Systems and methods for spatial reference sequencing

Abstract

Provided herein are methods, systems, compositions, and kits that can determine spatial information between a plurality of analytes in a sample by tagging such analytes with tags, where identities and / or locations of such tags are previously unknown prior to tagging. Each of a plurality of analyte sequences may be tagged with multiple spatial tags, such that each analyte sequence is associated with a set of two or more spatial tags. The sets of spatial tags may be analyzed to generate a map of analyte sequences. The map may comprise information about the respective absolute positions of each of a set of sequences with respect to a reference sequence. The map may comprise information about the respective probability cloud (or likely location) of each of a set of sequences with respect to a reference sequence.

Description

SYSTEMS AND METHODS FOR SPATIAL REFERENCE SEQUENCINGCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Pat. App. No. 63 / 575,548, filed April 5, 2024, which is entirely incorporated herein by reference for all purposes.BACKGROUND

[0002] Biological sample processing has various applications in the fields of molecular biology and medicine (e.g., diagnosis). For example, nucleic acid sequencing may provide information that may be used to diagnose a certain condition in a subj ect and in some cases tailor a treatment plan. Sequencing is widely used for molecular biology applications, including vector designs, gene therapy, vaccine design, industrial strain design and verification. Biological sample processing may involve a fluidics system and / or a detection system.

[0003] Despite the advance of sequencing technology, determining sequences with spatial resolution still requires laborious efforts.SUMMARY

[0004] Recognized herein is a need for methods, systems, compositions, and kits for the spatial mapping of a plurality of analyte sequences with respect to each other. Recognized herein is a need for methods, systems, compositions, and kits that can use tags, whose identities and / or locations are previously unknown. Provided herein are methods, systems, compositions, and kits that address at least the abovementioned needs. Beneficially, the provided systems, methods, kits, and compositions allow for the use of spatial tags which respective locations are not previously known or assayed prior to tagging the analyte sequences. This is advantageous over other systems which determine a spatial relationship between analyte sequences using an array of probes or other spatial tags with known locations, such as a system which has to deliberately generate such array with known locations and / or a system in which a predetermination step has to be performed to determine the identities and locations of the probes or other spatial tags in the array.

[0005] In an aspect, provided is a method for spatial mapping, comprising: (a) immobilizing a first set of beads comprising a plurality of first oligonucleotide molecules on a substrate, each of the first set of beads comprising a set of first oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the first set of beads; (b) loading a second set ofbeads comprising a plurality of second oligonucleotide molecules to the substrate comprising the first set of beads immobilized thereto, each of the second set of beads comprising a set of second oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the second set of beads, and capturing at least a subset of second oligonucleotide molecules of the second set of beads with at least a subset of first oligonucleotide molecules of the first set of beads: (c) extending the subset of first oligonucleotide molecules to generate a plurality of composite molecules on the first set of beads, each of the plurality of composite molecules comprising a first tag sequence derived from a spatial tag from the first set of beads and a second tag sequence derived from a spatial tag from the second set of beads; (d) loading a sample to the substrate comprising the first set of beads immobilized thereto, and capturing a plurality of analyte sequences from the sample with oligonucleotide molecules of the first set of beads, the oligonucleotide molecules comprising one or more of (i) at least a subset of the plurality of composite molecules or derivatives thereof, and / or (ii) an additional subset of first oligonucleotide molecules or derivatives thereof; and (e) generating spatially tagged analyte molecules, wherein each of the spatially tagged analyte molecules comprises a sequence of or derived from a spatial tag from the first set of beads.

[0006] In some embodiments, the method further comprises sequencing the spatially tagged analyte molecules or derivatives thereof, to generate sequencing data.

[0007] In some embodiments, the method further comprises using the sequencing data to generate a spatial map of the plurality of analyte sequences by identifying sets of associated spatial tags, where the spatial map comprises information about the respective locations or respective probability cloud of each of a set of analyte sequences with respect to a reference analyte sequence.

[0008] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.

[0009] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are amplified off the substrate prior to the sequencing.

[0010] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are released from the first set of beads or the second set of beads prior to the sequencing.

[0011] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.

[0012] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to a second substrate different from the substrate.

[0013] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are sequenced without being attached to any substrate.

[0014] In some embodiments, the method further comprises sequencing an additional subset of the plurality of composite molecules that did not capture any analyte sequence, or derivatives thereof.

[0015] In some embodiments, in (a) the first set of beads are immobilized to a plurality of individually addressable locations on the substrate.

[0016] In some embodiments, a first bead of the first set of beads and a second bead of the second set of beads are the same type of bead.

[0017] In some embodiments, a first bead of the first set of beads and a second bead of the second set of beads are different types of beads.

[0018] In some embodiments, the method further comprises, prior to (d), contacting the first set of beads with a plurality of capture sequence molecules comprising a capture sequence and a binding sequence, wherein the plurality of composite molecules bind to the binding sequence of the plurality of capture sequence molecules and is extended to generate an analyte capture sequence on a plurality of extended composite molecules, and wherein in (d) the plurality of analyte sequences are captured via the analyte capture sequence on the plurality of extended composite molecules.

[0019] In some embodiments, the analyte capture sequence comprises a poly-T sequence, a targeted sequence, a randomer sequence, or reverse complements thereof.

[0020] In some embodiments, the sample comprises a tissue sample, wherein the plurality of analyte sequences comprises a plurality of messenger ribonucleic acid (mRNA) transcript sequences or DNA sequences.

[0021] In some embodiments, the method further comprises fixing said sample.

[0022] In some embodiments, the method further comprises permeabilizing said sample.

[0023] In some embodiments, the first set of beads comprises at least 50 different spatial tags.

[0024] In some embodiments, the first set of beads comprises at least 100 different spatial tags.

[0025] In some embodiments, the first set of beads comprises at least 1000 different spatial tags.

[0026] In some embodiments, the first set of beads comprises at least 10,000 different spatial tags.

[0027] In some embodiments, the first set of beads immobilized to the substrate comprises at least 1.000,000 beads.

[0028] In some embodiments, the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.

[0029] In some embodiments, the first set of beads immobilized to the substrate comprises at least 1,000,000,000 beads.

[0030] In another aspect, provided is a method for spatial mapping, comprising: (a) immobilizing a first set of beads comprising a plurality of first oligonucleotide molecules on a substrate, each of the first set of beads compnsing a set of first oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the first set of beads; (b) loading a second set of beads comprising a plurality of second oligonucleotide molecules to the substrate comprising the first set of beads immobilized thereto, each of the second set of beads comprising a set of second oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the second set of beads, and capturing at least a subset of second oligonucleotide molecules of the second set of beads with at least a subset of first oligonucleotide molecules of the first set of beads; (c) extending the subset of first oligonucleotide molecules to generate a plurality of first composite molecules on the first set of beads or extending the subset of second oligonucleotide molecules to generate a plurality of second composite molecules on the second set of beads or both, each of the plurality of first or second composite molecules comprising a first tag sequence derived from a spatial tag from the first set of beads and a second tag sequence derived from a spatial tag from the second set of beads; (d) loading a sample to the substrate, and capturing a plurality of analyte sequences from the sample with oligonucleotide molecules of the second set of beads, the oligonucleotide molecules comprising one or more of (i) at least a subset of the plurality of second composite molecules or derivatives thereof, and / or (ii) an additional subset of second oligonucleotide molecules or derivatives thereof; and (e) generating spatially tagged analyte molecules, wherein each of the spatially tagged analyte molecules comprises a sequence of or derived from a spatial tag from the second set of beads.

[0031] In some embodiments, the method further comprises sequencing the spatially tagged analyte molecules or derivatives thereof, to generate sequencing data.

[0032] In some embodiments, the method further comprises using the sequencing data to generate a spatial map of the plurality' of analyte sequences by identifying sets of associated spatial tags, where the spatial map comprises information about the respective locations or respective probability cloud of each of a set of analyte sequences with respect to a reference analyte sequence.

[0033] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.

[0034] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are amplified off the substrate prior to the sequencing.

[0035] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are released from the first set of beads or the second set of beads prior to the sequencing.

[0036] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.

[0037] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to a second substrate different from the substrate.

[0038] In some embodiments, the spatially tagged analyte molecules or derivatives thereof are sequenced without being attached to any substrate.

[0039] In some embodiments, the method further comprises the plurality7of first composite molecules or derivatives thereof.

[0040] In some embodiments, in (a), the first set of beads are immobilized to a plurality7of individually addressable locations on the substrate.

[0041] In some embodiments, a first bead of the first set of beads and a second bead of the second set of beads are the same type of bead.

[0042] In some embodiments, a first bead of the first set of beads and a second bead of the second set of beads are different types of beads.

[0043] In some embodiments, the plurality of second oligonucleotide molecules comprises an analyte capture sequence, and wherein in (d) the plurality of analyte sequences is captured via the analyte capture sequence.

[0044] In some embodiments, the analyte capture sequence comprises a poly-T sequence, a targeted sequence, a randomer sequence, or reverse complements thereof.

[0045] In some embodiments, the sample comprises a tissue sample, wherein the plurality of analyte sequences comprises a plurality of messenger ribonucleic acid (mRNA) transcript sequences or DNA sequences.

[0046] In some embodiments, the method further comprises fixing said sample.

[0047] In some embodiments, the method further comprises permeabilizing said sample.

[0048] In some embodiments, the first set of beads comprises at least 50 different spatial tags.

[0049] In some embodiments, the first set of beads comprises at least 100 different spatial tags.

[0050] In some embodiments, the first set of beads comprises at least 1000 different spatial tags.

[0051] In some embodiments, the first set of beads comprises at least 10,000 different spatial tags.

[0052] In some embodiments, the first set of beads immobilized to the substrate comprises at least 1,000,000 beads.

[0053] In some embodiments, the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.

[0054] In some embodiments, the first set of beads immobilized to the substrate comprises at least 1,000,000,000 beads.

[0055] Another aspect of the present disclosure provides a non-transitoiy computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0056] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0057] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0058] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also "‘Figure” and “FIG.” herein) of which:

[0060] FIGs. 1A-1B illustrate examples of a geolocation bead. FIG. 1C illustrates example methods for synthesizing the geolocation bead constructs of FIGs. 1A-1B and for releasing first strands from such geolocation bead constructs.

[0061] FIGs. 2A-2B illustrate reagents and a workflow for generating a spatially tagged sequence using a bridge construct and multiple spatial tags.

[0062] FIG. 3 illustrates an example map that can be generated from a sample set of spatial tag data.

[0063] FIG. 4 illustrates a general workflow of a substrate-based spatial screening method.

[0064] FIG. 5A illustrates a spatial screening scheme using multiple substrates.

[0065] FIG. 5B illustrates an example particle comprising a plurality of oligonucleotide molecules including spatial tags.

[0066] FIG. 6 illustrates an example droplet with reagents of the present disclosure.

[0067] FIG. 7 illustrates an example solution environment with reagents of the present disclosure.

[0068] FIGs. 8A-8B illustrate examples of additional geolocations bead constructs.

[0069] FIG. 9A illustrates examples of additional bridge constructs. FIG. 9B illustrates examples of additional tagging schemes. FIGs. 9C-9D illustrate various bead release mechanisms.

[0070] FIG. 10 illustrates an example method for generating a tagged sequence under a 5’ approach.

[0071] FIG. 11A illustrates example constructs of a geolocation bead and a capture bead. FIG. 11B provides example capture complexes comprising capture beads.

[0072] FIG. 12 illustrates an example of additional capture bead and geolocation bead constructs.

[0073] FIG. 13 illustrates example individually addressable locations on different substrates.

[0074] FIGs. 14A, 14B, 14C, 14D, 14E, 14E, 14F and!4G illustrate different examples of cross-sectional surface profiles of a substrate.

[0075] FIGs. 15A-15B illustrate methods for loading beads onto a substrate. FIG. 15A illustrates a method for loading beads onto specific regions of a substrate. FIG. 15B illustrates a method for loading a subset of beads onto specific regions of a substrate.

[0076] FIG. 16 shows an example coating of a substrate with a hexagonal lattice of beads.

[0077] FIG. 17A shows an example system and method for loading a sample or a reagent onto a substrate. FIG. 17B shows another example system and method for loading a sample or a reagent onto a substrate.

[0078] FIG. 18 shows a flowchart for an example of a method for sequencing a nucleic acid molecule.

[0079] FIGs. 19A, 19B, 19C, andl9D illustrate an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs.

[0080] FIG. 20 illustrates an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs.

[0081] FIG. 21 illustrates an example of a substrate map using fiducial marker beads according to the methods described herein.

[0082] FIG. 22 illustrates a schematic for subjecting a reaction space to electrophoresis.

[0083] FIGs. 23A-23B illustrate an example method for preparing a geolocation bead library comprising two types of geolocation beads.

[0084] FIG. 24 illustrates a schematic bead-capture matrix from a top view and a cross-section side view in panels (A) and (B) respectively.

[0085] FIG. 25 illustrates an example workflow for bead-on-bead capture.

[0086] FIG. 26 illustrates an additional example workflow for bead-on-bead capture.

[0087] FIG. 27 illustrates an additional example workflow for bead-on-bead capture.

[0088] FIGs. 28A, 28B, and 28C illustrate an example molecular design for a pair of beads that can be used for bead-on-bead capture methods described herein. SEQ ID NOs for nucleic acid sequences in the upper left portion of FIG. 28A (from top to bottom): SEQ ID NOs: 1-5. SEQ ID NOs for nucleic acid sequences of 10 or more nucleotides in the upper right portion of FIG. 28A (from top to bottom): SEQ ID NOs:6-10. SEQ ID NOs for nucleic acid sequences in the bottom portion of FIG. 28A (from top to bottom): SEQ ID NOs: 11-14. SEQ ID NOs for nucleic acid sequences in the upper left portion of FIG. 28B (from top to bottom): SEQ ID NOs: 15-19. SEQ ID NOs for nucleic acid sequences of 10 or more nucleotides in the upperright portion of FIG. 28B (from top to bottom): SEQ ID NOs:20-24. SEQ ID NOs for nucleic acid sequences in the bottom portion of FIG. 28B (from top to bottom): SEQ ID NOs:25-28. SEQ ID NOs for nucleic acid sequences of 10 or more nucleotides in the upper portion of FIG. 28C (from top to bottom): SEQ ID NOs:29-34. SEQ ID NOs for nucleic acid sequences of 10 or more nucleotides in the lower portion of FIG. 28C (from top to bottom): SEQ ID NOs:35- 36.

[0089] FIG. 29 illustrates an example test of bead-on-bead capture on a surface.

[0090] FIG. 30 illustrates an additional example workflow for bead-on-bead capture.DETAILED DESCRIPTION

[0091] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0092] Provided herein are systems, methods, kits, and compositions for the spatial mapping of a plurality of analyte sequences with respect to each other. A plurality of composite molecules, each composite molecule comprising multiple (two or more) barcodes derived from multiple spatial tags, may be generated. When the composite molecules, or their derivatives, are sequenced, the association of the multiple barcodes in the single composite molecule may inform the relative positions of the multiple spatial tags with respect to each other to generate a map and / or probability cloud of the different spatial tags. An analyte may be tagged by one or more spatial tags, and such one or more spatial tags used to position the analyte on the map and / or probability cloud. In some cases, an analyte sequence or each of a plurality of analyte sequences may be tagged by a single spatial tag. In some cases, an analyte sequence or each of a plurality of analyte sequences may be tagged with multiple spatial tags, such that each analyte sequence is associated with a set of two or more spatial tags. The sets of spatial tags may be analyzed to generate a map of analyte sequences. The map may comprise information about the respective absolute positions of each of a set of sequences with respect to a reference sequence. Alternatively, or in addition, the map may comprise information about the respective probability7cloud (or likely location) of each of a set of sequences with respect to a reference sequence. Beneficially, the provided systems, methods, kits, and compositions allow for the use of spatial tags which respective locations are not previously known or assayed prior totagging the analyte sequences. This is advantageous over other systems which determine a spatial relationship between analyte sequences using an array of probes or other spatial tags with known locations, such as a system which has to deliberately generate such array with known locations and / or a system in which a pre-determination step has to be performed to determine the identities and locations of the probes or other spatial tags in the array. Methods for determining spatial relationships using spatial tags or barcodes whose location information is not previously known prior to tagging analytes are also described in U.S. Patent Pub. No. 2024 / 0043833A1 and Weinstein et al., Cell 178, 229-241 (2019), each of which is incorporated by reference herein in its entirety.

[0093] A method for the spatial mapping of a plurality of analyte sequences may comprise: (a) loading a plurality of geolocation beads onto a substrate to immobilize the plurality of geolocation beads onto a plurality of individually addressable locations on the substrate, where each geolocation bead comprises a spatial tag; (b) loading a sample onto the substrate (e.g., over the plurality of geolocation beads), where the sample retains a spatial relationship between a plurality of analyte sequences; (c) releasing spatial tags from the geolocation beads; (d) subjecting the sample to conditions sufficient to tag a set of at least two spatial tags to each of the plurality of analyte sequences (e.g., mRNA) to generate a plurality of spatially tagged sequences; and (e) generating a map of the plurality of analyte sequences by identifying sets of spatial tags from the plurality of spatially tagged sequences, where the map comprises information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence.

[0094] FIGs. 1A-1B illustrate examples of a geolocation bead. Referring to FIGs. 1A-1B, a geolocation bead 101 may comprise a plurality of oligonucleotide molecules (e.g., 103, 123) attached thereto. Though two oligonucleotide molecules are illustrated in these figures, any number of oligonucleotide molecules may be attached to the geolocation bead, for example on the order of 10, 102, 103, 104, 105, 106, 107, 108, or more. An oligonucleotide molecule 103 may be a partially or wholly double-stranded molecule, in which a first strand comprises a first attachment sequence 107, a spatial tag 109, and a second attachment sequence 111, and a second strand comprises complementary sequences 107’, 109’, and 111’ for the sequences in the first strand, respectively. Each oligonucleotide molecule of the plurality of oligonucleotide molecules on the geolocation bead 101 may comprise a common spatial tag 109. Different geolocation beads may comprise different spatial tags. For example, a first geolocation bead may comprise a first plurality of oligonucleotide molecules each comprising a first spatial tagand a second geolocation bead may comprise a second plurality' of oligonucleotide molecules each comprising a second spatial tag that is different form the first spatial tag. Thus, two nucleic acid molecules comprising the spatial tag, when identified (e.g., sequenced), may inform an operator that the two nucleic acid molecules were tagged by oligonucleotide molecules from the same geolocation bead.

[0095] In some cases, as illustrated in FIG. IB, an oligonucleotide molecule may comprise a unique molecular identifier (UMI) sequence (e.g., 115a, 115b). The unique molecular identifier sequence may be unique to each oligonucleotide molecule of the plurality of oligonucleotide molecules on the same geolocation bead. For example, oligonucleotide molecule 103 and oligonucleotide molecule 123 which is attached to the same geolocation bead 101 may comprise the same spatial tag 109 sequence, but different UMI sequences 115a, 115b, respectively. The UMI sequence may be disposed anywhere between the first attachment sequence 107 and the second attachment sequence 111. The UMI may be a nucleic acid sequence. The UMI may comprise at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34. 35. 36. 37, 38, 39, 40, 41,42. 43. 44, 45, 46, 47, 48. 49. 50. 60. 70, 80, 90, 100 or more bases. Alternatively or in addition, the UMI may comprise at most about 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer bases.

[0096] The first strands comprising the spatial tag 109 may be released from the geolocation bead 101 via various mechanisms. For example, in an example release mechanism, a strand displacing polymerase (e.g., a polymerase with relatively strong ability to strand displacement compared to polymerases that lack strand displacement activity' such as T4 and T7 DNA polymerases) and a primer may be provided under conditions sufficient to displace the first strand (comprising sequences 107, 109, 111) from the oligonucleotide molecule. Examples of strand displacing polymerases include, but are not limited to, Bst DNA polymerase, large fragment polymerase, and 029 polymerase. In some cases, for strand displacement in the 5’ to 3’ direction, the strand displacing polymerase may generate or leverage a flap at the 5’ end. As described elsewhere herein, where strand displacement is in the 3’ to 5’ direction, the strand displacing polymerase may generate or leverage a flap at the 3’ end. In another example release mechanism, an enzyme may be provided to digest the second strand (e.g., at least a portion thereof) to release the first strand. The enzy me may comprise 5’ to 3’ exonuclease activity'. Examples of enzymes with 5?to 3’ exonuclease activity include, but are not limited to, LambdaExonuclease, T7 Exonuclease, T5 Exonuclease, Exonuclease V, and Exonuclease VIII. The enzyme may comprise 3’ to 5’ exonuclease activity. Examples of enzymes with 3‘ to 5’ exonuclease activity include, but are not limited to. Exonuclease III and Exonuclease V. In one example, a 5’ end of the second strand may be phosphorylated and subjected to Lambada Exonuclease.

[0097] In some cases, the oligonucleotide molecules (e.g., 103, 123) may be immobilized to the bead 101 via a cross-link or other linker. The cross-link or other link may be cleaved to release the oligonucleotide molecules, or portion thereof, such as by applying one or more stimuli, including light stimuli, heat stimuli, chemical stimuli, magnetic stimuli, electrical stimuli, and other stimuli, or combination thereof. In some cases, the oligonucleotide molecules may be immobilized to the bead via a photo-crosslink. A photo-crosslink may be generated by a photo cross-linking reaction. In some instances, an oligodeoxynucleotide (ODN) comprising 3-cyanovinylcarbazole nucleoside (CNVK) can be subjected to photoirradiation conditions to photo-cross-link a target pyrimidine and theCNVK. In some instances, irradiation is provided at 366 nm for about 1 second for photo-cross-linking to thymine, and for up to about 25 seconds for photo-cross-linking to cytosine. In this example, irradiation provided at 312 nm for about 3 minutes can reverse the cross-link. Various other cross-linking reagents may be used to generate a cross-link (e.g., chemical cross-link). In some cases, heat may be applied to denature a double-stranded molecule to facilitate release of at least one of the strands (e.g., first strand) from the bead. The heat stimulus can be combined with cross-linking reactions. For example, the first strand may be released from the bead by applying a heat stimulus. In some cases, additionally, a primer may be cross-linked (e.g., photo-cross-linked) to the oligonucleotide molecule and extended, such as to prevent re-hybridization of the released strand to the remaining strand.

[0098] In some cases, the first strand may comprise one or more features (e.g., a blocking group) that prevent digestive activity by the enzyme on the first strand, such as illustrated as “X"’ for oligonucleotide molecule 123. In some cases, the first strand may comprise a cleavage site, such as illustrated as “U’" for oligonucleotide molecule 123. In some cases, the oligonucleotide molecule may be capped by an uracil, a ribonucleotide, or other modified nucleotide to facilitate digestion of the second strand to release the first strand. In some cases, the second strand may comprise one or more nicks (or nicks may be created) to facilitate strand displacement and / or digestive activity by an enzy me. In some instances, the second strand maycomprise one or more cleavable or digestible moieties (e.g., ribonucleotides, uracil, etc.) for cleavage or digestion by one or more enzymes (e.g., RNase HII).

[0099] In some examples, a USER cleavage reaction may be performed to process the cleavable or digestible moieties from the double stranded molecules and release the remaining molecule from the bead. In the USER cleavage reaction, a USER (uracil-specific excision reagent) enzyme may generate a nucleotide gap at a location of an uracil base (e.g., dU) in the molecule and facilitate cleavage.

[0100] FIGs. 9C-9D illustrate various bead release mechanisms. For all panels, shown is a bead 981 comprising a double-stranded oligonucleotide molecule 985, which comprises a first strand 982 and a second strand 983.

[0101] In panel (A) of FIG. 9C, the first strand 982 may comprise a cleavage site, denoted “U”, at or adjacent to the 5:terminus. The cleavage site may be cleaved to release the first strand from the bead. One or both strands may have one or more cleavage sites. In some cases, a USER enzyme mix may be used for the cleavage reaction. The USER enzyme mix may comprise uracil DNA glycosylase (UDG). which removes the sugar and creates an abasic site (AP site), and endonuclease (e.g., endonuclease VIII). which binds to the AP Site and cleaves. In some cases, to accelerate cleavage, the components of the USER enzy me mix may7be divided and provided independently and later combined. For example, with reference to the methods of FIG. 4, the UDG may be provided separately to the substrate 450 comprising a plurality of geolocation beads immobilized thereto, prior to loading of the sample (e.g., tissue), and the endonuclease may be provided separately to the sample (e.g., tissue). The cleavage reaction may take effect after the sample is loaded onto the substrate. Beneficially, the AP sites may be created as a substrate-priming step, prior to loading of the sample. In some cases, the endonuclease may be replaced with an APE1 enzyme in the USER enzyme mix, which cleaves multiple times and is Mg2+-dependent (as opposed to endonuclease VIII which is Mg2+- ind ependent). In an example, with reference to the methods of FIG. 4, the UDG and APE1 enzy me may be provided to the substrate 450 comprising a plurality of geolocation beads immobilized thereto, without Mg2+, and prior to loading of the sample, to prime the substrate and form the AP sites. The APE1 enzyme may bind to the AP sites, without cleavage activity due to the lack of the Mg2+. Separately, Mg2+may be provided separately to the sample (e.g., tissue). The cleavage reaction may take effect after the sample is loaded onto the substrate. It will be appreciated that different components of an enzyme mix may be provided to (1) the substrate, and (2) the sample, other than the specific examples (e.g., a first type of enzyme tothe substrate, and a second type of enzy me to the sample; two types of enzy mes to the substrate, and a catalyst or other reagent to the sample, or vice versa, etc.).

[0102] Panels (B)-(D) of FIGs. 9C-9D describe non-enzymatic release mechanisms. In panel (B) of FIG. 9C, the oligonucleotide molecule 985 may be coupled to (e.g., conjugated to) a desthiobiotin moiety 986 (a biotin analog which lacks the sulfur atom), and the geolocation bead 981 may be coupled to a streptavidin moiety’ 988. The desthiobiotin moiety’ 986 and the streptavidin moiety’ 988 may be bound together to couple the oligonucleotide molecule 985 to the geolocation bead 981, though at less binding strength (e.g., with disassociation constant (Kd) on the order of 10"11M) than that between streptavidin and biotin moieties (e.g., with Kd on the order of 10'15M). Upon provision of a high concentration of biotin moieties 987, the biotin-streptavidin bonds may displace the desthiobiotin-streptavidin bonds to release the oligonucleotide molecule 985 from the geolocation bead 981. Such non-enzymatic release mechanisms may be beneficial over enzymatic release mechanisms. For example, it may result in shorter release time (faster than enzymatic cleavage time, e.g., using USER cleave); it may reduce cost (biotin is cheaper compared to enzyme reagents); it may improve diffusion in hydrogel environments as biotins are much smaller in size than enzymes: it may reduce waste by permitting recycling of streptavidin-coupled beads (by extracting the biotin from the used geolocation beads, and attaching new desthiobiotin-conjugated oligonucleotide molecules).

[0103] In panel (C) of FIG. 9D, the first strand 982 may comprise one or more azobenzene (denoted "X’) and a cleavage site, denoted “U”, at or adjacent to the 5’ terminus. For example, the cleavage site may comprise 1, 2, 3, 4, or more uracil residues. Azobenezene is a lightsensitive molecule that changes between atrans-form and a cis-form under certain light and / or heat conditions. For example, azobenzene changes from trans-form to cis-form under UV light, and changes from cis-form to trans-form under VIS light and / or heat. Azobenzene may enable fast photoswitch of hybridization states of at least a segment of two strands of nucleic acid molecules. Azobenzene may be incorporated between the nucleotides of the first strand. The melting temperature (Tm) between the first strand 982 and the second strand 983 may be significantly reduced when the azobenzene is in cis-form, as compared to the Tmbetween the two strands without azobenzene and as compared to the Tmwhen the azobenzene is in transform, as it weakens the hydrogen bonds between the two strands. The Tmbetween the first strand the second strand may not vary’ as much when the azobenzene is in trans-form as compared to the Tmbetween the two strands without the azobenzene. Thus, providing UV light stimulus (e.g., 365 nm) to the geolocation bead 981 may change the azobenzene to cis-formand dehybridize, destabilize, or facilitate dehybridizing or destabilizing of the two strands at the location of the azobenzene incorporations. With reference to the methods described with respect to FIG. 4, after the substrate is loaded with a plurality of geolocation beads, the geolocation beads may be subjected to a USER enzyme mix, as described elsewhere herein, to form nicked strands (e.g., on strand 982) at the cleavage sites. The geolocation bead may be subjected to UV light to trigger the azobenzene, resulting in fast release of the nicked strands from the geolocation bead.

[0104] In panel (D) of FIG. 9D, the oligonucleotide molecule 985 may be coupled to (e.g., conjugated to) a azobenzene moiety' 992 at a 5' terminus, which azobenzene moiety is hydrophobic, and the bead 981 may be coupled to (e.g., conjugated to) an alpha-cyclodextrine (a-CD) moiety 991, which is a barrel protein with a hydrophilic outer shell and a hydrophobic core. The photoswitchable trans-form and cis-form of azobenzene has been described elsewhere herein. In trans-form, azobenzene may enter the a-CD core via hydrophobic interaction and thus bind the oligonucleotide molecule 985 and the bead 981 in a non-covalent bond. In cis-form, azobenzene may exit the a-CD core and detach from the a-CD, thus releasing the oligonucleotide molecule 985 from the geolocation bead 981. The transition from transform to cis-form of azobenzene may be triggered by UV light.

[0105] In some cases, the first strand may comprise a capture entity 113, such as illustrated for oligonucleotide molecule 123, which is configured for capture by a capturing entity. The capture entity may comprise or be biotin, a capture sequence (e.g., nucleic acid sequence) which may be hybridized to the second strand or which may be part of another nucleic acid molecule conjugated to the oligonucleotide molecule, a magnetic particle capable of capture by application of a magnetic field, a charged particle capable of capture by application of an electric field, a combination thereof, or one or more other mechanisms configured for, or capable of, capture by a capturing entity. The capturing entity may comprise or be streptavidin when the capture moiety comprises biotin, a complementary capture sequence when the capture entity comprises a capture sequence, an apparatus, system, or device configured to apply a magnetic field when the capture entity comprises a magnetic particle, an apparatus, system, or device configured to apply an electrical field when the capture entity comprises a charged particle, a combination thereof, and / or one or more other mechanisms configured to capture the capture entity. In some instances, the capturing group may comprise a secondary capture entity, for example, for subsequent capture by a secondary capturing entity. The secondary capture entity and secondary capturing entity may comprise any one or more of the capturingmechanisms described elsewhere herein (e.g., biotin and streptavidin, complementary capture sequences, etc.). In some instances, the secondary capture entity can comprise a magnetic particle (e.g., magnetic geolocation bead) and the secondary capturing entity can comprise a magnetic system (e.g., magnet, apparatus, system, or device configured to apply a magnetic field, etc.). In some instances, the secondary capture entity can comprise a charged particle (e.g., charged geolocation bead carrying an electrical charge) and the secondary’ capturing entity can comprise an electrical system (e.g., magnet, apparatus, system, or device configured to apply an electric field, etc.). In some instances, the capture moiety' comprises biotin, the capturing moiety comprises streptavidin coupled to a secondary’ capture entity7, a magnetic geolocation bead, and the secondary capturing entity7comprises a magnetic system.

[0106] The spatial tag (e.g., 109) may be a nucleic acid sequence. The spatial tag may comprise at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60,70, 80, 90, 100 or more bases. Alternatively or in addition, the spatial tag may comprise at most about 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41. 40. 39. 38. 37, 36, 35, 34, 33,32. 31. 30. 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16. 15. 14. 13. 12. 11. 10. 9, 8, 7.6, 5, 4, 3 or fewer bases. The spatial tag may be unique and common to a geolocation bead amongst a plurality7of geolocation beads. In some cases, the spatial tag may be substantially unique to a geolocation bead amongst a plurality of geolocation beads such that at least about 50%. 55%. 60%. 65%. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of geolocation beads in the plurality of geolocation beads comprise unique spatial tags not contained by any other geolocation bead in the plurality’ of geolocation beads. The methods, systems, compositions, and kits of the present disclosure may comprise any number of geolocation beads. In some cases, there may be a number of geolocation beads on the order of at least on the order of 102, 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, or more beads. In some cases, there may be at least a number of geolocation beads to correspond to a number of individually addressable locations available on the substrate to which the geolocation beads are loaded. Example arrays (e.g., possible distributions) of individually addressable locations 1301 on a substrate are illustrated in FIG. 13 (e.g.. from a top view). In FIG. 13, panel A shows a substantially rectangular substrate with regular linear arrays, panel B shows a substantially circular substrate with regular linear arrays, and panel C shows an arbitrarily shaped substrate with irregular arrays. Irregular or regular arrays may be disposed on any shape of substrate.

[0107] The first attachment sequence (e.g., 107) may be configured for attachment or coupling (e.g., hybridization) to a bridge construct as described elsewhere herein. The second attachment sequence (e.g., Ill) may be configured for attachment or coupling (e.g., hybridization) to the bridge construct. The first attachment sequence and the second attachment sequence may comprise different sequences. The first attachment sequence and the second attachment sequence may be or comprise the same sequence. The first attachment sequence and / or the second attachment sequence may comprise at least about 3. 4, 5, 6. 7, 8, 9. 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more bases. Alternatively or in addition, the first attachment sequence and / or the second attachment sequence may comprise at most about 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer bases.

[0108] In some cases, the first attachment sequence may be common to a plurality of geolocation beads such that each geolocation bead comprises the same first attachment sequence. In some cases, the second attachment sequence may be common to a plurality of geolocation beads such that each geolocation bead comprises the same second attachment sequence. In some cases, both the first attachment sequence and the second attachment sequence may be common to a plurality of geolocation beads such that each geolocation bead comprises both the same first attachment sequence and the second attachment sequence.

[0109] In some cases, the first attachment sequence may be common only to a subset of plurality of geolocation beads such that each geolocation bead in the subset comprises the same first attachment sequence, and one or more other subsets of the geolocation beads comprises a different first attachment sequence. In some cases, the second attachment sequence may be common only to a subset of plurality of geolocation beads such that each geolocation bead in the subset comprises the same second attachment sequence, and one or more other subsets of the geolocation beads comprises a different second attachment sequence. In some cases, a pair of the first attachment sequence and the second attachment sequence may be common only to a subset of plurality of geolocation beads such that each geolocation bead in the subset comprises the same pair of the first attachment sequence and the second attachment sequence, and one or more other subsets of the geolocation beads comprises a different pair of the first attachment sequence and the second attachment sequence (where either one of or both the first attachment sequence and the second attachment sequence are different). In some cases, aplurality of geolocation beads may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20. 30, 40, 50, 60, 70, 80. 90. 100. 1000, 10,000, or more subsets of geolocation beads within a plurality of geolocation beads with the same number of different pairs of first and second attachment sequences. Alternatively or in addition, a plurality of geolocation beads may comprise at most 10,000, 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 subsets of geolocation beads within a plurality of geolocation beads with the same number of different pairs of first and second attachment sequences.

[0110] In some cases, for a geolocation bead, there may be only one pair of first attachment sequence and second attachment sequence amongst the plurality of oligonucleotide molecules on the geolocation bead. In other cases, for a geolocation bead, there may be more than one pair of first attachment sequence and second attachment sequence amongst the plurality of oligonucleotide molecules on the geolocation bead.[OHl] The plurality of geolocation beads may be loaded onto a substrate. The substrate may comprise a plurality7of individually addressable locations. Upon or subsequent to loading, the plurality of geolocation beads may be immobilized to respective individually addressable locations on the substrate. All or a subset of individually addressable locations on the substrate may immobilize the plurality of geolocation beads. The identities of the spatial tags on the geolocation beads may be unknown. Alternatively, or in addition, the locations of the spatial tags on the geolocation beads may be unknown. In some cases, the identities of the spatial tags on the geolocation beads may be known, but their individual locations on the substrate may be unknown. The present disclosure may obviate the need to assay the immobilized beads to determine the identity-location information of the spatial tags prior to tagging of analyte sequences. Substrates and individually addressable locations are described in further detail elsewhere herein.

[0112] Subsequent to immobilization of the geolocation beads on the substrate, a sample may be loaded onto the substrate. The sample may retain, at least to some extent, a spatial relationship between a plurality of analyte sequences. In some instances, a tissue slice is loaded onto the substrate, where the tissue slice retains, at least to some extent, a spatial relationship between the transcripts contained therein. A sample may comprise a biological sample. The biological sample may be derived from a subject. The sample may comprise a plurality7of nucleic acid molecules (e.g., comprising analyte sequences), such as messenger RNA (mRNA) molecules. Samples that can be used in the present methods, systems, compositions, and kits are described in further detail elsewhere herein.

[0113] The spatial tags may be released from the geolocation beads, by releasing the first strands as described elsewhere herein, prior to, during, or subsequent to loading of the sample on the substrate. The releasing of the first strands from the geolocation beads may be referred to herein as ‘activation’ of the geolocation beads. Geolocation beads may be activated by providing one or more enzymes, as described elsewhere herein. In some cases, geolocation beads may be activated by providing one or more stimuli, such as heat, chemical, or light stimuli, a combination thereof, or any other stimuli. In some cases, for example, both heat and enzymes may be provided to activate the geolocation beads.

[0114] FIG. 1C illustrates example methods for synthesizing the geolocation bead constructs of FIGs. 1A-1B and for releasing first strands from such geolocation bead constructs. A geolocation bead 101 may comprise a partially double-stranded nucleic acid molecule attached thereto, the partially double-stranded nucleic acid molecule including a first strand and a second strand. The second strand can comprise complementary sequences (e.g., 107’, 109’, and 111’) for the sequences to be included in the first strand of the oligonucleotide molecule (e.g., 103) on the final geolocation bead construct. For example, the complementary’ sequences may be complementary to a first attachment sequence 107. a spatial tag sequence 109, and a second attachment sequence 111, and optionally a UMI (e.g., 115a, 115b) of the first strand of the oligonucleotide molecule 103. The first strand of the partially double-stranded nucleic acid molecule may comprise the second attachment sequence 111 bound to the complementary sequence on the second strand, for example at the 5’ end of the second strand. The first strand can comprise one or more features (e.g., a blocking group (“X”), cleavage sites (“U”)), as described with respect to oligonucleotide molecule 123. A primer sequence 131 comprising at least a portion of the first attachment sequence 107 may bind to the complementary sequence (e.g., 107’) in the second strand, extended to fill the gap between the primer sequence 131 and the second attachment sequence 111, and ligated 140 to generate oligonucleotide molecule 103 attached to the geolocation bead 101, as described elsewhere herein. During activation of the geolocation beads 150, the first strand comprising the spatial tag 109 may be released. In some instances, an enzyme configured for strand displacement and a primer may be provided to displace the first strand, and then the cleavage site may be cleaved to make the first strand accessible or able for downstream processing (e.g., able to bind to a bridge construct).

[0115] A bridge construct may be provided to the substrate prior to, during, or subsequent to release of the spatial tags. Alternatively, or in addition, the bridge construct may be provided to the substrate prior to, during, or subsequent to loading of the sample on the substrate.Altematively, or in addition, the bridge construct may be provided to the substrate prior to, during, or subsequent to rendering analyze sequences in the sample accessible by the bridge construct. The bridge construct may capture multiple spatial tags as well as an analyte sequence from the sample, to spatially tag the analyte sequence and generate a spatially tagged sequence. Prior to, during, or subsequent to release of the spatial tags, the analyte sequences in the sample may be rendered accessible for contact with a bridge construct, such as by allowing the bridge construct to cross a membrane of the sample (e.g., diffuse in), and / or by allowing the analyte sequences to cross a membrane of the sample (e.g., diffuse out) under conditions sufficient to retain, at least to some extent, a spatial relationship between the analyte sequences and retain, at least to some extent, a spatial relationship between the plurality of spatial tags. Upon release of the spatial tags, each of the spatial tags may begin diffusion from a respective point of origin (location of geolocation bead) towards a location of capture by the bridge construct. It will be appreciated that the distance that a spatial tag can travel between release and capture is a function of the rate of diffusion and various conditions (e.g., temperature, addition of viscous or crowding agents, concentration of various reagents, etc.). It will be appreciated that the distance that an analyte sequence (e.g., a messenger RNA (mRNA) molecule) can travel between its release from a sample, if released, and capture is a function of the rate of diffusion and various conditions (e.g., temperature, addition of viscous or crowding agents, concentration of various reagents, etc ). The systems, methods, kits, and compositions of the present disclosure provides reagents at concentrations and conditions sufficient to retain, at least to some extent, a spatial relationship between the analyte sequences and retain, at least to some extent, a spatial relationship between the plurality of spatial tags.

[0116] FIGs. 2A-2B illustrate reagents and a workflow for generating a spatially tagged sequence using a bridge construct and multiple spatial tags. Referring to FIG. 2A, provided and accessible for reaction are a bridge construct 201, an analyte sequence 221, and multiple spatial tags, including a first spatial tag molecule 231 and a second spatial tag molecule 241.

[0117] The first spatial tag molecule 231 may comprise a first spatial tag 233 disposed between a first pair of attachment sequences (e.g., first attachment sequence 207 and second attachment sequence 205). and the second spatial tag molecule 241 may comprise a second spatial tag 243 disposed between a second pair of attachment sequences (e.g., third attachment sequence 211 and fourth attachment sequence 209). The first and second spatial tags may be different. The first and second pairs of attachment sequences may be different. In FIG. 2A, the first spatial tag molecule 231 comprises, from 5?to 3’, the first attachment sequence 207, the first spatialtag 233, and the second atachment sequence 205. The second spatial tag molecule 241 comprises, from 5’ to 3‘. the third atachment sequence 211. the second spatial tag 243, and the fourth atachment sequence 209. The second spatial tag molecule 241 may comprise at a 5’ end a capture entity 213, as described elsewhere herein.

[0118] The bridge construct 201 may comprise a partially double-stranded molecule, where a first strand comprises a capture sequence 203 as an overhang and a binding sequence which binds to the second strand. The second strand may comprise a binding sequence which binds to the first strand, a first atachment binding sequence 207’, a second atachment binding sequence 205’, a third atachment binding sequence 211’, a fourth atachment binding sequence 209’, and spacer sequences. In FIG. 2A, the second strand comprises, from 5’ to 3’, the binding sequence, the second attachment binding sequence 205’. a spacer sequence, the first atachment binding sequence 207’, the fourth atachment binding sequence 209’, a spacer sequence, and the third atachment binding sequence 211’.

[0119] The capture sequence 203, while in FIG. 2A is denoted as a poly-T sequence (e.g., TTTTT) configured to capture a poly-A tail of the analyte sequence 221 (e.g., an mRNA sequence), may be any sequence configured to capture an analyte sequence. The analyte sequence, for example, may not have a poly-A tail. In some instances, the capture sequence may comprise a target sequence or a random sequence or any other sequence designed to capture an analyte sequence, or derivative thereof. The capture sequence may comprise a random n-mer sequence. In some examples, for targeted mRNA assays, the capture sequence may comprise a target mRNA sequence (or derivative thereof). In some examples, for targeted genomic DNA (gDNA) assays, the capture sequence may comprise a target gDNA sequence (or derivative thereof). In some examples, for antibody assays, the capture sequence may comprise a sequence configured to capture an oligonucleotide conjugated to one or more antibodies (e.g., DNA capture tags), or a derivative thereof. In some examples, for assays that utilize template switching reactions, the capture sequence may comprise a sequence configured to capture a product of a reverse transcription reaction, such as a polyG sequence. In some examples, for assays that utilize one or more probes, the capture sequence may comprise a sequence corresponding to a sequence of the probe, to a molecule associated with the probe, or derivative thereof. In some examples, the capture sequence may be part of a single strand portion, a double strand portion, or partially double-stranded complex. In some examples, the capture sequence may be part of a hybrid DNA / RNA complex.

[0120] In some examples, for a transposition assay concerning gDNA analytes, after a transposition reaction (e.g., subsequent to Tn5 transposase treatment of gDNA. where the Tn5 transposase comprises one or more barcode and / or adapter sequences), a partially doublestranded analyte may be generated. In some examples, wherein at least one end of the partially double-stranded analyte comprises an overhang comprising a barcode and / or adapter sequence. A bridge construct of the present disclosure (e.g., 201) may comprise a capture sequence (e.g., 203) that is configured to capture the overhang of the partially double-stranded analyte comprising the barcode and / or adapter sequence. One or more gap filling and / or ligation reactions may be performed to join the partially double-stranded bridge construct and transposition analyte.

[0121] Spacer sequences (e.g., “SP”) in the bridge construct may be the same sequence or different sequences. Spacer sequences may comprise a sequence of any length. For example, the spacer can be any internal spacer, e.g., C3 spacer, C12 spacer, spacer 9, spacer 18, etc. In some cases, the spacer sequence may be designed to not be complementary7to any spatial tag sequence, or portion thereof.

[0122] Referring to FIG. 2B, the bridge construct 201 may capture the analyte sequence 221 using the capture sequence 203 and be extended using the analyte sequence as a template. The bridge construct 201 may capture the first spatial tag molecule 231 using the second attachment binding sequence 205’ and the first attachment binding sequence 207’ which hybridizes with the second attachment sequence 205 and the first attachment sequence 207 of the first spatial tag molecule 231, respectively. Once the first spatial tag molecule 231 (e g., from FIG. 2A) is captured by the bridge construct 201, the first spatial tag 233 may be looped, as illustrated in FIG. 2B. The bridge construct 201 may capture the second spatial tag molecule 241 using the fourth attachment binding sequence 209’ and the third attachment binding sequence 211’ which hybridizes with the fourth attachment sequence 209 and the third attachment sequence 211 of the second spatial tag molecule 241, respectively. Once the second spatial tag molecule 241 (e.g., from FIG. 2A) is captured by the bridge construct 201, the second spatial tag 243 may be looped, as illustrated in FIG. 2B. A tagged complex 250 thus comprises the analyte sequence 221, first spatial tag 233, and second spatial tag 243. The tagged complex 250 may comprise the capture entity7213.

[0123] The sample may be incubated with the reagents (e.g., bridge constructs, spatial tags, etc.) for any period of time. For example, the sample may be incubated for at least about 1 minute (min), 2 min. 3 min, 4 min. 5 min, 6 min, 7 min. 8 min, 9 min. 10 min, 20 min, 30 min,40 min, 50 min, 60 min, 90 min, 120 min, 150 min, 180 min. 4 hours (h), 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h. 12 h or more. Alternatively or in addition, the sample may be incubated at most about 12 h, 1 1 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 180 min, 150 min, 120 min, 90 min, 60 min, 50 min, 40 min, 30 min 20 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min or less. Incubation may be performed at various reaction conditions (e.g., temperature, pH, salt concentration, etc.).

[0124] After sufficient time elapses, such that multiple bridge constructs have formed tagged complexes with various spatial tags and analyte sequences, e.g., including tagged complex 250, the tagged complexes may be recovered. In some cases, the tagged complex 250 may be captured using the capture entity 213, such as via a capturing entity. In some instances, the capture entity 213 comprises biotin and the capturing entity comprises streptavidin, where the streptavidin is coupled to a magnetic bead. In some instances, magnetic beads can be captured via applying a magnetic field. It will be appreciated that any capture mechanism may be used. The tagged complexes may be collected on or off the substrate.

[0125] The tagged complexes may be processed or repaired prior to, during, or subsequent to capture or isolation, for example to perform one or more of the following operations: cleaving at a cleavage site such as to remove a blocking group, ligating, denaturing, performing extension reactions, and other operations. Spatially tagged sequences (e.g., 260) may be generated. A spatially tagged sequence may comprise a sequence corresponding to the analyte sequence 221 (or portion thereof), a sequence corresponding to the first spatial tag sequence 233 and a sequence corresponding to the second spatial tag sequence 243. For example, in some cases, a spatially tagged sequence comprises the analyte sequence 221 (or portion thereof), a complement of the first spatial tag sequence, and a complement of the second spatial tag sequence. In another example, a spatially tagged sequence comprises a complement of the analyte sequence, first spatial tag sequence, and the second spatial tag sequence. The spatially tagged sequences may be subjected to library preparation, such as to attach one or more adapters, barcodes, such as to subject to amplification, etc., and sequencing to generate sequencing reads. Sequencing preparation and sequencing are described in further detail elsewhere herein.

[0126] It will be appreciated that while the workflow described herein illustrates a bridge construct that captures two spatial tags, the bridge construct may be designed to capture any number of spatial tags, for example, by including appropriate pairs of attachment binding sequences. In some instances, a bridge construct that is designed to capture five spatial tagscomprises five pairs of atachment binding sequences. These pairs of atachment binding sequences may include five of the same pair of atachment binding sequences, or two different pairs, three different pairs, four different pairs, or five different pairs of atachment binding sequences. Each pair of atachment binding sequences may correspond to (e.g., be complementary to) a pair of atachment sequences known to be in at least one geolocation bead in the plurality of geolocation beads loaded on the substrate. In some cases, a bridge construct may be designed to capture at least 2, 3. 4, 5, 6. 7, 8, 9. 10 or more spatial tags. Alternatively, or in addition, a bridge construct may be designed to capture at most 10, 9, 8, 7, 6, 5, 4, 3, or 2 spatial tags. A bridge construct may comprise only different pairs of attachment binding sequences, such that a first pair of attachment binding sequence is different from any other pair of atachment binding sequence in the bridge construct. Alternatively, a bridge construct may comprise a repeat of the same pair of atachment binding sequences, or a bridge construct may comprise a mixture of unique pair(s) of atachment binding sequences and overlapping pair(s) of atachment binding sequences.

[0127] Bridge constructs may be provided across the substrate such that they are available at all locations on the substrate where released spatial tags and analyte sequences are accessible. In some cases, the bridge constructs may be provided in uniform concentration across all locations. The bridge constructs may be provided in non-uniform concentrations across all locations. A solution comprising the bridge constructs may be dispensed to the substrate according to reagent dispensing mechanisms that are described in further detail elsewhere herein. All reagents, including initial loading of the geolocation beads and / or the sample may be dispensed to the substrate according to reagent dispensing mechanisms that are described elsewhere herein. For example, in some cases, the reagent may be spin-coated or otherwise dispensed to the substrate at a first location and subjected to move to a second location (e.g., radially outward) at high velocity reagent movement across the substrate to distribute the reagent across the substrate. In other cases, the reagent may be, at relatively lower velocities, be painted on the substrate such that there is minimum movement of the reagent from the location of dispensing to the location of immobilization and / or consumption of the reagent.

[0128] A map of the plurality of analyte sequences of the sample loaded to the substrate may then be generated by identifying sets of spatial tags from the spatially tagged sequences (e.g., sequencing reads thereof). The map may comprise information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence. Two spatially tagged sequences comprising the samespatial tag may indicate that the two spatially tagged sequences were within a certain range of proximity of each other. Furthermore, a spatially tagged sequence comprising a set of two or more spatial tags may indicate that the two or more spatial tags are within a certain range of proximity of each other. With multiple sets of two or more spatial tags, such a map of a network of spatial tags may be generated.

[0129] FIG. 3 illustrates an example map that can be generated from the following sets of spatial tags identified from spatially tagged sequences, where capital letters indicate different spatial tags:Analyte sequence 1 : [A, B] Analyte sequence 2: [A, C] Analyte sequence 3: [A, F] Analyte sequence 4: [B, C] Analyte sequence 5: [B, E] Analyte sequence 6: [C, D] Analyte sequence 7: [C, F]

[0130] For example, tag A was identified in sets with tags B. C, and F, indicating that it is likely that geolocation beads with spatial tags B, C, and F are each within a somewhat similar distance (e.g., radius) of the geolocation bead with spatial tag A. This is represented by a dotted circle around A in FIG. 3. Accordingly, analyte sequences 1, 2, and 3 are mapped to within the dotted circle. Tag B was identified in sets with tags A, C, and E, indicating that it is likely that geolocation beads attached with spatial tags A, C, and E were each within a certain distance (e.g., radius) of the geolocation bead with spatial tag B. This is represented by a dotted circle around B in FIG. 3. Accordingly, analyte sequences 1. 4, and 5 are mapped to within the dotted circle around B. Tag C was identified in sets with tags A, B, D, and F, indicating that it is likely that geolocation beads with spatial tags A, B, D, and F were each within a certain distance (e.g., radius) of the geolocation bead with spatial tag C. This is represented by a dotted circle around C in FIG. 3. Accordingly, analyte sequences 2, 4, 6, and 7 are mapped to within the dotted circle around C. In some cases, the lack of a set between two spatial tags may indicate that the geolocation beads with those two spatial tags were not located within a certain distance (e.g.. radius) of each other, such as [E, F], In some cases, for each geolocation bead, a maximum distance of diffusion of the spatial tag from the geolocation bead (from release to capture by the bridge construct) can be estimated to facilitate map generation. In some cases, a map of the plurality of geolocation beads may be generated prior to. concurrently with, or subsequent togenerating the map of the plurality of analyte sequences. As will be appreciated with more data, a more accurate and / or precise map may be generated. It will be appreciated that FIG. 3 is one example of a map, which is solely provided for illustration purposes. Many different maps that can be generated from the above-provided sample data.

[0131] The map may represent an estimate of a spatial relationship between different analyte references with respect to a reference analyte sequence, reference geolocation bead, or reference location, which may be selected arbitrarily. The estimate may include a best estimated location of an analyte sequence or a probability cloud of locations of an analyze sequence with respect to a reference. In some cases, the reference (e.g., reference analyte sequence, reference geolocation bead, reference location) may be selected to produce the most accurate or precise map. Computer systems may utilize one or more algorithms to generate the map. For example, in some cases, the one or more algorithms may perform triangulation or similar calculations. In some cases, the one or more algorithms may be able to solve complex problems. The one or more algorithms may include one or more machine learning algorithms.

[0132] FIG. 4 illustrates a general workflow of a substrate-based spatial screening method. A substrate 450 may be loaded with a plurality of geolocation beads 401 comprising a plurality of oligonucleotide molecules, as described elsewhere herein, such as the bead 101 described with respect to FIGs. 1A-1B. The substrate 450 may have immobilized thereto a plurality of the geolocation beads 401 on individually addressable locations. A sample 403 which retains, at least to some extent, a spatial relationship between a plurality of analyte sequences, such as tissue slices which retain a spatial relationship between transcripts, may be loaded onto the substrate 450 with the geolocation beads. At any point in time, a plurality’ of bridge constructs 405, as described elsewhere herein, such as bridge construct 201 described with respect to FIGs. 2A-2B, may be loaded onto the substrate. The geolocation beads may be activated to release the spatial tags (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli. The sample may also be subject to conditions sufficient to render analyte sequences, or derivatives thereof (e.g., complementary DNA to (cDNA) to mRNA, ligation products, etc.) accessible to the bridge constructs. On the substrate, a plurality of bridge constructs (e.g., a subset of a population of total bridge constructs loaded to the substrate) may each capture an analyte sequence and multiple spatial tags to generate tagged complexes (e.g., 250). A plurality of spatially tagged sequences 407 may be generated from the tagged complexes on or off the substrate, recovered (e.g., via the capture entity as described elsewhereherein), and prepared for sequencing. One or more ligation reactions may be performed on the substrate. From the sequencing information, a map of the plurality of analyte sequences may be generated by identifying sets of spatial tags from the plurality of spatially tagged sequences, where the map comprises information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence.

[0133] In an alternative workflow of a substrate-based spatial screening method, a sample which retains, at least to some extent, a spatial relationship between a plurality of analyte sequences, such as tissue slides which retain a spatial relationship between transcripts and / or single cells, may be loaded and immobilized to the substrate first. The sample may be immobilized on the substate in any design or pattern. In some examples, the samples are immobilized in hydrophilic and / or hydrophobic patterns. The sample may be processed prior to being loaded on the substrate, or while on the substrate, such as to subject the sample to fixation, permeabilization, antibody treatment, probe treatment, and / or any other sample processing reactions (e.g., reverse transcription, transposition, probe reactions, capture reactions, etc.). Such sample processing reaction(s) may be performed in any sequence and / or substantially simultaneously. At any point subsequent to loading the sample on the substrate, a plurality7of geolocation beads comprising a plurality7of oligonucleotide molecules, as described elsewhere herein, may be provided to the sample. For example, the geolocation beads may be provided substantially simultaneously with one or more sample processing reactions. At any point subsequent to loading the sample on the substrate, a plurality of bridge constructs, as described elsewhere herein (e.g., such as bridge construct 201 described with respect to FIGs. 2A-2B) may be loaded onto the substrate. The geolocation beads may be activated to release the spatial tags (e.g., via releasing the first strands) according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli. The sample may also be subject to conditions sufficient to render analyte sequences, or derivatives thereof (e.g., complementary DNA to (cDNA) to mRNA, ligation products, etc.)..) accessible to the bridge constructs. On the substrate, a plurality of bridge constructs (e.g., a subset of a population of total bridge constructs loaded to the substrate) may each capture an analyte sequence and multiple spatial tags to generate tagged complexes (e.g., 250). A plurality7of spatially tagged sequences may be generated from the tagged complexes on or off the substrate, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. One or more ligation reactions maybe performed on the substrate.From the sequencing information, a map of the plurality of analyte sequences may be generated by identifying sets of spatial tags from the plurality’ of spatially tagged sequences, where the map comprises information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence.

[0134] In some cases, a sample can be loaded onto a substrate in dilute concentrations to allow tagging of cellular contents of a plurality of cells with spatial tags from geolocation beads. The dilute concentration of the sample may allow for single cell resolution analysis. For example, a plurality’ of cells can be smeared onto a substrate at substantially dilute concentration. In some methods, a substrate may be loaded with a plurality of cells in a relatively dilute concentration, a plurality of geolocation beads as described elsewhere herein, and a plurality’ of bridge constructs as described elseyvhere herein. The plurality of cells may be provided in a concentration dilute enough that they are located relatively far apart on the substrate such as to prevent or make it extremely unlikely that, between the time of release and capture of spatial tags of geolocation beads by bridge constructs, a first spatial tag of a first geolocation bead located in proximity to a first cell can diffuse and / or a cellular analyte of the first cell can diffuse to be captured by a bridge construct along with a second spatial tag of a second geolocation bead located in proximity to a second cell. After loading onto the substrate, the analyte sequences and / or other cellular content in the cells may be rendered accessible to the bridge constructs. For example, the cells can be lysed. The geolocation beads may be activated to release the spatial tags (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzy mes and / or one or more stimuli (e.g., heat). The bridge constructs (e.g., a subset of a population of total bridge constructs or all of the bridge constructs) may each capture an analyte sequence and multiple spatial tags from the plurality of geolocation beads to generate tagged complexes (e.g., 250), for example as described with respect to FIG. 2B. A plurality of spatially tagged sequences (e.g., 260, 407) may be generated from the tagged complexes on or off the substrate, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information of the spatially tagged sequences, or derivatives thereof, an analyte sequence may be mapped to a cell by identify ing sets of spatial tags from the plurality of spatially tagged sequences. For example, where a first spatially tagged sequence is identified to include a first spatial tag and a second spatial tag, where a second spatially tagged sequence is identified to include a third spatial tag and a fourth spatial tag, and where a third spatiallytagged sequence is identified to include the second spatial tag and the third spatial tag, it can be inferred that all of the first spatial tag, second spatial tag. third spatial tag, and fourth spatial tag were present in proximity to each other on the substrate and therefore tagged analytes from the same cell, and thus it can also be inferred that any spatially tagged sequence found to have any one of the first, second, third, or fourth spatial tag originated from the same cell.

[0135] The methods described herein may further comprise methods for attenuation or prevention of long-distance diffusion by reagents, such as by attenuating diffusion altogether or by attenuating diffusion along a certain direction(s) on the substrate, and / or in emulsion or in solution, as described elsewhere herein. It may be undesirable for reagents to diffuse too far in a direction that is along an axis or plane contained in a final spatial map generated (e.g., x-y plane) as it may confuse proximity data that is later used to reconstruct the spatial map. The methods may prevent small particles that tend to diffuse relatively fast (e.g., DNA), compared to the duration of various reactions described herein (e.g., barcode activation by USER enzyme, capture, etc.), from diffusing too far from an originating location before tagging occurs, increasing the accuracy of a final spatial map. In some cases, diffusion can be attenuated by adding viscous reagents (e.g., PEG. etc.) and / or modulating one or more other reaction conditions (e.g., temperature).

[0136] In some cases, diffusion can be attenuated by encapsulating a reaction space in a gel, hydrogel (e.g., PEG hydrogel), or other mesh or matrix (e.g., polymer mesh matrix) to hinder particle movement therethrough. The encapsulation may be reversible. For example, the mesh or matrix may be degradable, such as after a certain period of time and / or upon application of one or more stimuli (e.g., chemical stimulus to induce, e.g., hydrolysis, enzymatic stimulus, photo stimulus, etc.). In an example, the reaction space comprising the sample and geolocation beads is crosslinked with a hydrophilic polymer to create a mesh that attenuates diffusion throughout the reaction space. The mesh may be nanoscale. In an example, a 4-arm PEG- acrylate macromer and PEG-dithiolglycolate crosslinker is used to form a PEG hydrogel that is degradable. In some cases, protein (e.g., bovine serum albumin (BSA) protein) or other solutes may be embedded or entrapped within the mesh network to increase a crowding effect to further attenuate diffusion.

[0137] Alternatively or in addition, the reaction space may be subjected to electrophoresis to accelerate movement of charged particles (e.g., DNA, mRNA, spatial tags, etc.) along a direction of the electric field, such as along the z-axis when an x-y plane spatial map is generated, to attenuate diffusion along non-z-axis directions. FIG. 22 illustrates a schematicfor subjecting the reaction space to electrophoresis. The geolocation beads 2205, each comprising the spatial tags 2206, and sample 2204 (e.g., 5-micron tissue) may be loaded onto a substrate 2201 as described elsewhere herein. An appropriate buffer 2203 (e.g., (Tris base / acetic acid / EDTA (TAE), Tris / borate / EDTA (TBE), etc.) may be added between two electrodes 2202a and 2202b that sandwich the sample-loaded substrate to facilitate electrophoresis. An electrode (conductive material) may be or comprise an indium tin oxide (ITO) slide. In some cases, the electrode is substantially transparent and conductive. The electric field may be activated with or prior to activation of the geolocation beads 2205 to release the spatial tags 2206. In some cases, the electric field may be activated after activation of the geolocation beads. The released spatial tags may be directed to diffuse primarily along a direction of the electric field (e.g.. along z-axis), instead of in the x- or y- axis directions. In an example, mRNA analytes and spatial tag molecules move toward the positive electrode. The electric field may be maintained until substantial completion of all tagging reactions. FIG. 22 illustrates a box around each geolocation bead to represent the diffusion cloud of the spatial tags (in the x-z plane). A reaction space may be both encapsulated in a mesh or matrix (e.g., PEG hydrogel) as described elsewhere herein and subjected to electrophoresis.

[0138] In some cases, the methods, systems, compositions, and kits described herein may comprise fiducial marker beads, or the use thereof. A fiducial marker bead may be used as a reference bead around which a spatial map may be generated according to the methods provided herein. In some cases, an absolute position of the fiducial marker bead on a substrate may be known, predetermined, and / or detectable. In some cases, where two or more fiducial marker beads are used, a location of one fiducial marker bead may be known, predetermined, and / or detectable with respect to at least one other fiducial marker bead. One or more of the geolocation beads on the substrate may be a fiducial marker bead. For example, a substrate may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more fiducial marker beads. Alternatively or in addition, a substrate may have at most about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 fiducial marker beads. In some cases, a ratio of the fiducial marker beads to total number of beads (including non-fiducial marker geolocation beads) may be at least about 1x1 O’10, lx 10"9, lx 10'8, lx IO’7, lx 10’6, lx 10‘5, lx 10'4, lx 10'3, lx 10'2, lx 10-1, 1 or more.

[0139] A fiducial marker bead may comprise a detectable feature, which distinguishes the fiducial marker bead from non-fiducial marker beads (e.g., remaining geolocation beads), the detectable feature being different than a spatial tag of the fiducial marker bead. In some cases,each fiducial marker bead loaded on a substrate may have a different detectable feature such that upon detection of a detectable feature, it can be identified to only one fiducial marker bead. In some cases, some or all of the fiducial marker beads loaded on a substrate may share a same detectable feature such that upon detection of that detectable feature, it can be identified to be any one of a larger group of fiducial marker beads. For example, multiple ty pes of fiducial marker beads may be used (e.g., one group of beads that have a red fluorophore and one group of beads that have a green fluorophore. etc.). In some cases, the detectable feature is an optically detectable feature, such as a fluorescently detectable feature or other feature detectable under interrogation at one or more wavelengths. The detectable feature may be detectable upon imaging. The detectable feature may be detectable by any detector described herein. The fiducial marker bead may be and behave the same as any variation of a geolocation bead described herein, comprising a spatial tag, except for additionally comprising the detectable feature. The spatial tag of a fiducial marker bead may be known or predetermined and associated with the detectable feature. For example, a first fiducial marker bead may fluoresce in red and comprise a first spatial tag known or predetermined to originate from the fiducial marker bead which fluoresces in red. and a second fiducial marker bead may fluoresce in green and comprise a second spatial tag known or predetermined to originate from the fiducial marker bead which fluoresces in green. In some cases, the spatial tag for each ty pe of fiducial marker bead can be the same (e.g.. 100 red fluorescing fiducial marker beads all have the same spatial tag), or different (e.g., 100 red fluorescing fiducial marker beads all have different spatial tags).

[0140] In any of the methods described herein, where a plurality7of geolocation beads are loaded or otherwise immobilized to a substrate, the method may comprise (A) loading or otherwise immobilizing a mixture of a plurality of geolocation beads and at least one fiducial marker bead onto the substrate, and (B) at any point subsequent to such loading or immobilizing, detecting respective location(s) of the at least one fiducial marker bead using respective detectable feature(s) of the at least one fiducial marker bead. In some instances, the methods may further comprise, prior to the loading or otherwise immobilizing, generating the fiducial marker bead, identifying a spatial tag of a fiducial marker bead (e.g., using a probe, sequencing, etc.), and / or mixing the plurality' of geolocation beads and the fiducial marker bead(s) to generate the mixture. The plurality of geolocation beads and the at least one fiducial marker bead may be loaded separately or substantially simultaneously onto the substrate. The fiducial marker bead(s) may be randomly dispersed on the substrate similar to any other geolocation beads as described elsewhere herein. In some cases, the detecting of the respectivedetectable feature(s) can comprise imaging the substrate to generate a real image of a substrate map in which locations of each fiducial marker bead are pinned. After sequencing reads corresponding to spatially tagged sequences are generated, a sequence comprising a first spatial tag, or complement thereof, which is known or predetermined to originate from a first fiducial marker bead may be pinned or superimposed to a first location of the first fiducial marker bead as detected in (B) (e.g., such as on the substrate map). Similarly, a sequence comprising a second spatial tag, or complement thereof, which is known or predetermined to originate from a second fiducial marker bead may be pinned or superimposed to a second location of the second fiducial marker bead as detected in (B) (e.g., such as on the substrate map), and so on. A spatial map may be generated where the absolute position of at the at least one fiducial marker bead (e.g., with respect to the substrate map) is grounded to be true, and locations of other geolocation beads (or probability cloud thereof) are determined around the fiducial marker beads. Alternatively, or in addition, a spatial map may be generated where the relative positions of at least two fiducial marker beads with respect to each other are grounded to be true, and locations of other geolocation beads (or probability cloud thereof) are determined around the fiducial marker beads. Alternatively, or in addition, a spatial map may be generated where the position of a fiducial marker bead is determined to be one of multiple distinct positions relative to the real image of the substrate map. Without fiducial marker beads, spatial image of the data may be reconstructed based on estimating global patterns from local proximity data. Beneficially, the fiducial marker beads may significantly reduce the computational complexity of generating accurate spatial maps. In some cases, the computational complexity may reduce as the number and / or types of fiducial marker beads increases. Further, the fiducial marker beads may assist reliable recovery of spatial images that have empty spaces or disjoint features which may not be available otherwise.

[0141] FIG. 21 illustrates an example of a substrate map using fiducial marker beads according to the methods described herein. A mixture of a first type of fiducial marker beads 2102 (e.g., red), a second type of fiducial marker beads 2103 (e.g., green), and non-fiducial marker beads 2101 are randomly loaded on a substrate 2100. The substrate 2100 is imaged to generate a real image of the substrate map. FIG. 21 illustrates a schematic of such a substrate map, where the locations of each fiducial marker beads are readily apparent. After performing the methods described herein, a sequence is identified to have been tagged by both a first type of fiducial marker bead spatial tag and a second type of fiducial marker bead spatial tag, and based on this data, and factoring in diffusion rates (e.g., see example circles in FIG. 21 marking diffusionclouds), locations where the first type of fiducial marker bead and the second type of fiducial marker bead are in close proximity are identified, and it is determined that the sequence was located in some proximity to region 2105 with respect to the substrate map.

[0142] Also provided herein are methods for spatial screening with multiple substrates. FIG. 5A illustrates a spatial screening scheme using multiple substrates. Applying a sample to multiple geo-loaded substrates simultaneously may allow for mapping analyte sequences in the sample to a three-dimensional (3D) spatial resolution and / or with increased two-dimensional (2D) resolution compared to applying a sample to only one geo-loaded substrate. A ‘geoloaded’ substrate may refer to a substrate of the present disclosure which has immobilized thereto a plurality of geolocation beads, according to methods described elsewhere herein.

[0143] A sample 505 may comprise multiple surfaces which can contact or interface a substrate, including at least a first surface 507a and a second surface 507b. In FIG. 5A, these are illustrated as a top surface and bottom surface which are opposite each other (e.g., of a tissue slice) but it will be appreciated that the multiple surfaces can have any orientation with respect to each other (e.g., sharing an edge), which allows for contact or interfacing with different substrates. A first geo-loaded substrate 551 which comprises a first plurality of geolocation beads 501 immobilized thereto may be applied to the first surface 507a and a second geo-loaded substrate 552 which comprises a second plurality of geolocation beads 503 immobilized thereto may be applied to the second surface 507b. The first plurality of geolocation beads 501 may comprise a first set of spatial tags which are completely unique from a second set of spatial tags comprised by the second plurality of geolocation beads 503, such that the two sets of spatial tags are mutually exclusive (no one spatial tag in the first plurality of geolocation beads 501 overlaps with another spatial tag from the second plurality of geolocation beads 503). Furthermore, a spatial tag may be designed and loaded to the substrates such that they are identifiable as originating from which plurality of geolocation beads and / or which substrate. The spatial tag may be identifiable as originating from one of multiple sets of plurality of geolocation beads without the full sequence or substantial portion of the full sequence of the spatial tag being known prior to loading. For example, a first set of geolocation beads may be designed such that each bead has a spatial tag that has a sequence starting with a certain base (e.g., “A”) or a certain sequence of bases (e.g., “ATA”), and a second set of geolocation beads may be designed such that each bead has a spatial tag that has a sequence starting with a different base (e.g., “T”) or a different sequence of bases (e.g., “ATT”). The first set of geolocation beads may be loaded to the first substrate (e.g., bottomsubstrate contacting the first surface 507a) and the second set of geolocation beads may be loaded to the second substrate (e.g.. top substrate contacting the second surface 507b). Once the final tagged sequences are sequenced, an operator may be able to map a spatial tag to the top substrate or the bottom substrate based on the first base or first sequence of bases identified. In this method, one need not know the full sequences of the geolocation beads, just any characterizing features (e.g., first base, first sequence of bases, last base, last sequence of bases) of the spatial tag and to which substrate such beads with the known characterizing features were loaded.

[0144] At any point in time, a plurality of bridge constructs (not illustrated in FIG. 5A) as described elsewhere herein (e.g., 405, 201) may be loaded onto the substrate. After applying the multiple geo-loaded substrates (e.g.. 551, 552) to the sample (505), the geolocation beads may be activated to release the spatial tags (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli. The sample may also be subject to conditions sufficient to render analyte sequences, or derivatives thereof (e.g., complementary DNA to (cDNA) to mRNA, ligation products, etc.) accessible to the bridge constructs. On one or more of the multiple substrates, a plurality of bridge constructs (e.g., a subset of a population of total bridge constructs loaded to the substrate) may each capture an analyte sequence and multiple spatial tags to generate tagged complexes (e.g., 250). For example, in this scheme, a bridge construct may capture multiple spatial tags from multiple geolocation beads on the first substrate (e.g., 551), capture multiple spatial tags from multiple geolocation beads on the second substrate (e.g., 552), or capture multiple spatial tags from at least one geolocation bead on the first substrate and at least one geolocation bead on the second substrate. Diffusion may occur in a vector that includes a z-axis component, such as to or away from one of the substrates (see axis illustration in FIG. 5A). In some cases, the reaction space may be subjected a directed force (e.g., magnetic force, electric force), to improve or focus diffusion directionality. A plurality of spatially tagged sequences 407 may be generated from the tagged complexes on or off the substrate, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information, a map of the plurality of analyte sequences may be generated by identifying sets of spatial tags from the plurality of spatially tagged sequences, where the map comprises information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to areference analyte sequence. If a directed force was applied during diffusion, such conditions may be used during modeling to improve the accuracy of the spatial maps that are generated.

[0145] In some cases where a first analyte sequence has been captured with multiple spatial tags from only the first substrate, in addition to being able to map out the relative two- dimensional positions on the first substrate when combined with other data, it may be inferred that the first analyte sequence was disposed closer to the first substrate than the second substrate. In some cases where a first analyte sequence has been captured with at least one spatial tag from the first substrate and at least one spatial tag from the second substrate, it may be inferred that (1) the first analyte sequence was disposed at a particular location on the z-axis where it was possible for both spatial tags from the first substrate and the second substrate to meet (or be captured by the bridge construct) within reaction diffusion conditions, for example not too close to the first substrate and not too close to the second substrate, and that (2) the geolocation bead(s) of the spatial tag(s) identified from the first substrate and the geolocation bead(s) of the spatial tag(s) identified form the second substrate are in proximity in the x-y axis. In this way, a map generated from the data (e.g.. sequencing information) collected or received from this multiple substrate scheme may have 3-D spatial resolution, such as to position an analyte sequence on a x-y-z or other 3-D coordinate system with respect to a reference point. Further, at least two 2-D maps may be generated from the data (e.g., sequencing information) collected or received from this multiple substrate scheme, one 2-D map corresponding to geolocation beads immobilized in the first substrate and one 2-D map corresponding to geolocation beads immobilized in the second substrate, to improve overall 2- D spatial resolution of the analyte sequences. When a sample provided between the two substrates are thin enough such that diffusion of reagents between the two substrates (e.g., from one substrate to the other substrate) is common throughout the layer of sample (e.g., along the z-axis), the scheme described herein may contribute more towards super 2-D resolution than the 3-D resolution.

[0146] In some cases, a subset or all of the geolocation beads may be designed to emit fluorescence to facilitate multi-channel (e.g., 2 channels, 3 channels, etc.) positional alignment, relative to tissue morphology (e.g., hematoxylin and eosin staining (H&E staining)).

[0147] While FIG. 5A illustrates an example with two substrates, it will be appreciated that more substrates may be applied where multiple surfaces are available. The different substrates may or may not be the same size and / or type. In some cases, miniature substrates may be applied to multiple surfaces of a sample which has one surface on a much larger substrate.

[0148] Also provided herein are methods for 3-D encoding of samples which may be used alternatively or in addition to the 3-D mapping workflows described with respect to FIG. 5A. A 3-D sample, such as a tissue slice, may be impregnated with a plurality of particles (e.g., nanoparticles), each comprising a plurality of spatial tags.

[0149] FIG. 5B illustrates an example of a particle 561 comprising a plurality of oligonucleotide molecules (e.g., 563). The particle may comprise any number of oligonucleotide molecules attached thereto, for example on the order of 10, 102, 103. 104, 105. or more. The oligonucleotide molecule 563 may correspond to the oligonucleotide molecule 103 described elsewhere herein, comprising the spatial tag. The particle may be a nanoparticle. In some cases, the particle may have a maximum dimension (e.g., diameter) of at least about 0.50 nanometers (nm). 0.55 nm, 0.60 nm, 0.65 nm, 0.70 nm, 0.75 nm, 0.80 nm, 0.85 nm, 0.90 nm, 0.95 nm, 1.0 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm or greater. Alternatively or in addition, a bead may have a maximum dimension of at most about 0.50 nanometers (nm), 0.55 nm, 0.60 nm, 0.65 nm. 0.70 nm. 0.75 nm. 0.80 nm. 0.85 nm. 0.90 nm. 0.95 nm, 1.0 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, or less. In one example, in a 3-dimensional space, estimating about 5-micron radius for a cell and about 0.01 -micron (or 10 nm) radius for a bead, approximately 2 million beads may fit in a cell to produce useful spatial information. A nanoparticle may comprise a dendrimer architecture with any useful generation, for example G3, G4, G5, G6, G7, etc. Example dendrimer species include, without limitation, polyamidoamine (PAMAM), polypropylene imine (PPI), polylysine, poly(propyl ether imine) (PEPIM), and viologen dendrimers. The oligonucleotide molecule may be attached to a surface group of a dendrimer.

[0150] A sample may be incubated with a plurality of such particles for sufficient time to allow for sufficient penetration of the sample with the particles across the sample volume. In some cases, such penetration may be accelerated by applying one or more directed forces, such as magnetic fields, electric fields, and / or pressure. Upon penetration, the particles may be activated to release the spatial tags (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein (e.g., with respect to geolocation beads and / or capture beads), such as by providing one or more enzymes and / or one or more stimuli. In someexamples, a photo or chemical stimulus is applied for the release of the spatial tags, as described with respect to FIG. 8A. At any point in time, a plurality of bridge constructs as described elsewhere herein may be provided to the sample. The sample may also be subject to conditions sufficient to render analyte sequences, or derivatives thereof (e.g., complementary DNA to (cDNA) to mRNA, ligation products, etc.) accessible to the bridge constructs. A plurality of bridge constructs may each capture an analyte sequence and multiple spatial tags to generate tagged complexes (e.g., 250). Diffusion may occur in a vector that includes a z-axis component, such as to or away from a substrate (see axis illustration in FIG. 5A). A plurality of spatially tagged sequences may be generated from the tagged complexes on or off the substrate, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information, a 3-D map of the plurality of analyte sequences may be generated by identifying sets of spatial tags from the plurality of spatially tagged sequences, where the map comprises information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence. If a directed force was applied, such conditions may be used during modeling to improve the accuracy of the spatial maps that are generated. Beneficially, use of such nano-dimension particles may permit significantly higher spatial resolution.

[0151] So far, methods, systems, kits, and compositions for mapping a spatial relationship between analyte sequences of a sample by loading geolocation beads on a substrate have been discussed. The geolocation beads and bridge constructs of the present disclosure may also be used in emulsion-based or solution-based reaction environments, off the substrate.

[0152] FIG. 6 illustrates an example droplet with reagents of the present disclosure. In an emulsion-based environment, a plurality of geolocation beads and bridge constructs, as described elsewhere herein, and a plurality of cells may be partitioned into a plurality of droplets to tag cellular contents of the plurality of cells with spatial tags within isolated reaction environments. The systems, methods, compositions, and kits described herein may be particularly beneficial where partitioning reagents into droplets in an emulsion are governed by the Poisson distribution. Droplet generation and partitioning systems and methods, as well as problems associated with Poisson distributions (e.g., waste of resources), are described in further detail in International Pub. No. WO2020 / 167656, which is entirely incorporated herein by reference for all purposes. It may be desirable to generate droplets that contain at most a single cell per droplet to provide isolated reaction environments for single cells. However, in doing so. there are many other droplets generated that contain no cell at all or in some casesmore than one cell. When there are multiple analytes that each need to be singly partitioned (e.g., cells and beads), the Poisson problem becomes multiple-fold and results in a large waste of resources. Beneficially, the systems, methods, compositions, and kits permit a droplet to include multiple beads (e.g., geolocation beads). For example, previously, if a droplet contains one cell and multiple beads, each bead comprising a bead-specific tag, cellular analytes tagged by two different bead-specific tags (e.g., spatial tags) within the same droplet may be distinguished and incorrectly classified as having originated from different cells. However, systems, methods, compositions, and kits of the present disclosure allow for the relation of the different bead-specific tags within the same droplet as having originated from the same droplet (and thus same cell) by using the geolocation and bridge constructs described herein.

[0153] A plurality of cells, a plurality of geolocation beads, and a plurality of bridge constructs are partitioned to generate a plurality of partitions. The plurality of partitions may comprise a plurality of droplets. Each bead in the plurality of geolocation beads may comprise a unique spatial tag such that no spatial tag of any bead overlaps with any other spatial tag of any other bead. Referring to FIG. 6, a droplet 601 of the plurality of droplets comprises a cell 603, a plurality of geolocation beads (e.g., geolocation bead 605), and a plurality of bridge constructs (e g., bridge construct 607). The geolocation bead 605 may correspond to any geolocation bead described herein (e.g., 101, 401). The bridge construct 607 may correspond to any bridge construct described herein (e.g., 201, 405). After partitioning, the analyte sequences and / or other cellular content in the cell 603 may be rendered accessible to the bridge constructs. For example, the cell 603 can be lysed. The geolocation beads may be activated to release the spatial tags (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli (e.g., heat). Within the droplet 601, the bridge constructs (e.g., a subset of a population of total bridge constructs in the droplet or all of the bridge constructs) may each capture an analyte sequence and multiple spatial tags from the plurality of geolocation beads to generate tagged complexes (e.g., 250), for example as described with respect to FIG. 2B. A plurality of spatially tagged sequences (e.g., 260, 407) may be generated from the tagged complexes inside or outside of the droplet, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information of the spatially tagged sequences, or derivatives thereof, an analyte sequence may be mapped to a cell by identifying sets of spatial tags from the plurality of spatially tagged sequences. For example, where a first spatially tagged sequence is identified to include a first spatial tag and a second spatial tag,where a second spatially tagged sequence is identified to include a third spatial tag and a fourth spatial tag. and where a third spatially tagged sequence is identified to include the second spatial tag and the third spatial tag, it can be inferred that all of the first spatial tag, second spatial tag, third spatial tag, and fourth spatial tag were present in the same droplet and therefore tagged analytes from the same cell, and thus it can also be inferred that any spatially tagged sequence found to have any one of the first, second, third, or fourth spatial tag originated from the same cell.

[0154] While FIG. 6 illustrates a droplet, the partition may be any type of partition, such as a well or micro-well. In some cases, reaction conditions may be adjusted to allow the method to be performed outside of partitions, such as in a solution-based environment. For example, the solution-based environment may comprise one or more of a diluted concentration of cells, higher viscosity, beads comprising cell affinity moieties (e.g., antibodies, lipophilic moieties, etc.), and high concentrations of beads, such that the beads are in large excess compared to other reagents (e g., cells).

[0155] As in the emulsion-based environment described with respect to FIG. 6, a solutionbased environment may allow tagging of cellular contents of a plurality of cells with spatial tags from geolocation beads. FIG. 7 illustrates an example solution environment with reagents of the present disclosure. A solution 701 may be provided to comprise a plurality7of cells (e.g., 703) in a relatively dilute concentration, a plurality of geolocation beads (e.g., geolocation bead 705). and a plurality of bridge constructs (e.g.. bridge construct 707). The plurality of cells may be provided in a concentration dilute enough that they are located relatively far apart within the solution such as to prevent or make it extremely unlikely that, between the time of release and capture of spatial tags of geolocation beads by bridge constructs, a first spatial tag of a first geolocation bead located in proximity to a first cell can diffuse and / or a cellular analyte of the first cell can diffuse to be captured by a bridge construct along with a second spatial tag of a second geolocation bead located in proximity to a second cell. The geolocation bead 705 may correspond to any geolocation bead described herein (e.g., 101, 401, 605). The bridge construct 707 may correspond to any bridge construct described herein (e g., 201. 405, 607). After being provided in the solution, the analyte sequences and / or other cellular content in the cells may7be rendered accessible to the bridge constructs. For example, the cells can be lysed. The geolocation beads may be activated to release the spatial tags (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli (e.g., heat). Within the solution701, the bridge constructs (e.g., a subset of a population of total bridge constructs or all of the bridge constructs) may each capture an analyte sequence and multiple spatial tags from the plurality of geolocation beads to generate tagged complexes (e.g., 250), for example as described with respect to FIG. 2B. A plurality of spatially tagged sequences (e.g., 260, 407) may be generated from the tagged complexes inside or outside of the solution, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information of the spatially tagged sequences, or derivatives thereof, an analyte sequence may be mapped to a cell by identifying sets of spatial tags from the plurality of spatially tagged sequences. For example, where a first spatially tagged sequence is identified to include a first spatial tag and a second spatial tag, where a second spatially tagged sequence is identified to include a third spatial tag and a fourth spatial tag, and where a third spatially tagged sequence is identified to include the second spatial tag and the third spatial tag, it can be inferred that all of the first spatial tag, second spatial tag, third spatial tag, and fourth spatial tag were present in proximity to each other in the solution and therefore tagged analytes from the same cell, and thus it can also be inferred that any spatially tagged sequence found to have any one of the first, second, third, or fourth spatial tag originated from the same cell. Even though the different cells are provided within a same solution, because the population of cells is so dilute it is unlikely that the same spatial tag (located in proximity with one cell than another cell) will be captured along with analyte sequences from more than one cell.

[0156] In some cases, the reaction conditions in the solution may be controlled such as to prevent long distance diffusion of reagents from a first location to a second location in the solution, such as by adding viscous reagents (e.g., PEG, etc.) and / or modulating various reaction conditions (e.g., temperature).

[0157] In any of the methods described herein, where a plurality of geolocation beads are provided in bulk solution, such as described with respect to FIG. 7, the method may comprise (A) providing a mixture of a plurality of geolocation beads and at least one fiducial marker bead onto the substrate, and (B) at any point subsequent to such providing, detecting respective location(s) of the at least one fiducial marker bead using respective detectable feature(s) of the at least one fiducial marker bead. In some instances, the methods may further comprise, pnor to the providing in solution, generating the fiducial marker bead, identifying a spatial tag of a fiducial marker bead (e.g., using a probe, sequencing, etc.), and / or mixing the plurality of geolocation beads and the fiducial marker bead(s) to generate the mixture. The plurality of geolocation beads and the at least one fiducial marker bead may be provided separately orsubstantially simultaneously in the solution. The fiducial marker bead(s) may be randomly dispersed in the solution similar to any other geolocation beads as described elsewhere herein. In some cases, the detecting of the respective detectable feature(s) can comprise imaging the solution across one or more planes. For example, various side images, top image, bottom images, etc., at various angles, may be generated of a container comprising the solution to generate one or more real images of the solution. In some cases, the one or more real images of the solution can be resolved into one or more 3D images. In some cases, the imaging may comprise 3D imaging. After sequencing reads corresponding to spatially tagged sequences are generated, a sequence comprising a first spatial tag, or complement thereof, which is known or predetermined to originate from a first fiducial marker bead may be pinned or superimposed to a first location of the first fiducial marker bead as detected in (B) (e.g., such as on the real images). Similarly, a sequence comprising a second spatial tag, or complement thereof, which is known or predetermined to originate from a second fiducial marker bead may be pinned or superimposed to a second location of the second fiducial marker bead as detected in (B), and so on.

[0158] FIGs. 8A-8B illustrate examples of additional geolocation bead constructs that can be used for any of the methods described herein. Referring to FIG. 8A, an oligonucleotide molecule 133 on geolocation bead 801 may be single-stranded, and comprise a first attachment sequence 107, a spatial tag 109, and a second attachment sequence 111. Optionally, the oligonucleotide molecule 133 may comprise a UMI sequence 115a. The different sequences are described in further detail elsewhere herein. The oligonucleotide molecule 133 may comprise a cleavage site, denoted “U” in FIG. 8. Upon application of a stimulus (e.g., heat, light) and / or providing one or more enzymes 850, the oligonucleotide molecule 133 may cleave to release a strand that comprises the first attachment sequence 107, the spatial tag 109, and the second attachment sequence 111, and optionally, the UMI sequence 115a. The cleavage site can be located to release such a strand, such as within the first attachment sequence 107 (as illustrated in FIG. 8A) or betw een the first attachment sequence 107 and the spatial tag 109.

[0159] Referring to FIG. 8B, panel (a), an oligonucleotide molecule 143 on geolocation bead 802 may be single-stranded and comprise tw o sets of a first attachment sequence, a spatial tag. and a second attachment sequence. For example, the oligonucleotide molecule 143 may comprise a third attachment sequence 171, a second spatial tag 173, and a fourth attachment sequence 175. Optionally, the oligonucleotide molecule 143 may comprise a UMI sequence 115a. There may be two cleavage sites, such as to separately or substantially simultaneouslyrelease a first strand that comprises the third attachment sequence 171, the second spatial tag 173, and the fourth attachment sequence 175, and a second strand that comprises the first attachment sequence 107, the spatial tag 109, and the second attachment sequence 111. Each of the segments (strands) may be capable of capture by the bridge constructs provided herein. In some cases, having multiple spatial tags on the same geolocation bead may increase spatial resolution. In some cases, the two cleavage sites for the two different capture-able strands may be cleaved at a controlled time to allow for further diffusion by one strand compared to the other strand. Though not illustrated in FIG. SB, the oligonucleotide molecule may be designed to have multiple UMIs, including many as releasable strands. It will be appreciated that, similarly, the geolocation bead may have any number of sets of the first attachment sequence, spatial tag, and third attachment sequence. The different sets can include the same spatial tag or different spatial tags, or mixtures thereof. The different sets can include the same pair of attachment sequences or different attachment sequences, or mixtures thereof.

[0160] Referring to FIG. 8B, panel (b), an oligonucleotide molecule 153 on geolocation bead 803 may be partially double-stranded and partially looped. A first strand may comprise a first attachment sequence 187, a spatial tag 189 which is looped (and not hybndized to the second strand), and a second attachment sequence 191. The second strand may comprise complementary sequences 107’, 109’ for the first and second attachment sequences 187, 191 in the first strand, respectively. A spacer sequence may be disposed between the complementary sequences 107’, 109’ which region corresponds to where the spatial tag 189 loops on the first strand. Beneficially, in this configuration, a complement of the spatial tag 189, which is not used in downstream operations, need not be synthesized. Example cleavage sites are indicated as “U” in the figure. For example, multiple cleavage sites in the bottom strand may be leveraged to cleave or nick and facilitate enzyme activity to release the first strand. The cleavage site in the first strand may be cleaved to release the first strand. The geolocation bead construct may exhibit reduced non-specific interactions.

[0161] FIG. 9A illustrates examples of additional bridge constructs that can be used for any of the methods described herein. Referring to FIG. 9A, panel (a), a bridge construct may comprise the capture entity 913, alternatively to or in addition to the capture entity of the oligonucleotide molecule on the geolocation bead (e g., capture entity 113 on oligonucleotide molecule 123 in FIG. 1A). Referring to FIG. 9A, panel (b), a bridge construct may be provided in two parts, a first part 911 and a second part 915. The first part 911 may be partially double-stranded and comprise, in a first strand, the capture sequence 203 as an overhang, and in a second strand,the second atachment binding sequence 205’. The second part 915 may comprise the first attachment binding sequence 207’ and the fourth atachment binding sequence 209’. The first part 911 of the bridge construct may capture the analyte sequence using the capture sequence and be extended using the analyte sequence as a template. The second atachment binding sequence of the first part of the bridge construct may capture a first spatial tag strand that comprises the first atachment sequence, first spatial tag, and the second atachment sequence. Then, the second part 915 of the bridge construct may hybridize to the first spatial tag strand by binding the first atachment binding sequence to the first atachment sequence. Then, the fourth atachment binding sequence of the second part of the bridge construct may capture a second spatial tag strand that comprises at least the fourth atachment sequence and the second spatial tag. Geolocation beads may be designed without the third atachment sequence, for example, as enabled by this two-part bridge construct.

[0162] FIG. 9B illustrates examples of additional tagging schemes using various bridge constructs. In FIG. 9B, panel (a), as described with respect to FIG. 9A, panel (b), a bridge construct may be provided in two parts, a first part and a second part. The first part may be partially double-stranded and comprise, in a first strand, the capture sequence 203 as an overhang, and in a second strand, a first handle 931, the first handle comprising an atachment binding sequence (e.g., second atachment binding sequence 205’) as an overhang. The second part may comprise a second handle 933 which comprises two or more attachment binding sequences (e.g., first attachment binding sequence 207’, fourth atachment binding sequence 209’). In operation, the first part of the bridge construct may capture the analyte sequence 932 using the capture sequence 203 and be extended using the analyte sequence as a template. The attachment binding sequence (e.g., second atachment binding sequence 205’) of the first part of the bridge construct may capture a first spatial tag strand (comprising a spatial tag and two or more atachment sequences) by binding to an atachment sequence (e.g., second atachment sequence 205) of the first spatial tag. The second part of the bridge construct may hybridize to the first spatial tag strand by binding to another atachment sequence (e.g., first atachment sequence 207) of the first spatial tag strand. An additional atachment binding sequence (e.g., fourth atachment binding sequence 209’) of the second handle may capture a second spatial tag strand 935 (comprising a spatial tag and two or more attachment sequences) by binding to a first atachment sequence of the second spatial tag strand (e. g. , the fourth atachment sequence 209). In some cases, a second atachment sequence 937 of the second spatial tag strand (e.g., third atachment sequence 211) may remain as an overhang, and unbound to the second handle.As described elsewhere herein, the second spatial tag strand may comprise a capture moiety (e.g., biotin) at a 5’ end. In some cases, the second attachment sequence 937 of the second spatial tag strand, which remains as an overhang, may alternatively or additionally function as a capture sequence, capture binding sequence, attachment sequence, attachment binding sequence, primer sequence, primer binding sequence, or other sequence during one or more downstream processes. Any geolocation bead and spatial tag construct, as described herein, may be used (e.g., 101, 801. 802, 803. etc.).

[0163] In panel (b) of FIG. 9B, as described with respect to FIG. 9A, panel (b), a bridge construct may be provided in two parts, a first part and a second part. The first part may be partially double-stranded and comprise, in a first strand, the capture sequence 203 as an overhang, and in a second strand, a first handle 941. The, the first handle 941 may comprise an attachment binding sequence, with a portion of it being an overhanging sequence. The second part may comprise a second handle 943, where the second handle 943 may comprise an attachment binding sequence. Functionally, the first part of the bridge construct may capture the analyte sequence 942 using the capture sequence 203 and be extended using the analyte sequence as a template. The overhang portion of the attachment binding sequence of the first part of the bridge construct may capture a first spatial tag strand (comprising a spatial tag and two or more attachment sequences) by binding to a first attachment sequence of the first spatial tag. The first attachment sequence of the first spatial tag may hybridize to a subsection of the overhang of the first part and be extended through a remaining section of the overhang. Then, the second part of the bridge construct may hybridize to the first spatial tag strand by binding a portion of the attachment binding sequence of the second handle 943 to a second attachment sequence of the first spatial tag strand. Then, a remaining portion of the second handle 943 may capture a second spatial tag strand 945 (comprising a spatial tag and two or more attachment sequences) by binding to a first attachment sequence of the second spatial tag strand. The first attachment of the second spatial tag may hybridize to a subsection of the remaining portion of the second handle 943 and be extended through the remaining section of the second handle. A second attachment sequence 947 of the second spatial tag strand may remain as an overhang and unbound to the second handle. As described elsewhere herein, the second spatial tag strand may comprise a capture moiety (e.g., biotin) at a 5’ end. In some cases, the second attachment sequence 947 of the second spatial tag strand, which remains as an overhang, may alternatively or additionally function as a capture sequence, capture binding sequence, attachment sequence,attachment binding sequence, primer sequence, primer binding sequence, or other sequence during one or more downstream processes.

[0164] FIGs. 19A-19D illustrate an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs. Referring to FIG. 19A, a bridge construct may comprise a partially double-stranded molecule, where a first strand comprises a capture sequence 1903 (e.g., polyT sequence) as an overhang and a binding sequence 1905 which binds to a second strand. Optionally, the first strand may comprise one or more additional functional sequences, such as a primer sequence, a barcode sequence, or a unique molecular identifier (UMI) sequence. In some cases, the barcode sequence may be unique to a sample (e.g., tissue), so as to be able to later attribute a tagged sequence back to the sample. The one or more additional functional sequences may be disposed between the capture sequence 1903 and the binding sequence 1905. In some cases, the binding sequence 1905 and the one or more additional functional sequences may together function as a barcode sequence for the bridge construct. In some cases, the barcode sequence for the bridge construct may be known. The second strand may comprise a binding sequence which binds to the first strand and an attachment binding sequence 1907’ (Hl’). Upon contact with an analyte sequence 1910 (e.g.. mRNA comprising polyA tail), the first strand of the bridge construct may capture (e.g., hybridize) the analyte sequence 1910 via the capture sequence 1903 (e.g., polyT sequence). Referring to FIG. 19B, at least two types of geolocation beads may be provided. A first type of geolocation bead 1901 may comprise a double stranded molecule, which in a first strand comprises, in a direction from proximal to distal to the bead, a first attachment sequence (Hl ), a first spatial tag sequence 1911 (BC 1), and a second attachment sequence (H2), and in a second strand comprises, in a direction from proximal to the distal to the bead, the complement of the first strand hybridized to the first strand (i.e., first complementary attachment sequence (HU), first spatial tag complementary sequence, and second complementary attachment sequence (H2’)). The second strand may be immobilized to the bead 1901. The first strand may comprise one or more cleavable moieties (e.g., uracil) in the second attachment sequence (H2), and the second strand may comprise one or more cleavable moieties in the first complementary attachment sequence (HU). The first attachment sequence (Hl) of the first strand may be complementary to the attachment binding sequence 1907’ (Hl’) of the bridge construct. A second ty pe of geolocation bead 1902 may comprise a double stranded molecule, which in a first strand comprises, in a direction from proximal to distal to the bead, a third attachment sequence (H2). a second spatial tag sequence 1912 (BC2), and a fourth attachment sequence(H3), and in a second strand comprises, in a direction from proximal to the distal to the bead, the complement of the first strand hybridized to the first strand (i.e., third complementary attachment sequence (H2’), first spatial tag complementary sequence, and fourth complementary attachment sequence (H3’)). The first strand may comprise, at an end (e.g., 5’ end), a capture moiety (e.g., biotin). The second strand may be immobilized to the bead 1902. The second strand may comprise one or more cleavable moieties in the third complementary attachment sequence (H2’). The third attachment sequence (H2) may correspond to the second attachment sequence (H2) of the first type of geolocation bead 1901. The two types of geolocation beads (e.g., 1901, 1902) may comprise any number of double stranded molecules, as described elsewhere herein with respect to different geolocation bead constructs.

[0165] Referring to FIG. 19C, the beads may be subjected to one or more stimuli to release the respective double stranded molecules from the beads, as described elsewhere herein. In some examples, alternatively or in addition, a USER cleavage reaction is performed to process the cleavable moieties from the double stranded molecules and release the remaining molecule from the bead. In the USER cleavage reaction, a USER (uracil-specific excision reagent) enzyme may generate a nucleotide gap at a location of the uracil base in the molecule and facilitate cleavage. Subsequent to such cleavage reaction, the double stranded molecule of the first type of geolocation bead 1901 may result in a partially double stranded molecule, where a first strand comprises the first attachment sequence (Hl) as an overhang and a second strand comprises the second complementary attachment sequence (H2’) as an overhang. Subsequent to such cleavage reaction, the double stranded molecule of the second type of geolocation bead 1902 may result in a partially double stranded molecule, where a first strand comprises the third attachment sequence (H2) as an overhang. The (a) loading of the geolocation beads, sample, and the bridge construct on a substrate, and (b) releasing of the spatial tags to allow diffusion before capture by the bridge constructs, are described in further detail elsewhere herein. Still referring to FIG. 19C, the bridge construct may capture the analyte sequence 1910 via the capture sequence 1903 and capture the first spatial tag 1911 (BC1) via the attachment binding sequence 1907’ (HE) which binds to the first attachment sequence (Hl). This complex may then capture the second spatial tag 1912 (BC2) via the second complementary attachment sequence (H2’) which binds to the third attachment sequence (H2), to generate a tagged complex. The captured molecules may be ligated. Referring to FIG. 19D, a reverse transcription reaction may be performed to generate a barcoded molecule which comprises the barcode molecule for the bridge construct, the first spatial tag 1911 (BC1), the second spatialtag 1912 (BC2), and the capture moiety (e.g., biotin). In some examples, the barcoded molecule comprises a barcoded cDNA molecule. Collection of the tagged complexes, as well as processing of such tagged complexes to generate the spatially tagged sequences (e.g., 1960), are described elsewhere herein.

[0166] FIG. 20 illustrates an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs. Referring to panel (A) of FIG. 20, a bridge construct may comprise a partially double-stranded molecule, where a first strand comprises a capture sequence 2003 (e.g., polyT sequence) as an overhang and a binding sequence 2005 which binds to a second strand. Optionally, the first strand may comprise one or more additional functional sequences, such as a primer sequence, a barcode sequence, or a unique molecular identifier (UMI) sequence. In some cases, the barcode sequence may be unique to a sample (e.g., tissue), so as to be able to later attribute a tagged sequence back to the sample. The one or more additional functional sequences may be disposed between the captures sequence 2003 and the binding sequence 2005. In some cases, the binding sequence 2005 and the one or more additional functional sequences may together function as a barcode sequence for the bridge construct. In some cases, the barcode sequence for the bridge construct may be known. The second strand may comprise, in order, a binding sequence which binds to the first strand, a first attachment binding sequence 2007’ (HT), a spacer sequence, a second attachment binding sequence 2009’, and the first attachment binding sequence 2007’. Spacer sequences in bridge constructs are described elsewhere herein. Upon contact with an analyte sequence 2010 (e.g., mRNA comprising polyA tail), the first strand of the bridge construct may capture (e.g., hybridize) the analyte sequence 2010 via the capture sequence 2003 (e.g., polyT sequence). The method may use one type of geolocation bead 2001. The geolocation bead 2001 may comprise a nucleic acid molecule which comprises, win a direction from proximal to distal to the bead, a first attachment sequence 2007 (Hl), a first spatial tag sequence 2011 (BC1), and a second attachment sequence 2009 (H2). In some cases, the nucleic acid molecule may comprise one or more additional functional sequences, such as a primer sequence, a barcode sequence or a UMI sequence. In some cases, the one or more additional sequences may be disposed between the first spatial tag sequence 2011 and the second attachment sequence 2009 (H2). The nucleic acid molecule may be single stranded. The strand may comprise one or more cleavable moieties (e.g., uracil) or cleavage sites proximal to the bead. The first attachment sequence 2007 (Hl) of the bead may be complementary to the first attachment binding sequence 2007’ (Hl ') of the bridge construct. The second attachment sequence 2009 (H2) of the bead may becomplementary to the second attachment binding sequence 2009’ of the bridge construct. The geolocation bead 2001 may comprise any number of oligonucleotide molecules, as described elsewhere herein with respect to different geolocation bead constructs.

[0167] Referring to panel (B) of FIG. 20, the beads may be subjected to one or more stimuli to release the respective oligonucleotide molecules from the beads, as described elsewhere herein. For example, the cleavage sites in the nucleic acid molecule may be cleaved to release the strands. The (a) loading of the geolocation beads, sample, and the bridge construct on a substrate, and (b) releasing of the spatial tags to allow diffusion before capture by the bridge constructs, are described in further detail elsewhere herein. The bridge construct may capture the analyte sequence 2010 via the capture sequence 2003 and capture the first spatial tag 2011 (BC1) via the first of the first attachment binding sequences 2007’ (Hl ’) which binds to the first attachment sequence 2007 (Hl) and the second attachment binding sequence 2009’ which binds to the second attachment sequence 2009 (H2). When bound to the bridge construct, the first spatial tag 2011 (BC1) segment may correspond to spacer sequence region of the bridge construct. This complex may then capture a second spatial tag 2021 (BC2) via the second of the first attachment binding sequences 2007’ (Hl ’) of the bridge construct which binds to the first attachment binding sequences 2007 of another spatial tag strand, to generate a tagged complex. The captured molecules may be ligated. Referring to panel (C) of FIG. 20, a reverse transcription reaction may be performed to generate a barcoded molecule which comprises at least the first spatial tag 2011 (BC1) and the second spatial tag 2021 (BC2). In some examples, the barcoded molecule comprises a barcoded cDNA molecule. Collection of the tagged complexes, as well as processing of such tagged complexes to generate the spatially tagged sequences (e.g., 2060), are described elsewhere herein.

[0168] Also provided herein are methods, systems, kits, and compositions that allow for a 5’ approach, as illustrated in FIG. 10. A primer sequence 1001 is provided to couple to an analyte sequence 1003 (e.g., mRNA sequence) (1051). For example, a primer sequence comprising a poly-T sequence captures a poly-A sequence at the 3’ end of a mRNA molecule. The primer sequence 1001 can be extended in a reverse transcription reaction to generate a cDNA transcript 1005 comprising an additional sequence 1031 (e.g.. polyC sequence) (1052). For example, the additional sequence 1031 may comprise bases that are added as a result of terminal transferase activity.

[0169] A 5’ template switching spatial tag oligonucleotide 1007 may be provided along with a spatial tag strand 1009. The template switching spatial tag oligonucleotide 1007 maycomprise a switch sequence 1033 at the 3’ end, which is configured to capture the additional sequence 1031. For example, the switch sequence 1033 comprises a polyG sequence or poly rG sequence. The template switching spatial tag oligonucleotide 1007 may comprise a first spatial tag. The spatial tag strand 1009 may comprise a second spatial tag. The template switching spatial tag oligonucleotide 1007 may attach to the spatial tag strand 1009 via complementary sequences on the 5' end of the template switching spatial tag oligonucleotide 1007 and the 5‘ end of the spatial tag strand 1009. respectively, to form complex 1011 (1055). The spatial tag strand 1009 may comprise a capture entity at the 3’ end such that an end of the complex 1011 comprises the capture entity, and the other end of the complex 1011 comprises the switch sequence 1033. The complex 1011 and the cDNA transcript 1005 may be provided to contact each other (1057, 1053), such that the switch sequence 1033 hybridizes to the additional sequence 1031 to form complex 1013. The cDNA transcript may then be extended to form complex 1015 (1059). The extended complex may be processed to generate a spatially tagged sequence which comprises the cDNA transcript, a complement of the first spatial tag, and the second spatial tag. Alternatively, the spatially tagged sequence may comprise a complement thereof. The complex 1015, or derivative thereof may be captured via the capture entity7. The sets of spatial tags identified in the spatially tagged sequences may be analyzed to generate a map of analyte sequences, in accordance with systems, methods, compositions, and kits described herein.

[0170] The spatial tag strand 1009 and the 5‘ template switching spatial tag oligonucleotide 1007 may be provided on geolocation beads in any of the different types of bead constructs described herein and configured for release of strands 1009 and 1007 instead of other spatial tag strands which are described herein (e.g., 231, 241 comprising spatial tags disposed between a pair of attachment sequences). For example, the spatial tag strand 1009 and the 5’ template switching spatial tag oligonucleotide 1007 may each be provided on different geolocation beads. Alternatively, they may be provided on the same bead. The primer sequence 1001 may be provided to the sample instead of bridge constructs which are described herein to facilitate the reverse transcription reactions prior to capture of the cDNA transcripts.

[0171] Also provided herein are alternative methods, systems, compositions, and kits for spatial screening. A plurality’ of geolocation beads and capture beads may be used. FIG. 11A illustrates example constructs of a geolocation bead 1101 and a capture bead 1121.

[0172] A geolocation bead 1101 may comprise a plurality of oligonucleotide molecules. An oligonucleotide molecule of the plurality of oligonucleotide molecules, illustrated in FIG. 11A,may comprise a first strand and a second strand, where one end 1106 of the oligonucleotide molecule is looped. The first strand may comprise a capture sequence 1102 (e.g., poly A), a spatial tag 1103, and a first primer sequence 1104. The first strand may be partially bound to a second strand which comprises sequences complementary to a portion of the capture sequence, the spatial tag, and a portion of the first primer sequence, respectively. A loop or hairpin may form at one end 1106 of the oligonucleotide, which does not form part of the first strand. Optionally, the capture sequence 1102 may be bound to a protecting sequence 1105 which is complementary to the capture sequence. Optionally, the oligonucleotide molecule may comprise a UMI sequence. Each of the plurality of oligonucleotide molecules may comprise a common spatial tag, and where there are UMI sequences, each of the plurality of oligonucleotide molecules may comprise a unique UMI sequence (different amongst the plurality of oligonucleotide molecules). The geolocation bead may comprise any number of oligonucleotide molecules, for example on the order of 10, 102, 103, 104, 105, 106, 107, 108, or more. The spatial tag may correspond to any spatial tag described herein.

[0173] To generate this oligonucleotide molecule on the geolocation bead 1101, an oligonucleotide molecule comprising the second strand and the loop, where the loop terminates in a blocking group (“X”) and a cleavage site (“U”), can be provided. At least a portion of the capture sequence 1102 may be bound to the second strand. At least a portion of the first primer sequence 1104 may be bound to the second strand, extended using the second strand as a template until reaching the portion of the capture sequence 1102 bound to the second strand, and ligated. Then, the cleavage site can be cleaved to free the 3’ end.

[0174] A capture bead 1121 may comprise a plurality' of oligonucleotide molecules. An oligonucleotide molecule of the plurality of oligonucleotide molecules, illustrated in FIG. 11A, may comprise a first strand and a second strand, where one end 1126 of the oligonucleotide molecule is looped. The first strand may comprise a capture sequence 1122 (e.g., polyT), a barcode sequence 1123, and a second primer sequence 1124. The first strand may be partially bound to a second strand which comprises sequences complementary to a portion of the capture sequence, the barcode sequence, and a portion of the second primer sequence, respectively. A loop or hairpin may form at one end 1126 of the oligonucleotide, which does not form part of the first strand. Optionally, the capture sequence 1122 may be bound to a protecting sequence 1125 which is complementary to the capture sequence. Optionally, the oligonucleotide molecule may comprise a UMI sequence. Each of the plurality' of oligonucleotide molecules may comprise a common barcode sequence, and where there are UMI sequences, each of theplurality of oligonucleotide molecules may comprise a unique UMI sequence (different amongst the plurality of oligonucleotide molecules). The capture bead may comprise any number of oligonucleotide molecules, for example on the order of 10, 102, 103, 104, 105, 106, 107, 108, or more. The barcode sequence may be a nucleic acid sequence. The barcode sequence may comprise at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. 41. 42. 43, 44, 45, 46, 47,48. 49, 50, 60, 70, 80, 90, 100 or more bases. Alternatively or in addition, the barcode sequence may comprise at most about 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39,38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewer bases.

[0175] To generate this oligonucleotide molecule on the capture bead 1121. an oligonucleotide molecule comprising the second strand and the loop, where the loop terminates in a blocking group (“X”) and a cleavage site (“U”), can be provided. At least a portion of the capture sequence 1122 may be bound to the second strand. At least a portion of the second primer sequence 1124 may be bound to the second strand, extended using the second strand as a template until reaching the portion of the capture sequence 1122 bound to the second strand, and ligated. Then, the cleavage site can be cleaved to free the 3’ end.

[0176] The oligonucleotide molecule on the geolocation bead and / or the oligonucleotide molecule on the capture bead may comprise a capture entity, as described elsewhere herein. The first strands on the oligonucleotide molecules on the beads may be released by release mechanisms described elsewhere herein, such as by providing an enzyme configured for strand displacement, digestion, and / or providing one or more stimuli.

[0177] In a substrate-based approach, a substrate may be loaded with a plurality of geolocation beads (e.g.. 1101) and a plurality of capture beads (e.g., 1121). Loading of beads are described elsewhere herein. The substrate may have immobilized thereto a plurality of geolocation beads and a plurality of capture beads on individually addressable locations. The two types of beads may be loaded onto the substrate in any ratio. In some cases, the ratio between the geolocation beads to the capture beads is about 1: 1. In some cases, the ratio of the geolocation beads to the capture beads is at least about 0.00001. 0.0001, 0.001. 0.01. 0.1, 1, 10, 100, 1000, 10000 or more. In some cases, the ratio of the geolocation beads to the capture beads is at most about 10000, 1000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or less.

[0178] A sample which retains, at least to some extent, a spatial relationship between a plurality of analyte sequences, such as tissue slices which retain a spatial relationship betweentranscripts, may be loaded onto the substrate. The geolocation beads and the capture beads may be activated to release the spatial tags and the barcode sequences, respectively (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli. The geolocation beads and the capture beads may be activated simultaneously or substantially simultaneously. The geolocation beads and the capture beads may be activated at different times, for example the capture beads first and then the geolocation beads next, or the geolocation beads first and the capture beads next. The same or different stimuli and / or enzy mes may activate the two beads. The sample may also be subject to conditions sufficient to render analyte sequences, or derivatives thereof (e.g., complementary' DNA to (cDNA) to mRNA, ligation products, etc.) accessible to the barcode sequences released from the capture beads.

[0179] On the substrate, a subset of a plurality of barcode sequences may each capture an analyte sequence from the sample, and another subset of a plurality7of barcode sequences may each capture a spatial tag sequence from the geolocation beads. FIG. 11B provides example capture complexes. A first capture complex 1131 comprises a first barcode strand 1151, released from a capture bead, hybridized to a first spatial tag strand 1152. released from a geolocation bead, and hybridized via capture sequence 1122 and 1102 of the first barcode strand the first spatial tag strand, respectively. The complex thus comprises the first primer sequence 1104, the spatial tag 1103, the barcode sequence 1123, and the second primer sequence 1124. A second capture complex 1132 comprises a first barcode strand 1151, released from a capture bead, hybridized to an analyte sequence 1130, released from the sample, and hybridized via capture sequence 1122 of the first barcode strand to a target sequence (e.g., poly A tail) of the analyte sequence 1130. The complex thus comprises the second primer sequence 1124, the analyte sequence 1130, and the barcode sequence 1123. A plurality of spatially tagged sequences may be generated from the two different types of capture complexes, which capture a spatial tag strand or which capture an analyte sequence, such as by extending one or more strands, on or off the substrate, recovered (e.g., via the capture entity' as described elsewhere herein), and prepared for sequencing. From the sequencing information, a map of the plurality of analyte sequences may be generated by identifying sets of a spatial tag and barcode sequence from the plurality of spatially tagged sequences, where the map comprises information about the respective locations or respective probability7cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence.

[0180] For example, where the sequence information yields a set of [analyte sequence 1, barcode sequence 1]: [spatial tag 5, barcode sequence 1]; [spatial tag 13, barcode sequence 1]; [analyte sequence 16, barcode sequence 1], it can be inferred that (1) the geolocation bead with spatial tag 5 and the geolocation bead with spatial tag 13 are in proximity to each other, that (2) each of the analyte sequence 1 and analyte sequence 16 is in proximity to both the geolocation bead with spatial tag 5 and the geolocation bead with spatial tag 13, and therefore that (3) analyte sequence 1 and analyte sequence 16 are in proximity to each other. More sets of data may be used to map out different analyte sequences and their relative locations.

[0181] In an emulsion-based approach, a plurality' of geolocation beads and a plurality of capture beads, as described elsewhere herein, and a plurality of cells may be partitioned into a plurality of droplets to barcode cellular contents of the plurality of cells within isolated reaction environments. Beneficially, the systems, methods, compositions, and kits permit a droplet to include multiple beads (e.g., geolocation beads), to allow for the relation of the different beadspecific tags within the same droplet as having originated from the same droplet (and thus same cell).

[0182] A plurality of cells, a plurality of geolocation beads, and a plurality of capture beads are partitioned to generate a plurality of partitions. The plurality of partitions may comprise a plurality of droplets. Each bead in the plurality' of geolocation beads may comprise a unique spatial tag such that no spatial tag of any bead overlaps with any other spatial tag of any other bead. Each bead in the plurality of capture beads may comprise a unique barcode sequence such that no barcode sequence of any bead overlaps with any other barcode sequence of any other bead. In some instances, a droplet comprises a cell, a plurality' of geolocation beads, and a plurality of capture beads. After partitioning, the analyte sequences and / or other cellular content in the cell may be rendered accessible to the barcode sequences of the capture beads. For example, the cell can be lysed. The geolocation beads and the capture beads may be activated to release the spatial tags and the barcode sequences, respectively (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli (e.g., heat). The release can be simultaneous or spaced for the two different types of beads. Within the droplet, a subset of a plurality of barcode sequences may each capture an analyte sequence from the sample, and another subset of a plurality of barcode sequences may each capture a spatial tag sequence from the geolocation beads, for example as described with respect to FIG. 11B, to generate barcoded complexes. A plurality of spatially tagged sequences may be generated from the barcodedcomplexes inside or outside of the droplet, recovered (e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information of the spatially tagged sequences, or derivatives thereof, an analyte sequence may be mapped to a cell by identifying sets of a spatial tag and barcode sequence from the plurality of spatially tagged sequences. For example, where a first spatially tagged sequence is identified to include a first spatial tag and a first barcode sequence, where a second spatially tagged sequence is identified to include the first spatial tag and a second barcode sequence, where a third spatially tagged sequence is identified to include a first analyte sequence and the first barcode sequence, and where a fourth spatially tagged sequence is identified to include a second analyte sequence and the second barcode sequence, it can be inferred that all of the first spatial tag, first barcode sequence, second barcode sequence, first analyte sequence, and second analyte sequence were present in the same droplet and therefore the first and second analyte sequences are from the same cell, and thus it can also be inferred that any spatially tagged sequence found to have any one of the first and second barcode sequence originated from the same cell. While this example is specific to a droplet, the partition may be any type of partition, such as a well or micro-well.

[0183] As in the emulsion-based environment, a solution-based environment may allow barcoding of cellular contents of a plurality of cells. A solution may be provided to comprise a plurality of cells in a relatively dilute concentration, a plurality7of geolocation beads, and a plurality of capture beads. The plurality of cells may be provided in a concentration dilute enough that they are located relatively far apart within the solution such as to prevent or make it extremely unlikely that, between the time of release and capture of spatial tags of geolocation beads by capture beads, a first spatial tag of a first geolocation bead located in proximity7to a first cell can diffuse and / or a cellular analyte of the first cell can diffuse to be captured by a barcode sequence of a capture bead located in proximity to a second cell. The geolocation beads and the capture beads may be activated to release the spatial tags and the barcode sequences, respectively (e.g., via releasing the first strands), according to various release mechanisms described elsewhere herein, such as by providing one or more enzymes and / or one or more stimuli (e.g., heat). The release can be simultaneous or spaced for the two different types of beads. Within the solution, a subset of a plurality of barcode sequences may each capture an analyte sequence from the sample, and another subset of a plurality of barcode sequences may each capture a spatial tag sequence from the geolocation beads, for example as described with respect to FIG. 11B, to generate barcoded complexes. A plurality of spatially tagged sequences may be generated from the barcoded complexes inside or outside of the solution, recovered(e.g., via the capture entity as described elsewhere herein), and prepared for sequencing. From the sequencing information of the spatially tagged sequences, or derivatives thereof, an analyte sequence may be mapped to a cell by identifying sets of a spatial tag and barcode sequence from the plurality of spatially tagged sequences. For example, where a first spatially tagged sequence is identified to include a first spatial tag and a first barcode sequence, where a second spatially tagged sequence is identified to include the first spatial tag and a second barcode sequence, where a third spatially tagged sequence is identified to include a first analyte sequence and the first barcode sequence, and where a fourth spatially tagged sequence is identified to include a second analyte sequence and the second barcode sequence, it can be inferred that all of the first spatial tag, first barcode sequence, second barcode sequence, first analyte sequence, and second analyte sequence were present in proximity to each other in the solution and therefore the first and second analyte sequences are from the same cell, and thus it can also be inferred that any spatially tagged sequence found to have any one of the first and second barcode sequence originated from the same cell.

[0184] In some cases, the reaction conditions in the solution may be controlled such as to prevent long distance diffusion of reagents from a first location to a second location in the solution, such as by adding viscous reagents (e.g., PEG, etc.) and / or modulating various reaction conditions (e.g., temperature).

[0185] FIG. 12 illustrates an example of additional capture bead and geolocation bead constructs. A capture bead 1221 may comprise a plurality of oligonucleotide molecules. An oligonucleotide molecule of the plurality of oligonucleotide molecules, illustrated in FIG. 12, may comprise a first strand and a second strand. The first strand may comprise a capture sequence 1222 (e.g., polyT), a barcode sequence 1223, and a second primer sequence 1224. The first strand may be partially bound to a second strand which comprises sequences complementary to a portion of the capture sequence, the barcode sequence, and a portion of the second primer sequence, respectively, and a primer binding sequence 1226’. Optionally, the capture sequence 1222 may be bound to a protecting sequence 1225 which is complementary’ to the capture sequence. Optionally, the oligonucleotide molecule may comprise a UMI sequence. In some cases, the capture sequence 1222 may be capped by a blocking group, denoted “X” and a cleavage site “U.” Such beads may be beneficial for suppressing artefacts from forming during extension. During activation of the beads, an enzyme configured for strand displacement and primer 1226 (e.g., displacement strand) may be provided to displace the first strand (e.g., in the 3?to 5?direction), and then the cleavagesite may be cleaved to make the first strand accessible or able to bind to the analyte sequence or the spatial tag strand. The cleavage site ”U" may be activatable by any one or more stimuli (e.g., light, heat, etc.) and / or one or more enzymes described herein. The oligonucleotide molecule may comprise a capture entity, as described elsewhere herein.

[0186] To generate this oligonucleotide molecule on the capture bead 1221, an oligonucleotide molecule comprising the second strand can be provided. At least a portion of the capture sequence 1222 may be bound to the second strand. At least a portion of the second primer sequence 1224 may be bound to the second strand, extended using the second strand as a template until reaching the portion of the capture sequence 1222 bound to the second strand, and ligated.

[0187] A geolocation bead 1231 may comprise a plurality of oligonucleotide molecules. An oligonucleotide molecule of the plurality of oligonucleotide molecules, illustrated in FIG. 12, may comprise a first strand and a second strand. The first strand may comprise a capture sequence 1232 (e.g., poly A), a spatial tag 1233, and a first primer sequence 1234. The first strand may be partially bound to a second strand which comprises sequences complementary to a portion of the capture sequence, the spatial tag. and a portion of the first primer sequence, respectively, and a primer binding sequence 1236’. Optionally, the capture sequence 1232 may be bound to a protecting sequence 1235, which is complementary to the capture sequence. Optionally, the oligonucleotide molecule may comprise a UMI sequence. In some cases, the capture sequence 1232 may be capped by a blocking group, denoted "X and a cleavage site “U.” During activation of the beads, an enzyme configured for strand displacement and primer 1236 (e.g., displacement strand) may be provided to displace the first strand (e.g., in the 3’ to 5’ direction), and then the cleavage site may be cleaved to make the first strand accessible or able to bind to a nucleic acid released from the capture bead. The cleavage siteCLU” may be activatable by any one or more stimuli (e.g., light, heat, etc.) and / or one or more enzymes described herein. The oligonucleotide molecule may comprise a capture entity, as described elsewhere herein.

[0188] To generate this oligonucleotide molecule on the capture bead 1231, an oligonucleotide molecule comprising the second strand can be provided. At least a portion of the capture sequence 1232 may be bound to the second strand. At least a portion of the first primer sequence 1234 may be bound to the second strand, extended using the second strand as a template until reaching the portion of the capture sequence 1232 bound to the second strand, and ligated.

[0189] A geolocation bead or capture bead may correspond to any bead described elsewhere herein. In some cases, a bead may have a maximum dimension (e.g.. diameter) of at least about 0.05 pm, 0.10 pm, 0.15 pm, 0.16 pm, 0.17 pm, 0.18 pm, 0.19 pm, 0.20 pm, 0.21 pm, 0.22 pm, 0.23 pm, 0.24 pm, 0.25 pm, 0.26 pm, 0.27 pm, 0.28 pm, 0.29 pm, 0.30 pm, 0.31 pm, 0.32 pm, 0.33 pm, 0.34 pm, 0.35 pm, 0.40 pm, 0.45 pm, 0.50 pm, 0.55 pm, 0.60 pm, 0.65 pm, 0.70 pm, 0.75 pm. 0.80 pm, 0.85 pm, 0.90 pm, 0.95 pm, 1.0 pm, 1.1 pm, 1.2 pm, 1.3 pm,1.4 pm, 1.5 pm, 1.6 pm. 1.7 pm, 1.8 pm. 1.9 pm, 2 pm, 3 pm. or greater. Alternatively or in addition, a bead may have a maximum dimension of at most about 0.05 pm, 0. 10 pm, 0. 15 pm,0.16 pm, 0.17 pm, 0.18 pm, 0.19 pm, 0.20 pm, 0.21 pm, 0.22 pm, 0.23 pm, 0.24 pm, 0.25 pm,0.26 pm, 0.27 pm, 0.28 pm, 0.29 pm, 0.30 pm, 0.31 pm, 0.32 pm, 0.33 pm, 0.34 pm, 0.35 pm,0.40 pm, 0.45 pm, 0.50 pm, 0.55 pm, 0.60 pm, 0.65 pm, 0.70 pm, 0.75 pm. 0.80 pm. 0.85 pm,0.90 pm, 0.95 pm, 1.0 pm, 1.1 pm, 1.2 pm, 1.3 pm, 1.4 pm, 1.5 pm, 1.6 pm, 1.7 pm, 1.8 pm, 1.9 pm, 2 pm, 3 pm, or less. In one example, in a 2-dimensional space, estimating about 5- micron radius for a cell and about 0.25-micron radius for a bead, approximately 100 beads may- fit in a cell to produce useful spatial information.

[0190] A geolocation bead and / or capture bead, such as described with respect to FIGs. 11A, 11B, andl2, may be a fiducial marker bead, as described elsewhere herein.Systems & Kits

[0191] Systems, kits, and compositions may comprise any or a combination of the reagents, such as the geolocation beads, capture beads, bridge constructs, enzymes, and primers described herein. In some cases, an index comprising a list of spatial tag sequences included in the geolocation beads may be provided. In some cases, an index comprising a list of barcode sequences included in the capture beads may be provided. The systems, kits, and compositions may include any reagent described herein, such as enzymes, viscosity or crowing agents, sequencing reagents, amplification reagents, and other reagents. A system may comprise any kit and / or reagent described herein. A system may comprise a state in which the provided kit has not been used, has been used, or is being used.

[0192] For example, a kit may comprise substrates, geolocation beads, capture beads, bridge constructs, primers and / or enzymes. A kit may comprise any substrate described herein, such as (i) a substrate that does not have any geolocation beads immobilized thereto, or (ii) a substrate comprising geolocation beads immobilized thereto, the geolocation beads comprising spatial tags. The kit may comprise indexed data comprising a list of spatial tag sequences included in the geolocation beads. A kit may comprise a plurality of geolocation beadscomprising spatial tag sequences. A kit may comprise a plurality of oligonucleotide molecules comprising spatial tag sequences. A kit may comprise a plurality of oligonucleotide molecules comprising capture sequences. A kit may comprise a plurality of oligonucleotide molecules each comprising both a spatial tag sequence and a capture sequence. A kit may comprise any sequencing reagent described herein. A kit may comprise any amplification reagent described herein.

[0193] In one example, a kit comprises (1) a substrate comprising a plurality of geolocation beads immobilized to a plurality of individually addressable locations on the substrate, wherein the plurality of geolocation beads comprises oligonucleotide molecules coupled thereto, wherein the oligonucleotide molecules comprise spatial tag molecules, wherein each geolocation bead of said plurality of geolocation beads comprises a unique spatial tag; (2) indexed data comprising a list of spatial tag sequences included in the geolocation beads; and (3) a second substrate comprising a second plurality of individually addressable locations configured to immobilize a second plurality' of geolocation beads. In some cases, the kit further comprises the second plurality of geolocation beads that are not immobilized to the second substrate. In some cases, the kit further comprises a reagent configured to release the oligonucleotide molecules from the plurality of geolocation beads. The reagent can comprise one or more of: (i) an enzy me mix comprising an enzy me configured to cleave or digest a cleavage site, wherein the oligonucleotide molecules comprises the cleavage site; (ii) biotin, wherein the oligonucleotide molecules are conjugated to a desthiobiotin moiety, wherein the plurality of geolocation beads comprises a streptavidin moiety bound to the desthiobiotin moiety; (iii) an enzyme mix comprising an enzyme configured to cleave or digest a cleavage site and UV light, wherein the oligonucleotide molecules comprises azobenzene and the cleavage site; and (iv) a light source configured to provide UV light, wherein the oligonucleotide molecules are conjugated to azobenzene, wherein the plurality of geolocation beads comprises an alpha-cyclodextrine (a-CD) moiety bound to the azobenzene. In some cases, the kit further comprises sequencing reagents, such as single-base nucleotide mixtures for each of the four base types (e.g.. A, C. G, T or U) or multi -base nucleotide mixtures (e.g., A&C, A&T, A&C&G, etc.). A single-base or multi-base nucleotide mixture may comprise a mixture of labeled and unlabeled nucleotides. A single-base or multi-base nucleotide mixture may comprise non-terminated nucleotides, in some cases comprising only non-terminated nucleotides (vs. terminated nucleotides). In some cases, the kit further comprises amplification reagents. Amplification reagents may comprise a polymerase, a nucleotide mixture, a primer,a buffer, or any combination thereof. In some cases, the kit further comprises a biological sample. In some cases, the biological sample may comprise a tissue. In some cases, the biological sample may be fixed and / or permeabilized. In some cases, the biological sample may be loaded on the substrate. In some cases, the kit further comprises fixing and / or permeabilizing reagents. In some cases, a bead of the plurality of geolocation beads may comprise at least 100,000 oligonucleotide molecules. In some cases, the at least 100,000 oligonucleotide molecules may comprise a spatial tag sequence of the spatial tag sequences that is common and unique to the geolocation bead amongst the plurality of geolocation beads. In some cases, an oligonucleotide molecule of the oligonucleotide molecules may comprise a capture sequence, wherein the capture sequence is configured to hybridize with a sequence of an analyte, or derivative thereof, of a biological sample. The capture sequence may be selected from, for example, apolyT sequence, a poly G sequence, a targeted mRNA sequence, a targeted gDNA sequence, a random n-mer sequence, and a probe sequence. The first and second substrate may be substantially identical in size, shape, and / or material. The first and second substrate may be different in size, shape, and / or material.

[0194] In another example, a kit comprises a substrate comprising a plurality of geolocation beads immobilized to a plurality of individually addressable locations on the substrate, wherein the plurality7of geolocation beads comprises oligonucleotide molecules coupled thereto, wherein the oligonucleotide molecules comprise spatial tag molecules, wherein each geolocation bead of said plurality of geolocation beads comprises a unique spatial tag, wherein the oligonucleotide molecules are releasable from the plurality of geolocation beads by one or more of the release mechanisms selected from the group consisting of: (a) providing an enzyme mix comprising an enzyme configured to cleave or digest a cleavage site, wherein the oligonucleotide molecules comprises the cleavage site; (b) providing biotin, wherein the oligonucleotide molecules are conjugated to a desthiobiotin moiety, wherein the geolocation bead comprises a streptavidin moiety bound to the desthiobiotin moiety7; (c) providing an enzy me mix comprising an enzyme configured to cleave or digest a cleavage site and UV light, wherein the oligonucleotide molecules comprises azobenzene and the cleavage site; and (d) providing UV light, wherein the oligonucleotide molecules are conjugated azobenzene, wherein the plurality7of geolocation beads comprises an alpha-cyclodextrine (a-CD) moiety bound to the azobenzene. In some cases, the kit may further comprise indexed data comprising a list of spatial tag sequences included in the geolocation beads. In some cases, the kit may further comprise a second substrate comprising a second plurality of individually addressablelocations configured to immobilize a second plurality of geolocation beads. In some cases, the kit may further comprise the second plurality of geolocation beads. In some cases, the kit further comprises a reagent configured to release the oligonucleotide molecules from the plurality of geolocation beads. The reagent can comprise one or more of: (i) an enzyme mix comprising an enzyme configured to cleave or digest a cleavage site, wherein the oligonucleotide molecules comprises the cleavage site; (ii) biotin, wherein the oligonucleotide molecules are conjugated to a desthiobiotin moiety, wherein the plurality of beads comprises a streptavidin moiety bound to the desthiobiotin moiety; (iii) an enzyme mix comprising an enzyme configured to cleave or digest a cleavage site and UV light, wherein the oligonucleotide molecules comprises azobenzene and the cleavage site; and (iv) a light source configured to provide UV light, wherein the oligonucleotide molecules are conjugated to azobenzene, wherein the plurality' of geolocation beads comprises an alpha-cyclodextrine (a-CD) moiety bound to the azobenzene. In some cases, the kit further comprises sequencing reagents, such as single-base nucleotide mixtures for each of the four base types (e.g., A, C, G, T or U) or multi-base nucleotide mixtures (e.g., A&C, A&T, A&C&G, etc.). A single-base or multi-base nucleotide mixture may comprise a mixture of labeled and unlabeled nucleotides. A singlebase or multi-base nucleotide mixture may comprise non-terminated nucleotides, in some cases comprising only non-terminated nucleotides (vs terminated nucleotides). In some cases, the kit further comprises amplification reagents. Amplification reagents may comprise a polymerase, a nucleotide mixture, a primer, a buffer, or any combination thereof. In some cases, the kit further comprises a biological sample. In some cases, the biological sample may comprise a tissue. In some cases, the biological sample may be fixed and / or permeabilized. In some cases, the biological sample may be loaded on the substrate. In some cases, the kit further comprises fixing and / or permeabilizing reagents. In some cases, a geolocation bead of the plurality of geolocation beads may comprise at least 100,000 oligonucleotide molecules. In some cases, the at least 100,000 oligonucleotide molecules may comprise a spatial tag sequence of the spatial tag sequences that is common and unique to the geolocation bead amongst the plurality of geolocation beads. In some cases, an oligonucleotide molecule of the oligonucleotide molecules may comprise a capture sequence, wherein the capture sequence is configured hybridize with a sequence of an analyte, or derivative thereof, of a biological sample. The capture sequence may be selected from, for example, a polyT sequence, a polyG sequence, a targeted mRNA sequence, a targeted gDNA sequence, a random n-mer sequence, and a probesequence. The first and second substrate may be substantially identical in size, shape, and / or material.

[0195] In one example, a system comprises a sequencing platform configured to (i) address individually addressable locations of substrates and (ii) rotate the substrates during dispensing of sequencing reagents to the substrates or during imaging of the substrates or during both. The sequencing platform may be any sequencing platform described herein. The system may further comprise any kit and / or reagent described herein (e.g., substrate, geolocation beads, bridge constructs, indexed data, reagent configured to release oligonucleotide molecules from a plurality of beads, sequencing reagent, amplification reagent, fixing and / or permeabilizing reagent, etc.). In some cases, the system may comprise a light source configured to provide light at desired frequencies (e.g.. UV light, fluorescent light, etc.).

[0196] In some instances, a sample is treated by a fixative or fixated, before it is loaded onto the substrate or provided in a reaction environment with one or more reagents of the present disclosure (e.g., geolocation beads, capture beads, etc.). Fixation, in some cases, may render the location of an analyte invariable. For example, an mRNA molecule may not diffuse away from its location in a tissue after fixation. In some case, permeabilization of a fixed biological sample may facilitate the contacting between the reagents and the analyte(s). In some cases, permeabilization of a sample may release an analyte. For example, permeabilization of a fixed tissue may release an mRNA vertically downward, e.g., via gravity, so that it can contact the reagents on the substrate. Once captured, the endogenous mRNA may be tagged with spatial tags and / or barcode sequences, as described elsewhere herein, which can be decoded or processed to determine spatial information. In some instances, a biological sample may be dissected, dissociated, digested, or degraded after an analyte is tagged, according to methods described herein. Such dissection, dissociation, digestion, or degradation of a biological sample may facilitate the processing of the processing of an analyte. Spatial information of an analyte may be retained and decoded via the tags disclosed herein, afterwards. For example, dissection, dissociation, digestion, or degradation of a biological sample may not remove the encoded location information of an analyte. The location of each analyte may be reconstructed digitally by decoding the tags and / or barcode sequences of the plurality of tagged analytes.

[0197] A plurality of spatially tagged sequences may be generated from the tagged and / or barcoded analytes on or off the substrate, such as by extending one or more strands of the tagged and / or barcoded analytes. For example, one or more extension reactions may be performed. For example, one or more ligation reactions may be performed. The plurality’ ofspatially tagged sequences may be prepared for sequencing. In some cases, one or more adapters may be attached to one or both ends of the spatially tagged sequences. The adaptercontaining spatially tagged sequences may be subjected to amplification reactions. In some cases, subsequent to amplification reactions, a plurality of beads may be collected, wherein each bead of at least a subset of beads (positive beads) comprises a colony of amplification products. For example, each positive bead may comprise, attached thereto, a plurality of nucleic acid molecules having sequence homology or sequence identity and comprising a sequence corresponding to a spatially tagged sequence. The plurality of beads may also comprise negative beads, or beads that do not have nucleic acid molecules comprising a sequence corresponding to a spatially tagged sequence. Optionally, the positive beads may be isolated from the negative beads. The plurality of beads or isolated positive beads may be loaded onto a substrate, as described elsewhere herein, and subjected to a method of sequencing nucleic acid molecules, as described elsewhere herein. Methods for processing analytes or templates (e.g., spatially tagged sequences) for sequencing to generate input material for substrates and sequencing systems described herein are described in International Patent Publication No. 2020 / 167656, which is entirely incorporated herein by reference for all purposes.

[0198] It will be appreciated that various methods for sample preparation or library preparation may be applied. It will be appreciated that various methods for barcoding the spatially tagged sequences may be applied. It will be appreciated that various method for amplification of the processed spatially tagged sequences may be applied. For example, an amplification may comprise a reverse transcription, primer extension, PCR, LCR, helicase-dependent amplification, asymmetric amplification, RCA, RPA, LAMP, NASBA, 3 SR, HCR, MDA, derivatives herein and thereof, or any combination herein and thereof. Amplification may comprise emulsion PCR (ePCR or emPCR). During downstream processing, such as during sample preparation or library preparation, any useful sequence may be appended to the spatially tagged sequence, or derivative thereof, such as flow cell attachment sequences, primer sequences, index sequences, barcode sequences, capture sequences, target sequences, etc. In some examples, a strand of a tagged complex 250 or a derivative thereof, may be captured via a primer molecule comprising a capture sequence (e.g., polyT). and the primer molecule extended. In another example, a strand of a tagged complex 250, or derivative thereof, may be subject to reverse transcription, and the transcript captured via a primer molecule comprising a capture sequence (e.g., polyG). In another example, a strand of a tagged complex 250, or derivative thereof, may be captured via a primer molecule comprising a capture sequence (e.g.,polyT), the primer molecule extended to generate a transcript comprising a polyC sequence at one end, and the transcript captured via a template switching oligonucleotide comprising a capture sequence (e.g., polyG). In another example, template switching reactions using one or more template switching sequences. In another example, a strand of a tagged complex 250, or derivative thereof, can be contacted with a single Tn5 adapter, reverse transcription performed, and PCR performed. In another example, a strand of a tagged complex 250, or derivative thereof, may be contacted with an enzyme to shear the nucleic acid molecule, a splint adapter may be ligated, and PCR performed. One or more downstream processes may comprise multiple rounds of PCR. One or more downstream processes may comprise enzy matic fragmentation. One or more downstream processes may comprise end repair of the A-tail.Bead-Capture Matrix

[0199] A method for spatial sequencing may comprise (a) immobilizing a first set of beads comprising a plurality of first oligonucleotide molecules on a substrate, each of the first set of beads comprising a set of first oligonucleotide molecules each comprising a spatial tag unique to the bead, (b) loading a second set of beads comprising a plurality of second oligonucleotide molecules to the substrate comprising the first set of beads immobilized thereto, each of the second set of beads comprising a set of second oligonucleotide molecules each comprising a spatial tag unique to the bead, and capturing at least a subset of second oligonucleotide molecules of the second set of beads with at least a subset of first oligonucleotide molecules of the first set of beads, (c) extending the subset of first oligonucleotide molecules to generate a plurality of composite molecules on the first set of beads, each of the plurality of composite molecules comprising a first tag sequence of a spatial tag from the first set of beads and a second tag sequence derived from a spatial tag from the second set of beads, (d) loading a sample to the substrate comprising the first set of beads immobilized thereto, and capturing a plurality of analyte sequences from the sample with oligonucleotide molecules of the first set of beads, the oligonucleotide molecules comprising one or more of (i) at least a subset of the plurality7of composite molecules or derivatives thereof, and / or (ii) an additional subset of first oligonucleotide molecules or derivatives thereof, and (e) generating spatially tagged analyte molecules, wherein each of the spatially tagged analyte molecules comprises a sequence of or derived from a spatial tag from the first set of beads. The method may comprise (f) sequencing the spatially tagged analyte molecules or derivatives thereof (e.g., reverse complements, adaptor-ligated, amplicons, otherwise processed, etc.), and optionally an additional subset of the plurality of composite molecules that did not capture any analyte sequence, or derivativesthereof, to generate sequencing data and using the sequencing data to generate a map of the plurality of analyte sequences by identifying sets of associated spatial tags, where the map comprises information about the respective locations or respective probability cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence. In some instances, the first set of beads may each comprise a spatial tag that is unique w ithin the first set of beads or unique within any set of beads. In some instances, the second set of beads may each comprise a spatial tag that is unique within the second set of beads or unique within any set of beads.

[0200] FIG. 24 illustrates a schematic bead-capture matrix from a top view and a cross-section side view- in panels (A) and (B) respectively. A first set of beads 2402 (“B#”) comprising a plurality of first oligonucleotide molecules 2403 may be immobilized to a substrate 2401. A second set of beads 2404 (“A#”) comprising a plurality of second oligonucleotide molecules 2405 may be loaded onto the substrate with the first set of beads. As seen in the illustration, at least a subset of the plurality of first oligonucleotide molecules 2405 on the first set of beads 2402 may capture at least a subset of the plurality of second oligonucleotide molecules 2405 on the second set of beads 2404, such as by hybridization between a pair of complementary sequences. In some cases, a single bead of the second set of beads (e.g., Al bead) may comprise second oligonucleotide molecules that are captured by first oligonucleotide molecules on multiple beads of the first set of beads (e.g., Bl and B2 beads). In some cases, a single bead of the first set of beads (e.g., B2 bead) may comprise first oligonucleotide molecules that capture second oligonucleotide molecules on multiple beads of the second set of beads (e.g., Al and A2 beads). It will be appreciated that while FIG. 24 illustrates the second set of beads directly above the first set of beads, the second set of beads may be in any orientation with respect to the first set of beads — for example, beads from the second set may also be in contact or near contact w ith the surface that beads from the first set are contacting.

[0201] FIG. 25 illustrates an example bead-on-bead capture scheme. Referring to panel (A), a first bead (“B”) 2502 can be immobilized on a substrate 2501. The first bead may comprise a plurality of first oligonucleotide molecules (e.g., 2503) (two illustrated) each comprising a first spatial tag (“BCb’?) unique to the first bead. The first spatial tag may be unique to the first bead amongst a plurality of first beads immobilized on the substrate. A first oligonucleotide molecule 2503 of the plurality of first oligonucleotide molecules may comprise from a first end to a second end, a first binding sequence (“P5 20’"), the first spatial tag fyBCb'’). and a first bead adapter sequence (“TS R1”). The first binding sequence may be configured to capture asecond binding sequence, such as via complementarity. In some cases, the first bead adapter sequencer may comprise one or more cleavable moieties (“U”) that may be used as a release mechanism. A second bead (“A”) 2504 can be loaded on the substrate, the second bead comprising a plurality of second oligonucleotide molecules (e.g., 2505) (one illustrated) each comprising a second spatial tag (“BCa”) unique to the second bead. The second spatial tag may be unique to the second bead amongst a plurality of second beads loaded to the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may partially or completely overlap with a set of second spatial tags contained by a plurality of second beads loaded on the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may not overlap with a set of second spatial tags contained by a plurality of second beads loaded on the substrate. A second oligonucleotide molecule 2505 of the plurality of second oligonucleotide molecules may comprise from a first end to a second end, the second binding sequence (“P5”’), the second spatial tag (“BCa”), and a second bead adapter sequence (“Pl”). The second binding sequence may comprise a portion that is complementary to the first binding sequence. In some cases, the second bead adapter sequence may comprise a third binding sequence. In some cases, the second bead adapter sequence may comprise an analyte capture sequence, such as a poly-A sequence, whose reverse complement may capture mRNA analytes or other sequences (e.g., targeted sequence, randomer sequence, or reverse complements thereol). In some cases, a 3’ end of the second oligonucleotide molecule may be blocked from extension, rendering the strand inextendible. Upon contact of a first oligonucleotide molecule (e.g., 2503) with a second oligonucleotide molecule (e.g., 2505), the first binding sequence of the first oligonucleotide molecule may anneal to the second binding sequence of the second oligonucleotide molecule, and the first oligonucleotide molecule extended to generate extended molecule 2507. The extended molecule may comprise, from a first end to a second end, a complement of the second bead adapter sequence (“Pl”’), a complement of the second spatial tag (“BCa”’), the first binding sequence (“P5_20”), the first spatial tag (“BCb”), and the first bead adapter sequence (“TS R1”).

[0202] Referring to panel (B), if the second bead adapter sequence does not comprise an analyte capture sequence (e.g., poly-A sequence) but comprises a third binding sequence, the extended molecule 2507 may be further extended with a capture sequence molecule 2509, the capture sequence molecule comprising, from a first end to a second end, a capture sequence (“Poly A”) and a fourth binding sequence (“P 1 *”). It will be appreciated that while the exampleillustrates poly A and polyT sequences, the capture sequence may correspond to (e.g., is or reverse complement of) any analyte capture sequence as described elsewhere herein. In some cases, a 3’ end of the capture molecule may be blocked from extension, rendering the strand inextendible. Upon contact of an extended molecule (e.g., 2507) and the capture sequence molecule (e.g., 2509), the fourth binding sequence of the capture sequence molecule may anneal to the third binding sequence of the extended molecule, and the extended molecule further extended to generate analyte capture molecule. The analyte capture molecule may comprise, from a first end to a second end, an analyte capture sequence 2508 (“TTT... ”), a complement of the second bead adapter sequence (“Pl”’), a complement of the second spatial tag (“BCa”’), the first binding sequence (“P5_20”), the first spatial tag (“BCb”), and the first bead adapter sequence (“TS Rl”). Optionally, a first oligonucleotide molecule (e.g.. 2503) of the first bead 2502 that does not capture any second oligonucleotide molecule from a second set of beads and thus is not extended, may also be extended by a second capture sequence molecule 2510. The second capture sequence molecule may comprise, from a first end to a second end, a capture sequence (“Poly A”) and a fifth binding sequence. In some cases, a 3’ end of the second capture molecule may be blocked from extension, rendering the strand inextendible. The fifth binding sequence may anneal to the first binding sequence of the first oligonucleotide molecule, and the first oligonucleotide molecule may be extended to generate a second analyte capture molecule. The second analyte capture molecule may comprise, from a first end to a second end. an analyte capture sequence (“TTT.. . ”), the first binding sequence (“P5_20”), the first spatial tag (“BCb”), and the first bead adapter sequence (“TS R1 ”). As such, the analyte capture molecule is a composite molecule that comprises an analyte capture sequence and information pertaining to two spatial tags, a first spatial tag from a first plurality of beads (immobilized to the surface) and a second spatial tag from a second plurality of beads (loaded to the substrate over the first plurality of beads). The second analyte capture molecule may comprise an analyte capture sequence and information pertaining to only one spatial tag from the first plurality of beads.

[0203] Alternatively, if the second bead adapter sequence of the second oligonucleotide molecules (e.g., 2505) comprise an analyte capture sequence (e.g., poly-A sequence), the extended molecule may already comprise the analyte capture sequence and extension via the capture sequence molecule 2509 is not needed.

[0204] Referring to panel (C), a sample comprising a plurality of analyte sequences may be loaded on the substrate. The sample may retain, at least to some extent, a spatial relationshipbetween the plurality of analyte sequences. In some instances, a tissue slice is loaded onto the substrate, where the tissue slice retains, at least to some extent, a spatial relationship between the transcripts contained therein. Samples that can be used in the present methods, systems, compositions, and kits are described in further detail elsewhere herein. A sequence (e.g., “AAA.. . ”) of an analyte sequence 2511 (“mRNA_AAA. . . ”) may anneal to the analyte capture sequence (e.g.. “TTT... ”) of the analyte capture molecule, and the analyte capture molecule may be extended to generate a tagged analyte molecule. A tagged analyte molecule may comprise, from a first end to a second end, a template sequence (“cDNA”), an analyte capture sequence (“TTT... ”), a complement of the second bead adapter sequence (“Pl ”’), a complement of the second spatial tag (“BCa”’), the first binding sequence (“P5_20”), the first spatial tag (“BCb”), and the first bead adapter sequence (“TS RI”). Where the analyte sequence is an mRNA sequence, the template sequence may correspond to a cDNA sequence. In some cases, the tagged analyte molecule may additionally comprise a template switching oligonucleotide (“TSO”) (e.g., polyC) sequence as a product of a reverse transcription reaction, which template switching oligonucleotide sequence may be used for downstream template switching operations described elsewhere herein. In some cases, a tagged analyte molecule (which can be used in a sequencing library) may comprise the structure comprising, from 5’ to 3’, the first bead adapter sequence (“TS_R1”), the first spatial tag (“BCb”), the first binding sequence (“P5_20”), a complement of the second spatial tag (“BCa”'), a complement of the second bead adapter sequence (“Pl”’), the analyte capture sequence (“TTT... ”). the template sequence (“cDNA”). In some cases, the tagged analyte molecule may comprise a template switching oligonucleotide sequence (“TSO”) at the 3’ end. In some cases, the tagged analyte molecule may comprise any alternative or additional functional sequence described elsewhere herein (e.g., barcode sequence, index sequence, UMI. adapter sequence, sequencer adapter sequence, primer sequence, etc.).

[0205] Optionally, where the first bead comprises the second analyte capture molecule, a sequence (e.g., “AAA.. . ”) of an analyte sequence (e.g., 2511) (“mRNA_AAA. .. ”) may anneal to the analyte capture sequence (e.g., “TTT... ”) of the second analyte capture molecule, and the second analyte capture molecule may be extended to generate a tagged analyte molecule. A tagged analyte molecule may comprise, from a first end to a second end, a template sequence (“cDNA”), an analyte capture sequence (“TTT... ”), the first binding sequence (“P5_20”), the first spatial tag (“BCb”), and the first bead adapter sequence (“TS RI”). Where the analyte sequence is an mRNA sequence, the template sequence may correspond to a cDNA sequence.In some cases, the tagged analyte molecule may additionally comprise a template switching oligonucleotide (“TSO") (e.g., polyC) sequence as a product of a reverse transcription reaction, which template switching oligonucleotide sequence may be used for downstream template switching operations described elsewhere herein.

[0206] The annealing between the analyte sequence and the analyte capture molecule (and / or the second analyte capture molecule) may occur prior to, during, or subsequent to release of the analyte capture molecule from the first bead. The analyte capture molecule, or derivative thereof (e.g., extension product comprising the template sequence), may be released from the first bead via any release mechanism described herein. In one example, the molecule is released from the first bead via USER cleavage of “U” cleavage sites in the first bead adapter sequence. The extension of the analyte capture molecule (and / or the second analyte capture molecule) may occur prior to, during, or subsequent to release of the analyte capture molecule from the first bead. The analyte sequence may be removed (e.g., by melting, denaturing, etc.) from the tagged analyte molecule prior to, during, or subsequent to release of the analyte capture molecule from the first bead.

[0207] The tagged analyte molecule may be subsequently processed as described elsewhere herein with respect to other spatially tagged sequences. For example, tagged analyte molecules may be subjected to library preparation, such as to attach one or more adapters, barcodes, such as to subject to amplification, etc., and sequencing to generate sequencing reads. Sequencing preparation and sequencing are described in further detail elsewhere herein. The sequencing data generated from sequencing may be processed and / or analyzed to generate a spatial map of the plurality7of analyte sequences in the sample.

[0208] FIG. 26 illustrates an additional example bead-on-bead capture scheme. Referring to panel (A), a first bead (“B”) 2602 can be immobilized on a substrate 2601. The first bead may comprise a plurality of first oligonucleotide molecules (e.g., 2603) each comprising a first spatial tag (“BCb”) unique to the first bead. The first spatial tag may be unique to the first bead amongst a plurality of first beads immobilized on the substrate. A first oligonucleotide molecule 2603 of the plurality of first oligonucleotide molecules may comprise from a first end to a second end, a first binding sequence (“X"), the first spatial tag (“BCb"), and a first bead adapter sequence. The first binding sequence may be configured to capture a second binding sequence, such as via complementarity7. In some cases, the first bead adapter sequencer may comprise one or more cleavable moieties (“U”) that may be used as a release mechanism. In some cases, a 3’ end of the first oligonucleotide molecule may be blocked from extension,rendering the strand inextendible. A second bead (“A”) 2604 can be loaded on the substrate, the second bead comprising a plurality of second oligonucleotide molecules (e.g., 2605) (two illustrated) each comprising a second spatial tag (“BCa”) unique to the second bead. The second spatial tag may be unique to the second bead amongst a plurality of second beads loaded to the substrate. In some cases, a set of first spatial tags contained by a plurality7of first beads immobilized on the substrate may partially or completely overlap with a set of second spatial tags contained by a plurality of second beads loaded on the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may not overlap with a set of second spatial tags contained by a plurality7of second beads loaded on the substrate. A second oligonucleotide molecule 2605 of the plurality of second oligonucleotide molecules may comprise from a first end to a second end, a second bead adapter sequence (“Pl”), the second spatial tag (‘'BCa”), and the second binding sequence (“PA26”). The second binding sequence may comprise a portion that is complementary to the first binding sequence. A first subset of the plurality7of second oligonucleotide molecules may capture first oligonucleotide molecules of the plurality of first beads. Upon contact of a first oligonucleotide molecule (e.g.. 2603) with a second oligonucleotide molecule (e.g.. 2605). the first binding sequence of the first oligonucleotide molecule may anneal to the second binding sequence of the second oligonucleotide molecule, and the second oligonucleotide molecule may be extended to generate composite molecule 2607. The composite molecule may comprise, from a first end to a second end, the second bead adapter sequence (“Pl”), the second spatial tag (“BCa”), the second binding sequence (“PA26”), a complement of the first spatial tag (“BCb”’), and a complement of the first bead adapter sequence.

[0209] Referring to panel (B), a second subset of the plurality7of second oligonucleotide molecules, mutually exclusive of the first subset of the plurality of second oligonucleotide molecules, may be extended with a capture sequence molecule 2609. The capture sequence molecule may comprise, from a first end to a second end, a capture sequence (“Poly A”) and a third binding sequence. It will be appreciated that while the example illustrates polyA and polyT sequences, the capture sequence may correspond to (e.g., is or reverse complement ol) any analyte capture sequence as described elsewhere herein. Upon contact of a second oligonucleotide molecule (e.g., 2605) and the capture sequence molecule (e.g., 2609), the third binding sequence of the capture sequence molecule may anneal to the second binding sequence of the second oligonucleotide molecule, and the second oligonucleotide molecule may be extended to generate an analyte capture molecule. The analyte capture molecule may comprise,from a first end to a second end, an analyte capture sequence 2608 (“TTT. . . ”), the second binding sequence (“PA26”), the second spatial tag (“BCa”). the second bead adapter sequence (“Pl”). As such, the first and second subsets of the plurality of second oligonucleotide molecules on a second bead (e.g., 2604) may be extended to generate at least two different sets of extended molecules, where a first set of extended molecules are composite molecules (e.g., 2607) that each comprises information pertaining to two spatial tags, a first spatial tag from a first plurality of beads (immobilized to the surface) and a second spatial tag from a second plurality of beads (loaded to the substrate over the first plurality of beads), and where a second set of extended molecules are analyte capture molecules which comprise an analyte capture sequence and information pertaining to only one spatial tag from the second plurality of beads.

[0210] Referring to panel (C), a sample comprising a plurality of analyte sequences may be loaded on the substrate. The sample may retain, at least to some extent, a spatial relationship between the plurality' of analyte sequences. In some instances, a tissue slice is loaded onto the substrate, where the tissue slice retains, at least to some extent, a spatial relationship between the transcripts contained therein. Samples that can be used in the present methods, systems, compositions, and kits are described in further detail elsewhere herein. A sequence (e.g.. “AAA.. . ”) of an analyte sequence 2611 (“mRNA_AAA. . . ”) may anneal to the analyte capture sequence (e.g., “TTT... ”) of the analyte capture molecule, and the analyte capture molecule may be extended to generate a tagged analyte molecule. A tagged analyte molecule may comprise, from a first end to a second end, a template sequence (“cDNA”). an analyte capture sequence (“TTT... ”), the second binding sequence (“PA26”), the second spatial tag (“BCa”), the second bead adapter sequence (“Pl”). Where the analyte sequence is an mRNA sequence, the template sequence may correspond to a cDNA sequence. In some cases, the tagged analyte molecule may additionally comprise a template switching oligonucleotide (“TSO”) (e.g., polyC) sequence at the 3’ end as a product of a reverse transcription reaction, which template switching oligonucleotide sequence may be used for downstream template switching operations described elsewhere herein. In some cases, the tagged analyte molecule may comprise any alternative or additional functional sequence described elsewhere herein (e.g., barcode sequence, index sequence, UMI, adapter sequence, sequencer adapter sequence, primer sequence, etc ).

[0211] The oligonucleotide molecules on the second bead (“A”) may be released from the second bead, including the different extended molecules (e.g., composite molecules comprising two spatial tag information, analyte capture molecules, tagged analyte molecules, etc.). Theoligonucleotide molecules on the first bead (“B”) may be released from the first bead. In some cases, the annealing between the analyte sequence and the analyte capture molecule may occur prior to, during, or subsequent to release of the analyte capture molecule from the second bead. An oligonucleotide may be released from a bead via any release mechanisms described elsewhere herein (e.g., USER cleavage). In some cases, the annealing between the analyte sequence and the analyte capture molecule may occur prior to, during, or subsequent to release of the first oligonucleotide molecules from the first bead. In some cases, the extension of the analyte capture molecule may occur prior to, during, or subsequent to release of the analyte capture molecule from the second bead. In some cases, the extension of the analyte capture molecule may occur prior to, during, or subsequent to release of the first oligonucleotide molecules from the first bead. The analyte sequence may be removed (e.g.. by melting, denaturing, etc.) from the tagged analyte molecule prior to, during, or subsequent to release of the analyte capture molecule from the second bead. The analyte sequence may be removed (e.g., by melting, denaturing, etc.) from the tagged analyte molecule prior to, during, or subsequent to release of the first oligonucleotide molecules from the first bead.

[0212] The tagged analyte molecules and the composite molecules derived from (e.g.. extended from, released from, etc.) the second bead may be subsequently processed as described elsewhere herein with respect to other spatially tagged sequences. For example, the molecules may be subjected to library preparation, such as to attach one or more adapters, barcodes, such as to subject to amplification, etc., and sequencing to generate sequencing reads. Sequencing preparation and sequencing are described in further detail elsewhere herein. The sequencing data generated from sequencing may be processed and / or analyzed to generate a spatial map of the plurality of analyte sequences in the sample.

[0213] FIG. 27 illustrates an additional example bead-on-bead capture scheme. Referring to panel (A), a first bead (“B”) 2702 can be immobilized on a substrate 2701. The first bead may comprise a plurality of first oligonucleotide molecules (e.g., 2703) each comprising a first spatial tag (“BCb”) unique to the first bead. The first spatial tag may be unique to the first bead amongst a plurality of first beads immobilized on the substrate. A first oligonucleotide molecule 2703 of the plurality of first oligonucleotide molecules may comprise from a first end to a second end, an analyte capture sequence (“TTT.. ”), a first binding sequence (“PA26”), the first spatial tag (“BCb”), and a first bead adapter sequence. The first binding sequence may be configured to capture a second binding sequence, such as via complementarity. In some cases, the first bead adapter sequencer may comprise one or more cleavable moieties (“U”) thatmay be used as a release mechanism. A second bead (' A") 2704 can be loaded on the substrate, the second bead comprising a plurality of second oligonucleotide molecules (e.g., 2705) each comprising a second spatial tag (“BCa”) unique to the second bead. The second spatial tag may be unique to the second bead amongst a plurality of second beads loaded to the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may partially or completely overlap with a set of second spatial tags contained by a plurality of second beads loaded on the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may not overlap with a set of second spatial tags contained by a plurality of second beads loaded on the substrate. A second oligonucleotide molecule 2705 of the plurality of second oligonucleotide molecules may comprise from a first end to a second end, a second bead adapter sequence (“Pl”), the second spatial tag (“BCa”), and the second binding sequence (“PA26”’). The second binding sequence may comprise a portion that is complementary to the first binding sequence. In some cases, the second bead adapter sequencer may comprise one or more cleavable moieties (“U”) that may be used as a release mechanism. Upon contact of a first oligonucleotide molecule (e.g.. 2703) with a second oligonucleotide molecule (e.g.. 2705), the first binding sequence of the first oligonucleotide molecule may anneal to the second binding sequence of the second oligonucleotide molecule, and the second oligonucleotide molecule may be extended to generate composite molecule 2707 attached to the second bead (“A”). The composite molecule may comprise, from a first end to a second end. the second bead adapter sequence (“Pl”), the second spatial tag (“BCa”), the second binding sequence (“PA26”’), a complement of the first spatial tag (“BCb”’), and a complement of the first bead adapter sequence (“X”’).

[0214] Referring to panel (B), the plurality of second beads (e.g., “A”) and / or composite molecules (e.g., 2707) from the second beads may be removed from the substrate and collected. The composite molecules may be released from the plurality of second beads via any release mechanism described herein. The plurality of first beads (e.g., “B”) comprising the first oligonucleotide molecules may remain immobilized to the substrate 2701.

[0215] Referring to panel (C), a sample comprising a plurality of analyte sequences may be loaded on the substrate. The sample may retain, at least to some extent, a spatial relationship between the plurality' of analyte sequences. In some instances, a tissue slice is loaded onto the substrate, where the tissue slice retains, at least to some extent, a spatial relationship between the transcripts contained therein. Samples that can be used in the present methods, systems, compositions, and kits are described in further detail elsewhere herein. A sequence (e.g.,“AAA.. . ”) of an analyte sequence 2711 (“mRNA_AAA. . . ”) may anneal to the analyte capture sequence (e.g.. “TTT... ”) of the first oligonucleotide molecule, and the first oligonucleotide molecule may be extended to generate a tagged analyte molecule. A tagged analyte molecule may comprise, from a first end to a second end, a template sequence ("cDNA"). an analyte capture sequence (“TTT... ”), the first binding sequence (“PA26”), the second spatial tag (“BCa”), and the second bead adapter sequence (“Pl”). Where the analyte sequence is an mRNA sequence, the template sequence may correspond to a cDNA sequence. In some cases, the tagged analyte molecule may additionally comprise a template switching oligonucleotide (“TSO”) (e.g., polyC) sequence at the 3’ end as a product of a reverse transcription reaction, which template switching oligonucleotide sequence may be used for downstream template switching operations described elsewhere herein. In some cases, the tagged analyte molecule may comprise any alternative or additional functional sequence described elsewhere herein (e.g., barcode sequence, index sequence, UMI, adapter sequence, sequencer adapter sequence, primer sequence, etc.).

[0216] The oligonucleotide molecules on the first bead (“B”) may be released from the first bead. In some cases, the annealing between the analyte sequence and the first oligonucleotide molecule may occur prior to, during, or subsequent to release of the first oligonucleotide molecules from the first bead. An oligonucleotide may be released from a bead via any release mechanisms described elsewhere herein (e.g., USER cleavage). In some cases, the extension of the first oligonucleotide molecule may occur prior to. during, or subsequent to release of the first oligonucleotide molecules from the first bead. The analyte sequence may be removed (e.g., by melting, denaturing, etc.) from the tagged analyte molecule prior to, during, or subsequent to release of the first oligonucleotide molecule from the first bead.

[0217] The tagged analyte molecules derived from the first bead (“B”) and the composite molecules derived from the second bead (“A”) may be subsequently processed as described elsewhere herein with respect to other spatially tagged sequences. For example, the molecules may be subjected to library preparation, such as to attach one or more adapters, barcodes, such as to subject to amplification, etc., and sequencing to generate sequencing reads. Sequencing preparation and sequencing are described in further detail elsewhere herein. The sequencing data generated from sequencing may be processed and / or analyzed to generate a spatial map of the plurality of analyte sequences in the sample.

[0218] FIG. 30 illustrates another example bead-on-bead capture scheme. Referring to panel (A) of FIG. 30, a first bead (“B”) 3002 can be immobilized on a substrate 3001. The first beadmay comprise a plurality of first oligonucleotide molecules (e.g., 3003) (two illustrated) each comprising a first spatial tag ("BCb") unique to the first bead. The first spatial tag may be unique to the first bead amongst a plurality of first beads immobilized on the substrate. A first oligonucleotide molecule 3003 of the plurality of first oligonucleotide molecules may comprise from a first end to a second end, a first binding sequence (“CapB”), the first spatial tag (“BCb’'), and a first bead adapter sequence (“PB”). The first binding sequence may be configured to capture a second binding sequence, such as via complementarity. In some cases, the first bead adapter sequencer may comprise one or more cleavable moieties (“U”) that may be used as a release mechanism. A second bead (“A”) 3004 can be loaded on the substrate, the second bead comprising a plurality of second oligonucleotide molecules (e.g.. 3005) (one illustrated) each comprising a second spatial tag (“BCa”) unique to the second bead. The second spatial tag may be unique to the second bead amongst a plurality of second beads loaded to the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may partially or completely overlap with a set of second spatial tags contained by a plurality of second beads loaded on the substrate. In some cases, a set of first spatial tags contained by a plurality of first beads immobilized on the substrate may not overlap with a set of second spatial tags contained by a plurality7of second beads loaded on the substrate. A second oligonucleotide molecule 3005 of the plurality of second oligonucleotide molecules may comprise from a first end to a second end, the second binding sequence (“CapA’'), the second spatial tag (“BCa”), and a second bead adapter sequence (“PA”). The second binding sequence (CapA) may comprise a portion that is complementary to the first binding sequence (CapB). The second binding sequence (CapA) may comprise an analyte capture sequence, such as a poly-T sequence which is configured to capture the poly-A tail of mRNA analytes or other analyte capture sequences (e.g., targeted sequence, randomer sequence, or reverse complements thereof). Thus, the second binding sequence may be capable of capturing both the first binding sequence on the first set of beads and analytes of interest. Upon contact of a first oligonucleotide molecule (e.g., 3003) with a second oligonucleotide molecule (e.g., 3005), the first binding sequence of the first oligonucleotide molecule may anneal to the second binding sequence of the second oligonucleotide molecule, and the first oligonucleotide molecule extended to generate a first composite extended molecule. The first composite extended molecule may comprise, from a first end to a second end, a complement of the second bead adapter sequence (“PA””), a complement of the second spatial tag (“BCa”’), the first binding sequence (“CapB”). the first spatial tag (“BCb”). and the first bead adapter sequence(“PB”). Alternatively, or in addition, the second oligonucleotide molecule that is bound to the first oligonucleotide molecule may be extended to generate a second composite extended molecule, which comprises the second spatial tag and a complement of the first spatial tag. Either or both of the first and second composite extended molecules, or derivatives thereof, may be sequenced and the pair of spatial tag sequences in a composite extended molecule maybe informative of a spatial relationship and / or spatial map, as described elsewhere herein.

[0219] Referring to panel (B) of FIG. 30, after the first and second plurality of beads are loaded on the substrate and beads from each set are hybridized, a sample comprising a plurality of analyte sequences may be loaded on the substrate. The sample may retain, at least to some extent, a spatial relationship between the plurality of analyte sequences. In some instances, a tissue slice is loaded onto the substrate, where the tissue slice retains, at least to some extent, a spatial relationship between the transcripts contained therein. Samples that can be used in the present methods, systems, compositions, and kits are described in further detail elsewhere herein. A sequence (e.g., “AAA... ’') of an analyte sequence 3011 (“mRNA”) may anneal to the second binding sequence comprising the analyte capture sequence (e.g., “TTT... ”) of a second oligonucleotide molecule (e.g.. 3005), and the second oligonucleotide molecule may be extended to generate a tagged analyte molecule. A tagged analyte molecule may comprise, from a first end to a second end, a template sequence (e.g., “cDNA”), the second binding sequence (e.g., “TTT... ”), the second spatial tag (“BCa”), and the second bead adapter sequence (“PA”). Where the analyte sequence is an mRNA sequence, the template sequence may correspond to a cDNA sequence. In some cases, the tagged analyte molecule may additionally be processed with a template switching oligonucleotide (“TSO”) using an appended sequence (e.g., polyC sequence) that is a product of a reverse transcription reaction, which template switching oligonucleotide sequence may be used for downstream template switching operations described elsewhere herein.

[0220] In some cases, a method may comprise (a) loading a first plurality of beads on a substrate, (b) loading a second plurality of beads on the substrate, (c) annealing beads from the first plurality of beads with beads from the second plurality of beads under appropriate conditions and then washing the substrate, (d) loading a sample on the substrate, (e) capturing analytes in the sample by annealing them with the binding sequence on the oligonucleotide molecules of the second plurality of beads, and performing extension reactions. The extension reactions may comprise reverse transcription reactions. The method may further comprise, (f) after extension of the oligonucleotide molecules on the beads, removing the analyte sequences(e.g., denaturing, stripping, NaOH treatment, etc.) from the beads, (g) generating second strands complementary to the extended oligonucleotide molecules using solution primers, (h) removing and collecting the synthesized second strands, and (i) collecting barcoded molecules from either or both bead type for spatial mapping. In some cases, the second set of beads may be removed from the first set of beads and the barcoded oligonucleotide molecules collected. In some cases, the barcoded oligonucleotide molecules may be released from the beads (either or both types). Alternatively or in addition, a primer may bind to the barcoded oligonucleotide molecules on either or both types of beads and extended to generate reverse complement copies of the barcoded oligonucleotide molecules which may be removed (e.g., denaturing, stripping, NaOH treatment, etc.) from the beads and then collected.

[0221] Alternatively, the second set of beads and the sample may be contacted to the first set of beads substantially simultaneously. In some cases, extension reactions of oligonucleotide molecules (e.g., after hybridization capture between beads or between a bead and an analyte) may be initiated after each annealing event — for example, the first set of beads and second set of beads hybridize between each other and the oligonucleotide molecules are extended, and then the analytes hybridize to the second oligonucleotide molecules on the beads and the second oligonucleotide molecules are extended. In some cases, extension reactions of oligonucleotide molecules may be initiated such that all extension reactions occur after all annealing occurrences — for example, the first set of beads and second set of beads are hybridized between each other and the analytes are hybridized to the second oligonucleotide molecules on the beads, and then all extensions occur.

[0222] The annealing between the analyte sequence and the second binding sequence may occur prior to, during, or subsequent to release of the second oligonucleotide molecule from the second bead. Any oligonucleotide molecule of any bead (e.g., first bead or second bead), or derivative thereof (e.g., extension product comprising the template sequence), may be released from the bead via any release mechanism described herein. In one example, the molecule is released from the bead via USER cleavage of “U” cleavage sites in the bead adapter sequence. The extension of an oligonucleotide molecule may occur prior to, during, or subsequent to release of the oligonucleotide molecule from the bead. The analyte sequence may be removed (e.g., by melting, denaturing, etc.) from the tagged analyte molecule prior to, during, or subsequent to release of oligonucleotide molecule, or derivative thereof, from the bead.

[0223] The tagged analyte molecule, or derivative thereof (e.g., template switched derivative, copy, reverse complement copy, amplicon, etc.), may be subsequently processed as described elsewhere herein with respect to other spatially tagged sequences. For example, tagged analyte molecules may be subjected to library preparation, such as to attach one or more adapters, barcodes, such as to subject to amplification, etc., and sequencing to generate sequencing reads. Sequencing preparation and sequencing are described in further detail elsewhere herein. The sequencing data generated from sequencing may be processed and / or analyzed to generate a spatial map of the plurality of analyte sequences in the sample. In this example, sequencing data (e.g., reads from) a collection of first composite extended molecules (comprising a first spatial tag from the first set of beads and a second spatial tag from the second set of beads), or derivatives thereof, may be used to inform and / or generate a spatial or relative map of first spatial tags, a spatial or relative map of second spatial tags, or both. Sequencing data (e.g., reads from) the tagged analyte molecules (comprising a second spatial tag from the second set of beads and a template sequence), or derivatives thereof, may be used to position the tagged analyte molecules on the spatial or relative map generated, thus mapping the analytes relative to each other.

[0224] It will be appreciated that for each of these workflows, the substrate can be washed after each appropriate step and / or before a next step, such as to remove non-reacted or nonimmobilized elements from the reaction environment.

[0225] It will be appreciated that the analyte capture sequence, while in FIGs. 25- 30 is denoted as a poly-T sequence (e.g., TTT .) configured to capture a poly-A tail of the analyte sequence (e.g., an mRNA sequence) or areverse complement thereof (e.g., AAA..), may be any sequence configured to capture an analyte sequence. The analyte sequence, for example, may not have a poly-A tail. In some instances, the capture sequence may comprise a target sequence or a random sequence or any other sequence designed to capture an analyte sequence, or derivative thereof. The capture sequence may comprise a random n-mer sequence. In some examples, for targeted mRNA assays, the capture sequence may comprise a target mRNA sequence (or derivative thereof). In some examples, for targeted genomic DNA (gDNA) assays, the capture sequence may comprise a target gDNA sequence (or derivative thereof). In some examples, for antibody assays, the capture sequence may comprise a sequence configured to capture an oligonucleotide conjugated to one or more antibodies (e.g., DNA capture tags), or a derivative thereof. In some examples, for assays that utilize template switching reactions, the capture sequence may comprise a sequence configured to capture a product of a reverse transcriptionreaction, such as a polyG sequence. In some examples, for assays that utilize one or more probes, the capture sequence may comprise a sequence corresponding to a sequence of the probe, to a molecule associated with the probe, or derivative thereof. In some examples, the capture sequence may be part of a single strand portion, a double strand portion, or partially double-stranded complex. In some examples, the capture sequence may be part of a hybrid DNA / RNA complex. In some examples, for a transposition assay concerning gDNA analytes, after a transposition reaction (e.g.. subsequent to Tn5 transposase treatment of gDNA, where the Tn5 transposase comprises one or more barcode and / or adapter sequences), a partially double-stranded analyte may be generated. In some examples, wherein at least one end of the partially double-stranded analyte comprises an overhang comprising a barcode and / or adapter sequence. The capture sequence may be configured to capture the overhang of the partially double-stranded analyte comprising the barcode and / or adapter sequence.

[0226] In some cases, an analyte capture molecule (and / or second analyte capture molecule) may comprise a UMI. For example, with respect to FIG. 25, the capture sequence molecule 2509 (and / or second capture sequence molecule 2510) may comprise a UMI sequence, or complement thereof, such that when the extended molecule is extended with the capture sequence molecule, the analyte capture molecule comprises the UMI. It will be appreciated that the UMI sequence may be added to the analyte capture molecule via any other method, such as via a separate extension reaction (separate from the capture sequence molecule extension). It will be appreciated that any other functional sequence may be added to the analyte capture molecule via one or more extension and / or ligation reactions.

[0227] The sequencing data may be used to construct an origin-destination (OD) matrix. For example, the information flow may be unidirectional, with event counting as interaction strength. The sequencing data may be used to construct a spatial map of different bead locations, such as locations of first beads with respect to first beads, second beads, and / or both. The sequencing data may be used to construct a spatial map of different bead locations, such as locations of second beads with respect to first beads, second beads, and / or both. The sequencing data may be used to construct a spatial map of different analyte sequences, by mapping the analyte sequences to bead position. The spatial map may indicate a location or probability cloud of locations for one or more beads and / or one or more analyte sequences.Sample processing systems

[0228] Despite the prevalence of sample processing systems and methods, such systems and methods may have low efficiency that can be time-intensive and wasteful of valuable resources, such as reagents. For example, prior microfluidic systems have utilized substrates containing numerous long, narrow channels. The typical flow cell geometry7for such substrates introduces a need to compromise between two competing requirements: 1) minimizing volume to minimize reagent usage; and 2) maximizing effective hydraulic diameter to minimize flow time. This trade-off may be especially important for washing operations, which may require large wash volumes and thus long amounts of time to complete. The tradeoff is illustrated by the Poiseuille equation that dictates flow in the laminar regime and is thus inherent to microfluidic systems that utilize such flow cell geometries. Such flow cell geometries may also be susceptible to contamination. Because such flow cell geometries allow for a finite, limited number of channels in the microfluidic systems, such finite number of channels may be shared between a plurality of different mixtures comprising different analytes, reagents, agents, and / or buffers. Contents of fluids flowing through the same channels may be contaminated. Thus, recognized herein is a need for methods and systems for sample processing and / or analysis with high efficiency.

[0229] Described herein are devices, systems, and methods for processing analytes using open substrates or open flow cell geometries that can address at least the abovementioned problems. The devices, systems and methods may be used to facilitate any application or process involving a reaction or interaction between an analyte and a fluid (e.g., a fluid comprising reagents, agents, buffers, other analytes, etc.). Such reaction or interaction may be chemical (e.g., polymerase reaction) or physical (e.g., displacement). The systems and methods described herein may benefit from higher efficiency, such as from faster reagent delivery7and lower volumes of reagents required per surface area. The systems and methods described herein may avoid contamination problems common to microfluidic channel flow cells that are fed from multiport valves which can be a source of carryover from one reagent to the next. The devices, systems, and methods may benefit from shorter completion time, use of fewer resources (e.g., various reagents), and / or reduced system costs. The open substrates or flow cell geometries may be used to process any analyte from any sample, such as but not limited to, nucleic acid molecules, protein molecules, antibodies, antigens, cells, and / or organisms, as described herein. The open substrates or flow cell geometries may be used for any application or process, such as, but not limited to, sequencing by synthesis, sequencing by ligation,amplification, proteomics, single cell processing, barcoding, and sample preparation, as described herein.

[0230] A sample processing system may comprise a substrate, and devices and systems that perform one or more operations with or on the substrate, which permit highly efficient dispensing of reagents onto the substrate, and highly efficient imaging of one or more analytes, or signals corresponding thereto, on the substrate, among other operations. The substrate may be an open substrate. The substrate may be substantially planar. The substrate may be textured and / or patterned. In some cases, the texture and / or pattern can distinguish individually addressable locations as described elsewhere herein. The sample processing system may comprise an imaging system comprising a detector. Substrates and detectors that can be used in the sample processing system are described in further detail in U.S. Patent Pub. No. 2021 / 0079464, which is entirely incorporated herein by reference for all purposes.

[0231] The term “biological sample,’’ as used herein, generally refers to any sample from a subject or specimen. The biological sample can be a fluid or tissue from the subject or specimen. The fluid can be blood (e.g., whole blood), saliva, urine, or sweat. The tissue can be from an organ (e.g., liver, lung, or thyroid), or a mass of cellular material, such as. for example, atumor. The biological sample can be a feces sample, collection of cells (e.g., cheek swab), or hair sample. The biological sample can be a cell-free or cellular sample. Examples of biological samples include nucleic acid molecules, amino acids, polypeptides, proteins, carbohydrates, fats, or viruses. For example, a biological sample may be a nucleic acid sample including one or more nucleic acid molecules, such as deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). The nucleic acid molecules may be cell-free or cell-free nucleic acid molecules, such as cell free DNA or cell free RNA. The nucleic acid molecules may be derived from a variety of sources including human, mammal, non-human mammal, ape, monkey, chimpanzee, reptilian, amphibian, avian, or plant sources. Further, samples may be extracted from variety of animal fluids containing cell free sequences, including but not limited to blood, serum, plasma, vitreous, sputum, urine, tears, perspiration, saliva, semen, mucosal excretions, mucus, spinal fluid, amniotic fluid, lymph fluid and the like. Cell free polynucleotides may be fetal in origin (via fluid taken from a pregnant subject) or may be derived from tissue of the subject itself.

[0232] The term “subject,” as used herein, generally refers to an individual from whom a biological sample is obtained. The subject may be a mammal or non-mammal. The subject may be an animal, such as a monkey, dog. cat, bird, or rodent. The subject may be a human. Thesubject may be a patient. The subject may be displaying a symptom of a disease. The subject may be asymptomatic. The subject may be undergoing treatment. The subject may not be undergoing treatment. The subject can have or be suspected of having a disease, such as cancer (e.g., breast cancer, colorectal cancer, brain cancer, leukemia, lung cancer, skin cancer, liver cancer, pancreatic cancer, lymphoma, esophageal cancer or cervical cancer) or an infectious disease. The subject can have or be suspected of having a genetic disorder such as achondroplasia, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-tooth, cri du chat, Crohn's disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile x syndrome. Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency, sickle cell disease, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria. Turner syndrome, velocardiofacial syndrome, WAGR syndrome, or Wilson disease.

[0233] The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “nucleic acid fragment,” “oligonucleotide” and “polynucleotide,” as used herein, generally refer to a polynucleotide that may have various lengths, such as either deoxyribonucleotides or deoxyribonucleic acids (DNA) or ribonucleotides or ribonucleic acids (RNA), or analogs thereof. Non-limiting examples of nucleic acids include DNA, RNA, genomic DNA or synthetic DNA / RNA or coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes. cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, and isolated RNA of any sequence. A nucleic acid molecule can have a length of at least about 10 nucleic acid bases (“bases”), 20 bases, 30 bases, 40 bases. 50 bases, 100 bases. 200 bases. 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 1 megabase (Mb), or more. A nucleic acid molecule (e.g., polynucleotide) can comprise a sequence of four natural nucleotide bases: adenine (A); cytosine (C); guanine (G): and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). A nucleic acid molecule mayinclude one or more nonstandard nucleotide(s), nucleotide analog(s) and / or modified nucleotide(s).

[0234] The term '‘nucleotide,” as used herein, generally refers to any nucleotide or nucleotide analog. The nucleotide may be naturally occurring or non-naturally occurring. The nucleotide analog may be a modified, synthesized or engineered nucleotide. The nucleotide analog may not be naturally occurring or may include a non-canonical base. The naturally occurring nucleotide may include a canonical base. The nucleotide analog may include a modified polyphosphate chain (e.g., triphosphate coupled to a fluorophore). The nucleotide analog may comprise a label. The nucleotide analog may be terminated (e.g., reversibly terminated). The nucleotide analog may comprise an alternative base.

[0235] Nonstandard nucleotides, nucleotide analogs, and / or modified analogs may include, but are not limited to, diaminopurine, 5 -fluorouracil, 5 -bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2 -thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine,1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5’- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine. 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine, ethynyl nucleotide bases, 1-propynyl nucleotide bases, azido nucleotide bases, phosphoroselenoate nucleic acids and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Additional, non-limiting examples of modifications include phosphate chains of greater length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties), modifications with thiol moieties (e.g., alpha- thio triphosphate and beta- thiotriphosphates) or modifications with selenium moieties (e.g.. phosphoroselenoate nucleic acids). Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and / or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone. Nucleic acid molecules mayalso contain amine -modified groups, such as aminoallyl-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo- programmed polymerases, or lower secondary structure. Nucleotide analogs may be capable of reacting or bonding with detectable moieties for nucleotide detection.

[0236] The term “analyte” may refer to molecules, cells, biological particles, or organisms. In some instances, a molecule may be a nucleic acid molecule, antibody, antigen, peptide, protein, or other biological molecule obtained from or derived from a biological sample. An analyte may originate from, and / or be derived from, a sample, such as a biological sample, such as from a cell or organism. An analyte may be synthetic. An analyte may be a biological analyte. For instance, the biological analyte may be a macromolecule, e.g., a nucleic acid, a carbohydrate, a protein, a lipid, etc. The biological analyte may comprise multiple macromolecular groups, e.g.. glycoproteins, proteoglycans, ribozymes, liposomes, etc. The biological analyte may be an antibody, antibody fragment, or engineered variant thereof, an antigen, a cell, a peptide, a polypeptide, etc. In some cases, the biological analyte comprises a nucleic acid molecule. The nucleic acid molecule may comprise at least about 10, 100, 1000, 10.000. 100.000, 1,000.000, 10,000,000. 100,000.000, 1,000.000,000 or more nucleotides. Alternatively, or in addition, the nucleic acid molecule may comprise at most about 1,000,000,000, 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1000, 100, 10 or fewer nucleotides. The nucleic acid molecule may have a number of nucleotides that is within a range defined by any two of the preceding values. In some cases, the nucleic acid molecule may also comprise a common sequence, to which an N-mer may bind. An N-mer may comprise 1, 2, 3, 4, 5, or 6 nucleotides and may bind the common sequence. In some cases, the nucleic acid molecules may be amplified to produce a colony of nucleic acid molecules attached to the substrate or attached to beads that may associate with or be immobilized to the substrate. In some instances, the nucleic acid molecules may be attached to beads and subjected to a nucleic acid reaction, e g., amplification, to produce a clonal population of nucleic acid molecules attached to the beads.

[0237] The term “processing an analyte,” as used herein, generally refers to one or more stages of interaction with one more samples. Processing an analyte may comprise conductinga chemical reaction, biochemical reaction, enzymatic reaction, hybridization reaction, polymerization reaction, physical reaction, any other reaction, or a combination thereof with, in the presence of, or on, the analyte. Processing an analyte may comprise physical and / or chemical manipulation of the analyte. For example, processing an analyte may comprise detection of a chemical change or physical change, addition of or subtraction of material, atoms, or molecules, molecular confirmation, detection of the presence of a fluorescent label, detection of a Forster resonance energy transfer (FRET) interaction, or inference of absence of fluorescence.

[0238] The term “sequencing,’’ as used herein, generally refers to a process for generating or identifying a sequence of a biological molecule, such as a nucleic molecule. Such sequence may be a nucleic acid sequence, which may include a sequence of nucleic acid bases. Sequencing may be single molecule sequencing or sequencing by synthesis, for example. Sequencing may be performed using analyte nucleic acid molecules immobilized on a support, such as a flow cell or one or more beads. Sequencing may refer to sequencing methodologies that do not require the analyte nucleic acid molecules to be directly or indirectly attached to a support (e.g., nanopore sequencing or other pore-based sequencing, etc.). In some cases, sequencing may comprise generating sequencing signals and / or sequencing reads from the analyte nucleic acid molecules.

[0239] The terms “amplifying.” “amplification,” and “nucleic acid amplification” are used interchangeably and generally refer to generating one or more copies of a nucleic acid or a template. For example, “amplification” of DNA generally refers to generating one or more copies of a DNA molecule. Moreover, amplification of a nucleic acid may be linear, exponential, or a combination thereof. Amplification may be emulsion based or may be nonemulsion based. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction (PCR), ligase chain reaction (LCR), helicase-dependent amplification, asymmetric amplification, rolling circle amplification (RCA), recombinase polymerase reaction (RPA), loop mediated isothermal amplification (LAMP), nucleic acid sequence based amplification (NASBA), self-sustained sequence replication (3SR), and multiple displacement amplification (MDA). Where PCR is used, any form of PCR may be used, with non-limiting examples that include real-time PCR, allelespecific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR. nested PCR. overlap-extension PCR. thermal asymmetricinterlaced PCR and touchdown PCR. Moreover, amplification can be conducted in a reaction mixture comprising various components (e.g., a primer(s). template, nucleotides, a polymerase, buffer components, co-factors, etc.) that participate or facilitate amplification. In some cases, the reaction mixture comprises a buffer that permits context independent incorporation of nucleotides. Non-limiting examples include magnesium-ion, manganese-ion and isocitrate buffers. Additional examples of such buffers are described in Tabor. S. et al. C.C. PNAS, 1989, 86. 4076-4080 and U.S. Patent Nos. 5.409,811 and 5,674,716. each of which is herein incorporated by reference in its entirety.

[0240] Useful methods for clonal amplification from single molecules include rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), which is incorporated herein by reference), bridge PCR (Adams and Kron, Method for Performing Amplification of Nucleic Acid with Two Primers Bound to a Single Solid Support, Mosaic Technologies, Inc. (Winter Hill, Mass.); Whitehead Institute for Biomedical Research, Cambridge, Mass., (1997); Adessi et al., Nucl. Acids Res. 28:E87 (2000); Pemov et al., Nucl. Acids Res. 33:el 1(2005); or U.S. Pat. No. 5.641,658, each of which is incorporated herein by reference), polony generation (Mitra et al., Proc. Natl. Acad. Sci. USA 100:5926-5931 (2003); Mitra et al., Anal. Biochem. 320:55-65(2003), each of which is incorporated herein by reference), and clonal amplification on beads using emulsions (Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817- 8822 (2003). which is incorporated herein by reference) or ligation to bead-based adapter libraries (Brenner et al., Nat. Biotechnol. 18:630-634 (2000); Brenner et al., Proc. Natl. Acad. Sci. USA 97: 1665-1670 (2000)); Reinartz, et al., Brief Funct. Genomic Proteomic 1 :95-104 (2002), each of which is incorporated herein by reference).

[0241] The terms "dispense" and “disperse’' may be used interchangeably herein. In some cases, dispensing may comprise dispersing and / or dispersing may comprise dispensing. Dispensing generally refers to distributing, depositing, providing, or supplying a reagent, solution, or other object, etc. Dispensing may comprise dispersing, which may generally refer to spreading.

[0242] The term “detector,” as used herein, generally refers to a device that is capable of detecting a signal, including a signal indicative of the presence or absence of one or more incorporated nucleotides or fluorescent labels. The detector may detect multiple signals. The signal or multiple signals may be detected in real-time during, substantially during a biological reaction, such as a sequencing reaction (e g., sequencing during a primer extension reaction), or subsequent to a biological reaction. In some cases, a detector can include optical and / orelectronic components that can detect signals. The term “detector"’ may be used in detection methods. Non-limiting examples of detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, acoustic detection, magnetic detection, and the like. Optical detection methods include, but are not limited to, light absorption, ultraviolet-visible (UV-vis) light absorption, infrared light absorption, light scattering, Rayleigh scattering, Raman scattering, surface-enhanced Raman scattering, Mie scattering, fluorescence, luminescence, and phosphorescence. Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy. Electrostatic detection methods include, but are not limited to, gel-based techniques, such as, for example, gel electrophoresis. Electrochemical detection methods include, but are not limited to, electrochemical detection of amplified product after high-performance liquid chromatography separation of the amplified products. A detector may be a continuous area scanning detector. For example, the detector may comprise an imaging array sensor capable of continuous integration over a scanning area wherein the scanning is electronically synchronized to the image of an object in relative motion. A continuous area scanning detector may comprise a time delay and integration (TDI) charge coupled device (CCD), Hybrid TDI, or complementary metal oxide semiconductor (CMOS) pseudo TDI device. For example, a continuous area scanning detector may comprise a TDI line-scan camera.

[0243] The term “open substrate,” as used herein, generally refers to a substrate in which any point on an active surface of the substrate is physically accessible from a direction normal to the substrate.

[0244] The systems and methods may utilize a substrate comprising a plurality of individually addressable locations. The plurality’ of individually addressable locations may be arranged as an array on the substrate. The plurality of individually addressable locations may be otherwise arranged, such as randomly or in any order, on the substrate. Each of the plurality of individually addressable locations, or each of a subset of such locations, may be capable of immobilizing thereto an analyte (e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.) or a reagent (e.g.. a nucleic acid molecule, a probe molecule, a barcode molecule, an antibody molecule, a primer molecule, a bead, etc.). For example, an analyte or reagent may be immobilized to an individually addressable location via a support, such as a bead. In some instances, a bead is immobilized to the individually addressable location, and the analyte or reagent is immobilized to the bead. In some cases, an individuallyaddressable location may immobilize thereto a plurality of analytes or a plurality of reagents. The plurality of analytes may be copies of a template analyte. For example, the plurality’ of analytes may have sequence homology or sequence identity. For example, the plurality- of analytes may be a clonal amplification colony. In other instances, the plurality of analytes may be different (e.g., comprise different sequences). In some examples, the plurality- of analytes is immobilized to the individually addressable location via a support, such as a bead. In some examples, a bead comprises a plurality of amplification products, as analytes, immobilized thereto, and the bead is immobilized to an individually addressable location on the substrate. In another example, the bead is immobilized to an individually addressable location on the substrate and is configured to capture or bind to a plurality of analytes. In another example, a plurality of reagents is immobilized to an individually addressable location on the substrate via a support, such as a bead. The plurality- of reagents may be configured for capturing or binding an analyte or another reagent. The plurality of reagents may be configured for release from the bead. The plurality- of reagents bound to the bead may be releasable prior to, during, or subsequent to capturing or binding, or otherwise interacting with, an analyte or another reagent. The substrate may immobilize a plurality of analytes or reagents across multiple individually addressable locations. The plurality of analytes or reagents may- be of the same ty pe of analyte or reagent (e.g., a nucleic acid molecule) or may be a combination of different types of analytes or reagents (e.g., nucleic acid molecules, protein molecules, etc.).

[0245] One or more surfaces of the substrate may be exposed to a surrounding open environment, and accessible from such surrounding open environment. For example, the array may be exposed and accessible from such surrounding open environment. In some cases, as described elsewhere herein, the surrounding open environment may be controlled and / or confined in a larger controlled environment.

[0246] Reagents may be dispensed to the substrate to multiple locations, and / or multiple reagents may be dispensed to the substrate to a single location, via different mechanisms. Reagent dispensing mechanisms disclosed herein may be applicable to sample dispensing. For example, a reagent may comprise the sample. The term ‘“loading onto a substrate,” as used in reference to a reagent or a sample herein, may refer to dispensing of the reagent or the sample to a surface of the substrate in accordance with any reagent dispensing mechanism described herein. In some cases, dispensing may be achieved via relative motion of the substrate and the dispenser (e.g., nozzle). For example, a reagent may be dispensed to the substrate at a first location, and thereafter travel to a second location different from the first location due to forces(e.g., centrifugal forces, centripetal forces, inertial forces, etc.) caused by motion of the substrate (e.g., rotational motion of the substrate, linear motion of the substrate, combination thereof, etc.). In another example, a reagent may be dispensed to a reference location, and the substrate may be moved relative to the reference location such that the reagent is dispensed to multiple locations of the substrate. In another example, a dispenser may be moved relative to the substrate to dispense the reagent at different locations, for example moved prior to, during, or subsequent to dispensing. In some instances, a reagent is ‘painted’ onto the substrate by moving the dispenser and / or the substrate relative to each other, along a desired path on the substrate. The open substrate geometry may allow for flexible and controlled dispensing of a reagent to a desired location on the substrate. In some cases, dispensing may be achieved without relative motion between the substrate and the dispenser. For example, multiple dispensers may be used to dispense reagents to different locations, and / or multiple reagents to a single location, or a combination thereof (e.g., multiple reagents to multiple locations). In some instances, an external force (e.g., involving a pressure differential, involving physical force, involving a magnetic force, involving an electrical force, etc.), such as wind, a fieldgenerating device, or a physical device, may be applied to one or more surfaces of the substrate to direct reagents to different locations across the substrate. In some instances, the method for dispensing reagents may comprise vibration. In some such instances, reagents may be distributed or dispensed onto a single region or multiple regions of the substrate (or a surface of the substrate). The substrate (or a surface thereof) may then be subjected to vibration, which may spread the reagent to different locations across the substrate (or the surface). Alternatively, or in conjunction, the method may comprise using mechanical, electric, physical, or other mechanisms to dispense reagents to the substrate. For example, the solution may be dispensed onto a substrate and a physical scraper (e.g., a squeegee) may be used to spread the dispensed material or spread the reagents to different locations and / or to obtain a desired thickness or uniformity across the substrate. Beneficially, such flexible dispensing may be achieved without contamination of the reagents. In some instances, where a volume of reagent is dispensed to the substrate at a first location, and thereafter travels to a second location different from the first location, the volume of reagent may travel in a path or paths, such that the travel path or paths are coated with the reagent. In some cases, such travel path or paths may encompass a desired surface area (e.g., entire surface area, partial surface area(s), etc.) of the substrate. In some instances, two or more reagents may be mixed on the surface of the substrate, such as by being dispensed at the same location and / or by directing a first reagent to travel to meetadditional reagent(s). In some instances, the mixture of reagents formed on the substrate may be homogenous or substantially homogenous. The mixture of reagents may be formed at a first location on the substrate prior to dispersing the mixing of reagents to other locations on the substrate, such as at locations to meet other reagents or analytes.

[0247] In some cases, the substrate may be rotatable about an axis. Analytes or reagents may be immobilized to the substrate during rotation. During one or more downstream processing operations, reagents may be dispensed onto the substrate prior to or during rotation of the substrate. When the substrate is rotated at a relatively high rotational velocity, high speed coating across the substrate may be achieved via tangential inertia directing unconstrained spinning reagents in a partially radial direction (that is, away from the axis of rotation) during rotation, a phenomenon commonly referred to as centrifugal force. This mode of directing reagents across a substrate may be herein referred to as centrifugal or inertial pumping. In some cases, the substrate may be rotated at relatively low velocities such that reagents dispensed to a certain location do not move to another location, or moves minimally, because of the rotation, to permit controlled dispensing of reagents to desired locations. Reagents dispensed on the substrate may or may not interact with analytes immobilized on the substrate. For example, when the analytes are nucleic acid molecules and when the reagents comprise nucleotides, the nucleic acid molecules may incorporate or otherwise react with one or more nucleotides. In another example, when the analytes are protein molecules and when the reagents comprise antibodies, the protein molecules may bind to or otherwise react with one or more antibodies. In another example, when the reagents comprise washing reagents, the substrate (and / or analytes on the substrate) may be washed of any unreacted (and / or unbound) reagents, agents, buffers, and / or other particles.

[0248] In some cases, the substrate may be movable in any vector or direction, as described elsewhere herein. For example, such motion may be non-linear (e.g., in rotation about an axis). In another example, such motion may be linear. In other examples, the motion may be a hybrid of linear and non-linear motion. The analytes may be immobilized to the substrate during any such motion. Reagents may be dispensed onto the substrate prior to, during, or subsequent to motion of the substrate. In some cases, inertial forces may direct unconstrained reagents across the substrate in any direction during any type of motion (e.g., rotational motion, non-rotational motion, linear motion, non-linear motion, accelerated motion, etc.) of the substrate.

[0249] One or more signals (such as optical signals) may be detected from a detection area on the substrate prior to, during, or subsequent to, the dispensing of reagents to generate anoutput. For example, the output may be an intermediate or final result obtained from processing of the analyte. Signals may be detected in multiple instances. The dispensing, rotating (or other motion), and / or detecting operations, in any order (independently or simultaneously), may be repeated any number of times to process an analyte. In some instances, the substrate may be washed (e.g., via dispensing washing reagents) between consecutive dispensing of the reagents. One or more detection operations can be performed within a desired time frame. For example, the detection operation can be performed within about 1 minute. 50 seconds, 40 seconds. 30 seconds, 20 seconds, 10 seconds or less than 10 seconds. In some instances, at least two detection operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds etc. In some instances, at least three detection operations can be performed within 1 minute, 50 seconds. 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds.

[0250] One or more dispensing operations can be performed w ithin a desired time frame. For example, the dispensing operation can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds. 10 seconds or less than 10 seconds. In some instances, at least two dispensing operations can be performed within 1 minute, 50 seconds, 40 seconds. 30 seconds, 20 seconds, 10 seconds or less than 10 seconds etc. In some instances, at least three dispensing operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds. Any operation or process of one or more methods disclosed herein may be performed within a desired time frame. In some instances, a combination of two or more operations or processes disclosed herein may be performed within a desired time frame. For example, the dispensing operation and the detection method may both be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds. 20 seconds, 10 seconds or less than 10 seconds. In some instances, at least two dispensing and detection operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds etc. In some instances, at least three dispensing and detection operations can be performed within 1 minute, 50 seconds, 40 seconds, 30 seconds, 20 seconds, 10 seconds or less than 10 seconds.

[0251] Systems and methods disclosed herein may obviate the need for barcoding of analytes (e.g., nucleic acid molecules), which may be time-consuming and expensive. For example, alternative or in addition to barcoding, the substrate and / or array of individually addressable locations may be spatially indexed to identify the analytes. Systems and methods disclosed herein may obviate the need for unique barcoding of individual analytes (e.g.,individual nucleic acid molecules) or individual samples. Systems and methods for spatial indexing are described in further detail in U.S. Patent Pub. No. 2021 / 0079464. which is entirely incorporated herein by reference for all purposes.

[0252] A substrate may be a solid substrate. The substrate may entirely or partially comprise one or more of rubber, glass, silicon, a metal such as aluminum, copper, titanium, chromium, or steel, a ceramic such as titanium oxide or silicon nitride, a plastic such as polyethylene (PE), low-density polyethylene (LDPE), high-density polyethylene (HDPE). polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamides, polycarbonates, polyesters, polyurethanes, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea-formaldehyde (UF), polyetheretherketone (PEEK), polyetherimide (PEI), polyimides, polylactic acid (PLA), furans, silicones, polysulfones, any mixture of any of the preceding materials, or any other appropriate material. The substrate may be entirely or partially coated with one or more layers of a metal such as aluminum, copper, silver, or gold, an oxide such as a silicon oxide (SixOy, where x, y may take on any possible values), a photoresist such as SU8, a surface coating such as an aminosilane or hydrogel, polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the preceding materials, or any other appropriate coating. A substrate may be fully or partially opaque to visible light. In some cases, a substrate may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% opaque to visible light. The substrate may have an opacity that is within a range defined by any two of the preceding values. A substrate may be fully or partially transparent to visible light. In some cases, a substrate may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% transparent to visible light. The substrate may have a transparency that is within a range defined by any two of the preceding values. In some cases, an illumination power (e.g., a laser power), during detection of a detection area of the substrate, may be adjusted based on the opacity or transparency of the substrate. The one or more layers of the substrate may have a thickness of at least 1 nanometer (nm), 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, Imicrometer (pm), 2 pm, 5 pm, 10 pm, 20 pm, 50 pm, 100 pm, 200 pm, 500 pm, or 1 millimeter (mm). The one or more layers may have a thickness that is within a range defined by any two of the preceding values. A surface of the substrate may bemodified to comprise any of the binders or linkers described herein. A surface of the substrate may be modified to comprise active chemical groups, such as amines, esters, hydroxyls, epoxides, and the like, or a combination thereof. In some instances, such binders, linkers, active chemical groups, and the like may be added as an additional layer or coating to the substrate.

[0253] The substrate may have the general form of a cylinder, a cylindrical shell or disk, a rectangular prism, or any other geometric form. The substrate may have a thickness (e.g., a minimum dimension) of at least 100 pm. 200 pm, 500 pm, 1 mm. 2 mm, 5 mm. or 10 mm. The substrate may have a thickness that is within a range defined by any two of the preceding values. The substrate may have a first lateral dimension (such as a width for a substrate having the general form of a rectangular prism or a radius for a substrate having the general form of a cylinder) of at least 1 mm. 2 mm, 5 mm, 10 mm, 20 mm, 50 mm. 100 mm. 200 mm. 500 mm, or 1,000 mm. The substrate may have a first lateral dimension that is within a range defined by any two of the preceding values. The substrate may have a second lateral dimension (such as a length for a substrate having the general form of a rectangular prism) or at least 1 mm. 2 mm, 5 mm, 10 mm, 20 mm, 50 mm, 100 mm, 200 mm, 500 mm, or 1,000 mm. The substrate may have a second lateral dimension that is within a range defined by any two of the preceding values.

[0254] A surface of the substrate may be planar. The surface of the substrate may be substantially planar. Substantially planar may refer to planarity at a micrometer level (e.g., a range of unevenness on the planar surface does not exceed the micrometer scale) or nanometer level (e.g., a range of unevenness on the planar surface does not exceed the nanometer scale). Alternatively, substantially planar may refer to planarity at less than a nanometer level or greater than a micrometer level (e.g., millimeter level). A surface of the substrate may be uncovered and may be exposed to an atmosphere. Alternatively, or in addition, a surface of the substrate may be textured or patterned. For example, the substrate may comprise grooves, troughs, hills, and / or pillars. The substrate may define one or more cavities (e.g., micro-scale cavities or nano-scale cavities). The substrate may define one or more channels. The substrate may have regular textures and / or patterns across the surface of the substrate. For example, the substrate may have regular geometric structures (e.g., wedges, cuboids, cylinders, spheroids, hemispheres, etc.) above or below a reference level of the surface. Alternatively, the substrate may have irregular textures and / or patterns across the surface of the substrate. For example, the substrate may have any arbitrary structure above or below a reference level of the substrate. In some instances, a texture of the substrate may comprise structures having a maximumdimension of at most about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%. 3%, 2%. 1%, 0.1%, 0.01%, 0.001%, 0.0001%. 0.00001% of the total thickness of the substrate or a layer of the substrate. In some instances, the textures and / or patterns of the substrate may define at least part of an individually addressable location on the substrate. A textured and / or patterned substrate may be substantially planar. FIGs. 14A-14G illustrate different examples of cross-sectional surface profiles of a substrate. FIG. 14A illustrates a cross-sectional surface profile of a substrate having a completely planar surface. FIG. 14B illustrates a cross-sectional surface profile of a substrate having semi-spherical troughs or grooves. FIG. 14C illustrates a cross-sectional surface profile of a substrate having pillars, or alternatively or in conjunction, wells. FIG. 14D illustrates a cross-sectional surface profile of a substrate having a coating. FIG. 14E illustrates a cross-sectional surface profile of a substrate having spherical particles. FIG. 14F illustrates a cross-sectional surface profile of FIG. 14B, with a first type of binders seeded or associated with the respective grooves. FIG. 14G illustrates a cross-sectional surface profile of FIG. 14B, with a second type of binders seeded or associated with the respective grooves.

[0255] The substrate may compnse an array. For instance, the array may be located on a lateral surface of the substrate. The array may be a planar array. The array may have the general shape of a circle, annulus, rectangle, or any other shape. The array may comprise linear and / or non-linear rows. The array may be evenly spaced or distributed. The array may be arbitrarily spaced or distributed. The array may have regular spacing. The array may have irregular spacing. The array may be a textured array. The array may be a patterned array. The array may comprise a plurality of individually addressable locations. The individually addressable locations may be arranged in any convenient pattern. For example, the individually addressable locations may be randomly oriented on the array. The plurality of individually addressable locations may form separate radial regions around a disk-shaped substrate. The plurality' of individually addressable locations may form a square, rectangle, disc, circular, annulus, pentagonal, hexagonal, heptagonal, octagonal, array, or any other pattern. One or more types of individually addressable locations may be generated. The types of individually addressable locations may be arrayed in any useful pattern, such as a square, rectangle, disc, annulus, pentagon, hexagon, radial pattern, etc. In some cases, the two types of individually addressable locations may have different chemical, physical, and / or biological properties (e.g., hydrophobicity, charge, color, topography, size, dimensions, geometry, etc.). For example, a first type of individually addressable location may bind a first type of biological analyte butnot a second type of biological analyte, and a second type of individually addressable location may bind the second ty pe of biological analyte but not the first type of biological analyte.

[0256] The analyte to be processed may be immobilized to the array. The array may comprise one or more binders described herein, such as one or more physical or chemical linkers or adaptors, which are coupled to a biological analyte. For instance, the array may comprise a linker or adaptor that is coupled to a nucleic acid molecule. Alternatively, or in addition, the biological analyte may be coupled to a bead, which bead may be immobilized to the array. In some cases, a subset of the array may not be coupled to a sample or analyte. In some cases, the array may be coupled to a sample or an analyte, but not all of the array may be processed. For example, the substrate may be coupled to a sample or analyte (e.g., comprising nucleic acid molecules), but the region of the array that is in proximity to the border of the array may not be subjected to further processing (e.g., detection). Similarly, other reagents may be immobilized to the array.

[0257] The individually addressable locations may comprise locations of analytes or groups of analytes that are accessible for manipulation. The manipulation may comprise placement, extraction, reagent dispensing, seeding, heating, cooling, or agitation. The manipulation may be accomplished through, for example, localized microfluidic, pipet, optical, laser, acoustic, magnetic, and / or electromagnetic interactions with the analyte or its surroundings.

[0258] In some cases, the individually addressable locations may be indexed, e.g., spatially, such that the analyte immobilized or coupled to each individually addressable location may be identified from a plurality of analytes immobilized to other individually addressable locations. For example, data corresponding to an indexed location, collected over multiple periods of time, may be linked to the same indexed location. In some cases, sequencing signal data collected from an indexed location, during iterations of sequencing-by-synthesis flows, are linked to the indexed location to generate a sequencing read for an analyte immobilized at the indexed location. In some embodiments, the individually addressable locations are indexed by demarcating part of the substrate. In some embodiments, the surface of the substrate is demarcated using etching. In some embodiments, the surface of the substrate is demarcated using a notch in the surface. In some embodiments, the surface of the substrate is demarcated using a dye or ink. In some embodiments, the surface of the substrate is demarcated by depositing a topographical mark on the surface. In some embodiments, a sample, such as a control nucleic acid sample, may be used to demarcate the surface of thesubstrate. As will be appreciated, a combination of positive demarcations and negative demarcations (lack thereof) may be used to index the individually addressable locations. In some embodiments, one or more reference objects (e.g., a reference bead that always emits a detectable signal during detection) are immobilized to any location(s) on the substrate, and the individually addressable locations are indexed with reference to the reference object. In some instances, a single reference point or axis (e.g., single demarcation) may be used to index all individually addressable locations. In some embodiments, each of the individually addressable locations is indexed. In some embodiments, a subset of the individually addressable locations is indexed. In some embodiments, the individually addressable locations are not indexed, and a different region of the substrate is indexed.

[0259] In some cases, an individually addressable location may comprise a distinct surface chemistry. The distinct surface chemistry may distinguish between different addressable locations. The distinct surface chemi stry may distinguish between different regions on the substrate. For example, a first location has a first affinity towards an object (e.g., a bead comprising nucleic acid molecules, e.g., amplicons, immobilized thereto) and a second location has a second, different affinity towards the object due to the distinct surface chemistries. The first location and the second location may or may not be located in the same region. The first location and the second location may or may not be disposed on the surface in alternating fashion. In another example, a first region (e.g., comprising a plurality of individually addressable locations) has a first affinity towards an object and a second region has a second, different affinity towards the object due to the distinct surface chemistries. A first location type or region t pe may comprise a first surface chemistry, and a second location type or region type may comprise a second surface chemi stry. In some cases, a third location type or region type may comprise a third surface chemistry. For example, a first location type or region type may comprise a positively charged surface chemistry and / or a hydrophobic surface chemistry, and a second location type or region type may comprise a negatively charged surface chemistry and / or a hydrophilic surface chemistry, as shown in FIG. 15A. The same object (e.g., a bead comprising nucleic acid molecules, e.g., amplicons, immobilized thereto) may have higher affinity towards a first location type or region type compared to a second location type or region type. The same object may be attracted towards a first location type or region type and repelled from a second location type or region type. In other examples, a first location type or region type comprising a first surface chemistry (e.g., a positively charged surface chemistry or a negatively charged surface chemistry) may interact with (e.g., have an affinity towards) a firstsample ty pe (e.g., a bead comprising nucleic acid molecules, e.g., amplicons, immobilized thereto) and exclude a second sample type (e.g., a bead lacking nucleic acid molecules, e.g., amplicons, immobilized thereto, e.g., entirely or in substantial volume), for example as illustrated in FIG. 15B. In some cases, a surface chemistry' may comprise an amine. In some cases, a surface chemistry may comprise a silane (e.g., tetramethylsilane). In some cases, the surface chemistry may comprise hexamethyldisilazane (HMDS). In some cases, the surface chemistry may comprise (3-aminopropyl)tri ethoxysilane (APTMS). In some cases, the surface chemistry may comprise a surface primer molecule or any oligonucleotide molecule that has any degree of affinity7towards another molecule.

[0260] Each individually addressable location may have the general shape or form of a circle, pit, bump, rectangle, or any other shape or form. An individually addressable location of a plurality of locations (e.g., alternating locations) may have an area. In some cases, a location may have an area of about 0.1 square micron (pm2), 0.2 pm2, 0.25 pm2, 0.3 pm2, 0.4 pm2, 0.5 pm2, 0.6 pm2, 0.7 pm2, 0.8 pm2, 0.9 pm2, 1 pm2, 1.1 pm2, 1.2 pm2, 1.25 pm2. 1.3 pm2, 1.4 pm2, 1.5 pm2, 1.6 pm2, 1.7 pm2, 1.75 pm2, 1.8 pm2, 1.9 pm2, 2 pm2, 2.25 pm2, 2.5 pm2, 2.75 pm2, 3 pm2, 3.25 pm2, 3.5 pm2, 3.75 pm2, 4 pm2, 4.25 pm2, 4.5 pm2, 4.75 pm2, 5 pm2, 5.5 pm2, or 6 pm2. A location may have an area that is yvithin a range defined by any two of the preceding values. A location may have an area that is less than about 0. 1 pm2or greater than about 6 pm2. In some cases, a location may have a width of about 0.1 micron (pm), 0.2 pm, 0.25 pm, 0.3 pm. 0.4 pm, 0.5 pm, 0.6 pm, 0.7 pm. 0.8 pm, 0.9 pm, 1 pm, 1.1 pm. 1.2 pm, 1.25 pm, 1.3 pm, 1.4 pm, 1.5 pm, 1.6 pm, 1.7 pm, 1.75 pm, 1.8 pm, 1.9 pm, 2 pm, 2.25 pm, 2.5 pm, 2.75 pm, 3 pm, 3.25 pm, 3.5 pm, 3.75 pm, 4 pm, 4.25 pm, 4.5 pm, 4.75 pm, 5 pm, 5.5 pm, or 6 pm. In some cases, a location may have a width that is within a range defined by any tyvo of the preceding values. A location may have a width that is less than about 0. 1 pm or greater than about 6 pm. Each individually addressable location may have a first lateral dimension (such as a radius for individually addressable locations having the general shape of a circle or a width for individually addressable locations having the general shape of a rectangle). In some cases, a first lateral dimension of a location may be at least 1 nanometer (nm). 2 nm, 5 nm, 10 nm, 20 nm, 50 nm. 100 nm, 200 nm. 500 nm, 1.000 nm. 2,000 nm, 5.000 nm, or 10,000 nm. The first lateral dimension may be within a range defined by any two of the preceding values. Each individually addressable location may have a second lateral dimension (such as a length for individually addressable locations having the general shape of a rectangle). The second lateral dimension may be at least 1 nanometer (nm). 2 nm, 5 nm, 10 nm, 20 nm, 50nm, 100 nm, 200 nm, 500 nm, 1,000 nm, 2,000 nm, 5,000 nm, or 10,000 nm. The second lateral dimension may be within a range defined by any two of the preceding values.

[0261] In some cases, the locations (e.g., of a same type) may be distributed on a substrate with a pitch determined by the distance between the center of a first location and the center of the closest or neighboring location (e g., of the same ty pe). Locations may be spaced with a pitch of about 0.1 micron (pm), 0.2 pm, 0.25 pm, 0.3 pm, 0.4 pm. 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm. 1.1 pm, 1.2 pm. 1.25 pm. 1.3 pm, 1.4 pm. 1.5 pm, 1.6 pm, 1.7 pm. 1.75 pm, 1.8 pm, 1.9 pm, 2 pm, 2.25 pm, 2.5 pm, 2.75 pm, 3 pm, 3.25 pm, 3.5 pm, 3.75 pm, 4 pm, 4.25 pm, 4.5 pm, 4.75 pm, 5 pm, 5.5 pm, 6 pm, 6.5 pm, 7 pm, 7.5 pm, 8 pm, 8.5 pm, 9 pm, 9.5 pm, or 10 pm. In some case the locations may be positioned with a pitch that is within a range defined by any two of the preceding values. The locations may be positioned with a pitch of less than about 0. 1 pm or greater than about 10 pm. In some cases, the pitch between any two locations of the same type may be determined as a function of a size of a loading object (e.g., bead). For example, where the loading object is a bead having a maximum diameter, the pitch may be at least about the maximum diameter of the loading object.

[0262] Indexing may be performed using a detection method and may be performed at any convenient or useful step. A substrate that is indexed, e.g., demarcated, may be subjected to detection, such as optical imaging, to locate the indexed locations, individually addressable locations, and / or the biological analyte. Imaging may be performed using a detection unit. Imaging may be performed using one or more sensors. Imaging may not be performed using the naked eye. The substrate that is indexed may be imaged prior to loading of the biological analyte. Following loading of the biological analyte onto the individually addressable locations, the substrate may be imaged again, e.g. to determine occupancy or to determine the positioning of the biological analyte relative to the substrate. In some cases, the substrate may be imaged after iterative cycles of nucleotide addition (or other probe or other reagent), as described elsewhere herein. The indexing of the substrate and known initial position (individually addressable location) of the biological analyte may allow for analysis and identification of the sequence information for each individually addressable location and / or position. Additionally, spatial indexing may allow for identification of errors that may occur, e.g., sample contamination, sample loss, etc.

[0263] The array may be coated with binders. For instance, the array may be randomly coated with binders. Alternatively, the array may be coated with binders arranged in a regular pattern (e.g., in linear arrays, radial arrays, hexagonal arrays etc.). The array may be coatedwith binders on at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%, 98%. or 99% of the number of individually addressable locations, or of the surface area of the substrate. The array may be coated with binders on a fraction of individually addressable locations, or of the surface areas of the substrate, which is within a range defined by any two of the preceding values. The binders may be integral to the array. The binders may be added to the array. For instance, the binders may be added to the array as one or more coating layers on the array.

[0264] The binders may be configured to immobilize analytes or reagents, such as through non-specific interactions, such as one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, physical interactions (for instance, adhesion to pillars or settling within wells), and the like. The binders may immobilize analytes or reagents through specific interactions. For instance, where the analyte or reagent is a nucleic acid molecule, the binders may comprise oligonucleotide adaptors configured to bind to the nucleic acid molecule. Alternatively, or in addition, such as to bind other types of analytes or reagents, the binders may comprise one or more of antibodies, oligonucleotides, nucleic acid molecules, aptamers, affinity binding proteins, hpids, carbohydrates, and the like. The binders may immobilize analytes or reagents through any possible combination of interactions. For instance, the binders may immobilize nucleic acid molecules through a combination of physical and chemical interactions, through a combination of protein and nucleic acid interactions, etc. The array may comprise an order of magnitude of at least about 10. 100, 103. 104, 105, 106. 107, 108, 109. 1010, 1011, or more binders. Alternatively, or in addition, the array may comprise an order of magnitude of at most about 1011, 1010, 109, 108, 107, 106, 105, 104, 103, 100, 10 or fewer binders. The array may have a number of binders that is within a range defined by any two of the preceding values. In some instances, a single binder may bind a single analyte (e.g., nucleic acid molecule) or single reagent. In some instances, a single binder may bind a plurality of analytes (e.g., plurality of nucleic acid molecules) or a plurality of reagents. In some instances, a plurality7of binders may bind a single analyte or a single reagent. Though examples herein describe interactions of binders with nucleic acid molecules, the binders may immobilize other molecules (such as proteins), other particles, cells, viruses, other organisms, or the like. Though examples herein describe interactions of binders with samples or analytes, the binders may similarly immobilize reagents.

[0265] In some instances, each location, or a subset of such locations, may have immobilized thereto an analyte (e.g.. a nucleic acid molecule, a protein molecule, acarbohydrate molecule, etc.) or reagent. In other instances, a fraction of the plurality of individually addressable location may have immobilized thereto an analyte or reagent. A plurality of analytes or reagents immobilized to the substrate may be copies of a template analyte ortemplate reagent. For example, the plurality of analytes (e.g., nucleic acid molecules) or reagents may have sequence homology. In other instances, the plurality of analytes or reagents immobilized to the substrate may not be copies. The plurality of analytes may be of the same type of analyte (e.g., a nucleic acid molecule) or reagent or may be a combination of different types of analytes or reagents (e.g., nucleic acid molecules, protein molecules, etc.).

[0266] In some instances, the array may comprise a plurality of types of binders. For example, the array may comprise different types of binders to bind different types of analytes or reagents. For example, the array may comprise a first type of binders (e.g., oligonucleotides) configured to bind a first ty pe of analyte (e.g., nucleic acid molecules) or reagent, and a second type of binders (e.g., antibodies) configured to bind a second type of analyte (e.g., proteins) or reagent, and the like. In another example, the array may comprise a first type of binders (e.g., first type of oligonucleotide molecules) to bind a first type of nucleic acid molecules and a second type of binders (e.g.. second type of oligonucleotide molecules) to bind a second type of nucleic acid molecules, and the like. For example, the substrate may be configured to bind different types of analytes or reagents in certain fractions or specific locations on the substrate by having the different types of binders in the certain fractions or specific locations on the substrate.

[0267] An array may have any number of individually addressable locations. For instance, the array may have at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000. 50,000, 100,000, 200,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 20,000.000, 50,000,000, 100.000,000, 200,000,000. 500,000.000, 1.000,000,000,2,000,000,000, 5,000,000,000, 10,000,000,000, 20,000,000,000, 50,000,000,000, or 100,000,000,000 individually addressable locations. The array may have a number of individually addressable locations that is within a range defined by any two of the preceding values. Each individually addressable location may be digitally and / or physically accessible individually (from the plurality of individually addressable locations). For example, each individually addressable location may be located, identified, and / or accessed electronically or digitally for mapping, sensing, associating with a device (e.g., detector, processor, dispenser, etc.), or otherwise processing. As described elsewhere herein, each individually addressable location may be indexed. Alternatively, the substrate may be indexed such that eachindividually addressable location may be identified during at least one step of the process. Alternatively, or in addition, each individually addressable location may be located, identified, and / or accessed physically, such as for physical manipulation or extraction of an analyte, reagent, particle, or other component located at an individually addressable location. In some instances, each individually addressable locations may have or be coupled to a binder, as described herein, to immobilize an analyte thereto. In some instances, only a fraction of the individually addressable locations may have or be coupled to a binder. In some instances, an individually addressable location may have or be coupled to a plurality of binders to immobilize an analyte or reagent thereto.

[0268] The analytes bound to the individually addressable locations may include, but are not limited to, molecules, cells, tissues, organisms, nucleic acid molecules, nucleic acid colonies, beads, clusters, polonies, DNA nanoballs, or any combination thereof (e.g., bead having attached thereto one or more nucleic acid molecules, e.g., one or more clonal populations of nucleic acid molecules). The analytes bound to the individually addressable locations may include any analyte described herein. The bound analytes may be immobilized to the array in a regular, patterned, periodic, random, or pseudo-random configuration, or any other spatial arrangement. In some embodiments, the analytes are bound to bead(s) which may then associate with or be immobilized to the substrate or regions of the substrate (e.g., individually addressable locations). In some embodiments, the analytes comprise a bead or a plurality of beads. In some cases, the bead or plurality of beads may comprise another analyte (e.g., nucleic acid molecule) or a clonal population of other analytes (e.g., a nucleic acid molecule that has been amplified on the bead). Such other analytes may be attached or otherwise coupled to the bead. For example, an analyte may comprise a plurality of beads, each bead having a clonal population of nucleic acid molecules attached thereto. In some cases, the bead is magnetic, and application of a magnetic field or using a magnet may be used to direct the analytes or beads comprising the analytes to the individually addressable locations. In some cases, the bead is electrically charged, and application of an electric field may be used to direct the analytes or beads comprising the analytes to the individually addressable locations. In some cases, a fluid may be used to direct the analyte to the individually addressable locations. The fluid may be a ferrofluid, and a magnet may be used to direct the fluid to the individually addressable locations. The individually addressable locations may alternatively or in conjunction comprise a material that is sensitive to a stimulus, e.g., thermal, chemical, or electrical or magnetic stimulus. For example, the individually addressable location maycomprise a photo-sensitive polymer or reagent that is activated when exposed to electromagnetic radiation. In some cases, a caged molecule may be used to reveal binding (e.g., biotin) moieties (e.g., binders) on the substrate. Subsequent exposure to a particular wavelength of light may result in un-caging of the binding moieties. A bead, e.g., with streptavidin, comprising the analyte may then associate with the uncaged binding moieties. In some cases, a subset of the individually addressable locations may not contain beads. In such cases, blank beads may be added to the substrate. The blank beads may then occupy the regions that are unoccupied by an analyte. In some cases, the blank beads have a higher binding affinity or avidity for the individually addressable locations than the beads comprising the analyte. In some cases, unoccupied locations, or binders at such locations, may be destroyed or rendered inactive. In some cases, unoccupied locations may be subjected to a process to remove any unbound analyte, e.g., aspiration, washing, air blasting etc. In some cases, the sample comprising the analyte may be loaded onto the substrate using a device, e.g., a microfluidic device, closed flow cell, etc. The loaded analyte may then associate with or be immobilized to the substrate or the individually addressable locations of the substrate. In such cases, the device may be removed following loading of the sample. Though examples herein describe immobilization of analytes to the substrate, similar mechanisms may immobilize reagents to the substrate. For example, reagents may comprise or be coupled to bead(s).

[0269] An analyte may be bound to any number of beads. Different analytes may be bound to any number of beads. The beads may be unique (i.e., distinct from each other). Any number of unique beads may be used. For instance, an order of magnitude of at least about 10, 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000 or more different beads may be used. Alternatively, or in addition, an order of magnitude of at most about 100,000,000,000. 10,000,000,000, 1,000,000,000, 100,000,000, 10,000,000, 1,000,000, 100,000, 10,000, 1000, 100, or 10 different beads may be used. A number of different beads can be within a range defined by any two of the preceding values. The beads may be distinguishable from one another using a property of the beads, such as color, reflectance, anisotropy, brightness, fluorescence, etc. As described elsewhere herein, in some cases, different beads may comprise different tags (e.g.. nucleic acid sequences) coupled thereto. For example, a bead may comprise an oligonucleotide molecule comprising a tag that identifies a head amongst a plurality of beads.

[0270] A sample may be diluted such that the approximate occupancy of the individually addressable locations is controlled. A sample may be diluted at least to a dilution of 1 : 1, 1:2,1:3, 1:4, 1 :5, 1 :6, 1:7, 1:8, 1:9, 1: 10, 1:20, 1 :30, 1:40, 1 :50, 1:60, 1:70, 1 :80, 1 :90, 1: 100, 1:200, 1:300, 1:400, 1 :500, 1:600, 1 :700. 1:800, 1 :900, 1 : 1000, 1: 10000, 1: 100000, 1: 1000000, 1 : 10000000, 1 : 100000000. Alternatively, a sample may be diluted at most to a dilution of 1: 1, 1 :2, 1 :3, 1:4, 1 :5, 1:6, 1:7, 1 :8, 1:9, 1 : 10, 1:20, 1:30, 1:40, 1 :50, 1 :60, 1:70, 1:80, 1 :90, 1: 100, 1:200, 1 :300, 1:400, 1 :500, 1:600, 1:700, 1:800, 1:900, 1 : 1000, 1: 10000, 1 : 100000, 1:1000000, 1: 10000000, 1 : 100000000. A dilution between any of these dilution values may also be used.

[0271] In some instances, a sample may comprise beads. Beads may be dispersed on a surface in any pattern, or randomly. Beads may be dispersed on one or more regions (e.g., a region having a particular surface chemistry ) of a surface. In some cases, beads may be dispersed on a surface or a region of a surface in a hexagonal lattice, as shown in FIG. 16, which illustrates in the right panel a zoomed out image of a portion of a surface, and in the left panel a zoomed in image of a section of the portion of the surface. In some instances, a sample comprising beads may be dispersed on a surface comprising distinct locations / regions differentiated by surface chemistry (e.g., as illustrated in FIG. 15A and FIG. 15B). For example, a sample comprising beads may be dispensed on a surface comprising positively charged locations / regions and / or hydrophobic locations / regions. The beads may have a high affinity for a first location type or region type (e g., positively charged). The beads may have a low' affinity for a second location type or region type (e.g., hydrophobic). A location may comprise no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 beads per location. In some embodiments, a bead may be substantially centered within an individually addressable location when immobilized. A location may have a width that is up to about 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, or 3 times the diameter (e.g., maximum diameter) of the bead. In some embodiments, a region may be spaced with a pitch determined by the distance between the center of a first location and the center of the closest or neighboring location of the same type. A location may be spaced with a pitch that is at least about 1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 2.2 times. 2.4 times, 2.6 times, 2.8 times, 3 times, 3.2 times, 3.4 times. 3.6 times, 3.8 times. 4 times, 4.2 times. 4.4 times, 4.6 times. 4.8 times, or 5 times the diameter (e g.,, maximum diameter) of the bead. In some cases, one or more of a location size, a location spacing, a bead affinity, a location surface chemistry' may be adjusted to reduce a deviation of a bead contact point from the center of a region. Though examplesherein describe a sample comprising beads, similarly, a reagent dispensed to the substrate may comprise beads.

[0272] A surface comprising a plurality of individually addressable locations may be loaded with beads. The beads may be loaded onto the surface at an occupancy determined by the number of locations of a given location type comprising at least one bead out of the total number of locations of the same location type. A surface comprising a plurality of locations may have occupancy of at least about 50%, 60%, 70%. 80%. 85%. 86%. 87%. 88%. 89%. 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99.5%, or 100%. For example, a surface may have at least about 90% of the locations of a given location ty pe loaded with at least one bead. Beads may land on the surface with a landing efficiency determined by the number of beads that bind to the surface out of the total number of beads dispensed on the surface. Beads may be dispensed onto a surface with a landing efficiency of at least about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100%. In some embodiments, one or more of a temperature, an incubation time, a surfactant, or a salt concentration of a solution comprising beads may be adjusted to increase bead occupancy. In some embodiments, one or more of a temperature, an incubation time, a surfactant, or a salt concentration of a solution comprising beads may be adjusted to increase bead loading efficiency.

[0273] In some cases, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%. 70%. 75%, 80%, 85%, 90%, or 95% of the available surface area of a substrate may be configured to accept a bead. In some instances, where less than 100% of the available surface area has loaded thereon a bead (e.g., has a bead immobilized thereto), negative space (e.g., locations with no bead immobilized thereto) may be used as a reference to identity7and / or index different individually addressable locations of the positive space (e.g., locations in which there is a bead). In some instances, a single individually addressable location in negative space is sufficient to index the entire substrate. The single individually addressable location in negative space will always remain Mark’ during imaging (e.g., during sequencing). In contrast, individually addressable locations in positive space will light up (e.g., be detectable, e.g., fluoresce) at different points in time (e.g., in more than 1 point in time) due to the present of analyte or reagent in the positive space. Thus, the single individually addressable location which is always 'dark’ may act as a reference against all other individually addressable locations. In other examples, multiple individually addressable locations in negative space may facilitate indexing of the substrate (e.g., serve as reference points). Alternatively, or in addition, a reference bead which is always ‘bright’ (e.g., always fluorescing regardless of time point)may be used as a reference to identify and / or index different individually addressable locations of the positive space. In such cases, even with 100% or substantially 100% of the available surface area loaded with beads, including the reference bead, the different individually addressable locations may be identified and / or indexed.

[0274] In some...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for spatial mapping, comprising:(a) immobilizing a first set of beads comprising a plurality of first oligonucleotide molecules on a substrate, each of the first set of beads comprising a set of first oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the first set of beads;(b) loading a second set of beads comprising a plurality of second oligonucleotide molecules to the substrate comprising the first set of beads immobilized thereto, each of the second set of beads comprising a set of second oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the second set of beads, and capturing at least a subset of second oligonucleotide molecules of the second set of beads with at least a subset of first oligonucleotide molecules of the first set of beads;(c) extending the subset of first oligonucleotide molecules to generate a plurality of composite molecules on the first set of beads, each of the plurality of composite molecules comprising a first tag sequence derived from a spatial tag from the first set of beads and a second tag sequence derived from a spatial tag from the second set of beads;(d) loading a sample to the substrate comprising the first set of beads immobilized thereto, and capturing a plurality of analyte sequences from the sample with oligonucleotide molecules of the first set of beads, the oligonucleotide molecules comprising one or more of (i) at least a subset of the plurality of composite molecules or derivatives thereof, and / or (ii) an additional subset of first oligonucleotide molecules or derivatives thereof; and(e) generating spatially tagged analyte molecules, wherein each of the spatially tagged analyte molecules comprises a sequence of or derived from a spatial tag from the first set of beads.

2. The method of claim 1, further comprising sequencing the spatially tagged analyte molecules or derivatives thereof, to generate sequencing data.

3. The method of claim 2, further comprising using the sequencing data to generate a spatial map of the plurality’ of analyte sequences by identifying sets of associated spatial tags, where the spatial map comprises information about the respective locations or respective probability cloud of each of a set of analyte sequences with respect to a reference analyte sequence.

4. The method of claims 2-3, wherein the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.

5. The method of claims 2-3, wherein the spatially tagged analyte molecules or derivatives thereof are amplified off the substrate prior to the sequencing.

6. The method of any one of claims 2-5, wherein the spatially tagged analyte molecules or derivatives thereof are released from the first set of beads or the second set of beads prior to the sequencing.

7. The method of any one of claims 2-6, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.

8. The method of any one of claims 2-6, wherein the spatially tagged analyte molecules or denvatives thereof are sequenced while attached to a second substrate different from the substrate.

9. The method of any one of claims 2-6, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced without being attached to any substrate.

10. The method of any one of claims 1-9, further comprising sequencing an additional subset of the plurality of composite molecules that did not capture any analyte sequence, or derivatives thereof.

11. The method of any one of claims 1-10. wherein in (a) the first set of beads are immobilized to a plurality of individually addressable locations on the substrate.

12. The method of any one of claims 1-11, wherein a first bead of the first set of beads and a second bead of the second set of beads are the same type of bead.

13. The method of any one of claims 1-11, wherein a first bead of the first set of beads and a second bead of the second set of beads are different types of beads.

14. The method of any one of claims 1-13, further comprising, prior to (d). contacting the first set of beads with a plurality of capture sequence molecules comprising a capture sequence and a binding sequence, wherein the plurality of composite molecules bind to the binding sequence of the plurality of capture sequence molecules and is extended to generate an analyte capture sequence on a plurality of extended composite molecules, and wherein in (d) theplurality of analyte sequences are captured via the analyte capture sequence on the plurality of extended composite molecules.

15. The method of claim 14, wherein the analyte capture sequence comprises a poly-T sequence, a targeted sequence, a randomer sequence, or reverse complements thereof.

16. The method of any one of claims 1-15, wherein the sample comprises a tissue sample, wherein the plurality of analyte sequences comprises a plurality of messenger ribonucleic acid (mRNA) transcript sequences or DNA sequences.

17. The method of any one of claims 1-16, further comprising fixing said sample.

18. The method of any one of claims 1-17, further comprising permeabilizing said sample.

19. The method of any one of claims 1-18, wherein the first set of beads comprises at least50 different spatial tags.

20. The method of any one of claims 1-19, wherein the first set of beads comprises at least100 different spatial tags.

21. The method of any one of claims 1-20. wherein the first set of beads comprises at least 1000 different spatial tags.

22. The method of any one of claims 1-21. wherein the first set of beads comprises at least 10,000 different spatial tags.

23. The method of any one of claims 1-22, wherein the first set of beads immobilized to the substrate comprises at least 1,000,000 beads.

24. The method of any one of claims 1-23, wherein the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.

25. The method of any one of claims 1-24, wherein the first set of beads immobilized to the substrate comprises at least 1,000,000,000 beads.

26. A method for spatial mapping, comprising:(a) immobilizing a first set of beads comprising a plurality of first oligonucleotide molecules on a substrate, each of the first set of beads comprising a set of first oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the first set of beads;(b) loading a second set of beads comprising a plurality of second oligonucleotide molecules to the substrate comprising the first set of beads immobilized thereto, each of the second set of beads comprising a set of second oligonucleotide molecules each comprising a spatial tag unique to the bead within at least the second set of beads, and capturing at least a subset of second oligonucleotidemolecules of the second set of beads with at least a subset of first oligonucleotide molecules of the first set of beads;(c) extending the subset of first oligonucleotide molecules to generate a plurality of first composite molecules on the first set of beads or extending the subset of second oligonucleotide molecules to generate a plurality7of second composite molecules on the second set of beads or both, each of the plurality7of first or second composite molecules comprising a first tag sequence derived from a spatial tag from the first set of beads and a second tag sequence derived from a spatial tag from the second set of beads;(d) loading a sample to the substrate, and capturing a plurality of analyte sequences from the sample with oligonucleotide molecules of the second set of beads, the oligonucleotide molecules comprising one or more of (i) at least a subset of the plurality of second composite molecules or derivatives thereof, and / or (ii) an additional subset of second oligonucleotide molecules or derivatives thereof; and(e) generating spatially tagged analyte molecules, wherein each of the spatially tagged analyte molecules comprises a sequence of or derived from a spatial tag from the second set of beads.

27. The method of claim 26, further comprising sequencing the spatially tagged analyte molecules or derivatives thereof, to generate sequencing data.

28. The method of claim 27, further comprising using the sequencing data to generate a spatial map of the plurality7of analyte sequences by identifying sets of associated spatial tags, where the spatial map comprises information about the respective locations or respective probability cloud of each of a set of analyte sequences with respect to a reference analyte sequence.

29. The method of claims 27-28, wherein the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.

30. The method of claims 27-28, wherein the spatially tagged analyte molecules or derivatives thereof are amplified off the substrate prior to the sequencing.

31. The method of any one of claims 27-30, wherein the spatially tagged analyte molecules or derivatives thereof are released from the first set of beads or the second set of beads prior to the sequencing.

32. The method of any one of claims 27-31, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.

33. The method of any one of claims 27-31, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to a second substrate different from the substrate.

34. The method of any one of claims 27-31, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced without being attached to any substrate.

35. The method of any one of claims 26-34, further comprising the plurality of first composite molecules or derivatives thereof.

36. The method of any one of claims 26-35, wherein in (a) the first set of beads are immobilized to a plurality of individually addressable locations on the substrate.

37. The method of any one of claims 26-36, wherein a first bead of the first set of beads and a second bead of the second set of beads are the same type of bead.

38. The method of any one of claims 26-36, wherein a first bead of the first set of beads and a second bead of the second set of beads are different types of beads.

39. The method of any one of claims 26-38, wherein the lurality of second oligonucleotide molecules comprises an analyte capture sequence, and wherein in (d) the plurality of analyte sequences is captured via the analyte capture sequence.

40. The method of claim 39, wherein the analyte capture sequence comprises a poly-T sequence, a targeted sequence, a randomer sequence, or reverse complements thereof.41 . The method of any one of claims 26-40, wherein the sample comprises a tissue sample, wherein the plurality7of analyte sequences comprises a plurality7of messenger ribonucleic acid (mRNA) transcript sequences or DNA sequences.

42. The method of any one of claims 26-41, further comprising fixing said sample.

43. The method of any one of claims 26-42, further comprising permeabilizing said sample.

44. The method of any one of claims 26-43, wherein the first set of beads comprises at least50 different spatial tags.

45. The method of any one of claims 26-44, wherein the first set of beads comprises at least100 different spatial tags.

46. The method of any one of claims 26-45, wherein the first set of beads comprises at least 1000 different spatial tags.

47. The method of any one of claims 26-46, wherein the first set of beads comprises at least 10,000 different spatial tags.

48. The method of any one of claims 26-47. wherein the first set of beads immobilized to the substrate comprises at least 1,000,000 beads.

49. The method of any one of claims 26-48, wherein the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.

50. The method of any one of claims 26-49, wherein the first set of beads immobilized to the substrate comprises at least 1,000,000.000 beads.

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