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WO2025213126A2 - Systèmes et procédés de séquençage de référence spatiale - Google Patents

Systèmes et procédés de séquençage de référence spatiale

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Publication number
WO2025213126A2
WO2025213126A2 PCT/US2025/023299 US2025023299W WO2025213126A2 WO 2025213126 A2 WO2025213126 A2 WO 2025213126A2 US 2025023299 W US2025023299 W US 2025023299W WO 2025213126 A2 WO2025213126 A2 WO 2025213126A2
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WO
WIPO (PCT)
Prior art keywords
beads
sequence
analyte
spatial
bead
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023299
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English (en)
Other versions
WO2025213126A3 (fr
Inventor
Ron SAAR DOVER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ultima Genomics Inc
Original Assignee
Ultima Genomics Inc
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Filing date
Publication date
Application filed by Ultima Genomics Inc filed Critical Ultima Genomics Inc
Publication of WO2025213126A2 publication Critical patent/WO2025213126A2/fr
Publication of WO2025213126A3 publication Critical patent/WO2025213126A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • Biological sample processing has various applications in the fields of molecular biology and medicine (e.g., diagnosis).
  • 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.
  • 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.
  • the method further comprises sequencing the spatially tagged analyte molecules or derivatives thereof, to generate sequencing data.
  • 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.
  • the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.
  • the spatially tagged analyte molecules or derivatives thereof are amplified off the substrate prior to the sequencing.
  • 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.
  • the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.
  • 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.
  • the method further comprises sequencing an additional subset of the plurality of composite molecules that did not capture any analyte sequence, or derivatives thereof.
  • the first set of beads are immobilized to a plurality of individually addressable locations on the substrate.
  • 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.
  • a first bead of the first set of beads and a second bead of the second set of beads are different types of beads.
  • the analyte capture sequence comprises a poly-T sequence, a targeted sequence, a randomer sequence, or reverse complements thereof.
  • 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.
  • mRNA messenger ribonucleic acid
  • the method further comprises fixing said sample.
  • the method further comprises permeabilizing said sample.
  • 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.
  • the first set of beads comprises at least 10,000 different spatial tags.
  • 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.
  • 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 oligonucle
  • the method further comprises sequencing the spatially tagged analyte molecules or derivatives thereof, to generate sequencing data.
  • 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.
  • the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.
  • 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.
  • the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.
  • the method further comprises the plurality 7 of first composite molecules or derivatives thereof.
  • the first set of beads are immobilized to a plurality 7 of individually addressable locations on the substrate.
  • 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.
  • the first set of beads comprises at least 50 different spatial tags.
  • 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.
  • the first set of beads comprises at least 10,000 different spatial tags.
  • the first set of beads immobilized to the substrate comprises at least 1,000,000 beads.
  • the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.
  • 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.
  • 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.
  • FIGs. 2A-2B illustrate reagents and a workflow for generating a spatially tagged sequence using a bridge construct and multiple spatial tags.
  • FIG. 3 illustrates an example map that can be generated from a sample set of spatial tag data.
  • FIG. 6 illustrates an example droplet with reagents of the present disclosure.
  • FIG. 7 illustrates an example solution environment with reagents of the present disclosure.
  • FIG. 9A illustrates examples of additional bridge constructs.
  • FIG. 9B illustrates examples of additional tagging schemes.
  • FIGs. 9C-9D illustrate various bead release mechanisms.
  • FIG. 10 illustrates an example method for generating a tagged sequence under a 5’ approach.
  • FIG. 11A illustrates example constructs of a geolocation bead and a capture bead.
  • FIG. 11B provides example capture complexes comprising capture beads.
  • FIG. 12 illustrates an example of additional capture bead and geolocation bead constructs.
  • 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.
  • FIG. 16 shows an example coating of a substrate with a hexagonal lattice of beads.
  • FIG. 18 shows a flowchart for an example of a method for sequencing a nucleic acid molecule.
  • FIGs. 19A, 19B, 19C, andl9D illustrate an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs.
  • FIG. 20 illustrates an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs.
  • FIG. 21 illustrates an example of a substrate map using fiducial marker beads according to the methods described herein.
  • the first strands comprising the spatial tag 109 may be released from the geolocation bead 101 via various mechanisms.
  • 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
  • a primer may be provided under conditions sufficient to displace the first strand (comprising sequences 107, 109, 111) from the oligonucleotide molecule.
  • strand displacing polymerases include, but are not limited to, Bst DNA polymerase, large fragment polymerase, and 029 polymerase.
  • 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.
  • the second strand may comprise 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the endonuclease may be replaced with an APE1 enzyme in the USER enzyme mix, which cleaves multiple times and is Mg 2+ -dependent (as opposed to endonuclease VIII which is Mg 2+ - ind ependent).
  • the UDG and APE1 enzy me may be provided to the substrate 450 comprising a plurality of geolocation beads immobilized thereto, without Mg 2+ , 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 Mg 2+ .
  • Panels (B)-(D) of FIGs. 9C-9D describe non-enzymatic release mechanisms.
  • 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" 11 M) than that between streptavidin and biotin moieties (e.g., with Kd on the order of 10' 15 M).
  • Kd disassociation constant
  • 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).
  • 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.
  • 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.
  • 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 (T m ) 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 T m between the two strands without azobenzene and as compared to the T m when the azobenzene is in transform, as it weakens the hydrogen bonds between the two strands.
  • the T m between the first strand the second strand may not vary’ as much when the azobenzene is in trans-form as compared to the T m between the two strands without the azobenzene.
  • providing UV light stimulus e.g., 365 nm
  • 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.
  • 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.
  • a-CD alpha-cyclodextrine
  • 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.
  • 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.
  • 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.
  • a capture sequence e.g., nucleic acid sequence
  • 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.
  • 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 capturing mechanisms described elsewhere herein (e.g., biotin and streptavidin, complementary capture sequences, etc.).
  • 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,
  • 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,
  • the spatial tag may be unique and common to a geolocation bead amongst a plurality 7 of 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.
  • 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.
  • 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.
  • the first attachment sequence may be common to a plurality of geolocation beads such that each geolocation bead comprises the same first attachment sequence.
  • the second attachment sequence may be common to a plurality of geolocation beads such that each geolocation bead comprises the same second attachment sequence.
  • 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.
  • 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.
  • 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.
  • 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).
  • a plurality 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.
  • 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.
  • first attachment sequence and second attachment sequence amongst the plurality of oligonucleotide molecules on the 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.
  • the plurality of geolocation beads may be loaded onto a substrate.
  • the substrate may comprise a plurality 7 of individually addressable locations.
  • 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.
  • 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.
  • 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 plurality 7 of nucleic acid molecules (e.g., comprising analyte sequences), such as messenger RNA (mRNA) molecules.
  • mRNA messenger RNA
  • 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.
  • 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.
  • 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.
  • 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.
  • the first strand comprising the spatial tag 109 may be released.
  • 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).
  • a bridge construct may be provided to the substrate prior to, during, or subsequent to release of the spatial tags.
  • the bridge construct may be provided to the substrate prior to, during, or subsequent to loading of the sample on the substrate.
  • 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.
  • 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.).
  • an analyte sequence e.g., a messenger RNA (mRNA) molecule
  • mRNA messenger RNA
  • 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.
  • FIGs. 2A-2B illustrate reagents and a workflow for generating a spatially tagged sequence using a bridge construct and multiple spatial tags.
  • a bridge construct 201 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.
  • 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.
  • the first spatial tag molecule 231 comprises, from 5 ? to 3’, the first attachment sequence 207, the first spatial tag 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.
  • 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.
  • 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’.
  • 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.
  • 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.
  • the capture sequence may comprise a target mRNA sequence (or derivative thereof).
  • 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.
  • 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.
  • 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 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.
  • Spacer sequences in the bridge construct may be the same sequence or different sequences. Spacer sequences may comprise a sequence of any length.
  • the spacer can be any internal spacer, e.g., C3 spacer, C12 spacer, spacer 9, spacer 18, etc.
  • the spacer sequence may be designed to not be complementary 7 to any spatial tag sequence, or portion thereof.
  • 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.
  • the first spatial tag molecule 231 e g., from FIG. 2A
  • 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.
  • the second spatial tag molecule 241 e.g., from FIG. 2A
  • 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 entity 7 213.
  • the sample may be incubated with the reagents (e.g., bridge constructs, spatial tags, etc.) for any period of time.
  • 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.
  • 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.).
  • the tagged complexes may be recovered.
  • the tagged complex 250 may be captured using the capture entity 213, such as via a capturing entity.
  • the capture entity 213 comprises biotin and the capturing entity comprises streptavidin, where the streptavidin is coupled to a magnetic bead.
  • 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.
  • 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.
  • 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.
  • 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.
  • a bridge construct that is designed to capture five spatial tags comprises 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.
  • 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.
  • 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.
  • 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 same spatial tag may indicate that the two spatially tagged sequences were within a certain range of proximity of each other.
  • 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.
  • 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]
  • 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.
  • 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.
  • analyte sequences 2, 4, 6, and 7 are mapped to within the dotted circle around C.
  • 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...
  • a maximum distance of diffusion of the spatial tag from the geolocation bead can be estimated to facilitate map generation.
  • a map of the plurality of geolocation beads may be generated prior to. concurrently with, or subsequent to generating 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.
  • 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.
  • the reference e.g., reference analyte sequence, reference geolocation bead, reference location
  • 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.
  • 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.
  • 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.
  • a plurality of bridge constructs 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 elsewhere herein), and prepared for sequencing.
  • One or more ligation reactions may be performed on the substrate.
  • 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.
  • 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.
  • a plurality 7 of geolocation beads comprising a plurality 7 of oligonucleotide molecules, as described elsewhere herein, may be provided to the sample.
  • the geolocation beads may be provided substantially simultaneously with one or more sample processing reactions.
  • 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.
  • a plurality of bridge constructs e.g., a subset of a population of total bridge constructs loaded to the substrate
  • a plurality 7 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.
  • One or more ligation reactions maybe performed on the substrate.
  • 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.
  • 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.
  • a plurality’ of cells can be smeared onto a substrate at substantially dilute concentration.
  • 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.
  • the analyte sequences and/or other cellular content in the cells may be rendered accessible to the bridge constructs.
  • 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
  • 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 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.
  • first spatially tagged sequence is identified to include a first spatial tag and a second spatial tag
  • second spatially tagged sequence is identified to include a third spatial tag and a fourth spatial tag
  • third spatially tagged sequence is identified to include the second spatial tag and the third spatial tag
  • 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.
  • a final spatial map generated e.g., x-y plane
  • 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.
  • diffusion can be attenuated by adding viscous reagents (e.g., PEG. etc.) and/or modulating one or more other reaction conditions (e.g., temperature).
  • 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.
  • 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.).
  • 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.
  • a 4-arm PEG- acrylate macromer and PEG-dithiolglycolate crosslinker is used to form a PEG hydrogel that is degradable.
  • protein e.g., bovine serum albumin (BSA) protein
  • BSA bovine serum albumin
  • other solutes may be embedded or entrapped within the mesh network to increase a crowding effect to further attenuate diffusion.
  • 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 schematic for 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
  • ITO indium tin oxide
  • 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...
  • 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.
  • 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.
  • an absolute position of the fiducial marker bead on a substrate may be known, predetermined, and/or detectable.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the detecting of the respective detectable 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.
  • 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).
  • 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.
  • a plurality of bridge constructs 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.
  • a plurality of bridge constructs may each capture an analyte sequence and multiple spatial tags to generate tagged complexes (e.g., 250).
  • 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.
  • 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.
  • 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.
  • the scheme described herein may contribute more towards super 2-D resolution than the 3-D resolution.
  • 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)).
  • multi-channel e.g., 2 channels, 3 channels, etc.
  • tissue morphology e.g., hematoxylin and eosin staining (H&E staining)
  • FIG. 5A illustrates an example with two substrates
  • more substrates may be applied where multiple surfaces are available.
  • the different substrates may or may not be the same size and/or type.
  • miniature substrates may be applied to multiple surfaces of a sample which has one surface on a much larger substrate.
  • 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.
  • 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, 10 2 , 10 3 . 10 4 , 10 5 . 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • PAMAM polyamidoamine
  • PPI polypropylene imine
  • PPIM polylysine
  • PPIM poly(propyl ether imine)
  • viologen dendrimers include, without limitation, polyamidoamine (PAMAM), polypropylene imine (PPI), polylysine, poly(propyl ether imine) (PEPIM), and viologen dendrimers.
  • PAMAM polyamidoamine
  • PPI polypropylene imine
  • PPIM polylysine
  • PPIM poly(propyl ether imine)
  • 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.
  • such penetration may be accelerated by applying one or more directed forces, such as magnetic fields, electric fields, and/or pressure.
  • 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.
  • a photo or chemical stimulus is applied for the release of the spatial tags, as described with respect to FIG. 8A.
  • 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.
  • geolocation beads and bridge constructs of the present disclosure may also be used in emulsion-based or solution-based reaction environments, off the substrate.
  • FIG. 6 illustrates an example droplet with reagents of the present disclosure.
  • 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 cases more 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).
  • beads e.g., geolocation beads
  • 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.
  • 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.
  • 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).
  • the analyte sequences and/or other cellular content in the cell 603 may be rendered accessible to the bridge constructs.
  • 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).
  • the analyte sequences and/or other cellular content in the cells may 7 be rendered accessible to the bridge constructs.
  • 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).
  • the bridge constructs e.g., a subset of a population of total bridge constructs or all of the bridge constructs
  • a plurality of spatially tagged sequences 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.
  • 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).
  • viscous reagents e.g., PEG, etc.
  • modulating various reaction conditions e.g., temperature
  • 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.
  • 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.
  • the one or more real images of the solution can be resolved into one or more 3D images.
  • 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).
  • 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.
  • 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.
  • the oligonucleotide molecule 143 may comprise a third attachment sequence 171, a second spatial tag 173, and a fourth attachment sequence 175.
  • the oligonucleotide molecule 143 may comprise a UMI sequence 115a.
  • 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.
  • the oligonucleotide molecule may be designed to have multiple UMIs, including many as releasable strands.
  • 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.
  • 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.
  • Example cleavage sites are indicated as “U” in the figure.
  • 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.
  • 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).
  • a second atachment sequence 937 of the second spatial tag strand e.g., third atachment sequence 211
  • the second spatial tag strand may comprise a capture moiety (e.g., biotin) at a 5’ end.
  • the second attachment sequence 937 of the second spatial tag strand 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.).
  • 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.
  • 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.
  • 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.
  • 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.
  • the second spatial tag strand may comprise a capture moiety (e.g., biotin) at a 5’ end.
  • FIGs. 19A-19D illustrate an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs.
  • 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.
  • 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.
  • 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.
  • the binding sequence 1905 and the one or more additional functional sequences may together function as a barcode sequence for the bridge construct.
  • 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’).
  • 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).
  • 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
  • a fourth attachment sequence (H3) 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.
  • the beads may be subjected to one or more stimuli to release the respective double stranded molecules from the beads, as described elsewhere herein.
  • a USER cleavage reaction is performed to process the cleavable moieties from the double stranded molecules and release the remaining molecule from the bead.
  • 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.
  • 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.
  • 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 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.
  • 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 spatial tag 1912 (BC2), and the capture moiety (e.g., biotin).
  • 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.
  • FIG. 20 illustrates an additional workflow for spatially encoding analytes using geolocation beads and bridge constructs.
  • 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.
  • 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.
  • 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 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).
  • the nucleic acid molecule may comprise one or more additional functional sequences, such as a primer sequence, a barcode sequence or a UMI sequence.
  • 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 be complementary 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • a primer sequence 1001 is provided to couple to an analyte sequence 1003 (e.g., mRNA sequence) (1051).
  • an analyte sequence 1003 e.g., mRNA sequence
  • 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).
  • the additional sequence 1031 may comprise bases that are added as a result of terminal transferase activity.
  • 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 may comprise a switch sequence 1033 at the 3’ end, which is configured to capture the additional sequence 1031.
  • 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.
  • the spatially tagged sequence may comprise a complement thereof.
  • the complex 1015, or derivative thereof may be captured via the capture entity 7 .
  • 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.
  • 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).
  • 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.
  • FIG. 11A illustrates example constructs of a geolocation bead 1101 and a capture bead 1121.
  • 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.
  • the capture sequence 1102 may be bound to a protecting sequence 1105 which is complementary to the capture sequence.
  • 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, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or more.
  • the spatial tag may correspond to any spatial tag described herein.
  • 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.
  • the capture bead may comprise any number of oligonucleotide molecules, for example on the order of 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 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,
  • 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,
  • 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.
  • 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.
  • the ratio between the geolocation beads to the capture beads is about 1: 1.
  • 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.
  • 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.
  • 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 between transcripts, 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.
  • 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.
  • a subset of a plurality of barcode sequences may each capture an analyte sequence from the sample, and another subset of a plurality 7 of 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.
  • 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 probability 7 cloud (or likely location) of each of a set of analyte sequences with respect to a reference analyte sequence.
  • 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.
  • 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.
  • 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).
  • 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.
  • a droplet comprises a cell, a plurality' of geolocation beads, and a plurality of capture beads.
  • the analyte sequences and/or other cellular content in the cell may be rendered accessible to the barcode sequences of the capture beads.
  • 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.
  • 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 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.
  • first spatially tagged sequence is identified to include a first spatial tag and a first barcode sequence
  • second spatially tagged sequence is identified to include the first spatial tag and a second barcode sequence
  • third spatially tagged sequence is identified to include a first analyte sequence and the first barcode sequence
  • fourth spatially tagged sequence is identified to include a second analyte sequence and the second barcode sequence
  • 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 plurality 7 of 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 proximity 7 to 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.
  • 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.
  • first spatially tagged sequence is identified to include a first spatial tag and a first barcode sequence
  • second spatially tagged sequence is identified to include the first spatial tag and a second barcode sequence
  • third spatially tagged sequence is identified to include a first analyte sequence and the first barcode sequence
  • fourth spatially tagged sequence is identified to include a second analyte sequence and the second barcode sequence
  • 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).
  • viscous reagents e.g., PEG, etc.
  • modulating various reaction conditions e.g., temperature
  • 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’.
  • the capture sequence 1222 may be bound to a protecting sequence 1225 which is complementary’ to the capture sequence.
  • the oligonucleotide molecule may comprise a UMI sequence.
  • the capture sequence 1222 may be capped by a blocking group, denoted “X” and a cleavage site “U.”
  • X blocking group
  • U cleavage site
  • Such beads may be beneficial for suppressing artefacts from forming during extension.
  • an enzyme configured for strand displacement and primer 1226 e.g., displacement strand
  • primer 1226 e.g., displacement 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.
  • 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.
  • 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’.
  • the capture sequence 1232 may be bound to a protecting sequence 1235, which is complementary to the capture sequence.
  • the oligonucleotide molecule may comprise a UMI sequence.
  • the capture sequence 1232 may be capped by a blocking group, denoted "X and a cleavage site “U.”
  • an enzyme configured for strand displacement and primer 1236 e.g., displacement strand
  • 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 site CL 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.
  • 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.
  • 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..
  • a bead may have a maximum dimension of at most about 0.05 pm, 0. 10 pm, 0. 15 pm,
  • 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, 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.
  • an index comprising a list of spatial tag sequences included in the geolocation beads may be provided.
  • 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.
  • 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 beads comprising 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 here
  • 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 azobenzen
  • 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).
  • the kit further comprises amplification reagents.
  • Amplification reagents may comprise a polymerase, a nucleotide mixture, a primer, a buffer, or any combination thereof.
  • the kit further comprises a biological sample.
  • the biological sample may comprise a tissue.
  • the biological sample may be fixed and/or permeabilized.
  • the biological sample may be loaded on the substrate.
  • the kit further comprises fixing and/or permeabilizing reagents.
  • a bead of the plurality of geolocation beads may comprise at least 100,000 oligonucleotide molecules.
  • 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.
  • 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.
  • a kit comprises a substrate comprising a plurality of geolocation beads immobilized to a plurality of individually addressable locations on the substrate, wherein the plurality 7 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, 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 desthiobio
  • the kit may further comprise indexed data comprising a list of spatial tag sequences included in the geolocation beads.
  • the kit may further comprise a second substrate comprising a second plurality of individually addressable locations configured to immobilize a second plurality of geolocation beads.
  • the kit may further comprise the second plurality of geolocation beads.
  • 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.
  • 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).
  • the kit further comprises amplification reagents.
  • Amplification reagents may comprise a polymerase, a nucleotide mixture, a primer, a buffer, or any combination thereof.
  • the kit further comprises a biological sample.
  • the biological sample may comprise a tissue.
  • the biological sample may be fixed and/or permeabilized.
  • the biological sample may be loaded on the substrate.
  • the kit further comprises fixing and/or permeabilizing reagents.
  • a geolocation bead of the plurality of geolocation beads may comprise at least 100,000 oligonucleotide molecules.
  • 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.
  • 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 probe sequence.
  • the first and second substrate may be substantially identical in size, shape, and/or material.
  • 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.).
  • the system may comprise a light source configured to provide light at desired frequencies (e.g.. UV light, fluorescent light, etc.).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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’ of spatially 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.
  • each bead of at least a subset of beads comprises a colony of amplification products.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • a template switching oligonucleotide comprising a capture sequence e.g., polyG
  • template switching reactions using one or more template switching sequences can be contacted with a single Tn5 adapter, reverse transcription performed, and PCR performed.
  • 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.
  • 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
  • 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 derivatives thereof, 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.
  • sequencing the spatially tagged analyte molecules or derivatives thereof e.g., reverse complements, adaptor-ligated, amplicons, otherwise processed, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • a single bead of the second set of beads e.g., Al bead
  • a single bead of the first set of beads 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).
  • 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.
  • FIG. 25 illustrates an example bead-on-bead capture scheme.
  • 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 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.
  • 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.
  • 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.
  • the second bead adapter sequence may comprise a third binding sequence.
  • 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).
  • 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).
  • a 3’ end of the second oligonucleotide molecule may be blocked from extension, rendering the strand inextendible.
  • 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”).
  • 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 *”).
  • 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.
  • 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”).
  • 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.
  • a 3’ end of the second capture molecule may be blocked from extension, rendering the strand inextendible.
  • 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.
  • the second bead adapter sequence of the second oligonucleotide molecules e.g., 2505
  • 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.
  • the template sequence may correspond to a cDNA sequence.
  • 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.
  • TSO template switching oligonucleotide
  • 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
  • the molecule is released from the first bead via USER cleavage of “U” cleavage sites in the first bead adapter sequence.
  • the tagged analyte molecule may be subsequently processed as described elsewhere herein with respect to other spatially tagged sequences.
  • 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 7 of analyte sequences in the sample.
  • FIG. 26 illustrates an additional example bead-on-bead capture scheme.
  • 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 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.
  • a set of first spatial tags contained by a plurality 7 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.
  • first oligonucleotide molecule e.g.. 2603
  • second oligonucleotide molecule e.g.. 2605
  • 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.
  • a second subset of the plurality 7 of 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.
  • 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”).
  • the first and second subsets of the plurality of second oligonucleotide molecules on a second bead 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.
  • 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)
  • 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.
  • TSO template switching oligonucleotide
  • 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 ).
  • 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.).
  • the oligonucleotide molecules on the first bead (“B”) may be released from the first bead.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 27 illustrates an additional example bead-on-bead capture scheme.
  • 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..
  • the first binding sequence may be configured to capture a second binding sequence, such as via complementarity.
  • 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") 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.
  • 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.
  • the second bead adapter sequencer may comprise one or more cleavable moieties (“U”) that may be used as a release mechanism.
  • U cleavable moieties
  • 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.
  • 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.
  • 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 sequence e.g., “AAA.. . ” of an analyte sequence 2711 (“mRNA_AAA. . . ”) may anneal to the analyte capture sequence (e.g..
  • TTTT... 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”).
  • the template sequence may correspond to a cDNA sequence.
  • 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.
  • TSO template switching oligonucleotide
  • 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.).
  • 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.
  • 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.
  • 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.
  • FIG. 30 illustrates another example bead-on-bead capture scheme.
  • a first bead (“B”) 3002 can be immobilized on a substrate 3001.
  • the first bead may 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.
  • 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...
  • the second spatial tag may be unique to the second bead amongst a plurality of second beads loaded to the substrate.
  • 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.
  • 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 7 of 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).
  • the second binding sequence may be capable of capturing both the first binding sequence on the first set of beads and analytes of interest.
  • a first oligonucleotide molecule e.g., 3003
  • 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”).
  • 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.
  • 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.
  • 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.
  • 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”).
  • the template sequence may correspond to a cDNA sequence.
  • 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.
  • TSO template switching oligonucleotide
  • 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.
  • the second set of beads may be removed from the first set of beads and the barcoded oligonucleotide molecules collected.
  • the barcoded oligonucleotide molecules may be released from the beads (either or both types).
  • 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.
  • 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.
  • 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
  • derivative thereof e.g., extension product comprising the template sequence
  • 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.
  • the tagged analyte molecule, or derivative thereof e.g., template switched derivative, copy, reverse complement copy, amplicon, etc.
  • 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.
  • 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.
  • 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.
  • the capture sequence may comprise a target mRNA sequence (or derivative thereof).
  • the capture sequence may comprise a target gDNA sequence (or derivative thereof).
  • 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.
  • the capture sequence may comprise a sequence configured to capture a product of a reverse transcription reaction, such as a polyG sequence.
  • the capture sequence may comprise a sequence corresponding to a sequence of the probe, to a molecule associated with the probe, or derivative thereof.
  • the capture sequence may be part of a single strand portion, a double strand portion, or partially double-stranded complex.
  • the capture sequence may be part of a hybrid DNA/RNA complex.
  • 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.
  • the capture sequence may be configured to capture the overhang of the partially double-stranded analyte comprising the barcode and/or adapter sequence.
  • an analyte capture molecule may comprise a UMI.
  • 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.
  • 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.
  • analyte may refer to molecules, cells, biological particles, or organisms.
  • 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.
  • the biological analyte may be a macromolecule, e.g., a nucleic acid, a carbohydrate, a protein, a lipid, etc.
  • 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.
  • 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.
  • 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.
  • a detector can include optical and/or electronic 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.
  • open substrate 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.
  • 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..
  • analyte e.g., a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.
  • a reagent e.g.
  • an analyte or reagent may be immobilized to an individually addressable location via a support, such as a bead.
  • a bead is immobilized to the individually addressable location, and the analyte or reagent is immobilized to the bead.
  • an individually addressable location may immobilize thereto a plurality of analytes or a plurality of reagents. The plurality of analytes may be copies of a template analyte.
  • the plurality’ of analytes may have sequence homology or sequence identity.
  • 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).
  • the plurality- of analytes is immobilized to the individually addressable location via a support, such as a bead.
  • 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.
  • the bead is immobilized to an individually addressable location on the substrate and is configured to capture or bind to a plurality of analytes.
  • 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.).
  • One or more surfaces of the substrate may be exposed to a surrounding open environment, and accessible from such surrounding open environment.
  • the array may be exposed and accessible from such surrounding open environment.
  • the surrounding open environment may be controlled and/or confined in a larger controlled environment.
  • 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.).
  • forces e.g., centrifugal forces, centripetal forces, inertial forces, etc.
  • motion of the substrate e.g., rotational motion of the substrate, linear motion of the substrate, combination thereof, etc.
  • 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.
  • 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.
  • 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.
  • 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).
  • an external force e.g., involving a pressure differential, involving physical force, involving a magnetic force, involving an electrical force, etc.
  • wind e.g., a fieldgenerating device, or a physical device
  • the method for dispensing reagents may comprise vibration.
  • 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).
  • the method may comprise using mechanical, electric, physical, or other mechanisms to dispense reagents to the substrate.
  • 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.
  • a physical scraper e.g., a squeegee
  • such flexible dispensing may be achieved without contamination of the reagents.
  • the volume of reagent may travel in a path or paths, such that the travel path or paths are coated with the reagent.
  • travel path or paths may encompass a desired surface area (e.g., entire surface area, partial surface area(s), etc.) of the substrate.
  • 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 meet additional reagent(s).
  • 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.
  • the substrate may be rotatable about an axis.
  • Analytes or reagents may be immobilized to the substrate during rotation.
  • reagents may be dispensed onto the substrate prior to or during rotation of the substrate.
  • 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.
  • 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.
  • 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.
  • 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.
  • the substrate may compnse an array.
  • 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.
  • 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.).
  • 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.
  • 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.
  • 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.
  • the individually addressable locations are indexed by demarcating part of the substrate.
  • 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 the substrate. As will be appreciated, a combination of positive demarcations and negative demarcations (lack thereof) may be used to index the individually addressable locations.
  • one or more reference objects are immobilized to any location(s) on the substrate, and the individually addressable locations are indexed with reference to the reference object.
  • a single reference point or axis e.g., single demarcation
  • each of the individually addressable locations is indexed.
  • a subset of the individually addressable locations is indexed.
  • the individually addressable locations are not indexed, and a different region of the substrate is indexed.
  • 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 may have an area. In some cases, a location may have an area of about 0.1 square micron (pm 2 ), 0.2 pm 2 , 0.25 pm 2 , 0.3 pm 2 , 0.4 pm 2 , 0.5 pm 2 , 0.6 pm 2 , 0.7 pm 2 , 0.8 pm 2 , 0.9 pm 2 , 1 pm 2 , 1.1 pm 2 , 1.2 pm 2 , 1.25 pm 2 .
  • 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 pm 2 or greater than about 6 pm 2 .
  • 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.
  • a location may have a width that is within a range defined by any tyvo 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, 50 nm, 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.
  • the locations 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the array may be coated with binders.
  • the array may be randomly coated with binders.
  • 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 coated with 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.
  • 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.
  • the binders may comprise oligonucleotide adaptors configured to bind to the nucleic acid molecule.
  • 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.
  • 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, 10 3 . 10 4 , 10 5 , 10 6 . 10 7 , 10 8 , 10 9 .
  • the array may comprise an order of magnitude of at most about 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 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.
  • a single binder may bind a single analyte (e.g., nucleic acid molecule) or single reagent.
  • a single binder may bind a plurality of analytes (e.g., plurality of nucleic acid molecules) or a plurality of reagents.
  • a plurality 7 of binders may bind a single analyte or a single reagent.
  • the binders may immobilize other molecules (such as proteins), other particles, cells, viruses, other organisms, or the like.
  • the binders may similarly immobilize reagents.
  • each location may have immobilized thereto an analyte (e.g.. a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.) or reagent.
  • analyte e.g. a nucleic acid molecule, a protein molecule, a carbohydrate molecule, etc.
  • 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.
  • the plurality of analytes (e.g., nucleic acid molecules) or reagents may have sequence homology.
  • the array may comprise a plurality of types of binders.
  • the array may comprise different types of binders to bind different types of analytes or reagents.
  • 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.
  • a first type of binders e.g., oligonucleotides
  • a second type of binders e.g., antibodies
  • 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.
  • 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.
  • An array may have any number of individually addressable locations.
  • 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,
  • 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 each individually addressable location may be identified during at least one step of the process.
  • 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.
  • each individually addressable locations may have or be coupled to a binder, as described herein, to immobilize an analyte thereto.
  • only a fraction of the individually addressable locations may have or be coupled to a binder.
  • an individually addressable location may have or be coupled to a plurality of binders to immobilize an analyte or reagent thereto.
  • 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.
  • 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).
  • the analytes comprise a bead or a plurality of beads.
  • 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.
  • an analyte may comprise a plurality of beads, each bead having a clonal population of nucleic acid molecules attached thereto.
  • 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.
  • 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.
  • 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.
  • the individually addressable location may comprise a photo-sensitive polymer or reagent that is activated when exposed to electromagnetic radiation.
  • 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.
  • a subset of the individually addressable locations may not contain beads.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a reagent dispensed to the substrate may comprise beads.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • 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.
  • negative space e.g., locations with no bead immobilized thereto
  • 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).
  • 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.
  • the single individually addressable location which is always 'dark’ may act as a reference against all other individually addressable locations.
  • multiple individually addressable locations in negative space may facilitate indexing of the substrate (e.g., serve as reference points).
  • a reference bead which is always ‘bright’ e.g., always fluorescing regardless of time point
  • 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.
  • the different individually addressable locations may be identified and/or indexed.
  • beads may be dispensed to the substrate according to one or more systems and methods shown in FIGs. 17A-17B.
  • a solution comprising beads may be dispensed from a dispense probe 1701 (e.g., a nozzle) to a substrate 1703 (e.g.. a wafer) to form a layer 1705.
  • the dispense probe may be positioned at a fixed height (“Z”) above the substrate.
  • the beads are retained in the layer 1705 by electrostatic retention and may immobilize to the substrate at respective individually addressable locations.
  • a set of beads in the solution may each comprise a population of amplified products (e.g., nucleic acid molecules) immobilized thereto, which amplified products accumulate to a negative charge on the bead with affinity to a positive charge.
  • the beads may comprise reagents that have a negative charge.
  • the substrate comprises alternating surface chemistry between distinguishable locations, in which a first location type comprises APTMS earn ing a positive charge with affinity towards the negative charge of the amplified bead (e g., a bead comprising amplified products immobilized thereto, and as distinguished from a negative bead which does not the comprise the same) or other bead comprising the negative charge, and a second location type comprises HMDS which has lower affinity and/or is repellant of the amplified bead or other bead comprising the negative charge.
  • a first location type comprises APTMS earn ing a positive charge with affinity towards the negative charge of the amplified bead (e g., a bead comprising amplified products immobilized thereto, and as distinguished from a negative bead which does not the comprise the same) or other bead comprising the negative charge
  • a second location type comprises HMDS which has lower affinity and/or is repellant of the amplified bead or other bead compris
  • FIG. 17B illustrates a reagent (e.g., beads) being dispensed along a path on an open surface of the substrate.
  • a reagent solution may be dispensed from a dispense probe (e.g., a nozzle).
  • a solution may be dispensed from a plurality of dispense probes. For example, a first reagent in a solution may be dispensed from a first dispense probe, a second reagent in a solution may be dispensed from a second dispense probe, and a third reagent in a solution may be dispensed from a third dispense probe.
  • the reagents dispensed from different dispense probes may combine on the substrate to form a homogenous or substantially homogenous solution.
  • the dispense probe may be positioned at a fixed height above a substrate (e.g., a wafer).
  • the reagent may be dispensed on the surface in any desired pattern or path. This may be achieved by moving one or both of the substrate and the dispense nozzle.
  • the reagent e.g., beads
  • the substrate and/or the dispense probe may have angular and/or linear velocity with respect to each other.
  • the substrate may be configured to move in any vector with respect to a reference point.
  • the systems, devices, and apparatus described herein may further comprise a motion unit configured to move the substrate.
  • the motion unit may comprise any mechanical component, such as a motor, rotor, actuator, linear stage, drum, roller, pulleys, etc., to move the substrate. Such components may be mechanically connected to the substrate directly or indirectly via intermediary components (e.g., gears, stages, actuators, discs, pulleys, etc.).
  • the motion unit may be automated. Alternatively, or in addition, the motion unit may receive manual input.
  • the substrate may be configured to move with any velocity. In some instances, the substrate may be configured to move with different velocities during different operations described herein.
  • the substrate may be configured to move with a velocity that varies according to a time-dependent function, such as a ramp, sinusoid, pulse, or other function or combination of functions.
  • the time-varying function may be periodic or aperiodic.
  • the substrate may be configured to move along an x-axis, a y-axis, a z-axis, or any combination thereof, where an x-axis and y-axis are substantially parallel to a surface plane of the substrate and a z-axis is substantially normal to the surface plane of the substrate.
  • a solution may be provided to the substrate prior to or during motion of the substrate to inertially direct the solution across the array on the substrate.
  • the surface-reagent exchange may comprise washing, in which each subsequent pulse comprises a reduced concentration of the surface reagent.
  • the solution may have a temperature different than that of the substrate, thereby providing a source or sink of thermal energy to the substrate or to an analyte or reagent located on the substrate.
  • the thermal energy may provide a temperature change to the substrate or to the analyte or reagent.
  • the temperature change may be transient.
  • the temperature change may enable, disable, enhance, or inhibit a chemical reaction, such as a chemical reaction to be carried out upon the analyte.
  • the chemical reaction may comprise denaturation, hybridization, or annealing of nucleic acid molecules.
  • the chemical reaction may comprise a step in a polymerase chain reaction (PCR). bridge amplification, or other nucleic acid amplification reaction.
  • PCR polymerase chain reaction
  • bridge amplification or other nucleic acid amplification reaction.
  • the temperature change may modulate, increase, or decrease a signal detected from the analyte (or from probes in the solution).
  • the surface of the substrate may be in fluid communication with at least one fluid nozzle (of a fluid channel).
  • the surface may be in fluid communication with the fluid nozzle via a non-solid gap, e.g., an air gap.
  • the surface may additionally be in fluid communication with at least one fluid outlet.
  • the surface may be in fluid communication with the fluid outlet via an airgap.
  • the nozzle may be configured to direct a solution to the array.
  • the outlet may be configured to receive a solution from the array.
  • the solution may be directed to the array using one or more dispensing nozzles. For example, the solution may be directed to the array using at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 dispensing nozzles.
  • the solution may be directed to the array using a number of nozzles that is within a range defined by any two of the preceding values.
  • different reagents e.g., nucleotide solutions of different types, different probes, washing solutions, etc.
  • Each nozzle may be connected to a dedicated fluidic line or fluidic valve, which may further prevent contamination.
  • a type of reagent may be dispensed via one or more nozzles.
  • the one or more nozzles may be directed at or in proximity to a center of the substrate. Alternatively, the one or more nozzles may be directed at or in proximity to a location on the substrate other than the center of the substrate.
  • one or more nozzles may be directed closer to the center of the substrate than one or more of the other nozzles.
  • one or more nozzles used for dispensing washing reagents may be directed closer to the center of the substrate than one or more nozzles used for dispensing active reagents.
  • the one or more nozzles may be arranged at different radii from the center of the substrate.
  • Two or more nozzles may be operated in combination to deliver fluids to the substrate more efficiently.
  • One or more nozzles may be configured to deliver fluids to the substrate as a jet, spray (or other dispersed fluid), and/or droplets.
  • One or more nozzles may be operated to nebulize fluids prior to delivery’ to the substrate.
  • the fluids may be delivered as aerosol particles.
  • a surfactant may be added to the solution, or a surfactant may be added to the surface to facilitate uniform coating or to facilitate sample loading efficiency.
  • a surfactant may be added to the solution, or a surfactant may be added to the surface to facilitate uniform coating or to facilitate sample loading efficiency.
  • Such optimization may prevent the solution from exiting the substrate along a relatively focused stream or travel path such that the fluid only contacts the substrate at partial surface areas (as opposed to the entire surface area) — in such cases, a significantly larger volume of reagents may have to be dispensed to achieve uniform and full coating of the substrate.
  • Such optimization may also prevent the solution from scattering or otherwise reflecting or bouncing off the substrate upon contact and disturbing the surface fluid.
  • the thickness of the solution may be adjusted using mechanical, electric, physical, or other mechanisms. For example, the solution may be dispensed onto a substrate and subsequently leveled using, e.g., a physical scraper such as a squeegee, to obtain a desired thickness of uniform
  • the solution may be heated prior to being dispensed on the substrate.
  • the solution may be at a higher temperature than the ambient temperature.
  • the solution may be heated to about 25° C, 26° C, 27° C, 28° C, 29° C, 30° C, 31° C, 32° C, 33° C, 34° C, 35° C, 36° C, 37° C, 38° C, 39° C, 40° C, 45° C, 50° C, 55° C, 60° C, 65° C, 70° C, 75° C, 80° C, 85° C, 90° C, 95° C prior to dispensing.
  • a solution may be heated to a temperature that is within a range defined by any two of the preceding values.
  • One or more solutions dispensed on a surface may undergo a reaction on the surface.
  • a first solution e.g., comprising a reactant
  • a second solution e.g., comprising an enzyme
  • One or more solutions dispensed on a surface may deactivate or quench a chemical reaction.
  • a quenching solution e.g., comprising EDTA or an acid
  • a solution (e.g., a solution comprising a reactant, a solution comprising an enzyme, or a quenching solution) may be dispensed on the surface in a pattern (e.g., a spiral pattern).
  • a quenching solution is dispensed on the surface in the same pattern as a solution comprising a reactant, thereby maintaining a substantially constant reaction time at each point on the surface to which a solution is dispensed.
  • a quenching solution is dispensed on the surface in the same pattern as a solution comprising an enzyme, thereby maintaining a substantially constant reaction time at each point on the surface to which a solution is dispensed.
  • one or more solutions dispensed on a surface may activate or catalyze a chemical reaction.
  • an activating solution e.g., comprising catalysts, enzymes, primers, etc.
  • a reaction e.g., in the same dispense pattern
  • a variety of methods may be employed to dispense one or more solutions onto a substrate to ensure a substantially similar reaction time across an area of the substrate in contact with the one or more solutions.
  • a solution may be spin-coated onto a surface by dispensing the solution at or near the axis of rotation of a rotating substrate such that the centrifugal force of the rotating substrate facilitates the outward spread of the solution away from the axis of rotation.
  • Spin-coating may be well-suited for dispensing one or more solutions that initiate or quench a reaction that occurs on a time scale that is slow relative to the dispensing time.
  • one or more solutions may be delivered directly to the reaction site without substantial displacement of the one or more solution from the point of deliver ⁇ '.
  • Methods of direct delivery' of a solution to the reaction site may include aerosol delivery of the solution, applying the solution using an applicator, curtain-coating the solution, slot-die coating, dispensing the solution from a translating dispense probe, dispensing the solution from an array of dispense probes, dipping the substrate into the solution, or contacting the substrate to a sheet comprising the solution.
  • Aerosol delivery may comprise delivering a solution to the substrate in aerosol form by directing the solution to the substrate using a pressure nozzle or an ultrasonic nozzle.
  • Applying the solution using an applicator may comprise contacting the substrate with an applicator comprising the solution and translating the applicator relative to the substrate.
  • Apply ing the solution using an applicator may comprise painting the substrate.
  • the solution may be applied in a pattern by translating the applicator, rotating the substrate, translating the substrate, or a combination thereof.
  • the pattern may be a spiral pattern.
  • the pattern may be a circular pattern.
  • Curtain-coating may comprise dispensing the solution from a dispense probe to the substrate in a continuous stream (e.g., a curtain or a flat sheet) and translating the dispense probe relative to the substrate.
  • a solution may be curtain-coated in a pattern by translating the dispense probe, rotating the substrate, translating the substrate, or a combination thereof.
  • the pattern may be a spiral pattern.
  • the pattern may be a circular pattern.
  • Slot-die coating may comprise dispensing the solution from a dispense probe positioned near the substrate such that the solution forms a meniscus between the substrate and the dispense probe and translating the dispense probe relative to the substrate.
  • a solution may be slot-die coated in a patern by translating the dispense probe, rotating the substrate, translating the substrate, or a combination thereof.
  • the patern may be a spiral patern.
  • the patern may be a circular patern.
  • Dispensing the solution from a translating dispense probe may comprise translating the dispense probe relative to the substrate in a patern (e.g., a spiral patern, a circular patern, a linear patern, a striped pattern, a cross-hatched patern, or a diagonal patern).
  • Dispensing the solution from an array of dispense probes may comprise dispensing the solution from an array of nozzles (e.g...
  • Dipping the substrate into the solution may comprise dipping the substrate into a reservoir comprising the solution.
  • the reservoir may be a shallow reservoir to reduce the volume of the solution required to coat the substrate.
  • Contacting the substrate to a sheet comprising the solution may comprise bringing the substrate in contact with a sheet of material (e.g., a porous sheet or a fibrous sheet) permeated with the solution.
  • the solution may be transferred to the substrate.
  • the sheet of material may be a single-use sheet. In some embodiments, the sheet of material may be a reusable sheet.
  • a solution may be incubated on the substrate.
  • the solution may be incubated on the substrate under conditions that maintain a layer of fluid on the surface.
  • the solution may be incubated for at least about 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, or 90 min.
  • the incubation time may be within a range defined by any two of the preceding values. In some cases, the incubation may be for more than 90 minutes.
  • the layer of fluid may maintain a film thickness of at least 10 nanometers (nm), 20 nm, 50 nm, 100 nm, 200 nm. 500 nm, 1 micrometer (pm), 2 pm, 5 pm, 10 pm, 20 pm, 50 pm, 100 pm. 200 pm, 500 pm. or 1 mm during incubation.
  • One or more of the temperature of the chamber, the humidity of the chamber, the motion of the substrate, or the composition of the fluid may be adjusted such that the layer of fluid is maintained during incubation.
  • the substrate or a surface thereof may comprise other features that aid in solution or reagent retention on the substrate or thickness uniformity of the solution or reagent on the substrate.
  • the surface may comprise a raised edge (e.g., a rim) which may be used to retain solution on the surface.
  • the surface may comprise a rim near the outer edge of the surface, thereby reducing the amount of the solution that flows over the outer edge.
  • the solution may comprise any sample or any analyte disclosed herein.
  • the solution may comprise any reagent disclosed herein.
  • the solution may be a reaction mixture comprising a variety of components.
  • the solution may comprise a plurality of probes configured to interact with the analyte.
  • the probes may have binding specificity to the analyte.
  • the probes may not have binding specificity to the analyte.
  • a probe may be configured to permanently couple to the analyte.
  • a probe may be configured to transiently couple to the analyte.
  • a nucleotide probe may be permanently incorporated into a growing strand hybridized to a nucleic acid molecule analyte.
  • a nucleotide probe may transiently bind to the nucleic acid molecule analyte.
  • Transiently coupled probes may be subsequently removed from the analyte. Subsequent removal of the transiently coupled probes from an analyte may or may not leave a residue (e.g., chemical residue) on the analyte.
  • a type of probe in the solution may depend on the type of analyte.
  • a probe may comprise a functional group or moiety configured to perform specific functions.
  • a probe may comprise a label (e.g., dye).
  • a probe may be configured to generate a detectable signal (e.g., optical signal), such as via the label, upon coupling or otherwise interacting with the analyte.
  • a probe may be configured to generate a detectable signal upon activation (e.g., application of a stimulus).
  • a nucleotide probe may comprise reversible terminators (e.g., blocking groups) configured to terminate polymerase reactions (until unblocked).
  • the solution may comprise other components to aid, accelerate, or decelerate a reaction between the probe and the analyte (e.g., enzymes, catalysts, buffers, saline solutions, chelating agents, reducing agents, other agents, etc.).
  • the solution may be a washing solution.
  • a washing solution may be directed to the substrate to bring the washing solution in contact with the array after a reaction or interaction between reagents (e.g., a probe) in a reaction mixture solution with an analyte immobilized on the array.
  • the washing solution may wash away any free reagents from the previous reaction mixture solution.
  • the solution may comprise a cleaving agent, such as to cleave a label and/or a blocking group, and/or otherwise act on a cleavage site (e.g., to cleave a sequence).
  • the probe may be configured to interact with any other reagent described herein, for example a reagent immobilized to an individually addressable location.
  • a reagent immobilized to an individually addressable location for example a reagent immobilized to an individually addressable location.
  • an analyte in one processing experiment may be used as a reagent for another processing experiment.
  • the different processing experiments may be performed on the same substrate or different substrates.
  • a bead comprising an oligonucleotide molecule comprising a barcode sequence may be immobilized to an individually addressable location on the substrate, the oligonucleotide molecule may be interrogated as the analyte by one or more probes (e.g., so as to identify the barcode sequence, such as to index the individually addressable location with the barcode sequence), a sample may be loaded onto the substrate (e.g., such as over the bead), and then the bead comprising the oligonucleotide molecule immobilized to the individually addressable location may be used to capture another analyte (e.g., nucleic acid molecule, e.g., mRNA transcript) at the individually addressable location (e.g., so as to tag the other analyte with the barcode sequence).
  • analyte e.g., nucleic acid molecule, e.g., mRNA transcript
  • the tagged analyte may be collected from the substrate, processed (e.g.. released from the bead and amplified on another bead such that the other bead comprises an amplification product of the tagged analyte), and it or its derivative reloaded onto another substrate for interrogation by one or more probes such as to determine a sequence of the tagged analyte or its derivative.
  • a detectable signal such as an optical signal (e.g., fluorescent signal) may be generated upon reaction between a probe in the solution and the analyte.
  • the signal may originate from the probe and/or the analyte.
  • the detectable signal may be indicative of a reaction or interaction between the probe and the analyte.
  • the detectable signal may be a non-optical signal.
  • the detectable signal may be an electronic signal.
  • the detectable signal may be detected by one or more sensors.
  • an optical signal may be detected via one or more optical detectors in an optical detection scheme described elsewhere herein.
  • the signal may be detected during motion of the substrate.
  • the signal may be detected following termination of the motion of the substrate.
  • the signal may be detected while the analyte is in fluid contact with the solution.
  • the signal may be detected following washing of the solution.
  • the signal may be muted, such as by cleaving a label from the probe and/or the analyte, and/or modifying the probe and/or the analyte. Such cleaving and/or modification may be affected by one or more stimuli, such as exposure to a chemical, an enzyme, light (e.g., ultraviolet light), or temperature change (e.g., heat).
  • the signal may otherwise become undetectable by deactivating or changing the mode (e.g., detection wavelength) of the one or more sensors, or terminating or reversing an excitation of the signal.
  • detection of a signal may comprise capturing an image or generating a digital output (e.g., between different images).
  • the additional signals detected may provide incremental, or final, data about the analyte during the processing.
  • the analyte is a nucleic acid molecule and the processing is sequencing
  • additional signals detected for each iteration may be indicative of a base in the nucleic acid sequence of the nucleic acid molecule.
  • the operations may be repeated 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, or 1,000,000,000 cycles to process the analyte.
  • a different solution may be directed to the substrate for each cycle.
  • 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, or 1,000,000,000 solutions may be directed to the substrate.
  • a washing solution may be directed to the substrate between each cycle (or at least once during each cycle). For instance, a washing solution may be directed to the substrate after each type of reaction mixture solution is directed to the substrate.
  • the washing solutions may be distinct.
  • the washing solutions may be identical. For example, 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, or 1,000,000,000 washing solutions may be directed to the substrate.
  • the nucleic acid molecule may be subjected to a primer extension reaction under conditions sufficient to incorporate or specifically bind at least one nucleotide from the plurality of nucleotides into a growing strand that is complementary to the nucleic acid molecule.
  • a signal indicative of incorporation or binding of at least one nucleotide may be detected, thereby sequencing the nucleic acid molecule.
  • Embodiment 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
  • Embodiment 9 The method of any one of embodiments 2-6, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced without being attached to any substrate.
  • Embodiment 10 The method of any one of embodiments 1-9, further comprising sequencing an additional subset of the plurality of composite molecules that did not capture any analyte sequence, or derivatives thereof.
  • Embodiment 12 The method of any one of embodiments 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.
  • Embodiment 21 The method of any one of embodiments 1-20, wherein the first set of beads comprises at least 1000 different spatial tags.
  • Embodiment 23 The method of any one of embodiments 1-22. wherein the first set of beads immobilized to the substrate comprises at least 1,000,000 beads.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.
  • Embodiment 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 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 oligonucleot
  • Embodiment 28 The method of embodiment 27, 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.
  • Embodiment 29 The method of embodiments 27-28, wherein the spatially tagged analyte molecules or derivatives thereof are amplified on the substrate prior to the sequencing.
  • Embodiment 30 The method of embodiments 27-28. wherein the spatially tagged analyte molecules or derivatives thereof are amplified off the substrate prior to the sequencing.
  • Embodiment 31 The method of any one of embodiments 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.
  • Embodiment 32 The method of any one of embodiments 27-31. wherein the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to the substrate.
  • Embodiment 33 The method of any one of embodiments 27-31, wherein the spatially tagged analyte molecules or derivatives thereof are sequenced while attached to a second substrate different from the substrate.
  • Embodiment 36 The method of any one of embodiments 26-35, wherein in (a) the first set of beads are immobilized to a plurality of individually addressable locations on the substrate.
  • Embodiment 37 The method of any one of embodiments 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.
  • Embodiment 39 The method of any one of embodiments 26-38, wherein the plurality of second oligonucleotide molecules comprises an analyte capture sequence, and wherein in (d) the plurality' of analyte sequences areis captured via the analyte capture sequence.
  • Embodiment 40 The method of embodiment 39, wherein the analyte capture sequence comprises a poly-T sequence, a targeted sequence, a randomer sequence, or reverse complements thereof.
  • Embodiment 41 The method of any one of embodiments 26-40. 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.
  • mRNA messenger ribonucleic acid
  • Embodiment 42 The method of any one of embodiments 26-41, further comprising fixing said sample.
  • Embodiment 45 The method of any one of embodiments 26-44. wherein the first set of beads comprises at least 100 different spatial tags.
  • Embodiment 46 The method of any one of embodiments 26-45, wherein the first set of beads comprises at least 1000 different spatial tags.
  • Embodiment 49 The method of any one of embodiments 26-48, wherein the first set of beads immobilized to the substrate comprises at least 100,000,000 beads.
  • Embodiment 50 The method of any one of embodiments 26-49, wherein the first set of beads immobilized to the substrate comprises at least 1.000,000,000 beads.

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Abstract

La présente invention concerne des procédés, des systèmes, des compositions et des kits qui peuvent déterminer des informations spatiales entre une pluralité d'analytes dans un échantillon par marquage desdits analytes avec des étiquettes, les identités et/ou les emplacements desdites étiquettes étant préalablement inconnus avant le marquage. Chaque séquence d'une pluralité de séquences d'analytes peut être marquée avec de multiples marqueurs spatiaux, de sorte que chaque séquence d'analytes est associée à un ensemble d'au moins deux marqueurs spatiaux. Les ensembles d'étiquettes spatiales peuvent être analysés pour générer une carte de séquences d'analytes. La carte peut comprendre des informations concernant les positions absolues respectives de chaque séquence d'un ensemble de séquences par rapport à une séquence de référence. La carte peut comprendre des informations concernant le nuage de probabilité respectif (ou l'emplacement probable) de chaque séquence d'un ensemble de séquences par rapport à une séquence de référence.
PCT/US2025/023299 2024-04-05 2025-04-04 Systèmes et procédés de séquençage de référence spatiale Pending WO2025213126A2 (fr)

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