comprising a polypeptide-based material. Non-limiting examples of hydrogels comprising polypeptide-based materials include transmembrane segments of spider silk and human muscle L-type calcium channels (e.g., ) of a combination of synthetic peptide-based materials containing a repeating arginine-alanine-aspartic acid-alanine sequence (RADARADARADARADA) (e.g. ), EAK16 (AEAEAKAKAEAEAKAK), KLD12 (KLDLKLDLKLDL) and PGMATRIX TM An amphipathic 16-residue peptide.

[0410] In some embodiments, the hydrogel in the conditionally removable coating is a stimulus-responsive hydrogel. The stimulus-responsive hydrogel can undergo a gel-to-solution and / or gel-to-solid transition when one or more external triggers (e.g., a releasing agent) are applied. See, for example, Willner, Acc. Chem. Res. 50: 657-658, 2017, which is incorporated herein by reference in its entirety. Non-limiting examples of stimulus-responsive hydrogels include thermoresponsive hydrogels, pH-responsive hydrogels, light-responsive hydrogels, redox-responsive hydrogels, analyte-responsive hydrogels, or combinations thereof. In some embodiments, the stimulus-responsive hydrogel can be a multi-stimulus-responsive hydrogel.

[0411] A "releasing agent" or "external trigger" is an agent that, when applied to the conditionally removable coating, causes the conditionally removable coating to be removed from the substrate. External triggers or releasing agents can include physical triggers, such as thermal, magnetic, ultrasonic, electrochemical, and / or light stimulation, as well as chemical triggers, such as pH, redox reactions, supramolecular complexes, and / or biocatalytically driven reactions. See, for example, Echeverria et al., Gels (2018), 4, 54; doi: 10.3390 / gels4020054, which is incorporated herein by reference in its entirety. The type of "releasing agent" or "external trigger" can depend on the type of conditionally removable coating. For example, a conditionally removable coating characterized by having a redox-responsive hydrogel can be removed upon application of a releasing agent comprising a reducing agent, such as dithiothreitol (DTT). As another example, a pH-responsive hydrogel can be removed upon application of a releasing agent that changes pH.

[0412] (d) Array

[0413] In many methods described herein, features (as further described below) are collectively positioned on a substrate."array" is a specific arrangement of a plurality of features that are irregular or form a regular pattern. Each feature in an array is different from each other based on its relative spatial position. Generally speaking, at least two of the plurality of features in an array include different capture probes (e.g., any example of capture probes described herein).

[0414] Array can be used for measuring a large amount of analytes simultaneously.In some embodiments, oligonucleotide is used at least in part to create array.For example, one or more copies of the oligonucleotide of single species (for example, capture probe) can correspond to or be directly or indirectly attached to the given feature in the array.In some embodiments, the given feature in the array includes two or more oligonucleotides (for example, capture probe).In some embodiments, two or more oligonucleotides (for example, capture probe) that are directly or indirectly connected to given feature on the array include common (for example, identical) spatial bar code.

[0415] A "feature" is an entity that serves as a support or repository for various molecular entities used in sample analysis. Examples of features include, but are not limited to, beads, dots of any two-dimensional or three-dimensional geometry (e.g., inkjet dots, mask dots, squares on a grid), wells, and hydrogel pads. In some embodiments, a feature is directly or indirectly attached or affixed to a substrate. In some embodiments, the feature is not directly or indirectly attached or affixed to a substrate, but is, for example, arranged within a closed or partially closed three-dimensional space (e.g., a well or pit).

[0416] In addition to the features described above, a variety of other features can be used to form the arrays described herein. For example, in some embodiments, features formed by inkjet printing, screen printing, or electrostatic deposition of polymers and / or biopolymers on a substrate can be used to form an array. For example, inkjet printing of biopolymers is described in PCT Patent Application Publication No. WO 2014 / 085725. For example, inkjet printing of polymers is described in de Gans et al., Adv Mater. 16(3): 203-213 (2004). Electrostatic deposition methods for polymers and biopolymers are described in, for example, Hoyer et al., Anal. Chem. 68(21): 3840-3844 (1996). The entire contents of each of the above references are incorporated herein by reference.

[0417] As another example, in some embodiments, features are formed from metal microparticles or nanoparticles. Suitable methods for depositing such particles to form arrays are described, for example, in Lee et al., Beilstein J. Nanotechnol. 8: 1049-1055 (2017), the entire contents of which are incorporated herein by reference.

[0418] As another example, in some embodiments, features are formed by magnetic particles assembled on a substrate. Examples of methods and particles for assembling such arrays are described in Ye et al., Scientific Reports 6: 23145 (2016), the entire contents of which are incorporated herein by reference.

[0419] As another example, in some embodiments, a feature corresponds to a region of a substrate into which one or more optical markers have been incorporated and / or which has been altered by a process such as permanent photobleaching. Suitable substrates for implementing features in this manner include, for example, a variety of polymers. Methods for forming such features are described, for example, in Moshrefzadeh et al., Appl. Phys. Lett. 62:16 (1993), the entire contents of which are incorporated herein by reference.

[0420] As another example, in some embodiments, the features may correspond to colloidal particles that assemble (e.g., via self-assembly) to form an array. Suitable colloidal particles are described, for example, in Sharma, Resonance 23(3): 263-275 (2018), the entire contents of which are incorporated herein by reference.

[0421] As another example, in some embodiments, features can be formed by photopolymerizing a point array of a monomer solution on a substrate. In particular, two-photon and three-photon polymerization can be used to produce features of relatively small (e.g., submicron) size. Suitable methods for preparing features on a substrate in this manner are described in Nguyen et al., Materials Today 20(6): 314-322 (2017), the entire contents of which are incorporated herein by reference.

[0422] In some embodiments, the features are directly or indirectly attached or affixed to a liquid permeable substrate. In some embodiments, the features are directly or indirectly attached or affixed to a biocompatible substrate. In some embodiments, the features are directly or indirectly attached or affixed to a substrate that is a hydrogel.

[0423] Figure 12 Depicted are exemplary arrangements of barcoded features in an array. From left to right, Figure 12 Shown are (L) a slide comprising six spatially barcoded arrays, (C) a magnified schematic diagram of one of the six spatially barcoded arrays showing a grid of barcoded features associated with a biological sample, and (R) a magnified schematic diagram of a portion of the array showing the specific identities of multiple features in the array (labeled as ID578, ID579, ID560, etc.).

[0424] As used herein, the term "bead array" refers to an array comprising a plurality of beads as features in an array. In some embodiments, the beads are attached to a substrate. For example, the beads can be optionally attached to a substrate such as a microscope slide and close to a biological sample (e.g., a tissue section comprising a cell). The beads can also be suspended in a solution and deposited on a surface (e.g., a film, a tissue section, or a substrate (e.g., a microscope slide)).

[0425] Examples of bead arrays on or within a substrate include beads positioned in wells, such as BeadChip arrays (available from Illumina Inc., San Diego, California), arrays used in the 454 LifeSciences (a subsidiary of Roche AG, Basel, Switzerland) sequencing platform, and arrays used in the Ion Torrent sequencing platform (a subsidiary of Life Technologies, Carlsbad, California). Examples of bead arrays are described, for example, in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, 6,210,891, 6,258,568, and 6,274,320; U.S. Patent Application Publication Nos. 2009 / 0026082; 2009 / 0127589; 2010 / 0137143; and 2010 / 0282617; and PCT Patent Application Publication Nos. WO 00 / 063437 and WO 2016 / 162309, the entire contents of which are incorporated herein by reference.

[0426] In some embodiments, a bead array comprises a plurality of beads. For example, a bead array can comprise at least 10,000 beads (e.g., at least 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads, at least 10,000,000 beads). In some embodiments, the plurality of beads comprises beads of a single type (e.g., substantially uniform in size, shape, and other physical properties, such as translucency). In some embodiments, the plurality of beads comprises two or more different types of beads.

[0427] In some embodiments, a bead array is formed when beads are embedded in a hydrogel layer, wherein the hydrogel polymerizes and fixes the relative bead positions. The bead array can be pre-equilibrated and combined with a reaction buffer and an enzyme (e.g., a reverse transcription mixture). In some embodiments, the bead array is frozen.

[0428] A "flexible array" comprises a plurality of spatial barcoding features attached to or embedded in a flexible substrate (e.g., a membrane or tape) placed on a biological sample. In some embodiments, a flexible array comprises a plurality of spatial barcoding features embedded in a hydrogel substrate. To form such an array, the features of the microarray are copied into a hydrogel, and the size of the hydrogel is reduced by removing water. These steps can be performed multiple times. For example, in some embodiments, a method for preparing a high-density spatial barcoding array may include copying a plurality of features from a microarray into a first hydrogel, wherein the first hydrogel is in contact with the microarray; reducing the size of the first hydrogel including the copied features by removing water to form a first contracted hydrogel including the copied features; copying the features in the first contracted hydrogel into a second hydrogel, wherein the second hydrogel is in contact with the first hydrogel; and reducing the size of the second hydrogel including the copied features by removing water to form a second contracted hydrogel including the copied features, thereby generating a high-density spatial barcoding array. The result is a high-density flexible array including spatial barcoding features.

[0429] In some embodiments, spatially barcoded beads can be loaded onto a substrate (eg, a hydrogel) to create high-density self-assembled bead arrays.

[0430] Flexible array can be pre-balanced, merges reaction buffer and functional concentration enzyme (for example, reverse transcription mixture).In some embodiments, flexible bead array can be stored for a long time (for example, several days) or frozen until standby.In some embodiments, the permeabilization of biological sample (for example, tissue section) can be carried out by adding enzyme / detergent before contacting with flexible array.Flexible array can be placed directly on sample, or indirectly contact with biological sample (for example, between biological sample and flexible bead array, there is an intermediate layer or material).In some embodiments, once flexible array is applied to sample, reverse transcription and targeted capture of analyte can be carried out on solid microspheres or round beads of the first size and the round beads of the second size.

[0431] A "microcapillary array" is a series of array features demarcated by microcapillaries. A "microcapillary channel" is a separate partition created by microcapillaries. For example, a microcapillary channel can be fluidically isolated from other microcapillary channels, such that the fluid or other contents of one microcapillary channel in the array are separated from the fluid or other contents of an adjacent microcapillary channel in the array. The density and arrangement of the microcapillaries can be any suitable density or arrangement of discrete sites.

[0432] In some embodiments, the microtubule array is processed to produce conditions that are conducive to loading. An example is to use a corona rod (BD-20AC, Electro Technic Products) to produce a hydrophilic surface. In some embodiments, features (e.g., beads attached to capture probes) are loaded onto the microtubule array so that the exact position of the features in the array is known. For example, a capture probe comprising a spatial barcode can be placed into a microtubule channel so that the spatial barcode can identify the position of the barcode sequence from which the barcoded nucleic acid molecule is derived.

[0433] In some embodiments, when using a random distribution to distribute features, empirical testing can be performed to generate loading / distribution conditions that favor a single feature per microcapillary channel. In some embodiments, it can be desirable to achieve distribution conditions that favor only a single feature per microcapillary channel (e.g., a bead). In some embodiments, it can be desirable to achieve distribution conditions that favor more than one feature per microcapillary channel (e.g., a bead) by flowing the feature through the microcapillary channel.

[0434] In some embodiments, the microcapillary array is placed in contact with the sample (e.g., on top or below) so that the microcapillaries containing features (e.g., beads that may include capture probes) are in contact with the biological sample. In some embodiments, the biological sample is placed on the exposed side of the microcapillary array and mechanical pressure is applied to move the biological sample into the microcapillary channels to create a fluid-isolated reaction chamber containing the biological sample.

[0435] In some embodiments, a biological sample is partitioned by contacting a microcapillary array with the biological sample, thereby creating a microcapillary channel comprising beads and a portion of the biological sample. In some embodiments, the portion of the biological sample contained in the microcapillary channel is one or more cells. In some embodiments, after the one or more cells are added to the microcapillary channel, features are introduced into the microcapillary array by flow.

[0436] In some embodiments, reagents are added to the microcapillary array. The added reagents may include enzyme reagents and reagent mixtures for nucleic acid amplification. In some embodiments, the reagents include reverse transcriptase, ligase, one or more nucleotides, and any combination thereof. After the reagents are added to the microcapillary channels, one or more microcapillary channels may be sealed, for example, using silicone oil, mineral oil, a non-porous material, or a cap.

[0437] In some embodiments, after incubation for a certain time and at a certain temperature or temperature range (e.g., after a hybridization or amplification reaction), the reagent solution is removed from each microcapillary channel. The reagent solutions can be processed separately for sequencing or combined for sequencing analysis.

[0438] In some embodiments, some or all features in an array comprise capture probes. In some embodiments, an array can comprise capture probes attached directly or indirectly to a substrate.

[0439] The capture probe comprises a capture domain (e.g., a nucleotide sequence) that can specifically bind (e.g., hybridize) to a target analyte (e.g., mRNA, DNA, or protein) within a sample. In some embodiments, the binding (e.g., hybridization) of the capture probe to the target can be detected and quantified by detecting a visual signal (e.g., a fluorophore, a heavy metal (e.g., silver ion), or a chemiluminescent marker) that has been incorporated into the target. In some embodiments, the intensity of the visual signal is related to the relative abundance of each analyte in the biological sample. Because an array can contain tens of thousands or millions of capture probes (or more), a feature array with capture probes can interrogate many analytes in parallel.

[0440] In some embodiments, the substrate includes one or more capture probes designed to capture analytes from one or more organisms. In a non-limiting example, the substrate may include one or more capture probes designed to capture mRNA from one organism (e.g., human) and one or more capture probes designed to capture DNA from a second organism (e.g., bacteria).

[0441] Capture probes can be attached to substrates or features using a variety of techniques. In some embodiments, capture probes are directly attached to features fixed on an array. In some embodiments, capture probes are fixed to a substrate by chemical fixation. For example, chemical fixation can occur between a functional group on the substrate and a corresponding functional element on the capture probe. Exemplary corresponding functional elements in the capture probe can be inherent chemical groups of the capture probe, such as hydroxyl groups, or functional elements can be introduced into the capture probe. An example of a functional group on the substrate is an amine group. In some embodiments, the capture probe to be fixed includes a functional amine group or is chemically modified to include a functional amine group. Means and methods for such chemical modification are well known in the art.

[0442] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end. In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe is immobilized on a feature by its 5′ end and comprises, from the 5′ end to the 3′ end: a barcode (e.g., spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs) and a capture domain.

[0443] In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and / or UMI), a second functional domain, and a capture domain. In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end and comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end and does not comprise a spatial barcode. In some embodiments, the capture probe is immobilized on a feature or substrate by its 5′ end and does not comprise a UMI. In some embodiments, the capture probe comprises a sequence for initiating a sequencing reaction.

[0444] In some embodiments, a capture probe is immobilized on a feature or substrate by its 3′ end. In some embodiments, a capture probe is immobilized on a feature or substrate by its 3′ end and comprises, from its 3′ end to its 5′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, a capture probe is immobilized on a feature or substrate by its 3′ end and comprises, from its 3′ end to its 5′ end: one barcode (e.g., spatial barcode or UMI) and one capture domain. In some embodiments, a capture probe is immobilized on a feature or substrate by its 3′ end and comprises, from its 3′ end to its 5′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, a capture probe is immobilized on a feature or substrate by its 3′ end and comprises, from its 3′ end to its 5′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.

[0445] The positioning of the functional groups within the capture probe to be fixed can be used to control and shape the binding behavior and / or orientation of the capture probe, for example, the functional group can be placed at the 5' or 3' end of the capture probe or within the sequence of the capture probe. In some embodiments, the capture probe can also include a substrate (e.g., a support attached to the capture probe, a support attached to a feature, or a support attached to a substrate). Typical substrates for the capture probe to be fixed include portions capable of binding to the capture probe (e.g., binding to amine-functionalized nucleic acids). Examples of such substrates are carboxyl, aldehyde, or epoxy supports.

[0446] In some embodiments, the substrate to which the capture probe can be immobilized can be chemically activated, for example, by activating available functional groups on the substrate. The term "activated substrate" relates to a material in which interactive or reactive chemical functional groups are established or enabled by a chemical modification procedure. For example, a substrate comprising carboxyl groups can be activated prior to use. In addition, some substrates contain functional groups that react with specific moieties already present in the capture probe.

[0447] In some embodiments, a covalent bond is used to couple the capture probe directly to the substrate. In some embodiments, the capture probe is indirectly coupled to the substrate by a joint (i.e., a chemical joint) that separates the "first" nucleotide of the capture probe from the substrate. In some embodiments, the capture probe is not directly bound to the array, but interacts indirectly, such as by being bound to a molecule that itself is directly or indirectly bound to the array. In some embodiments, the capture probe is indirectly attached to the substrate (e.g., via a solution comprising a polymer).

[0448] In some embodiments where the capture probe is indirectly immobilized on an array feature, for example by hybridization to a surface probe capable of binding the capture probe, the capture probe may further comprise an upstream sequence capable of hybridizing to the 5' end of the surface probe (in the 5' direction of a sequence that hybridizes to a nucleic acid, e.g., RNA from a tissue sample). Separately, the capture domain of a capture probe may be considered a capture domain oligonucleotide, which may be used in the synthesis of the capture probe in embodiments where the capture probe is indirectly immobilized on an array.

[0449] In some embodiments, the substrate is composed of an inert material or matrix (e.g., a glass slide) that has been functionalized, for example, by treating with a material comprising a reactive group capable of immobilizing the capture probe. For example, see WO 2017 / 019456, the entire contents of which are incorporated herein by reference. Non-limiting examples include polyacrylamide hydrogels supported on an inert substrate (e.g., a glass slide; see WO 2005 / 065814 and U.S. Patent Application No. 2008 / 0280773, the entire contents of which are incorporated herein by reference).

[0450] In some embodiments, a functionalized biomolecule (e.g., a capture probe) is immobilized on a functionalized substrate using a covalent method. Methods of covalent attachment include, for example, condensation of amines and activated carboxylates (e.g., N-hydroxysuccinimide esters); condensation of amines and aldehydes under reductive amination conditions; and cycloaddition reactions, such as Diels-Alder [4+2] reactions, 1,3-dipolar cycloaddition reactions, and [2+2] cycloaddition reactions. Methods of covalent attachment also include, for example, click chemistry reactions, including [3+2] cycloaddition reactions (e.g., Huisgen 1,3-dipolar cycloaddition reactions and copper (I)-catalyzed azide-alkyne cycloaddition reactions (CuAAC)); sulfhydryl-ene reactions; Diels-Alder reactions and reverse electron demand Diels-Alder reactions; [4+1] cycloadditions of isonitriles and tetrazines; nucleophilic ring opening of small carbocycles (e.g., epoxide opening of amino oligonucleotides). Methods of covalent attachment also include, for example, maleimide and thiol; and p-nitrophenyl ester-functionalized oligonucleotides and polylysine-functionalized substrates. Methods of covalent attachment also include, for example, disulfide bond reactions; free radical reactions (see, for example, U.S. Patent No. 5,919,626, the entire contents of which are incorporated herein by reference); and hydrazide-functionalized substrates (e.g., wherein the hydrazide functional group is directly or indirectly attached to the substrate) and aldehyde-functionalized oligonucleotides (see, for example, Yershov et al., (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, the entire contents of which are incorporated herein by reference).

[0451] In some embodiments, a functionalized biomolecule (e.g., a capture probe) is immobilized on a functionalized substrate using a photochemical covalent method. Methods for photochemical covalent attachment include, for example, immobilization of anthracene-conjugated oligonucleotides (see, e.g., Koch et al., (2000) Bioconjugate Chem. 11, 474-483, the entire contents of which are incorporated herein by reference).

[0452] In some embodiments, non-covalent methods are used to immobilize functionalized biomolecules (e.g., capture probes) on functionalized substrates. Methods for non-covalent attachment include, for example, biotin-functionalized oligonucleotides and streptavidin-treated substrates (see, e.g., et al., (1993) Analytical Biochemistry 209, 278-283 and Gilles et al., (1999) Nature Biotechnology 17, 365-370, the entire contents of which are incorporated herein by reference).

[0453] In some embodiments, oligonucleotides (e.g., capture probes) can be attached to a substrate or feature according to the methods described in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287, 5,807,522, 5,837,860, and 5,472,881; U.S. Patent Application Publication Nos. 2008 / 0280,773 and 2011 / 0059,865; Shalon et al. (1996) Genome Research, 639-645; Rogers et al. (1999) Analytical Biochemistry 266, 23-30; Stimpson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379-6383; Beattie et al., (1995) Clin. Chem. 45, 700-706; Lamture et al., (1994) Nucleic Acids Research 22, 2121-2125; Beier et al., (1999) Nucleic Acids Research 27, 1970-1977; Joos et al., (1997) Analytical Biochemistry 247, 96-101; Nikiforov et al., (1995) Analytical Biochemistry 227, 201-209; Timofeev et al., (1996) Nucleic Acids Research 24, 3142-3148; Chrisey et al., (1996) Nucleic Acids Research24, 3031-3039; Guo et al., (1994) Nucleic Acids Research Running and Urdea (1990) BioTechniques 8, 276-279; Fahy et al. (1993) Nucleic Acids Research 21, 1819-1826; Zhang et al. (1991) 19, 3929-3933; and Rogers et al. (1997) Gene Therapy 4, 1387-1392. The entire contents of each of the above documents are incorporated herein by reference.

[0454] Array can be prepared by a variety of methods. In some embodiments, by synthesizing (for example, in situ synthesis) oligonucleotides on the array or by inkjet printing or lithography to prepare the array. For example, the light-directed synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid phase DNA synthesis. In order to achieve photolithographic synthesis, a synthetic linker modified with a photochemical protecting group can be attached to a substrate, and a photolithographic mask (applied to a specific area of the substrate) and light can be used to modify the photochemical protecting group, thereby producing an array with a local photodeprotection effect. Many of these methods are known in the art, and are described in, for example, Miller et al. "Basic concepts of microarrays and potential applications in clinical microbiology", Clinical microbiology reviews 22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676, all of which are incorporated herein by reference.

[0455] In some embodiments, the array is "dotted" or "printed" with oligonucleotides, and then these oligonucleotides (e.g., capture probes) are attached to the substrate. Oligonucleotides can be applied by non-contact or contact printing. Non-contact printers can use the same method as computer printers (e.g., bubble jet or inkjet) to spray small droplets of probe solution onto substrates. This dedicated inkjet printer can spray oligonucleotide solution drops (instead of ink) of nanoliter to picoliter volume onto substrates. In contact printing, each printing needle directly applies the oligonucleotide solution to a specific position on the surface. Oligonucleotides can be attached to the substrate surface by the electrostatic interaction of the negative charge of the DNA phosphate backbone with the positively charged coating on the substrate surface or by the ultraviolet cross-linking covalent bond between the thymine base in the DNA and the amine group on the treated substrate surface. In some embodiments, the substrate is a glass slide. In some embodiments, oligonucleotides (e.g., capture probes) are attached to chemical substrates by covalent bonds, such as epoxy silane, aminosilane, lysine, polyacrylamide, etc.

[0456] These arrays can also be prepared using in situ synthesis methods. In some embodiments, these arrays can be prepared using photolithography. This method generally relies on UV masking on a substrate and photodirected combinatorial chemical synthesis to selectively synthesize probes directly on the array surface, one nucleotide at a time per site, simultaneously for multiple sites. In some embodiments, the substrate contains covalent linker molecules with photoremovable protecting groups on their free ends. UV light is directed through a photolithographic mask to deprotect and activate selected sites bearing hydroxyl groups, which initiates coupling with incoming protected nucleotides that attach to the activated sites. The mask is designed to select exposed sites, thereby specifying the coordinates on the array to which each nucleotide can attach. This process can be repeated, with a new mask applied to activate a different set of sites and couple a different base, allowing the construction of any oligonucleotide at each site. This process can be used to synthesize thousands of different oligonucleotides. In some embodiments, maskless array synthesizer technology can be used. This uses a programmable micromirror array to create a digital mask that reflects the desired UV pattern to remove protection from features.

[0457] In some embodiments, the inkjet spotting process can also be used for in situ oligonucleotide synthesis. Different nucleotide precursors plus catalysts can be printed onto a substrate, followed by coupling and deprotection steps. This method relies on printing picoliter volumes of nucleotides onto the array surface in repeated rounds of base-by-base printing, which extends the length of the oligonucleotide probes on the array.

[0458] Arrays can also be prepared by electric field active hybridization to control the transport of nucleic acids. When a positive current is applied to one or more test sites on the array, negatively charged nucleic acids can be transported to specific sites or features. The surface of the array can include binding molecules, such as streptavidin, which forms a bond (such as streptavidin-biotin bond) when the electronically addressed biotinylated probe arrives at its targeted position. The positive current is then removed from the activated feature, and a new test site can be activated by the targeted application of a positive current. Repeat this process until all sites on the array are covered.

[0459] Arrays for spatial analysis can be generated by various methods as described herein. In some embodiments, the array has a plurality of capture probes comprising spatial barcodes. These spatial barcodes and their relationship to the position on the array can be determined. In some cases, such information is easy to obtain because oligonucleotides are spotted, printed or synthesized on the array in a predetermined pattern. In some cases, the spatial barcode can be decoded by methods described herein (e.g., by in situ sequencing, by various labels associated with the spatial barcode, etc.). In some embodiments, an array can be used as a template to generate a subarray. Therefore, the spatial barcode can be transferred to a subarray with a known pattern.

[0460] In some embodiments, the array of the probe comprising barcode can be produced by connecting a plurality of oligonucleotides.In some instances, the plurality of oligonucleotides comprise the part of barcode, and generate complete barcode when connecting the plurality of oligonucleotides.For example, the first oligonucleotide comprising the first portion of barcode can be attached to substrate (for example, using any method oligonucleotide is attached to substrate as described herein), then, the second oligonucleotide comprising the second portion of barcode can be connected to the first oligonucleotide to generate complete barcode.The first, second and any other part of barcode can be used for increasing the diversity of barcode.In the example that the second oligonucleotide is also attached to substrate before connecting, the first and / or second oligonucleotide can be attached to substrate via the surface joint comprising cracking site.After connecting, by cracking the oligonucleotide linearization connected at cracking site.

[0461] In order to increase the diversity of the barcode, a plurality of second oligonucleotides comprising two or more different barcode sequences can be connected to a plurality of first oligonucleotides comprising the same barcode sequence, thereby generating two or more different types of barcodes. In order to achieve selective connection, the first oligonucleotide attached to the substrate comprising the first portion of the barcode can be initially protected with a protecting group (e.g., a photocleavable protecting group), and the protecting group can be removed before connecting between the first oligonucleotide and the second oligonucleotide. In the case where the barcoded probes on the array are generated by connecting two or more oligonucleotides, a concentration gradient of the oligonucleotides can be applied to the substrate so that different combinations of oligonucleotides are incorporated into the barcoded probes according to their positions on the substrate.

[0462] Barcode probes on an array can also be generated by adding single nucleotides to existing oligonucleotides on the array, for example, using a polymerase that functions in a template-independent manner. Single nucleotides can be added to existing oligonucleotides in a concentration gradient, thereby generating probes of varying lengths depending on the probe's location on the array.

[0463] Arrays can also be prepared by modifying existing arrays, for example, by modifying the oligonucleotides attached to the array. For example, probes can be generated on an array comprising oligonucleotides, and oligonucleotides are attached to the array at 3 ' ends and have free 5 ' ends. The oligonucleotides can be oligonucleotides synthesized in situ, and can include barcodes. The length of the oligonucleotides can be less than 50 nucleotides (nt) (for example, less than 45, 40, 35, 30, 25, 20, 15 or 10 nt). In order to produce probes using these oligonucleotides, primers (for example, constant sequences shared by oligonucleotides) can be used that are complementary to the portion of the oligonucleotides and hybridize with the oligonucleotides and extend (using oligonucleotides as templates) to form duplexes and produce 3 ' overhangs. Therefore, 3 ' overhangs make other nucleotides or oligonucleotides be added to the duplex. Capture probes can be produced (for example, by a connection mediated by a splint oligonucleotide) by, for example, adding one or more oligonucleotides to the end of the 3 ' overhang, wherein the oligonucleotides added can include the sequence of the capture domain or the part of the sequence.

[0464] In the case where the oligonucleotides on the existing array include recognition sequences that can hybridize to the splint oligonucleotides, probes can also be generated by directly attaching other oligonucleotides to the existing oligonucleotides via the splint oligonucleotides. The recognition sequence can be located at the free 5' end or the free 3' end of the oligonucleotides on the existing array. The recognition sequence useful for the method of the present invention may not contain restriction enzyme recognition sites or secondary structures (e.g., hairpins) and may include a high content of guanine and cytosine nucleotides, thereby having high stability.

[0465] Bead arrays can be generated by attaching beads (e.g., barcoded beads) to a substrate in a regular pattern or irregular arrangement. Beads can be attached to selective regions on a substrate, for example, by selectively activating regions on the substrate to allow attachment of the beads. Activating the selective regions on the substrate can include activating a coating (e.g., a photocleavable coating) or polymer applied to the substrate. Beads can be attached iteratively, for example, a subset of beads can be attached at a time, and the same process can be repeated to attach the remaining beads. Alternatively, all beads can be attached to the substrate in one step.

[0466] Barcoded beads or beads comprising a plurality of barcoded probes can be generated by first preparing a plurality of barcoded probes on a substrate, depositing a plurality of beads on the substrate, and using the probes on the substrate as a template to generate probes attached to the beads.

[0467] Large-scale commercial manufacturing methods allow millions of oligonucleotides to be attached to an array. Commercial arrays include those from Roche NimbleGen (Wisconsin) and Affymetrix (Thermo Fisher Scientific).

[0468] In some embodiments, the array can be prepared according to the methods described in WO 2012 / 140224, WO 2014 / 060483, WO 2016 / 162309, WO 2017 / 019456, WO 2018 / 091676, and WO 2012 / 140224, and U.S. Patent Application No. 2018 / 0245142. The entire contents of the above documents are incorporated herein by reference.

[0469] In some embodiments, the features on the array include beads. In some embodiments, two or more beads are dispersed on a substrate to create an array, wherein each bead is a feature on the array. The beads can optionally be dispersed into wells on the substrate, for example, such that each well only accommodates a single bead.

[0470] A "bead" is a particle. A bead can be porous, non-porous, solid, semi-solid, and / or combinations thereof. In some embodiments, the bead is dissolvable, breakable, and / or degradable, while in certain embodiments, the bead is non-degradable.

[0471] The beads can generally be any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, rectangular, amorphous, circular, cylindrical, and variations thereof. A cross section (e.g., a first cross section) can correspond to the diameter or maximum cross-sectional dimension of the bead. In some embodiments, the bead can be approximately spherical. In these embodiments, the first cross section can correspond to the diameter of the bead. In some embodiments, the bead can be approximately cylindrical. In such embodiments, the first cross section can correspond to the diameter, length, or width of the approximately cylindrical bead.

[0472] Beads can be of uniform or non-uniform size. "Polydispersity" generally refers to the heterogeneity of molecular or particle size. The polydispersity index (PDI) of beads can be calculated using the equation PDI=Mw / Mn, where Mw is the weight-average molar mass and Mn is the number-average molar mass. In certain embodiments, beads can be provided as a plurality of beads or bead populations having a relatively monodisperse size distribution. When it is desired to provide a relatively consistent amount of reagent, maintaining relatively consistent bead characteristics (e.g., size) contributes to overall consistency.

[0473] In some embodiments, the beads provided herein can have a size distribution having a coefficient of variation in cross-sectional dimension of less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or less. In some embodiments, the plurality of beads provided herein have a polydispersity index of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less.

[0474] In some embodiments, the diameter or maximum dimension of the beads may be no greater than 100 μm (e.g., no greater than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm).

[0475] In some embodiments, the plurality of beads has an average diameter of no greater than 100 μm. In some embodiments, the plurality of beads has an average diameter or largest dimension of no greater than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm.

[0476] In some embodiments, the volume of the beads may be at least about 1 μm 3 , for example at least 1 μm 3 , 2μm 3 , 3μm 3 , 4μm 3 , 5μm 3 , 6μm 3 , 7μm 3 , 8μm 3 , 9μm 3 , 10μm 3 , 12μm 3 , 14μm 3 , 16μm 3 , 18μm 3 , 20μm 3 , 25μm 3 , 30μm 3 , 35μm 3 , 40μm 3 , 45μm 3 , 50μm 3 , 55μm 3 , 60μm 3 , 65μm 3 , 70μm 3 , 75μm 3 , 80μm 3 , 85μm 3 , 90μm 3 , 95μm3 , 100μm 3 , 125μm 3 , 150μm 3 , 175μm 3 , 200μm 3 , 250μm 3 , 300μm 3 , 350μm 3 , 400μm 3 , 450μm 3 、μm 3 , 500μm 3 , 550μm 3 , 600μm 3 , 650μm 3 , 700μm 3 , 750μm 3 , 800μm 3 , 850μm 3 , 900μm 3 , 950μm 3 , 1000μm 3 , 1200μm 3 , 1400μm 3 , 1600μm 3 , 1800μm 3 , 2000μm 3 , 2200μm 3 , 2400μm 3 , 2600μm 3 , 2800μm 3 , 3000μm 3 or larger.

[0477] In some embodiments, the beads may have a diameter of about 1 μm. 3 with 100μm 3 The volume between, for example, about 1 μm 3 and 10μm 3 Between, about 10μm 3 With 50μm 3 Between or about 50μm 3 with 100μm 3 In some embodiments, the beads may comprise approximately 100 μm 3 with 1000μm 3 The volume between, for example, about 100 μm 3 With 500μm 3 Between or about 500μm 3 with 1000μm 3 In some embodiments, the beads may comprise about 1000 μm3 with 3000μm 3 The volume between, for example, about 1000 μm 3 with 2000μm 3 Between or about 2000μm 3 with 3000μm 3 In some embodiments, the beads may comprise about 1 μm 3 with 3000μm 3 The volume between, for example, about 1 μm 3 with 2000μm 3 Between, about 1μm 3 with 1000μm 3 Between, about 1μm 3 With 500μm 3 Between or about 1μm 3 and 250μm 3 The volume between.

[0478] The beads may comprise one or more cross-sections that are the same or different. In some embodiments, the beads may have a first cross-section that is different from a second cross-section. The beads may have a first cross-section that is at least about 0.0001 microns, 0.001 microns, 0.01 microns, 0.1 microns, or 1 micron. In some embodiments, the beads may comprise at least about 1 micron (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80μm, 85μm, 90μm, 100μm, 120μm, 140μm, 160μm, 180μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1 millimeter (mm) or larger cross section (e.g., a first cross section). In some embodiments, the beads may comprise a cross-section (e.g., a first cross-section) between about 1 μm and 500 μm, such as between about 1 μm and 100 μm, between about 100 μm and 200 μm, between about 200 μm and 300 μm, between about 300 μm and 400 μm, or between about 400 μm and 500 μm. For example, the beads may comprise a cross-section (e.g., a first cross-section) between about 1 μm and 100 μm. In some embodiments, the beads may have a second cross-section of at least about 1 μm. For example, beads may include at least about 1 micron (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm , 16μm, 17μm, 18μm, 19μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm , 80μm, 85μm, 90μm, 100μm, 120μm, 140μm, 160μm, 180μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 millimeter (mm) or larger second cross-section.In some embodiments, the beads may comprise a second cross-section between about 1 μm and 500 μm, e.g., between about 1 μm and 100 μm, between about 100 μm and 200 μm, between about 200 μm and 300 μm, between about 300 μm and 400 μm, or between about 400 μm and 500 μm. For example, the beads may comprise a second cross-section between about 1 μm and 100 μm.

[0479] In some embodiments, the beads can be nanoscale (e.g., the beads can have a diameter or maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm)) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less). The plurality of beads can have an average diameter or average maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less). In some embodiments, the diameter or size of the beads is about the size of a single cell (e.g., a single cell being assessed).

[0480] In some embodiments, the beads can be gel beads. A "gel" is a semi-rigid material that is permeable to liquids and gases. Exemplary gels include, but are not limited to, gels having colloidal structures, such as agarose; polymeric network structures, such as gelatin; hydrogels; and cross-linked polymer structures, such as polyacrylamide, SFA (e.g., see U.S. Patent Application Publication No. 2011 / 0059865, incorporated herein by reference in its entirety), and PAZAM (e.g., see U.S. Patent Application Publication No. 2014 / 0079923, incorporated herein by reference in its entirety).

[0481] Depending on the intended use, gel can be configured to various shapes and sizes. In some embodiments, gel is prepared and configured to gel beads (e.g., gel beads comprising capture probes attached to or associated with the gel beads). The gel beads can be hydrogel beads. The hydrogel beads can be formed by molecular precursors, such as polymers or monomeric substances.

[0482] In some embodiments, the hydrogel beads 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 repeating units). Cross-linking may be achieved through covalent, ionic, and / or induced interactions and / or physical entanglement.

[0483] The semi-solid beads may be liposomal beads.

[0484] Solid beads can include metals, including but not limited to iron oxide, gold, and silver. In some embodiments, the beads can be silica beads. In some embodiments, the beads can be rigid. In some embodiments, the beads can be flexible and / or compressible.

[0485] Beads can be macromolecules. Beads can be formed from bound nucleic acid molecules. Beads can be formed by covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. The polymer or monomer can be natural or synthetic. The polymer or monomer can be or include, for example, a nucleic acid molecule (e.g., DNA or RNA).

[0486] The beads can be rigid, or can be flexible and / or compressible. The beads can include a coating comprising one or more polymers. Such a coating can be destroyed or dissolved. In some embodiments, the beads include a spectral or optical marker (e.g., a dye) attached directly or indirectly (e.g., via a joint) to the beads. For example, the beads can be prepared as colored preparations (e.g., beads showing different colors in the visible spectrum) that can change color (e.g., colorimetric beads) to form beads of different colors (e.g., opaque and / or transparent beads) when a desired stimulus (e.g., heat and / or chemical reaction) is applied.

[0487] The beads may comprise natural and / or synthetic materials. For example, the beads may comprise natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include, but are not limited to, proteins, sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ipagua gum, gum arabic, agar, gelatin, shellac, karaya gum, xanthan gum, corn gum, guar gum, karaya gum, agarose, alginic acid, sodium alginate, or natural polymers thereof. Examples of synthetic polymers include, but are not limited to, acrylics, resins, nylons, silicones, spandex, viscose rayon, polycarboxylates, polyvinyl acetates, polyacrylamides, polyacrylates, polyethylene glycols, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, polychlorotrifluoroethylene, polyethylene oxide, polyethylene terephthalate, polyethylene, polyisobutylene, polymethyl methacrylate, polyoxymethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polyvinyl fluoride, and / or combinations thereof (e.g., copolymers). Beads can also be formed from materials other than polymers, including, for example, lipids, micelles, ceramics, glass-ceramics, composites, metals, and / or other inorganic materials.

[0488] In some embodiments, the beads are degradable beads. Degradable beads may include one or more substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.) with labile bonds, such that when the beads / substances are exposed to an appropriate stimulus, the labile bonds are broken and the beads degrade. The labile bonds may be chemical bonds (e.g., covalent bonds, ionic bonds) or may be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.). In some embodiments, the cross-linking agent used to generate the beads may include labile bonds. When exposed to appropriate conditions, the labile bonds may break and the beads may degrade. For example, when polyacrylamide gel beads containing the cysteamine cross-linker are exposed to a reducing agent, the disulfide bonds of the cysteamine may be broken and the beads may degrade.

[0489] Degradation can refer to the dissociation of bound or entrained materials (e.g., disulfide linkers, primers, other oligonucleotides, etc.) from the beads, regardless of whether the physical beads themselves are structurally degraded. For example, due to changes in the chemical environment, entrained materials can be released from the beads by the permeabilization pressure difference. For example, changes in the pore size of the beads due to the permeabilization pressure difference can generally occur without structural degradation of the beads themselves. In some embodiments, the increase in pore size due to the permeabilization swelling of the beads can release materials entrained within the beads. In some embodiments, the permeabilization shrinkage of the beads due to pore size shrinkage can cause the beads to better retain entrained materials.

[0490] Any suitable reagent of degradable pearl can be used. In some embodiments, the change of temperature or pH value can be used for the heat-sensitive or pH-sensitive bond in the degradation pearl. In some embodiments, chemical degradation agent can be used for the chemical bond in the degradation pearl by oxidation, reduction or other chemical changes. For example, chemical degradation agent can be a reducing agent, such as DTT, wherein DTT can degrade the disulfide bond formed between the cross-linking agent and the gel precursor, thereby degrading the pearl. In some embodiments, a reducing agent can be added to degrade the pearl, which can cause the pearl to release its content. The example of reducing agent can include but is not limited to dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (disulfide butylamine or DTBA), tris (2-carboxyethyl) phosphine (TCEP) or its combination.

[0491] Any chemical agent can be used to initiate degradation of the beads. Examples of chemical agents include, but are not limited to, pH-mediated changes in the integrity of components within the beads, degradation of bead components by cleavage of cross-links, and disaggregation of bead components.

[0492] In some embodiments, the beads can be formed from a material that includes a degradable chemical crosslinker, such as N,N'-bis-(acryloyl)cystamine (BAC) or cystamine. Degradation of such a degradable crosslinker can be achieved by various mechanisms. In some instances, the beads can be contacted with a chemical degradation agent that can cause oxidation, reduction, or other chemical changes. For example, the chemical degradation agent can be a reducing agent, such as dithiothreitol (DTT). Other examples of reducing agents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.

[0493] In some embodiments, exposure to an aqueous solution (e.g., water) can trigger hydrolytic degradation, thereby initiating degradation of the beads. Application of a thermal stimulus can also induce the beads to release their contents. Changes in temperature can cause various changes in the beads. For example, heat can liquefy solid beads. Changes in heat can cause the beads to melt, partially degrading the beads. In some embodiments, heat can increase the internal pressure of the bead components, causing the beads to rupture or explode. Heat can also act on the thermosensitive polymers used to construct the beads.

[0494] In the case of using degradable beads, it is beneficial to avoid exposing the beads to one or more stimuli that cause the degradation before a given time, so as to avoid premature degradation of the beads and problems caused by such degradation, including, for example, poor flow properties and aggregation. For example, in the case of beads including reducible cross-linking groups (e.g., disulfide groups), it is desirable to avoid contacting the beads with reducing agents (e.g., DTT or other disulfide cleavage reagents). In these embodiments, in some embodiments, the treatment of the beads described herein will not contain reducing agents, such as DTT. Since reducing agents are generally provided in commercial enzyme preparations, it is desirable to provide an enzyme preparation that does not contain reducing agents (or does not contain DTT) when treating the beads described herein. Examples of such enzymes include, for example, polymerase preparations, reverse transcriptase preparations, ligase preparations, and many other enzyme preparations that can be used to treat the beads described herein. The term "reducing agent-free" or "DTT-free" preparation refers to a preparation having a lower range of about 1 / 10, about 1 / 50, or about 1 / 100 of the material used to degrade the beads. For example, for DTT, a reducing agent-free formulation can have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001 mM DTT. In some embodiments, the amount of DTT can be undetectable.

[0495] Compared to non-degradable beads, when appropriate stimulation is applied to the beads, degradable beads can be used to release the attached capture probes (e.g., nucleic acid molecules, spatial barcode sequences and / or primers) from the beads more quickly. For example, for substances bound to the inner surface of porous beads or in the case of encapsulated substances, when the beads degrade, the substance can have greater mobility and accessibility to other substances in the solution. In some embodiments, substances can also be attached to degradable beads via degradable linkers (e.g., disulfide linkers). The degradable linkers can respond to the same stimulation as the degradable beads, or the two degradable substances can respond to different stimulations. For example, capture probes with one or more spatial barcodes can be attached to polyacrylamide beads including cystamine by disulfide bonds. When spatial barcoded beads are exposed to a reducing agent, the beads degrade and release capture probes with one or more spatial barcode sequences when the disulfide bonds between the capture probe and the beads and the disulfide bonds of cystamine in the beads are broken.

[0496] Adding multiple types of unstable bonds to beads can produce beads that can respond to various stimuli. Each type of unstable bond can be sensitive to relevant stimuli (e.g., chemical stimuli, light, temperature, pH, enzymes, etc.) so that the release of the reagent attached to the beads by each unstable bond can be controlled by applying appropriate stimulation. Some non-limiting examples of unstable bonds that can be coupled to precursors or beads include ester bonds (e.g., cleavage with acid, alkali, or hydroxylamine), vicinal diol bonds (e.g., cleavage by sodium periodate), Diels-Alder bonds (e.g., cleavage by thermal cracking), sulfone bonds (e.g., cleavage by alkali), silyl ether bonds (e.g., cleavage by acid), glycosidic bonds (e.g., cleavage by amylase), peptide bonds (e.g., cleavage by protease), or phosphodiester bonds (e.g., cleavage by nuclease (e.g., DNA enzyme)). The bond can be targeted by other nucleic acid molecules, such as restriction enzymes (e.g., restriction endonucleases) for cleavage. This functionality can be used to control the release of reagents from beads. In some embodiments, another reagent comprising an unstable bond can be connected to the beads via, for example, the activated functional groups of the above-mentioned beads after the gel beads are formed. In some embodiments, the gel beads comprising unstable bonds are reversible. In some embodiments, gel beads having reversible unstable bonds are used to capture one or more regions of interest of a biological sample. For example, but not limited to, beads comprising heat-labile bonds can be heated by a light source (e.g., a laser), which causes changes in the gel beads that contribute to capturing biological samples in contact with the gel beads. Capture probes having one or more releasable, cleavable, or reversibly attached spatial barcodes of beads described herein include capture probes that are released or releasable by the cleavage of the connection between the capture probe and the beads, or capture probes that are released by the degradation of the underlying beads themselves, so that the capture probes having one or more spatial barcodes are to be touched by other reagents or become touchable by other reagents, or both.

[0497] Beads can have different physical properties. The physical properties of beads can be used to characterize beads. Non-limiting examples of the physical properties of beads include size, shape, roundness, density, symmetry and hardness. For example, beads can have different sizes. By using a microfluidic channel network configured to provide beads of a specific size (for example, based on channel size, flow rate, etc.), beads of different sizes can be obtained. In some embodiments, the beads have different hardness values, which can be obtained by changing the polymer concentration used to generate the beads. In some embodiments, the physical properties of the capture probe can be used to make the spatial barcode attached to the beads optically detectable. For example, nucleic acid origami, such as deoxyribonucleic acid (DNA) origami, can be used to generate optically detectable spatial barcodes. To this end, a nucleic acid molecule or multiple nucleic acid molecules can be folded to form two-dimensional and / or three-dimensional geometric shapes. Different geometric shapes can be detected optically.

[0498] In some embodiments, special types of nanoparticles having one or more different physical properties can be used to make the beads physically distinguishable. For example, Janus particles having hydrophilic and hydrophobic surfaces can be used to provide unique physical properties.

[0499] In some embodiments, the beads are capable of identifying multiple analytes (e.g., nucleic acids, proteins, chromatin, metabolites, drugs, gRNAs, and lipids) from a single cell. In some embodiments, the beads are capable of identifying a single analyte (e.g., mRNA) from a single cell.

[0500] The pore size of the beads can be adjusted. For example, the pore size can be selected to retain denatured nucleic acids. The pore size can be selected to maintain diffusion permeability to exogenous chemicals such as sodium hydroxide (NaOH) and / or endogenous chemicals such as inhibitors. The beads can be formed from biocompatible and / or biochemically compatible materials and / or materials that maintain or enhance cell viability. The beads can be formed from materials that can be thermally, chemically, enzymatically, and / or optically depolymerized.

[0501] In some embodiments, beads can be non-covalently loaded with one or more agents. Bead...

Claims

1. A non-diagnostic method for spatial analysis of genomic DNA present in a biological sample, the method comprising: providing an array comprising a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises a spatial barcode and a capture domain; permeabilizing the biological sample under conditions sufficient to cause transposon insertion into genomic DNA in the biological sample, wherein the permeabilization of the biological sample is performed under enzymatic permeabilization conditions, wherein the enzymatic permeabilization conditions comprise contacting the biological sample with pepsin, collagenase, or proteinase K; or contacting the biological sample with both collagenase and proteinase K; providing a transposon sequence and a transposase to the biological sample under conditions wherein the transposon sequence is inserted into genomic DNA; allowing the transposase to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA; contacting a biological sample comprising fragmented genomic DNA with the array under conditions wherein the capture probes interact with the fragmented genomic DNA; and The positions of the capture probes on the array are correlated with positions in the biological sample, allowing spatial analysis of fragmented genomic DNA.

2. The method of claim 1, wherein an array comprising a plurality of capture probes is provided on a substrate.

3. The method of any one of claims 1-2, wherein the capture probe is attached directly or indirectly.

4. The method of claim 1, wherein the array comprising a plurality of capture probes is provided on a feature on a substrate, said feature comprising beads, spots of any two-dimensional or three-dimensional geometry, wells, and hydrogel pads.

5. The method of claim 2, wherein the substrate comprises a microfluidic channel.

6. The method of claim 1, wherein the capture probe further comprises one or more of a cleavage domain, a functional domain, and a unique identifier, or a combination thereof.

7. The method of claim 1, further comprising a migration step, wherein the fragmented genomic DNA is migrated to a substrate.

8. The method of claim 7, wherein the migration step is an active migration step comprising applying an electric field to the fragmented genomic DNA.

9. The method of claim 7, wherein the migration step is a passive migration step comprising diffusion.

10. The method of any one of claims 7-9, wherein migrating fragmented genomic DNA from a biological sample comprises exposing the biological sample and features to heat, the features comprising beads, spots of any two-dimensional or three-dimensional geometry, wells, and hydrogel pads.

11. The method of claim 2, wherein the biological sample is immobilized on the substrate.

12. The method of claim 1 , wherein the transposase is a dimer comprising a first monomer complexed with a first adaptor comprising a transposon end sequence and a sequence complementary to a capture domain, wherein a second monomer complexed with a second adaptor comprising a transposon end sequence and a second adaptor sequence, wherein the transposase ligates the first adaptor and the second adaptor to the fragmented genomic DNA. The method of claim 12 , wherein the first adaptor and the second adaptor have a 5′ end and a 3′ end, wherein the 5′ end is phosphorylated in situ.

14. The method of claim 12 or 13, wherein the 5' end of the first adaptor complexed with the first monomer and the 5' end of the second adaptor complexed with the second monomer are phosphorylated before fragmenting the DNA.

15. The method of claim 13, wherein the step of phosphorylating the 5' end of the first adaptor complexed with the first monomer and the 5' end of the second adaptor complexed with the second monomer comprises contacting the first monomer-first adaptor complex and the second monomer-second adaptor complex with a polynucleotide kinase in the presence of ATP.

16. The method of claim 1, wherein the capture domain of the capture probe comprises a sequence that hybridizes to another sequence that is complementary to the capture domain of the first adaptor.

17. The method of claim 12, wherein the capture probe is a partially double-stranded molecule comprising a first strand comprising a capture domain hybridized to a second strand, and wherein the first strand serves as a template for ligating the first adaptor to the second strand.

18. The method of claim 12, wherein the first adaptor sequence that hybridizes to the capture probe and is complementary to the capture domain or a portion thereof serves as a template for ligating and joining the 5' end of the first adaptor to the 3' end of the capture probe.

19. The method of claim 1, wherein the capture probe comprises a surface probe and a splint oligonucleotide, and wherein the splint oligonucleotide comprises a sequence that is complementary to the hybridization domain of the surface probe or a portion thereof.

20. The method of claim 19, wherein the splint oligonucleotide comprises a capture domain having a sequence complementary to the first adaptor or a portion thereof.

21. The method of claim 19 or 20, wherein the splint oligonucleotide hybridizes to the first adaptor or a portion thereof and to the hybridization domain of the surface probe or a portion thereof.

22. The method of claim 19, wherein ligation is performed in the presence of a splint oligonucleotide, thereby ligating the surface probe of the capture probe and the first adaptor.

23. The method of claim 12, wherein the fragmented genomic DNA hybridized to the capture probe via the first adaptor is an extension template for generating an extended capture probe comprising a sequence of a spatial barcode and a sequence complementary to the fragmented genomic DNA.

24. The method of claim 23, wherein the capture probes hybridized to the fragmented genomic DNA are extended using a DNA polymerase. The method of claim 24 , wherein the DNA polymerase has strand displacement activity.

26. The method of claim 1, further comprising gap repairing single-strand breaks in the fragmented genomic DNA.

27. The method of claim 12, wherein the sequence complementary to the capture probe is a unique sequence.

28. The method of claim 1, wherein the capture probe is ligated to the fragmented genomic DNA by DNA ligase.

29. The method of claim 1, wherein the transposase is Tn5 transposase or a functional derivative thereof.

30. The method of claim 29, wherein the Tn5 transposase comprises a sequence at least 80% identical to SEQ ID NO:

1.

31. The method of claim 1, wherein the transposase is Mu transposase or a functional derivative thereof.

32. The method of claim 31 , wherein the Mu transposase comprises a sequence at least 80% identical to SEQ ID NO:

2.

33. The method of claim 12, wherein the transposon end sequence comprises a sequence that is at least 80% identical to SEQ ID NO:

8.

34. The method of claim 12, wherein the transposon end sequence comprises a sequence at least 80% identical to any one of SEQ ID NOs: 9-14.

35. The method of claim 1, wherein the enzymatic permeabilization conditions comprise contacting the biological sample with an acidic solution comprising an enzyme.

36. The method of claim 35, wherein the pepsin comprises a sequence at least 80% identical to SEQ ID NO: 3 or 4.

37. The method of any one of claims 35-36, wherein the enzymatic permeabilization conditions comprise contacting the biological sample with: (i) collagenase; (ii) proteinase K; or (iii) Both.

38. The method of claim 37, wherein the collagenase comprises a sequence that is at least 80% identical to SEQ ID NO: 5 or 6.

39. The method of claim 37, wherein the proteinase K enzyme comprises a sequence at least 80% identical to SEQ ID NO:

7.

40. The method of claim 24 or 25, wherein the DNA molecules are generated using fragmented genomic DNA hybridized to the capture probes as an extension template.

41. The method of claim 17 or 18, wherein the DNA molecules are generated using fragmented genomic DNA hybridized to the capture probes as a ligation template.

42. The method of claim 40, comprising the step of analyzing DNA molecules.

43. The method of claim 42, wherein the step of analyzing the DNA molecules comprises sequencing.

44. The method of claim 1, wherein a spatial barcode of the capture probe is associated with the fragmented genomic DNA to spatially analyze the fragmented genomic DNA, the fragmented genomic DNA being associated with the capture probe.

45. The method of claim 1, further comprising a step wherein the biological sample is imaged either before or after contacting the biological sample with a substrate.

46. A kit for use in a non-diagnostic method for spatial analysis of nucleic acids in a biological sample as defined in any one of claims 1 to 45, wherein the kit comprises: (i) an array having a plurality of capture probes thereon; (ii) one or more biological sample permeabilization reagents, wherein the permeabilization reagents comprise pepsin, collagenase, or proteinase K; or both collagenase and proteinase K; (iii) one or more transposases; (iv) one or more reverse transcriptases; and (v) one or more lytic enzymes.

47. A non-diagnostic method for spatial analysis of genomic DNA and RNA present in a biological sample, the method comprising: Providing an array, wherein the array comprises a plurality of capture probes, wherein a first capture probe in the plurality of capture probes comprises a spatial barcode and a first capture domain, and wherein a second capture probe in the plurality of capture probes comprises a spatial barcode and a second capture domain; permeabilizing the biological sample under conditions sufficient to cause transposon insertion into genomic DNA in the biological sample, wherein the permeabilization of the biological sample is performed under enzymatic permeabilization conditions, wherein the enzymatic permeabilization conditions comprise contacting the biological sample with pepsin, collagenase, or proteinase K; or contacting the biological sample with both collagenase and proteinase K; providing a transposon sequence and a transposase to the biological sample under conditions wherein the transposon sequence is inserted into genomic DNA; allowing the transposase to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA; contacting a biological sample comprising fragmented genomic DNA and RNA with the array under conditions wherein the first capture domain interacts with the fragmented genomic DNA and the second capture domain interacts with the RNA; and The position of the first capture probe on the array is correlated with a position in the biological sample, and the position of the second capture probe on the array is correlated with a position in the biological sample, thereby spatially analyzing fragmented genomic DNA and RNA at positions in the biological sample.

48. The method of claim 47, wherein the RNA is mRNA.

49. The method of claim 47 or 48, wherein the first capture domain and the second capture domain are identical.

50. The method of claim 49, wherein the first capture domain and the second capture domain comprise homopolymeric poly(T) sequences.

51. The method of claim 47, wherein the first capture domain and the second capture domain are different.

52. The method of claim 51, wherein the first capture domain comprises a random sequence and the second capture domain comprises a poly(T) sequence.

53. The method of claim 47, wherein an array comprising a plurality of capture probes is provided on a substrate.

54. The method of claim 47, wherein the array comprising a plurality of capture probes is provided on a feature comprising beads, spots of any two-dimensional or three-dimensional geometry, wells, and hydrogel pads.

55. The method of claim 54, wherein the feature comprises a first capture probe, a second capture probe, or both.

56. The method of claim 47, wherein the first capture probe, the second capture probe, or both are attached directly or indirectly.

57. The method of claim 47, wherein the array comprising a plurality of capture probes is provided on a feature on a substrate, said feature comprising beads, spots of any two-dimensional or three-dimensional geometry, wells, and hydrogel pads.

58. The method of claim 53, wherein the substrate comprises a microfluidic channel.

59. The method of claim 47, wherein the first capture probe, the second capture probe, or both comprise one or more of a cleavage domain, a functional domain, and a unique identifier, or a combination thereof.

60. The method of claim 47, comprising a migration step, wherein the fragmented genomic DNA and RNA migrate to a substrate.

61. The method of claim 60, wherein the migrating step is an active migrating step.

62. The method of claim 60, wherein the migrating step is a passive migrating step.

63. The method of any one of claims 60-62, wherein migrating the fragmented genomic DNA and RNA from the biological sample comprises exposing the biological sample to heat.

64. The method of claim 53, wherein the biological sample is immobilized on the substrate.

65. The method of claim 47, wherein the fragmented genomic DNA is repaired by ligating the breaks with a ligase.

66. The method of claim 47, further comprising gap repairing single-strand breaks in the fragmented genomic DNA.

67. The method of claim 47, wherein a sequence complementary to the first capture domain of the first capture probe is introduced to the fragmented genomic DNA.

68. The method of claim 47, wherein the first capture domain of the first capture probe hybridizes to a sequence complementary to the capture domain that is introduced to the fragmented genomic DNA.

69. The method of claim 53, wherein the random sequence of the first capture domain hybridizes to fragmented genomic DNA.

70. The method of claim 47, wherein the second capture domain of the second capture probe hybridizes to a complementary sequence in the mRNA.

71. The method of claim 67, wherein the sequence complementary to the first capture domain and the complementary sequence in the mRNA are homopolymeric sequences.

72. The method of claim 71, wherein the homopolymeric sequence is a poly(A) sequence.

73. The method of claim 47, comprising extending the first capture probe using fragmented genomic DNA as an extension template, and extending the second capture probe using RNA as an extension template.

74. The method of claim 73, wherein the first capture probe is extended using a DNA polymerase.

75. The method of claim 74, wherein the second capture probe is extended using reverse transcriptase.

76. The method of claim 47, wherein the transposase is Tn5 transposase or a functional derivative thereof.

77. The method of claim 76, wherein the Tn5 transposase comprises a sequence at least 80% identical to SEQ ID NO:

1.

78. The method of claim 47, wherein the transposase is Mu transposase or a functional derivative thereof.

79. The method of claim 78, wherein the Mu transposase comprises a sequence at least 80% identical to SEQ ID NO:

2.

80. The method of claim 47, wherein the transposase is complexed with an adaptor comprising a transposon end sequence.

81. The method of claim 80, wherein the transposon end sequence comprises a sequence that is at least 80% identical to SEQ ID NO:

8.

82. The method of claim 80, wherein the transposon end sequence comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 9-14.

83. The method of claim 47, wherein the enzymatic permeabilization conditions comprise contacting the biological sample with an acidic solution comprising an enzyme.

84. The method of claim 83, wherein the pepsin comprises a sequence at least 80% identical to SEQ ID NO: 3 or 4.

85. The method of any one of claims 82-84, wherein the enzymatic permeabilization conditions comprise contacting the biological sample with: (i) collagenase; (ii) proteinase K; or (iii) Both.

86. The method of claim 85, wherein the collagenase comprises a sequence at least 80% identical to SEQ ID NO: 5 or 6.

87. The method of claim 85, wherein the proteinase K enzyme comprises a sequence at least 80% identical to SEQ ID NO:

7.

88. The method of claim 47, wherein the step of analyzing the DNA molecules comprises sequencing.

89. The method of claim 47, wherein the step of associating the spatial barcode of the first capture probe with the fragmented genomic DNA spatially analyzes the fragmented genomic DNA, wherein the fragmented genomic DNA is associated with the first capture probe.

90. The method of claim 47, wherein the spatial barcode of the second capture probe is associated with the mRNA associated with the second capture probe to spatially analyze the mRNA.

91. The method of claim 47, further comprising a step wherein the biological sample is imaged before or after contacting the biological sample with a substrate.

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