Reusable surface chemistry for spatial substrates

Abstract

The present disclosure provides substrates, compositions, and methods for re-generating and reusing a substrate for spatially tagging a target molecule in a sample.

Description

REUSABLE SURFACE CHEMISTRY FOR SPATIAL SUBSTRATES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No.63 / 739,259, filed December 27, 2024, which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (File Name:4213_005PC01_SequenceListing_ST26; Size: 4,783 bytes; Date of Creation: December 17, 2025) filed with the application is herein incorporated by reference in its entirety.BACKGROUND

[0003] Substrates designed for spatial transcriptomics are typically surfaces with bound nucleic acid capture oligonucleotides on the surface of a substrate (e.g., a flowcell, array, slide, etc.) which become chemically modified during a spatial genomics assay. These modifications currently preclude the reuse of a substrate for multiple rounds of sample processing, necessitating the manufacture of a fresh substrate for every run.

[0004] Reusing a spatial substrate is particularly desirable since each substrate has a subsequent “map” associated with it. When a user needs a new substrate (whether that is a flowcell, array, or slide) it also needs a new accompanying map with that substrate in order to trace back the spatial information. If a user can reuse a substrate, it is convenient from a materials perspective and also a process perspective as the manufacturer does not have to create a new map. Therefore, there exists a need in the art for reusable spatial substrates.BRIEF SUMMARY

[0005] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of thenucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation: a spatial barcode sequence, a restriction enzyme recognition sequence, and a capture sequence; contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe; extending the capture sequence to produce an extended nucleic acid probe; contacting the substrate with an oligonucleotide that is complementary to the restriction enzyme recognition sequence under conditions wherein the oligonucleotide hybridizes to the restriction enzyme recognition sequence of the nucleic acid probe; contacting the substrate with a restriction enzyme, wherein the restriction enzyme cleaves the extended nucleic acid probe; contacting the substrate with a regeneration oligonucleotide; and extending the nucleic acid probe against the regeneration probe.

[0006] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe and a second nucleic acid probe comprises a spatial barcode probe, wherein the capture probe comprises, in a 5' to 3' orientation: a restriction enzyme recognition sequence, and a capture sequence, and wherein the spatial barcode probe comprises, in a 5' to 3' orientation: a spatial barcode sequence, and a template switch oligonucleotide (TSO), contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe and extending the capture sequence to produce an extended nucleic acid probe comprising first strand cDNA; contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence; extending the capture probe from the free 3’ end using the spatial barcode probe as template to generate an extended capture probe; contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe; contacting the substrate with an oligonucleotide that is complementary to the restriction enzyme recognition sequence under conditions wherein the oligonucleotide hybridizes to the restriction enzyme recognition sequence of the capture probe, and contacting the substratewith a restriction enzyme, wherein the restriction enzyme cleaves the extended capture probe; contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the capture probe, wherein the regeneration oligonucleotide comprises a sequence that is complementary to the restriction enzyme recognition sequence, and a sequence that is complementary to the capture sequence; and extending the nucleic acid probe against the regeneration oligonucleotide.

[0007] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe and a second nucleic acid probe comprises a spatial barcode probe, wherein the capture probe comprises, in a 5' to 3' orientation: a first restriction enzyme recognition sequence, and a capture sequence, and wherein the spatial barcode probe comprises, in a 5' to 3' orientation: a spatial barcode sequence, a second restriction enzyme recognition sequence, and a template switch oligonucleotide (TSO), contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe and extending the capture sequence to produce an extended nucleic acid probe comprising first strand cDNA; contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence; extending the capture probe from the free 3 ’ end using the spatial barcode probe as template to generate an extended capture probe; extending the spatial barcode from the free 3’ end using the capture probe as a template to generate an extended spatial barcode probe; contacting the substrate with a first primer oligonucleotide under conditions wherein the first primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized first primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe; contacting the substrate with a second primer oligonucleotide under conditions wherein the second primer oligonucleotide hybridizes to the extended spatial barcode probe, and extending the hybridized second primer oligonucleotide to generate an oligonucleotide that is complementary to the extended spatial barcode probe; contacting the substrate with a first oligonucleotide that is complementary to the first restriction enzyme recognition sequence under conditionswherein the first oligonucleotide hybridizes to the first restriction enzyme recognition sequence of the capture probe, and contacting the substrate with a first restriction enzyme, wherein the first restriction enzyme cleaves the extended capture probe at the first restriction enzyme recognition sequence; contacting the substrate with a second oligonucleotide that is complementary to the second restriction enzyme recognition sequence under conditions wherein the second oligonucleotide hybridizes to the second restriction enzyme recognition sequence of the spatial barcode probe, and contacting the substrate with a second restriction enzyme, wherein the second restriction enzyme cleaves the extended spatial barcode probe at the second restriction enzyme recognition sequence; contacting the substrate with a first regeneration oligonucleotide under conditions wherein the first regeneration oligonucleotide hybridizes to the capture probe, wherein the first regeneration oligonucleotide comprises a sequence that is complementary to the first restriction enzyme recognition sequence, and a sequence that is complementary to the capture sequence, and extending the capture probe using the first regeneration oligonucleotide as a template; and contacting the substrate with a second regeneration oligonucleotide under conditions wherein the second regeneration oligonucleotide hybridizes to the spatial barcode probe, wherein the second regeneration oligonucleotide comprises a sequence that is complementary to the second restriction enzyme recognition sequence, and a sequence that is complementary to the TSO sequence, and extending the spatial barcode probe using the second regeneration oligonucleotide as a template.

[0008] In some aspects, a spatially tagged second strand cDNA is generated from the extended nucleic acid probe, and wherein the spatially tagged second strand cDNA is dehybridized from the extended nucleic acid probe sequenced. In some aspects, spatially tagged second strand cDNA is generated using a template switching oligonucleotide (TSO) approach or a random priming approach.

[0009] In some aspects, the nucleic acid probe further comprises a sequencing by synthesis (SBS) sequence. In some aspects, the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI). In some aspects, the SBS sequence is SBS 12 or a complement thereof (SBS 12'). In some aspects, the SBS sequence is SBS3 or a complement thereof (SBS3'). In some aspects, the nucleic acid probe further comprises a flowcell clustering sequence, and wherein the flowcell clustering sequence is selected from the group consisting of P5, P5', P7, P7'. In some aspects, the spatial barcode probe is blocked at the 3’ end.

[0010] In some aspects, the first regeneration oligonucleotide comprises a sequence that is complementary to the first restriction enzyme recognition sequence and a sequence that is complementary to the capture sequence, and the second regeneration oligonucleotide comprises a sequence that is complementary to the second restriction enzyme recognition sequence and a sequence that is complementary to the TSO. In some aspects, first and second oligonucleotides are removed prior to contacting the substrate with the first and second regeneration oligonucleotides. In some aspects, the oligonucleotide that is complementary to the restriction enzyme recognition sequence is removed prior to contacting the substrate with the regeneration oligonucleotide.

[0011] In some aspects, the capture probe and the spatial barcode probe further comprise a primer sequence. In some aspects, the spatial barcode probe further comprises a primer sequence and a UMI sequence.

[0012] In some aspects, the substrate is a solid surface, glass slide, flowcell, an array, or beads. In some aspects, the nucleic acid probes are arranged in specific locations on the substrate. In some aspects, the spatial barcode sequence is correlated with a positional location on the substrate. In some aspects, the plurality of nucleic acid probes comprises a plurality of clusters of nucleic acid probes, wherein the nucleic acid probes of each cluster comprise a unique spatial barcode. In some aspects, the spatial barcode sequence spatially tags the nucleic acid probe.

[0013] In some aspects, the capture sequence comprises a poly thymidine (polyT) sequence. In some aspects, the capture sequence comprises a target-specific capture sequence, a randomer, or a semi-randomer.

[0014] In some aspects, the method further comprises contacting the target molecule with an enzyme to degrade the target molecule. In some aspects, the method further comprises removing the target molecule by dehybridization or by contacting the target molecule with NaOH.

[0015] In some aspects, the restriction enzyme is a rare cutter enzyme, a type II restriction enzyme, or a frequent cutter enzyme. In some aspects, the spatial location of the target molecule is determined.

[0016] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of thenucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation: a spatial barcode, and a capture sequence; contacting a sample with the nucleic acid probes on the substrate under conditions wherein the target molecules from the sample hybridize to the capture sequences of the nucleic acid probes; extending the nucleic acid probe to produce an extended nucleic acid probe that comprises first strand cDNA, or portions thereof, and the spatial barcode sequences, thereby spatially tagging the target nucleic acids of the biological sample, providing a blocking oligonucleotide that is complementary to the capture sequence and contacting the substrate with a blocking oligonucleotide under conditions wherein the blocking oligonucleotide hybridizes to the capture sequence, and contacting the target nucleic acids with an exonuclease.

[0017] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a spatial barcode, and a capture sequence, and wherein a second acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises in a 5' to 3' orientation: a spatial barcode sequence, and a TSO sequence; contacting a sample with the nucleic acid probes on the substrate under conditions wherein the target molecules from the sample hybridize to the capture sequences of the nucleic acid probes; extending the capture probe to produce a capture probe that comprises first strand cDNA, or portions thereof; contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence and extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA and a spatial barcode sequence complement (SBC’); contacting the substrate with a first blocking oligonucleotide under conditions wherein the first blocking oligonucleotide hybridizes to the capture sequence of the capture probe, contacting the substrate with a second blocking oligonucleotide under conditions wherein the second blocking oligonucleotide hybridizes to the TSO of the spatial barcode probe, and contacting the target nucleic acids with an exonuclease.

[0018] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a spatial barcode, and a capture sequence, and wherein a second acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises in a 5' to 3' orientation: a spatial barcode sequence, and a TSO sequence; contacting a sample with the nucleic acid probes on the substrate under conditions wherein the target molecules from the sample hybridize to the capture sequences of the nucleic acid probes; extending the capture probe to produce a capture probe that comprises first strand cDNA, or portions thereof; contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence and extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA and a spatial barcode sequence complement (SBC’); extending the spatial barcode from the free 3’ end using the capture probe as a template to generate an extended spatial barcode probe comprising second strand cDNA and the spatial barcode sequence; contacting the substrate with a first blocking oligonucleotide under conditions wherein the first blocking oligonucleotide hybridizes to the capture sequence of the capture probe, contacting the substrate with a second blocking oligonucleotide under conditions wherein the second blocking oligonucleotide hybridizes to the TSO of the spatial barcode probe, and contacting the target nucleic acids with an exonuclease.

[0019] In some aspects, a spatially tagged second strand cDNA is generated from the extended nucleic acid probe, and wherein the spatially tagged second strand cDNA is dehybridized from the extended nucleic acid probe and sequenced. In some aspects, the spatially tagged second strand cDNA is generated using a template switching oligonucleotide (TSO) approach or a random priming approach.

[0020] In some aspects, the nucleic acid probe further comprises a sequencing by synthesis (SBS) sequence. In some aspects, the SBS sequence is SBS12, or a complement thereof (SBS12'). In some aspects, the SBS sequence is SBS3 or a complement thereof (SBS3'). In some aspects, the nucleic acid probe further comprises a flowcell clustering sequence. Insome aspects, the flowcell clustering sequence is selected from the group consisting of P5, P5', P7, P7'. In some aspects, the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

[0021] In some aspects, the method further comprises contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe, wherein the complementary copy of the extended capture probe comprises second strand cDNA and the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended capture probe is dehybridized from the extended capture probe and sequenced.

[0022] In some aspects, the method further comprises contacting the substrate with a first primer oligonucleotide under conditions wherein the first primer oligonucleotide hybridizes to the extended capture probe and extending the hybridized first primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe, wherein the complementary copy of the extended capture probe comprises second strand cDNA and the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended capture probe is dehybridized from the extended capture probe and sequenced, and contacting the substrate with a second primer oligonucleotide under conditions wherein the second primer oligonucleotide hybridizes to the extended capture probe and extending the hybridized second primer oligonucleotide to generate an oligonucleotide that is complementary to the extended spatial barcode probe, wherein the complementary copy of the extended spatial barcode probe comprises first strand cDNA and the spatial barcode sequence complement, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended spatial barcode probe is dehybridized from the extended spatial barcode probe and sequenced.

[0023] In some aspects, the capture probe further comprises a primer sequence. In some aspects, the spatial barcode probe further comprises a primer sequence and a UMI sequence. In some aspects, the first strand cDNA nucleic acid sequence that is complementary to the target molecule that is hybridized to the capture sequence, or a portion thereof. In some aspects, the substrate is a solid surface, glass slide, flowcell, an array, or a bead. In some aspects, the substrate comprises a plurality of nucleic acid probes,and wherein the nucleic acid probes are arranged in specific locations on the substrate. In some aspects, the spatial barcode correlates to a positional location on the substrate. In some aspects, the plurality of nucleic acid probes comprises a plurality of clusters of nucleic acid probes, wherein the nucleic acid probes of each cluster comprise a unique spatial barcode. In some aspects, the spatial barcode spatially tags the nucleic acid probe.

[0024] In some aspects, the capture sequence comprises a poly-thymidine (polyT) sequence. In some aspects, the capture sequence comprises a target-specific capture sequence, a randomer, or a semi-randomer.

[0025] In some aspects, the exonuclease is exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, or a polymerase. In some aspects, the spatial location of the target molecule is determined.

[0026] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: grafting a first moiety to a surface of the substrate, binding a second moiety to the first moiety, wherein the second moiety comprises a capture sequence and a spatial barcode sequence, binding a target nucleic molecule to the capture sequence of the second moiety and generating first strand cDNA, decoupling the second moiety from the first moiety thereby regenerating the substrate.

[0027] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: grafting a first moiety to a surface of the substrate, attaching primer sequences to the surface of the substrate, binding a second moiety to the first moiety, wherein the second moiety comprises a capture sequence and a spatial barcode sequence, binding a target nucleic molecule to the capture sequence of the second moiety and generating first strand cDNA, decoupling the second moiety from the first moiety thereby regenerating the substrate.

[0028] In some aspects, the method further comprises clustering the spatial barcode and linearizing. The method of any one of claims 53-55, wherein the first moiety comprises alkyne-PEG-biotin. In some aspects, the alkyne-PEG-biotin is grafted to the azides of a polymer coating the surface of the substrate. In some aspects, the second moiety comprises 5’ biotinylated P5 and P7 oligonucleotides coupled to streptavidin. In some aspects, a reagent is used to decouple the second moiety from the first moiety. In some aspects, the reagent comprises hot formamide.

[0029] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation: a nuclease recognition sequence, an adapter sequence, a spatial barcode sequence, and a capture sequence, contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture sequence of the nucleic acid probe, extending the nucleic acid probe using the target molecule as a template to produce an extended nucleic acid probe comprising the nucleic acid probe and a first strand cDNA, contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the capture sequence of the nucleic acid probe, and extending the regeneration oligonucleotide using the nucleic acid probe as a template to generate an oligonucleotide that is complementary to the nucleic acid probe, contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the extended nucleic acid probe, producing a first portion of the extended nucleic acid probe and a second portion of the extended nucleic acid probe, wherein the first portion of the extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the nucleic acid probe comprises the adapter sequence, the spatial barcode sequence, the capture sequence, and the first strand cDNA, and extending the first portion of the nucleic acid probe from the free 3’ end using the regeneration oligonucleotide as a template, thereby regenerating the nucleic acid probe.

[0030] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation: a spatial barcode sequence, a nuclease recognition sequence, and optionally a linker, contacting the nucleic acid probes with a customizable oligonucleotide under conditions wherein the customizable oligonucleotide hybridizes to the capture sequence of the nucleic acid probe, extending the nucleic acid probe using the customizable oligonucleotide as a template to produce a custom nucleic acid probe, contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes tothe custom nucleic acid probe, extending the custom nucleic acid probe using the target molecule as a template to produce an extended custom nucleic acid probe comprising the nucleic acid probe and a first strand cDNA, contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the extended custom nucleic acid probe, contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the extended custom nucleic acid probe, producing a first portion of the extended custom nucleic acid probe and a second portion of the extended custom nucleic acid probe, wherein the first portion of the extended custom nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the extended custom nucleic acid probe comprises the first strand cDNA, and extending the first portion of the nucleic acid probe from the free 3’ end using the regeneration oligonucleotide as a template, thereby regenerating the nucleic acid probe.

[0031] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation: a first nuclease recognition sequence, a first adapter sequence, a spatial barcode sequence, a second nuclease recognition sequence, and a capture sequence, contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe, extending the nucleic acid probe using the target molecule as a template to produce a first extended nucleic acid probe comprising a first strand cDNA, contacting the substrate with a first oligonucleotide and extending the first oligonucleotide using the first extended nucleic acid probe as a template to generate an extended first oligonucleotide, wherein: the extended first oligonucleotide is complementary to the first extended nucleic acid probe, the extended first oligonucleotide and the first extended nucleic acid probe form a double-stranded oligonucleotide, and the extended first oligonucleotide comprises a second strand cDNA, contacting the substrate with a first nuclease that is specific to the first nuclease recognition sequence, wherein the first nuclease cleaves the first extended nucleic acid probe comprised in the doublestranded oligonucleotide, producing a first portion of the first extended nucleic acid probe and a second portion of the first extended nucleic acid probe, wherein the first portion of the first extended nucleic acid probe is immobilized on the substrate at the 5’ end, andwherein the second portion of the first extended nucleic acid probe comprises the first adapter sequence, the spatial barcode, the capture sequence, and the first strand cDNA, extending the first portion of the first extended nucleic acid probe from the free 3’ end to generate a second extended nucleic acid probe, using the extended first oligonucleotide as a template, contacting the substrate with a second nuclease that is specific to the second nuclease recognition sequence, wherein the second nuclease cleaves the second extended nucleic acid probe, producing a first portion of the second extended nucleic acid probe and a second portion of the second extended nucleic acid probe, wherein the first portion of the second extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the second extended nucleic acid probe contains the first strand cDNA, removing the extended first oligonucleotide from the first portion of the second extended nucleic acid probe, contacting the substrate with a second oligonucleotide under conditions wherein the second oligonucleotide hybridizes to the first portion of the second extended nucleic acid probe, and extending the first portion of the second extended nucleic acid probe from the free 3’ end using the second oligonucleotide as a template, thereby regenerating the nucleic acid probe.

[0032] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation: a nuclease recognition sequence, a sequencing-by-synthesis (SBS) sequence, a spatial barcode sequence, and a capture sequence contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture sequence of the nucleic acid probe, extending the nucleic acid probe using the target molecule as a template to produce an extended nucleic acid probe comprising a first strand cDNA, contacting the substrate with a oligonucleotide under conditions wherein the oligonucleotide hybridizes to the extended nucleic acid probe, contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the extended nucleic acid probe, producing a first portion of the extended nucleic acid probe and a second portion of the extended nucleic acid probe, wherein the first portion of the extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the extended nucleic acid probe contains the first strand cDNA, extending the first portionof the extended nucleic acid probe from the free 3’ end using the oligonucleotide as a template, thereby regenerating the nucleic acid probe, contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the nucleic acid probe, producing a first portion of the nucleic acid probe and a second portion of the nucleic acid probe, wherein the first portion of the nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion comprises the SBS sequence, the spatial barcode sequence, and the capture sequence of the nucleic acid probe, and extending the first portion of the nucleic acid probe from the free 3’ end, using the oligonucleotide as a template to regenerate the nucleic acid probe.

[0033] In some aspects, the present disclosure provides a method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising: providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a nuclease enzyme recognition site, and a capture sequence, wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode, and a template switch oligonucleotide (TSO) sequence, wherein the spatial barcode probe is 3’ blocked such that extension of the spatial barcode probe is prevented; contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture sequence of the capture probe and extending the capture sequence to comprise first strand cDNA using the target molecule as a template; extending the capture probe using the hybridized target molecule as a template to generate an extended capture probe comprising first strand cDNA; hybridizing a template switch oligonucleotide complement (TSO’) to the capture probe; hybridizing the TSO’ sequence and the TSO sequence and extending the capture probe using the TSO sequence as template to generate an extended capture probe that comprises first strand cDNA and a sequence that is complementary to the spatial barcode sequence (SBC’); contacting the substrate with a nuclease that hybridizes to the nuclease recognition site of the extended capture probe; contacting the substrate with a nuclease, wherein the nuclease cleaves the extended capture probe at the nuclease recognition sequence to generate a first portion of the capture probe and a second portion of the capture probe; contacting the substrate with a regeneration oligonucleotide under conditions wherein the regenerationoligonucleotide hybridizes to the first portion capture probe, wherein the regeneration oligonucleotide comprises a sequence that is complementary to at least a portion of the nuclease recognition sequence and a sequence that is complementary to the capture sequence; and extending the first portion of the capture probe against the template to thereby regenerate the capture probe.

[0034] In some aspects, the nucleic acid probe further comprises a sequencing-by-synthesis (SBS) sequence. In some aspects, the SBS sequence is SBS12 or a complement thereof (SBS12'). In some aspects, the SBS sequence is SBS3 or a complement thereof (SBS3'). In some aspects, the nucleic acid probe further comprises a flow cell clustering sequence. In some aspects, the flow cell clustering sequence is selected from the group consisting of P5, P5', P7, P7'. In some aspects, the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

[0035] In some aspects, customizable oligonucleotide comprises: a sequence that is complementary to the nuclease recognition sequence, a linker, and a sequence that is complementary to a capture sequence. In some aspects, the customizable oligonucleotide further comprises an identifier sequence, wherein the identifier sequence is located between the capture sequence and the linker.

[0036] In some aspects, extending the first portion of the extended custom nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the extended custom nucleic acid probe. In some aspects, the regeneration oligonucleotide is not bound to the surface of the substrate. In some aspects, the regeneration oligonucleotide is removed. In some aspects, after the second portion of the extended custom nucleic acid probe is collected, the second portion of the extended custom nucleic acid probe is amplified and sequenced. In some aspects, the first oligonucleotide comprises a randomer and optionally a second adapter sequence.

[0037] In some aspects, the first nuclease specifically targets the first nuclease recognition sequence, and the second nuclease specifically targets the second nuclease recognition sequence. In some aspects, the second nuclease also cleaves the extended first oligonucleotide. In some aspects, extending the first portion of the first nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the first nucleic acid probe. In some aspects, extending the first portion of the second nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the second nucleic acid probe.

[0038] In some aspects, the first or second oligonucleotide is not bound to the surface of the substrate. In some aspects, the second oligonucleotide is removed. In some aspects, the nucleic acid probe further comprises a sequencing-by-synthesis (SBS) sequence. In some aspects, the SBS sequence is SBS12 or a complement thereof (SBS12'). In some aspects, the SBS sequence is SB S3 or a complement thereof (SB S3'). In some aspects, nucleic acid probe further comprises a flow cell clustering sequence. In some aspects, the flow cell clustering sequence is selected from the group consisting of P5, P5', P7, P7'. In some aspects, the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

[0039] In some aspects, the oligonucleotide comprises: a nucleic acid sequence that is complementary to the nuclease recognition sequence, a nucleic acid sequence that is complementary to the SBS sequence a nucleic acid sequence that is complementary to the spatial barcode sequence, and a nucleic acid sequence that is complementary to the capture sequence.

[0040] In some aspects, extending the first portion of the nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the nucleic acid probe. In some aspects, the second portion of the nucleic acid probe is collected.

[0041] In some aspects, extending the first portion of the extended nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the extended nucleic acid probe. In some aspects, the second portion of the extended nucleic acid probe is collected, and sequenced. In some aspects, extending the first portion of capture probe from the free 3’ end results in the displacement of at least a portion of the second portion of capture probe. In some aspects, the regeneration oligonucleotide is removed.

[0042] In some aspects, after the second portion of the capture probe is collected, the second portion of the capture probe is amplified and sequenced. In some aspects, the capture probe further comprises an adapter primer sequence. In some aspects, the spatial barcode probe further comprises a primer sequence and a UMI sequence.

[0043] In some aspects, the method further comprises determining the spatial location of the target molecule. In some aspects, the method further comprises contacting the substrate with a single-stranded DNA-specific 3 ’-5’ exonuclease before reusing the substrate.

[0044] In some aspects, the exonuclease is a Thermus thermophilus exonuclease. In some aspects, the nuclease is a Cas nickase or a Cas nickase variant. In some aspects, the Casnickase is Cas9 nickase (Cas9n), Streptococcus pyogenes Cas9 nickase (spCas9n), Streptococcus pyogenes Cas9 High Fidelity nickase (spCas9HFn), Staphylococcus aureus Cas9 nickase (SaCas9n), Staphylococcus aureus Cas9 High Fidelity nickase (SaCas9HFn), Casl2a nickase (Casl2an) or a variant thereof. In some aspects, the Cas9 nickase is used in conjunction with a tracerRNA:crRNA or a single-guide RNA. In some aspects, the nuclease is a nicking endonuclease. In some aspects, the nicking endonuclease is Nt.BstNBI, Nt.BstSEI, Nt.BspQI, Nt.BbvCI, Nt.AlwI, Nb.BsrDI, Nb.BsmI, Nt.CviPII, Nb.BtsI, Nb.BbvCI, Nb.BssSI, or Nt.BsmAI. In some aspects, the nuclease is a homing endonuclease modified to have nickase activity. In some aspects, the homing endonuclease modified to have nickase activity is an I-Scel nickase, an I-Anil nickase, or an I-Dmol nickase. In some aspects, the nuclease is a chimeric nickase.

[0045] The method of claim 111, wherein the chimeric nickase comprises a DNA binding molecule fused to a DNA nicking domain. In some aspects, the nuclease is a Zinc finger nickase. In some aspects, the nuclease is a transcription activator-like effector (TALE) nickase. In some aspects, the TALE nickase is an engineered TALE nickase comprising a TALE repeat domain fused with a FokI nuclease domain or a MutH nicking variant. In some aspects, the nuclease is a rare-cutter nickase. In some aspects, the nuclease is a uracil or Oxo-G dependent repair enzyme. In some aspects, the nuclease is USER, EndoQ, FPG, or OGG.

[0046] In some aspects, the substrate is a solid surface. In some aspects, the solid surface is planar. In some aspects, the substrate is a glass slide, a flow cell, an array, or beads. In some aspects, the nucleic acid probes are arranged in specific locations on the substrate. In some aspects, the spatial barcode correlates to a positional location on the substrate. In some aspects, the plurality of nucleic acid probes comprise a plurality of clusters of nucleic acid probes, wherein the nucleic acid probes of each cluster comprise a unique spatial barcode. In some aspects, the spatial barcode spatially tags the nucleic acid probe.

[0047] In some aspects, the capture sequence comprises a poly-thymidine (poly-T) sequence or a target-specific capture sequence. In some aspects, the capture sequence comprises a randomer or a semi-randomer.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a schematic diagram illustrating a method to regenerate nucleic acid probes on the surface of a substrate using a restriction enzyme.

[0049] Figure 2 is a schematic diagram illustrating a method to regenerate nucleic acid probes on the surface of a substrate using an exonuclease.

[0050] Figure 3 is a series of schematic diagrams illustrating a method to regenerate nucleic acid probes on the surface of a substrate using a dual oligonucleotide approach and a restriction enzyme. Figure 3 A shows mRNA captured on the capture probe and first strand cDNA synthesis by reverse transcriptase. Figure 3B shows template switching by hybridizing the 3’ end of the capture probe to the 3’ end of the spatial barcode probe using a template switch oligonucleotide (“TSO”) that was added to the 3’ end of the capture probe. Figure 3C shows the generation of second strand cDNA. Figure 3D shows the removal and amplification of second strand cDNA. Figure 3E shows a restriction enzyme cutting the extended capture probe. Figure 3F shows hybridizing the regeneration oligonucleotide to the cut capture probe to regenerate the capture probe. Figure 3G shows the regenerated capture probe and regenerated spatial barcode oligonucleotide.

[0051] Figure 4 is a series of schematic diagrams illustrating a method to regenerate nucleic acid probes on the surface of a substrate using a dual oligonucleotide approach and a restriction enzyme. Figure 4A shows mRNA captured on the capture probe and first strand cDNA synthesis by reverse transcriptase. Figure 4B shows template switching by hybridizing the 3’ end of the capture probe to the 3’ end of the spatial barcode probe using a template switch oligonucleotide (“TSO”) that was added to the 3’ end of the capture probe. Figure 4C shows the generation of second strand cDNA of both the capture probe and the spatial barcode probe. Figure 4D shows the removal and amplification of second strand cDNA molecules. Figure 4E shows restriction enzymes cutting the extended capture probe and the extended spatial barcode probe. Figure 4F shows hybridizing the regeneration oligonucleotides to the cut capture probe and the cut spatial barcode probe to regenerate the capture probe and the spatial barcode probe. Figure 4G shows the regenerated capture probe and regenerated spatial barcode oligonucleotide.

[0052] Figure 5 is a series of schematic diagrams illustrating a method to regenerate nucleic acid probes on the surface of a substrate using a dual oligonucleotide approach and an exonuclease. Figure 5A shows that after second strand cDNA has been generated, removedand amplified, complementary blocker oligonucleotides were added to the capture probe and the spatial barcode probe and an exonuclease was added. Figure 5B shows the regeneration of the spatial barcode probe and capture probe.

[0053] Figure 6 is a series of schematic diagrams illustrating a method to regenerate nucleic acid probes on the surface of a substrate using a dual oligonucleotide approach and an exonuclease. Figure 6A shows that after second strand cDNA molecules have been generated, removed and amplified, complementary blocker oligonucleotides were added to the capture probe and the spatial barcode probe and an exonuclease was added. Figure 6B shows the regeneration of the spatial barcode probe and capture probe.

[0054] Figure 7 is a schematic diagram illustrating a method to prevent degradation of unbound capture probes and to prevent second strand synthesis using a complementary blocking oligonucleotide.

[0055] Figure 8 is a schematic diagram illustrating a blocking oligonucleotide bound to a capture probe.

[0056] Figure 9 illustrates a flow of example operations for using and reusing a flowcell, and example structures formed using such operations.

[0057] Figure 10 illustrates a method for flowcell reuse with biotin / streptavidin complexes.

[0058] Figure 11 is a graph showing the natural run-to-run contamination without extra reagents of reusable flowcells.

[0059] Figure 12 is a series of graphs illustrates a method for substrate regeneration using biotin / streptavidin complexes. Figure 12A illustrates grafting alk-PEG-biotin and P5 / Gz to the surface of the substrate, clustering the spatial barcodes and linearizing, and binding the biotin / streptavidin complexes to the alk-PEG-biotin. Figure 12B shows first strand cDNA generation and removing the biotin / streptavidin complexes to regenerate the substrate. Figure 12C shows binding new biotin / streptavidin complexes to start the process again

[0060] Figure 13 illustrates a method for substrate regeneration that involves generating 1st strand cDNA for processing.

[0061] Figures 14A, 14B, and 14C illustrate a method for substrate regeneration using customizable oligonucleotides to capture a target molecule and generating 1st strand cDNA for processing.

[0062] Figures 15A and 15B illustrate a method for substrate regeneration that involves amplifying 1st strand cDNA for processing and generating 2nd strand cDNA.

[0063] Figures 16A, 16B, 16C, and 16D illustrate a method for substrate regeneration using customizable oligonucleotides and amplifying 2nd strand cDNA for processing.

[0064] Figure 17 illustrates a method for flow cell regeneration using a long oligonucleotide to regenerate the substrate, and generating 1st strand cDNA for processing.

[0065] Figure 18 illustrates a method for flow cell regeneration using a long oligonucleotide to regenerate the substrate, generating 1st stand cDNA for processing, and generating a pool of long oligonucleotides to use for flow cell regeneration.

[0066] Figure 19 illustrates a method for substrate regeneration using two oligonucleotide probes (e.g., a capture probe and a spatial barcode probe) and a nuclease. Figure 19A illustrates mRNA capture by the capture probe, first strand cDNA synthesis, and extension of the capture probe. Figure 19B shows hybridization of the regeneration oligonucleotide, cleaving the capture probe with a nuclease, and then extending the capture probe with DNA polymerase using the hybridized regeneration oligonucleotide as a template.DETAILED DESCRIPTION

[0067] The present disclosure provides compositions, substrates, and methods for generating reusable substrates for spatially tagging a target molecule in a sample. To help sequencing become more sustainable, the present disclosure is directed to generating reusable substrates by applying different regeneration methods. The reusable substrates can be used for the localized detection of a target molecule within a tissue sample.A. Definitions

[0068] The terminology used in the present disclosure is for the purpose of describing particular aspects only and is not intended to be limiting.

[0069] As used in this specification and the enumerated paragraphs herein, the singular forms "a," "an," and "the" include plural reference unless the context dictates otherwise.

[0070] As used herein, the terms “substantially,” "about," and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20-25 percent (%), for example, within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range of values.

[0071] As used herein, the term “substrate” refers to a material used as a support for compositions described herein. In some aspects, the substrate can be a solid support. Anyvariety of solid supports can be used in a method, composition, or apparatus of the present disclosure.

[0072] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g., due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene, and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, 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. In some aspects, the substrate is glass. Other suitable substrate materials may include polymeric materials, silicon, quartz (fused silica), boro float glass, silica, silica-based materials, carbon, metals including gold, optical fiber or optical fiber bundles, or sapphire. The particular material can be selected based on properties desired for a particular use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will utilize radiation of the desired wavelength, such as one or more of the techniques set forth herein. Conversely, it may be desirable to select a material that does not pass radiation of a certain wavelength (e.g., being opaque, absorptive, or reflective). This can be useful for the formation of a mask to be used during the manufacture of the structured substrate, or to be used for a chemical reaction or analytical detection carried out using the structured substrate. Other properties of a material that can be exploited are inertness or reactivity to certain reagents used in a downstream process, ease of manipulation, or low cost during a manufacturing process manufacture. Further examples of materials that can be used in the structured substrates or methods of the present disclosure are described in US Pat. App. Pub. No. 2012 / 0316086 Al and 2013 / 0116153, the entire contents of each are incorporated by reference herein. In some aspects, the solid support is a flow cell as described herein below.

[0073] Other example substrate materials can include metal oxide, organo-silicate (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An exampleof POSS is described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776- 778, which is incorporated by reference in its entirety. In some examples, substrates used in the present application include silica-based substrates, such as glass, fused silica, or other silica-containing material. In some examples, silica-based substrates can include silicon, silicon dioxide, silicon nitride, or silicone hydride. In some examples, substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, and poly(methyl methacrylate). Example plastic materials include poly(methyl methacrylate), polystyrene, cyclic olefin polymer substrates, COCs, and epoxies In some examples, the substrate is or includes a silica-based material or plastic material or a combination thereof. In particular examples, the substrate has at least one surface including glass or a silicon- based polymer. In some examples, the substrates can include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface including a metal oxide.

[0074] In some aspects, the surface includes a tantalum oxide or tin oxide. Acrylamides, enones, or acrylates may also be utilized as a substrate material or component. Other substrate materials can include, but are not limited to gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers, and copolymers. In some examples, the substrate and / or the substrate surface can be, or include, quartz. In some other examples, the substrate and / or the substrate surface can be, or include, a semiconductor, such as GaAs or ITO. The foregoing lists are intended to illustrate, but not limit, the present application. Substrates can include a single material or a plurality of different materials. Substrates can be composites or laminates. In some examples, the substrate includes an organo-silicate material.

[0075] Substrates can be flat, round, spherical, rod-shaped, or any other suitable shape.Substrates may be rigid or flexible. In some examples, a substrate is a glass slide, an array, a bead, or a flow cell. Substrates can be non-patterned, textured, or patterned on one or more surfaces of the substrate. In some examples, the substrate is patterned. Such patterns may include posts, pads, wells, ridges, channels, or other three-dimensional concave or convex structures. Patterns may be regular or irregular across the surface of the substrate. Patterns can be formed, for example, by nanoimprint lithography or by the use of metal pads that form features on non-metallic surfaces, for example.

[0076] As used herein, the term “bead” refers to a small body made of a rigid or semi-rigid material. The body can have a shape characterized, for example, as a sphere, oval, microsphere, or other recognized particle shape whether having regular or irregular dimensions. Example materials that are useful for beads include, without limitation, glass; plastic such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane or polytetrafluoroethylene (TEFLON®, from Chemours); polysaccharides or cross-linked polysaccharides such as agarose or Sepharose; nylon; nitrocellulose; resin; silica or silica-based materials including silicon and modified silicon; carbon-fiber, metal; inorganic glass; optical fiber bundle, or a variety of other polymers. Example beads include, without limitation, controlled pore glass beads, paramagnetic beads, thoria sol, Sepharose beads, nanocrystals, and others known in the art as described, for example, in Microsphere Detection Guide from Bangs Laboratories, Fishers Ind. Beads may also be coated with a polymer that has a functional group that can attach to an oligonucleotide.

[0077] A solid support can include a collection of beads. The beads can be suspended in a solution or they can be located on the surface of a substrate. Examples of arrays having beads located on a surface include those wherein beads are located in wells such as a BeadChip array (Illumina Inc., San Diego Calif.), substrates used in sequencing platforms from 454 LifeSciences (a subsidiary of Roche, Basel Switzerland) or substrates used in sequencing platforms from Ion Torrent (a subsidiary of Life Technologies, Carlsbad Calif.). Other solid supports having beads located on a surface are described in U.S. Pat. Nos.6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; or 6,274,320; US Pat. App. Publ. Nos. 2009 / 0026082 Al; 2009 / 0127589 Al; 2010 / 0137143 Al; or 2010 / 0282617 Al or PCT Publication No. WO 2000 / 63437, each of which is incorporated herein by reference. Several of the above references describe methods for attaching nucleic acids to beads before loading the beads in or on a solid support. It will, however, be understood that the oligonucleotides can be made first and then attached to the beads which can then be loaded onto an array and used in a method set forth herein. In some aspects, the oligonucleotides are released from the beads and attached to a solid support (for example, and without limitation, a flow cell).

[0078] In some aspects, a substrate described herein forms at least part of a flow cell or is located in or coupled to a flow cell. Flow cells may include a flow chamber that is divided into a plurality of lanes or a plurality of sectors. As used herein, the term “flow cell” isintended to mean a vessel having a chamber where a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some aspects, the chamber is configured for detection of the reaction that occurs in the chamber. For example, the chamber can include one or more transparent surfaces allowing optical detection of tissue samples, optically labeled molecules, or the like in the chamber. Exemplary flow cells include, but are not limited to those used in a nucleic acid sequencing apparatus such as flow cells for the Genome Analyzer®, MiSeq®, NextSeq®, or HiSeq® platforms commercialized by Illumina, Inc. (San Diego, Calif.); or for the SOLiD™ or Ion Torrent™ sequencing platform commercialized by Life Technologies (Carlsbad, Calif.). Exemplary flow cells and methods for their manufacture and use are also described, for example, in WO 2014 / 142841 Al; U.S. Pat. App. Pub. No.2010 / 0111768 Al and U.S. Pat. No. 8,951,781, each of which is incorporated herein by reference.

[0079] In some aspects, the solid supports typically used for bead arrays are used without beads. For example, nucleic acids, such as the oligonucleotides described herein, can be attached directly to the wells or gel material in wells. Thus, the above references are illustrative of materials, compositions, or apparatus that can be modified for use in the methods and compositions set forth herein.

[0080] A solid support used in a method set forth herein can include an array of beads, wherein different oligonucleotides are attached to different beads in the array. In various aspects, each bead can be attached to a different oligonucleotide and the beads can be randomly distributed on the solid support in order to effectively attach the different nucleic acid probes to the solid support.

[0081] Optionally, the solid support can include wells having dimensions that accommodate no more than a single bead. In such a configuration, the beads may be attached to the wells due to forces resulting from the fit of the beads in the wells. It is also possible to use attachment chemistries or adhesives to hold the beads in the wells.

[0082] As described herein, oligonucleotides that are attached to beads can comprise or consist of barcode sequences. According to methods provided herein, a population of beads can be configured such that each bead is attached to only one type of oligonucleotide comprising a plurality of barcodes, and many different beads (each with a different oligonucleotide) are present in the population.

[0083] Optionally, the substrate can include a gel coating. Attachment of nucleic acids to a solid support via a gel is exemplified by flow cells available commercially from Illumina Inc. (San Diego, CA) or described in US Pat. App. Pub. Nos. 2011 / 0059865 Al, 2014 / 0079923 Al, or 2015 / 0005447 Al; or PCT Publ. No. WO 2008 / 093098, each of which is incorporated herein by reference. Exemplary gels that can be used in the methods and apparatus set forth herein include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide, or SFA (see, for example, US Pat. App. Pub. No.2011 / 0059865 Al, which is incorporated herein by reference).

[0084] In some aspects, the surface of the substrate can include PAZAM (see, for example, US Pat. App. Publ. Nos. 2014 / 0079923 Al, or 2015 / 0005447 Al, each of which is incorporated herein by reference). PAZAM can include azide moieties which may be reacted with moieties in molecules to couple the molecules to the surface. For example, molecules may include alkynes (such as alkyne, BCN, or DBCO) as moieties, PEG1 to PEG10 as linkers, and biotin as a moiety. In the non-limiting example in which PEG4 is the linker, molecules may be referred to as alkyne-PEG4-biotin molecules. Once attached to the surface via a reaction between moieties, biotin is available to be reacted. In some aspects, biotin is then reacted with an active site of a pre-incubated streptavidin-dual biotin- P5 / P7 complex, binding complexes to the surface. Each streptavidin-dual biotin-P5 / P7 complex can include a streptavidin central molecule and at least one oligonucleotide, e.g., one or more P5 and / or P7 oligonucleotides which are functionalized to dual biotin to which a respective active site of streptavidin binds. At this point, oligonucleotides can be used for appropriate clustering and sequencing processes (not specifically illustrated). To initiate regeneration and reuse of the flowcell, a reagent such as hot formamide or ethylene glycol is introduced to decouple the streptavidin central molecule from biotin, for example by denaturing the streptavidin. A nuclease digest is also performed such that nuclease digests polynucleotides in the flowcell, e.g., oligonucleotides and any polynucleotides coupled thereto, into nucleotides. At this point, or at a later time, another set of complexes can be introduced and the cycle repeated.

[0085] In some aspects, a solid support can be configured as an array of features to which nucleic acids can be attached. As used herein, the term "feature" means a location in an array for a particular species of molecule. A feature can contain only a single molecule or it can contain a population of several molecules of the same species. Features of an arrayare typically discrete. The discrete features can be contiguous or they can have spaces between each other. The size of the features and / or spacing between the features can vary such that arrays can be high-density, medium-density, or lower-density. High-density arrays are characterized as having sites separated by less than about 15 pm. Mediumdensity arrays have sites separated by about 15 to 30 pm, while low-density arrays have sites separated by greater than 30 pm. An array can have, for example, sites that are separated by less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, or 0.5 pm. An apparatus or method of the present disclosure can be used to detect an array at a resolution sufficient to distinguish sites at the above densities or density ranges. Exemplary features include without limitation, beads (or other particles) in or on a substrate, droplets, wells in a substrate, projections from a substrate, ridges on a substrate, or channels in a substrate.

[0086] Features may be present on a solid support before contacting the solid support with nucleic acid probes. For example, in aspects where probes are attached to a support via hybridization to primers, the primers can be attached at the features, whereas interstitial areas outside of the features substantially lack any of the primers. Nucleic acid probes can be captured at preformed features on a solid support, and optionally amplified on the solid support, using methods set forth in US Pat. No. 8,895,249, US Pat. No. 8,778,849, or US Pat App. Pub. No. 2014 / 0243224 Al, each of which is incorporated herein by reference. Alternatively, a solid support may have a lawn of primers or may otherwise lack features. In this case, a feature can be formed by virtue of the attachment of a nucleic acid probe to the solid support. Optionally, the captured nucleic acid probe can be amplified on the solid support such that the resulting cluster becomes a feature. Although the attachment is exemplified above as a capture between a primer and a complementary portion of a probe, it will be understood that capture moieties other than primers can be present at pre-formed features or as a lawn. Other exemplary capture moieties include, but are not limited to, chemical moieties capable of reacting with a nucleic acid probe to create a covalent bond or receptors capable of binding non-covalently to a ligand on a nucleic acid probe.

[0087] In some aspects, the step of attaching nucleic acid probes to a solid support can be carried out by providing a fluid that contains a mixture of different nucleic acid probes and contacting this fluidic mixture with the solid support. The contact can result in the fluidic mixture being in contact with a surface to which many different nucleic acid probes from the fluidic mixture will attach. Thus, the probes have random access to the surface (whether the surface has pre-formed features configured to attach the probes or a uniform surfaceconfigured for attachment). Accordingly, the probes can be randomly located on the solid support.

[0088] The total number and variety of different probes that end up attached to a surface can be selected for a particular application or use. For example, in aspects where a fluidic mixture of different nucleic acid probes is contacted with a solid support for purposes of attaching the probes to the support, the number of different probe species can exceed the occupancy of the solid support for probes. Thus, the number and variety of different probes that attach to the solid support can be equivalent to the probe occupancy of the solid support. Alternatively, the number and variety of different probe species on the solid support can be less than the occupancy (i.e. there will be redundancy of probe species such that the solid support may contain multiple features having the same probe species). Such redundancy can be achieved, for example, by contacting the solid support with a fluidic mixture that contains a number and variety of probe species that is substantially lower than the probe occupancy of the solid support.

[0089] Attachment of the nucleic acid probes can be mediated by hybridization of the nucleic acid probes to complementary primers that are attached to the solid support, chemical bond formation between a reactive moiety on the nucleic acid probe and the solid support (examples are set forth in US Pat. No. 8,895,249, US Pat. No. 8,778,849, or US Pat App. Pub. No. 2014 / 0243224 Al, each of which is incorporated herein by reference), affinity interactions of a moiety on the nucleic acid probe with a solid support-bound moiety (e.g. between known receptor-ligand pairs such as streptavidin-biotin, antibody-epitope, lectin-carbohydrate and the like), physical interactions of the nucleic acid probes with the solid support (e.g. hydrogen bonding, ionic forces, van der Waals forces and the like), or other interactions known in the art to attach nucleic acids to surfaces.

[0090] The features can be present in any of a variety of desired formats. For example, the features can be wells, pits, channels, ridges, raised regions, pegs, posts, or the like. In some aspects, the features can contain beads. However, in particular aspects, the features need not contain a bead or particle.

[0091] As used herein, the term “array” refers to a population of sites that can be differentiated from each other according to relative location. Different molecules that are at different sites of an array can be differentiated from each other according to the locations of the sites in the array. An individual site of an array can include one or more molecules of a particular type. For example, a site can include a single nucleic acid molecule havinga particular sequence or a site can include several nucleic acid molecules having the same sequence (and / or complementary sequence, thereof). The sites of an array can be different features located on the same substrate. The sites of an array can be separate substrates each bearing a different molecule. Different molecules attached to separate substrates can be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.

[0092] As used herein, the term "pitch," when used in reference to features of an array, is intended to refer to the center-to-center spacing for adjacent features. A pattern of features can be characterized in terms of average pitch. The pattern can be ordered such that the coefficient of variation around the average pitch is small or the pattern can be random in which case the coefficient of variation can be relatively large. In either case, the average pitch can be, for example, at least about 10 nm, 0.1 pm, 0.5 pm, 1 pm, 5 pm, 10 pm, 100 pm or more. Alternatively or additionally, the average pitch can be, for example, at most about 100 pm, 10 pm, 5 pm, 1 pm, 0.5 pm 0.1 pm or less. Of course, the average pitch for a particular pattern of features can be between one of the lower values and one of the upper values selected from the ranges above.

[0093] High-density arrays are characterized as having an average pitch of less than about 15 pm. Medium-density arrays have an average pitch of about 15 to 30 pm, while low- density arrays have an average pitch greater than 30 pm. An array useful in the invention can have an average pitch that is less than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm or 0.5 pm. The average pitch values and ranges set forth above or elsewhere herein are intended to apply to ordered arrays or random arrays. In particular aspects, features on a solid support can each have an area that is larger than about 100 nm2, 250 nm2, 500 nm2, 1 pm2, 2.5 pm2, 5 pm2, 10 pm2, 100 pm2or 500 pm2. Alternatively or additionally, features can each have an area that is smaller than about 1 mm2500 pm2100 pm225 pm2, 10 pm25 pm21 pm2500 nm2, or 100 nm2. The above ranges can describe the apparent area of a bead or other particle on a solid support when viewed or imaged from above.

[0094] A solid support can include or can be made by the methods set forth herein to attach, a plurality of different nucleic acid probes. For example, a solid support can include at least 10, 100, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109or more different probes. Alternatively or additionally, a solid support can include at most 1 x 109, 1 x 108, 1 x 107,1 x 106, 1 x IO5, 1 x 104, 1 x 103, 100, or fewer different probes. It will be understood that each of the different probes can be present in several copies, for example, when the probes have been amplified to form a cluster. Thus, the above ranges can describe the number of different nucleic acid clusters on a solid support. It will also be understood that the above ranges can describe the number of different barcodes, target capture sequences, or other sequence elements set forth herein as being unique to particular nucleic acid probes. Alternatively or additionally, the ranges can describe the number of extended probes or modified probes created on a solid support using a method set forth herein.

[0095] As used herein, terms such as “covalently coupled” or “covalently bonded” refer to the forming of a chemical bond that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently coupled molecule refers to a molecule that forms a chemical bond, as opposed to a non-covalent bond such as electrostatic interaction.

[0096] As used herein, the term “linker” is intended to mean a portion of a molecule via which one element is attached to another element. For example, a linker may attach a first reactive moiety to a second reactive moiety. Linkers may be covalent.

[0097] As used herein, the term “different," when used in reference to nucleic acids, means that the nucleic acids have nucleotide sequences that are not the same as each other. Two or more nucleic acids can have nucleotide sequences that are different along their entire length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different along a substantial portion of their length. For example, two or more nucleic acids can have target nucleotide sequence portions that are different for the two or more molecules while also having a universal sequence portion that is the same for the two or more molecules. The term can be similarly applied to proteins that are distinguishable as different from each other based on amino acid sequence differences.

[0098] As used herein, “complementary” means that an oligonucleotide comprises a sequence of nucleotides that can form a double-stranded structure by matching base pairs with another oligonucleotide or part thereof. As used herein “substantially complementary” means that the oligonucleotide has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% overall sequence identity to the complementary sequence.

[0099] As used herein, the terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and to include naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific fashion or capable of being used as atemplate for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g. found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)). A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine, or guanine and a ribonucleic acid can have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art. The terms "probe" or "target," when used in reference to a nucleic acid or sequence of a nucleic acid, are intended as semantic identifiers for the nucleic acid or sequence in the context of a method or composition set forth herein and do not necessarily limit the structure or function of the nucleic acid or sequence beyond what is otherwise explicitly indicated. The terms "probe" and "target" can be similarly applied to other analytes such as proteins, small molecules, cells, or the like.

[0100] As used herein, a “primer” is a nucleic acid molecule that can hybridize to a target sequence, such as an adapter attached to a library fragment. As one example, an amplification primer can serve as a starting point for template amplification and cluster generation. As another example, a synthesized nucleic acid (template) strand may include a site to which a primer e.g., a sequencing primer) can hybridize in order to prime the synthesis of a new strand that is complementary to the synthesized nucleic acid strand. Any primer can include any combination of nucleotides or analogs thereof. In some examples, the primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases long and can include a variety of non-natural nucleotides. In various aspects, the sequencing primer is a short strand, ranging from 5 to 60 bases, from 10 to 60 bases, from 10 to 20 bases, from 10 to 30 bases, from 10 to 40 bases, from 10 to 50 bases, or from 20 to 40 bases.

[0101] As used herein, the term “clustering primer sequence” or “clustering sequence” refers to a nucleotide sequence in solution and / or immobilized on a surface that is used for amplifying the template polynucleotides to create identical copies of the same templates (i.e., clusters). Examples of clustering primer sequences may include but are not limited toP5 primer, P5' primer, P7 primer, P7' primer, Pl 5 primer, Pl 5' primer, Pl 7' primer, Pl 7' primer, B15 primer, and Bl 5’ primer as described herein. Clustering primers, their sequences, and their uses are also described, e.g., in WO2019222264, incorporated by reference herein in its entirety.

[0102] As used herein, the term “adapter” refers generally to any linear nucleic acid molecule that can be ligated to an oligonucleotide of the disclosure. In some aspects, adapters include two reverse complementary oligonucleotides forming a double-stranded structure. In some aspects, an adapter includes two oligonucleotides that are complementary at one portion and mismatched at another portion, forming a Y-shape or fork-shaped adapter that is double-stranded at the complementary portion and has two floppy overhangs at the mismatched portion. In some aspects, adapters are copied onto the library molecules using templated polymerase synthesis (e.g., second strand cDNA synthesis as described herein). In some aspects, adapters are ligated to a first complementary strand of the disclosure. In some aspects, an adapter comprises two oligonucleotides that are double-stranded at one portion and single-stranded at another portion, forming an adapter with an overhang. In some aspects, an adapter comprises a B15 nucleotide sequence. In some aspects, the adapter that comprises a B15 nucleotide sequence is referred to herein as Adpl. In some aspects, an adapter comprises a P7 nucleotide sequence. In some aspects, the adapter that comprises a P7 nucleotide sequence is referred to herein as Adp2. In some aspects, an adapter comprises a sequence that is complementary to a primer. In some aspects, an adapter comprises a sequence that is complementary to a P5 primer or a P5’ primer. In some aspects, an adapter comprises a sequence complementary to a P7 primer or a P7’ primer. In some aspects, an adapter comprises a sequence complementary to a B 15 primer or a Bl 5’ primer.

[0103] As used herein, the terms “sample” or “tissue” or “tissue sample” are intended to mean an aggregation of cells, and, optionally, intercellular matter. In some aspects, the cells in a tissue are not free-floating in solution and instead are attached to each other to form a multicellular structure. Exemplary tissue types include muscle, nerve, epidermal, and connective tissues. In some aspects, the tissue sample is from a human.

[0104] In some aspects, the sample is one or more cells. The cell(s) can be individual and free from any tissue or multicellular structure at the time contact is made with the solid support. For example, the cell(s) can be present in a fluid (e.g. when a plurality of different cells are present the fluid can be a fluidic mixture of the different cells) and the fluid canbe contacted with the solid support to which the different probes are attached. Any of a variety of cells can be used including, for example, those from a prokaryote, archaea, or eukaryote. One or more cells used in a method, composition, or apparatus of the present disclosure can be a single-celled organism or a multicellular organism. Exemplary organisms from which one or more cells can be obtained include, but are not limited to a mammal, plant, algae, nematode, insect, fish, reptile, amphibian, fungi, or Plasmodium falciparum. Exemplary species are set forth previously herein or known in the art.

[0105] Aspects of the present disclosure can also use one or more subcellular components as a tissue sample. For example, a fluidic mixture can include one or more nuclei, Golgi apparatus, mitochondria, chloroplasts, membrane fractions, vesicles, endoplasmic reticulum, or other components known in the art. Other useful types of tissue samples are one or more viruses or viroids. It will be understood that a tissue sample can be a homogeneous culture or population of the above cells, subcellular components, viruses, or viroids. Alternatively, the tissue sample can be a non-homogenous collection of cells, subcellular components, viruses, or viroids, for example, derived from several different organisms in a community or ecosystem. An exemplary community is the collection of bacteria present in the digestive system, lung, or other organ of a multicellular organism such as a mammal.

[0106] One or more cells, subcellular components, viruses, or viroids that are contacted with a solid support in a method set forth herein can be attached to the solid support. Attachment can be achieved using methods known in the art such as those exemplified herein with respect to attachment of nucleic acids to a solid support. In some aspects, attachment is selective for specific types of cells, subcellular components, viruses, or viroids. For example, the solid support can include antibodies or other receptors that are selective for epitopes or ligands present on one or a subset of different cells, subcellular components, viruses, or viroids present in a fluidic mixture. In other aspects, the attachment of cells, subcellular components, viruses, or viroids can be mediated by non-selective moieties such as chemical moieties that are broadly reactive.

[0107] In some aspects, one or more cells, subcellular components, viruses, or viroids that have been contacted with a solid support can be lysed to release target nucleic acids. Lysis can be carried out using methods known in the art such as those that employ one or more chemical treatments, enzymatic treatments, electroporation, heat, hypotonic treatments, sonication, or the like. Exemplary lysis techniques are set forth in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).

[0108] In some aspects, the tissue sample may be fixed, sectioned, and mounted on a surface. The tissue can be derived from a multicellular organism such as those exemplified above in regard to cells. A tissue section can be contacted with a solid support, for example, by laying the tissue on the surface of the solid support. The tissue can be freshly excised from an organism or it may have been previously preserved for example by freezing, embedding in a material such as paraffin (e.g. formalin fixed paraffin embedded samples), formalin fixation, infiltration, dehydration, or the like. The methods disclosed herein may be performed before or after staining the tissue sample. For example, following hematoxylin and eosin staining, a tissue sample may be spatially analyzed in accordance with the methods provided herein. A method may include analyzing the histology of the sample (e.g., using hematoxylin and eosin staining) and then spatially analyzing the tissue.

[0109] In some aspects, the sample can be fixed by deep freezing the sample at a temperature suitable to maintain or preserve the integrity of the tissue structure. A fixed or embedded tissue sample can be sectioned, i.e., thinly sliced, using known methods.

[0110] Optionally, a tissue section can be attached to a solid support, for example, using techniques and compositions exemplified herein with regard to attaching nucleic acids, cells, viruses, beads, or the like to a solid support. As a further option, a tissue can be permeabilized and the cells of the tissue lysed when the tissue is in contact with a solid support. Any of a variety of treatments can be used such as those set forth above in regard to lysing cells. Target nucleic acids that are released from a tissue that is permeabilized can be captured by nucleic acid probes on the surface of the substrate.[OHl] As used herein, the term “immobilized” refers to the state of two things being joined, fastened, adhered, attached, connected, or bound to each other. For example, an analyte, such as a nucleic acid, can be immobilized on a material, such as a bead, gel, or surface, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions. In some aspects, a covalent attachment can be used, but all that is required is that the oligonucleotides remain stationary or attached to a surface under conditions inwhich it is intended to use the surface, for example, in applications requiring nucleic acid capture, amplification, and / or sequencing. Oligonucleotides to be used as capture oligonucleotides can be immobilized such that a 3'-end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence.

[0112] Exemplary covalent linkages include, for example, those that result from the use of click chemistry techniques. Exemplary non-covalent linkages include, but are not limited to, non-specific interactions (e.g., hydrogen bonding, ionic bonding, van der Waals interactions etc.) or specific interactions (e.g., affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). Exemplary linkages are set forth in U.S. Pat. Nos. 6,737,236; 7,259,258; 7,375,234 and 7,427,678; and US Pat. Pub. No. 2011 / 0059865 Al, each of which is incorporated herein by reference.

[0113] As used herein, “hybridize” is intended to mean noncovalently associating a first oligonucleotide to a second oligonucleotide along the lengths of those polymers to form a double-stranded “duplex” or “complex.” For example, two DNA oligonucleotide strands may associate through complementary base pairing. The strength of the association between the first and second oligonucleotides increases with the complementarity between the sequences of nucleotides within those oligonucleotides. The strength of hybridization between oligonucleotides may be characterized by a temperature of melting (Tm) at which 50% of the duplexes have oligonucleotide strands that disassociate from one another. Oligonucleotides that are “partially” hybridized to one another means that they have sequences that are complementary to one another, but such sequences are hybridized with one another along only a portion of their lengths to form a partial duplex. Oligonucleotides with an “inability” to hybridize include those that are physically separated from one another such that an insufficient number of their bases may contact one another in a manner so as to hybridize with one another.

[0114] As used herein, the term “plurality” is intended to mean a population of two or more members, which may all be the same or two or more members may be different. Pluralities may range in size from small, medium, large, to very large. The size of a small plurality may range, for example, from a few members to tens of members. Medium-sized pluralities may range, for example, from tens of members to about 100 members or hundreds of members. Large pluralities may range, for example, from about hundreds of members toabout 1,000 members, to thousands of members, and up to tens of thousands of members. Very large pluralities may range, for example, from tens of thousands of members to about hundreds of thousands, a million, millions, tens of millions, and up to or greater than hundreds of millions of members. Therefore, a plurality may range in size from two to well over one hundred million members as well as all sizes, as measured by the number of members, in between and greater than the above example ranges. Accordingly, the definition of the term is intended to include all integer values greater than two. An upper limit of a plurality may be set, for example, by the theoretical diversity of bead types in an array.

[0115] As used herein, the term “attached” refers to the state of two things being joined, fastened, adhered, connected, or bound to each other. For example, an oligonucleotide can be attached to a material, such as a bead, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0116] As used herein, the terms “semi-random” or “semi-randomer” refer to a nucleotide sequence that comprises or consists of a partially pre-determined nucleotide sequence combined with a random nucleotide sequence.

[0117] As used herein, the terms “target” or “target molecule” refer to any molecule found within a cell. For example, a target molecule can include, but is not limited to, a nucleic acid, a nucleic acid sequence, RNA, mRNA, siRNA, rRNA, tRNA, DNA, cDNA, genomic DNA, a protein, an oligonucleotide, or a polynucleotide. A target molecule may be a nucleic acid sequence to be analyzed. A target molecule may include nucleotide sequences additional to a target sequence to be analyzed. For example, a target molecule may include one or more adapters, including an amplification adapter that functions as a primer binding site, which flank(s) a target molecule sequence that is to be analyzed. In particular examples, target molecules may have different sequences than one another but may have first and second adapters that are the same as one another. The two adapters that may flank a particular target molecule sequence may have the same sequence as one another, or complementary sequences to one another, or the two adapters may have different sequences. Thus, species in a plurality of target molecules may include regions of known sequence that flank regions of unknown sequence that are to be evaluated by, for example,sequencing (e.g., SBS). In some examples, the target molecule carries an amplification adapter at a single end, and such adapter may be located at either the 3' end or the 5' end of the target polynucleotide. Target molecules may be used without any adapter, in which case a primer binding sequence may come directly from a sequence found in the target polynucleotide.

[0118] The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein.The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description, the terms may be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species.

[0119] An oligonucleotide is a polymer comprised of nucleotides. In some aspects, oligonucleotides may be of any length and include, in various aspects, DNA oligonucleotides, RNA oligonucleotides, analogs thereof, or a combination thereof. In some aspects, an oligonucleotide is single-stranded, double-stranded, or partially doublestranded.

[0120] In some aspects, nucleotides may include naturally occurring nucleotides and functional analogs thereof. Examples of functional analogs are those that are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). An analog structure can have an alternate sugar moiety including any of a variety known in the art. Nucleotides can include native or non-native bases. A native DNA can include one or more of adenine, thymine, cytosine and / or guanine, and a native RNA can include one or more of adenine, uracil, cytosine and / or guanine. Any non-native base may be used, such as a locked nucleic acid (LNA) and a bridged nucleic acid (BNA). Example modified nucleotides include inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5- hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15- halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5 '-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates.

[0121] In some aspects, oligonucleotides also include those having at least one modified internucleotide linkage. In some aspects, the oligonucleotide is all or in part a peptide nucleic acid. Other modified intemucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those comprising one or more universal bases. "Universal base" refers to molecules capable of substituting for binding to any one of A, C, G, T and U in nucleic acids by forming hydrogen bonds without significant structure destabilization. Examples of universal bases include but are not limited to 5’- nitroindole-2’ -deoxyriboside, 3 -nitropyrrole, inosine and hypoxanthine.

[0122] In some aspects, an oligonucleotide of the disclosure, or a modified form thereof, is generally about 5 nucleotides to about 150 nucleotides in length. In further aspects, an oligonucleotide of the disclosure is about 5 to about 125 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 90 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 45 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 35 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, about 5 to about 15 nucleotides in length, about 5 to about 10 nucleotides in length, about 10 to about 150 nucleotides in length, about 10 to about 125 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 90 about 10 to about 50 nucleotides in length, about 10 to about 45 nucleotides in length, about 10 to about 40 nucleotides in length, about 10 to about 35 nucleotides in length, about 10 to about 30 nucleotides in length, about 10 to about 25 nucleotides in length, about 10 to about 20 nucleotides in length, about 10 to about 15 nucleotides in length, and all oligonucleotides intermediate in length of the sizes specifically disclosed to the extent that the oligonucleotide is able to achieve the desired result. Accordingly, in some aspects, an oligonucleotide of the disclosure is or is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 or more nucleotides in length. In some aspects, an oligonucleotide of the disclosure is less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or more nucleotides in length. In some aspects, the length of an oligonucleotide (such as a primer) of the disclosure is between about 5 base pairs (bp) and 40 bp, or between about 5 bp and 35 bp, or between about 5 bp and 30 bp, or between about 10 bp and 35 bp, or between about 10 bp and 30 bp, or between about 20 bp and 40 bp, or between about 20 bp and 35 bp, or between about 20 bp and 30 bp, or between about 9 and 20 bp or between about 5 and 15 bp, or between about 9 and 15 bp in length. In some aspects, the length of an oligonucleotide (such as a primer) of the disclosure is about 10 bp, 13 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, or 40 bp. As described herein, in various aspects the oligonucleotide may be a P5 primer, a P5’ primer, a P7 primer, or a P7’ primer.

[0123] As used herein, a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind a primer and a single-stranded target polynucleotide, and can sequentially add nucleotides to the growing primer to form a “complementary copy” polynucleotide having a sequence that is complementary to that of the target polynucleotide. DNA polymerases may bind to the target polynucleotide and then move down the target polynucleotide sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing polynucleotide strand. DNA polymerases may synthesize complementary DNA molecules from DNA templates. RNA polymerases may synthesize RNA molecules from DNA templates (transcription). Other RNA polymerases, such as reverse transcriptases, may synthesize cDNA molecules from RNA templates. Still, otherRNA polymerases may synthesize RNA molecules from RNA templates, such as RdRP. Polymerases may use a short RNA or DNA strand (primer), to begin strand growth. Some polymerases may displace the strand upstream of the site where they are adding bases to a chain. Such polymerases may be said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase.

[0124] Example DNA polymerases include Bst DNA polymerase, 9° Nm DNA polymerase, Phi29 DNA polymerase, DNA polymerase I (E. colt), DNA polymerase I (Large), (KI enow) fragment, Klenow fragment (3 '-5' exo-), T4 DNA polymerase, T7 DNA polymerase, Deep VentR™ (exo-) DNA polymerase, Deep VentR™ DNA polymerase, DyNAzyme™ EXT DNA, DyNAzyme™ II Hot Start DNA Polymerase, Phusion™ High- Fidelity DNA Polymerase, Therminator™ DNA Polymerase, Therminator™ II DNA Polymerase, VentR® DNA Polymerase, VentR® (exo-) DNA Polymerase, RepliPHI™ Phi29 DNA Polymerase, rBst DNA Polymerase, rBst DNA Polymerase (Large), Fragment (IsoTherm™ DNA Polymerase), MasterAmp™ AmpliTherm™, DNA Polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNA polymerase, Tbr DNA polymerase, DNA polymerase Beta, ThermoPhi DNA polymerase, and Isopol™ SD+ polymerase. In specific, nonlimiting examples, the polymerase is selected from a group consisting of Bst, Bsu, and Phi29. Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3' and / or 5' exonuclease activity.

[0125] Example RNA polymerases include RdRps (RNA dependent, RNA polymerases) that catalyze the synthesis of the RNA strand complementary to a given RNA template. Example RdRps include polioviral 3Dpol, vesicular stomatitis virus L, and hepatitis C virus NS5B protein. Example RNA Reverse Transcriptases. A non-limiting example list to include are reverse transcriptases derived from Avian Myelomatosis Virus (AMV), Murine Moloney Leukemia Virus (MMLV) and / or the Human Immunodeficiency Virus (HIV), telomerase reverse transcriptases such as (hTERT), SuperScript™ III, SuperScript™ IV Reverse Transcriptase, ProtoScript® II Reverse Transcriptase.

[0126] As used herein, the term "extend" or “extension” when used in reference to a nucleic acid, is intended to mean addition of at least one nucleotide to the nucleic acid. In some aspects, one or more nucleotides can be added to the 3' end of a nucleic acid, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase, or reverse transcriptase(RT)). Chemical or enzymatic methods can be used to add one or more nucleotide to the 3' or 5' end of a nucleic acid. An extension reaction, in which nucleotides are added to the 3' end of an oligonucleotide (e.g., a primer) is performed in the presence of a polymerase, such as a DNA or RNA polymerase. In some aspects, the polymerase is a non-thermostable isothermal strand displacement polymerase. In some aspects, the extension reaction is carried out by recombinase polymerase amplification (RPA). RPA comprises three core enzymes - a recombinase, a single-stranded DNA binding protein (SSB) and a stranddisplacing polymerase. As described in Daher et al. (Rana K Daher, Gale Stewart, Maurice Boissinot, Michel G Bergeron, Recombinase Polymerase Amplification for Diagnostic Applications, Clinical Chemistry, Volume 62, Issue 7, 1 July 2016). One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (e.g., ligase catalysis) methods. A nucleic acid can be extended in a template directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended.

[0127] As used herein, the term “primer” is defined as a polynucleotide to which nucleotides may be added via a free 3' OH group. A primer may include a 3' block inhibiting polymerization until the block is removed. A primer may include a modification at the 5' terminus to allow a coupling reaction or to couple the primer to another moiety. A primer may include one or more moieties, such as 8-oxo-G, which may be cleaved under suitable conditions, such as UV light, chemistry, enzyme, or the like. The primer length may be any suitable number of bases long and may include any suitable combination of natural and non-natural nucleotides. A target polynucleotide may include an “amplification adapter” or, more simply, an “adapter,” that hybridizes to (has a sequence that is complementary to) a primer and may be amplified so as to generate a complementary copy polynucleotide by adding nucleotides to the free 3' OH group of the primer.

[0128] As used herein, the term “double-stranded,” when used in reference to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in the polynucleotide are hydrogen bonded to respective nucleotides in a complementary polynucleotide. A double-stranded polynucleotide also may be referred to as a “duplex.”

[0129] As used herein, the term “single-stranded,” when used in reference to a polynucleotide, means that essentially none of the nucleotides in the polynucleotide are hydrogen bonded to a respective nucleotide in a complementary polynucleotide.

[0130] As used herein, the term “cluster” refers to a population of nucleic acids that is attached to a solid support.

[0131] The method of present disclosure can include a step of performing a nucleic acid detection to determine the barcode sequence of the nucleic acid probes that are located on the solid support. In many aspects, the probes are randomly located on the solid support and the nucleic acid detection reaction provides information to locate each of the different probes. Exemplary nucleic acid detection methods include, but are not limited to nucleic acid sequencing of a probe, hybridization of nucleic acids to a probe, ligation of nucleic acids that are hybridized to a probe, extension of nucleic acids that are hybridized to a probe, extension of a first nucleic acid that is hybridized to a probe followed by ligation of the extended nucleic acid to a second nucleic acid that is hybridized to the probe, or other methods known in the art such as those set forth in US Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0132] In some aspects, the methods of the present disclosure can include sequencing to spatially detect a target molecule. In some aspects, sequencing techniques, such as sequencing-by-synthesis (SBS) techniques, can be used in the disclosed methods. SBS can be carried out as follows. To initiate a first SBS cycle, one or more labeled nucleotides, DNA polymerase, SBS primers, etc., can be contacted with one or more features on a solid support (e.g. feature(s) where nucleic acid probes are attached to the solid support). Those features where SBS primer extension causes a labeled nucleotide to be incorporated can be detected. Optionally, the nucleotides can include a reversible termination moiety that terminates further primer extension once a nucleotide has been added to the SBS primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for aspects that use reversible termination, a deblocking reagent can be delivered to the solid support (before or after detection occurs).

[0133] Washes can be carried out between the various delivery steps. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with a composition, apparatus, or method of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), PCT Publ. Nos. WO 91 / 06678, WO 04 / 018497 or WO 07 / 123744; US Pat. Nos. 7,057,026, 7,329,492,7,211,414, 7,315,019 or 7,405,281, and US Pat. App. Publ. No. 2008 / 0108082, each of which is incorporated herein by reference.

[0134] Other sequencing procedures that use cyclic reactions can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into a nascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); or US Pat. Nos. 6,210,891, 6,258,568, or 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated can be detected via luciferase-produced photons. Thus, the sequencing reaction can be monitored via a luminescence detection system. Excitation radiation sources used for fluorescence-based detection systems are not necessary for pyrosequencing procedures. Useful fluidic systems, detectors, and procedures that can be used for the application of pyrosequencing to apparatus, compositions, or methods of the present disclosure are described, for example, in PCT Pat. App. Publ. No. W02012 / 058096, US Pat. App. Publ. No. 2005 / 0191698 Al, or US Pat. Nos. 7,595,883 or 7,244,559, each of which is incorporated herein by reference.

[0135] In some aspects, sequencing-by-ligation reactions can also be used. Examples of sequencing-by-ligation reactions include, for example, those described in Shendure et al. Science 309:1728-1732 (2005); or US Pat. Nos. 5,599,675 or 5,750,341, each of which is incorporated herein by reference. Some aspects can include sequencing-by-hybridization procedures as described, for example, in Bains et al., Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); or PCT Pat. App. Publ. No. WO 1989 / 10977, each of which is incorporated herein by reference. In both sequencing-by-ligation and sequencing-by- hybridization procedures, target nucleic acids (or amplicons thereof) that are present at sites of an array are subjected to repeated cycles of oligonucleotide delivery and detection. Compositions, apparatus, or methods set forth herein or in references cited herein can be readily adapted for sequencing-by- ligation or sequencing-by-hybridization procedures. Typically, the oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described with regard to SBS procedures herein or in references cited herein.

[0136] Some sequencing aspects can utilize methods involving the real-time monitoring of DNA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and y-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs). Techniques and reagents for FRET -based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), each of which is incorporated herein by reference.

[0137] Some sequencing aspects include the detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on the detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies and Thermo Fisher subsidiary) or sequencing methods and systems described in US Pat app. Publ. Nos. 2009 / 0026082 Al; 2009 / 0127589 Al; 2010 / 0137143 Al; or US 2010 / 0282617 Al, each of which is incorporated herein by reference

[0138] In some aspects, nucleic acid hybridization techniques are also useful methods for determining barcode sequences. In some cases, combinatorial hybridization methods can be used such as those used for decoding of multiplex bead arrays (see e.g. US Pat. No.8,460,865, which is incorporated herein by reference). Such methods utilize labeled nucleic acid decoder probes that are complementary to at least a portion of a barcode sequence. A hybridization reaction can be carried out using decoder probes having known labels such that the location where the labels end up on the solid support identifies the nucleic acid probes according to the rules of nucleic acid complementarity. In some cases, pools of many different probes with distinguishable labels are used, thereby allowing a multiplex decoding operation. The number of different barcodes determined in a decoding operation can exceed the number of labels used for the decoding operation. For example, decoding can be carried out in several stages where each stage constitutes hybridization with a different pool of decoder probes. The same decoder probes can be present in different pools but the label that is present on each decoder probe can differ from pool to pool (i.e. each decoder probe is in a different "state" when in different pools). Various combinations of these states and stages can be used to expand the number of barcodes that can be decoded well beyond the number of distinct labels available for decoding. Such combinatorialmethods are set forth in further detail in US Pat. No. 8,460,865 or Gunderson et al., Genome Research 14:870-877 (2004), each of which is incorporated herein by reference.

[0139] The present disclosure is generally directed to methods for regenerating reusable substrates for spatially tagging a target molecule. In some aspects, the substrate can include at least one nucleic acid probe. In some aspects, the substrate can include a plurality of nucleic acid probes. In some aspects, the nucleic acid probes can include, among other things, a spatial barcode, a restriction enzyme recognition sequence, and a capture sequence.B. The Surface

[0140] The present disclosure provides, in various aspects, methods of preparing an immobilized library of target nucleic acids of a biological sample comprising providing a surface (e.g., an Illumina flow cell (Illumina Inc., San Diego Calif.)) comprising pluralities of oligonucleotides (e.g., probes). In some aspects, the nucleic acid probes are arranged in specific locations on the substrate.

[0141] In some aspects, one or more probes immobilized on a surface comprises a spacer.The term "spacer" as used herein means a moiety that serves to increase distance of an oligonucleotide from the surface. In some aspects, the spacer provides sufficient distance from the surface to enable a reverse transcriptase (RT) to access the capture nucleotide sequence (e.g., a polyT capture nucleotide sequence) of the capture oligonucleotide. In some aspects, the spacer when present is an organic moiety. In some aspects, the spacer is a polymer, including but not limited to a water-soluble polymer, a nucleic acid, a polypeptide, an oligosaccharide, a carbohydrate, a lipid, or a combination thereof. In some aspects, an oligonucleotide (e.g., a capture oligonucleotide, a spatially barcoded oligonucleotide) comprises 1, 2, 3, 4, 5, or more spacer moi eties. In some aspects, the spacer is an oligonucleotide spacer. An oligonucleotide spacer may have any sequence that does not interfere with the desired function of the oligonucleotide. In various aspects, the bases of the oligonucleotide spacer are all adenylic acids, all thymidylic acids, all cytidylic acids, all guanylic acids, all uridylic acids, or all some other modified base. In various aspects, the length of the spacer is or is equivalent to at least about 2 nucleotides, at least about 3 nucleotides, at least about 4 nucleotides, at least about 5 nucleotides, 5-10 nucleotides, 10-20 nucleotides, nucleotides, 10-30 nucleotides, or greater than 30 nucleotides.

[0142] As used herein, the term “immobilized” refers to the state of two things being joined, fastened, adhered, attached, connected, or bound to each other. For example, an analyte, such as a nucleic acid, can be immobilized on a material, such as a bead, gel, or surface, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of pairs of electrons between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of pairs of electrons and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions. In various aspects, covalent attachment can be used, but all that is required is that the oligonucleotides remain stationary or attached to a surface under conditions in which it is intended to use the surface, for example, in applications requiring nucleic acid capture, amplification, and / or sequencing. Oligonucleotides to be used as probes can be immobilized such that a 3'-end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence.

[0143] Exemplary covalent linkages include, for example, those that result from the use of click chemistry techniques. Exemplary non-covalent linkages include, but are not limited to, non-specific interactions (e.g., hydrogen bonding, ionic bonding, van der Waals interactions etc.) or specific interactions (e.g., affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). Exemplary linkages are set forth in U.S. Pat. Nos. 6,737,236; 7,259,258; 7,375,234 and 7,427,678; and US Pat. Pub. No. 2011 / 0059865 Al, each of which is incorporated herein by reference.

[0144] As used herein, the term "extend," when used in reference to a nucleic acid, is intended to mean addition of at least one nucleotide to the nucleic acid. In particular aspects, one or more nucleotides can be added to the 3' end of a nucleic acid, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase, or reverse transcriptase (RT)). Exemplary reverse transcriptase enzymes of the present disclosure include, but are not limited to, Maxima H- (Thermo Fisher Scientific Inc.) and Superscript IV (Thermo Fisher Scientific Inc.). Chemical or enzymatic methods can be used to add one or more nucleotide to the 3' or 5' end of a nucleic acid. An extension reaction, in which nucleotides are added to the 3' end of an oligonucleotide (e.g., a primer) is performed in the presence of a polymerase, such as a DNA or RNA polymerase. In some aspects, the polymerase is a non-thermostable isothermal strand displacement polymerase. Suitable non-thermostable strand displacement polymerasesaccording to the present disclosure can be found, for example, through New England BioLabs, Inc. and include phi29, Bsu, Klenow, DNA Polymerase I (E. colt), and Therminator. In some aspects, the extension reaction is carried out by recombinase polymerase amplification (RPA). RPA comprises three core enzymes - a recombinase, a single-stranded DNA binding protein (SSB) and a strand-displacing polymerase. As described in Daher et al. (Rana K Daher, Gale Stewart, Maurice Boissinot, Michel G Bergeron, Recombinase Polymerase Amplification for Diagnostic Applications, Clinical Chemistry, Volume 62, Issue 7, 1 July 2016). One or more oligonucleotides can be added to the 3' or 5' end of a nucleic acid, for example, via chemical or enzymatic (e.g., ligase catalysis) methods. A nucleic acid can be extended in a template directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended.Nucleic Acid Probes

[0145] As used herein, the terms “probe,” and “nucleic acid probe,” refer to an oligonucleotide having a nucleotide sequence that is capable of specifically annealing to a single-stranded polynucleotide sequence to be analyzed or subjected to a nucleic acid interrogation under conditions encountered in a primer annealing step of, for example, an amplification or sequencing reaction.

[0146] In some aspects, the nucleic acid probes which comprise a capture sequence. In some aspects, the nucleic acid probes comprise a spatial barcode sequence. In some aspects, the nucleic acid probes comprise a capture sequence and a spatial barcode sequence. In some aspects, the capture probe comprises additional nucleic acid sequences. For example, the nucleic acid probe can also include a sequencing by synthesis (SBS) sequence. In some aspects, the SBS sequence is SBS12 or a complement thereof (SBS12'). In some aspects, the SBS sequence is SBS3 or a complement thereof (SBS3').

[0147] In some aspects, the nucleic acid probe can also contain a flowcell clustering sequence. In some aspects, the flowcell clustering sequence is P5, P5', P7, or P7'. As used herein, the terms “P5” and “P7” may be used when referring to examples of adapters. The terms “P5'” (P5 prime) and “P7'” (P7 prime) refer to the complement of P5 and P7, respectively. It will be understood that any suitable adapter can be used in the methods presented herein and that the use of P5 and P7 are exemplary aspects only. In some aspects, the nucleic acid probe comprises an adapter sequence. In some aspects, the adaptersequence Adpl comprises a “P5”,”P7”, “B15”, “P5”’ (P5 prime), “P7”’ (P7 prime), “B15”’ (Bl 5 prime), “Pl 5”, or “Pl 7” nucleotide sequence. In some aspects, the adapter sequence Adpl comprises P5-A14ME. In some aspects, the adapter sequence Adpl comprises ME'V2B15'. In various aspects, the adapter sequence Adp2 comprises a “P5”,”P7”, “Bl 5”, “P5”’ (P5 prime), “P7”’ (P7 prime), “B15”’ (B15 prime), “P15”, or “P17” nucleotide sequence. In some aspects, a first nucleic acid probe comprises a first adapter sequence (Adpl), and a second nucleic acid probe comprises a second adapter sequence (Adp2). In some aspects, Adpl and Adp2 are the same sequence. In some aspects, Adpl and Adp2 are different sequences.

[0148] Uses of adapters such as P5 and P7 or their complements on flowcells are known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, each of which is incorporated herein by reference in its entirety. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be useful in the methods presented herein for hybridization to a complementary sequence and amplification of a sequence. One of skill in the art will understand how to design and use primer sequences that are suitable for the capture and / or amplification of nucleic acids as presented herein.

[0149] In some aspects, the nucleic acid probes can include a unique molecular identifier (UMI) or a single molecule identifier (SMI). As used herein, the term “unique molecular identifier” or “UMI” refers to a molecular tag, either random, non-random, or semi-random, that may be attached to a nucleic acid. When incorporated into a nucleic acid, a UMI can be used to correct for subsequent amplification bias by directly counting unique molecular identifiers (UMIs) that are sequenced after amplification. A UMI can be attached to similar nucleic acids, e.g., adapters, making each nucleic acid unique. As used herein, the term “single molecular identifier” or “SMI” refers to a molecular tag, either random, nonrandom, or semi-random, that may be attached to a nucleic acid. In various aspects, a SMI is a unique molecular identifier (UMI). When incorporated into a nucleic acid, a SMI can be used to correct for subsequent amplification bias by directly counting single molecular identifiers (SMIs) that are sequenced after amplification. A SMI (e.g., a UMI) can be attached to similar nucleic acids, e.g., adapters, making each nucleic acid unique. SMIs(e.g., UMIs) may also be used to uniquely tag individual molecules (e.g., individual mRNA molecules) in a sample (e.g., individual mRNA molecules in a tissue sample, cell sample, or sample library). In some aspects, a UMI is a random nucleotide sequence (e.g., N9).

[0150] In some aspects, the nucleic acid probes can also contain primer binding sites. In some aspects, a nucleic acid probe contains first and second universal primer binding sites, they will be located at the ends of the probe. In some aspects, it may be desirable to remove at least one of the primer binding sites from the nucleic acid probe or amplicons produced from the probe. Accordingly, the nucleic acid probes can optionally include a cleavage site between the target capture sequence and one of the universal primer binding sequences. In this case, a cleavage reaction can be performed to separate the universal primer binding site from the target capture sequence. Generally, the portion of the probe (or its amplicons) that contains the target capture sequence will be attached to the solid support resulting in removal of the primer binding site from the solid support and retention of the target capture sequence. Thus, the cleaved probe can be used for hybridizing target nucleic acids and the cleaved probe can be extended using the method set forth previously herein.

[0151] In some aspects, a nucleic acid probe can include one more cleavage sites. In some aspects, a nucleic acid probe can include two different cleavage sites. In aspects where the nucleic acid probe includes primer binding sites, a first cleavage site can be located between a first primer binding site and one or more other sequence elements of the probe. A second cleavage site can be located between a second primer binding site and one or more other sequence elements of the probe. The cleavage sites can be reactive to different cleavage reactions such that each one can be selectively cleaved without necessarily cleaving the other. Accordingly, the first cleavage site can be cleaved prior to modifying the probe (for example, prior to producing an extended probe), thereby separating the first primer binding site from one or more other sequence elements that remain attached to a solid support. The second cleavage site can be cleaved after modifying the probe (for example, after producing the extended probe), thereby releasing the modified probe for subsequent detection.

[0152] Alternatively, a nucleic acid probe can include the first cleavage site and a primer that is used to capture or amplify the nucleic acid probe can include the second cleavage site. In this configuration, the first cleavage site can be located between a first primer binding site and one or more other sequence elements of the probe such that cleavage separates the first primer binding site from one or more other sequence elements of the probe that remain attached to a solid support. Again, this first cleavage step will typicallybe carried out prior to modifying the probe (for example, prior to producing an extended probe). A second cleavage step can be carried out to cleave the second cleavage site after modifying the probe (for example, after producing the extended probe), thereby releasing the modified probe for subsequent detection. Thus, this cleavage site is useful for the release of modified probes (e.g. extended probes) to detect the sequence information and determine what sequences are present in a biological specimen and where the sequences are present in the specimen.

[0153] In some aspects, one or more probes that are contacted with a solid support in a method set forth herein can include a sequencing primer binding site.

[0154] Accordingly, a modified probe (e.g. extended probe) can be detected in a sequencing technique that includes a step of hybridizing a sequencing primer to the sequencing primer binding site. The sequencing primer binding site can be located in the probe such that cleavage of a modified version of the probe (e.g. an extended probe) will yield a released probe that includes the sequencing primer binding site. The sequencing primer binding site can be a universal sequencing primer binding site such that a plurality of different probes (e.g. having different barcode and / or target sequences) will have the same sequencing primer binding site.

[0155] In some aspects, the nucleic acid probe comprises a template switching oligonucleotide (TSO) sequence. In some aspects, the nucleic acid probe does not comprise a template-switching oligonucleotide (TSO) sequence.

[0156] In some aspects, the nucleic acid probe comprises a restriction enzyme recognition sequence. In some aspects, the nucleic acid probe comprises an adapter sequence and / or a primer sequence. In some aspects, the nucleic acid probe comprises a nuclease recognition sequence.

[0157] In some aspects, the nucleic acid probe is a capture probe. In some aspects, the capture probe comprises a capture sequence. In some aspects, for example in a single oligonucleotide approach, the capture probe comprises a capture sequence and a spatial barcode sequence. In some aspects, for example in a dual oligonucleotide approach, the capture probe does not comprise a spatial barcode sequence.

[0158] In some aspects, the nucleic acid probe is a spatial barcode probe. In some aspects, the spatial barcode probe comprises a spatial barcode sequence. In some aspects, the spatial barcode probe does not comprise a capture sequence.Spatial Barcode Sequences and Spatial Barcode Probes

[0159] In some aspects, the nucleic acid probe comprises at least one spatial barcode. As used herein, the term “barcode,” “spatial barcode,” or “spatial tag,” is intended to mean a series of nucleotides in an oligonucleotide that can be used to identify the oligonucleotide, a spatial address on a surface, a characteristic of the oligonucleotide, or a manipulation that has been carried out on the oligonucleotide. In some aspects, the spatial barcode correlates to a positional location on the substrate. The spatial barcode can be unique to a positional location on the substrate. In some aspects, the spatial barcode tags the nucleic acid probe.

[0160] In some aspects, the barcode sequence is a naturally occurring nucleotide sequence or a nucleotide sequence that does not occur naturally in the organism from which the barcoded nucleic acid was obtained. In some aspects, the barcode sequence is unique to a single nucleic acid species in a population or a barcode sequence can be shared by several different nucleic acid species in a population. For example, each nucleic acid capture probe in a population on a substrate for spatial capture of nucleic acids in a biological sample, e.g., a permeabilized tissue sample, a cell suspension, can include different barcode sequences from all other nucleic acid capture probes in the population. Alternatively, each nucleic acid probe in a population can include different barcode sequences from some or most other nucleic acid capture probes in a population. For example, each capture probe in a population can have a barcode that is present for several different capture probes in the population even though the capture probes with the common barcode differ from each other at other sequence regions along their length. In various aspects, one or more barcode sequences that are used with a biological tissue are not present in the genome, transcriptome, or other nucleic acids of the tissue sample. For example, barcode sequences can have less than 80%, 70%, 60%, 50%, or 40% sequence identity to the nucleic acid sequences in a particular tissue.

[0161] Barcode sequences can be any of a variety of lengths. Longer sequences can generally accommodate a larger number and variety of barcodes for a population. Generally, all probes in a plurality will have the same length barcode (albeit with different sequences), but it is also possible to use different length barcodes for different probes. A barcode sequence can be at least 2, 4, 6, 8, 10, 12, 15, 20 or more nucleotides in length. Alternatively or additionally, the length of the barcode sequence can be at most 20, 15, 12, 10, 8, 6, 4, or fewer nucleotides. Examples of barcode sequences that can be used are setforth, for example in, US Pat. App. Publ. No. 2014 / 0342921 Al and US Pat. No. 8,460,865, each of which is incorporated herein by reference.

[0162] In some aspects, the spatial barcode probe is blocked at its 3’ end to prevent extension of the spatial barcode probe. For example, extension can be blocked by modifying the 3’ end of the spatial barcode probe to include 3'ddC, 3' Inverted dT, 3' C3 spacer, and 3' phosphorylation.

[0163] In some aspects, a nucleic acid probe is a spatial barcode probe. In aspects where the method comprises a spatial barcode probe, the spatial barcode probe comprises a spatial barcode sequence. In some aspects, the spatial barcode probe does not comprise a capture sequence. In some aspects, the spatial barcode probe comprises, in a 5’ to 3’ direction, a primer sequence (e.g., Adp2), a spatial barcode sequence, an optional UMI, and a TSO. In some aspects, the spatial barcode probe can comprise additional sequences, such as a restriction enzyme recognition sequence, and a nuclease recognition sequence, for example.Capture Sequences and Capture Probes

[0164] In some aspects, the nucleic acid probes hybridize to target nucleic acids of a biological sample via a capture sequence. In some aspects, the nucleic acid probe can comprise a capture sequence. As used herein, a “capture sequence” refers to a nucleic acid sequence that is generally complementary to a target molecule. In some aspects, the capture sequence hybridizes to its complementary target molecule thereby capturing the target molecule. In some aspects, each of the plurality of capture probes comprises the same capture sequence. In some aspects, the plurality of capture probes comprises multiple, different capture nucleotide sequences. In some aspects, the multiple, different capture nucleotide sequences comprise one or more gene-specific capture sequences, one or more universal capture sequences, or a combination thereof. In some aspects, a plurality of different nucleic acid probes can include different target capture sequences that hybridize to different target nucleic acid sequences from a tissue sample. Different target capture sequences can be used to selectively bind to one or more desired target molecules in a tissue sample.

[0165] In some aspects, the capture sequence is a gene-specific nucleic acid sequence. As used herein, the term "gene-specific" or "target specific" when used in reference to a capture probe or other nucleic acid is intended to mean a capture probe or other nucleic acid that includes a nucleotide sequence specific to a targeted nucleic acid, e.g., a nucleic acid froma tissue sample, namely a sequence of nucleotides capable of selectively annealing to an identifying region of a targeted nucleic acid. Gene-specific capture probes can have a single species of oligonucleotide, or can include two or more species with different sequences. Thus, the gene-specific capture probes can be two or more sequences, including 3, 4, 5, 6, 7, 8, 9 or 10 or more different sequences. The gene-specific capture probes can comprise a gene-specific capture primer sequence and a universal capture probe sequence. Other sequences such as sequencing primer sequences and the like also can be included in a genespecific capture primer.

[0166] In some aspects, the capture sequence can be a universal capture sequence. As used herein, the terms "universal capture sequence" or “universal sequence” refer to a series of nucleotides that is common to two or more nucleic acid molecules even if the molecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of molecules can allow the capture of multiple different nucleic acids using a population of universal capture nucleic acids that are complementary to the universal sequence. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication or amplification of multiple different nucleic acids using a population of universal primers that are complementary to the universal sequence. Thus, a universal capture nucleic acid or a universal primer includes a sequence that can hybridize specifically to a universal sequence. Target nucleic acid molecules may be modified to attach universal adapters, for example, at one or both ends of the different target sequences. In some aspects, the universal capture sequence can be a random nucleic acid sequence, or a semi-random nucleic acid sequence. In some aspects the universal capture sequence is a random nucleotide sequence or a nonself complementary semi-random sequence.

[0167] In some aspects, the capture sequence hybridizes to an adaptor region in a nucleic acid sequencing library. In some aspects, the capture sequence can contain a Poly A sequence. In some aspects, the capture sequence can contain a Poly T sequence. As used herein, the terms “Poly T,” or “Poly A” refer to a nucleic acid sequence that comprises two or more thiamine (T) or adenine (A) bases, respectively. A Poly T or Poly A sequence can include at least about 2, 5, 8, 10, 12, 15, 18, 20, 40, 50, 60 or more of the T or A bases. In some aspects, a Poly T sequence can have between 10-60 nucleotides. In some aspects, a Poly T sequence can have between 20-40 nucleotides. In some aspects, the disclosure contemplates use of a "polyTVN" sequence, wherein “T” is a capture nucleotide sequence,“V” is adenine (A), cytosine (C), or guanine (G), and “N” is adenine (A), cytosine (C), guanine (G), or thymine (T) The polyTVN sequence is used, in some aspects, to bias reverse transcription to the base of the poly A tail on the mRNA molecule.

[0168] Any of a variety of target nucleic acids can be captured and analyzed in a method set forth herein including, but not limited to, messenger RNA (mRNA), copy DNA (cDNA), genomic DNA (gDNA), ribosomal RNA (rRNA) or transfer RNA (tRNA). Particular target sequences can be selected from databases and appropriate capture sequences designed using techniques and databases known in the art.

[0169] In some aspects, the multiple, different capture nucleotide sequences comprise one or more gene-specific capture sequences, one or more universal capture sequences, or a combination thereof. In some aspects, the capture nucleotide sequence is a poly-T sequence, a poly-A sequence, a gene-specific capture sequence, or a universal capture sequence, or any combination thereof.

[0170] In some aspects, the capture nucleotide sequences are extended following hybridization of the capture oligonucleotide to the target nucleic acid. In some aspects, the extending of the capture nucleotide sequence is performed using a reverse transcriptase. In some aspects, the extending of the capture nucleotide sequence is performed using a terminal deoxynucleotidyl transferase (TdT). In some aspects, the extending of the capture nucleotide sequence using a terminal deoxynucleotidyl transferase (TdT) occurs in the presence of only one type of substrate (e.g., the extending is performed in the presence of only riboguanosine). In some aspects, the extending of the capture nucleotide sequence using a terminal deoxynucleotidyl transferase (TdT) occurs in the presence of a locked nucleic acid (LNA) riboguanosine. In some aspects, the target nucleic acids are polyadenylated prior to hybridization of the target nucleic acids to the capture nucleotide sequences. In some aspects, the target nucleic acids are polyadenylated using a poly(A) polymerase. In some aspects, the target nucleic acids are polyadenylated using chemical ligation or enzymatic ligation.

[0171] In some aspects, a nucleic acid probe is a capture probe. In aspects where the method comprises a capture probe, the capture probe comprises a capture sequence. In some aspects, the capture probe does not comprise a spatial barcode sequence. In some aspects, the capture probe comprises, in a 5’ to 3’ direction, a primer sequence (e.g., Adpl), and a capture sequence. In some aspects, the capture probe can comprise additionalsequences, such as restriction enzyme recognition sequences, and nuclease recognition sequences.

[0172] In some aspects, the nucleic acid probe comprises a capture sequence and a spatial barcode sequence.

[0173] In some aspects, the plurality of nucleic acid probes comprises at least one capture probe and one spatial barcode probe. In this aspect, the capture probe comprises a capture sequence and the spatial barcode probe comprises a spatial barcode sequence (i.e., the capture sequence and the spatial barcode sequence are on separate probes). For example, in some aspects, the surface comprises (i) a plurality of capture probes, wherein one or more of the plurality of capture probes comprises a first adapter (Adpl) sequence that is immobilized on the surface of the substrate and a capture sequence that is configured to bind to the target nucleic acids of the biological sample; and (ii) a plurality of spatially barcoded probes, wherein one or more of the plurality of spatially barcoded probes comprises a second adapter (Adp2) sequence that is immobilized on the surface, a spatial barcode, and a template switching oligonucleotide (TSO) sequence.Other OligonucleotidesRegeneration Oligonucleotides

[0174] In some aspects, the method further requires contacting substrate with a regeneration oligonucleotide. In some aspects, the regeneration oligonucleotide is a single stranded nucleotide molecule. In some aspects, the regeneration oligonucleotide is a double stranded nucleotide molecule. In some aspects, the regeneration oligonucleotide comprises DNA. In some aspects, the regeneration oligonucleotide comprises RNA. In some aspects, the regeneration oligonucleotide is used to regenerate the nucleic acid probe by using the regeneration oligonucleotide as a template. For example, the nucleic acid probe is extended by any suitable method using the regeneration oligonucleotide as a template.

[0175] In some aspects, the regeneration oligonucleotide comprises sequences that are complementary to the desired sequences of the nucleic acid probe. For example, in some aspects, a regeneration oligonucleotide comprises a sequence that is complementary to the SBS sequence, a sequence that is complementary to the restriction enzyme recognition sequence, and a sequence that is complementary to the capture sequence. Thus, when the regeneration oligonucleotide is hybridized to the nucleic acid probe, the nucleic acid probecan be extended to comprise the complement of the sequences contained in the regeneration oligonucleotide.

[0176] In some aspects, the regeneration oligonucleotide comprises a sequence that is complementary to a primer sequence, a sequence that is complementary to a restriction enzyme recognition sequence, and a sequence that is complementary to a capture sequence. In some aspects, the regeneration oligonucleotide comprises a sequence that is complementary to a to a restriction enzyme recognition sequence, and a sequence that is complementary to a TSO.Template Switch Oligonucleotides

[0177] As used herein, a “template switch oligo” or “TSO” refers to an oligonucleotide useful in a method of DNA sequencing in which the oligonucleotide hybridizes to untemplated cytosine (C) nucleotides added to the end of a target RNA or DNA template by a reverse transcriptase during reverse transcription. For example, the TSO comprises a poly G sequence that binds the poly C sequence added to the target template. In some aspects, the TSO comprises 2-5 guanosines that hybridizes to the untemplated cytosine nucleotides. In some embodiments, the 2-5 guanosines are riboguanosines, or modified or locked nucleic acids. In some aspects, the TSO comprises rGrGrG.

[0178] As used herein, “a sequence that is complementary to the TSO,” “TSO complement,” or “TSO”’ refers to a series of untemplated cytosine (C) nucleotides added to the end of a target RNA or DNA template by a reverse transcriptase during reverse transcription.Blocking Oligonucleotides

[0179] In some aspects, after the target mRNA is captured by the capture probes and first- strand cDNA is synthesized, any unbound capture probes are typically digested with an exonuclease. The degradation of unbound capture probes prevents the substrate from being reused. In some aspects, the present disclosure provides a method for regenerating reusable substrates for spatially tagging a target molecule. In some aspects, the method includes adding a blocking oligonucleotide to the substrate to prevent the degradation of unbound capture probes. In some aspects, the blocking oligonucleotide can inhibit second strand synthesis.

[0180] As used herein, the term “blocking oligonucleotide” refers to an oligonucleotide that hybridizes to a nucleic acid probe. The blocking oligonucleotide comprises at least one nucleotide. In some aspects, the blocking oligonucleotide is 1 to 200 nucleotides long. In some aspects, the blocking oligonucleotide comprises more than 200 nucleotides.

[0181] As used herein, the term “unbound capture probe” or “unextended capture probe” refers to a capture probe that did not bind a target molecule. For example, an unbound capture probe can mean a capture probe that did not bind to mRNA released from a tissue sample.

[0182] In some aspects, the blocking oligonucleotide hybridizes directly to unbound capture probes. In some aspects, the blocking oligonucleotide can universally hybridize to all unbound capture probes. In some aspects, the blocking oligonucleotide can hybridize to specific unbound capture probes.

[0183] In some aspects, the blocking oligonucleotide is complementary to a nucleic acid probe. In some aspects, the blocking oligonucleotide is complementary to a capture probe. In some aspects, the blocking oligonucleotide hybridizes to all or a portion of the capture probe. In some aspects, the blocking oligonucleotide is complementary to the capture sequence, or a portion thereof. In some aspects, the blocking oligonucleotide hybridizes to the capture sequence and the SBS sequence regions of the capture probe, or a portion thereof. In some aspects, the blocking oligonucleotide comprises (1) a sequence that is complementary to the SBS sequence of the capture probe, and (2) a sequence that is complementary to the capture sequence. In some aspects, the blocking oligonucleotide can comprise a PolyA sequence. In some aspects, a PolyA sequence of the blocking oligonucleotide hybridizes to a PolyT sequence of the capture probe. In some aspects, a PolyA sequence of the blocking oligonucleotide can be longer than a PolyT sequence of the capture probe and extend past the capture probe. For example, a PolyA sequence of the blocking oligonucleotide can be about 30 nucleotides long, whereas a PolyT sequence of the capture probe can be 20 nucleotides long. In some aspects, the blocking oligonucleotide prevents degradation of the capture probe.

[0184] In some aspects, the blocking oligonucleotide is complementary to the TSO sequence, or a portion thereof, and prevents degradation of the spatial barcode probe.

[0185] In some aspects, the blocking oligonucleotide can extend past the SBS sequence region through the inclusion of a linker. In some aspects, the blocking oligonucleotide canextend past the spatial barcode sequence region of a capture probe. Suitable linkers are well known in the art and in many cases are even commercially available.

[0186] In some aspects, a blocking oligonucleotide can incorporate a 5’ modification or overhang to inhibit or block ligation. In some aspects, a blocking oligonucleotide can incorporate 3’ modifications to block extension of the blocking oligonucleotide.

[0187] In some aspects, a blocking oligonucleotide can comprise a synthetic or modified backbone. In some aspects, the synthetic or modified backbone of the blocking oligonucleotide can inhibit chemistry on double-stranded DNA.

[0188] In some aspects, a blocking oligonucleotide can incorporate at least one biotin molecule. In some aspects, the biotin molecule can be used for purification and / or removal.

[0189] A method for regenerating a substrate for spatially tagging a target molecule in a sample can include the steps of (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation comprises a spatial barcode, and a capture sequence, (b) contacting a sample with the nucleic acid probes on the substrate, (c) hybridizing the capture sequences of the nucleic acid probes to target molecules from the sample that are proximal to the nucleic acid probes, (d) extending the capture sequences to produce extended probes that comprise sequences of the target molecules, or portions thereof, and copies of the spatial tag sequences, thereby spatially tagging the target nucleic acids of the biological sample, (e) contacting the substrate with a blocking oligonucleotide that is complementary to all or a portion of at least one capture probe, wherein the blocking oligonucleotide hybridizes to at least one unbound capture probe preventing degradation of the unbound capture probe. In some aspects, the blocking oligonucleotide can inhibit second strand synthesis.

[0190] The present disclosure further provides a reusable substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and an oligonucleotide, wherein the nucleic acid probe comprises, in a 5' to 3' orientation, a spatial barcode and a capture domain, and wherein the oligonucleotide inhibits second strand synthesis. In some aspects, the nucleic acid probe can contain additional components, as discussed above.C. Contacting the Surface with a Target Molecule

[0191] A disclosed method can include a step of hybridizing nucleic acid probes, that are on a substrate, to target nucleic acids that are from portions of the sample that are proximal to the probes. In some aspects, a biological sample (e.g., a tissue section) can be attached to a surface, for example, using techniques and compositions described in, for example, U.S. Patent No. 11,390,912, incorporated by reference herein in its entirety. In some aspects, a tissue sample can be permeabilized and the cells of the tissue lysed when the tissue is in contact with a surface. Target nucleic acids that are released from a tissue that is permeabilized can be captured by capture oligonucleotides on the surface. In some aspects, the biological sample is digested.

[0192] In some aspects, the tissue sample is treated to improve the capture of the target molecule by the nucleic acid probes. For example, a tissue sample can be treated to remove embedding material (e.g. to remove paraffin or formalin) from the sample prior to release, capture, or modification of nucleic acids. This can be achieved by contacting the sample with an appropriate solvent (e.g. xylene and ethanol washes). Treatment can occur prior to contacting the tissue sample with a substrate set forth herein or the treatment can occur while the tissue sample is on the substrate. Exemplary methods for manipulating tissues for use with solid supports to which nucleic acids are attached are set forth in US Pat. App. Publ. No. 2014 / 0066318 Al, which is incorporated herein by reference.

[0193] Generally, a target nucleic acid will diffuse from a region of the sample to an area of the substrate that is in proximity to that region of the specimen. Here the target nucleic acid will interact with nucleic acid probes that are proximal to the region of the specimen from which the target nucleic acid was released. A target-probe hybrid complex can form where the target nucleic acid encounters a complementary target capture sequence on a nucleic acid probe. The location of the target-probe hybrid complex will generally correlate with the region of the sample from where the target nucleic acid was derived. In multiplex aspects, the substrate will include a plurality of nucleic acid probes, the sample will release a plurality of target nucleic acids and a plurality of target-probe hybrids will be formed on the solid support. The sequences of the target nucleic acids and their locations on the substrate will provide spatial information about the nucleic acid content of the sample. Although the example above is described in the context of target nucleic acids that are released from a sample, it will be understood that the target nucleic acids need not be released. Rather, the target nucleic acids may remain in contact with the sample, forexample, when they are attached to an exposed surface of the sample in a way that the target nucleic acids can also bind to appropriate nucleic acid probes on the substrate.

[0194] In some aspects, the biological tissue is removed from the surface after generation of a complementary template-switching oligonucleotide (TSO’) sequence on the first complementary strands. In some aspects, the biological tissue is removed from the surface after formation of the first complementary strands. In some aspects, the digestion of the biological sample occurs after generation of the first complementary strands. In some aspects, digestion of the biological sample occurs after generation of the first complementary strands but prior to generation of second complementary strands. In some aspects, the target nucleic acids (e.g., RNA) are removed from the surface. In some aspects, removal of target nucleic acids from the surface occurs after generation of the first complementary strands. In some aspects, removal of the target nucleic acids occurs after generation of the first complementary strands but prior to generation of second complementary strands. In some aspects, removal of the target nucleic acids from the surface is achieved by changing a condition. In further aspects, the condition is temperature, pH, formamide concentration, Mg+ based RNA fragmentation, RNAse digestion, or a combination thereof.

[0195] In some aspects, the substrate can contain a plurality of nucleic acid probes, and the tissue sample can release a plurality of target molecules, thereby forming a plurality of target molecule-probe complexes on the substrate. The sequences of the target molecules and their locations on the support will provide spatial information about the nucleic acid content of the tissue sample.

[0196] In some aspects, the nucleic acid probes are randomly located on the solid support.The identity and location of the nucleic acid probes may have been decoded prior to contacting the tissue sample with the solid support. Alternatively, the identity and location of the nucleic acid probes can be determined after contacting the solid support with the tissue sample.D. Probe Extension and First-Strand cDNA Generation

[0197] In some aspects, after a target molecule is hybridized to a capture sequence, the nucleic acid probe is extended to produce an extended probe. In some aspects, the capture sequence is extended to form an extended probe. In some aspects, the hybridized target molecule is used as a template to extend the nucleic acid probe. In some aspects, apolymerase is added to extend the nucleic acid probe. In some aspects, the extended nucleic acid probe comprises a nucleic acid sequence that is complementary to the target molecule that is hybridized to the capture sequence. In some aspects, extending the capture sequence produces first strand cDNA.

[0198] In aspects where the nucleic acid probes comprise barcode sequences, the resulting extended probes can include the barcode sequences and sequences complementary to the target molecules. The extended probes can be spatially tagged versions of the target molecules from the tissue sample.

[0199] The nucleic acid probes can be extended by using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the nucleic acid probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the nucleic acid probe via chemical or enzymatic (e.g., ligase catalysis) methods. A nucleic acid can be extended in a template-directed manner, whereby the product of extension is complementary to a template nucleic acid that is hybridized to the nucleic acid that is extended. In some aspects, a DNA primer is extended by a reverse transcriptase using an RNA template, thereby producing a cDNA. Thus, an extended probe made in a method set forth herein can be a reverse transcribed DNA molecule. All or part of the target molecule that is hybridized to a nucleic acid probe can be copied by extension. DNA molecule. Exemplary methods for extending nucleic acids are set forth in US Pat. App. Publ. No. US 2005 / 0037393 Al or US Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0200] The sequences of the extended probes identify what nucleic acids are in the tissue sample and where in the tissue sample the target molecules are located. It will be understood that other sequence elements that are present in the nucleic acid probes can also be included in the extended probes. Such elements include, for example, primer binding sites, cleavage sites, other tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like.

[0201] In some aspects, the target molecule is removed from the substrate after first strand cDNA synthesis. The target molecule can be removed by any suitable method known in the art. For example, in a method where the target molecule is mRNA, the target molecule can be removed by treating the substrate with 0.1 N NaOH. In other aspects, the target moleculecan be removed by dehybridizing the target molecule from the nucleic acid probe. For example, the target molecule can be dehybridized by, for example, altering the pH, adding salt, or elevating the temperature.

[0202] Although the methods of the present disclosure are exemplified by an aspect where probes that are hybridized to target nucleic acids are extended to copy at least a portion of the target nucleic acid, it will be understood that the probes can be modified in alternative ways. The probes that are hybridized to target nucleic acids can be subjected to a reaction that creates a target-specific modification of the probe. A target-specific modification will result only when the probe interacts with a target nucleic acid, for example, via complementary -based hybridization. In many aspects, the target-specific modification will be specific to the sequence of the particular target nucleic acid that interacts with the probe. Examples of useful target-specific modifications include but are not limited to, insertion or addition of a sequence by ligation or transposition (see, for example, US Pat. App. Publ. No. 2010 / 0120098 Al, incorporated herein by reference), chemical modifications such as psoralen crosslinking or addition of a detectable tag moiety, modifications by nucleic acid enzymes, ligation of a hairpin linker, or other modifications set forth in the nucleic acid assays of US Pat. App. Publ. No. US 2005 / 0037393 Al or US Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0203] In some aspects, following formation of a first extended probe (which comprises first strand cDNA), a plurality of template switching oligonucleotides is hybridized to the plurality of non-templated nucleotides of the first extended probe such that each of the plurality of template switching oligonucleotides that is hybridized to the plurality of non- templated nucleotides of the first extended probe is positioned at the terminus of the target nucleic acids that is distal to the surface. The plurality of non-templated nucleotides on the first extended probe is then extended using the template switching oligonucleotides as template, thereby generating a complementary template-switching oligonucleotide (TSO’) sequence on the first complementary strands. In some aspects comprising a templateswitching reaction, the disclosure contemplates that the template switching reaction occurs concurrently with the reverse transcription reaction that is used to generate the first extended probes. For example, the plurality of template switching oligonucleotides may be added directly into the reverse transcription mix.

[0204] Next, the complementary template-switching oligonucleotide (TSO’) sequence is hybridized on one or more of the first complementary strands to the template switchingoligonucleotide (TSO) sequence of one or more of the plurality of spatially barcoded probes, and the template-switching oligonucleotide binding site is extended on the one or more of the first extended probes using the one or more of the plurality of spatially barcoded probes as template, thereby generating a spatially barcoded first strand cDNA comprising a sequence complementary to the spatial barcode (SBC’) and a sequence complementary to the second adapter (Adp2’) sequence on the one or more of the first complementary strands, thereby preparing the immobilized library of target nucleic acids. In some aspects, the template switching oligonucleotide (TSO) sequence of one or more or all of the plurality of spatially barcoded probes is 3' blocked such that extension is blocked.

[0205] In some aspects, an Adp2 primer that is hybridized to the sequence complementary to the second adapter (Adp2’) sequence on the one or more of the first extended probes is extended, thereby generating one or more second complementary strands, which comprise second strand cDNA. In some aspects, the one or more first extended probes and / or the one or more second complementary strands are amplified, thereby generating a plurality of hybridized first extended probes and second complementary strands, or portions thereof. In some aspects, the second complementary strands are removed (eluted) from the surface, after which the second complementary strands are subjected to PCR for amplification.E. Second Strand cDNA synthesis

[0206] In some aspects, second complementary strands comprising spatially tagged second-strand cDNA are generated from the first extended probes. For example, the second strand cDNA can be generated using the extended nucleic acid probe as a template, and so, the second strand cDNA molecule can have, among other things, a sequence that is complementary to the barcode sequence of the nucleic acid probe, and a sequence that can be correlated with the target molecule. In some aspects, the second strand cDNA molecule is dehybridized from the extended probe and subsequently sequenced. The second strand cDNA molecule can be dehybridized by any suitable method known in the art. In some aspects, as described above, the spatially tagged second strand cDNA molecule is generated using a template-switching oligonucleotide (TSO) approach or a random priming approach.

[0207] In some aspects, the second strand cDNA molecule is dehybridized from the extended nucleic acid probe and is sequenced. In some aspects, the second strand cDNA molecule is tagged with the spatial barcode sequence. In some aspects, the second strand cDNA molecule contains a sequence that is identical to or complementary to a portion, orall, of the target molecule sequence. The spatially tagged second strand cDNA molecule can be dehybridized from the extended nucleic acid probe by any method known in the art. The spatially tagged second strand cDNA molecule can be synthesized by any method known in the art. For example, the second strand cDNA molecule can be sequenced by SBS sequencing. In other aspects, the second strand cDNA molecule can be sequenced by Sanger sequencing (i.e., chain termination sequencing), Next Generation sequencing, single-molecule real-time sequencing, ion semiconductor sequencing, combinatorial probe anchor synthesis, sequencing by ligation, nanopore sequencing, or pyrosequencing. The sequence of the cDNA molecule can be analyzed to determine the spatial location of the target molecule.F. Methods of regenerating a substrateRestriction Enzyme-Mediated

[0208] In some aspects, the present disclosure provides a method of regenerating and reusing a substrate for spatially tagging a target molecule in a sample wherein the method comprises a restriction enzyme. In some aspects, the nucleic acid probes further comprise a restriction enzyme recognition sequence. In some aspects, the restriction enzyme recognition sequence is located between the spatial barcode and the capture sequence of the nucleic acid probes. In some aspects, the restriction enzyme recognition sequence is located between the adapter sequence and the capture sequence of the nucleic acid probes. In some aspects, the restriction enzyme recognition sequence is located between the spatial barcode sequence and the TSO sequence.

[0209] In some aspects, the restriction enzyme recognition sequence is a nucleic acid sequence that is recognized by a restriction enzyme. In some aspects, a restriction enzyme is added to the substrate and cleaves the nucleic acid probe at the restriction enzyme recognition sequence. As used herein, the term “restriction enzyme” or “restriction endonuclease” is an enzyme that recognizes a specific nucleotide sequence (i.e., a target sequence) in a target nucleic acid, and will cleave the target nucleic acid at or near every target site, leaving a blunt or a staggered end.

[0210] In some aspects, the restriction enzyme will cleave double-stranded DNA. In some aspects, the restriction enzyme will cleave single-stranded DNA.

[0211] In some aspects, the restriction enzyme is a type Ils restriction enzyme. Type Ils restriction enzymes are endonucleases that have a recognition sequence that is distant fromthe restriction site. In other words, Type Ils restriction endonucleases cleave outside of the recognition sequence to one side. Examples of type II restriction enzymes are NmeAlll (GCCGAG(21 / 19) and FokI, Alwl, Mme I. There are Type Ils enzymes that cut outside the recognition sequence at both sides.

[0212] In some aspects, the restriction enzyme recognizes a rare recognition sequence ( / .< ., rare cutter). Restriction enzymes typically have recognition sequences that vary in number of nucleotides from 4 (such as Msel) to 6 (EcoRI) and even 8 (Notl). Rare cutters are restriction endonucleases that have a relatively long recognition sequence. In some aspects, rare cutters have 6 or more nucleotides that they recognize and subsequently cut.

[0213] In some aspects, the restriction enzyme is a frequent cutter. The term ‘frequent’ in this respect is typically used in relation to the term ‘rare’. Frequent cutting endonucleases (aka frequent cutters) are restriction endonucleases that have a relatively short recognition sequence. In some aspects, frequent cutters typically have 4 or 5 nucleotides that they recognize and subsequently cut.

[0214] Examples of restriction enzymes include, but are not limited to, methylationsensitive restriction enzymes (MSRE), isoschizomers, and restriction fragments.

[0215] Suitable restriction enzymes and their recognition sequences are well known in the art and in many cases are even commercially available (e.g. from New England Biolabs, Beverley MA; ThermoFisher, Waltham, MA or Sigma Aldrich, St. Louis MO). A particularly useful restriction enzyme will break a bond in a nucleic acid strand at a site that is 3'-remote to its binding site in the nucleic acid, examples of which include Type II or Type Ils restriction enzymes. In some aspects, an oligonucleotide that is complementary to the restriction enzyme recognition sequence hybridizes to the restriction enzyme recognition sequence on the nucleic acid probe.

[0216] In some aspects, the present disclosure provides a method for regenerating a substrate for spatially tagging a target molecule in a sample can include the steps of (a) providing a substrate that includes a plurality of nucleic acid probes immobilized on the substrate at the 5' end of the nucleic acid probe, wherein the nucleic acid probes comprises, in a 5' to 3' orientation, (1) a spatial barcode, (2) a restriction enzyme recognition sequence, and (3) a capture sequence; (b) contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe; (c) extending the capture sequence to produce an extended nucleic acid probe; (d) contacting the substrate with a first oligonucleotide that is complementary to the restrictionenzyme recognition sequence under conditions wherein the first oligonucleotide hybridizes to the restriction enzyme recognition sequence of the nucleic acid probe; (e) contacting the substrate with a restriction enzyme, wherein the restriction enzyme cleaves the extended nucleic acid probe; (f) contacting the substrate with a second oligonucleotide; and (g) extending the nucleic acid probe against the template.

[0217] In some aspects, first strand cDNA is generated during step (c), wherein the cDNA is generated using the hybridized target molecule as a template. In some aspects, in step (d) after the nucleic acid probe is extended using the target molecule as a template, a first oligonucleotide is added to the substrate. The first oligonucleotide is complementary to the restriction enzyme recognition sequence, and the first oligonucleotide hybridizes to the restriction enzyme recognition sequence on the nucleic acid probe.

[0218] In some aspects, in step (e), a restriction enzyme can be added to the substrate, and the restriction enzyme can cleave the extended nucleic acid probe. In some aspects, the restriction enzyme can cleave the nucleic acid probe in such a way that the spatial barcode portion of the probe remains attached to the substrate. The restriction enzyme can cleave the nucleic acid probe in an area on the nucleic acid probe that releases the capture sequence with the first strand cDNA that is complementary to the target molecule.

[0219] In some aspects, in step (f), a second oligonucleotide is then added to the substrate, and hybridizes to the remaining nucleic acid probe. To facilitate hybridization, the second oligonucleotide can include a sequence complementary to the remaining probe immobilized to the substrate. In some aspects, the second oligonucleotide can also include a sequence that is complementary to the capture sequence which can be used as a template sequence to regenerate the original capture sequence.

[0220] In some aspects, in step (g), the nucleic acid probe is extended against the template.In some aspects, the first oligonucleotide is removed from the substrate prior to this step. In some aspects, the nucleic acid probe is extended with a polymerase. This allows the nucleic acid probe to be re-generated.

[0221] In some aspects, the barcoded cDNA molecules are released from the array and analyzed, for example, by high throughput next-generation sequencing (NGS), such as sequencing-by-synthesis (SBS).

[0222] In some aspects, the present disclosure further provides method of regenerating a substrate for spatially tagging a target molecule in a sample, wherein the target molecule is hybridized to a nucleic acid probe, comprising (a) providing a substrate comprising at leastone nucleic acid probe, wherein the nucleic acid probe comprises a spatial barcode, a restriction enzyme recognition sequence, and a capture sequence, (b) contacting the substrate with a first oligonucleotide that is complementary to the restriction enzyme recognition sequence under conditions wherein the first oligonucleotide hybridizes to the restriction enzyme recognition sequence of the nucleic acid probe, (c) contacting the substrate with a restriction enzyme under conditions wherein the restriction enzyme cleaves the extended nucleic acid probe, (d) contacting the substrate with a second nucleic acid probe, and (e) extending the nucleic acid probe against the template.

[0223] The present disclosure further provides a reusable substrate for spatially tagging a target molecule. In some aspects, the substrate comprises a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation, a spatial barcode, a restriction enzyme recognition sequence, and a capture sequence.

[0224] In some aspects, the substrate comprises a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises a spatial barcode, a restriction enzyme recognition sequence, and a capture sequence.

[0225] In some aspects, the present disclosure further provides method of regenerating a substrate for spatially tagging a target molecule in a sample comprising (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a restriction enzyme recognition sequence, and a capture sequence, wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode, and a template switch oligonucleotide (TSO) sequence; (b) contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture probe; (c) extending the capture sequence to comprise first strand cDNA; (d) hybridizing a template switch oligonucleotide (TSO’) that is complementary to the TSO sequence to the capture probe; (e) hybridizing the TSO’ sequence and the TSO sequence; (f) extending the capture probe using the TSO sequenceas template to generate an extended capture probe; (g) contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe; (h) contacting the substrate with an oligonucleotide (RE’) that is complementary to the restriction enzyme recognition sequence under conditions wherein the RE’ hybridizes to the restriction enzyme recognition sequence of the extended capture probe; (i) contacting the substrate with a restriction enzyme, wherein the restriction enzyme cleaves the extended capture probe; (j) contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the capture probe, wherein the regeneration oligonucleotide comprises a RE’ sequence, and a sequence that is complementary to the capture sequence; (k) extending the nucleic acid probe against the template to thereby regenerate the capture probe.

[0226] In some aspects, the present disclosure further provides method of regenerating a substrate for spatially tagging a target molecule in a sample comprising (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a restriction enzyme recognition sequence, and a capture sequence; wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode, and a template switch oligonucleotide (TSO) sequence, wherein the spatial barcode probe is 3’ blocked such that extension of the spatial barcode probe is prevented; (b) contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture probe; (c) extending the capture sequence to comprise first strand cDNA; (d) hybridizing a template switch oligonucleotide (TSO’) that is complementary to the TSO sequence to the capture probe; (e) hybridizing the TSO’ sequence and the TSO sequence; (f) extending the capture probe using the TSO sequence as template to generate an extended capture probe; (g) contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe; (h) contacting the substrate with an oligonucleotide (RE’) that iscomplementary to the restriction enzyme recognition sequence under conditions wherein the RE’ hybridizes to the restriction enzyme recognition sequence of the extended capture probe; (i) contacting the substrate with a restriction enzyme, wherein the restriction enzyme cleaves the extended capture probe; (j) contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the capture probe, wherein the regeneration oligonucleotide comprises a RE’ sequence, and a sequence that is complementary to the capture sequence; (k) extending the nucleic acid probe against the template to thereby regenerate the capture probe.

[0227] In some aspects, the present disclosure further provides method of regenerating a substrate for spatially tagging a target molecule in a sample comprising (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a first restriction enzyme recognition sequence, and a capture sequence; wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode, a second restriction enzyme recognition sequence, and a template switch oligonucleotide (TSO) sequence; (b) contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture probe; (c) extending the capture sequence to comprise first strand cDNA; (d) hybridizing a template switch oligonucleotide (TSO’) that is complementary to the TSO sequence to the capture probe; hybridizing the TSO’ sequence and the TSO sequence; (e) extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA, a spatial barcode sequence complement (SBC’) and optionally additional sequences; (f) extending the spatial barcode using the capture probe as a template, generating an extended spatial barcode probe which comprises second strand cDNA and the spatial barcode sequence, and optionally additional sequences; (g) contacting the substrate with a first primer oligonucleotide under conditions wherein the first primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized first primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe; (h) contacting the substrate with a second primer oligonucleotide under conditions wherein the second primer oligonucleotide hybridizes to the extended spatial barcode probe, and extending the hybridized secondprimer oligonucleotide to generate an oligonucleotide that is complementary to the extended spatial barcode probe; (i) contacting the substrate with a first oligonucleotide (RET) that is complementary to the first restriction enzyme recognition sequence under conditions wherein the REF hybridizes to the first restriction enzyme recognition sequence of the extended capture probe; (j) contacting the substrate with a second oligonucleotide (RE2’) that is complementary to the second restriction enzyme recognition sequence under conditions wherein the RE2’ hybridizes to the second restriction enzyme recognition sequence of the extended spatial barcode probe; (k) contacting the substrate with a first restriction enzyme, wherein the first restriction enzyme cleaves the extended capture probe; (1) contacting the substrate with a second restriction enzyme, wherein the second restriction enzyme cleaves the extended spatial barcode probe; (m) contacting the substrate with a first regeneration oligonucleotide under conditions wherein the first regeneration oligonucleotide hybridizes to the capture probe, wherein the first regeneration oligonucleotide comprises a REF sequence, and a sequence that is complementary to the capture sequence; (n) extending the capture probe using the first regeneration oligonucleotide as a template to thereby regenerate the capture probe; (o) contacting the substrate with a second regeneration oligonucleotide under conditions wherein the second regeneration oligonucleotide hybridizes to the spatial barcode probe, wherein the second regeneration oligonucleotide comprises a RE2’ sequence, and a sequence that is complementary to the TSO sequence; (p) extending the spatial barcode probe using the second regeneration oligonucleotide as a template to thereby regenerate the spatial barcode probe.Exonucl ease-Medi ated

[0228] As used herein, “exonuclease” refers to an enzyme that removes nucleotides from the end of a polynucleotide molecule. Suitable exonucleases are well known in the art and in many cases are even commercially available. Examples of exonucleases include, but are not limited to, exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, and exonuclease VI. In some aspects, the exonuclease can be a polymerase, which has 3’ to 5’ exonuclease activity. In some aspects, an exonuclease is added to the substrate to degrade the first strand cDNA that has been incorporated into the capture probe during a functional assay.

[0229] In some aspects, the present disclosure provides a method for regenerating a substrate for spatially tagging a target molecule in a sample can include the steps of (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation comprises a spatial barcode, and a capture sequence, (b) contacting a sample with the nucleic acid probes on the substrate, (c) hybridizing the capture sequences of the nucleic acid probes to target molecules from the sample that are proximal to the nucleic acid probes, (d) extending the capture sequences to produce extended probes that comprise first strand cDNA, or portions thereof, and copies of the spatial tag sequences, thereby spatially tagging the target nucleic acids of the biological sample, and (e) contacting the target nucleic acids with an exonuclease.

[0230] In some aspects, an oligonucleotide (i.e., a “complementary blocker”) is added to the substrate before adding an exonuclease. In some aspects, the complementary blocker is complementary to the nucleic acid probe. The complementary blocker can hybridize to any portion, or all, of the nucleic acid probe. For example, the complementary blocker can hybridize to the capture sequence of the nucleic acid probe. In other aspects, the complementary blocker can hybridize to the capture sequence and to a portion, or all, of the SBS sequence on the nucleic acid probe. In some aspects, the complementary blocker hybridizes to a capture sequence, (e.g., a polyT / 20xT site) in the nucleic acid probe. In some aspects, the complementary blocker is added to the substrate to protect the nucleic acid probe from degradation by the exonuclease.

[0231] In some aspects, an exonuclease is added to the substrate. Any suitable exonuclease can be used. As described above, suitable exonucleases are known in the art. In some aspects, an exonuclease can degrade the first strand cDNA. In a method that includes a complementary blocker that is hybridized to the nucleic acid probe, the exonuclease degrades the first strand cDNA molecule, and optionally other nucleic acids, protecting the nucleic acid probe from degradation. The complementary blocker prevents the exonuclease from degrading the entire nucleic acid probe.

[0232] In some aspects, after the exonuclease degrades the first strand cDNA, the exonuclease can be removed from the substrate. The exonuclease can be removed from the substrate by any suitable method known in the art.

[0233] In some aspects, after the exonuclease is removed from the substrate, the complementary blocker can be dehybridized and removed from the substrate by any suitable method known in the art. For example, For example, the exonuclease can be dehybridized by, for example, altering the pH, adding salt, or elevating the temperature.

[0234] Once the complementary blocker is removed from the nucleic acid probe, the original nucleic acid probe remains. Thus, the nucleic acid probe is regenerated and can be used for subsequent processing.

[0235] In some aspects, the present disclosure also provides a method of spatially tagging a target molecule comprising (a) providing a substrate comprising at least one nucleic acid probe, wherein the nucleic acid probe comprises a spatial barcode sequence, and a capture sequence, (b) contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe, (c) extending the capture sequence to produce an extended nucleic acid probe comprising first strand cDNA, (d) providing an oligonucleotide that is complementary to the capture sequence, (e) contacting the substrate with a blocking oligonucleotide under conditions wherein the blocking oligonucleotide hybridizes to the capture sequence, and (f) contacting the extended probe with an exonuclease, wherein the exonuclease degrades the cDNA of the extended nucleic acid probe.

[0236] In some aspects, the present disclosure provides a method for regenerating a substrate for spatially tagging a target molecule in a sample can include the steps of (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a capture sequence; wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode sequence and a template switch oligonucleotide (TSO) sequence, wherein the spatial barcode probe is 3’ blocked such that extension of the spatial barcode probe is prevented; (b) contacting a sample with the nucleic acid probes on the substrate, (c) hybridizing the capture sequences of the capture probes to target molecules from the sample that are proximal to the nucleic acid probes, (d) extending the capture sequences of the capture probes using the hybridized target molecule as a template to produce extended capture probes that comprise first stand cDNA, or portions thereof; (e) hybridizing a template switch oligonucleotide (TSO’) that iscomplementary to the TSO sequence of the spatial barcode probe to the capture probe; (f) hybridizing the TSO’ sequence of the extended capture probe and the TSO sequence of the spatial barcode probe; (g) extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA, a spatial barcode sequence complement (SBC’) and optionally additional sequences; (h) contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe, wherein the complementary copy of the extended capture probe comprises second strand cDNA and the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended capture probe is optionally removed for analysis and further processing; (i) contacting the substrate with a first oligonucleotide (i.e., a “first complementary blocker”) that is complementary to the capture sequence under conditions wherein the first complementary blocker hybridizes to the capture sequence of the extended capture probe; (j) contacting the substrate with a second oligonucleotide (i.e., a “second complementary blocker”) that is complementary to the TSO sequence (TSO’) under conditions wherein the second complementary blocker hybridizes to the TSO sequence of the extended spatial barcode probe; (k) contacting the substrate with a exonuclease, wherein the exonuclease degrades the extended capture probe from the 3 ’ end of the extended capture probe until the exonuclease is blocked by the first complementary blocker hybridized to the capture sequence, and wherein the spatial barcode probe is not degraded because the second complementary blocker is hybridized to the spatial barcode probe; (1) removing the first and second complementary blockers thereby regenerating the capture probe and the spatial barcode probe.

[0237] In some aspects, the present disclosure provides a method for regenerating a substrate for spatially tagging a target molecule in a sample can include the steps of (a) providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a capture sequence; wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode sequence and a templateswitch oligonucleotide (TSO) sequence; (b) contacting a sample with the nucleic acid probes on the substrate, (c) hybridizing the capture sequences of the capture probes to target molecules from the sample that are proximal to the nucleic acid probes, (d) extending the capture sequences of the capture probes using the hybridized target molecule as a template to produce extended capture probes that comprise first stand cDNA, or portions thereof; (e) hybridizing a template switch oligonucleotide (TSO’) that is complementary to the TSO sequence of the spatial barcode probe to the capture probe; (f) hybridizing the TSO’ sequence of the extended capture probe and the TSO sequence of the spatial barcode probe; (g) extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA, a spatial barcode sequence complement (SBC’), and optionally additional sequences; (h) extending the spatial barcode probe using the capture probe as template to generate an extended spatial barcode probe comprising second strand cDNA, a spatial barcode sequence, and optionally additional sequences; (i) contacting the substrate with a first primer oligonucleotide (e.g., Adp2) under conditions wherein the first primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized first primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe, wherein the complementary copy of the extended capture probe comprises second strand cDNA and the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended capture probe is optionally removed for analysis and further processing; (j) contacting the substrate with a second primer oligonucleotide (e.g., Adl2) under conditions wherein the second primer oligonucleotide hybridizes to the extended spatial barcode probe, and extending the hybridized second primer oligonucleotide to generate an oligonucleotide that is complementary to the extended spatial barcode probe, wherein the complementary copy of the extended spatial barcode probe comprises first strand cDNA and the complement of the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended spatial barcode probe probe is optionally removed for analysis and further processing; (k) contacting the substrate with a first oligonucleotide (i.e., a “first complementary blocker”) that is complementary to the capture sequence under conditions wherein the first complementary blocker hybridizes to the capture sequence of the extended capture probe; (1) contacting the substrate with a second oligonucleotide (i.e., a “second complementary blocker”) that is complementary tothe TSO sequence (TSO’) under conditions wherein the second complementary blocker hybridizes to the TSO sequence of the extended spatial barcode probe; (m) contacting the substrate with a exonuclease, wherein the exonuclease degrades the extended capture probe from the 3’ end of the extended capture probe until the exonuclease is blocked by the first complementary blocker hybridized to the capture sequence, and wherein the exonuclease degrades the extended spatial barcode probe from the 3’ end of the extended spatial barcode probe until the exonuclease is blocked by the second complementary blocker hybridized to the TSO sequence; (n) removing the first and second complementary blockers thereby regenerating the capture probe and the spatial barcode probe.

[0238] In some aspects, the present disclosure further provides a reusable substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and an exonuclease, wherein the nucleic acid probe comprises, in a 5' to 3' orientation, a spatial barcode and a capture domain. In some aspects, the nucleic acid probe can contain additional components, as discussed above.

[0239] In some aspects, the present disclosure further provides a reusable substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and an exonuclease, wherein the nucleic acid probe comprises a spatial barcode and a capture domain.

[0240] In some aspects, the present disclosure further provides a composition comprising at least one nucleic acid probe, wherein the nucleic acid probe comprises a spatial barcode sequence and capture domain, an oligonucleotide that comprises a sequence that is complementary to the capture domain, and an exonuclease.Biotin / Streptavidin Complex-Mediated

[0241] In some aspects, molecules are bound (e.g., covalently) to the surface of the substrate in order to provide a moiety to which complexes can be reversibly coupled and decoupled in subsequent steps so as to provide flowcell reusability. For example, in Figure 9, each molecule 120 includes moiety 121 and moiety 130. Moiety 121 can react with moiety 111, attached to the surface 110, thereby binding molecule 120 to surface 110. Moiety 111 and moiety 121 can include any suitable pair of reactive moieties that form a linkage that is substantially irreversible during operations such as described with referenceto Figure 9. For example, moiety 111 and moiety 121 can include an amine-NHS pair, an amine-imidoester pair, an amine-pentofluorophenyl ester pair, an amine-hydroxymethyl phosphine pair, an amine-carboxylic acid pair, a thiol-maleimide pair, a thiol -haloacetyl pair, a thiol-pyridyl disulfide pair, a thiol-thiosulfonate pair, a thiol-vinyl sulfone pair, an aldehyde- hydrazide pair, an aldehyde-alkoxyamine pair, a hydroxy-isocyanate pair, an azide-alkyne pair, an azide-phosphine pair, an azide-cyclooctyne pair, an azide-norbornene pair, a transcycloctene- tetrazine pair, a norbornene-tetrazine pair, an oxime, a SpyTag- SpyCatcher pair, a SNAP -tag-O6- benzylguanine pair, a CLIP -tag-O2-benzylcytosine pair, or a sortase coupling. Non-limiting examples of moiety pairs 111, 121 that can be used to couple molecule 120 to surface 110 are shown in Table 1 below.Table 1><" >>>"

[0242] As noted above, each molecule 120 includes a moiety 130. Moiety 130 can be coupled to moiety 121 of that molecule via a suitable element, such as a linker 122. Nonlimiting examples of linkers include an alkyl chain or a polymer. Non-limiting examples of polymers for use in linker 122 include poly ether, polyamide, polyester, polyaryl, poly(ethylene glycol) (PEG) or the like. Illustratively, linker 122 can include PEG having between 1 and 10 ethylene glycol units, e.g., PEG1 to PEG10, illustratively PEG2 to PEG6, such as PEG4.

[0243] Moiety 130 can be used to reversibly couple complex 140 to the surface 110, so as to reversibly couple an oligonucleotide to the surface. For example, complex 140 can include moiety 150 which couples to moiety 130, and at least one oligonucleotide 160coupled to moiety 150. In some aspects, complex 140 includes central molecule 151 (such as a protein), that includes one or more active sites to which other elements can be coupled. For example, oligonucleotide 160 can be coupled to a first active site of central molecule 151, e.g., via covalent or non-covalent interaction between a moiety coupled to the oligonucleotide and the first active site (in this, note that active sites can be considered moi eties). In some aspects, moiety 150 can correspond to a second active site on the central molecule 151.

[0244] Oligonucleotide 160 can be single-stranded, and optionally can be bound to moiety 150 via a suitable element, such as a linker (which can be configured similarly as linker 122). Oligonucleotide 160 can be or include a capture primer such as can be used for seeding and / or amplifying a template polynucleotide.

[0245] Complex 140 can be coupled to surface 110 via reaction between moiety 130 coupled to surface 110 and moiety 150 of complex 140. Moiety 130 and moiety 150 can include any suitable pair of reactive moieties that couple to one another in a way that is substantially irreversible during certain operations such as described with reference to Figure 9 (e.g., during seeding, amplifying, and sequencing template polynucleotides), and subsequently can be decoupled from one another during one or more other operations such as described with reference to Figure 9 (e.g., when regenerating the flowcell for reuse after sequencing). In some aspects, moiety 150 can form a non-covalent bond with moiety 130, which can facilitate subsequent removal of complex 140 from the flowcell so that the flowcell can be reused in a manner such as described herein.

[0246] In some aspects, moiety 130 can be selected so as to bond to an active site (moiety 150) of molecule 151. For example, moiety 130 and molecule 151 can include a biotin / streptavidin pair, a polyhistidine-tag (His-tag) / transition metal pair, a DIG / anti-DIG pair, a c-myc / anti-cmyc pair, a GST / glutathione pair, or a FLAG / anti-FLAG pair. Nonlimiting examples of pairs of moiety 130 and central molecule 151 that can be used to couple molecule 120 to complex 140 are shown in Table 2 below. Additionally, nonlimiting examples of reagents that can be used to decouple molecule 120 from complex 140 are shown in Table 2.Table 2

[0247] In some aspects, molecule 151 is or includes a protein having multiple active sites (moieties 150). Oligonucleotide 160 can be coupled to a moiety that is coupled to a firstone of the active sites (e.g., via its own moiety 130 such as exemplified in Table 2), and moiety 130 of molecule 120 can be coupled to a second one of the active sites. Illustratively, molecule 151 can be or include streptavidin or related protein (e.g., neutravidin, avidin, or Strep-Tactin); oligonucleotide 160 can be coupled to biotin or related moiety (e.g., desthiobiotin, dual-biotin, or Strep-tag) that is coupled to a first active site of the molecule 151; and moiety 130 can be or include another biotin or related moiety (e.g., desthiobiotin, dual -biotin, or Strep-tag) that is coupled to a second active site of the molecule 151. Optionally, complex 140 can include multiple oligonucleotides 160, e.g., coupled to different active sites of molecule 151. The oligonucleotides can have the same sequences as one another (e.g., can all be P5, or can all be P7). Alternatively, the oligonucleotides can have different sequences than one another (e.g., can be a mixture of P5 and P7). In nonlimiting examples where molecule 151 includes streptavidin or related protein, the protein can be incubated ahead of time with multiple P5 and / or P7 oligonucleotides 160 which are coupled to respective biotins or related moieties to form a complex which can referred to herein as “streptavidin-dualbiotin-P5 / P7”. In some aspects, the resulting complex 140 can include one, two, or three oligonucleotides 160, leaving at least one available active site available to bind with biotin or related moiety at surface 110 in a manner such as illustrated in Figure 9. As another example, molecule 151 can be or include His-tag which is coupled directly or indirectly to oligonucleotide 160, moiety 130 can be or include a transition metal that is coupled to an active site of the His-tag. As another example, molecule 151 can be or include a transition metal which is coupled directly or indirectly to oligonucleotide 160, moiety 130 can be or include a His-tag with an active site that is coupled to the transition metal.

[0248] Oligonucleotides 160 can be used for seeding, clustering, and sequencing processes (not specifically illustrated), e.g., can be used as primers to generate clusters of amplicons that can be sequenced using sequencing-by-synthesis. Following such sequencing, the flowcell can be regenerated and then reused. For example, reagent 170 can be introduced to decouple complex 140 from moiety 130. For example, reagent 170 can interfere with the bond between moiety 150 and moiety 130 (illustratively, by denaturing central molecule 151, or by competing with the bond between moiety 150 and moiety 130) thereby decoupling complex 140 from moiety 130.

[0249] In some aspects, a nuclease digest is also performed such that nuclease 180 digests polynucleotides in the flowcell (e.g., oligonucleotides 160, and any polynucleotidescoupled thereto), into nucleotides 190. In some aspects, nuclease 180 includes any appropriate nuclease. For example, nuclease 180 can include a polymerase, illustratively a DNA polymerase that has 3' to 5' exonuclease activity. In some aspects, additionally, or alternatively, nuclease 180 can include an exonuclease (such as Exonuclease I, also referred to as Exol). In some aspects, additionally, or alternatively, nuclease 180 can include a nonspecific dsDNA nuclease (such as DNasel). In some aspects, additionally, or alternatively, nuclease 180 can include Micrococcal Nuclease (MNase). MNase digests 5'- phosphodiester bonds of DNA and RNA, yielding 3 '-phosphate mononucleotides and oligonucleotides. Further details regarding MNase are found in the following references, the entire contents of which are incorporated by reference herein: Cuatrecasas et al., “Catalytic properties and specificity of the extracellular nuclease of Staphylococcus aureus,” J. Biol. Chem. 242(7): 1541-1547 (1967); Craig et al., “Plasmid cDNA- directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system,” Nucleic Acids Res. 20(19): 4987-4985 (1992); and O’Neill et al., “Immunoprecipitation of native chromatin: NChIP,” Methods 31(1): 86-82 (2003).

[0250] In some aspects, nuclease 180 can include a combination of different nucleases, such as a combination of DNasel and Exol. In some aspects, nuclease 180 can consist essentially of MNase. MNase can be particularly potent, giving significantly less contamination from run to run when compared to a combination of DNasel and Exol (so the single enzyme MNase can in some aspects perform better than the 2 enzymes together), as shown in Figure 11.

[0251] At any suitable time after using reagent 170 and nuclease 180, the surface 110 can be washed, a new set of molecules 140 can be introduced, and the cycle repeated. The new set of complexes 140 can be of the same type or of a different type than the original set of complexes 140. Illustratively, the new set of complexes can include oligonucleotides having the same sequence as oligonucleotides 160, or having one or more different sequences than oligonucleotides 160.

[0252] Additionally, kits are provided herein that include one or more elements such as described with reference to Figure 9. For example, a kit can include a plurality of molecules 120 configured to be coupled to surface 110. Each molecule 120 includes first moiety 130. The kit can include a plurality of complexes 140. Each complex 140 includes second moiety 150, which can couple to first moiety 130. Each complex 140 also includes oligonucleotide 160 which is coupled to second moiety 150. The kit can include a reagent 170 to decouplefirst moiety 130 and second moiety 150 from one another. The kit can include at least one nuclease 180 to digest polynucleotides. Note, however, that kits need not necessarily include molecules 120. Instead, the flowcell can come with a surface that already includes first moiety 130 which is ready to be coupled to complexes 140 in the kit.

[0253] Figure 10 illustrates a method of reusing a flowcell with biotin / streptavidin complexes. For example, Figure 10 shows biotin grafted onto the surface of the substrate which captures streptavidin complexes with P5 / P7 primers bound. In some aspects, the complex can be a streptavidin-dualbiotin molecule. In some aspects, alkyne-PEG-biotin can be grafted to the substrate. In some aspects, 5’ biotinylated P5 and P7 oligonucleotides can bind to the alkyne-PEG-biotin via streptavidin. In some aspects, the streptavidin and 5’ biotinylated P5 and P7 oligonucleotides can be stripped from the alkyne-PEG-biotin after processing. In some aspects, after previous streptavidin and 5’ biotinylated P5 and P7 oligonucleotides can be stripped from the alkyne-PEG-biotin, fresh 5’ biotinylated P5 and P7 oligonucleotides can be coupled to the alkyne-PEG-biotin via streptavidin.

[0254] In some aspects, a nuclease can be used in between sequencing runs. For example, Figure 11 illustrates the reduction of run-to-run contamination using a nuclease and additionally the improvement with MNase over DNasel. Data illustrated here was collected using a HiSeqX instrument sequencing a human DNA library followed by a second run sequencing a PhiX library after regenerating the flowcell either without a nuclease step or with a nuclease as will be described. The percentage alignment human reads within PhiX reads in the second run was used to determine how much DNA contamination remained on the flowcell between the first and second runs. Figure 11 shows that there is natural run-to- run contamination without extra reagents for reuseable flow cells.

[0255] It can be seen that reusing the flowcell without using a nuclease to digest polynucleotides between runs resulted in a percent alignment of about 3.4%, which was an unacceptably high level of contamination in the second run. In comparison, reusing the flowcell using a combination of DNAsel and Exol to digest the previous runs’ nucleotides resulted in a significantly lower level of contamination in the second run, about 0.012%. From this, it can be understood that using a nuclease between sequencing runs can reduce or inhibit contamination from the previous sequencing run. In still further comparison, reusing the flowcell using only MNase to digest the previous runs’ nucleotides resulted in an even lower level of contamination in the second run, about 0.0019%. From this, it can be understood that using MNase can significantly reduce contamination from run to run.For example, MNase was observed here to provide about six times less contamination than the combination of DNasel and Exol. As such, MNase can be a particularly useful nuclease for use in reducing run-to-run contamination when reusing a flowcell.

[0256] In some aspects, the present disclosure provides substrate comprising at least one nucleic acid probe, wherein the nucleic acid probe comprises: a spatial barcode, and a capture sequence; and an oligonucleotide that inhibits second strand synthesis. In some aspects, the present disclosure provides a method of regenerating a substrate comprising: (a) providing a substrate comprising at least one nucleic acid probe, wherein the nucleic acid probe comprises: a spatial barcode sequence, and a capture sequence; (b) contacting the substrate with an oligonucleotide that is complementary to the capture sequence, wherein the oligonucleotide hybridizes to the capture sequence; (c) and preventing second strand synthesis. In some aspects, the oligonucleotide is complementary to the capture sequence. In some aspects, the oligonucleotide hybridizes to the capture sequence. In some aspects, the nucleic acid probe further comprises a first strand cDNA generated from a target molecule in a sample. In some aspects, the oligonucleotide extends past the spatial barcode region through the inclusion of a linker. In some aspects, the linker comprises a Poly(A) sequence. In some aspects, oligonucleotide incorporates a 5’ modification or overhang to inhibit ligation. In some aspects, oligonucleotide comprises a modified or synthetic backbone. In some aspects, the oligonucleotide incorporates biotin. In some aspects, the oligonucleotide incorporates 3’ modifications to block extension of a blocking oligonucleotide. In some aspects, the oligonucleotide is complementary to the capture sequence, the substrate further comprises a streptavidin complex with bound P5 / P7 oligonucleotides. In some aspects, alkyne-PEG-biotin is grafted to the substrate. In some aspects, 5’ biotinylated P5 and P7 oligonucleotides bind to the alkyne-PEG-biotin via streptavidin. In some aspects, the streptavidin and 5’ biotinylated P5 and P7 oligonucleotides are stripped from the alkyne-PEG-biotin after processing. In some aspects, fresh 5’ biotinylated P5 and P7 oligonucleotides are coupled to the alkyne-PEG-biotin via streptavidin. In some aspects, the streptavidin and 5’ biotinylated P5 and P7 oligonucleotides are stripped with HiSeqX PE from the alkyne-PEG-biotin after processing. In some aspects, a DNase is used between rounds of processing. In some aspects, the substrate is a flowcell, an array, or beads. In some aspects, the flowcell further comprises a reversible linkage. In some aspects, a surface of the flowcell comprising a first moiety is generated by grafting a molecule containing the first moiety onto the surface. Insome aspects, the first moiety is grafted onto the azides of a polymer coating the surface of the flowcell. In some aspects, the first moiety is a molecule containing biotin. In some aspects, a second moiety is used to bind surface primers to the first moiety. In some aspects, the second moiety is a streptavidin-dualbiotin molecule. In some aspects, the reversible linkage reactions are reversed using a reagent. The method of claim 35, wherein the reagent is hot formamide.

[0257] In some aspects, the present disclosure provides a method of regenerating a flowcell comprising: (a) grafting a first moiety to a surface of a flowcell, and (b) binding a second moiety to the first moiety. In some aspects, the first moiety is alkyne-PEG-biotin. In some aspects, the alkyne-PEG-biotin is grafted to the azides of a polymer coating the surface of the flowcell. In some aspects, the second moiety is 5’ biotinylated P5 and P7 oligonucleotides coupled to streptavidin. In some aspects, the second moiety is decoupled from the first moiety after processing. In some aspects, a reagent is used to decouple the second moiety from the first moiety. In some aspects, the reagent is hot formamide.

[0258] In some aspects, the present disclosure provides a method of regenerating a substrate comprising: (a) grafting a first moiety to a surface of the substrate, (b) binding clustering primers to the surface of the substrate, and (c) binding a second moiety to the first moiety. In some aspects, the method further comprises clustering the spatial barcode and linearizing. In some aspects, the method further comprises binding streptavidin-biotin- 20T-SBS12 complex to the alkyne-PEG-biotin that is grafted on the surface of the substrate. In some aspects, the method further comprises contacting a sample with the substrate, and hybridizing target nucleic acids the capture sequences of the oligonucleotide probes that are conjugated to the biotin molecules of the streptavidin-biotin-20T-SBS12 complex. In some aspects, the method further comprises generating first stand cDNA using the hybridized target nucleic acid as a template, and collecting the first strand cDNA for further processing and analysis. In some aspects, the method further comprises removing the streptavidin-biotin-20T-SBS12 complexes from the surface of the substrate. In some aspects, the method further comprises binding new streptavidin-biotin-20T-SBS12 molecules the surface, thereby regenerating the substrate.

[0259] In some aspects, the clustering primers bound to the surface of the substrate are P5 and Gz. In some aspects, the streptavidin-biotin-20T-SBS12 complex comprises oligonucleotide probes comprising a capture sequence (e.g., 20T sequence) and a sequencing by synthesis primer (e.g., SBS12) conjugated to biotin molecules, which werebound to streptavidin molecules. In some aspects, the streptavidin-biotin-20T-SBS12 complex comprises additional elements. In some aspects, only one oligonucleotide probe comprising a capture sequence and a sequencing by synthesis primer are conjugated to the biotin molecules. In some aspects, one, two, three, four, five, six, seven, eight, nine, ten, or more than ten oligonucleotide probes comprising a capture sequence and a sequencing by synthesis primer are conjugated to the biotin molecules. In some aspects, only one biotin molecules is bound to the streptavidin molecule. In some aspects, one, two, three, four, five, six, seven, eight, nine, ten, or more than ten biotin molecules are bound to the streptavidin molecule. In some aspects, first strand cDNA is generated with reverse transcriptase. In some aspects, second strand cDNA can be generated using the first strand cDNA as a template, and the second strand cDNA can be removed and collected for further processing and analysis. In some aspects, the streptavidin-biotin molecules are removed from the surface of the substrate using hot formamide. In some aspects, this process is repeated of regenerating the substrate is repeated one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more than twenty times.Nuclease-mediated

[0260] In some aspects, the methods disclosed herein use a nicking reaction. As used herein, the term “nuclease,” “endonuclease,” or “restriction enzyme” is an enzyme that recognizes a specific nucleotide sequence (i.e., a nuclease recognition sequence) in a target oligonucleotide and will cleave the target oligonucleotide at or near every target site, leaving a blunt or a staggered end. In some aspects, a nuclease is added to the substrate and cleaves the nucleic acid probe or an oligonucleotide at a specific nuclease recognition sequence.

[0261] The final product of the nicking reaction could either be a nick (i.e., missing of a single phosphodiester bond) or a gap (i.e., missing of one or more nucleotides) and may be in the form of a 3 ’ OH that can directly be extended by the DNA polymerase or may initially be blocked (e.g. as a 3’-P group), which could then be de-blocked to form an extendable 3 ’-OH group using an appropriate enzymatic or chemical approach.

[0262] In some aspects, the nucleic acid probes or oligonucleotides can contain one or more nuclease recognition sequences. The term “nuclease recognition sequence” refers to a nucleic acid sequence that is recognized by a nuclease. In some aspects, the nucleaserecognition sequence is about 5, 6, or 7 base pairs long. In some aspects, the nuclease recognition sequence is greater than 7 base pairs long. In some aspects, the nuclease recognition sequence is about 12-20 base pairs long.

[0263] In some aspects, the nuclease will cleave double-stranded DNA. In some aspects, the nuclease will cleave single-stranded DNA.

[0264] In some aspects, the nuclease is a type Ils restriction enzyme. Type Ils restriction enzymes are endonucleases that have a recognition sequence that is distant from the restriction site. For example, Type Ils restriction endonucleases cleave outside of the recognition sequence to one side. Examples of type II restriction enzymes are NmeAlll (GCCGAG(21 / 19) and FokI, Alwl, Mme I. There are Type Ils enzymes that cut outside the recognition sequence at both sides.

[0265] In some aspects, the nuclease recognition sequence is a rare cutter sequence. In some aspects, the nuclease recognizes the rare recognition sequence (i.e., rare cutter sequence). In some aspects, nucleases can have recognition sequences that vary in number of nucleotides, for example, from 4 (such as Msel) to 6 (EcoRI) and even 8 (Notl) base pairs. Rare cutters are nucleases that have a relatively long recognition sequence. In some aspects, rare cutters have 6 or more nucleotides that they recognize and subsequently cut.

[0266] In some aspects, the nuclease is a frequent cutter. The term “frequent cutter” refers to nucleases that have a relatively short recognition sequence. In some aspects, for example, frequent cutters typically have 4 or 5 nucleotides that they recognize and subsequently cut.

[0267] Examples of nucleases include, but are not limited to, methylation-sensitive restriction enzymes (MSRE), isoschizomers, and restriction fragments.

[0268] Suitable nucleases and their recognition sequences are well known in the art and in many cases are even commercially available (e.g. from New England Biolabs, Beverley MA; ThermoFisher, Waltham, MA or Sigma Aldrich, St. Louis MO). A particularly useful restriction enzyme will break a bond in a nucleic acid strand at a site that is 3'-remote to its binding site in the nucleic acid, examples of which include Type II or Type Ils restriction enzymes.

[0269] In some aspects, the nuclease is a Cas nickase or a Cas nickase variant. Many Cas enzymes, such as Cas9, introduce staggered cuts on both strands. These Cas enzymes are engineered to have the nickase activity (e.g., a Cas9-D10A nickase, which cuts the target strand, a Cas9-H840A nickase, which cuts the non-target strand, and a Casl2a-R1226A nickase). In aspects where the nickase is a Cas nickase or variant thereof, the nickase canthe Cas nickase can be, for example, Cas9 nickase (Cas9n), Streptococcus pyogenes Cas9 nickase (spCas9n), Streptococcus pyogenes Cas9 High Fidelity nickase (spCas9HFn), Staphylococcus aureus Cas9 nickase (SaCas9n), Staphylococcus aureus Cas9 High Fidelity nickase (SaCas9HFn), Casl2a nickase (Casl2an) or a variant thereof. In addition, the Cas9 nickase can be used in conjunction with a tracerRNA:crRNA or a single-guide RNA.

[0270] In some aspects, the nuclease is a nicking endonuclease. It has been previously demonstrated that the restriction enzyme Nt.BspQI, which has a long seven-base pair recognition sequence, and Sequenase 2.0, in the presence of single-stranded DNA binding proteins, can linearly amplify templates up to five thousand nucleotides in length. See Joneja, A. and X. Huang, “Linear Nicking Endonuclease-mediated Strand Displacement DNA Amplification,” Anal Biochem. (2011) 414.1: 58-69, which is incorporated by reference in its entirety.

[0271] In some aspects, the nicking endonuclease is Nt.BstNBI, Nt.BstSEI, Nt.BspQI, Nt.BbvCI, Nt.AlwI, Nb.BsrDI, Nb.BsmI, Nt.CviPII, Nb.BtsI, Nb.BbvCI, Nb.BssSI, or Nt.BsmAI.

[0272] Some of the currently available nicking endonucleases have been engineered from naturally occurring restriction enzymes by inactivating or replacing one of two subunits that each cleave one DNA strand, or by applying selection strategies to isolate mutant variants of enzymes that nick one strand specifically. It has been possible in some instances to isolate single subunits of heterodimeric enzymes that possess strand-specific nicking activity. These engineered nicking enzymes interact with short recognition sequences that are usually seven or fewer base pairs in length. Protein engineering of homing enzymes has the potential to create sequence- and strand-specific DNA endonucleases with high target specificity. Homing endonucleases are a class of enzymes that recognize DNA target sequences greater than 14 base pairs in length. The LScel homing enzyme was engineered with site- and strand-specific DNA nicking activity and has an 18-base pair recognition sequence. See Niu et al., “Engineering Variants of the I-Scel Homing Endonuclease with Strand-specific and Site-specific DNA-nicking Activity, J. Mol. Biol. (2008) 382: 188-202, which is incorporated by reference in its entirety.

[0273] Accordingly, in some aspects, the nuclease of the methods disclosed herein is a homing endonuclease modified to have nickase activity. For example, the homing endonuclease modified to have nickase activity is an I-Scel nickase, an LAnil nickase, or an I-Dmol nickase. In some aspects, the nuclease is a homing endonuclease and thenuclease recognition sequence is greater than 7 base pairs long. For example, the nuclease recognition sequence may be between 12 and 45 base pairs long. In some aspects, the nuclease recognition sequence is 18 base pairs long. In some aspects, the nuclease recognition sequence is 15 base pairs long. In some aspects, prolonged incubation with homing endonuclease modified to have nickase activity can result in a double stranded cut.

[0274] In some aspects, the nuclease is a chimeric nickase. In some aspects, the chimeric nickase comprises a DNA binding molecule fused to a DNA nicking domain.

[0275] In some aspects, the nuclease is a Zinc finger nickase.

[0276] In some aspects, the nuclease is a transcription activator-like effector (TALE) nickase. In some aspects, the TALE nickase is an engineered TALE nickase comprising a TALE repeat domain fused with a FokI nuclease domain or a MutH nicking variant.

[0277] The nicking can be performed via any of the available methods known in the art.For example, the nicking can be achieved via enzymatic methods. In some aspects, the nuclease is a uracil or Oxo-G dependent repair enzyme. For example, the nuclease is USER, EndoQ, FPG, or OGG. In some aspects, the nicking is accomplished via chemical methods, for example using CCL1.Substrate Regeneration with First Strand cDNA

[0278] In some aspects, a method of regenerating a substrate for spatially tagging a target molecule in a sample comprises generating, collecting, and analyzing first stand cDNA. The method can first include preparing a substrate by attaching nucleic acid probes to the surface of a substrate (e.g., a flow cell). The nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end. The nucleic acid probes can comprise, in a 5' to 3' orientation, a nuclease recognition sequence, an adapter sequence, a spatial barcode sequence, and a capture sequence. The nucleic acid probes can also comprise additional sequences.

[0279] In some aspects, the method further comprises contacting a tissue sample containing target molecules with the substrate and the target molecule hybridizes to the capture sequences of the nucleic acid probes. In some aspects, the target molecule is mRNA. In some aspects, the method can first include treating the tissue sample to improve the capture of the target molecule by the nucleic acid probes.

[0280] In some aspects, after the target molecule is hybridized to the capture sequence of the nucleic acid probe, the nucleic acid probe is extended to comprise first strand cDNA,using the target molecule as a template. The nucleic acid probe can be extended to include the first stand cDNA using any suitable method, for example, by using a polymerase or reverse transcriptase such as a DNA polymerase, such as Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase. Extending the nucleic acid probe using the hybridized target molecule as a template, generates an extended nucleic acid probe that comprises first stand cDNA. The first strand cDNA has a sequence that is complementary to the target molecule that is hybridized to the capture sequence.

[0281] In some aspects, the target molecule is removed from the extended nucleic acid probe using any suitable method known in the art. For example, in aspects where the target molecule is mRNA, RNase H can be used to remove the hybridized target mRNA. In some aspects, an alkaline medium can be used to remove the hybridized target mRNA. For example, a NaOH solution or a KOH solution can be used.

[0282] In some aspects, the method further comprises contacting the substrate with a regeneration oligonucleotide, and the regeneration oligonucleotide hybridizes to the extended nucleic acid probe. The regeneration oligonucleotide comprises, at least, a sequence that is complementary to the capture sequence of the nucleic acid probe. In some aspects, for example, the regeneration oligonucleotide comprises a PolyA sequence, a randomer sequence, a semi-randomer sequence, or a target specific sequence. In some aspects, the regeneration oligonucleotide is not attached to the surface of the substrate. In some aspects, the regeneration oligonucleotide comprises additional sequences.

[0283] In some aspects, after the regeneration oligonucleotide hybridizes to the extended nucleic acid probe, the regeneration oligonucleotide is extended from the 3’ end, using any suitable method known in the art, to produce an oligonucleotide that is complementary to the nucleic acid probe. For example, in some aspects, the regeneration oligonucleotide is extended using a DNA polymerase, such as Sequenase version 2.0, Klenow Fragment exo- , Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase.

[0284] In some aspects, the method further comprises adding a nuclease that is specific to the nuclease recognition sequence of the nucleic acid probe to the substrate. In some aspects, the nuclease cleaves the extended nucleic acid probe at the nuclease recognition sequence, and produces a first portion of the extended nucleic acid probe and a second portion of the extended nucleic acid probe. The first portion of the extended nucleic acid probe is attached to the surface of the substrate at the 5’ end. The second portion of theextended nucleic acid probe comprises the first strand cDNA, the capture sequence, the spatial barcode sequence, and the adapter sequence.

[0285] In some aspects, the first portion of the extended nucleic acid probe is extended from the free 3’ end, by using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the first portion of the extended nucleic acid probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the first portion of the extended nucleic acid probe via chemical or enzymatic (e.g., ligase catalysis) methods. The first portion of the extended nucleic acid probe can be extended in a template- directed manner, whereby the product of extension is complementary to the regeneration oligonucleotide that is hybridized to the nucleic acid that is extended. Exemplary methods for extending nucleic acids are set forth in US Pat. App. Publ. No. US 2005 / 0037393 Al or US Pat. No. 8,288,103 or 8,486,625, each of which is incorporated herein by reference.

[0286] In some aspects, a DNA polymerase is used to extend the first portion of the extended nucleic acid probe. The DNA polymerase can be a strand-displacing DNA polymerase, or a non-strand displacing polymerase. In aspects where the first portion of the extended nucleic acid probe is extended using a strand-displacing DNA polymerase, the DNA polymerase can be Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase. In aspects where the first portion of the extended nucleic acid probe is extended using a non-strand displacing DNA polymerase, the non-strand displacing DNA polymerase can be used with a helicasedependent amplification (HD A) system or a T4 replisome.

[0287] The first portion of the extended nucleic acid probe is extended using the hybridized regeneration oligonucleotide as a template. Extending the first portion of the extended nucleic acid probe regenerates the nucleic acid probe.

[0288] Extending the first portion of the extended nucleic probe also displaces the second portion of the extended nucleic acid probe. In some aspects, the second portion of the extended nucleic acid probe comprises, at least, the first strand cDNA and the spatial barcode. In some aspects, the displaced second portion of the extended nucleic acid probe can be collected, amplified, and sequenced. The second portion of the extended nucleic acid probe can be analyzed to determine the spatial location of the target molecule.

[0289] The second portion of the extended nucleic acid probes can be collected, amplified, sequenced, and analyzed by any suitable method known in the art, for example, high throughput next-generation sequencing (NGS), such as sequencing-by-synthesis (SBS).

[0290] The sequences of the second portion of the extended nucleic acid probes identify the nucleic acids in the tissue sample, and where in the tissue sample the target molecules are located. It will be understood that other sequence elements that are present in the nucleic acid probes can also be included in the second portion of the extended nucleic acid probes. Such elements include, for example, primer binding sites, cleavage sites, other or additional spatial barcodes or tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like.

[0291] In some aspects, the regeneration oligonucleotide is removed from the regenerated nucleic acid probe, by any suitable method known in the art, to allow for additional rounds of processing.

[0292] In some aspects, the substrate is contacted with a single-stranded DNA-specific 3’- 5’ exonuclease before reusing the substrate. In some aspects, the exonuclease is a Thermus thermophiles exonuclease.Customizable Substrate Regeneration with First Strand cDNA

[0293] In some aspects, a method of substrate regeneration can comprise generating customized nucleic acid probes, and generating a collecting first strand cDNA. In some aspects, a method of regenerating a substrate for spatially tagging a target molecule in a sample can comprise the following process: the method can first include preparing a substrate by attaching nucleic acid probes to the surface of a substrate (e.g., a flow cell). The nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end. The nucleic acid probes attached to the substrate contain, in a 5' to 3' orientation, a spatial barcode sequence, a nuclease recognition sequence, and optionally a linker. In some aspects, the nucleic acid probe can comprise additional sequences.

[0294] In some aspects, the user can make a custom nucleic acid probe. A custom nucleic acid probe, comprises, at least, a customized capture sequence. To make the custom nucleic acid probes, the nucleic acid probes that are attached to the substrate are contacted with a customizable oligonucleotide under conditions wherein the customizable oligonucleotide hybridizes to the nucleic acid probe. The customizable oligonucleotide can comprise a sequence that is complementary to the nuclease recognition sequence, a linker, and acapture sequence. In some aspects, the capture sequence is complementary to a target molecule capture sequence. The target molecule capture sequence is the sequence that will capture the target molecule. For example, if the target molecule capture sequence is a PolyT sequence, the customizable oligonucleotide will comprise a PolyT sequence. Because the custom nucleic acid probe is generated by extending the nucleic acid probe using the custom oligonucleotide as a template, the custom nucleic acid will have a PolyA sequence and capture target molecules with a PolyT sequence.

[0295] In some aspects, the customizable oligonucleotide further comprises additional sequences. For example, in some aspects, the customizable oligonucleotide further comprises an identifier sequence, wherein the identifier sequence is located between the capture sequence and the linker.

[0296] In some aspects, the nucleic acid probe is extended using the customizable oligonucleotide as a template to produce a custom nucleic acid probe. The nucleic acid probe can be extended by any suitable method known in the art, using the hybridized custom oligonucleotide as a template. For example, DNA polymerase can be used to extend the nucleic acid probes to generate a custom nucleic acid probe. In some aspects, the DNA polymerase is Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase.

[0297] Extending the nucleic acid probe using the custom oligonucleotide as a template produces a custom nucleic acid probe that comprises, in a 5' to 3' orientation, a spatial barcode sequence, a nuclease recognition sequence, optionally a linker, and a customized capture sequence. In some aspects, the custom nucleic acid probe contains additional sequences. Figure 14A illustrates the process of making a custom nucleic acid probe.

[0298] In some aspects, the method further comprises contacting a sample containing target molecules with the substrate, and the target molecule hybridizes to the capture sequences of the custom nucleic acid probes. In some aspects, the target molecule is mRNA. In some aspects, the method can first include treating the tissue sample to improve the capture of the target molecule by the nucleic acid probes.

[0299] In some aspects, after the target molecule is hybridized to the capture sequence of the custom nucleic acid probe, the custom nucleic acid probe is extended to comprise first strand cDNA, using the target nucleic acid as a template. The custom nucleic acid probe can be extended to include the first stand cDNA using any suitable method, such as using a polymerase or reverse transcriptase, for example. The first strand cDNA will have asequence that is complementary to the target molecule that is hybridized to the capture sequence. Extending the custom nucleic acid probe using the hybridized target molecule as a template, generates an extended custom nucleic acid probe that comprises first strand cDNA.

[0300] In some aspects, the target molecule is removed from the extended custom nucleic acid probe using any suitable method known in the art. For example, in aspects where the target molecule is mRNA, RNase H can be used to remove the hybridized target mRNA. In some aspects, an alkaline medium can be used to remove the hybridized target mRNA. For example, a NaOH solution or a KOH solution can be used.

[0301] In some aspects, the method further comprises contacting the substrate with a regeneration oligonucleotide, and the regeneration oligonucleotide hybridizes to the extended custom nucleic acid probe. In some aspects, the regeneration oligonucleotide comprises, at least, a sequence that is complementary to the nuclease recognition sequence. In aspects where the custom nucleic acid probe contains a linker sequence, the regeneration oligonucleotide can optionally comprise a sequence that is complementary to the linker sequence. In some aspects, the regeneration oligonucleotide contains additional sequences. In some aspects, the regeneration oligonucleotide is not attached to the surface of the substrate.

[0302] In some aspects, the method further comprises adding a nuclease that is specific to the nuclease recognition sequence of the custom nucleic acid probe to the substrate. The nuclease cleaves the extended custom nucleic acid probe at the nuclease recognition sequence, and produces a first portion of the extended custom nucleic acid probe and a second portion of the extended custom nucleic acid probe. The first portion of the extended custom nucleic acid probe is attached to the surface of the substrate at the 5’ end. The second portion of the extended custom nucleic acid probe comprises at least the first strand cDNA.

[0303] In some aspects, the method further comprises extending the first portion of the extended custom nucleic acid probe from the free 3’ end, by using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the first portion of the extended custom nucleic acid probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the first portion of the extended customnucleic acid probe via chemical or enzymatic (e.g., ligase catalysis) methods. The first portion of the extended custom nucleic acid probe can be extended in a template-directed manner, whereby the product of extension is complementary to a regeneration oligonucleotide that is hybridized to the nucleic acid that is extended.

[0304] In some aspects, the first portion of the extended custom nucleic acid probe is extended using the hybridized regeneration oligonucleotide as a template. Extending the first portion of the extended custom nucleic acid probe regenerates the original nucleic acid probe. Extending the first portion of the extended custom nucleic probe also displaces the second portion of the extended custom nucleic acid probe. The second portion of the extended custom nucleic acid probe comprises at least the first strand cDNA and the spatial barcode. In some aspects, the displaced second portion of the extended custom nucleic acid probe can be collected and analyzed. In some aspects, the second portion of the extended custom nucleic acid probe is analyzed to determine the spatial location of the target molecule. For example, the second portion of the custom extended nucleic acid probes can be collected, amplified, sequenced, and analyzed by any suitable method known in the art, for example, high throughput next-generation sequencing (NGS), such as sequencing-by- synthesis (SBS).

[0305] In some aspects, the sequences of the second portion of the extended custom nucleic acid probes re used to identify the nucleic acids in the tissue sample, and where in the tissue sample the target molecules are located. In some aspects, the other sequence elements that are present in the custom nucleic acid probes can also be included in the second portion of the extended custom nucleic acid probes. Such elements can include, for example, primer binding sites, cleavage sites, other or additional spatial barcodes or tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like.

[0306] In some aspects, the regeneration oligonucleotide is removed from the regenerated nucleic acid probe, by any suitable method known in the art, to allow for additional rounds of processing.

[0307] In some aspects, the substrate is contacted with a single-stranded DNA-specific 3’- 5’ exonuclease before reusing the substrate. In some aspects, the exonuclease is a Thermus thermophiles exonuclease.Substrate Regeneration with Second Strand cDNA

[0308] In some aspects, a method of regenerating a substrate for spatially tagging a target molecule in a sample comprises regenerating the substrate, and generating second strand cDNA. In some aspects, the method first includes preparing a substrate by attaching nucleic acid probes to the surface of a substrate (e.g., a flow cell). In some aspects, he nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end, and contain, in a 5' to 3' orientation, a first nuclease recognition sequence, a first adapter sequence, a spatial barcode sequence, a second nuclease recognition sequence, and a capture sequence.

[0309] In some aspects, the method further comprises contacting a sample containing at least one target molecule (e.g., mRNA) with the substrate and the target molecule hybridizes to the capture sequences of the nucleic acid probes.

[0310] In some aspects, after the target molecule is hybridized to the capture sequence of the nucleic acid probe, the nucleic acid probe is extended to comprise first strand cDNA, using the target molecule as a template. The nucleic acid probe can be extended to include the first stand cDNA using any suitable method, for example, by using a polymerase or reverse transcriptase. The first strand cDNA will have a sequence that is complementary to the target molecule that is hybridized to the capture sequence. Extending the nucleic acid probe using the hybridized target molecule as a template, generates a first extended nucleic acid probe that comprises first strand cDNA. In some aspects, the target molecule is removed from the first extended nucleic acid probe using any suitable method known in the art.

[0311] In some aspects, the substrate is contacted with a first oligonucleotide. The first oligonucleotide can comprise a randomer, and optionally, a second adapter sequence. In some aspects, the first oligonucleotide can comprise additional sequences. In some aspects, the first oligonucleotide is not bound to the surface of the substrate.

[0312] In some aspects, the first oligonucleotide is extended from the 3’ end, using any suitable method known in the art, to produce an extended first oligonucleotide that is complementary to the first extended nucleic acid probe.

[0313] In some aspects, the first oligonucleotide is extended using a template switch oligonucleotide (TSO) approach. For example, a reverse transcriptase adds the TSO complement to the 5’ end of the transcript. In some aspects, the substrate is contacted with a first-strand synthesis mix that includes a reverse transcriptase and a TSO under conditionsto generate an oligonucleotide comprising a cDNA that is complementary to the first extended nucleic acid probe and a TSO complement hybridized to the 5’ end of the first cDNA. The reverse transcriptase incorporates un-templated cytosine nucleotides at the 5' end of the first cDNA. The TSO includes a sequence that hybridizes to the un-templated cytosine nucleotides. The TSO hybridizes to the un-templated cytosine nucleotides and the reverse transcriptase is extended to generate the complement of the TSO attached to the ‘5 end of the cDNA.

[0314] Extending the first oligonucleotide generates an extended first oligonucleotide. The extended first oligonucleotide is complementary to the first extended nucleic acid probe, and comprises second strand cDNA. The extended first oligonucleotide and the extended first nucleic acid probe form a double stranded oligonucleotide.

[0315] In some aspects, the method further comprises a first nuclease that is specific to the first nuclease recognition sequence of the nucleic acid probe is added to the substrate. The first nuclease cleaves the first extended nucleic acid probe at the first nuclease recognition sequence, and produced a first portion of the first extended nucleic acid probe and a second portion of the first extended nucleic acid probe. The first portion of the first extended nucleic acid probe remained attached to the surface of the substrate at the 5’ end. The second portion of the first extended nucleic acid probe comprises the first strand cDNA. The second portion of the first extended nucleic acid probe can further comprise the capture sequence, the spatial barcode sequence, and the first adapter sequence. In some aspects, the second portion of the first extended nucleic acid probe can comprise additional sequences.

[0316] In some aspects, the first portion of the first extended nucleic acid probe is extended from the free 3’ end, by using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the nucleic acid probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the nucleic acid probe via chemical or enzymatic (e.g., ligase catalysis) methods. In some aspects, the first portion of the first extended nucleic acid probe can be extended in a template-directed manner, whereby the product of extension is complementary to the extended first oligonucleotide that is hybridized to the nucleic acid that is extended.

[0317] In some aspects, the first portion of the first extended nucleic acid probe is extended using the extended first oligonucleotide as a template. Extending the first portion of thefirst extended nucleic acid probe generates the second extended nucleic acid probe, which is complementary to the extended first oligonucleotide and is identical to the first extended nucleic acid probe.

[0318] In some aspects, extending the first portion of the first extended nucleic probe also displaces at least a portion the second portion of the first extended nucleic acid probe. In some aspects, the entire second portion of the first extended nucleic acid probe is displaced. The second portion of the first extended nucleic acid probe comprises at least the first strand cDNA and the spatial barcode. In some aspects, the displaced second portion of the first extended nucleic acid probe can be collected, amplified, and sequenced. The second portion of the first extended nucleic acid probe can be analyzed to determine the spatial location of the target molecule. For example, the second portion of the extended first nucleic acid probe can be collected, amplified, sequenced, and analyzed by any suitable method known in the art, for example, high throughput next-generation sequencing (NGS), such as sequencing- by-synthesis (SBS).

[0319] The sequences of the second portion of the first extended nucleic acid probes identify the nucleic acids in the tissue sample and where in the tissue sample the target molecules are located. In some aspects, other sequence elements that are present in the nucleic acid probes are also included in the second portion of the first extended nucleic acid probes. Such elements include, for example, primer binding sites, cleavage sites, other or additional spatial barcodes or tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like. In some aspects, the second portion of the extended first nucleic acid probe can be amplified by additional rounds of nicking and extension.

[0320] In some aspects the method further comprises adding a second nuclease that is specific to the second nuclease recognition sequence to the substrate. In some aspects, the second nuclease cleaved the double stranded oligonucleotide (comprising the second extended nucleic acid probe and the extended first oligonucleotide) at the second nuclease recognition sequence. This produces a first portion of the second extended nucleic acid probe and a second portion of the second extended nucleic acid probe. In some aspects, the second nuclease can also cleave the extended first oligonucleotide to produce a staggered, double stranded cut. The second nuclease can be any suitable nuclease known in the art. Suitable nucleases are described above. For example, the second nuclease can be Tai I. Inthe aspects where the second nuclease makes a double stranded cut, the second nuclease can also be a CRISPR-CAS system, a nuclease, or a zinc finger nuclease, for example.

[0321] In some aspects, the second nuclease only makes a single stranded cut. For example, the second nuclease may only cleave the second extended nucleic acid probe at the second nuclease recognition site, and leave the extended first oligonucleotide intact. In this aspect, where the nuclease makes a single-stranded cut, the nuclease may be any nuclease known in the art to make a single stranded cut.

[0322] In some aspects, the first portion of the second extended nucleic acid probe remained attached to the surface of the substrate at the 5’ end, and contains the spatial barcode sequence. The second portion of the second extended nucleic acid probe comprises, among other things, the first strand cDNA.

[0323] In some aspects, remaining extended first oligonucleotide was dehybridized from the first portion of the second extended nucleic acid probe. The remaining extended first oligonucleotide can be dehybridized by any suitable method known in the art.

[0324] In some aspects, a second oligonucleotide was added to the substrate, and the second oligonucleotide hybridized to the first portion of the second extended nucleic acid probe. The second oligonucleotide comprises at least a sequence that is complementary to the second nuclease recognition site, and a sequence that is complementary to the capture sequence of the nucleic acid probe. The second oligonucleotide can comprise additional sequences. In some aspects, the second oligonucleotide is not bound to the surface of the substrate.

[0325] In some aspects, the method further comprises extending the first portion of the second extended nucleic acid probe from the free 3’ end, using any suitable method known in the art. The first portion of the extended nucleic acid probe is extended using the hybridized second oligonucleotide as a template. Extending the first portion of the extended nucleic acid probe regenerates the nucleic acid probe. This process can be repeated for several additional rounds of processing.

[0326] In some aspects, the substrate is contacted with a single-stranded DNA-specific 3’- 5’ exonuclease before reusing the substrate. In some aspects, the exonuclease is a Thermus thermophiles exonuclease.Customizable Substrate Regeneration with Second Strand cDNA

[0327] In some aspects, a method of substrate regeneration comprises generating customized nucleic acid probes, and generating second strand cDNA. In some aspects, a method of regenerating a substrate for spatially tagging a target molecule in a sample comprises the following process: the method first includes preparing a substrate by attaching nucleic acid probes to the surface of a substrate (e.g., a flow cell). The nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end, and contain, in a 5' to 3' orientation, a first adapter sequence, a spatial barcode sequence, a nuclease recognition sequence, and optionally a linker.

[0328] In some aspects, the user makes a custom nucleic acid probe. In some aspects, a custom nucleic acid probe, comprises, at least, a customized capture sequence. In some aspects, to make the custom nucleic acid probes, the nucleic acid probes that are attached to the substrate are contacted with a customizable oligonucleotide under conditions wherein the customizable oligonucleotide hybridizes to the nucleic acid probe, and the nucleic acid probe attached to the substrate is extended using the customizable oligonucleotide as a template to produce a custom nucleic acid probe. In some aspects, the customizable oligonucleotide comprises a sequence that is complementary to the nuclease recognition sequence, a sequence complementary to a linker (if the nucleic acid probe comprises a linker), and a capture sequence. In some aspects, the capture sequence is complementary to a target molecule capture sequence. The target molecule capture sequence is the sequence that will capture the target molecule. For example, if the target molecule capture sequence is a PolyT sequence, the customizable oligonucleotide will comprise a PolyT sequence. Because the custom nucleic acid probe is generated by extending the nucleic acid probe using the custom oligonucleotide as a template, the custom nucleic acid will have a PolyA sequence and capture target molecules with a PolyT sequence.

[0329] In some aspects, the customizable oligonucleotides also further comprise additional sequences. For example, the customizable oligonucleotide can further comprise an identifier sequence, wherein the identifier sequence is located between the capture sequence and the linker.

[0330] In some aspects, to make a custom nucleic acid probe, the nucleic acid probe is extended by any suitable method known in the art, using the hybridized custom oligonucleotide as a template. Extending the nucleic acid probe using the custom oligonucleotide as a template produces a custom nucleic acid probe that comprises, in a 5'to 3' orientation, a spatial barcode sequence, a nuclease recognition sequence, optionally a linker, and a customized capture sequence. In some aspects, the custom nucleic acid probe contains additional sequences. Figure 16A illustrates the process of making a custom nucleic acid probe.

[0331] In some aspects, the method further comprises contacting a tissue sample containing target molecules with the substrate, and the target molecule hybridizes to the capture sequence of the custom nucleic acid probes. In some aspects, the target molecule is mRNA. In some aspects, the method can first include treating the tissue sample to improve the capture of the target molecule by the nucleic acid probes.

[0332] In some aspects, after the target molecule is hybridized to the capture sequence of the custom nucleic acid probe, the custom nucleic acid probe is extended to comprise first strand cDNA, using the target nucleic acid as a template. The custom nucleic acid probe can be extended to include the first stand cDNA using any suitable method, such as using a polymerase or reverse transcriptase. The first strand cDNA will have a sequence that is complementary to the target molecule that is hybridized to the capture sequence. Extended the custom nucleic acid probe using the hybridized target molecule as a template, generates an extended custom nucleic acid probe that comprises first strand cDNA. In some aspects, the target molecule is removed from the extended nucleic acid probe using any suitable method known in the art.

[0333] In some aspects, the method further comprises contacting the substrate with a first oligonucleotide. In some aspects, the first oligonucleotide comprises a randomer, a second nuclease recognition sequence, and optionally, a second adapter sequence. In some aspects, the first oligonucleotide is not bound to the surface of the substrate.

[0334] In some aspects, the first oligonucleotide is extended from the 3’ end, using any suitable method known in the art, to produce an oligonucleotide that is complementary to the extended custom nucleic acid probe.

[0335] In some aspects, the first oligonucleotide is extended using a template switch oligonucleotide (TSO) approach. For example, a reverse transcriptase adds the TSO complement to the 5’ end of the transcript. In some aspects, the substrate is contacted with a first-strand synthesis mix that includes a reverse transcriptase and a TSO under conditions to generate an oligonucleotide comprising a cDNA that is complementary to the extended custom nucleic acid probe and a TSO complement hybridized to the 5’ end of the first cDNA. The reverse transcriptase incorporates untemplated cytosine nucleotides at the 5'end of the cDNA. The TSO includes a sequence that hybridizes to the untemplated cytosine nucleotides. The TSO hybridizes to the untemplated cytosine nucleotides and the reverse transcriptase is extended to generate the complement of the TSO attached to the ‘5 end of the cDNA.

[0336] Extending the first oligonucleotide generates an extended first oligonucleotide. The extended first oligonucleotide is a complement to the extended custom nucleic acid probe and comprises second strand cDNA. The extended first oligonucleotide and the extended custom nucleic acid probe form a double stranded oligonucleotide.

[0337] In some aspects, a first nuclease that is specific to the first nuclease recognition sequence is added to the substrate. In some aspects, the nuclease cleaves the extended custom nucleic acid probe at the first nuclease recognition sequence, and produces a first portion of the extended custom nucleic acid probe and a second portion of the extended custom nucleic acid probe. The first portion of the extended custom nucleic acid probe remained attached to the surface of the substrate at the 5’ end. The second portion of the extended custom nucleic acid probe comprises the first strand cDNA. The second portion of the extended custom nucleic acid probe can further comprise the capture sequence, the spatial barcode sequence, and the first adapter sequence. In some aspects, the second portion of the extended custom nucleic acid probe can further comprise additional sequences.

[0338] In some aspects, the first portion of the extended custom nucleic acid probe is extended from the free 3’ end, by using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the first portion of the extended custom nucleic acid probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the first portion of the extended custom nucleic acid probe via chemical or enzymatic (e.g., ligase catalysis) methods. The first portion of the extended custom nucleic acid probe can be extended in a template-directed manner, whereby the product of extension is complementary to the extended first oligonucleotide that is hybridized to the first portion of the extended custom nucleic acid probe.

[0339] In some aspects, the first portion of the extended custom nucleic acid probe can be extended with a DNA polymerase. The DNA polymerase can be a strand-displacing DNA polymerase, or a non-strand displacing polymerase. Examples of stand displacing DNApolymerases include Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase. In aspects where the first portion of the extended custom nucleic acid probe is extended using a non-strand displacing DNA polymerase, the non-strand displacing DNA polymerase can be used with a helicasedependent amplification (HD A) system or a T4 replisome.

[0340] In some aspects, the first portion of the extended custom nucleic acid probe is extended using the extended first oligonucleotide as a template. Extending the first portion of the extended custom nucleic acid probe regenerates the extended custom nucleic acid probe, which is complementary to the extended first oligonucleotide.

[0341] In some aspects, extending the first portion of the extended custom nucleic probe also displaces at least a portion the second portion of the extended custom nucleic acid probe. In some aspects, the entire second portion of the extended custom nucleic acid probe is displaced. The second portion of the extended custom nucleic acid probe comprises at least the first strand cDNA. In some aspects, the displaced second portion of the extended custom nucleic acid probe can be collected, amplified, and sequenced. The second portion of the extended custom nucleic acid probe can be analyzed to determine the spatial location of the target molecule. The second portion of the extended custom nucleic acid probe can be collected, amplified, sequenced, and analyzed by any suitable method known in the art, for example, high throughput next-generation sequencing (NGS), such as sequencing-by- synthesis (SBS). In some aspects, the sequences of the second portion of the extended nucleic custom acid probes can identify the nucleic acids in the tissue sample, and where in the tissue sample the target molecules are located. In some aspects, other sequence elements that are present in the nucleic acid probes can also be included in the second portion of the extended custom nucleic acid probes. Such elements include, for example, primer binding sites, cleavage sites, other or additional spatial barcodes or tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like.

[0342] In some aspects, the second portion of the extended custom nucleic acid probe can be amplified by additional rounds of nicking and extension.

[0343] In some aspects, a second nuclease that is specific to the second nuclease recognition sequence of the first oligonucleotide is added to the substrate. The second nuclease cleaves the extended first oligonucleotide at the second nuclease recognitionsequence. This produces a first portion of the extended first oligonucleotide and a second portion of the extended first oligonucleotide.

[0344] In some aspects, the second portion of the extended first oligonucleotide comprises at least the second strand cDNA. The second portion of the extended first oligonucleotide can also comprise additional sequences. For example, the second portion of the extended first oligonucleotide can also comprise a sequence that is complementary to the capture sequence, and a sequence that is complementary to the spatial barcode sequence.

[0345] In some aspects, the first portion of the extended first oligonucleotide is extended using the extended custom nucleic acid probe as a template. The first portion of the extended first oligonucleotide can be extended using any suitable method known in the art. In some aspects, extending the first portion of the extended first oligonucleotide also displaces at least a portion of the second portion of the extended first oligonucleotide. In some aspects, the entire second portion of the extended first oligonucleotide is displaced. In some aspects, the displaced second portion of the extended first oligonucleotide comprises, among other things, the second strand cDNA and a sequence complementary to the spatial barcode sequence. In some aspects, the displaced second portion of the extended first oligonucleotide can be collected and analyzed.

[0346] In some aspects, the regenerated extended first oligonucleotide is dehybridized from the extended custom nucleic acid probe. In some aspects, the extended first oligonucleotide can be collected and analyzed after it is removed from the extended custom nucleic acid probe.

[0347] In some aspects, a second oligonucleotide is added, and the second oligonucleotide hybridizes to the extended custom nucleic acid probe. In some aspects, the second oligonucleotide comprises a sequence that is complementary to the first nuclease recognition sequence of the nucleic acid probe, and a sequence that is complementary to the linker sequence, if the extended nucleic acid probe comprises a linker sequence. The second oligonucleotide can comprise additional sequences, such as a sequence that is complementary to the spatial barcode sequence of the extended nucleic acid probe. In some aspects, the second oligonucleotide is not bound to the surface of the substrate.

[0348] In some aspects, the first nuclease that is specific to the first nuclease recognition sequence of the extended custom nucleic acid probe is added to the substrate. Any suitable nuclease can be used. In some aspects, a first nuclease cleaves the extended custom nucleic acid probe at the first nuclease recognition sequence, and generates a first portion of theextended custom nucleic acid probe and a second portion of the custom extended nucleic acid probe. The first portion of the extended custom nucleic acid probe remained attached to the surface of the substrate at the 5’ end. The second portion of the extended custom nucleic acid probe comprises, among other things, the first strand cDNA.

[0349] In some aspects, the first portion of the extended custom nucleic acid probe can be extended from the free 3’ end, using any suitable method known in the art. In some aspects, the first portion of the extended custom nucleic acid probe is extended using the second oligonucleotide as a template. Extending the first portion of the extended custom nucleic acid probe regenerates the nucleic acid probe.

[0350] In some aspects, extending the first portion of the extended custom nucleic probe also displaces the second portion of the extended custom nucleic acid probe. The second portion of the extended custom nucleic acid probe comprises, among other things, the first strand cDNA. In some aspects, the displaced second portion of the extended custom nucleic acid probe can be collected and analyzed.

[0351] This process of regenerated the nucleic acid probe and, in some aspects, this process is repeated for several rounds to analyze the same or different target molecules. In some aspects, after the nucleic acid probe is regenerated, the custom oligonucleotides can be hybridized to the nucleic acid probes to generate the custom nucleic acid probes, thus restarting this process.

[0352] In some aspects, the second oligonucleotide is removed after the nucleic acid probe is regenerated, to allow for additional rounds of processing.

[0353] In some aspects, the substrate is contacted with a single-stranded DNA-specific 3’- 5’ exonuclease before reusing the substrate. In some aspects, the exonuclease is a Thermus thermophiles exonuclease.Substrate Regeneration with Long Oligonucleotides

[0354] In some aspects, a method of regenerating a substrate for spatially tagging a target molecule in a sample comprises regenerating the substrate using a long oligonucleotide, and collecting and analyzing first stand cDNA. In some aspects, the method first includes preparing a substrate by attaching nucleic acid probes to the surface of a substrate (e.g., a flow cell). In some aspects, the nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end, and comprise, in a 5' to 3' orientation, a nuclease recognition sequence, a sequencing by synthesis (SBS) sequence, a spatial barcodesequence, and a capture sequence. The nucleic acid probes can comprise additional sequences.

[0355] In some aspects, the method further comprises contacting a tissue sample containing target molecules with the substrate and the target molecule hybridizes to the capture sequences of the nucleic acid probes. In some aspects, the target molecule is mRNA. In some aspects, the method can first include treating the tissue sample to improve the capture of the target molecule by the nucleic acid probes.

[0356] In some aspects, after the target molecule is hybridized to the capture sequence of the nucleic acid probe, the nucleic acid probe is extended to comprise first strand cDNA, using the target molecule as a template. The nucleic acid probe can be extended to include the first stand cDNA using any suitable method, such as by using a polymerase or reverse transcriptase. In some aspects, the first strand cDNA will have a sequence that is complementary to the target molecule that is hybridized to the capture sequence. In some aspects, the target molecule is removed from the extended nucleic acid probe after first strand cDNA synthesis using any suitable method known in the art..

[0357] In some aspects, the method further comprises contacting the substrate with a long oligonucleotide, and the long oligonucleotide hybridizes to the extended nucleic acid probe. In some aspects, the long oligonucleotide comprises, a sequence that is complementary to the nuclease recognition sequence, a sequence that is complementary to the SBS sequence, a sequence that is complementary to the spatial barcode sequence, and a sequence that is complementary to the capture sequence of the nucleic acid probe. In some aspects, the long oligonucleotide is not bound to the surface of the substrate.

[0358] In some aspects, a nuclease that is specific to the nuclease recognition sequence of the nucleic acid probe is added to the substrate. The nuclease can be any suitable nuclease known in the art. In some aspects, the nuclease cleaves the extended nucleic acid probe at the nuclease recognition sequence, and produces a first portion of the extended nucleic acid probe and a second portion of the extended nucleic acid probe. In some aspects, the first portion of the extended nucleic acid probe remained attached to the surface of the substrate at the 5’ end. In some aspects, the second portion of the extended nucleic acid probe comprises the first strand cDNA, the capture sequence, the spatial barcode sequence, and the adapter sequence.

[0359] In some aspects, the first portion of the extended nucleic acid probe is extended from the free 3’ end, by using methods exemplified herein or otherwise known in the artfor amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the first portion of the extended nucleic acid probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the first portion of the extended nucleic acid probe via chemical or enzymatic (e.g., ligase catalysis) methods. In some aspects, the first portion of the extended nucleic acid probe can be extended in a template-directed manner, whereby the product of extension is complementary to the long oligonucleotide that is hybridized to the nucleic acid probe that is extended.

[0360] In some aspects, the first portion of the extended nucleic acid probe is extended with a polymerase. In some aspects, the polymerase is a DNA polymerase. In some aspects, the DNA polymerase can be a strand-displacing DNA polymerase, or a non-strand displacing polymerase. Examples of strand-displacing DNA polymerases include Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase. In aspects where the first portion of the extended nucleic acid probe is extended using a non-strand displacing DNA polymerase, the non-strand displacing DNA polymerase can be used with a helicase-dependent amplification (HDA) system or a T4 replisome.

[0361] In some aspects, the first portion of the extended nucleic acid probe is extended using the hybridized long oligonucleotide as a template. Extending the first portion of the extended nucleic acid probe regenerates the nucleic acid probe.

[0362] In some aspects, extending the first portion of the extended nucleic probe also displaces at least a portion of the second portion of the extended nucleic acid probe. In some aspects, the entire second portion of the extended nucleic acid probe is displaced. The second portion of the extended nucleic acid probe comprises at least the first strand cDNA and the spatial barcode. In some aspects, the displaced second portion of the extended nucleic acid probe can be collected, amplified, and sequenced. In some aspects, the second portion of the extended nucleic acid probe can be analyzed to determine the spatial location of the target molecule. For example, the second portion of the extended nucleic acid probes can be collected, amplified, sequenced, and analyzed by any suitable method known in the art, for example, high throughput next-generation sequencing (NGS), such as sequencing- by-synthesis (SBS).The sequences of the second portion of the extended nucleic acid probes can identify the nucleic acids that are in the tissue sample, and where in the tissuesample the target molecules are located. In some aspects, other sequence elements that are present in the nucleic acid probes are also included in the second portion of the extended nucleic acid probes. Such elements include, for example, primer binding sites, cleavage sites, other or additional spatial barcodes or tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like.

[0363] In some aspects, the regeneration oligonucleotide is removed from the regenerated nucleic acid probe, by any suitable method known in the art, to allow for additional rounds of processing.

[0364] In some aspects, the substrate is contacted with a single-stranded DNA-specific 3’- 5’ exonuclease before reusing the substrate. In some aspects, the exonuclease is a Thermus thermophiles exonuclease.

[0365] In some aspects, the displaced second portion of the nucleic acid probe can be collected and used to make additional long oligonucleotides for use in this process. This is advantageous because these long oligonucleotides have the desired complementary sequences (for example, a sequence complementary to the spatial barcode sequence) to the nucleic acid probes. Additional long oligonucleotides can be made by amplifying the collected second portion of the nucleic acid probe using any suitable method known in the art, for example, PCR either on or off the substrate. In some aspects, this process can also use RE’ primers and Poly-A primers.Dual Oligonucleotide / Nuclease

[0366] In some aspects, the present disclosure further provides method of regenerating a substrate for spatially tagging a target molecule in a sample comprising providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation: a nuclease enzyme recognition site, and a capture sequence; wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation: a spatial barcode, and a template switch oligonucleotide (TSO) sequence, wherein the spatial barcode probe is 3’ blocked such that extension of the spatial barcode probe is prevented. In some aspects, the capture probe comprises additional or other sequences, such as aprimer sequence (e.g., Adpl). In some aspects, the spatial barcode probe comprises additional or other sequences, such as a primer sequence (Adp2) and a UMI.

[0367] In some aspects, the method further comprises contacting a tissue sample containing target molecules with the substrate and the target molecule hybridizes to the capture sequences of the nucleic acid probes. In some aspects, the target molecule is mRNA. In some aspects, the method can first include treating the tissue sample to improve the capture of the target molecule by the nucleic acid probes.

[0368] In some aspects, after the target molecule is hybridized to the capture sequence of the nucleic acid probe, the nucleic acid probe is extended to comprise first strand cDNA, using the target molecule as a template. The nucleic acid probe can be extended to include the first stand cDNA using any suitable method, for example, by using a polymerase or reverse transcriptase such as a DNA polymerase, such as Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase. Extending the nucleic acid probe using the hybridized target molecule as a template, generates an extended nucleic acid probe that comprises first stand cDNA. The first strand cDNA has a sequence that is complementary to the target molecule that is hybridized to the capture sequence.

[0369] In some aspects, the target molecule is removed from the extended nucleic acid probe using any suitable method known in the art. For example, in aspects where the target molecule is mRNA, RNase H can be used to remove the hybridized target mRNA. In some aspects, an alkaline medium can be used to remove the hybridized target mRNA. For example, a NaOH solution or a KOH solution can be used.

[0370] In some aspects, the method further comprises hybridizing a template switch oligonucleotide (TSO’) that is complementary to the TSO sequence to the capture probe, hybridizing the TSO’ sequence and the TSO sequence, and extending the capture probe using the TSO sequence as template to generate an extended capture probe that comprises first strand cDNA and a sequence that is complementary to the spatial barcode sequence (SBC’).

[0371] In some aspects, the method further comprises contacting the substrate with a regeneration oligonucleotide, and the regeneration oligonucleotide hybridizes to the extended nucleic acid probe. The regeneration oligonucleotide comprises, at least, a sequence that is complementary to at least a portion of the nuclease recognition sequence. In some aspects, for example, the regeneration oligonucleotide comprises a PolyA- Ill -sequence, a randomer sequence, a semi-randomer sequence, or a target specific sequence, and a primer sequence complement (e.g, Adpl’).. In some aspects, the regeneration oligonucleotide is not attached to the surface of the substrate. In some aspects, the regeneration oligonucleotide comprises additional sequences.

[0372] In some aspects, the method further comprises adding a nuclease that is specific to the nuclease recognition sequence of the nucleic acid probe to the substrate. In some aspects, the nuclease cleaves the extended capture probe at the nuclease recognition sequence, and produces a first portion of the extended capture probe and a second portion of the extended capture probe. The first portion of the extended capture probe is attached to the surface of the substrate at the 5’ end. The second portion of the extended capture probe comprises first strand cDNA, the capture sequence, the spatial barcode sequence complement (SBC’), and primer sequences (Adpl and Adp2’).

[0373] In some aspects, the first portion of the extended capture probe is extended from the free 3’ end, by using methods exemplified herein or otherwise known in the art for amplification of nucleic acids or sequencing of nucleic acids. For example, one or more nucleotides can be added to the 3' end of the first portion of the extended capture probe, for example, via polymerase catalysis (e.g. DNA polymerase, RNA polymerase or reverse transcriptase). One or more oligonucleotides can be added to the 3’ end of the first portion of the extended capture probe via chemical or enzymatic (e.g., ligase catalysis) methods. The first portion of the extended capture probe can be extended in a template-directed manner, whereby the product of extension is complementary to the regeneration oligonucleotide that is hybridized to the probe that is extended.

[0374] In some aspects, a DNA polymerase is used to extend the first portion of the extended capture probe. The DNA polymerase can be a strand-displacing DNA polymerase, or a non-strand displacing polymerase. In aspects where the first portion of the extended nucleic acid probe is extended using a strand-displacing DNA polymerase, the DNA polymerase can be Sequenase version 2.0, Klenow Fragment exo-, Bst Large Fragment, Vent exo-, 9° Nm, or any suitable DNA polymerase. In aspects where the first portion of the extended capture probe is extended using a non-strand displacing DNA polymerase, the non-strand displacing DNA polymerase can be used with a helicasedependent amplification (HD A) system or a T4 replisome.

[0375] The first portion of the extended capture probe is extended using the hybridized regeneration oligonucleotide as a template. Extending the first portion of the extended capture probe thereby regenerates the capture probe.

[0376] Extending the first portion of the extended capture probe also displaces the second portion of the extended capture probe. In some aspects, the second portion of the extended capture probe comprises, the first strand cDNA and the spatial barcode complement. In some aspects, the second portion of the extended capture probe comprises additional sequence, such as the capture sequence, primer sequences, the TSO complement, and optionally a UMI complement. In some aspects, the displaced second portion of the extended capture probe can be collected, amplified, and sequenced. The second portion of the extended capture probe can be analyzed to determine the spatial location of the target molecule. The second portion of the extended capture probes can be collected, amplified, sequenced, and analyzed by any suitable method known in the art, for example, high throughput next-generation sequencing (NGS), such as sequencing-by-synthesis (SBS).

[0377] The sequences of the second portion of the extended capture probes identify the nucleic acids in the tissue sample, and where in the tissue sample the target molecules are located. In some aspects, other sequence elements that are present in the capture probes and the complement of sequence elements in the spatial barcode probe can also be included in the second portion of the extended capture probes. Such elements include, for example, primer binding sites, cleavage sites, other or additional spatial barcodes or tag sequences (e.g. sample identification tags), capture sequences, recognition sites for nucleic acid binding proteins or nucleic acid enzymes, or the like.

[0378] In some aspects, the regeneration oligonucleotide is removed from the regenerated nucleic acid probe, by any suitable method known in the art, to allow for additional rounds of processing.

[0379] In some aspects, the substrate is contacted with a single-stranded DNA-specific 3’- 5’ exonuclease before reusing the substrate. In some aspects, the exonuclease is a Thermus thermophiles exonuclease.EXAMPLESExample 1

[0380] Referring to Figure 1, a substrate was prepared by attaching nucleic acid probes to the surface of a substrate (e.g., a flowcell). The nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end. The nucleic acid probes contain, in a 5' to 3' orientation, a spatial barcode sequence, a SBS sequence (e.g., SB SI 2), a restriction enzyme recognition sequence, and a capture sequence comprising a polyT sequence.

[0381] A sample was contacted with the substrate and target nucleic acids (e.g., mRNA) hybridized to the capture sequences of the nucleic acid probes. Generally, a target nucleic acid will diffuse from a region of the sample to an area of the substrate that is in proximity to that region of the specimen. Here the target nucleic acid will interact with nucleic acid probes that are proximal to the region of the specimen from which the target nucleic acid was released. A target-probe hybrid complex can form where the target nucleic acid encounters a complementary target capture sequence on a nucleic acid probe. The location of the target-probe hybrid complex will generally correlate with the region of the sample from where the target nucleic acid was derived.

[0382] After the target nucleic acid is hybridized to the capture sequence of the nucleic acid probe, the first strand cDNA is generated using the target nucleic acid as a template. The first strand cDNA can be extended using any suitable method, such as using a polymerase or reverse transcriptase. The first strand cDNA will have a sequence that is complementary to the target molecule that is hybridized to the capture sequence.

[0383] In some aspects, the target molecule is dehybridized from the nucleic acid probe, and second strand cDNA can be generated using the extended nucleic acid probe as a template. Accordingly, the second strand cDNA molecule can have, among other things, a sequence that is complementary to the barcode sequence of the nucleic acid probe, and a sequence that can be correlated with the target molecule. The second strand cDNA molecule can be dehybridized from the extended nucleic acid probe and subsequently sequenced. The second strand cDNA molecule can be dehybridized by any suitable method known in the art, such as altering the pH, adding salt, or elevating the temperature. The sequenced second strand cDNA can be analyzed to identify the spatial location of a target molecule in a sample.

[0384] Next, a first oligonucleotide that is complementary to the restriction enzyme recognition sequence is added to the substrate. The oligonucleotide binds to the restriction enzyme recognition sequence, forming a double-stranded molecule at the restriction enzyme recognition sequence.

[0385] A restriction enzyme is added that cleaves the nucleic acid probe at the restriction enzyme recognition sequence (i.e., the cut site). The restriction enzyme cleaves the nucleic acid and the first strand cDNA, or any other transcripts, at the restriction enzyme recognition sequence.

[0386] Next, a second oligonucleotide that contains (1) a sequence that is complementary to the remaining nucleic acid probe, and (2) a template to regenerate the original capture sequence is hybridized to the remaining probe. A polymerase is added, which extends the remaining nucleic acid probe against the template, and a second oligonucleotide, yielding a regenerated nucleic acid probe.

[0387] The second oligonucleotide is then removed from the nucleic acid probe, and the fresh, re-generated nucleic acid probe can be reused for subsequent rounds of processing. This process of re-generating the nucleic acid probe can be repeated several times.Example 2

[0388] Referring to Figure 2, a substrate was prepared by attaching nucleic acid probes to the surface of a substrate (e.g., a flowcell). The nucleic acid probes are attached and immobilized to the surface of the substrate by their 5’ end. The nucleic acid probes contain, in a 5' to 3' orientation, a spatial barcode sequence, a SBS sequence (e.g., SB SI 2), and a capture sequence comprising a polyT sequence.

[0389] A sample was contacted with the substrate and target nucleic acids (e.g., mRNA) hybridized to the capture sequences of the nucleic acid probes. Generally, a target nucleic acid will diffuse from a region of the sample to an area of the substrate that is in proximity to that region of the specimen. Here the target nucleic acid will interact with nucleic acid probes that are proximal to the region of the specimen from which the target nucleic acid was released. A target-probe hybrid complex can form where the target nucleic acid encounters a complementary target capture sequence on a nucleic acid probe. The location of the target-probe hybrid complex will generally correlate with the region of the sample from where the target nucleic acid was derived.

[0390] After the target nucleic acid is hybridized to the capture sequence of the nucleic acid probe, the first strand cDNA is generated using the target nucleic acid as a template. The first strand cDNA can be extended using any suitable method, such as using a polymerase or reverse transcriptase. The first strand cDNA will have a sequence that is complementary to the target molecule that is hybridized to the capture sequence.

[0391] In some aspects, the target molecule is dehybridized from the nucleic acid probe, and second strand cDNA can be generated using the extended nucleic acid probe as a template. Accordingly, the second strand cDNA molecule can have, among other things, a sequence that is complementary to the barcode sequence of the nucleic acid probe, and a sequence that can be correlated with the target molecule. The second strand cDNA molecule can be dehybridized from the extended nucleic acid probe and subsequently sequenced. The second strand cDNA molecule can be dehybridized by any suitable method known in the art, such as altering the pH, adding salt, or elevating the temperature. The sequenced second strand cDNA can be analyzed to identify the spatial location of a target molecule in a sample.

[0392] Next, an oligonucleotide (i.e., a complementary blocker) that is complementary to all or a portion of the capture sequence on the nucleic acid probe is hybridized to the capture sequence. The complementary blocker prevents degradation of the nucleic acid probe by an exonuclease.

[0393] After the complementary blocker is hybridized to the nucleic acid probe, an exonuclease is added, which degrades the first strand cDNA on the nucleic acid probe. The exonuclease stops degrading the cDNA on the nucleic acid probe when it is blocked by the complementary blocker oligonucleotide. Next, the exonuclease is removed, and the complementary oligonucleotide is removed by any suitable method, such as dehybridization. This results in a refreshed nucleic acid probe that can be reused for additional rounds of processing. This process of re-generating the nucleic acid probes can be repeated several times.Example 3

[0394] Referring to Figures 3A-3G, a substrate was prepared by attaching nucleic acid probes to the surface of a substrate (e.g., a flowcell). Two nucleic acid probes, a capture probe and a spatial barcode probe, were attached and immobilized to the surface of the substrate by their 5’ end. The nucleic acid probes comprise several nucleic acid regions.For example, the capture probes comprised, in a 5' to 3' orientation, a primer sequence (e.g., Adpl), a restriction enzyme recognition sequence, and a capture sequence (e.g., a poly-T sequence). The spatial barcode probe comprised a primer sequence (e.g., Adp2), a spatial barcode sequence, an optional unique molecular identifier (“UMI”) sequence, and a template switch oligonucleotide (“TSO”). The spatial barcode probe was blocked at its 3’ end to prevent extension of the spatial barcode probe. The primer sequences of the capture probe and the spatial barcode probe can be the same sequence or different sequences.

[0395] A sample was contacted with the substrate and target nucleic acids (e.g., mRNA) hybridized to the capture sequence of the capture probes. Generally, a target nucleic acid will diffuse from a region of the sample to an area of the substrate that is in proximity to that region of the specimen. Here the target nucleic acid will interact with the capture probes that are proximal to the region of the specimen from which the target nucleic acid was released. A target-probe hybrid complex can form where the target nucleic acid encounters a complementary target capture sequence on a nucleic acid probe. The location of the target-probe hybrid complex will generally correlate with the region of the sample from where the target nucleic acid was derived.

[0396] As shown in Figure 3 A, after the target nucleic acid was hybridized to the capture probe, first strand cDNA was generated using reverse transcriptase and the target nucleic acid as a template. First strand cDNA can be extended using any suitable method, such as using a polymerase or reverse transcriptase. The first strand cDNA has a sequence that is complementary to the target molecule that is hybridized to the capture sequence. The target molecule can be optionally dehybridized from the nucleic acid probe.

[0397] As shown in Figure 3B, a TSO was added to the 3’ end of the extended capture probe. The TSO added to the 3’ end of the capture probe is complementary to the TSO on the 3’ end of the spatial barcode probe. The TSO of the capture probe and the TSO of the spatial barcode probe were hybridized and the capture probe was extended by DNA polymerase using the spatial barcode sequence as a template. This resulted in the capture probe now comprising, in a 5’ to 3’ direction, a primer sequence (e.g., Adpl), a restriction enzyme recognition sequence, and a capture sequence (e.g., a poly-T sequence), first strand cDNA that is complementary to the target molecule sequence, a TSO, a sequence that is complementary to the spatial barcode probe’s UMI (optional), a sequence that is complementary to the spatial barcode sequence of the spatial barcode probe, and a sequence that is complementary to the primer sequence of the spatial barcode probe (e.g., a sequencethat is complementary to Adp2). As shown in Figure 3C, Adp2 primers were hybridized to the extended capture probe at the complementary Adp2 sequence, and second strand cDNA was generated with DNA polymerase.

[0398] As shown in Figure 3D, heat or alkaline denaturation was used to remove the copy of the extended capture probe comprising second strand cDNA from the substrate. The removed second strand cDNA can be amplified and used in library prep, and / or sequenced to obtain spatial gene expression information.

[0399] As shown in Figure 3E, a sequence that is complementary to the restriction enzyme recognition sequence is hybridized to the restriction enzyme recognition sequence. Next, a restriction enzyme was added that cleaves the extended capture probe at the restriction enzyme recognition sequence (i.e., the cut site). The restriction enzyme cleaves the capture probe so that the first strand cDNA can be removed and optionally collected and processed.

[0400] As shown in Figure 3F, an oligonucleotide (i.e„ a regeneration oligonucleotide) was hybridized to remaining portion of the capture probe. The regeneration oligonucleotide comprised a primer sequence (optional, can be all or part of the reverse complement of the Adpl sequence), a sequence that is complementary to all or a portion of the restriction enzyme recognition sequence of the capture probe, and a sequence that is complementary to the desired capture sequence (e.g., a poly-A sequence). The capture probe was extended with DNA polymerase using the regeneration oligonucleotide as a template. Figure 3G shows the regenerated capture probe and spatial barcode probe, which can be used for additional rounds of processing.Example 4

[0401] Referring to Figures 4A-4G, a substrate was prepared by attaching nucleic acid probes to the surface of a substrate (e.g., a flowcell). Two nucleic acid probes, a capture probe and a spatial barcode probe, were attached and immobilized to the surface of the substrate by their 5’ end. The nucleic acid probes comprise several nucleic acid regions. For example, the capture probes comprised, in a 5' to 3' orientation, a primer sequence (e.g., Adpl), a restriction enzyme recognition sequence (“RE1” in Figure 4), and a capture sequence (e.g., a poly-T sequence). The spatial barcode probe comprised a primer sequence (e.g., Adp2), a spatial barcode sequence, an optional UMI sequence, a restriction enzyme recognition sequence (“RE2” in Figure 4), and a template switch oligonucleotide (“TSO”). The primer sequences of the capture probe and the spatial barcode probe can be the samesequence or different sequences. The sequences of the restriction enzyme recognition sequences of the capture probe and the spatial barcode probe can be the same sequence or different sequences.

[0402] A sample was contacted with the substrate and target nucleic acids (e.g., mRNA) hybridized to the capture sequence of the capture probes. Generally, a target nucleic acid will diffuse from a region of the sample to an area of the substrate that is in proximity to that region of the specimen. Here the target nucleic acid will interact with the capture probes that are proximal to the region of the specimen from which the target nucleic acid was released. A target-probe hybrid complex can form where the target nucleic acid encounters a complementary target capture sequence on a nucleic acid probe. The location of the target-probe hybrid complex will generally correlate with the region of the sample from where the target nucleic acid was derived.

[0403] As shown in Figure 4A, after the target nucleic acid was hybridized to the capture probe, first strand cDNA was generated using reverse transcriptase and the target nucleic acid as a template. First strand cDNA can be extended using any suitable method, such as using a polymerase or reverse transcriptase. The first strand cDNA has a sequence that is complementary to the target molecule that is hybridized to the capture sequence. The target molecule can be optionally dehybridized from the nucleic acid probe.

[0404] As shown in Figure 4B, a TSO was added to the 3’ end of the extended capture probe. The TSO added to the 3’ end of the capture probe is complementa...

Claims

WHAT IS CLAIMED IS:

1. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation:i. a spatial barcode sequence,ii. a restriction enzyme recognition sequence, and iii. a capture sequence;b. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe;c. extending the capture sequence to produce an extended nucleic acid probe; d. contacting the substrate with an oligonucleotide that is complementary to the restriction enzyme recognition sequence under conditions wherein the oligonucleotide hybridizes to the restriction enzyme recognition sequence of the nucleic acid probe;e. contacting the substrate with a restriction enzyme, wherein the restriction enzyme cleaves the extended nucleic acid probe;f. contacting the substrate with a regeneration oligonucleotide; and g. extending the nucleic acid probe against the regeneration probe.

2. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe and a second nucleic acid probe comprises a spatial barcode probe, wherein the capture probe comprises, in a 5' to 3' orientation:i. a restriction enzyme recognition sequence, and ii. a capture sequence,and wherein the spatial barcode probe comprises, in a 5' to 3' orientation:i. a spatial barcode sequence, andii. a template switch oligonucleotide (TSO),b. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe and extending the capture sequence to produce an extended nucleic acid probe comprising first strand cDNA;c. contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence;d. extending the capture probe from the free 3’ end using the spatial barcode probe as template to generate an extended capture probe;e. contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe;f. contacting the substrate with an oligonucleotide that is complementary to the restriction enzyme recognition sequence under conditions wherein the oligonucleotide hybridizes to the restriction enzyme recognition sequence of the capture probe, and contacting the substrate with a restriction enzyme, wherein the restriction enzyme cleaves the extended capture probe;g. contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the capture probe, wherein the regeneration oligonucleotide comprises a sequence that is complementary to the restriction enzyme recognition sequence, and a sequence that is complementary to the capture sequence; andh. extending the nucleic acid probe against the regeneration oligonucleotide.

3. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprisesa capture probe and a second nucleic acid probe comprises a spatial barcode probe, wherein the capture probe comprises, in a 5' to 3' orientation:i. a first restriction enzyme recognition sequence, and ii. a capture sequence,and wherein the spatial barcode probe comprises, in a 5' to 3' orientation:i. a spatial barcode sequence,ii. a second restriction enzyme recognition sequence, and iii. a template switch oligonucleotide (TSO),b. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe and extending the capture sequence to produce an extended nucleic acid probe comprising first strand cDNA;c. contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence;d. extending the capture probe from the free 3’ end using the spatial barcode probe as template to generate an extended capture probe;e. extending the spatial barcode from the free 3’ end using the capture probe as a template to generate an extended spatial barcode probe;f. contacting the substrate with a first primer oligonucleotide under conditions wherein the first primer oligonucleotide hybridizes to the extended capture probe, and extending the hybridized first primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe;g. contacting the substrate with a second primer oligonucleotide under conditions wherein the second primer oligonucleotide hybridizes to the extended spatial barcode probe, and extending the hybridized second primer oligonucleotide to generate an oligonucleotide that is complementary to the extended spatial barcode probe;h. contacting the substrate with a first oligonucleotide that is complementary to the first restriction enzyme recognition sequence under conditions wherein the first oligonucleotide hybridizes to the first restriction enzyme recognition sequence of the capture probe, and contacting the substrate with a first restriction enzyme, wherein the firstrestriction enzyme cleaves the extended capture probe at the first restriction enzyme recognition sequence;i. contacting the substrate with a second oligonucleotide that is complementary to the second restriction enzyme recognition sequence under conditions wherein the second oligonucleotide hybridizes to the second restriction enzyme recognition sequence of the spatial barcode probe, and contacting the substrate with a second restriction enzyme, wherein the second restriction enzyme cleaves the extended spatial barcode probe at the second restriction enzyme recognition sequence;j. contacting the substrate with a first regeneration oligonucleotide under conditions wherein the first regeneration oligonucleotide hybridizes to the capture probe, wherein the first regeneration oligonucleotide comprises a sequence that is complementary to the first restriction enzyme recognition sequence, and a sequence that is complementary to the capture sequence, and extending the capture probe using the first regeneration oligonucleotide as a template; andk. contacting the substrate with a second regeneration oligonucleotide under conditions wherein the second regeneration oligonucleotide hybridizes to the spatial barcode probe, wherein the second regeneration oligonucleotide comprises a sequence that is complementary to the second restriction enzyme recognition sequence, and a sequence that is complementary to the TSO sequence, and extending the spatial barcode probe using the second regeneration oligonucleotide as a template.

4. The method of claim 1, wherein a spatially tagged second strand cDNA is generated from the extended nucleic acid probe, and wherein the spatially tagged second strand cDNA is dehybridized from the extended nucleic acid probe between steps (c) and (d) and sequenced.

5. The method of claim 1 , wherein the spatially tagged second strand cDNA is generated using a template switching oligonucleotide (TSO) approach or a random priming approach.

6. The method of claim 1, wherein the nucleic acid probe further comprises a sequencing by synthesis (SBS) sequence.

7. The method of claim 1, wherein the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

8. The method of claim 6, wherein the SBS sequence is SBS12 or a complement thereof (SBS12').

9. The method of claim 6, wherein the SBS sequence is SBS3 or a complement thereof (SB S3').

10. The method of claim 1, wherein the nucleic acid probe further comprises a flowcell clustering sequence, and wherein the flowcell clustering sequence is selected from the group consisting of P5, P5', P7, P7'.

11. The method of claim 2, wherein the spatial barcode probe is blocked at the 3 ’ end.

12. The method of claim 3, wherein:a. the first regeneration oligonucleotide comprises a sequence that is complementary to the first restriction enzyme recognition sequence and a sequence that is complementary to the capture sequence, andb. the second regeneration oligonucleotide comprises a sequence that is complementary to the second restriction enzyme recognition sequence and a sequence that is complementary to the TSO.

13. The method of claim 3 or 12, wherein the first and second oligonucleotides are removed prior to contacting the substrate with the first and second regeneration oligonucleotides.

14. The method of claim 1 or 2, wherein the oligonucleotide that is complementary to the restriction enzyme recognition sequence is removed prior to contacting the substrate with the regeneration oligonucleotide.

15. The method of claim 2 or 3, wherein the capture probe and the spatial barcode probe further comprise a primer sequence.

16. The method of claim 2 or 3, wherein the spatial barcode probe further comprises a primer sequence and a UMI sequence.

17. The method of any one of clams 1-16, wherein the substrate is a solid surface, glass slide, flowcell, an array, or beads.

18. The method of any one of clams 1-17, wherein the nucleic acid probes are arranged in specific locations on the substrate.

19. The method of any one of clams 1-18, wherein the spatial barcode sequence is correlated with a positional location on the substrate.

20. The method of any one of clams 1-19, wherein the plurality of nucleic acid probes comprises a plurality of clusters of nucleic acid probes, wherein the nucleic acid probes of each cluster comprise a unique spatial barcode.

21. The method of any one of clams 1-20, wherein the spatial barcode sequence spatially tags the nucleic acid probe.

22. The method of any one of clams 1-21, wherein the capture sequence comprises a poly thymidine (polyT) sequence.

23. The method of any one of clams 1-22, wherein the capture sequence comprises a targetspecific capture sequence, a randomer, or a semi-randomer.

24. The method of any one of clams 1-23, further comprising contacting the target molecule with an enzyme to degrade the target molecule.

25. The method of any one of clams 1-24, further comprising removing the target molecule by dehybridization or by contacting the target molecule with NaOH.

26. The method of any one of clams 1-25, wherein the restriction enzyme is a rare cutter enzyme, a type II restriction enzyme, or a frequent cutter enzyme.

27. The method of any one of clams 1-26, wherein the spatial location of the target molecule is determined.

28. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation:i. a spatial barcode, andii. a capture sequence;b. contacting a sample with the nucleic acid probes on the substrate under conditions wherein the target molecules from the sample hybridize to the capture sequences of the nucleic acid probes;c. extending the nucleic acid probe to produce an extended nucleic acid probe that comprises first strand cDNA, or portions thereof, and the spatial barcode sequences, thereby spatially tagging the target nucleic acids of the biological sample,d. providing a blocking oligonucleotide that is complementary to the capture sequence and contacting the substrate with a blocking oligonucleotide under conditions wherein the blocking oligonucleotide hybridizes to the capture sequence, ande. contacting the target nucleic acids with an exonuclease.

29. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation:i. a spatial barcode, andii. a capture sequence,and wherein a second acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises in a 5' to 3' orientation:i. a spatial barcode sequence, andii. a TSO sequence;b. contacting a sample with the nucleic acid probes on the substrate under conditions wherein the target molecules from the sample hybridize to the capture sequences of the nucleic acid probes;c. extending the capture probe to produce a capture probe that comprises first strand cDNA, or portions thereof;d. contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence and extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA and a spatial barcode sequence complement (SBC’);e. contacting the substrate with a first blocking oligonucleotide under conditions wherein the first blocking oligonucleotide hybridizes to the capture sequence of the capture probe,f. contacting the substrate with a second blocking oligonucleotide under conditions wherein the second blocking oligonucleotide hybridizes to the TSO of the spatial barcode probe, andg. contacting the target nucleic acids with an exonuclease.

30. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation:i. a spatial barcode, andii. a capture sequence,and wherein a second acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises in a 5' to 3' orientation:iii. a spatial barcode sequence, andiv. a TSO sequence;b. contacting a sample with the nucleic acid probes on the substrate under conditions wherein the target molecules from the sample hybridize to the capture sequences of the nucleic acid probes;c. extending the capture probe to produce a capture probe that comprises first strand cDNA, or portions thereof;d. contacting the substrate with a template switch oligonucleotide complement (TSO’) under conditions wherein the TSO’ is added to the free 3’ end of capture probe and hybridizing the TSO’ sequence and the TSO sequence and extending the capture probe using the spatial barcode probe as template to generate an extended capture probe comprising first strand cDNA and a spatial barcode sequence complement (SBC’);e. extending the spatial barcode from the free 3’ end using the capture probe as a template to generate an extended spatial barcode probe comprising second strand cDNA and the spatial barcode sequence;f. contacting the substrate with a first blocking oligonucleotide under conditions wherein the first blocking oligonucleotide hybridizes to the capture sequence of the capture probe,g. contacting the substrate with a second blocking oligonucleotide under conditions wherein the second blocking oligonucleotide hybridizes to the TSO of the spatial barcode probe, andh. contacting the target nucleic acids with an exonuclease.

31. The method of claim 28, wherein a spatially tagged second strand cDNA is generated from the extended nucleic acid probe, and wherein the spatially tagged second strand cDNA is dehybridized from the extended nucleic acid probe and sequenced.

32. The method of claim 31, wherein the spatially tagged second strand cDNA is generated using a template switching oligonucleotide (TSO) approach or a random priming approach.

33. The method of claim 28, wherein the nucleic acid probe further comprises a sequencing by synthesis (SBS) sequence.

34. The method of claim 33, wherein the SBS sequence is SBS12, or a complement thereof (SBS12').

35. The method of claim 33, wherein the SBS sequence is SBS3 or a complement thereof (SB S3').

36. The method of claim 28, wherein the nucleic acid probe further comprises a flowcell clustering sequence.

37. The method of claim 36, wherein the flowcell clustering sequence is selected from the group consisting of P5, P5', P7, P7'.

38. The method of claim 28, wherein the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

39. The method of claim 29, further comprising contacting the substrate with a primer oligonucleotide under conditions wherein the primer oligonucleotide hybridizes to the extended capture probe after step (d), and extending the hybridized primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe, wherein the complementary copy of the extended capture probe comprises second strand cDNA and the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended capture probe is dehybridized from the extended capture probe and sequenced.

40. The method of claim 30, further comprising:a. contacting the substrate with a first primer oligonucleotide under conditions wherein the first primer oligonucleotide hybridizes to the extended capture probe and extending the hybridized first primer oligonucleotide to generate an oligonucleotide that is complementary to the extended capture probe, wherein the complementary copy of theextended capture probe comprises second strand cDNA and the spatial barcode sequence, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended capture probe is dehybridized from the extended capture probe and sequenced, andb. contacting the substrate with a second primer oligonucleotide under conditions wherein the second primer oligonucleotide hybridizes to the extended capture probe and extending the hybridized second primer oligonucleotide to generate an oligonucleotide that is complementary to the extended spatial barcode probe, wherein the complementary copy of the extended spatial barcode probe comprises first strand cDNA and the spatial barcode sequence complement, thereby spatially tagging the target nucleic acids of the biological sample, and wherein the complementary copy of the extended spatial barcode probe is dehybridized from the extended spatial barcode probe and sequenced.

41. The method of claim 29 or 30, wherein the capture probe further comprises a primer sequence.

42. The method of claim 29 or 30, wherein the spatial barcode probe further comprises a primer sequence and a UMI sequence.

43. The method of any one of claims 28-30, wherein the first strand cDNA nucleic acid sequence that is complementary to the target molecule that is hybridized to the capture sequence, or a portion thereof.

44. The method of any one of claims 28-43, wherein the substrate is a solid surface, glass slide, flowcell, an array, or a bead.

45. The method of any one of claims 28-44, wherein the substrate comprises a plurality of nucleic acid probes, and wherein the nucleic acid probes are arranged in specific locations on the substrate.

46. The method of any one of claims 28-45, wherein the spatial barcode correlates to a positional location on the substrate.

47. The method of any one of claims 28-46, wherein the plurality of nucleic acid probes comprises a plurality of clusters of nucleic acid probes, wherein the nucleic acid probes of each cluster comprise a unique spatial barcode.

48. The method of any one of claims 28-47, wherein the spatial barcode spatially tags the nucleic acid probe.

49. The method of any one of claims 28-48, wherein the capture sequence comprises a polythymidine (polyT) sequence.

50. The method of any one of claims 28-49, wherein the capture sequence comprises a targetspecific capture sequence, a randomer, or a semi-randomer.

51. The method of any one of claims 28-50, wherein the exonuclease is exonuclease I, exonuclease II, exonuclease III, exonuclease IV, exonuclease V, exonuclease VI, or a polymerase.

52. The method of any one of claims 28-51, wherein the spatial location of the target molecule is determined.

53. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. grafting a first moiety to a surface of the substrate,b. binding a second moiety to the first moiety, wherein the second moiety comprises a capture sequence and a spatial barcode sequence,c. binding a target nucleic molecule to the capture sequence of the second moiety and generating first strand cDNA,d. decoupling the second moiety from the first moiety thereby regenerating the substrate.

54. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. grafting a first moiety to a surface of the substrate,b. attaching primer sequences to the surface of the substrate,c. binding a second moiety to the first moiety, wherein the second moiety comprises a capture sequence and a spatial barcode sequence,d. binding a target nucleic molecule to the capture sequence of the second moiety and generating first strand cDNA,e. decoupling the second moiety from the first moiety thereby regenerating the substrate.

55. The method of claim 54, wherein the method further comprises clustering the spatial barcode and linearizing.

56. The method of any one of claims 53-55, wherein the first moiety comprises alkyne-PEG- biotin.

57. The method of claim 56, wherein the alkyne-PEG-biotin is grafted to the azides of a polymer coating the surface of the substrate.

58. The method of any one of claims 53-57, wherein the second moiety comprises 5’ biotinylated P5 and P7 oligonucleotides coupled to streptavidin.

59. The method of any one of claims 53-58, wherein a reagent is used to decouple the second moiety from the first moiety.

60. The method of claim 59, wherein the reagent comprises hot formamide.

61. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation:i. a nuclease recognition sequence,ii. an adapter sequence,iii. a spatial barcode sequence, andiv. a capture sequence,b. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture sequence of the nucleic acid probe,c. extending the nucleic acid probe using the target molecule as a template to produce an extended nucleic acid probe comprising the nucleic acid probe and a first strand cDNA,d. contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the capture sequence of the nucleic acid probe, and extending the regeneration oligonucleotide using the nucleic acid probe as a template to generate an oligonucleotide that is complementary to the nucleic acid probe,e. contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the extended nucleic acid probe, producing a first portion of the extended nucleic acid probe and a second portion of the extended nucleic acid probe, wherein the first portion of the extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the nucleic acid probe comprises the adapter sequence, the spatial barcode sequence, the capture sequence, and the first strand cDNA, andf. extending the first portion of the nucleic acid probe from the free 3’ end using the regeneration oligonucleotide as a template, thereby regenerating the nucleic acid probe.

62. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation:i. a spatial barcode sequence,ii. a nuclease recognition sequence, andiii. optionally a linker,b. contacting the nucleic acid probes with a customizable oligonucleotide under conditions wherein the customizable oligonucleotide hybridizes to the capture sequence of the nucleic acid probe,c. extending the nucleic acid probe using the customizable oligonucleotide as a template to produce a custom nucleic acid probe,d. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the custom nucleic acid probe, e. extending the custom nucleic acid probe using the target molecule as a template to produce an extended custom nucleic acid probe comprising the nucleic acid probe and a first strand cDNA,f. contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the extended custom nucleic acid probe,g. contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the extended custom nucleic acid probe, producing a first portion of the extended custom nucleic acid probe and a second portion of the extended custom nucleic acid probe, wherein the first portion of the extended custom nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the extended custom nucleic acid probe comprises the first strand cDNA, andh. extending the first portion of the nucleic acid probe from the free 3’ end using the regeneration oligonucleotide as a template, thereby regenerating the nucleic acid probe.

63. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation:i. a first nuclease recognition sequence,ii. a first adapter sequence,iii. a spatial barcode sequence,iv. a second nuclease recognition sequence, andv. a capture sequence,b. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the nucleic acid probe,c. extending the nucleic acid probe using the target molecule as a template to produce a first extended nucleic acid probe comprising a first strand cDNA,d. contacting the substrate with a first oligonucleotide and extending the first oligonucleotide using the first extended nucleic acid probe as a template to generate an extended first oligonucleotide, wherein:i. the extended first oligonucleotide is complementary to the first extended nucleic acid probe,ii. the extended first oligonucleotide and the first extended nucleic acid probe form a double-stranded oligonucleotide, andiii. the extended first oligonucleotide comprises a second strand cDNA, e. contacting the substrate with a first nuclease that is specific to the first nuclease recognition sequence, wherein the first nuclease cleaves the first extended nucleic acid probe comprised in the double-stranded oligonucleotide, producing a first portion of the first extended nucleic acid probe and a second portion of the first extended nucleic acid probe, wherein the first portion of the first extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the first extended nucleic acid probe comprises the first adapter sequence, the spatial barcode, the capture sequence, and the first strand cDNA,f. extending the first portion of the first extended nucleic acid probe from the free 3’ end to generate a second extended nucleic acid probe, using the extended first oligonucleotide as a template,g. contacting the substrate with a second nuclease that is specific to the second nuclease recognition sequence, wherein the second nuclease cleaves the second extended nucleic acid probe, producing a first portion of the second extended nucleic acid probe and a second portion of the second extended nucleic acid probe, wherein the first portion of the second extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the second extended nucleic acid probe contains the first strand cDNA,h. removing the extended first oligonucleotide from the first portion of the second extended nucleic acid probe,i. contacting the substrate with a second oligonucleotide under conditions wherein the second oligonucleotide hybridizes to the first portion of the second extended nucleic acid probe, andj . extending the first portion of the second extended nucleic acid probe from the free 3’ end using the second oligonucleotide as a template, thereby regenerating the nucleic acid probe.

64. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, and wherein the nucleic acid probe comprises, in a 5' to 3' orientation:i. a nuclease recognition sequence,ii. a sequencing-by-synthesis (SBS) sequence,iii. a spatial barcode sequence, andiv. a capture sequenceb. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture sequence of the nucleic acid probe,c. extending the nucleic acid probe using the target molecule as a template to produce an extended nucleic acid probe comprising a first strand cDNA,d. contacting the substrate with a oligonucleotide under conditions wherein the oligonucleotide hybridizes to the extended nucleic acid probe,e. contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the extended nucleic acid probe, producing a first portion of the extended nucleic acid probe and a second portion of the extended nucleic acid probe, wherein the first portion of the extended nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion of the extended nucleic acid probe contains the first strand cDNA,f. extending the first portion of the extended nucleic acid probe from the free 3’ end using the oligonucleotide as a template, thereby regenerating the nucleic acid probe, g. contacting the substrate with a nuclease, wherein the nuclease cleaves the nuclease recognition sequence of the nucleic acid probe, producing a first portion of the nucleic acid probe and a second portion of the nucleic acid probe, wherein the first portion of the nucleic acid probe is immobilized on the substrate at the 5’ end, and wherein the second portion comprises the SBS sequence, the spatial barcode sequence, and the capture sequence of the nucleic acid probe, andh. extending the first portion of the nucleic acid probe from the free 3’ end, using the oligonucleotide as a template to regenerate the nucleic acid probe.

65. A method of regenerating a substrate for spatially tagging a target molecule in a sample, comprising:a. providing a substrate comprising a plurality of nucleic acid probes, wherein each nucleic acid probe of the plurality of nucleic acid probes is immobilized on the substrate at the 5' end of the nucleic acid probe, wherein a first nucleic acid probe comprises a capture probe, wherein the capture probe comprises in a 5' to 3' orientation:i. a nuclease enzyme recognition site, andii. a capture sequence,wherein a second nucleic acid probe comprises a spatial barcode probe, wherein the spatial barcode probe comprises, in a in a 5' to 3' orientation:iii. a spatial barcode, andiv. a template switch oligonucleotide (TSO) sequence,wherein the spatial barcode probe is 3’ blocked such that extension of the spatial barcode probe is prevented;b. contacting the substrate with a sample comprising a target molecule under conditions wherein the target molecule hybridizes to the capture sequence of the capture probe and extending the capture sequence to comprise first strand cDNA using the target molecule as a template;c. extending the capture probe using the hybridized target molecule as a template to generate an extended capture probe comprising first strand cDNA;d. hybridizing a template switch oligonucleotide complement (TSO’) to the capture probe;e. hybridizing the TSO’ sequence and the TSO sequence and extending the capture probe using the TSO sequence as template to generate an extended capture probe that comprises first strand cDNA and a sequence that is complementary to the spatial barcode sequence (SBC’);f. contacting the substrate with a nuclease that hybridizes to the nuclease recognition site of the extended capture probe;g. contacting the substrate with a nuclease, wherein the nuclease cleaves the extended capture probe at the nuclease recognition sequence to generate a first portion of the capture probe and a second portion of the capture probe;h. contacting the substrate with a regeneration oligonucleotide under conditions wherein the regeneration oligonucleotide hybridizes to the first portion capture probe, wherein the regeneration oligonucleotide comprises a sequence that is complementary to at least a portion of the nuclease recognition sequence and a sequence that is complementary to the capture sequence; andi. extending the first portion of the capture probe against the template to thereby regenerate the capture probe.

66. The method of claim 61, wherein the nucleic acid probe further comprises a sequencing- by-synthesis (SBS) sequence.

67. The method of claim 66, wherein the SBS sequence is SBS 12 or a complement thereof (SBS12').

68. The method of claim 66, wherein the SBS sequence is SBS3 or a complement thereof (SB S3').

69. The method of claim 61, wherein the nucleic acid probe further comprises a flow cell clustering sequence.

70. The method of claim 69, wherein the flow cell clustering sequence is selected from the group consisting of P5, P5', P7, P7'.

71. The method of claim 61, wherein the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

72. The method of claim 62, wherein the customizable oligonucleotide comprises:a. a sequence that is complementary to the nuclease recognition sequence, b. a linker, andc. a sequence that is complementary to a capture sequence.

73. The method of claim 72, wherein the customizable oligonucleotide further comprises an identifier sequence, wherein the identifier sequence is located between the capture sequence and the linker.

74. The method of claim 62, wherein extending the first portion of the extended custom nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the extended custom nucleic acid probe.

75. The method of claim 62, wherein the regeneration oligonucleotide is not bound to the surface of the substrate.

76. The method of claim 62, wherein the regeneration oligonucleotide is removed after step (h).

77. The method of claim 62, wherein after the second portion of the extended custom nucleic acid probe is collected, the second portion of the extended custom nucleic acid probe is amplified and sequenced.

78. The method of claim 63, wherein the first oligonucleotide comprises a randomer and optionally a second adapter sequence.

79. The method of claim 63, wherein the first nuclease specifically targets the first nuclease recognition sequence, and the second nuclease specifically targets the second nuclease recognition sequence.

80. The method of claim 63, wherein the second nuclease also cleaves the extended first oligonucleotide.

81. The method of claim 63, wherein extending the first portion of the first nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the first nucleic acid probe.

82. The method of claim 63, wherein extending the first portion of the second nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the second nucleic acid probe.

83. The method of claim 63, wherein the first or second oligonucleotide is not bound to the surface of the substrate.

84. The method of claim 63, wherein the second oligonucleotide is removed after step (1).

85. The method of claim 63, wherein the nucleic acid probe further comprises a sequencing- by-synthesis (SBS) sequence.

86. The method of claim 85, wherein the SBS sequence is SBS 12 or a complement thereof (SBS12').

87. The method of claim 86, wherein the SBS sequence is SB S3 or a complement thereof (SB S3').

88. The method of claim 63, wherein the nucleic acid probe further comprises a flow cell clustering sequence.

89. The method of claim 88, wherein the flow cell clustering sequence is selected from the group consisting of P5, P5', P7, P7'.

90. The method of claim 63, wherein the nucleic acid probe further comprises a unique molecular identifier (UMI) or a single molecule identifier (SMI).

91. The method of claim 64, wherein the oligonucleotide comprises:a. a nucleic acid sequence that is complementary to the nuclease recognition sequence,b. a nucleic acid sequence that is complementary to the SBS sequence c. a nucleic acid sequence that is complementary to the spatial barcode sequence, andd. a nucleic acid sequence that is complementary to the capture sequence.

92. The method of claim 64, wherein extending the first portion of the nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the nucleic acid probe.

93. The method of claim 64, wherein the second portion of the nucleic acid probe is collected.

94. The method of claim 64, wherein extending the first portion of the extended nucleic acid probe from the free 3’ end results in the displacement of at least a portion of the second portion of the extended nucleic acid probe.

95. The method of claim 94, wherein the second portion of the extended nucleic acid probe is collected, and sequenced.

96. The method of claim 65, wherein extending the first portion of capture probe from the free 3’ end results in the displacement of at least a portion of the second portion of capture probe.

97. The method of claim 65, wherein the regeneration oligonucleotide is removed after step (i).

98. The method of claim 65, wherein after the second portion of the capture probe is collected, the second portion of the capture probe is amplified and sequenced.

99. The method of claim 65, wherein the capture probe further comprises an adapter primer sequence.

100. The method of claim 65, wherein the spatial barcode probe further comprises a primer sequence and a UMI sequence.

101. The method of any one of claims 61-100, further comprising determining the spatial location of the target molecule.

102. The method of any one of claims 61-101, further comprising contacting the substrate with a single-stranded DNA-specific 3’-5’ exonuclease before reusing the substrate.

103. The method of claim 102, wherein the exonuclease is a Thermus thermophilus exonuclease.

104. The method of any one of claims 61-103, wherein the nuclease is a Cas nickase or a Cas nickase variant.

105. The method of claim 104, wherein the Cas nickase is Cas9 nickase (Cas9n), Streptococcus pyogenes Cas9 nickase (spCas9n), Streptococcus pyogenes Cas9 High Fidelity nickase (spCas9HFn), Staphylococcus aureus Cas9 nickase (SaCas9n), Staphylococcus aureus Cas9 High Fidelity nickase (SaCas9HFn), Casl2a nickase (Casl2an) or a variant thereof.

106. The method of claim 105, wherein the Cas9 nickase is used in conjunction with a tracerRNA:crRNA or a single-guide RNA.

107. The method of any one of claims 61-103, wherein the nuclease is a nicking endonuclease.

108. The method of claim 107, wherein the nicking endonuclease is Nt.BstNBI, Nt.BstSEI, Nt.BspQI, Nt.BbvCI, Nt.AlwI, Nb.BsrDI, Nb.BsmI, Nt.CviPII, Nb.BtsI, Nb.BbvCI, Nb.BssSI, orNt.BsmAI.

109. The method of any one of claims 61-103, wherein the nuclease is a homing endonuclease modified to have nickase activity.

110. The method of claim 109, wherein the homing endonuclease modified to have nickase activity is an I-Scel nickase, an I-Anil nickase, or an I-Dmol nickase.

111. The method of any one of claims 61-103, wherein the nuclease is a chimeric nickase.

112. The method of claim 111, wherein the chimeric nickase comprises a DNA binding molecule fused to a DNA nicking domain.

113. The method of any one of claims 61-103, wherein the nuclease is a Zinc finger nickase.

114. The method of any one of claims 61-103, wherein the nuclease is a transcription activatorlike effector (TALE) nickase.

115. The method of claim 114, wherein the TALE nickase is an engineered TALE nickase comprising a TALE repeat domain fused with a FokI nuclease domain or a MutH nicking variant.

116. The method of any one of claims 61-103, wherein the nuclease is a rare-cutter nickase.

117. The method of any one of claims 61-103, wherein the nuclease is a uracil or Oxo-G dependent repair enzyme.

118. The method of claim 117, where in the nuclease is USER, EndoQ, FPG, or OGG.

119. The method of any one of claims 61-118, wherein the substrate is a solid surface.

120. The method of claim 119, wherein the solid surface is planar.

121. The method of any one of claims 61-118, wherein the substrate is a glass slide, a flow cell, an array, or beads.

122. The method of any one of claims 61-121, wherein the nucleic acid probes are arranged in specific locations on the substrate.

123. The method of any one of claims 61-122, wherein the spatial barcode correlates to a positional location on the substrate.

124. The method of any one of claims 61-123, wherein the plurality of nucleic acid probes comprise a plurality of clusters of nucleic acid probes, wherein the nucleic acid probes of each cluster comprise a unique spatial barcode.

125. The method of any one of claims 61-124, wherein the spatial barcode spatially tags the nucleic acid probe.

126. The method of any one of claims 61-125, wherein the capture sequence comprises a polythymidine (poly-T) sequence or a target-specific capture sequence.

127. The method of any one of claims 61-125, wherein the capture sequence comprises a randomer or a semi-randomer.

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