Spatial omics based on barcode diffusion

Abstract

Provided herein are compositions, kits, and methods for collecting, integrating, and analyzing various aspects of information from tissue at cellular or subcellular levels. The information includes spatial mappings from the nucleic acid barcodes to reconstruct locations of nodes in the sample where the nucleic acid barcodes are generated. In some workflows, light-based techniques are incorporated into additional layers for selective spatial tagging of areas. In a further step, such tags are optionally analyzed by high throughput imaging or next generation sequencing.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 390,498, filed on July 19, 2022, and U.S. Provisional Application No. 63 / 509,190, filed on June 20, 2023, the entire contents of which are incorporated herein by reference.

[0003] background

[0004] Understanding how molecules are arranged in cells, and how cells are arranged in tissues, is essential for explaining, predicting, and engineering biological states. Advances in microscopy have made it possible to reconstruct subcellular structures with high resolution (e.g., electron microscopy), but the composition of biomolecules in such structures remains difficult to capture. Several methods are capable of recording paired molecular proximity (FRET, APR, DNAnanoscope, DNA microscopy), but these methods are typically limited to specific biomolecules (e.g., two proteins detected with antibodies) and cannot be generalized for measurement of arbitrary biomolecule distributions. Therefore, there is a need for methods for measuring the spatial position and conformation of biomolecules from angstrom scale to supercellular interactions at millimeter scale.

[0005] Brief description

[0006] The present invention provides a method for generating biological information, which includes providing a node factory at a node position on a biological sample, wherein the node factory contains one or more node nucleic acids, and wherein each node nucleic acid contains: a node barcode region, one or more flanking node barcode hybridization regions and optionally a node primer region; amplifying the one or more node nucleic acids to generate multiple concatemers of node nucleic acids, wherein the multiple concatemers diffuse away from the node position over time; cleaving the multiple concatemers into more than one node nucleic acid; attaching two or more node nucleic acids generated by the node factory to generate multiple node nucleic acids; and analyzing the frequency of association of the two or more node nucleic acids, wherein the association frequency provides information for spatial mapping of the biological sample.

[0007] The present invention provides a method for generating biological information, which includes: depositing a template nucleic acid at a node position on a biological sample, wherein the template nucleic acid comprises: a node barcode region; and one or more flanking node barcode hybridization regions; amplifying the template nucleic acid to generate a node nucleic acid, wherein the node nucleic acid diffuses away from the node position over time; depositing a factory target nucleic acid onto the biological sample, wherein the factory target nucleic acid comprises: a target binding region; and one or more flanking target binding hybridization regions; and attaching at least one of the node nucleic acids to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node nucleic acid and the factory target nucleic acid provides biological information for spatially mapping the biological sample.

[0008] The present invention provides a method for information storage, which includes: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a template nucleic acid at a node position on a surface, wherein the template nucleic acid comprises: a node barcode region, each corresponding to one of the more than one barcodes; and one or more flanking node barcode hybridization regions; amplifying the template nucleic acid to generate a node nucleic acid; depositing a factory target nucleic acid onto the surface, wherein the factory target nucleic acid comprises: a target binding region; one or more flanking target binding hybridization regions; optionally, a target primer region; and attaching at least one of the node nucleic acids to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node nucleic acid and the factory target nucleic acid provides the stored information.

[0009] Provided herein is a method for information storage, the method comprising: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a first oligonucleotide onto a surface, wherein the first oligonucleotide comprises: optionally, a surface binding region; a first barcode region corresponding to a barcode in the more than one barcodes; and one or more flanking first oligonucleotide hybridization regions, wherein the surface binding region and the one or more flanking second oligonucleotide hybridization regions optionally comprise photoreactive nucleobases; optionally selectively irradiating the surface to form covalently linked oligonucleotides; depositing a second oligonucleotide on the surface, wherein the second oligonucleotide comprises: a second barcode region corresponding to a barcode in the more than one barcodes; and one or more flanking second oligonucleotide hybridization regions, wherein the one or more flanking second oligonucleotide hybridization regions comprise photoreactive nucleobases, and wherein at least one of the one or more flanking first oligonucleotide hybridization regions is complementary to at least one of the one or more flanking second oligonucleotide hybridization regions; and selectively irradiating the surface to covalently link the second oligonucleotide to the first oligonucleotide; and optionally repeating the second depositing step and irradiating step one or more times, thereby generating concatemers.

[0010] The present invention provides a method for generating biological information, which includes depositing a node factory comprising a matrix at a node position on a biological sample, wherein each node factory contains more than one node nucleic acid, and wherein each node nucleic acid contains: a node barcode region; one or more flanking node barcode hybridization regions; optionally, a node primer region, a target binding region, a target barcode region; one or more flanking target binding hybridization regions; and optionally, a target primer region, which releases at least one node nucleic acid from the node factory, wherein the release provides biological information for spatially mapping the biological sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments utilizing the principles of the present invention, wherein:

[0013] Figure 1A The concentration gradient in the diffusion cloud of the barcode chain generated from the node is illustrated.

[0014] Figure 1B The expansion of the diffusion cloud over time for four individual node locations generated from the barcode is illustrated.

[0015] Figure 1C Illustrated are possible combinations of barcodes used to identify a chain generated from a node factory, including a node type barcode, a node barcode, and a unique identifier.

[0016] Figure 2 Exemplary pairs or multiple concatenations generated from overlapping diffuse clouds are illustrated.

[0017] Figure 3 Representative modes of concatemerization are shown, including ligation, splinted ligation, cross-junction synthesis, ligation and cross-interstrand crosslink (ICL) synthesis, cross-ICL and nick synthesis, and paired copies.

[0018] Figure 4 Illustration of reconstruction of 2D or 3D spatial arrangements of node positions calculated from concatemerization of node barcodes in overlapping diffusion clouds.

[0019] Figure 5A Schematic representation of the generation of a promoter activation node barcode protected by a photocage.

[0020] Figure 5B Illustration of promoter-activated nodal barcode generation using photocrosslinking.

[0021] Figure 6The diagram shows the binding of a promoter or primer to the barcode strand of a template node.

[0022] Figure 7 Amplification of the template node barcode strand containing the promoter region is shown.

[0023] Figure 8 Amplification of the node barcode strand comprising primers is shown.

[0024] Fig. 9 Amplification of the node barcode strand containing primers and nicking sites is shown.

[0025] Fig.10 Amplification of the node barcode strand by primer exchange reaction (PER) is shown.

[0026] Fig.11 Amplification of a node barcode strand is illustrated, wherein the hybridized primer comprises a domain upstream of the primer domain, and wherein the upstream domain is included in the resulting transcript.

[0027] Fig.12 Node barcode templates comprising unique modifier domains 3' or 5' to the node barcode domain are depicted.

[0028] Fig.13A and Fig. 13B Diagram shows the process workflow incorporating sample preparation, light-based in situ barcoding of populations of cells, and transcript extraction and sequencing.

[0029] Fig. 13C Illustration of the workflow provided for fractional spatial barcoding.

[0030] Fig.14A and Fig. 14B The diagram shows Fig. 13C Schemes for fraction barcoding during or after the workflow described in .

[0031] Fig.15 Illustrated are exemplary combinations of node barcode strands and target barcode strands that also include unique identifiers.

[0032] Fig.16 Possible combinations and barcode sequences in concatemers are provided.

[0033] Fig.17 A photo-directed cross-linking reaction as described herein is illustrated.

[0034] Fig.18 Diagram shows an iterative barcode addition method in which a barcode strand is added to the 5' end of the cDNA.

[0035] Fig.19A Illustration of photo-directed cross-linking used to generate barcoded concatemers.

[0036] Fig.19B Data reconstruction from barcode concatemers is shown.

[0037] Fig. 20 The diagram illustrates the process of cross-linking.

[0038] Fig.21 Schematic representation of the workflow for cross-linking across barcode concatemers to generate continuous nucleic acids.

[0039] Fig. 22 The combination of unique identifiers from different regions within the node barcode diffusion cloud and amplified node barcode chains is shown.

[0040] Fig.23A Illustrated is polymerization of a node barcode strand comprising primers and node barcodes hybridized to an immobilized target nucleic acid strand comprising a target barcode, unique identifier and linker, affinity region, modification, crosslinking and / or PCR primer to generate a double-stranded nucleic acid comprising regions from both strands.

[0041] Fig. 23B Illustrated is polymerization of a node barcode strand comprising a node barcode hybridized to an immobilized target nucleic acid strand comprising a target barcode, unique identifier and linker, affinity region, modification, crosslinking and / or PCR primer to generate a double-stranded nucleic acid comprising regions from both strands.

[0042] Fig.23C Illustration of polymerization of a node barcode strand comprising a primer, a node barcode, and a non-hybridizing domain 3' to the hybridization sequence hybridized to an immobilized target nucleic acid strand comprising a target barcode, a unique identifier and linker, affinity region, modification, crosslinking and / or PCR primer, wherein the target nucleic acid strand is extended to generate a single-stranded combined sequence.

[0043] Fig.23D Illustration of polymerization of a node barcode chain without primers, comprising a node barcode and a non-hybridizing domain 3' to a hybridization sequence, hybridized to an immobilized target nucleic acid chain comprising a target barcode, a unique identifier and linker, affinity region, modification, crosslinking and / or PCR primer, wherein the target nucleic acid chain is extended to generate a single-stranded combined sequence.

[0044] Fig.23E Illustration of polymerization of a node barcode strand comprising a primer and a node barcode hybridized to an immobilized target nucleic acid strand comprising a non-hybridizing domain 3' to a hybridization sequence, a target barcode, a unique identifier and a linker, an affinity region, modifications, crosslinks and / or PCR primers, wherein the node barcode strand is extended to generate a single-stranded combined sequence.

[0045] Fig.23FIllustration of polymerization of a primer-free, node barcode-containing node barcode chain hybridized to an immobilized target nucleic acid chain containing a non-hybridizing domain 3' to a hybridization sequence, a target barcode, a unique identifier and linker, an affinity region, modifications, crosslinks and / or PCR primers, wherein the target nucleic acid chain is extended to generate a single-stranded combined sequence.

[0046] Figure 24A-24C An exemplary embodiment of in situ proximity recording of target RNA transcripts using cDNA is illustrated, wherein a node barcode nucleic acid comprising a first primer, a node barcode, and a hybridization sequence is hybridized to a target nucleic acid comprising a hybridization sequence, a cDNA complementary to the target RNA, a unique identifier, and a second PCR primer.

[0047] Fig.24A The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0048] Fig. 24B Illustrated is polymerization in which the node barcode strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the target nucleic acid to generate a single-stranded nucleic acid containing regions from both strands.

[0049] Fig.24C Illustrated is polymerization in which the target nucleic acid strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the node barcode strand to generate a single-stranded nucleic acid containing regions from both strands.

[0050] Figure 24D-Figure 24F An exemplary embodiment of in situ proximity recording of target RNA transcripts using cDNA is illustrated, wherein a node barcode nucleic acid comprising a first primer, a unique identifier, and a node barcode is hybridized to a target nucleic acid comprising a cDNA complementary to the target RNA, a unique identifier, and a second PCR primer.

[0051] Fig.24D The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0052] Fig.24E Illustrated is a polymerization in which a node barcode strand comprises a non-hybridizing domain 3' to a hybridizing sequence and a target nucleic acid is extended to generate a single-stranded nucleic acid comprising regions from both strands.

[0053] Fig.24F Illustrated is a polymerization in which the target nucleic acid strand contains a non-hybridizing domain 3' to the hybridization sequence and the node barcode strand is extended to generate a single-stranded nucleic acid containing regions from both strands.

[0054] Figure 25A-Figure 25CAn exemplary embodiment of in situ proximity recording of target RNA transcripts using ISH probes is illustrated, wherein a node barcode nucleic acid comprising a first primer, a unique identifier, and a node barcode hybridizes to a target nucleic acid comprising a cDNA complementary to the target RNA, a unique identifier, and a second PCR primer.

[0055] Fig.25A The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0056] Fig.25B Illustrated is polymerization in which the node barcode strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the target nucleic acid to generate a single-stranded nucleic acid containing regions from both strands.

[0057] Fig.25C Illustrated is polymerization in which the target nucleic acid strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the node barcode strand to generate a single-stranded nucleic acid containing regions from both strands.

[0058] Figure 25D-Figure 25F An exemplary embodiment of in situ proximity recording of target RNA transcripts using an ISH probe is illustrated, wherein a node barcode nucleic acid comprising a first primer, a unique identifier, a node barcode, and a hybridization sequence is hybridized to a target nucleic acid comprising a hybridization sequence, an ISH probe complementary to the target RNA, a unique identifier, and a second PCR primer.

[0059] Fig.25D The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0060] Fig.25E Illustrated is polymerization in which the node barcode strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the target nucleic acid to generate a single-stranded nucleic acid containing regions from both strands.

[0061] Fig.25F Illustrated is polymerization in which the target nucleic acid strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the node barcode strand to generate a single-stranded nucleic acid containing regions from both strands.

[0062] Figure 26A-26C An exemplary embodiment of in situ proximity recording of a protein by binding an antibody to a target protein is illustrated, wherein the antibody is conjugated to a hybridization sequence, and a node barcode nucleic acid comprising a first primer and a node barcode is hybridized to a target nucleic acid comprising a barcode complementary to the conjugated hybridization sequence, a unique identifier, and a second PCR primer.

[0063] Fig.26A The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0064] Fig.26B The polymerization of extending a target nucleic acid to generate a single-stranded nucleic acid containing regions from both strands is illustrated.

[0065] Fig.26C The polymerization of extending a node barcode strand to generate a single-stranded nucleic acid containing regions from both strands is illustrated.

[0066] Figure 26D-Figure 26F An exemplary embodiment of in situ proximity recording of a protein by binding an antibody to a target protein is illustrated, wherein the antibody is conjugated to a target nucleic acid, wherein a node barcode nucleic acid comprising a first primer and a node barcode is hybridized to a target nucleic acid comprising a barcode, a unique identifier, and a second PCR primer.

[0067] Fig.26D The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0068] Fig.26E Illustrated is polymerization in which the node barcode strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the target nucleic acid to generate a single-stranded nucleic acid containing regions from both strands.

[0069] Fig.26F Illustrated is polymerization in which the target nucleic acid strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the node barcode strand to generate a single-stranded nucleic acid containing regions from both strands.

[0070] Figure 26G-Figure 26I An exemplary embodiment of in situ proximity recording of a protein by binding an antibody to a target protein is illustrated, wherein the antibody is conjugated to a target nucleic acid, wherein a node barcode nucleic acid comprising a first primer, a unique modifier, a node barcode, and a hybridization sequence is hybridized to a target nucleic acid comprising a hybridization sequence, a barcode, a unique identifier, and a second PCR primer.

[0071] Figure 26G The polymerization of extending both strands to generate a double-stranded nucleic acid comprising a region from both strands is illustrated.

[0072] Fig.26H Illustrated is polymerization in which the node barcode strand contains a non-hybridizing domain 3' to the hybridization sequence, which extends the target nucleic acid to generate a single-stranded nucleic acid containing regions from both strands.

[0073] Fig.26I The polymerization of extending a node barcode strand to generate a single-stranded nucleic acid containing regions from both strands is illustrated.

[0074] Figure 27A-27GAn exemplary embodiment of in situ proximity recording of target RNA or DNA transcripts using cDNA or ISH probes is illustrated, wherein a node barcode nucleic acid comprising a first primer, a node barcode, and a hybridization domain is hybridized to a target nucleic acid comprising a hybridization domain and a cDNA or ISH probe complementary to the target RNA, and the probe is photocrosslinked to a strand comprising the barcode and an additional unique identifier and a second PCR primer.

[0075] Fig.27A An exemplary hybridization scheme is illustrated.

[0076] Fig.27B An exemplary hybridization scheme is illustrated, wherein the node barcode strand also comprises a non-hybridizing domain 3' to the hybridizing domain.

[0077] Fig.27C An exemplary hybridization scheme is illustrated wherein the cDNA / ISH probe strand further comprises a non-hybridizing region 3' to the hybridization domain.

[0078] Fig.27D An exemplary hybridization scheme is illustrated, wherein the hybridization domain comprises a first PCR primer and a node barcode domain, and wherein the node barcode strand further comprises a non-hybridizing region 3' to the hybridization domain.

[0079] Fig.27E An exemplary hybridization scheme is illustrated wherein the node barcode strand also comprises a unique modification.

[0080] Fig.27F An exemplary hybridization scheme is illustrated, wherein the node barcode strand further comprises a unique modification, and wherein the node barcode strand further comprises a non-hybridizing domain 3' to the hybridizing domain.

[0081] Figure 27G An exemplary hybridization scheme is illustrated, wherein the node barcode strand further comprises a unique modification, and wherein the cDNA / ISH probe strand further comprises a non-hybridizing domain 3' to the hybridizing domain.

[0082] Fig.28A The node barcode strand is illustrated comprising PCR primers, the node barcode region, and a poly-T 3' tail that serves as a primer for reverse transcription of mRNA sequences.

[0083] Fig.28B Diagram shows a node barcode strand comprising PCR primers, unique modifiers, the node barcode region, and a poly-T 3' tail that serves as a primer for reverse transcription of mRNA sequences.

[0084] Fig.28C Diagram shows the node barcode strand comprising PCR primers, the node barcode region, and the 5N3G 3' tail that serves as a primer for reverse transcription of the mRNA sequence.

[0085] Fig.28D Diagram shows the node barcode strand comprising PCR primers, unique modifiers, the node barcode region, and a 5N3G 3' tail that serves as a primer for reverse transcription of mRNA sequences.

[0086] Fig.29A Illustration of a node barcode strand containing a docking sequence, a node barcode region, and a poly-T3' tail that serves as a primer for reverse transcription of an mRNA sequence, followed by photo-directed barcoding to photo-crosslink a strand containing a PCR primer, a unique identifier, and another barcode sequence.

[0087] Fig.29B Illustration of a node barcode strand containing a docking sequence, a unique modifier, a node barcode region, and a poly-T 3' tail that serves as a primer for reverse transcription of an mRNA sequence, followed by photo-directed barcoding to photo-crosslink a strand containing a PCR primer, a unique identifier, and another barcode sequence.

[0088] Fig.29C Illustration of a node barcode strand containing a docking sequence, a node barcode region, and a 5N3G 3' tail that serves as a primer for reverse transcription of an mRNA sequence, followed by photo-directed barcoding to photo-crosslink a strand containing a PCR primer, a unique identifier, and another barcode sequence.

[0089] Fig.29D Illustration of a node barcode strand containing a docking sequence, a unique modifier, a node barcode region, and a 5N3G 3' tail that serves as a primer for reverse transcription of an mRNA sequence, followed by photo-directed barcoding to photo-crosslink a strand containing a PCR primer, a unique identifier, and another barcode sequence.

[0090] Fig.30 The diagram shows the coding combination of four barcode chains using the binary coding method.

[0091] Fig.31 The diagram shows the encoding combination of two barcode chains using the quaternary encoding method.

[0092] Fig.32 The diagram shows the encoding combination of two barcode chains using the fractional encoding method.

[0093] Fig.33 The process workflow of the technology stack based on DNA array is illustrated. Digital data is converted into nucleic acids and then undergoes molecular writing via DNA lithography. Subsequently, the options for reading include molecular reading via imaging or molecular reading via next generation sequencing. After error correction, the generated information is converted back into digital data.

[0094] Fig.34A sample tray design with input and output ports is illustrated.

[0095] Fig.35 The workflow for photo-directed oligonucleotide-by-oligonucleotide synthesis is shown, with m different barcode sequences encoding data for every n rounds, and log_2(m^n) bits of information can be encoded with only m*n rounds of photopatterning (left part of the image). Within each spatially programmable pixel, many copies of the same "biased polymer" sequence are assembled (right part of the image).

[0096] Fig.36A The readout workflow using a cross-junction splicing reaction is illustrated, copies of the encoded concatemers can be made using a DNA polymerase and then sent for sequencing.

[0097] Fig.36B Diagrammatically illustrates a readout workflow using concatemers that can be directly imaged using strands labeled with complementary fluorophores.

[0098] Fig.37A is a schematic diagram illustrating the workflow of in situ node chain / node nucleic acid generation directed by oligonucleotide hybridization.

[0099] Fig.37B and Fig.37C is a schematic diagram illustrating the node chain generation padlock used in the cleat connection (which forms the node template when connected). Fig.37B Padlock probe generation Fig.37E Use Fig.37C Padlock probe generation Fig.37F The image in .

[0100] Fig.37D Schematic diagram illustrating rolling circle amplification (RCA) concatemers with fluorescent probe detection (upper panel) and the strands released using cleavage oligonucleotides of different lengths (lower panel), which reconstitute double-stranded restriction enzyme sites.

[0101] Fig.37E is a schematic diagram illustrating the cleavage of oligonucleotides of different lengths and different distances from the recognition site sequence.

[0102] Fig.37F are images showing spot intensity after cleavage with various oligonucleotides and restriction enzymes compared to controls (Og = oligonucleotide, RE = restriction enzyme). Scale bar: 100 μm.

[0103] Figure 37G are images illustrating probe signals for concatemers and cleaved concatemers before and after wash steps.

[0104] Fig.37His a line graph illustrating the mean spot intensity for the "after cleavage, before wash", "after wash" and "reprobe" samples divided by the mean spot intensity for the "after cleavage, before wash" condition, which is the baseline signal prior to diffusion. This yields a "normalized mean spot intensity" value for each condition. The no restriction enzyme control (-BtsI) is plotted separately from the cleavage condition (+BtsI) in the line graph to show the difference in spot fluorescence retention with and without BtsI cleavage.

[0105] Fig.38A is a schematic diagram illustrating the release of diffuse node strands from a concatemer by cleavage of the hairpin secondary structure through double-stranded cleavage or nicking of the hairpin structure.

[0106] Fig.38B is a diagram for clamping connections to generate Fig.38C The chain of nodes in the image generates a schematic diagram of a padlock.

[0107] Fig.38C are images illustrating cleavage at the hairpin using a double-strand cutter (restriction enzyme) or nicking enzyme compared to a no enzyme control. Temperature cycles and wash steps were applied from 8°C to 55°C. Scale bar: 100 μm.

[0108] Fig.39A is an image illustrating the generation of concatemer density by nodal chaining of ligation parameters, showing the difference in spot (concatemer) density across SplintR ligase concentrations in reactions with 2 different incubation times - 1 hour and overnight (-16 hours). Scale bar: 100 μm.

[0109] Fig.39B is a diagram for clamping connections to generate Fig.39C The chain of nodes in the line graph generates a schematic diagram of a padlock.

[0110] Fig.39C is a graph illustrating quantification of spots per field of view for the lower ligase concentration condition with resolvable spots. The higher ligase concentration condition was excluded from quantification because individual spots could not be resolved under the imaging conditions.

[0111] Fig.40A is a diagram for clamping connections to generate Fig.40B The chain of nodes in the image generates a schematic diagram of a padlock.

[0112] Fig.40B The images are for illustrating detection by hybridization probe and detection by anti-DIG antibody. Scale bar: 100 μm.

[0113] Fig.41A is a schematic diagram illustrating an exemplary isothermal method for generating barcoded node chains using reverse transcriptase and nickase.

[0114] Fig.41B is a schematic diagram illustrating a template chain.

[0115] Fig.41C is a gel illustrating gel products from templates of varying sizes and starting structures amplified using an isothermal reaction with reverse transcriptase and nickase.

[0116] Fig.42A is a schematic diagram illustrating an isothermal method for in situ generation of barcoded node strands using reverse transcriptase and nickase on concatemeric templates.

[0117] Fig.42B The diagram is used for clamping connections to generate Fig.42D and Fig.42E Schematic diagram of the padlock generated by the chain of nodes in the bar graph and gel image.

[0118] Fig.42C is a schematic diagram illustrating an isothermal method for in situ generation of barcoded node strands using reverse transcriptase and nickase on concatemeric templates.

[0119] Fig.42D is a bar graph illustrating qPCR quantification of in situ strand generation from concatemer templates showing a large increase in strand concentration compared to a control lacking the essential components of the reaction: nickase and reverse transcriptase (Pol).

[0120] Fig.42E is a gel illustrating the size range of products from purified, unamplified cell supernatant and from PCR amplification. (PCR products are amplified to completion and the intensity does not reflect input abundance). The "mock" lane corresponds to a synthetic DNA strand with the size and sequence structure of the expected amplicon generated by the reaction. The gel shows that the sizes correspond between the expected control and the reaction products. N = nickase; P = RT polymerase.

[0121] Fig.43A Diagrammatically, a workflow is presented for releasing two types of strands from a single nodal source for priming reverse transcription of cellular mRNA and conjugation to another nodal nucleic acid.

[0122] Fig.43B is a schematic diagram of concatemers of target nucleic acids capable of generating conjugations to cDNAs and also generating node nucleic acids for use in a node-node handshake.

[0123] Fig.43C is a schematic diagram of nucleic acids generated from two different RCA factories (including two types of node nucleic acids and two types of target nucleic acids).

[0124] Figure 43D-Figure 43F is a diagram illustrating the 'one-step' release of the target nucleic acid hybridized to the target mRNA ( Fig.43D), the association of reverse transcriptase ( Fig.43E ) and the generation of factory target nucleic acid-cDNA concatemers ( Fig.43F ) schematic diagram.

[0125] Figure 43G and Figure 43H is a diagram illustrating the sequential release of two types of combined nodes and nucleic acids to hybridize via RT primer sequences on the target nucleic acid ( Figure 43G ) and the cleavage oligonucleotide hybridizes to the cleavage site between the node nucleic acid and the target nucleic acid ( Figure 43H ) schematic diagram.

[0126] Fig.43I Is a bar graph quantifying relative qPCR Ct from input cDNA in samples receiving externally generated target nucleic acid, target nucleic acid generated by RCA, and without Phi29.

[0127] Fig.43J is an image of an agarose gel showing the size range of amplified cDNA; lane 1 is the sample contacted with Phi29 and reverse transcriptase, lane 2 is the sample contacted with Phi29 but not with reverse transcriptase, and lane 3 is the sample not contacted with Phi29 but with reverse transcriptase.

[0128] Figure 43K is a bar graph showing the percentage of node-strand barcoded cDNAs that conform to the expected amplicon structure based on NGS reads in samples that received externally generated target nucleic acids, samples that did not receive reverse transcriptase, and samples with target nucleic acids generated by directed reverse transcription.

[0129] Figure 43L is a bar graph illustrating the fraction of reads attributed to Type 1 and Type 2 templates in samples that received externally generated node nucleic acids or samples that received Type 1 and Type 2 target nucleic acids generated in situ.

[0130] Figure 43M-Figure 43O is a schematic diagram showing the progress of hybridization of handshake sequences from two node nucleic acids and extension of both nucleic acids to generate a double-stranded node handshake.

[0131] Figure 43P is a bar graph showing relative Ct values ​​from different transcription and release conditions, where generations with sequential barcode release and one-step release show high input cDNA counts, reflected by low relative Ct, and samples without RCA or padlock show low cDNA counts, reflected by high relative Ct.

[0132] Figure 43Q are images of agarose gels run with samples from one step and sequential release of generated nucleic acids compared to a mock template of expected size.

[0133] Figure 43R is a bar graph showing genes mapped using samples that received externally generated target nucleic acid, samples without reverse transcriptase, or samples from target nucleic acid generated in situ.

[0134] Figure 43S is a compilation of alignments of the generated node nucleic acids, showing consistent alignments of 1) Handshake PCR Primer 1, 2) Handshake PCR Primer 2, 3) Handshake Sequence, 4) Node 1 ID + first 10 bases of Handshake Sequence, and 5) last 10 bases of Handshake Sequence + Node 2 ID across 1000 reads.

[0135] Fig.44A is a schematic diagram showing the structure of a padlock probe sequence used in RCA to generate a concatemer having a nicking site on a secondary hairpin structure.

[0136] Fig.44B The target hybridization method is illustrated, including cleavage of the target nucleic acid by nicking at the double-stranded hairpin region, hybridization to mRNA, and reverse transcription of the mRNA.

[0137] Fig.44C The CNVK barcode sequences are shown to crosslink target nucleic acids both in concatemer form (in this case with a hairpin secondary structure) and to target nucleic acids hybridized to mRNA.

[0138] Figure 44D-Figure 44E are images of cells treated with nuclear stain and spatial barcodes with attached fluorophores, indicating crosslinking to both cleaved and uncleaved concatemers.

[0139] Fig.45A is a schematic diagram showing the use of different padlock probes to target different cells in a mixed culture.

[0140] Fig.45B is a schematic diagram of the four padlock probe structures used to generate two types of mouse-targeting nucleic acids and two types of human-targeting nucleic acids.

[0141] Fig.45C It comes from it comes from Fig.45B Schematic representation of a single repeat of the concatemer generated with an associated cleavage oligonucleotide at the restriction site of the padlock probe described.

[0142] Fig.45D and Fig.45E is an image of HeLa and 3T3 co-culture; Fig.45D This is an image of HeLa cells stained with FAM. Fig.45E These are images of 3T3 stained with cy3.

[0143] Fig.45F is a schematic diagram of the combined RT and handshake nucleic acid, indicating the two primer regions.

[0144] Figure 45G is an image of an agarose gel showing the Fig.45F Each primer region mentioned in the table generates replicates of similar size.

[0145] Figure 45H is a bar graph presenting the fraction of cDNA reads that mapped to human or mouse using human-targeted and mouse-targeted padlock probes.

[0146] Fig.45I is a compilation of approximately 10,000 reads of sequences associated between handshake sequences generated by mouse and human directed padlock probes.

[0147] Figure 46A-Figure 46T is a graph reconstructed from nodes based on the simulated barcoding simulation.

[0148] Details

[0149] Provided herein are compositions, devices, methods and systems for use in proximity coding in samples.Such processes may include the deposition of nucleic acids at node positions. Unique barcodes are generated at node positions in samples. Barcodes may diffuse therefrom to generate clouds of different barcodes. These barcodes may interact with nearby biomolecules to form recognizable sequences, revealing information about the local biomolecular environment of the node. In embodiments where barcode clouds overlap, the barcodes in the overlapping portions are optionally cascaded into unique and recognizable sequences. Analysis of cascaded barcodes allows mapping of specific sequences between individual node positions. Spatial analysis using methods described herein may provide resolution that is not limited by light diffraction or sample dimensions. Spatial analysis may optionally be combined with visual selection methods that may directly link multidimensional and high-resolution cell phenotypes (including morphology, protein markers, spatial organization) to transcriptomes, genomes or proteome spectra of different sample types. The methods described herein are applicable to analyzing tissues, synthetic tissues, cells separated or cultured or cell states in solutions. Such compositions, systems and methods are also described herein for use in information data storage purposes.

[0150] Diffusion Marking

[0151] Provided herein are methods for generating nucleic acid chains from a template chain that subsequently diffuse away from a point source (referred to herein as a node position). As used herein, a chain comprises a nucleic acid or nucleotide chain. Interactions between nucleic acids generated from different node positions or interactions with target molecules can provide additional information about the relative proximity of the tagged agent. In some embodiments, the node nucleic acid comprises a node barcode. In other embodiments, the node barcode provides information specific to the node position, thereby allowing downstream means for node position identification. The generation mechanism described herein can produce more than one copy of a nucleic acid chain comprising a unique barcode. The generated chain can diffuse away from the generation site, thereby producing a diffusion region or cloud. In some embodiments, the diffusion cloud comprises a concentration gradient surrounding each node. In some embodiments, the concentration of the generated node barcode chain decreases as a factor of the distance from the generation site. As Figure 1A As depicted in , a node acts as a factory for generating node barcode chains. In some embodiments, the node factory contains copies of node nucleic acids. Copies of node nucleic acids are released from the node factory to diffuse away from the node location. In some embodiments, the node factory contains template nucleic acids, which contain sequences complementary to the node nucleic acids. According to the methods described herein, copies of node nucleic acids are generated from the node factory and diffuse away from the node location. As the chains diffuse out from the nodes, a cloud comprising a concentration gradient is generated. The concentration of node barcode chains at a point in the center of the diffuse cloud will be much higher than the concentration at a point away from the center. The lowest concentration of node barcode chains exists at the edge of the cloud. Figure 1B Diffusion timelines for four node positions are shown. Node barcode chains are generated within a set time period or continuously. Over time, the generated chains diffuse away from the node position, generating a diffusion cloud. In some embodiments, the diffusion clouds of the generated chains are different and do not overlap. In some embodiments, the diffusion cloud expands to overlap with an adjacent diffusion cloud. In some embodiments, the node barcode chain is single-stranded. In some embodiments, the node barcode chain is double-stranded. In some embodiments, the node nucleic acid includes RNA. In some embodiments, the node nucleic acid includes DNA. In some embodiments, each type of node can be identified by a node type barcode encoded in the template chain. In some embodiments, the node type barcode is consistent across node types. In some embodiments, each node can be identified by a node barcode encoded in the template chain. In some embodiments, the node barcode is specific to an individual node. In some embodiments, each chain generated by a node can be identified by a unique modifier. In some embodiments, the unique modifier is specific to each nucleic acid chain. Exemplary combinations of identification sequences are shown in Figure 1C The intervening space between the marker sequences may contain nothing, but may contain linkers, primers, flanking L or R sequences or promoters.

[0152] In some embodiments, the diffusion node barcode chains in overlapping diffusion clouds are combined by concatenation. Figure 2 As shown in FIG. 1 , a concatemer can contain two or more different node barcode chains. The concatemers can be paired or can include more than one concatemer. Figure 3 As shown in FIG, concatemers of node barcode chains can be formed by ligation, splinting, cross-linking, ligation and cross-linking interchain crosslink (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. In some embodiments, the concatenation of barcodes is sequential with the generation of node barcodes. In some embodiments, the concatenation of barcodes is simultaneous with the generation of node barcodes. An exemplary reconstruction of relative node positions based on the frequency of node barcodes is shown in FIG. Figure 4 In some embodiments, the spatial orientation of the node position is determined by mapping the sequencing concatemer. In some embodiments, the concatemer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more different node barcodes. In some embodiments, the spatial orientation is one-dimensional, two-dimensional or three-dimensional.

[0153] In some embodiments, the node barcode chain contains typical DNA bases, RNA bases, or any combination thereof. The bases can be edited or modified in situ using enzymatic editing or any other means after generation. The bases can also incorporate modified nucleotides, such as dUTP, fluorophore-labeled dNTP, fluorophore-labeled NTP, 2'-O-Me NTP, atypical bases, or otherwise modified dNTPs or NTPs.

[0154] In some embodiments, the node barcode comprises a series of random bases (e.g., NNNNN, NNNNNNNNNNN, NNNNNNNNNNNNNNN, DDDDDD, DDDDDDDD, HHHHH, HHHHHHHHH, wherein "N" represents base A, C, G, or T / U; "D" represents base A, G, or T / U; and "H" represents base A, C, or T / U) or any combination of random bases and non-random bases. The length of the node barcode can be between 1nt and 50nt or longer. More than one node barcode can be present on each node barcode chain. In some embodiments, the node barcode is about 1 to about 10 nucleotides, about 10 to about 20 nucleotides, about 20 to about 30 nucleotides, about 30 to about 40 nucleotides, about 40 to about 50 nucleotides, or more than 50 nucleotides. In some embodiments, the node barcode is about 3 to about 30 nucleotides. In some embodiments, the node barcode includes a three-letter code.

[0155] The node barcode chain comprises a node barcode region. In some embodiments, the node barcode chain also comprises one or more flanking regions, referred to herein as "L" and "R" regions. In some embodiments, the flanking regions comprise PCR primers, chain barcodes, affinity sequences, hybridization sequences complementary to downstream components, or other sequences capable of fixation. In some embodiments, the affinity sequence comprises additional barcodes, aptamers, DNA enzymes, RNA enzymes, ISH probes, primers, or any combination thereof. In some embodiments, the flanking regions comprise binding sites for fluorophore-labeled oligonucleotides.

[0156] The flanking region can be 1 to about 500 nucleotides. In some embodiments, the flanking region is about 1 to about 50 nucleotides, about 50 to 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300 nucleotides, about 300 to about 350 nucleotides, about 350 to about 400 nucleotides, about 400 to about 450 nucleotides, about 450 to about 500 nucleotides. In some embodiments, the flanking region is about 10 to about 40 nucleotides.

[0157] In some embodiments, the node barcode chain also includes a unique modifier. The unique modifier includes a universal base or other base modification to produce heterogeneity in the node barcode chain. The unique modifier may include a universal base or other base modification to produce heterogeneity in the node barcode sequence. In some embodiments, each node barcode chain includes a unique modifier to allow more quantitative measurements. In some embodiments, the node barcode or unique modifier also includes a targetable sequence for base editing. In some embodiments, the node nucleic acid chain is edited using CRISPR technology. In some embodiments, the targetable sequence is adjusted over time.

[0158] The total length of the node barcode chain can affect the characteristics including quality, diffusion rate, diffusion limitation and sequencing efficiency. In some embodiments, the full node barcode chain is about 150 to about 200 nucleotides. In some embodiments, the length of the full node barcode chain is about 20 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 350, about 350 to about 400, about 400 to about 450, about 450 to about 500, about 500 to about 550, about 550 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, about 900 to about 1000, about 100 to about 1500 or about 1500 to about 2000 nucleotides.

[0159] In some embodiments, the node nucleic acid chain includes bases beyond the 5' or 3' end of the complementary template sequence. Additional bases can be added by reaction heterogeneity, enzyme activity or other means. In some embodiments, the T7 RNA polymerase includes 1 to 3 bases (G) from the end of the promoter sequence on the 5' end of the transcribed RNA chain.

[0160] Node fixation and barcode assembly

[0161] Provided herein are methods including node fixation and barcode assembly. In some embodiments, the node position is spatially restricted in the sample to provide position data. In some embodiments, the node position is transiently fixed in the sample. In some embodiments, the node position is permanently fixed in the sample. In some embodiments, the node position diffuses in the sample. In some embodiments, the node position is incorporated into the expression system. In some embodiments, the expression system is contained in the genome. In some embodiments, the genome is a viral genome. In some embodiments, the polymerase is a part of the node barcode chain generation mechanism. In some embodiments, the polymerase is a mixture of DNA polymerase, RNA polymerase, engineered polymerase, polymerase, or any combination thereof. The polymerase may have strand displacement activity or no strand displacement activity. The polymerase may have exonuclease activity or no exonuclease activity. The polymerase may be tethered, hybridized, cross-linked, or photocross-linked to or near a substrate or sample, a promoter, or a template strand to localize synthesis to the region. In some embodiments, the template strand or a strand complementary to a domain on the template strand contains additional sequence domains to allow tethering in a sequence-specific manner. In some embodiments, the template strand, complementary sequence, promoter or primer sequence is fixed in vitro on a bead, on a surface, on a slide, in LNP, AAV, lentivirus, other viruses, in an exosome, or in situ on a sample. Fixation can be via attachment, conjugation or hybridization to a target or substrate or by, for example, direct incorporation into the sequence of the target by a transposon. Fixation can be achieved by affinity reactions including hybridization, binding, crosslinking, photocrosslinking, or any combination thereof. The fixation method can be specific to a specific subcellular, cell, cell type specificity, or supercellular structure, such as a membrane, mitochondria, protein, nucleic acid, lipid, protein-protein complex, protein-RNA complex, or any combination thereof. In some embodiments, the template strand is randomly dispersed throughout the substrate by random binding, nonspecific binding, conjugation to a surface, incorporation into a hydrogel, or any other means or combination thereof. In some embodiments, the template strand binds to multiple targets. In some embodiments, multiple targets are general classes of molecules, such as proteins, lipids, or carbohydrates. In some embodiments, multiple targets are specific classes of molecules, such as, for example, kinases, antibodies, or phospholipids. In some embodiments, the target or targets also bind to another set of multiple targets. For example, the template strand can bind to a kinase, which in turn binds to ADP or ATP and a substrate. Therefore, multiple kinases can bind to multiple substrates. In another example, the template strand binds to ATP, which will bind to multiple kinases. In some embodiments, the template strand is focused in a specific area by depositing reagents on a specific area, spatially confined crosslinking, photocrosslinking, or any other means or combination thereof. The template strand can be crosslinked or photocrosslinked in situ, with optional washing or additional sample preparation steps after attachment or fixation.Using focused light on a specific region of interest (ROI), cross-linked template strands can be photocross-linked only to a specific spatial region. In order to adapt to attachment chemistry, one or more template strands can contain additional sequences complementary to the fixed strands at the 5' or 3' ends. In some embodiments, the PCR primer domain is included in the 5' or 3' domain of the template strand and / or primer strand. One or more strands can contain phosphorothioate, 2'O methyl, reverse dT or other modifications to protect them from exonuclease activity.

[0162] In some embodiments, assembly of the node complex is based on hybridization (e.g., DNA-DNA, DNA-RNA, RNA-RNA, or any variation or combination thereof), immobilization (as described above), or via assembly / chain tandem methods, including ligation, splint ligation, cross-linking synthesis, ligation and cross-linking (ICL) synthesis, cross-ICL and nicking synthesis, primer exchange reaction protein (Reactin) (PER), asymmetric PCR, Gibson assembly, transposase-based tandem (e.g., Tn5 transposition of RNA-DNA hybrids), or any variation or combination thereof.

[0163] In some embodiments, the node generated by the nucleic acid is attached to a target molecule. The target can be a DNA molecule, RNA molecule, protein, lipid, antibody or other molecule that has been conjugated to, hybridized to, cross-linked to, photocross-linked to or directly attached to a nucleic acid chain on the same or different sequence backbone that has or can be made to have a single-stranded overhang at its 3' end or 5' end.

[0164] Diffusion from the node can be regulated by adjusting the sample environment, nucleic acid chain length, any other factor affecting diffusion, or any combination thereof. Diffusion in the solution is controlled by adjusting the parameters that affect the rate at which molecules can move through the solution. Parameters affecting diffusion include viscosity, time, temperature, crowding agents, pH, electric fields, physical characteristics, or any combination thereof. In some embodiments, the diffusion of nucleic acids is regulated by adding molecular crowding agents. Crowding agents can include dextran sulfate, glycerol, polymers, crystals. In some embodiments, the diffusion of nucleic acids is enhanced by heating a population of tissue samples or cells provided herein. In some embodiments, diffusion is enhanced by heating to more than 30°C, more than 35°C, more than 40°C, more than 45°C, more than 50°C. In some embodiments, diffusion is enhanced by heating to about 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C.

[0165] In some embodiments, the sample environment varies in viscosity, crowding agents, fixation, polymerization of reagents in solution, concentration of reagents in solution, attachment of molecules to endogenous scaffolds in the sample, attachment of molecules to externally introduced scaffolds, or any other method that affects diffusion, fixation, or mobility in the sample. In some embodiments, the nodes are embedded in a substrate or medium. The viscosity of the sample can be adjusted, for example, with a hydrogel or any excipient that increases the poise measure of the environment. In some embodiments, the sample medium has a viscosity greater than 1 centipoise (cP). In some embodiments, the sample medium has a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP. In some embodiments, the crowding agent comprises dextran sulfate, glycerol, a polymer, a crystal, or any combination thereof. Sample fixation can be accomplished by heating, microwaves, cryopreservation, or chemical means. Chemical fixation includes cross-linking fixatives, such as aldehydes, including formaldehyde or glutaraldehyde; precipitating fixatives, such as alcohols, including ethanol, methanol or acetone; oxidants, such as osmium tetroxide, potassium dichromate, chromic acid and potassium permanganate; mercury, such as B-5 and Zenker fixatives; picrates; or hepes-glutamate buffer-mediated organic solvent protection (HOPE) fixatives. In some embodiments, the sample is crystallized, for example, by silver fixation. In some embodiments, the node is attached to the sample substrate using any of the methods described herein.

[0166] In some embodiments, diffusion is changed by adjusting features including reaction time, temperature, chemistry, electromagnetic force or adapting physical features. Physical features can include subcellular, cellular or supercellular structures, or physical features are incorporated to block, inhibit or bind node barcode chains. In some embodiments, samples are protected from physical interference during barcode node generation. In some embodiments, barcode generation is performed on an isolation platform to minimize vibration or interference. In some embodiments, nucleases such as RNA enzymes, DNA enzymes, exonucleases or endonucleases are used to limit the distance of node nucleic acid diffusion. In some embodiments, node nucleic acids are tethered to long supports, which limits the distance they can diffuse. In some embodiments, when concatemers are elongated, concatemers can be tethered to rolling circle amplification complexes. In some embodiments, concatemers grown from point sources can be used to generate extendable ends that can flexibly bind to biomolecules near them without freely diffusing as separate chains in solution. In some embodiments, diffusion is initiated by regulating features. In some embodiments, diffusion is initiated before, during or after adding node nucleic acids. In some embodiments, diffusion is stopped by regulating features. In some embodiments, diffusion is stopped before, during, or after the addition of the node nucleic acid.

[0167] In some embodiments, the target can diffuse toward the node. In some embodiments, the sample includes fixed nodes and diffuse targets. In some embodiments, the sample includes a mixed population of nodes, wherein a subpopulation of nodes generates diffuse products and another subpopulation of nodes generates fixed nucleic acids.

[0168] In some embodiments, the node nucleic acid chain comprises one or more photolabile protecting groups. Such photoactivation or provision of "photocage" provides controlled initiation of node nucleic acid production. Synthesis can be time-limited. In some embodiments, photocaging allows spatial control of node nucleic acid chain generation. In some embodiments, photocaging is controlled by targeted optical microscopy. Without limitation, photolabile protecting groups include (2-nitrophenyl) ethyl groups, 2-nitrobenzyl-based groups, carbonyl-based groups, benzyl-based groups, coumarin-based groups, boron dipyrromethene (BODIPY)-based groups, cyanine-based groups, ortho-nitrobenzyl (NB)-based groups, or any variation or combination thereof.

[0169]

[0170] In some embodiments, the promoter sequence comprises a photocage protecting group. Upon light activation, the promoter binds to the template strand, allowing replication to proceed. Figure 5AAn exemplary embodiment is shown in which the template node barcode strand is fixed to the sample via a conjugated binding moiety. A promoter protected with a photolabile protecting group is added to the sample. Simultaneously with or subsequent to the addition of the promoter, the sample is exposed to light, releasing the photolabile protecting group, allowing the promoter to hybridize with a complementary sequence to initiate replication.

[0171] In some embodiments, the sequence comprises a photocrosslinker, providing light-controlled synthesis. In some embodiments, activation of the photocrosslinker induces or increases synthesis. In some embodiments, activation of the photocrosslinker reduces or stops synthesis. Exemplary photocrosslinkers include aryl azides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diaza heterocycles, psoralen derivatives, or any combination thereof. In some embodiments, the photocrosslinker is 3-cyanovinylcarbazole nucleoside (CNVK).

[0172] In some embodiments, the promoter comprises a photocrosslinker. Figure 5B An exemplary embodiment is shown in which the promoter sequence comprises a CNVK group. Simultaneously or subsequently to the addition of the promoter, the sample is exposed to light to crosslink the promoter to the complementary region of the template strand.

[0173] In some embodiments, the generation of node nucleic acid chains is regulated over time using one or more methods of time tracking or gating of node nucleic acid chain generation alone or in any combination. Degraded NTPs and / or dNTPs can replace standard NTPs / dNTPs or be used in combination with standard NTPs / dNTPs. The incorporation of hot start NTPs and / or dNTPs allows regulation or monitoring by temperature control. The addition of base editing enzymes or mutagens provides mutations of node barcodes and unique modifiers over time.

[0174] In some embodiments, the sample or substrate temperature is regulated during the generation of the node barcode strands. The temperature can be kept constant, or it can be cycled, for example between 12°C and 42°C, between 4°C and 95°C, or any temperature therebetween. Temperature regulation can be by any means, such as a PCR machine, a flat-top incubator, a water bath, a piezoelectric device, a heated chamber around a microscope, a heated buffer flowing through the sample or substrate, or any other means or combination thereof.

[0175] Node barcode chain composition

[0176] In addition to the node barcode region, the node barcode chain can include additional domains, modifications or other additions. Such elements can guide chain generation, provide fixation of the template nucleic acid chain or provide its spatial identification. In some embodiments, the node position in the sample can include one or more nucleic acid chains. The node position can be fixed on a substrate, in a substrate or in a sample. In some embodiments, the node position includes a template nucleic acid chain, and a polymerase can generate a copy of the node barcode chain thereon. In some embodiments, the generated node barcode chain diffuses away from the node position.

[0177] like Figure 6 As shown in FIG. 1 , the template nucleic acid strand encodes the promoter / primer hybridization region (“promoter * / Primer * ”), left and right flank areas (“L * ” and “R * ”) and node barcode regions (node ​​barcode * ). In some embodiments, the primer hybridizes to the hybridization region on the template strand, and the polymerase drives the extension of the node barcode strand. In some embodiments, synthesis can be achieved through a promoter region and transcription. In some embodiments, synthesis can rely on another enzyme to start de novo synthesis on the template strand.

[0178] One or more template strands in the node complex may comprise additional domains or modifications. In some embodiments, additional domains or modifications may facilitate fixation in or on a substrate or in a sample. In some embodiments, additional domains or modifications are used for spatial identification. In some embodiments, the template strand further comprises biotin, an antibody, a fluorophore, a dye, an aptamer, a probe, a barcode, or any combination thereof. In some embodiments, additions or modifications are inserted into the template strand.

[0179] In some embodiments, the node barcode strand consists of a promoter site ( Figure 7 The promoter on the middle template strand * ”) template strand. The promoter is a specific regulatory DNA sequence that exists upstream of the transcription start site. The promoter controls gene transcription by providing binding sites for RNA polymerase and other transcription factors. Figure 7As shown in, in some embodiments, the promoter region is upstream of any functional region of the node barcode template. In some embodiments, the promoter region comprises one or more functional regions of the node barcode template, or up to the entire length of the node barcode template. In some embodiments, the template strand forms a secondary structure. In some embodiments, the secondary structure is a hairpin. Transcription of the template strand is achieved with a polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the DNA polymerase includes Phi29, Pol I, Pol II, Pol III, Pol IV, Pol V, T4, In some embodiments, the polymerase is an RNA polymerase. In some embodiments, the RNA polymerase comprises T7, T3, SP6, or any combination thereof. In some embodiments, the polymerase comprises an exonuclease activity that degrades the encountered downstream strand via a 5' to 3' exonuclease activity. In some embodiments, the node barcode sequence is flanked by additional sequences (e.g., L and R domains) that accommodate downstream chemistry for target proximity and node barcode proximity recording.

[0180] In some embodiments, the method for generating a node barcode chain includes using a primer that is bound to a template chain and extended by one or more polymerases to produce a copy of the template chain. In some embodiments, the primer is a short nucleotide sequence for amplifying the target DNA. The primer is used as the starting sequence for the synthesis of the new chain. In some embodiments, the generation is achieved by other enzymes such as recombinases, helicases, single-stranded binding proteins or nickases. Fig. 9 An exemplary amplification of a template node nucleic acid using primers that hybridize to complementary regions of a template strand is shown. In some embodiments, the primer region is upstream of any functional region of a node barcode template. In some embodiments, the primer region comprises one or more functional regions of a node barcode template, or up to the entire length of the node barcode template. The strand is extended with a DNA or RNA polymerase. In some embodiments, the polymerase comprises a recombinase, a helicase, SSB-assisted polymerization, or any variation or combination thereof. Fig. 9The figure shows an exemplary amplification of a template node nucleic acid using a nickase-assisted synthesis. The primer hybridizes with a complementary region of the template strand. In some embodiments, the primer binds to another region on the template strand and forms a secondary structure. In some embodiments, the secondary structure is a hairpin. During or after chain extension, the nickase introduces a single-stranded cut in the extended chain. In some embodiments, the nickase is Cas9 or any variation thereof. In some embodiments, the polymerase includes a recombinase, a helicase, SSB-assisted polymerization or any variation thereof or combination. In some embodiments, amplification is achieved by rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branched amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER), primer exchange reaction (PER), or any variation or combination thereof. In some embodiments, the polymerase comprises a strand displacement polymerase comprising Phi29, Bst polymerase, or any combination or variation thereof.

[0181] The amplification methods described herein are interchangeable and stackable. In some embodiments, the amplification products from the described amplification are used as templates in subsequent rounds of amplification by the same or different methods. In some embodiments, the amplification products from one method are amplified simultaneously by another method. In some embodiments, the same amplification method is used in multiple rounds.

[0182] Rolling circle amplification

[0183] Rolling circle amplification (RCA) is a method of generating multiple copies of a nucleic acid in a continuous chain from a circular template. RCA amplifies a template nucleic acid into long, repetitive single-stranded oligonucleotides. Typically, RCA includes the formation of a circular template nucleic acid. In some embodiments, the template nucleic acid or padlock probe is annealed to a ligation template or nucleic acid splint. In some embodiments, the splint and template nucleic acid include DNA or RNA. The template nucleic acid is circularized using a ligase. In some embodiments, the ligase includes T4 ligase, Ligase or any combination thereof. In some embodiments, the primer sequence, polymerase and deoxynucleotide triphosphate (dNTP) are incubated with the template nucleic acid to generate a concatemer of single-stranded DNA comprising repeated copies complementary to the circular template. In some embodiments, the polymerase includes Phi29, Bst, Vent exo-DNA polymerase or T7 RNA polymerase.

[0184] In some embodiments, the nucleic acid comprising the splint sequence is a region of nucleic acid in a sample. In some embodiments, the splint sequence is a region of genomic DNA, mRNA, cDNA or other nucleic acid in a sample. In some embodiments, the splint sequence is on the mRNA encoding a gene. In some embodiments, the splint sequence is a regulatory RNA. In some embodiments, the splint sequence is a circular RNA (circRNA), a ribosomal RNA (rRNA), a 16S ribosomal RNA (16S rRNA), a transfer RNA (tRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a non-coding RNA, a long non-coding RNA (lncRNA) or a micro RNA (miRNA).

[0185] Hybridization with nucleic acids in the sample allows the node factory to be deposited at a fixed position. In some embodiments, the padlock probe hybridizes with the splint sequence at a fixed position in the sample. In some embodiments, the splint sequence is located on the surface, in a cell, in a nucleus, or in a tissue. In this case, the splint sequence serves as an anchor or tether for the RCA machine. In some embodiments, the padlock probe hybridizes with the splint sequence on the nucleic acid encoding the gene in the sample.

[0186] In some embodiments, RCA generates at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 copies of the template.

[0187] In some embodiments, the attachment template or splint is conjugated to a surface, a protein, a small molecule, a nucleic acid, a lipid, or any combination thereof. In some embodiments, the surface comprises glass, a bead, a microplate, a scaffold, or another substrate. In some embodiments, the protein comprises a receptor, a ligand, an antibody, or any functional fragment thereof.

[0188] In some embodiments, concatemers generated by the node factory provide extendable ends that flexibly bind to biomolecules in their vicinity. The "tethered" generated ends of the RCA concatemers diffuse locally in the sample rather than freely diffusing.

[0189] The RCA template can also encode restriction sites, allowing endonuclease cleavage of the generated concatemers. In some embodiments, restriction sites are encoded on the RCA template. In some embodiments, single-stranded specific nucleases cleave nucleic acids at single-stranded restriction sites. In some embodiments, the template encodes the recognition site of double-stranded nucleases, wherein the nucleases perform double-stranded cleavage at the recognition site. In some embodiments, oligonucleotides comprising sequences complementary to the region encoding the recognition sequence are deposited on the concatemers and allowed to hybridize with nucleic acids. In some embodiments, endonucleases are then added to the concatemers to perform double-stranded cleavage at the restriction site. In some embodiments, endonucleases can be added simultaneously with RCA polymerases. In some embodiments, RCA occurs simultaneously with digestion. In some embodiments, concatemers form a secondary hairpin structure in the region comprising the recognition sequence, thereby providing a double-stranded region for endonuclease cleavage. In some embodiments, double-stranded specific endonucleases perform single-stranded cleavage in double-stranded sequences. In some embodiments, concatemers form a secondary hairpin structure in the region comprising the recognition sequence, thereby providing a double-stranded region for endonuclease cleavage. In some embodiments, double-stranded restriction enzymes cut across a single base pair, generating blunt ends. In some embodiments, double-stranded restriction enzymes perform staggered cuts across different base pairs generating sticky ends. In some embodiments, cleavage is performed by DNA enzymes, RNA enzymes, or CRISPR endonucleases for cleaving concatemers.

[0190] In some embodiments, a single-stranded barcode node strand is generated using a primer exchange reaction (PER). A hairpin oligonucleotide or a double-stranded construct is used to template the stepwise synthesis of single-stranded DNA. Fig.10 As shown in , the primer binds to the short 3' overhang on the hairpin and is extended by the strand displacement polymerase until a "terminator" is reached. The terminator is any modification on the template strand that stops the polymerase from progressing. The three-way branch migration mediated by random breathing of the DNA strand causes the newly synthesized product to shift from the hairpin or double-stranded construct. The new strand can then dissociate from the template. Multiple hairpins or constructs can be included to template multiple synthesis steps. In some embodiments, the hairpin or double-stranded template comprises a 3' overhang. In some embodiments, the single-stranded barcode chain is generated using asymmetric PCR or any strategy for producing self-replicating nucleic acids. Some methods described herein include the use of additional enzymes or PER with a strand displacement polymerase, thereby eliminating the use of a high temperature denaturation step during the synthesis of the node barcode chain.

[0191] Nucleic acids are generated by methods described herein. In some embodiments, nucleic acids are generated by isothermal reactions. In some embodiments, the addition of nucleotides does not require thermal cycling. In some embodiments, synthesis includes thermal cycling. In some embodiments, synthesis of nucleic acids includes replication or transcription. In some embodiments, the synthesis method includes cross-linking synthesis.

[0192] In some embodiments, the amplification of the node barcode strand comprising primers further comprises an additional domain. Fig.11 Exemplary embodiments are shown where the primers additionally include PCR primers, unique identifier sequences, any other functional or non-functional domains, or any combination thereof. Primers with additional domains can be used to generate longer node barcode chains that also contain these domains.

[0193] The template strand may additionally contain one or more unique modifiers. In some embodiments, the unique modifier comprises one or more universal bases, atypical bases, or polymerases that can pair with random or erroneous bases to produce other bases or modifications of a random sequence that is specific or semi-specific to a particular node barcode strand. The unique modifier domain may contain a mixture of these and normal nucleotides, and one or more unique modifier domains may be present that are dispersed throughout the node barcode strand sequence. Therefore, although the chains generated at each node should have the same node barcode sequence, they may have different unique modifier sequences. Fig.12 Exemplary embodiments are shown where the unique modifier region is 3' to the node barcode region or 5' to the node barcode region. In some embodiments, the unique modifier information is used downstream after next generation sequencing to deduplicate reads and obtain more quantitative information about the number of neighboring recorded events.

[0194] handshake sequence

[0195] In some embodiments, the flanking region on the node nucleic acid includes a hybridization domain. In some embodiments, the hybridization domain is reverse complementary to the flanking region of another node nucleic acid. In some embodiments, the node nucleic acid also includes a primer. The hybridized node nucleic acid has primers on both hybridized strands, allowing the two strands to extend to generate a double-stranded nucleic acid. These hybridization domains or handshake sequences allow node nucleic acids from different node factories to hybridize. The hybridization of node nucleic acids from different node factories generates multiple node nucleic acids. Sequencing of the combined node nucleic acids provides spatial triangulation information or handshakes between node positions. The combination of handshake sequences between node nucleic acids provides triangulation information of vertex or node positions. In some embodiments, the target nucleic acid / cDNA complex and the handshake node nucleic acid are amplified and sequenced by PCR. In some embodiments, by sequencing the incompletely digested concatemers, the original association between the target nucleic acid and the node nucleic acid on each concatemer is cataloged to achieve the association between the barcoded cDNA and the node nucleic acid. Exemplary embodiments of node nucleic acid hybridization are described in Examples 12 and Figure 43M-Figure 43O Available in.

[0196] Order of Operations

[0197] The synthesis, diffusion, cleavage, and binding of barcoded nucleic acids are not restricted in time order, and not even all steps need to be performed. In some embodiments, the node factory generates nucleic acids, accompanied by diffusion and target binding of the generated nucleic acids. In some embodiments, the node factory generates nucleic acids, accompanied by cleavage, diffusion, and target binding of the generated nucleic acids. In some embodiments, the node factory generates nucleic acids, diffusion occurs after generation, and target binding occurs after diffusion. In some embodiments, the node factory generates nucleic acids, followed by simultaneous cleavage, diffusion, and target binding of the generated nucleic acids. In some embodiments, factory generation, cleavage, diffusion, and target binding of nucleic acids occur sequentially. In some embodiments, factory generation is followed by tethered diffusion and target binding of the generated concatemers, followed by cleavage of the concatemers.

[0198] Imaging nodes or diffuse clouds

[0199] In some embodiments, nodes, node concatemers or node nucleic acid diffusion clouds are visualized by incorporating one or more dyes or stains. In some embodiments, dyes or stains include fluorophore-labeled NTPs, fluorescently labeled probes, dyes or stains as described elsewhere herein. In some embodiments, dyes or stains are inserted into nucleic acids. In some embodiments, modified nucleotides, dyes or stains are added during the synthesis of node barcode chains. In some embodiments, node barcode chains are hybridized with complementary fluorophore-labeled oligonucleotides. In some embodiments, modified nucleotides are selected from fluorophore-labeled, DIG-labeled or biotin-labeled dNTP / NTPs. In some embodiments, modified nucleotides are selected from EdU, BrdU or PdU.

[0200] Fixed nucleic acid

[0201] In some embodiments, the displacement of barcoded nucleic acid includes applying reagents to release the nucleic acid containing barcodes from the surface. The reagents for releasing the nucleic acid containing barcodes can include RNA enzymes, DNA enzymes and / or proteases. In some embodiments, record extraction and sequencing include the displacement of barcoded nucleic acid. Optionally, displacement is achieved by applying RNA hydrolases. In some embodiments, RNA hydrolases are RNA enzymes H. The cross-linking of continuous nucleic acids can also achieve the displacement of barcoded nucleic acids. Enzymatic displacement and synthesis reactions can be combined or performed successively. After the sequence has been extracted from the sample, the sample can be further imaged or other determinations (e.g., mass spectrometry, H&E staining). Subsequent polymerase chain reaction (PCR) amplification and optional purification are followed by optional library preparation and next generation sequencing. The resulting sequence analysis is merged with the captured imaging data to provide integrated spatial and sequence-based information output.

[0202] The analysis using the methods described herein can be applied to fixed nucleic acids. In some embodiments, fixed nucleic acids are fixed to substrates. In some embodiments, fixation is carried out via formalin / formaldehyde treatment. In some embodiments, fixation is carried out via glyoxal or oxalaldehyde treatment. The analysis using the methods described herein can be applied to any fixed nucleic acids. In some embodiments, nucleic acids, proteins, or any combination thereof are characterized using the described methods. In some embodiments, fixed nucleic acids are fixed by affinity reactions, and the affinity reactions include hybridization, conjugation, binding, cross-linking, photocross-linking, initiation and extension, or any combination thereof. In some embodiments, affinity reactions are specific to specific subcellular, cell, cell type specific or supercellular structures such as membranes, mitochondria, genomic DNA, proteins, nucleic acids, lipids, protein-protein complexes, protein-RNA complexes, or any combination thereof. Therefore, in some embodiments, fixed nucleic acids are bound to multiple targets in the sample. In some embodiments, the target or multiple targets are also bound to another set of multiple targets. In some instances, the template strand is bound to a kinase, and the kinase is then bound to ADP or ATP and a substrate. In such a case, multiple kinases are bound to multiple substrates. In another example, the template strand is bound to ATP, which will bind to a variety of kinases. In some embodiments, fixation is randomly dispersed throughout the substrate by random binding, non-specific binding or conjugation to the surface, limiting or randomly incorporating into the hydrogel or focusing in a specific area. In some embodiments, focused deposition is carried out by depositing the reagent on a specific area, spatially confined crosslinking or light crosslinking as described herein. Fixed nucleic acids can be conjugated to affinity molecules or fixed to a support, substrate, surface, bead or column. The method described herein can be used for in situ hybridization (ISH) probes, DNA conjugated to antibodies, DNA encoding libraries, cDNA libraries, CRISPR libraries, viral libraries and / or genomes, nucleic acids covalently linked to transparent hydrogels, nucleic acid conjugated proteins, aptamers, guide RNAs, nucleic acids located on surfaces (e.g., glass slides or microarrays) or sequences generated in situ (e.g., by in vitro transcription (IVT)) are analyzed for profiles. The method described herein can be used for peptide libraries, protein libraries, nano antibody libraries, aptamer libraries or antibody libraries for profile analysis. In some embodiments, proteins or variants thereof are directly attached to their respective encoding mRNA transcripts, such as by ribosome stalling during translation. When identifying associated proteins using methods as described herein, the attached mRNA transcripts can be considered as nucleic acid "tags". In some embodiments, the sample is fixed before and / or after the node barcode chain is generated.

[0203] In some embodiments, the fixed nucleic acid is conjugated to an affinity molecule. In some embodiments, the affinity molecule comprises a component of a ligand / receptor complex. In some embodiments, the affinity molecule comprises an antibody or a component thereof. In some embodiments, the antibody component is an antibody fragment, a nanobody or other affinity reagent. In some embodiments, the antibody fragment as described herein is a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, an IgG fragment, an Fc fragment or any combination thereof. In some embodiments, the antibody is an IgA, IgG or IgM antibody or a functional fragment thereof.

[0204] In some embodiments, the fixed nucleic acid is conjugated to the support. In some embodiments, the support is biodegradable. In some embodiments, the support is non-biodegradable. In some embodiments, the support is biocompatible. In some embodiments, the support includes a synthetic material. In some embodiments, the synthetic material includes silicone, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polylactic acid-co-glycolic acid (PLGA) or any combination thereof. In some embodiments, the support includes natural materials. In some embodiments, natural materials include derivatives of the extracellular matrix, collagen, fibrin, polysaccharides (including chitosan or glycosaminoglycans (GAGs)) or any combination thereof. In some embodiments, glycosaminoglycans (GAGs) include hyaluronic acid.

[0205] In some embodiments, the immobilized nucleic acid is incorporated into a hydrogel. In some embodiments, the hydrogel comprises poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol (PEG), methacrylic acid (MAA), PEG-PEGMA, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), acrylamide / acrylic acid copolymer, linear cationic polyallylammonium chloride, poly(N-isopropylacrylamide) (PNIPAM), chitosan, acrylate-modified PEG and acrylate-modified hyaluronic acid, heparin, amine-terminated functionalized 4-arm star-PEG, or any combination thereof.

[0206] In some embodiments, fixed nucleic acid is conjugated to a surface comprising a flexible material. In some embodiments, flexible materials include but are not limited to modified nylon, unmodified nylon, nitrocellulose, polypropylene or any combination thereof. In some embodiments, the surface comprises a rigid material. In some embodiments, the rigid material includes but is not limited to glass, fused silica, silicon, silicon dioxide, silicon nitride, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate and blends thereof), metals (e.g., gold, platinum) or any combination thereof. The surface described herein can be made of a material including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass or any combination thereof.

[0207] In some embodiments, the immobilized nucleic acid is conjugated to a bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the bead is an immunoaffinity bead.

[0208] In some embodiments, the immobilized nucleic acid comprises a surface binding region. In some embodiments, the surface binding region hybridizes with a complementary sequence on the surface. In some embodiments, the surface binding region comprises an activated molecule that interacts with a molecule or coating on the surface. In some embodiments, the surface binding region comprises a conjugated molecule that interacts with a molecule or coating on the surface.

[0209] In some embodiments, multiple copies of fixed nucleic acid are conjugated to beads. In some embodiments, conjugation is reversible conjugation. In some embodiments, reversible conjugation is photocleavable, photoactivated (e.g., azobenzene modification), thermally unstable, chemically cleavable (e.g., by adjusting salt concentration or pH), enzymatically cleavable (e.g., by restriction enzymes or endonucleases) or otherwise reversible. In some embodiments, beads comprising conjugated nucleic acids are deposited at node positions on a surface or sample. The conjugated barcodes are released from the beads by cleavage. The released barcodes diffuse away from the node positions. In some embodiments, fixed nucleic acids are encapsulated and can be reversibly released. In some embodiments, the encapsulates are in LNP, beads, AAV, slow viruses, or exosomes.

[0210] In the methods described herein, the sample is in a suspension, solution or hydrogel. In some embodiments, the hydrogel includes poly (2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol (PEG), methacrylic acid (MAA), PEG-PEGMA, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone (PVP), acrylamide / acrylic acid copolymer, linear cationic polyallyl ammonium chloride, poly (N-isopropylacrylamide) (PNIPAM), chitosan, acrylate-modified PEG and acrylate-modified hyaluronic acid, heparin, amine-terminal functionalized 4-arm star-PEG or any combination thereof.

[0211] The detection resolution using the barcoding methods described herein is not limited by light diffraction. Such barcoding methods allow for the detection of samples with very high density as well as very low density targets. Additionally, the barcoding methods described herein can be applied in two or three dimensions without losing resolution or requiring significant adjustments to the method.

[0212] Applications of the methods described herein include, but are not limited to, identification of drug targets, identification of biomarkers, profiling, characterization of cell and disease models, characterization of differentiation states and cell states, tissue mapping, and multidimensional analysis.

[0213] Sample preparation

[0214] Provided herein is a method for preparing a sample for a downstream bioanalysis step. In some embodiments, as provided herein, the sample includes a colony of cells. In some embodiments, provided herein is a sample including a tissue sample. In some embodiments, provided herein is a sample including a laminar cell culture. Non-limiting examples of samples that can be used include: intact tissue, dissected tissue, dissociated cells, organoids, engineered tissues, cultured cells, subcellular compositions, cell suspensions, organs, organelles, tissue biopsies, frozen tissues, tissue sections, tissue blocks, membrane-bound structures, cell-free systems or lysates, formalin-fixed paraffin-embedded (FFPE) samples, exosomes, nuclei, fixed nuclei, fixed exosomes, protein complexes, RNPs or any combination thereof. In some embodiments, provided herein is a sample including a colony of cultured cells. Cultured cells may include, but are not limited to, primary cell cultures, dissociated cells, dissected cells, cell lines and / or transformed cells of passage. In some embodiments, provided herein is a colony of cells including engineered tissues. In some embodiments, engineered tissues include colonies of cells differentiated in vitro by humans. In some embodiments, the cells of human in vitro differentiation are derived from human induced pluripotent stem cells (hiPSC), embryonic stem cells or adult stem cells. In some embodiments, the cells of human in vitro differentiation are in vitro differentiated cardiomyocytes, in vitro differentiated neurons, in vitro differentiated hepatocytes, in vitro differentiated endothelial cells, in vitro differentiated epithelial cells, in vitro differentiated smooth muscle cells, in vitro differentiated skeletal muscle cells or in vitro differentiated nephrocytes. In some embodiments, the colony of cells provided herein includes retinal cells. In some embodiments, the colony of cells provided herein is obtained from a subject. In some embodiments, the sample is unfixed or fixed. In some embodiments, the subject is a mammal. In some embodiments, mammals are primates, non-human primates, humans, mice, rats, goats, rabbits, dogs, camelids or cats.

[0215] In some embodiments, the colony of cells includes a homogeneous colony of cells (e.g., cells of the same type or the same species). In some embodiments, the colony of cells includes a heterogeneous colony of cells (e.g., cells of different types, different species, or any combination thereof). In some embodiments, the colony of cells includes cells from a species. In some embodiments, the colony of cells includes cells from two or more species. In some embodiments, the colony of cells includes a cell type. In some embodiments, the colony of cells includes cells from two or more different cell types. In some embodiments, the colony of cells includes cells from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 1000 or more species. In some embodiments, the population of cells includes cells from at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 250, at least 500, at least 1000, or more different cell types.

[0216] In some embodiments, the sample used in the methods described herein is a tissue sample. In some embodiments, the tissue sample provided herein includes connective tissue, epithelial tissue, muscle tissue or neural tissue. In some embodiments, the tissue sample provided herein includes a combination of tissues. Organs include different types of tissues and have specific functions. In some embodiments, the combination of tissues is an organ. In some embodiments, the combination of tissues is an organoid. In some embodiments, the tissue sample is obtained from a subject. In some embodiments, the tissue sample provided herein is obtained from: brain, eye, lung, liver, bladder, kidney, heart, stomach, intestine, lymph node, skeletal muscle, smooth muscle, pharynx, larynx, artery, liver, gallbladder, bone, spleen, vein, pancreas, reproductive organ, tumor, infected tissue or any combination thereof. In some embodiments, the tissue sample provided herein is a complete organ or biopsy. In some embodiments, the biopsy includes tissue with or suspected of having abnormal proliferation or growth. In some embodiments, the biopsy includes cancer cells.

[0217] In some embodiments, the colony of cells described herein includes one or more microorganisms. In some embodiments, the colony of cells includes prokaryotic cells or eukaryotic cells. In some embodiments, the colony of cells provided herein includes bacterial cells, fungal cells, archaeal cells, eukaryotic cells, or any combination thereof. In some embodiments, the colony of cells includes mammalian cells. In some embodiments, mammalian cells are cells of humans, non-human primates, primates, pigs, horses, sheep, cats, rats, mice, dogs, llamas, rabbits, or goats. In some embodiments, the colony of cells also includes viruses. In some embodiments, the virus is a pathogenic virus. In some embodiments, the colony of cells includes a viral vector. In some embodiments, the colony of cells has been genetically modified. Non-limiting examples of genetic modification include base editing, gene silencing (e.g., by CRISPR / Cas9 system), gene insertion, gene deletion.

[0218] In some embodiments, the colony of cells is fixed and then frozen. In some embodiments, the colony of cells is frozen and then fixed after thawing and / or slicing. In some embodiments, the colony of fixed cells is sliced ​​and then fixed for the second time. In some embodiments, the colony of cells is stored at room temperature after fixation. In some embodiments, the colony of cells is fixed with organic solvents such as alcohol and acetone. In some embodiments, the colony of cells (e.g., tissue) is embedded in paraffin and then fixed. In some embodiments, the colony of cells is fixed with a cross-linking agent. In some embodiments, the cross-linking agent includes formalin, formaldehyde, paraformaldehyde, disulfide-bis (succinimidyl propionate) (DSP) or any combination thereof. In some embodiments, a permeabilization step is performed. In some embodiments, permeabilization is not performed. In some embodiments, permeabilization occurs simultaneously with fixation. In some embodiments, permeabilization is performed after fixation. In some embodiments, permeabilization agents include acetone, alcohol, detergents or any combination thereof.

[0219] In some embodiments, the colony of cells for analysis as provided herein is a living cell. In other embodiments, living cells are directly contacted with a barcode sequence, or indirectly contacted via a connecting molecule. In some embodiments, the connecting molecule binds to a cell surface receptor. In some embodiments, the connecting molecule binds to a cell surface molecule. In some embodiments, the cell surface molecule is a protein or a nucleic acid. The nucleic acid may be RNA or DNA. The protein may be a cell receptor. In some embodiments, the cell surface molecule is located at a cell adhesion point. In some embodiments, the barcode sequence is introduced into a living cell intracellularly. In some embodiments, the barcode sequence is attached to a lipid nanoparticle (LNP) as a carrier for delivery to a cell. In some embodiments, AAV, lentivirus, exosomes or other encapsulations are carriers for delivering a barcode sequence to a cell. In some embodiments, cells are transfected or transformed to receive a barcode sequence. In some embodiments, cells are mechanically perturbed so that the cell can take up a barcode sequence.

[0220] In some embodiments, the samples described herein include nucleic acids. In some embodiments, the samples include nucleic acids directly or indirectly bound or tethered to a surface. In some embodiments, the samples include nucleic acids in solution. In some embodiments, the samples include nucleic acids in a hydrogel, a swollen hydrogel, a biological sample embedded in a hydrogel, a biological sample embedded in a swollen hydrogel, with or without sample clarification. In some embodiments, the samples include nucleic acids in a scaffold. In some embodiments, the nucleic acids are attached directly or indirectly to a synthetic matrix. In some embodiments, the synthetic matrix includes beads or a semisolid gel.

[0221] In some embodiments, the samples described herein include cells. In some embodiments, the cells are bound to a surface. In some embodiments, the cells are in solution. In some embodiments, the cells are connected to a hydrogel. In some embodiments, the cells are connected to a synthetic scaffold. In some embodiments, the cells are directly or indirectly attached to the synthetic scaffold. In some embodiments, the synthetic scaffold includes beads or semisolid gels. In some embodiments, the synthetic scaffold includes extracellular matrix proteins. In some embodiments, the extracellular matrix proteins include collagen or fibronectin.

[0222] In some embodiments, provided herein is a sample frozen at -5 ° C to -200 ° C. In some embodiments, provided herein is a sample frozen at about -5 ° C, about -10 ° C, about -15 ° C, about -20 ° C, about -25 ° C, about -30 ° C, about -35 ° C, about -40 ° C, about -45 ° C, about -50 ° C, about -55 ° C, about -60 ° C, about -65 ° C, about -70 ° C, about -75 ° C, about -80 ° C, about -85 ° C, about -90 ° C, about -95 ° C, about -100 ° C or any combination thereof. In some embodiments, provided herein is a tissue sample or a colony of cells that are rapidly frozen. In some embodiments, provided herein is a tissue sample or a colony of cells that are frozen by slow or balanced freezing.

[0223] In some cases, fixation may affect protein biochemistry, masking epitopes. In some embodiments, the samples described herein are also treated with an antigen retrieval method. In some embodiments, the antigen retrieval method is protease-induced epitope retrieval (PIER). In some embodiments, the antigen retrieval method is heat-induced epitope retrieval (HIER). In some embodiments, the antigen retrieval method is any other method capable of reversing epitope masking. In some embodiments, the antigen retrieval step is combined with a permeabilization step. In some embodiments, the antigen retrieval step is performed independently.

[0224] In some embodiments, the samples provided herein are fixed on a microscope slide or a cover glass. In some embodiments, the microscope slide is a chamber slide. In some embodiments, the chamber slide comprises one or more chambers. In some embodiments, the slide comprises polystyrene. In some embodiments, the slide comprises glass. In some embodiments, the cover glass comprises glass. In some embodiments, the cover glass comprises UV transparent material. In some embodiments, the cover glass comprises quartz.

[0225] In some embodiments, slide includes coating to promote the colony of tissue sample or cell provided herein to adhere to the slide surface. In some embodiments, coating includes poly-L-lysine or poly-D-lysine. In some embodiments, coating includes gelatin. In some embodiments, coating includes 3-aminopropyltriethoxysilane (APES). In some embodiments, adhesive tape is used to adhere the sample to the slide. In some embodiments, adhesive tape is polyvinylidene chloride, polyester / silicone, polypropylene film, cellophane, synthetic rubber-resin hot melt, low-density polyethylene film, polyester film or any combination thereof.

[0226] In some embodiments, the sample is embedded in a hydrogel. In some embodiments, the hydrogel comprises a synthetic gel or a polyepoxide. In some embodiments, the embedded sample is generated at the tissue-hydrogel matrix. In some embodiments, the embedded sample provides for the expansion of a population of tissue samples or cells provided herein. In some embodiments, the hydrogel is a natural hydrogel, a synthetic hydrogel, or a synthetic / natural hybrid hydrogel. In some embodiments, the natural hydrogel is a protein, a polysaccharide, a protein / polysaccharide, DNA, or any combination thereof. In some embodiments, the protein hydrogel comprises collagen, elastin, fibrin, silk, lysozyme, (Corning Inc., Corning, NY), genetically engineered proteins, or any combination thereof. In some embodiments, the polysaccharide comprises hyaluronic acid (HA), alginate, chitosan, dextran, or any combination thereof. In some embodiments, the protein / polysaccharide comprises collagen / HA, laminin / cellulose, fibrin / alginate, gelatin / agarose, chitosan, alginate, dextran, or any combination thereof. In some embodiments, the DNA hydrogel comprises X-DNA, Y-DNA, T-DNA, linear plasmid DNA, or any combination thereof. In some embodiments, the synthetic hydrogel is a non-biodegradable hydrogel, a biodegradable hydrogel, a bioactive hydrogel, or any combination thereof. In some embodiments, the non-biodegradable hydrogel comprises poly(2-hydroxyethyl methacrylate) (PHEMA), poly(2-hydroxypropyl methacrylate) (PHPMA), poly(N-isopropylacrylamide) (PNIPAm), poloxamer ( In some embodiments, the biodegradable hydrogel comprises degradable polyethylene glycol (PEG), polypropylene fumarate-PEG (PPF-PEG), poly(2-hydroxyethyl methacrylate)-poly(ε-caprolactone) (PHEMA-PCL), a synthetic peptide, or any combination thereof. In some embodiments, the bioactive hydrogel comprises a cell-adhesive hydrogel, an enzyme-sensitive hydrogel, a growth factor-carrying hydrogel, another bioactive hydrogel, or any combination thereof. In some embodiments, the synthetic / natural hybrid hydrogel comprises PEG / dextran, heparin, HA, chondroitin sulfate (CS), one or more proteins, PNIPAm / protein, chitosan, alginate, synthetic peptide / protein, polysaccharide, PVA / DNA, Pluronic / dextran, PHPMA / protein, or any combination thereof.

[0227] In some embodiments, the sample includes cells. In some embodiments, the sample includes cells from a suspension. In some embodiments, the sample includes a complete organism. In some embodiments, the complete organism is a multicellular organism. In some embodiments, the complete organism is a unicellular organism. Exemplary multicellular organisms include but are not limited to roundworms (Caenorhabditis elegans), zebrafish (zebrafish) (Daniorerio) and fruit flies (Drosophila). Exemplary unicellular organisms include but are not limited to bacteria and yeast. In some embodiments, the sample includes tissue. In some embodiments, the tissue of the slice is sliced. In some embodiments, the thickness of the slice is up to about 5um, about 10um, about 15um, about 20um, about 25um, about 30um, about 35um, about 40um, about 45um, about 50um or larger.

[0228] In some embodiments, the samples described herein are imaged using a microscope. In some embodiments, the microscope used in the methods described herein comprises a compound microscope, a fluorescence microscope, a confocal microscope, a light sheet microscope, a Raman microscope, or a digital microscope.

[0229] Barcoding workflow

[0230] Features in sample preparation, in situ imaging and barcoding, and transcript extraction and sequencing are further described herein. An exemplary workflow for analyzing biological cells is Fig.13A Provided in. In short, the step of sample preparation (e.g., a population of tissue samples or cells) is performed. The sample preparation step may include culturing cells or obtaining tissues or organoids, followed by imaging. Dyes and / or probes may be applied to the sample to enhance the visualization of cells and / or structures therein. Optional staining and / or imaging subsequently provide morphological and structural information of the sample to assist in region selection, cell segmentation, and mask generation. The sample is then optionally permeabilized. In some embodiments, permeabilization is performed prior to imaging the sample or simultaneously with fixation of the sample. In some embodiments, permeabilization is performed to analyze intracellular biomolecules.

[0231] Following the workflow, for in situ analysis of biological cells, reverse transcription of RNA is performed, optionally followed by tailing of the resulting cDNA. Spatially directed barcoding within selected regions of interest is then performed using a photomask, optionally with the regions selected for masking based on earlier sample imaging. Optionally, the biological cells are further imaged to reveal incorporation of the barcode. Following Fig.13AThe process described in, record extraction and sequencing include displacement of barcoded nucleic acids. The displacement is optionally achieved by applying RNase H. The cross-linking of continuous nucleic acids can also achieve displacement of barcoded nucleic acids. Enzymatic displacement and synthesis reactions can be combined or performed sequentially. After the sequence has been extracted from the sample, the sample can be further imaged or otherwise measured (e.g., mass spectrometry, H&E staining). Subsequent polymerase chain reaction (PCR) amplification and optional purification are followed by optional library preparation and next generation sequencing. The resulting sequence analysis is merged with the captured imaging data to provide an integrated spatial and sequence-based information output.

[0232] Another exemplary workflow is shown in Fig. 13B In brief, a sample preparation step (e.g., a tissue sample or a colony of cells) is performed. The sample preparation step may include culturing cells or obtaining tissues or organoids, followed by imaging. Dyes and / or probes may be applied to the sample to enhance visualization of the cells and / or structures therein. Subsequent imaging provides morphological and structural information of the sample to assist in region selection, cell segmentation, and mask generation. The sample is then optionally permeabilized.

[0233] like Fig. 13B As described in, the barcoding of the sample includes target binding. In some embodiments, the target binding is affinity binding. Affinity binding may include binding to a ligand, an epitope, or any other specific recognition binding. In some embodiments, the target binding is hybridization. Hybridization may include hybridization of nucleic acid chains, including but not limited to deoxyribonucleic acid (DNA) chains, ribonucleic acid (RNA) chains, peptide nucleic acid (PNA) chains, locked nucleic acid (LNA) chains, and combinations thereof. For example, hybridization may include hybridization of RNA to RNA, DNA to DNA, RNA to DNA, RNA to PNA, DNA to PNA, PNA to PNA, or any combination thereof.

[0234] The fractional spatial barcoding workflow is shown in Fig. 13C In the present invention, the sample preparation and optional imaging steps include methods similar to the previous workflow. In this case, a unique ratio of barcodes or barcode arrangements is assigned to a specific cell or region of interest (ROI). The duration or power of the applied radiation energy is controlled so as to define the fraction of available targets that will be crosslinked with the contacted barcodes. Fig.14A is an exemplary depiction of controllably increasing illumination power or time to induce more crosslinking events in a specific region (in this case, cells). Multiple rounds of fractional barcode crosslinking can be applied across a sample, allowing cells or regions of interest to be distinguished and identified. Fig. 14BThe figure shows an exemplary combination of four barcode sequences applied across cells or regions. Each cell or region of interest can be assigned a unique segmentation of a barcode sequence in the barcode sequence read, so that after subsequent single cell or single region barcoding and sequencing, the original spatial position of the cell can be restored based on the score of the read of each barcode measured with next generation sequencing. In some cases, after sample dissociation, the cell or region is barcoded separately. In some embodiments, before sequencing, a sorting method for separating different regions is applied, such as separately collecting barcoding, microfluidics, barcoding beads or other single cell barcoding technologies. Each cell or ROI can be identified based on the score of the light cross-linked barcode in the read corresponding to a node or a combination thereof.

[0235] The generated node barcode strands described herein can be combined with a target sequence. In some embodiments, the target sequence comprises one or more cDNAs, nucleic acid barcodes, ISH probes, other unique identifier sequences, or any combination thereof. Fig.15 An exemplary embodiment of a node barcode chain is shown in combination with a target barcode and a unique identifier to record proximity information about which node is close to a molecule of interest. Complementary regions on the chain hybridize, and the chain is extended using the polymerization method described herein. The proximity information can be used to computationally reconstruct the original spatial orientation of the node position in the sample or substrate. The spatial orientation can be mapped based on the frequency of the node barcodes present in the target nucleic acid and the tandem nucleic acid.

[0236] Any combination of conjugated nucleic acids comprising node nucleic acids, factory target nucleic acids, and target barcodes is contemplated herein. The methods described herein are not limited to the order of conjugation of the barcoded nucleic acids. Additionally, in various embodiments, the barcoded strands described herein comprise any combination, order, and repetition of target barcodes, node barcodes, factory target barcodes, node type barcodes, unique modifiers, or any other identifier described herein. An exemplary order and number of barcodes is depicted in Fig.16 middle. Fig.16 The intervening spaces between the barcodes depicted in the sequence are optionally free of anything, including adapters, primers, L or R flanking sequences, or promoter sequences. Fig.16 As shown in, a "target identification sequence" refers to any sequence used to identify a "target" (or more targets) being analyzed. In some embodiments, the target sequence is the target itself, for example, the fixed nucleic acid can be a sequence attached to the target or the reverse complement of the attached sequence. In some embodiments, an ISH probe, aptamer or nucleic acid conjugated to a protein is an attached sequence. In some embodiments, the target sequence can be stacked to multiple levels. For example, a node barcode is combined with a probe sequence, and the probe sequence hybridizes with a nucleic acid conjugated to an antibody that binds to a target protein.

[0237] dyeing

[0238] Provided herein is a method for image capture and enhancement thereof, which incorporates the application of imaging agents.Imaging agents can be used to distinguish cells, cell types and / or subcellular structures.In some embodiments, a tissue sample or a colony of cells provided herein is contacted with an imaging agent.In some embodiments, an imaging agent is a dye or stain.In some embodiments, a dye or stain includes 7-AAD, acridine orange, bismarck brown, calcein, CFSE, fuchsin, coomassie blue, cresyl violet, crystal violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hoechst stain, iodine, malachite green, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide (official name: osmium tetroxide), propidium iodide, resazurin, rhodamine, safranin, trypan blue, tetrazolium salts or any combination thereof.

[0239] In some embodiments, the sample is visualized with immunofluorescence (IF) staining. In some embodiments, the IF staining reagent includes fluorophore-conjugated streptavidin. In some embodiments, the imaging agent is a fluorophore. Exemplary fluorophores for inclusion in the workflow described herein include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthylfluorescein (pH 10); 5-carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5-carboxyfluorescein); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; ABQ; acid fuchsin; ACMA (9-amino-6-chloro-2-methoxyacridine); acridine orange; acridine red; acridine yellow; Acriflavin; Acriflavin Feulgen SITSA; aequorin (photoprotein); Alexa Fluor 350 TM Alexa Fluor 430 TM ;Alexa Fluor488 TM Alexa Fluor 532 TM ; Alexa Fluor 546 TM ; Alexa Fluor 568 TM Alexa Fluor 594 TM ;Alexa Fluor 633 TM;Alexa Fluor 647 TM Alexa Fluor 660 TM Alexa Fluor 680 TM ; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aniline Blue; Anthrocylstearate; APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7GLL; Atabrine; ATTO-TAG TM CBQCA; ATTO-TAG TM FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); BG-647; Bimane; bisbenzamide; Blancophor FFG; Blancophor SV; BOBO TM -1;BOBO TM -3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy FI; Bodipy FL ATP; Bodipy Fl-ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X,SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO TM -1; BO-PRO TM-3; Brilliant Sulphoflavin FF; Calcium Crimson; Calcium Crimson Blue TM ; Calcium Green; Calcium Green-1Ca2+ dye; Calcium Green-2Ca2+; Calcium Green-5NCa2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcium Fluorescent White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue TM ;CascadeYellow;catecholamine;CFDA;CFP-cyan fluorescent protein;chlorophyll;chromomycin A;CMFDA;coelenterazine;coelenterazine cp;coelenterazine f;coelenterazine fcp;coelenterazine h;coelenterazine hep;coelenterazine ip;coelenterazine O;coumarin phalloidin;CPM methylcoumarin;CTC;Cy2 TM ; Cy3.1 8; Cy3.5 TM ;Cy3 TM ;Cy5.1 8;Cy5.5 TM ;Cy5 TM ;Cy7 TM ; cyan GFP; cyclic AMP fluorescent sensor (FiCRhR); d2; Dabcyl; dansyl; dansylamide; dansylcadaverine; dansyl chloride; dansylDHPE; dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (dihydrofluorescein diacetate); DDAO; DHR (dihydrorhodamine 123); di-4-ANEPPS; di-8-ANEPPS (non-ratio); DiA (4-di-16-ASP); DIDS; dihydrorhodamine 123 (DHR); DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium homodimer-1 (EthD-1); Euchrysin; Europium (III) chloride; Europium; EYFP; Eva Green; Fast Blue; FDA; Feulgen (para-purpurin); FITC; FL-645; Flazo Orange; Fluo-3; Fluo-4; Fluorescein diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1-43 TM ;FM 4-46;FuraRed TM(high pH); Fura-2, high calcium; Fura-2, low calcium; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GFP (S65T); GFP red shift (rsGFP); GFP wild type, no-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbene (FluoroGold); Serotonin; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751; Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine rhodamine; Lissamine rhodamine B; LOLO-1; LO-PRO-1; Fluorescent Yellow; Mag Green; Magdala Red (Phloxin B); Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxilon Brilliant Flavin 10GFF; Maxilon Brilliant Flavin 8GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromodiamine; Monobromodiamine (mBBr-GSH); Monochlorodiamine; MPS (Methyl Green Pyronine Stilbene));NBD;NBD amine;Nile Red;Nitrobenzoxadidole;Norepinephrine;Nuclear Fast Red;Nuclear Yellow;Nylosan Brilliant Iavin E8G;Oregon Green TM ;Oregon Green 488-X;OregonGreen TM488; Oregon Green TM 500; Oregon Green TM 514; Pacific Blue; Feulgen; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); MagdalaRed; Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26; PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufm; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B 540; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Red; R-phycoerythrin (PE); Red-shifted GFP (rsGFP, S65T); S65A; S65C; S65L; S65T; Sapphire GFP; Serotonin; Sevron BrilliantRed 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; SevronYellow L; sgBFP TM ;sgBFP TM (super glow BFP); sgGFP TM ;sgGFP TM(super glow GFP); SITS; SITS (primulina); SITS (stilbene isothiosulphonic acid); SPQ (6-methoxy-N-(3-sulfopropyl)-quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYBR Gold; SYBR Green I; SYBR Green II; SYBR Safe; Tetracycline; Tetramethylrhodamine; Texas Red TM ;Texas Red-X TM Conjugates; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazolyl Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (calcium fluorescent white); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC (Tetramethyl Rodamine IsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; XL665; X-rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; and YOYO-3. Many suitable forms of these fluorescent compounds are available and can be used.

[0240] In some embodiments, sample imaging provides a basis for identifying a cell region of interest (ROI). In some embodiments, the ROI includes a barcoded region targeted for spatial definition of cDNA in the sample. In some embodiments, the ROI is based on the morphology of the sample, such as a cell, a cell structure, a tissue layer, or a subcellular structure. In some embodiments, the ROI has regular or irregular boundaries. In some embodiments, the ROI describes the boundaries of the barcoded space defined in the sample. In some embodiments, the ROI is based on a grid. In some embodiments, the ROI is two-dimensional (2D). In some embodiments, the ROI is three-dimensional (3D).

[0241] The photomask provides a template to control the area in the sample that receives the radiated light. In some embodiments, a photomask is generated to allow radiation of one or more ROIs. In some embodiments, one or more photomasks are generated. In some embodiments, a photomask associated with each ROI is generated. In some embodiments, each cross-linking step includes a unique photomask. In some embodiments, the photomask is manually or machine generated. In some embodiments, a photomask is generated according to sample imaging as previously described herein.

[0242] In-situ barcoding

[0243] Barcoding provides information tags in samples. In some embodiments, DNA barcodes are short segments of DNA where a single sequence can be used to identify or distinguish samples, cells, ROIs, cell types, targets, target types, nodes, node types, proximal molecules, spatial information, node information, or a combination thereof. In some cases, barcodes are incorporated into longer lengths of nucleic acid for further processing. Further processing can include extraction, amplification, sequencing, analysis, and any combination thereof.

[0244] In some embodiments described herein, the method includes barcoding the target in the sample. In some embodiments, the target is a nucleic acid, a protein, an antigen, a lipid, a sugar, a cell structure, a scaffold, or any combination thereof. In some embodiments, the target is a nucleic acid generated in situ. In some embodiments, cDNA is generated from the nucleic acid in the sample by in situ transcription. In some embodiments, in situ transcription is reverse transcription. In some embodiments, RNA is generated in situ from a partially or completely double-stranded DNA by transcription. In some embodiments, overhangs are added to cDNA by adding multiple A tails. In some embodiments, then the tissue sample or cell colony provided herein is barcoded by adding one or more nucleic acid barcodes.

[0245] In some embodiments, the target nucleic acid is an amplification product. Nucleic acid amplification can be performed at a constant temperature ("isothermal") to reduce the use of complex external equipment. In some embodiments, the target nucleic acid is generated by isothermal amplification. In some embodiments, isothermal amplification is performed at ambient temperature and / or room temperature. In some embodiments, ambient temperature and / or room temperature are about 20°C to 25°C. In some embodiments, isothermal amplification is rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), strand displacement amplification (SDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branch amplification method (RAM), nucleic acid sequence-based amplification (NASBA). In some embodiments, the target nucleic acid is generated by exchange reaction signal amplification (SABER). In some embodiments, the target nucleic acid is generated by a synthetic reaction. In some embodiments, the target nucleic acid is generated by a primer exchange reaction (PER) or RCA. In some embodiments, PER is a PER assay based on terminal deoxynucleotidyl transferase (Tdt). In some embodiments, the target nucleic acid is a long repeat single-stranded RNA or DNA sequence. In some embodiments, amplification is performed at multiple temperatures (e.g., in one cycle). In some embodiments, amplification is performed using non-isothermal, linear, or exponential amplification methods. In some embodiments, amplification is PCR. In some embodiments, PCR is asymmetric PCR. In some embodiments, amplification is isothermal. Exemplary isothermal amplification methods include strand displacement amplification (SDA), rolling circle amplification (RCA), whole genome amplification (WGA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and multiple displacement amplification (MDA).

[0246] Nucleic acids provided herein can be non-specifically or specifically conjugated to another molecule in the sample. In some embodiments, nucleic acids are conjugated to proteins, epitopes, lipids, liposomes, droplets, beads, another nucleic acid LNP, AAV, exosomes or slow viruses. In some embodiments, barcodes are conjugated to molecules. In some embodiments, barcodes include nucleic acids conjugated to antibodies, antibody fragments, proteins, nanobodies, small molecules, nucleic acid therapeutics, lipids, nanoparticles, lipid nanoparticles or other affinity reagents. In some embodiments, antibody fragments as described herein are F (ab ') 2 fragments, Fab ' fragments, Fab fragments, Fv fragments, IgG fragments, Fc fragments or any combination thereof. In some embodiments, lipids are emulsions, liposomes, particles or any combination thereof. In some embodiments, antibodies are IgA, IgG or IgM antibodies or their functional fragments. In some embodiments, tissue samples provided herein or cell colonies are imaged after barcoding.

[0247] In some embodiments, the target nucleic acid is conjugated to a molecule. In some embodiments, the target nucleic acid and / or the molecule conjugated to the target nucleic acid is detected by proximity ligation assay (PLA). PLA allows in situ detection of protein-protein interactions at a distance of approximately less than 40 nanometers. In some embodiments, the target nucleic acid is a PLA oligonucleotide. In some embodiments, the barcode nucleic acid provided herein also comprises a PLA oligonucleotide. In some embodiments, the target nucleic acid comprises a first PLA oligonucleotide, wherein the first PLA oligonucleotide is conjugated to a first protein of interest. In some embodiments, the first PLA oligonucleotide is hybridized with a second PLA oligonucleotide, wherein the second PLA oligonucleotide is conjugated to a second protein of interest. In some embodiments, the first protein of interest or the second protein of interest is selected from the group consisting of: antibodies, antibody fragments, cell surface receptors, cell surface proteins and intracellular proteins, mitochondrial proteins, nuclear proteins, transcription factors or scaffold proteins. In some embodiments, the first PLA oligonucleotide and the second PLA oligonucleotide are connected to form circular DNA. In some embodiments, circular DNA is amplified. In some embodiments, circular DNA includes a barcode chain provided herein. In some embodiments, the circular DNA is contacted with a barcode strand provided herein.In a PLA reaction, the barcodes can be used to identify unique protein-protein or nucleic acid and protein interactions within a sample.

[0248] In some embodiments, the concatemers described herein are attached to proteins. In other embodiments, proteases are applied to release concatemers from surfaces. In some embodiments, the barcodes described herein are attached to proteins. In other embodiments, proteases are applied to release barcodes from surfaces. Exemplary proteases for inclusion include, but are not limited to, proteinase K. In some embodiments, proteases are applied prior to antigen repair reactions (e.g., reverse transcription), prior to barcoding reactions, prior to concatemerization reactions, and / or prior to concatemer repair reactions.

[0249] In some embodiments, the target nucleic acid is DNA. In some embodiments, the DNA is nuclear DNA. In some embodiments, the DNA is mitochondrial DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the target nucleic acid is an intron. In some embodiments, the target nucleic acid is an exon. In some embodiments, RNA is messenger RNA (mRNA). In some embodiments, the target nucleic acid is a regulatory RNA. In some embodiments, RNA is circular RNA (circRNA), ribosomal RNA (rRNA), 16S ribosomal RNA (16S rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), non-coding RNA, long non-coding RNA (lncRNA) or micro RNA (miRNA).

[0250] In some embodiments, the target nucleic acid is exposed to a DNA or RNA polymerase. In some embodiments, a polymerase refers to an enzyme that performs template-directed synthesis of a polynucleotide. In some embodiments, a DNA polymerase is isolated from or derived from Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, or a modified form thereof. In some embodiments, polymerases include, but are not limited to, Klenow fragment (New England Biolabs Inc.), Taq DNA polymerase (QIAGEN), 9°N TM DNA polymerase (NewEngland Biolabs Inc.), Deep Vent TM DNA polymerase (New England Biolabs Inc.), Manta DNA polymerase (Enzymatics), Bst DNA polymerase (New England Biolabs Inc.), or phi29 DNA polymerase (New England Biolabs Inc.). In some embodiments, the polymerase is DNA-dependent. In some embodiments, the polymerase is RNA-dependent. In some embodiments, the polymerase is a reverse transcriptase.

[0251] In some embodiments, in situ reverse transcription includes the incorporation of primers. In some embodiments, primers describe short nucleic acid sequences. In some embodiments, primers are DNA. In some embodiments, primers are RNA. In some embodiments, primers have regions complementary to DNA or RNA chains. In some embodiments, primers provide free 3'-OH, and DNA polymerase starts synthesizing deoxyribonucleotide chains at this free 3'-OH. In some embodiments, primers contain about 5 to about 50 or more nucleotides. In some embodiments, primers contain about 10 to about 30 nucleotides. In some embodiments, primers as described herein include 3' sequences NNNNNNN (7 N), NNNNGGG (5 N and 3 G), NNNNNCCC (5 N and 3 C), NNNNNAAA (5 N and 3 A) or NNNNNTTT (5 N and 3 T).

[0252] In some embodiments, the primer is attached to an affinity molecule, a scaffold, a substrate, a surface, a bead, or a column. In some embodiments, the primer is in suspension, solution, or a hydrogel.

[0253] In embodiments, 3' overhangs are added to cDNA transcripts in the tailing step. In some embodiments, tailing is achieved by using terminal transferase and dXTP (dATP, dCTP, dGTP, dTTP, or any combination thereof). Adding ddXTP (ddATP, ddCTP, ddGTP, ddTTP, or any combination thereof) or another terminating nucleotide at the 3' end prevents subsequent extension during the later cross-linking step. In some embodiments, ddXTP or another terminating nucleotide is included at low concentrations to randomly terminate the 3' end. In some embodiments, 3' overhangs are added using other strategies. In some embodiments, overhangs are added by ligation.

[0254] The barcodes provided herein generally encode non-genetic data. In some embodiments, the barcode chain is a single-stranded nucleic acid. The hybridization domain is introduced into the barcode chain to allow the barcode to be arbitrarily arranged by iterative hybridization, crosslinking and washing steps. In some embodiments, the nucleic acid barcode chain comprises a barcode domain. In some embodiments, the length of the barcode domain is about 5 to about 10 bases. In some embodiments, the barcode domain flanks a group of two alternating hybridization domains. In some embodiments, the barcode domain flanks a unique hybridization domain. In some embodiments, the barcode domain flanks a group of 3, 4, 5, 6 or more alternating hybridization domains. In some embodiments, the hybridization domain is a crosslinking domain. In some embodiments, the hybridization domain is a complementary strand. In some embodiments, a group of two alternating hybridization domains comprises a crosslinking strand and a complementary strand. In some embodiments, the nucleic acid barcode comprises a barcode domain connected to (i) a crosslinking strand comprising a photoactivated nucleotide and (ii) a complementary strand. In some embodiments, the length of the alternating hybridization domain can be at least 5, at least 10, at least 15, or at least 20 bases. In some embodiments, the alternating hybridization domain comprises bases that hybridize to the hybridization domain of the second nucleic acid barcode. In some embodiments, the cross-linked strand of one nucleic acid barcode hybridizes to the complementary strand of another nucleic acid barcode.

[0255] In some embodiments, the barcodes provided herein are deposited onto the sample at a concentration of about 20 nM to about 500 nM. In some embodiments, the barcodes provided herein are deposited onto the sample at a concentration of up to about 1 nM, 10 nM, 20 nM, 100 nM, 250 nM, 500 nM, 1000 nM or more. The binding of the barcode to the target region can be stable or transient.

[0256] In some embodiments, the length of the nucleic acid barcode domain is about 5-10 bases. In some embodiments, the length of the nucleic acid barcode domain is 4, 5, 6, 7, 8, 9, 10 or more bases. In some embodiments, the length of the nucleic acid barcode domain is about 10 or about 10-15 bases. Each unique barcode domain can be assigned a position value of "0" or "1". In some embodiments, the tandem string of nucleic acid barcodes is equivalent to a string of 0 and 1.

[0257] In some embodiments, the cross-linked chain is a single-stranded nucleic acid having a length of 200 nucleotides or less. In some embodiments, the length of the cross-linked chain is 5-100, 5-75, 5-50, 5-25, 5-15 or 5-10 nucleotides. In some embodiments, the length of the cross-linked chain can be 10-100, 10-50, 10-25, 10-20, 15-25 or 15-25 nucleotides. In some embodiments, the length of the cross-linked chain can be 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, the cross-linked chain comprises at least one photoreactive nucleotide. In some embodiments, the cross-linked chain comprises at least one CNVK or CNVD nucleotide. In some embodiments, the cross-linked chain is complementary to the complementary chain, wherein the cross-linked chain is a component of the first nucleic acid barcode and the complementary chain is a component of the second nucleic acid barcode. In some embodiments, the cross-linked chain is DNA or RNA. In some embodiments, the cross-links are also functionalized with moieties such as fluorophores, quantum dots, biotin, streptavidin, functional chemical groups, acrylamide, maleimide, lipids, metal isotopes, and other tags or nanoparticles.

[0258] In some embodiments, the length of the nucleic acid barcode chain is at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides. In some embodiments, the length of the nucleic acid barcode chain is 20 nucleotides. In some embodiments, the nucleic acid barcode chain comprises a barcode domain having a length of 2 to 10 nucleotides, a cross-linked chain having a length of 2 to 12 nucleotides, and a complementary chain having a length of 2 to 12 nucleotides. In some embodiments, the nucleic acid barcode chain comprises the following structure in the 5' to 3' direction: 5'-cross-linked chain to barcode domain to complementary chain-3'. In some embodiments, the nucleic acid barcode chain comprises the following structure in the 5' to 3' direction: 5'-complementary chain to barcode domain to cross-linked chain-3'.

[0259] The cross-linked strands can be designed to stably bind or transiently bind to the complementary strand. In some embodiments, the cross-linked strands can be covalently or non-covalently bound to the complementary strand. In some embodiments, the cross-linked strands can be complementary to the complementary strand. The cross-linked strands can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% complementary to the complementary strand. In addition, the cross-linked strands can bind to complementary domains of different nucleic acid barcodes. In some embodiments, the cross-linked strands can bind to complementary domains of nucleic acids that contain their own cross-linked strands but do not contain barcode strands.

[0260] The nucleic acid barcode chains can be connected in series and covalently fixed together by photochemical cross-linking reactions. In some embodiments, the cross-linking reaction is light-driven. In some embodiments, the cross-linking reaction is chemically driven. In some embodiments, the cross-linking reaction is pH-driven. In some embodiments, the cross-linking reaction can be carried out in an aqueous solution. In some embodiments, a photoreactive cross-linking agent is used in the reaction. In some embodiments, a photoreactive nucleobase is used in the reaction. In such embodiments, the cross-linked chain of the nucleic acid barcode includes at least one photoreactive nucleobase. In some embodiments, the photoreactive nucleobase can be any modified nucleobase that can form a crosslink with another nucleobase in the presence of light. In some embodiments, the photoreactive nucleobase is a modified pyrimidine or purine nucleobase. In some embodiments, the photoreactive nucleobase includes 2-hydroxycarbazole, 3-cyanovinylcarbazole phosphoramidite (CNVK) or 3-cyanovinylcarbazole modified d-threonine alcohol (CNVD). In some embodiments, the photoreactive nucleobase comprises a vinyl, acrylate, N-hydroxysuccinimide, amine, carboxylate, or thiol chemical group. In some embodiments, the photoreactive nucleobase comprises bromodeoxyuridine. In some embodiments, the CNVK crosslinking base exhibits the highest crosslinking efficiency with thymine (T) bases located adjacent to bases on the complementary strand and can be directly incorporated into the DNA hybridization domain itself as a base substitution. In some embodiments, the crosslinking reaction is performed using light of 365 nm wavelength and is completed within 1 second, such as Fig.17 In some embodiments, more than one photocrosslinker molecule is incorporated so that different barcodes contain different photocrosslinker molecules that can crosslink at different wavelengths. Such changes in the barcodes allow methods to change crosslinking events at one or more regions of interest. In some embodiments, a reverse crosslinking reaction is performed. For example, applying a wavelength of 312nm for a few minutes can provide crosslinking reversal. In some embodiments, a series of crosslink-then-decrosslinking reactions are performed to form concatemers. In some embodiments, molecules are deposited on a substrate provided herein to locally generate light wavelengths to support photosensitive crosslinking reactions.

[0261] The cross-linking reaction can be designed to use any wavelength of visible light or ultraviolet light. In some embodiments, the cross-linking reaction can be completed within 0.1, 0.25, 0.5, 1, 5 or 10 seconds. In some embodiments, the cross-linking reaction can be completed within 0.5, 1, 5, 10, 20, 30, 40, 50 or 60 minutes. In some embodiments, the cross-linking reaction has a negligible effect on the bases adjacent to the photoreactive nucleobase. In some embodiments, other photochemical nucleic acid cross-linking agents (including psoralen) are used in combination with nucleic acid barcodes.

[0262] In some embodiments, a cross-linking reaction is applied to a nucleic acid barcode strand that is hybridized to the 5' end of a cDNA. In some embodiments, a cross-linking reaction is applied to a nucleic acid barcode strand that is hybridized to the 3' end of a cDNA. In some embodiments, additional barcode strands are iteratively added to the 5' or 3' end of a cDNA-nucleic acid barcode structure using binding and cross-linking as previously described. Fig.18 Shown is a process involving iterative addition of a barcode strand to the 5' end of a cDNA according to the methods described herein.

[0263] In some embodiments, the nucleic acid probe comprises a barcode and a photosensitive cross-linking molecule. In some embodiments, the nucleic acid probe comprises a barcode and a CNVK molecule. In some embodiments, the nucleic acid probe comprises a barcode, a CNVK molecule, and a fluorophore. In some embodiments, the nucleic acid probe hybridizes with a factory target nucleic acid. In some embodiments, the nucleic acid probe hybridizes with a node nucleic acid. In some embodiments, the nucleic acid probe hybridizes with a concatemer of a factory target nucleic acid, a concatemer node nucleic acid, or a concatemer of both a target binding and a node nucleic acid.

[0264] Cleavage oligonucleotide is a short oligonucleotide of about 10 to about 50 nucleotides in length, which hybridizes with a restriction site on a single-stranded nucleic acid, producing a double-stranded region around the restriction site. In some embodiments, the function of an endonuclease requires a double-stranded recognition sequence. In some embodiments, a short cleavage oligonucleotide is cross-linked with a concatemer to generate a hybrid node chain carrying a node barcode. In some embodiments, a cleavage oligonucleotide is attached to a single-stranded nucleic acid using light-directed light cages. In some embodiments, the generation of a stable, covalently linked structure with an in-situ generated node chain allows the use of additional functionalizations that can be used to synthesize oligonucleotides.

[0265] In some embodiments, the cleavage oligonucleotide is modified before, during, or after hybridization. In some embodiments, the cleavage oligonucleotide is modified to incorporate one or more additional functional groups into the oligonucleotide.

[0266] In some embodiments, the cross-linking reaction occurs simultaneously with hybridization. In some embodiments, cycles of hybridization and cross-linking are repeated without a washing step to remove uncross-linked barcode strands.

[0267] In some embodiments, light-directed cross-linking is selectively applied to cell samples. Cross-linking is selectively performed by shielding areas from radiation, allowing cross-linking only in unshielded areas. A photomask provides a template to shield the area receiving radiated light in the sample. In some embodiments, a photomask is generated to allow radiation of one or more ROIs. In some embodiments, the photomask is manually or machine generated. In some embodiments, a photomask is generated according to sample imaging as previously described herein. In some embodiments, a photomask is applied to the sample before radiation.

[0268] In some embodiments, in addition to or in place of a photomask, light is focused on one or more ROIs as described herein. In some embodiments, a digital micromirror device (DMD) is used to focus the light. In some embodiments, a confocal microscope is used to focus the light. In some embodiments, the confocal microscope is a laser scanning confocal microscope. In some embodiments, a DVD recorder is used to focus the light. In some embodiments, a Blu-ray disc recorder is used to focus the light.

[0269] In some embodiments, light is focused on the surface of a colony of a tissue sample or cell provided herein. In some embodiments, light is focused on a plane comprising at least a portion of a colony of a tissue sample or cell provided herein. In some embodiments, light is focused on one or more planar surfaces on or within a colony of a tissue sample or cell provided herein. In some embodiments, the cross-linking of light orientation described herein is applied to the surface of a colony of a tissue sample or cell provided herein. In some embodiments, the cross-linking of light orientation described herein is applied to multiple planes within a colony of a tissue sample or cell provided herein. In some embodiments, the cross-linking of light orientation described herein is applied to multiple planes of a colony of a three-dimensional tissue sample or cell provided herein.

[0270] In some embodiments, the radiation is provided at a specific wavelength or wavelength range. In some embodiments, the radiation is ultraviolet (UV) radiation. In some embodiments, the radiation is provided at a wavelength in the range of about 300 nanometers (nm) to about 450 nm. In some embodiments, the radiation is provided at a wavelength of about 300 nm, about 305 nm, about 310 nm, about 315 nm, about 320 nm, about 325 nm, about 330 nm, about 335 nm, about 340 nm, about 345 nm, about 350 nm, about 355 nm, about 360 nm, about 365 nm, about 370 nm, about 375 nm, about 380 nm, about 385 nm, about 390 nm, about 395 nm, about 400 nm, about 405 nm, about 410 nm, about 415 nm, about 420 nm, about 425 nm, about 430 nm, about 435 nm, about 440 nm, about 445 nm, about 450 nm, or any combination thereof. In some embodiments, the radiation is provided at a wavelength of about 365 nm. In some embodiments, the radiation is provided at a wavelength of about 405 nm.

[0271] In some embodiments, the radiation is provided for a specific amount of time. In some embodiments, the radiation is provided for about 1 second to up to 10 seconds. In some embodiments, the radiation is provided for about 100 milliseconds (ms) to up to 10 seconds. In some embodiments, the radiation is provided for at least about 10 ms or more, at least about 100 ms or more, at least about 500 ms or more, at least about 600 ms or more, at least about 700 ms or more, at least about 800 ms or more, at least about 900 ms or more, at least about 1 second or more, at least about 2 seconds or more, at least about 3 seconds or more, at least about 4 seconds or more, at least about 5 seconds or more, at least about 6 seconds or more, at least about 7 seconds or more, at least about 8 seconds or more, at least about 9 seconds or more, at least about 10 seconds or more, at least about 11 seconds or more, at least about 12 seconds or more, at least about 13 seconds or more, at least about 14 seconds or more, at least about 15 seconds or more, at least about 16 seconds or more, at least about 17 seconds or more, at least about 18 seconds or more, at least about 19 seconds or more, at least about 20 seconds or more, or at least about 30 seconds or more. The order and frequency of the barcode types in the concatemer are not specified or limited. Concatemers optionally include a combination of target barcodes, node barcodes.

[0272] Iterative addition of nucleic acid barcodes to cDNA produces nucleic acid concatemers. In some embodiments, the nucleic acid concatemers include at least three nucleic acid barcode chains. The nucleic acid concatemers may include nucleic acid barcode chains covalently linked to each other via photoreactive nucleotides. In some embodiments, the nucleic acid concatemers may include at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 nucleic acid barcode chains. In some embodiments, the nucleic acid concatemers may include at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 barcode domains, each of which contains data (e.g., each barcode domain can be uniquely / independently assigned a bit value). An embodiment of digital data storage using light-directed serial connections of barcodes is shown in Figure 19A-19B The concatemer of nucleic acid barcodes can include at least two, at least three, at least four, at least five, at least ten, at least 15, at least 20, at least 25, at least 30, or at least 50 nucleic acid barcodes. The order and frequency of barcode types in the concatemer are not specified or limited. The concatemer optionally includes a combination of target barcodes and node barcodes.

[0273] The number of additional barcode strands that can be added is not limited. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more additional barcode strands can be added. In some embodiments, 75, 100, 150, 200, 250 or more additional barcode strands can be added.

[0274] In some embodiments, the CNVK-labeled chain can hybridize 1 to 10 bases or 10 to 20 bases with the docking chain. In some embodiments, salt concentration, temperature, denaturant concentration or chain concentration affect the stability of chain binding. In some embodiments, the stability of chain binding is adjusted so that the average binding of the CNVK-labeled chain is less than 1 second, between 1 and 10 seconds or less than 1 minute. In some embodiments, the CNVK-labeled chain can have an increased homology domain (20-40 bases, more than 40 bases) with the docking chain. In some embodiments, salt is increased or the temperature is lowered, so as to adjust the chain binding to be longer than one minute on average. In some embodiments, conditions can be adjusted to determine the average binding time of the CNVK-labeled chain and the docking chain.

[0275] Nucleic acid barcodes optionally include additional detectable labels, such as fluorophores, luminescent and bioluminescent markers (e.g., biotin, luciferases (e.g., bacteria, fireflies, click beetles, etc.), luciferin, and aequorin), radioactive labels (e.g., 3 H. 125 I. 35S. 14 C or 32 P), enzymes (e.g., β-galactosidase, glucuronidase, phosphatase (e.g., alkaline phosphatase), peroxidase (e.g., horseradish peroxidase), and cholinesterase), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads. In some embodiments, the barcodes described herein are modified to include the addition of small molecules, antibodies, enzymes (e.g., Cas9), or metal isotopes.

[0276] In some embodiments, a nucleic acid described herein is covalently linked to at least one other nucleic acid through a single photoresponsive element of a hybridization domain.

[0277] The concatemers of nucleic acid barcodes can be in various positions on the substrates provided herein. For example, the concatemers can be linear, have two different orientations, or have multiple orientations in different directions. The barcodes can be positioned along x, y, and z coordinates in space.

[0278] Methods as described herein include applying reagents (including barcode strands, wash sequences, etc.) to a sample. In some embodiments, the application method includes traditional microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers for depositing new solutions (e.g., Echo Liquid Beckman Coulter, Indianapolis, IN), an inkjet printer with improved content, an automated liquid handler, optionally incorporating a pipette, a robotic arm for handling slides between solution baths, or any combination thereof. In some embodiments, the application method is performed on a stage. In some embodiments, the application method is performed off a stage.

[0279] The sample can be treated to prevent or reduce non-specific binding of the barcode strand. In some embodiments, before applying the barcode strand, a blocking agent is added to a population of tissue samples or cells provided herein. In some embodiments, while applying the barcode strand, a blocking agent is added to a population of tissue samples or cells provided herein. In some embodiments, the blocking agent includes non-specific nucleic acid sequences, sheared salmon sperm DNA, tRNA, yeast tRNA, single-stranded oligonucleotides, double-stranded oligonucleotides, polysaccharides, charged polysaccharides, negatively charged polysaccharides, negatively charged molecules, charged molecules, bovine serum albumin (BSA), skim milk powder, detergents, non-ionic polymer surfactants (e.g., poly(ethylene oxide) / poly(propylene oxide) triblock copolymers - PLURONIC TM F-127, F-68), crowding agent or any combination thereof. In some embodiments, ribosome depletion or targeted depletion of high-abundance sequences occurs before any of the following: reverse transcription, barcoding, sequence extraction, cross-binding synthesis or PCR amplification. In some embodiments, ribosome depletion or targeted depletion of high-abundance sequences occurs after PCR amplification. In some embodiments, an in situ hybridization (ISH) probe targeting a high-abundance sequence is included before or during reverse transcription to prevent specific high-abundance sequences from acting as binding sites for reverse transcription primers. In some embodiments, ISH probes are combined with nuclease activity (e.g., RNase H) to specifically digest high-abundance sequences in situ before reverse transcription. In some embodiments, double-stranded specific nucleases (e.g., dsDNA enzymes) are applied after PCR to specifically enrich high-abundance sequences.

[0280] After the radiation and cross-linking reaction, in some embodiments, the uncross-linked barcodes are removed. In some embodiments, the uncross-linked barcodes are removed by washing. In some embodiments, the uncross-linked barcodes are not removed before additional cross-linking cycles. In some embodiments, the sample is washed with a chemical denaturant solution. In some embodiments, the chemical denaturant solution comprises one or more salts. In some embodiments, the salt is NaCl. In some embodiments, the salt concentration is 0.1M to 2M. In some embodiments, the salt concentration is about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1.0M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2.0M. In some embodiments, the chemical denaturant is formamide. In some embodiments, the chemical denaturant is ethylene carbonate. In some embodiments, the formamide solution is 50%-60% formamide. In some embodiments, the denaturant solution is in PBS buffer. In some embodiments, the denaturant solution comprises a detergent. In some embodiments, the concentration of the detergent in the denaturant solution is about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 2%, about 3%, about 4%, or about 5% by volume. In some embodiments, the detergent is a polysorbate. In some embodiments, the polysorbate is polysorbate 20 ( 20, Merck KGaA, Darmstadt, Germany) or polysorbate 80 ( 80, Merck KGaA, Darmstadt, Germany). In some embodiments, the detergent is a nonionic surfactant. In some embodiments, the nonionic surfactant is a nonionic polyoxyethylene detergent. In some embodiments, the detergent is polyethylene glycol (PEG), 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (CAS No.: 9002-93-1), N,N-dimethyltetradecylamine N-oxide (TDAO, CAS No.: 3332-27-2). In some embodiments, the detergent is Triton X-100, Nereid, TDAO or a combination thereof. In some embodiments, washing is performed at room temperature. In some embodiments, washing is performed at a temperature higher or lower than room temperature. In some embodiments, washing is performed at 4°C, 16°C, 25°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C or higher.

[0281] As previously described herein, the barcodes optionally include a detectable label. After washing to remove uncrosslinked DNA barcodes, the bound barcodes can be visualized. In some embodiments, visualization is performed by colorimetric, fluorescent, ultraviolet or other means.

[0282] The density of the barcoded cDNA target can be measured by the number of unique sequences detected. In some embodiments, the density of the barcoded cDNA target is measured in one square micrometer (μm 2 ) of a tissue sample, a population of cells, or a population of cells. In some embodiments, the density of barcoded cDNA targets is in a cubic micrometer (μm 3 ) of a tissue sample, a population of cells, an organoid, or an organ. In some embodiments, the methods provided herein generate barcodes per square micrometer (μm 2 ) about 55 tags or barcodes. In some embodiments, the methods provided herein generate 2 about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2250, about 2500, about 2750, about 3000, about 3500, about 4000, about 4500, about 5000 tags.

[0283] In some embodiments, the methods provided herein generate2 Up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, up to about 50, up to about 55, up to about 60, up to about 65, up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, up to about 250, up to about 300, up to about 350, up to about 400, up to about 450, up to about 500 , up to about 550, up to about 600, up to about 650, up to about 700, up to about 800, up to about 900, up to about 1000, up to about 1200, up to about 1400, up to about 1600, up to about 1800, up to about 2000, up to about 2250, up to about 2500, up to about 2750, up to about 3000, up to about 3500, up to about 4000, up to about 4500, up to about 5000 tags.

[0284] In some embodiments, the methods provided herein generate 3 ), about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2250, about 2500, about 2750, about 3000, about 3500, about 4000, about 4500, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000 tags.

[0285] In some embodiments, the methods provided herein generate 3), up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, up to about 35, up to about 40, up to about 45, up to about 50, up to about 55, up to about 60, up to about 65, up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, up to about 250, up to about 300, up to about 350, up to about 400, up to about 450, up to about 500, up to about 550, up to about 600, up to about 650 , up to about 700, up to about 800, up to about 900, up to about 1000, up to about 1200, up to about 1400, up to about 1600, up to about 1800, up to about 2000, up to about 2250, up to about 2500, up to about 2750, up to about 3000, up to about 3500, up to about 4000, up to about 4500, up to about 5000, up to about 6000, up to about 7000, up to about 8000, up to about 9000, up to about 10,000 tags.

[0286] Fractional barcoding

[0287] Adjusting the radiation of a sample can control the amount of crosslinking therein. In some embodiments, radiation for a longer duration or higher intensity increases the number of crosslinks formed in a 1D, 2D, or 3D region. In some embodiments, crosslinking can be controllably increased in a specific region, cell, or on a sample or strand in a test tube. Intensity is measured in watts per square centimeter (W / cm 2 ) measured by radiation. In some embodiments, the fraction of crosslinks formed in a specific area or volume can be precisely controlled by controlling the radiation exposure time and power level (for example, on a microscope, using an LED or other light source on a sample or test tube). Such regulation allows for regional specific photosensitive crosslinking events of barcodes. In some embodiments, selective radiation control allows for preselected gradation ("gradation (fractionation)") of any given barcode (or label) to attach it to a specific region of interest (tissue region, cell, subcellular region, micropore, tube, etc.). In some embodiments, the light intensity is about 1 to about 5000 W / cm^2. In some embodiments, the light intensity is up to about 1 W / cm^2. In some embodiments, the light intensity is up to about 5000 W / cm^2. In some embodiments, the light intensity is at least 5000 W / cm^2. In some embodiments, the light intensity exceeds 5000 W / cm^2. The above intensity is the intensity when the intensity is focused on a plane light. Similar intensities can be applied in 3D, in which case W / cm^3 amounts are applied. When irradiating a solution, the volume of the sample is irradiated. In such a case, the intensity per volume unit is applied, such as with a UV gun.

[0288] In some embodiments, the duration or power of the radiation exposure can allow the fraction of crosslinks formed in a specific region of interest (ROI) to be controllably adjusted. In other words, the fraction of sequences crosslinked to the barcode in a specific ROI can be controlled by the radiation exposure time or intensity level. In some embodiments, up to about 100% of the barcodes are crosslinked after radiation. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the barcode sequences in the ROI are photocrosslinked with one or more target sequences after radiation. In some embodiments, about 10% of the barcodes in the ROI are photocrosslinked with the target sequence after radiation. In some embodiments, about 20% of the barcodes in the ROI are photocrosslinked with the target sequence after radiation. In some embodiments, about 30% of the barcodes in the ROI are photocrosslinked with the target sequence after radiation. In some embodiments, about 40% of the barcodes in the ROI are photocrosslinked to the target sequence after irradiation. In some embodiments, about 50% of the barcodes in the ROI are photocrosslinked to the target sequence after irradiation. In some embodiments, about 60% of the barcodes in the ROI are photocrosslinked to the target sequence after irradiation. In some embodiments, about 70% of the barcodes in the ROI are photocrosslinked to the target sequence after irradiation. In some embodiments, about 80% of the barcodes in the ROI are photocrosslinked to the target sequence after irradiation. In some embodiments, about 90% of the barcodes in the ROI are photocrosslinked to the target sequence after irradiation.

[0289] The population within the sample can be encoded for identification using the fractional application of one or more barcodes. Binary, ternary, quaternary or any smaller fractional encoding can be used. In some embodiments, any combination of fractional applications of different barcodes that allow barcode fractions to be distinguished can be used. In some embodiments, one or more barcodes are applied in a defined fractional amount in an ROI, generating a unique combination of one or more barcodes in each ROI. The fractional barcodes can be read using sequencing technology, fluorescent imaging, spectrophotometry or any other detection method. In an exemplary embodiment, one ROI in the sample contains 40% barcode 1+20% barcode 2+20% barcode 3, while another ROI contains 20% barcode 1+40% barcode 2+20% barcode 3. The fractions do not need to all add up to 100%, but should be sufficiently identifiable in the readout so that they can be reliably deconvoluted.

[0290] Transcript extraction and sequencing

[0291] The barcode concatemers generated can be displaced from the tissue sample or cell population provided herein. The concatemers can be synthesized into continuous nucleic acid chains. These steps can occur successively or simultaneously. In some embodiments, continuous nucleic acid chains are collected. In some embodiments, nucleic acid chains are then amplified and sequenced. Sequencing data can be combined with earlier imaging data to provide spatially limited sequencing information.

[0292] Provided herein is a method for concatemer replacement. In some embodiments, the concatemer bound to RNA is replaced from a tissue sample or a population of cells provided herein. In some embodiments, the concatemer is chemically replaced. In some embodiments, the concatemer is enzymatically replaced. In some embodiments, the concatemer is replaced using an enzyme (e.g., RNase H) that specifically cleaves RNA. In some embodiments, the concatemer is replaced using an enzyme that specifically cleaves DNA, a DNA enzyme (e.g., DNA enzyme I). In some embodiments, the concatemer is physically replaced. In some embodiments, the concatemer is replaced by a cross-linking process. In some embodiments, cross-linking is performed before replacing the barcoded DNA. In some embodiments, cross-linking is performed after replacing the barcoded DNA. In some embodiments, replacement and cross-linking occur simultaneously.

[0293] Methods for cross-linking synthesis are provided herein. Cross-linking synthesis is performed directly in situ, or during / after displacement. The basic strategy for cross-linking synthesis is depicted in Fig. 20 In. Two nucleic acids that have hybridized together to form a knot are shown. A primer binds to the front of the knot on the first nucleic acid (left). A strand displacement polymerase is used to copy the x domain (also referred to herein as the junction domain) until it reaches the terminator (shown in black and also referred to herein as the closed domain). The new x domain competes with the old x domain during random walk branch migration. Eventually, the new x domain can bind to the exposed x* domain on the second template strand (right, also referred to herein as the synthesis zone). Polymerization can then continue on the second template strand, copying along the new main strand.

[0294] In some embodiments, cross-linking is performed to produce DNA, RNA, PNA or LNA. In some embodiments, the method of cross-linking includes contacting the target nucleic acid or barcode provided herein with a strand displacement polymerase. In some embodiments, cross-linking is performed at room temperature (e.g., at least about 20°C up to 25°C). In some embodiments, cross-linking is performed at a temperature below room temperature (e.g., about 15°C, about 10°C, about 4°C). In some embodiments, cross-linking is performed at a temperature above room temperature (e.g., about 30°C, about 37°C). In some embodiments, nucleic acid amplification or cross-linking is performed at a constant temperature. In some embodiments, nucleic acid amplification or cross-linking is performed at different or varying temperatures.

[0295] Provided herein are methods in which a polymerase is prevented or stopped from moving along the backbone of a template strand and / or copying a template strand by a blocking domain. In some embodiments, the blocking domain comprises a T domain known to increase the number of double-stranded nucleic acids. m In some embodiments, the modified nucleotides include, but are not limited to, locked nucleic acids (LNA), 2'-O-methoxy-ethyl (2'-MOE) nucleotides, 2,6-diaminopurine, G-clamps (analogs of C with 4 hydrogen bonds), and guanidino G-clamp nucleotides, etc.

[0296] When the nucleic acid template comprises two or more crossover junctions, the first nucleic acid of the first crossover junction can be the second nucleic acid of the second crossover junction. Likewise, the second nucleic acid of the first crossover junction can be the first nucleic acid of the second crossover junction.

[0297] Note that in some embodiments, the nucleic acids described herein comprise a barcode domain. In an exemplary embodiment, the second nucleic acid of the junction comprises a barcode domain at the 5' end. In some embodiments, one of the first synthesis region or the second synthesis region of the nucleic acid comprises a barcode domain.

[0298] The compositions and methods described herein can be used to synthesize a specified sequence of any length. Fig.21 An exemplary embodiment is illustrated in which multiple barcode strands are hybridized together and replicated through a crossover reaction to form a longer sequence. Each junction between barcode strands displays the same domain motif, whereby the strand domain copied on the 3' end of the growing strand before reaching the terminator (b, c, d, e) can cross the crossover junction and bind to the complementary sequence (b*, c*, d*, e*) exposed on the next barcode strand. Any sequence can be added in the template region between the motif domains (shown in gray) to enable assembly of longer sequences.

[0299] In some embodiments, at least one closed domain comprises a poly monomer stretch. For example, the closed domain comprises a stretch of poly A, poly T, poly C, or poly G.

[0300] In some embodiments, the closed domain may include covalent crosslinking of two barcode chains. In some embodiments, covalent crosslinking is photocrosslinking or chemical crosslinking. In some embodiments, covalent crosslinking includes crosslinking the nucleotides at the 5' end of the first barcode chain with the nucleotides at the 3' end of the second template chain. In some embodiments, crosslinking is at an oligonucleotide or a single nucleic acid backbone bond (backbone linkage). In some embodiments, crosslinking is by a phosphodiester bond. In some embodiments, crosslinking reaction is between CNVK nucleotides and complementary nucleotides. In some embodiments, crosslinked nucleotides serve as terminators of polymerases.

[0301] In some embodiments, the closed region comprises any desired nucleotide sequence or number of nucleotides. In some embodiments, each closed domain can have any length. In some embodiments, the length of each closed domain can be 1 nucleotide to 100 nucleotides. In some embodiments, the length of each closed domain is independently one, two, three, four, five, six, seven, eight, nine or ten nucleotides. Note that the closed domain can be a single nucleotide.

[0302] In some embodiments, in situ hybridization (ISH) is applied. The ISH step generally involves depositing small nucleic acids (e.g., 15-40mer). In some embodiments, nucleic acid probes identify one or more regions of the genome. In some embodiments, the nucleic acid probes of ISH identify RNA. In the case of smISH, many small nucleic acids identify RNA together, wherein the combination of a single probe results in a weak signal, but the ability to obtain signals from the collection of all probes (directly or via the connection of a label to the probe) is robust. In some embodiments, before or after antigen repair (e.g., amplification reaction), ISH or improved ISH reactions are applied to samples described herein. In other embodiments, the probes for ISH are also used as barcodes or for the formation of concatemers for barcode binding, followed by methods described herein.

[0303] In some embodiments, fluorescent in situ hybridization (FISH) is applied. The FISH step generally involves depositing small nucleic acids (e.g., 15-40mer) with a label (e.g., a fluorescent label). In some embodiments, the nucleic acid fluorescent probe of FISH identifies one or more regions of the genome. In some embodiments, the nucleic acid fluorescent probe of FISH identifies RNA. In the case of smFISH, many small nucleic acids identify RNA together, wherein the combination of a single fluorescent probe results in a weak signal, but the signal from the collection of all probes is robust. In some embodiments, FISH or modified FISH reactions are applied to samples described herein before or after antigen repair (e.g., an amplification reaction). In other embodiments, the probe for FISH is also used as a barcode or for the formation of concatemers for barcode binding, followed by methods described herein.

[0304] Amplification methods are provided herein. In some embodiments, method includes PCR amplification. PCR amplification uses DNA polymerase to synthesize DNA from deoxynucleotide substrates on single-stranded templates. In some embodiments described herein, PCR methods include real-time PCR, quantitative real-time PCR (Q-RT PCR), reverse transcriptase PCR (RT-PCR), multiple PCR, nested PCR, long-range PCR (Long-range PCR), single-cell PCR, rapid cycle PCR, methylation-specific PCR (MSP), hot start PCR, high-fidelity PCR, in situ PCR, variable number of tandem repeats (VNTR) PCR, asymmetric PCR, PCR based on repetitive sequences, overlap extension PCR, assembly PCR, sequence-specific PCR (Intersequence-specific PCR, ISSR), connection-mediated PCR, methylation-specific PCR, miniprimer PCR (Miniprimer PCR), solid phase PCR, touchdown PCR (Touch down PCR) or any combination thereof. In some embodiments, reverse transcriptase is applied to amplification described herein. In some embodiments, reverse transcriptase is MuLV reverse transcriptase or avian myeloblastosis virus (Avian myeloblastosis virus, AMV) reverse transcriptase. In some embodiments, DNA polymerase is applied to amplification described herein. In some embodiments, DNA polymerase is T7 DNA polymerase, thermophilic eubacterial microorganism Thermus aquaticus DNA polymerase, PfuDNA polymerase or Bst DNA polymerase. In some embodiments, DNA ligase is applied to the connection chain. In some embodiments, DNA ligase is EC 6.5.1.1. In some embodiments, DNA ligase is T4 DNA ligase.

[0305] In some embodiments, the barcode sequence or a primer complementary to such a sequence comprises a polymerase promoter sequence. In some embodiments, the promoter is an RNA polymerase promoter. In some embodiments, the polymerase promoter sequence includes, but is not limited to, a T7 RNA polymerase promoter region (SEQ ID NO: 1: TAATACGACTCACTATAG), a T3 RNA polymerase promoter region (SEQ ID NO: 2: AATTAACCCTCACTAAAG), or an SP6 RNA polymerase promoter region (SEQ ID NO: 3: ATTTAGGTGACACTATAG).

[0306] Provided herein are methods for preparing amplicons. In some embodiments, the amplicons are purified. In some embodiments, the amplicons are analyzed by gel electrophoresis. In some embodiments, the amplicons are separated. In some embodiments, the amplicons are fragmented. In some embodiments, the amplicons are end-repaired. In some embodiments, the amplicons are covalently linked to adapters. In some embodiments, the amplicons are tagged.

[0307] Provided herein is a method for sequencing. In some embodiments, sequencing includes next generation sequencing (NGS) technology. In some embodiments, sequencing includes massive parallel sequencing. In some embodiments, sequencing includes chain termination sequencing, nanopore sequencing, connection sequencing, combined probe anchor synthesis, synthetic sequencing, pyrophosphate sequencing, ion semiconductor or single molecule real-time sequencing.

[0308] Provided herein is a method for protein analysis. In some embodiments, the protein recovered from the sample described herein is analyzed by mass spectrometry, which is an analytical tool for measuring the mass-to-charge ratio (m / z) of one or more molecules present in the sample. Such measurement can be used to calculate the molecular weight of the sample components.

[0309] Provided herein is a method for spatially indexing the transcribed nucleic acid in the region of interest (ROI) selected in cells and tissues. In some embodiments, the transcribed nucleic acid is a reverse transcribed DNA or a transcribed RNA. In some embodiments, the barcode sequence from the sequencing read provides spatial index data. In some embodiments, the spatial index data is combined with the imaging method described earlier herein to provide an association between cell phenotype information and transcriptome information.

[0310] In some embodiments, DNA strands are pooled across multiple ROIs. In some embodiments, DNA strands from a single ROI are collected in a region-specific manner. In some embodiments, DNA strands from a single cell are pooled individually. In some embodiments, DNA from a single cell or other defined ROI is individually barcoded and sequenced.

[0311] Provided herein is a method for information labeling with nucleic acid. As described herein, information labeling provides non-genetic information. In some embodiments, non-genetic information includes spatial information in the sample. In some embodiments, the sample is a two-dimensional sample. In some embodiments, the sample is a three-dimensional sample. In some embodiments, non-genetic information includes sample information, such as tube number, hole number, sample source, charge, quality or other sample characteristics, or any combination thereof. In some embodiments, non-genetic information includes digital information. The method provided herein allows barcodes to be deposited on the surface. Such barcodes can convey information about the identity, substrate, timing, position, intensity, charge, affinity, etc. of the target. Therefore, barcodes provide nucleic acid media for digital information storage. The method provided herein for barcode deposition includes but is not limited to applying force to transfer the barcode from a reservoir to the sample. Exemplary forces for applying include laminar pressure, capillary pressure, slipstream pressure, magnetic force, electrostatic force, creeping force, acoustic force, vibration force, centripetal force, centrifugal force, or any combination thereof. In some embodiments, a buffer exchange process is used for barcode deposition. In some embodiments, an electrophoresis process is used for barcode deposition. In such a method, the migration of charged particles (e.g., nucleic acid barcode molecules) in an electric field is due to electrophoresis, and the electrode is close to the substrate containing the cell. In some embodiments, electrophoresis is applied to remove nucleic acids from the surface. In some embodiments, electrophoresis is applied to remove the concatemers described herein from the surface. The electrode can generate a uniform field across the surface, or a specific electrode can be used for each region of interest. In some embodiments, temperature changes are used for barcode deposition. In other embodiments, the barcode sequence includes a temperature-sensitive feature, and the increase in temperature makes hybridization areas that are unavailable at lower temperatures available. In some embodiments, the ROI includes a heating unit or electrode adjacent to the ROI that is sufficient to increase the temperature at the ROI. In other embodiments, the barcode includes a melting temperature, and the ROI is heated to reach the barcode melting temperature, thereby allowing selective hybridization of one or more barcodes.

[0312] The exemplary methods described herein can also be combined to incorporate any combination of the described methods. For example, a sample barcoded with iterative construction of barcodes is also tagged using the fractional barcoding method described herein.

[0313] In some embodiments, the barcode is deposited in a preselected pattern to a ROI across a surface provided herein. In some embodiments, the pattern forms a grid of repeated shapes across the region, and alternating shapes are preselected for barcode deposition. In some embodiments, the pattern forms a grid of repeated shapes across the region, and less than all shapes are preselected for barcode deposition. In some embodiments, the shape is a square, rectangle, triangle, ellipse, diamond or sphere.

[0314] Provided herein are methods in which cells (or subcellular compartments such as nuclei) of a sample are sorted by defined features before or after barcoding or labeling. In some embodiments, cells are sorted before each iteration of barcoding. In some embodiments, cells are sorted before any barcoding step. In some embodiments, cells are sorted after all barcoding steps. In some embodiments, cells are sorted in one step based on features (e.g., surface receptors). In other embodiments, cells are sorted in a second step based on a second feature (e.g., gene expression). In some embodiments, cells are sorted based on a first feature, and cells are barcoded using methods described herein. In other embodiments, barcoded cells are further sorted based on a second feature, and are optionally further barcoded.

[0315] Provided herein are methods for nucleic acid barcoding, the methods comprising cell sorting. In some embodiments, cell sorting is performed using fluorescence activated cell sorting (FACS) analysis. In some embodiments, cell sorting is performed using a microfluidic device. In some embodiments, cell sorting is performed using flow cytometry. In some embodiments, cell sorting is performed using magnetic cell sorting. In some embodiments, cell nuclei or multi-cellular arrangements are sorted rather than single cells.

[0316] The diffused node barcode strand can interact with a nucleic acid barcode from a target molecule. The target molecule comprises one or more cDNAs, nucleic acid barcodes, ISH probes, other unique identifier sequences, or any combination thereof. The combination of the node barcode and the target identifier generates a record of the spatial correlation between the node position and the target. Fig. 22An exemplary embodiment of a node barcode in a diffusion cloud combined with a target nucleic acid having a different unique identifier is shown. The node barcode 1512 is generated in a chain and diffuses out of the node. The node barcode chain is combined with the target nucleic acid chain, each containing a different unique identifier (UMI 5389, UMI 8942, and UMI 3548 in the figure). All combined nucleic acids contain the same node barcode sequence and have different target unique identifiers. In some embodiments, the target nucleic acid is diffused or fixed to a molecule of interest.

[0317] In the methods described herein, nucleic acids can be combined to provide proximity information. In some embodiments, the combined nucleic acids are generated by connecting a node barcode chain to a target molecule or a unique identifier. Connections can include splint molecules. Connections can include extending one or more chains by polymerization. In some embodiments, sequences are assembled using primer exchange reaction (PER), cross-linking, asymmetric PCR, Gibson assembly, transposase-based tandem (e.g., using Tn5 transposition for RNA-DNA hybrids) or any combination thereof. Figure 23A-23F An exemplary embodiment of a polymerization-based proximity record ligation reaction is shown. Fig.23A Shown is a node barcode chain comprising primers and node barcodes hybridized to an immobilized target nucleic acid comprising a target barcode, a unique identifier and a linker, an affinity region, modifications, crosslinking and / or PCR primers. The two chains are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig. 23B Shown is a node barcode chain without primers, containing a node barcode, hybridized to an immobilized target nucleic acid containing a target barcode, a unique identifier and a linker, an affinity region, modifications, crosslinking and / or PCR primers. The two chains are extended by polymerization to generate a double-stranded combined sequence. The double-stranded combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.23C A node barcode chain comprising a primer, a node barcode, and a non-hybridizing domain 3' of a hybridization sequence is shown hybridized to an immobilized target nucleic acid comprising a target barcode, a unique identifier and a linker, an affinity region, a modification, a crosslinking and / or a PCR primer. The target nucleic acid chain is extended by polymerization to generate a sequence of a single-stranded combination. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.23D A node barcode chain without primers, a non-hybridizing domain comprising a node barcode and a 3' hybridization sequence is shown hybridized to an immobilized target nucleic acid comprising a target barcode, a unique identifier and a linker, an affinity region, modifications, crosslinking and / or PCR primers. The target nucleic acid chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.23EA node barcode chain comprising primers and node barcodes is shown hybridized to an immobilized target nucleic acid comprising a non-hybridizing domain 3' of a hybridization sequence, a target barcode, a unique identifier and a linker, an affinity region, modifications, crosslinking and / or PCR primers. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.23F Shown is a node barcode chain without primers, comprising a node barcode, hybridized to an immobilized target nucleic acid comprising a non-hybridizing domain 3' of a hybridization sequence, a target barcode, a unique identifier and a linker, an affinity region, modifications, crosslinking and / or PCR primers. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS.

[0318] The unique identification sequence is optionally added as an intrinsic part of the template sequence or externally. In some embodiments, the unique identifier is incorporated into the sequence as an oligonucleotide added to the generated chain. In some embodiments, external addition occurs before replication. In some embodiments, external addition occurs after chain generation.

[0319] Example Applications

[0320] The methods described herein provide proximity records of various types of targets. In some embodiments, the target is any other substance to which a DNA, RNA, protein or nucleic acid can be fixed. Detection can be by cDNA synthesis, ISH probe binding, antibody detection, any other means for detecting target nucleic acid, or any combination thereof. The proximity record of a node barcode and a target molecule can occur on a target molecule that has been barcoded separately (previously, simultaneously or subsequently) using other assays (e.g., by binding, crosslinking, photocrosslinking, hybridization, tag fragmentation).

[0321] Figure 24A-24F An exemplary embodiment of in situ proximity recording of target RNA transcripts using cDNA is shown. Fig.24A A node barcode strand that also contains a first PCR primer is shown hybridized to a target barcode strand that contains a cDNA, a unique identifier, and a second PCR primer. The two strands are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig. 24B A node barcode chain comprising a first PCR primer, a node barcode, a hybridization sequence, and a non-hybridization domain 3' of the hybridization sequence is shown hybridized to a target barcode chain comprising a cDNA, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.24C A node barcode chain hybridized to a target barcode chain further comprising a first PCR primer and a hybridization sequence is shown, wherein the target barcode chain comprises a hybridization domain 3' to the hybridization sequence, a cDNA sequence, a unique identifier, and a second PCR primer. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.24D A node barcode strand comprising a first PCR primer and a unique modifier is shown hybridized to a target barcode strand comprising a cDNA, a unique identifier, and a second PCR primer. The two strands are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.24E A node barcode chain comprising a first PCR primer, a unique modifier, a node barcode, a hybridization sequence, and a non-hybridization domain 3' of the hybridization sequence is shown hybridized to a target barcode chain comprising a cDNA, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.24F A node barcode chain hybridized to a target barcode chain further comprising a first PCR primer and a hybridization sequence is shown, wherein the target barcode chain comprises a hybridization domain 3' to the hybridization sequence, a cDNA sequence, a unique identifier, and a second PCR primer. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS.

[0322] Figure 25A-Figure 25F An exemplary embodiment of proximity recording of target RNA transcripts using ISH probes is shown. Fig.25A A node barcode strand that also contains a first PCR primer is shown hybridized to a target barcode strand that contains an ISH probe, a unique identifier, and a second PCR primer. The two strands are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.25B A node barcode chain comprising a first PCR primer, a node barcode, a hybridization sequence, and a non-hybridization domain 3' of the hybridization sequence is shown hybridized to a target barcode chain comprising an ISH probe, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.25CA node barcode chain comprising a first PCR primer, a node barcode, and a hybridization sequence hybridized to a target barcode chain is shown, wherein the target barcode chain comprises a non-hybridizing domain 3' of the hybridization sequence, an ISH probe, a unique identifier, and a second PCR primer. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.25D A node barcode strand comprising a first PCR primer and a unique modifier is shown hybridized to a target barcode strand comprising an ISH probe, a unique identifier, and a second PCR primer. The two strands are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.25E A node barcode chain comprising a first PCR primer, a unique modifier, a node barcode, a hybridization sequence hybridized to a target barcode chain, and a non-hybridization domain 3' of the hybridization sequence is shown, the target barcode chain comprising an ISH probe, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.25F A node barcode chain comprising a first PCR primer, a unique modifier, a node barcode, and a hybridization sequence hybridized to a target barcode chain is shown, wherein the target barcode chain comprises a non-hybridizing domain 3' of the hybridization sequence, an ISH probe, a unique identifier, and a second PCR primer. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS.

[0323] Figure 26A-26C An exemplary embodiment of in situ proximity recording of a protein is shown. An antibody conjugated to a hybridization sequence binds the protein by affinity binding. The antibody-conjugate hybridizes to a barcode sequence on a chain, wherein the barcode sequence chain also contains a unique identifier and a primer. Fig.26A A node barcode strand comprising a first PCR primer, a node barcode, and hybridized to a target barcode strand comprising a barcode, a unique identifier, and a second PCR primer is shown. The two strands are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.26B A node barcode chain comprising a first PCR primer, a node barcode, and a non-hybridized domain 3' of a hybridization sequence hybridized to a target barcode chain is shown, the target barcode chain comprising a barcode, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a sequence of a single-stranded combination. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.26CA node barcode chain comprising a first PCR primer, a node barcode, and a hybridization domain that hybridizes with a target barcode chain is shown, wherein the target barcode chain comprises a non-hybridization domain 3' of the hybridization sequence, a barcode, a unique identifier, and a second PCR primer. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS.

[0324] Figure 26D-Figure 26F An exemplary embodiment of in situ proximity recording of a protein is shown, wherein an antibody conjugated to a target nucleic acid binds the protein by affinity binding. Fig.26D A node barcode strand also comprising a first PCR primer is shown hybridized to a target barcode strand conjugated to an antibody comprising a barcode, a unique identifier, and a second PCR primer. The two strands are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.26E A node barcode chain further comprising a first PCR primer, a node barcode, a hybridization sequence hybridized to a target barcode chain conjugated to an antibody, and a non-hybridized domain 3' to the hybridization sequence is shown, wherein the target barcode chain conjugated to the antibody comprises a barcode, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.26F A node barcode chain comprising a first PCR primer, a node barcode, and a hybridization sequence hybridized to an antibody-conjugated target barcode chain comprising a non-hybridizing domain 3' to the hybridization sequence, a barcode, a unique identifier, and a second PCR primer is shown. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS.

[0325] Figure 26G-Figure 26I An exemplary embodiment of in situ proximity recording of a protein is shown, wherein an antibody conjugated to a target nucleic acid binds the protein by affinity binding. Figure 26G A node barcode chain is shown that also includes a first PCR primer and a unique modifier that hybridizes to a target barcode chain conjugated to an antibody, and the target barcode chain conjugated to an antibody includes a barcode, a unique identifier, and a second PCR primer. The two chains are extended by polymerization to generate a sequence of a double-stranded combination. The sequence of the double-stranded combination is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.26HA node barcode chain comprising a first PCR primer, a unique modifier, a node barcode, a hybridization sequence hybridized to an antibody-conjugated target barcode chain, and a non-hybridized domain 3' of the hybridization sequence is shown, wherein the antibody-conjugated target barcode chain comprises a barcode, a unique identifier, and a second PCR primer. The target barcode chain is extended by polymerization to generate a sequence of a single-stranded combination. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS. Fig.26I A node barcode chain further comprising a first PCR primer, a unique modifier, a node barcode, and a hybridization sequence hybridized to an antibody-conjugated target barcode chain is shown, wherein the antibody-conjugated target barcode chain comprises a non-hybridizing domain 3' to the hybridization sequence, a barcode, a unique identifier, and a second PCR primer. The node barcode chain is extended by polymerization to generate a single-stranded combined sequence. The combined sequence is then processed by extraction and / or PCR and / or library preparation and / or NGS.

[0326] Figure 27A-27G An exemplary embodiment of proximity recording of a target RNA transcript using a cDNA transcript is shown. Fig.27A Shown is a node barcode strand comprising a first PCR primer, a node barcode, and a hybridization domain that hybridizes to a target barcode strand comprising a hybridization domain and a cDNA or ISH probe complementary to a target RNA or DNA sequence photo-crosslinked to the target barcode, a unique identifier, and a second PCR primer. Fig.27B Shown is a node barcode strand comprising a first PCR primer, a node barcode, a hybridization domain that hybridizes to a target barcode strand and a non-hybridization domain 3' to the hybridization domain, wherein the target barcode strand comprises a hybridization domain and a cDNA or ISH probe complementary to a target RNA or DNA sequence photo-crosslinked to the target barcode, a unique identifier, and a second PCR primer. Fig.27C Shown is a node barcode strand comprising a first PCR primer, a node barcode, and a hybridization domain that hybridizes to a target barcode strand, the target barcode strand comprising a non-hybridizing domain 3' to the hybridization domain and a cDNA or ISH probe complementary to a target RNA or DNA sequence photo-crosslinked to the target barcode, a unique identifier, and a second PCR primer. Fig.27D Shown is a node barcode strand comprising a first PCR primer, a node barcode, and a non-hybridizing domain 3' of the node barcode region that hybridizes to a target barcode strand, wherein the target barcode strand comprises a hybridizing domain complementary to the node barcode and the first primer, a cDNA or ISH probe region complementary to a target RNA or DNA sequence photo-crosslinked to the strand comprising the target barcode, a unique identifier, and a second PCR primer. Fig.27EShown is a node barcode strand comprising a first PCR primer, a unique modifier, a node barcode, and a hybridization domain that hybridizes to a target barcode strand comprising a hybridization domain and a cDNA or ISH probe region complementary to a target RNA or DNA photo-crosslinked to the strand comprising the barcode, a unique identifier, and a second PCR primer. Fig.27F Shown is a node barcode strand comprising a first PCR primer, a unique modifier, a node barcode, a hybridization domain that hybridizes to a target barcode strand, and a non-hybridizing region 3' to the hybridization domain, wherein the target barcode strand comprises a hybridization domain and a cDNA or ISH probe region complementary to a target RNA or DNA photo-crosslinked to the barcode, a unique identifier, and a second PCR primer. Figure 27G Shown is a node barcode strand comprising a first PCR primer, a unique modifier, a node barcode, and a hybridization domain that hybridizes to a target barcode strand comprising a non-hybridizing region 3' to the hybridization domain and a cDNA or ISH probe region complementary to the target RNA or DNA photo-crosslinked to the barcode, a unique identifier, and a second PCR primer.

[0327] In some embodiments, the node barcode strand is used as a primer on the target nucleic acid strand. Transcription from the primer region generates a template cDNA region on the node barcode strand. Figure 28A-Figure 28D An exemplary embodiment of using a node barcode strand as a primer for reverse transcribing a target mRNA is shown. Fig.28A A node barcode strand comprising a PCR primer and a node barcode and a 3' poly-T domain that hybridizes to a poly-A tail on an mRNA target is shown. Reverse transcription of the poly-T domain generates a cDNA strand extension associated with the node barcode strand. Fig.28B A node barcode strand comprising a PCR primer, unique modifiers and node barcodes and a 3' poly-T domain that hybridizes to a poly-A tail on an mRNA target is shown. Reverse transcription of the poly-T domain generates a cDNA strand extension associated with the node barcode strand. Fig.28C A node barcode strand comprising PCR primers and node barcodes and a random sequence (5N3G) used for priming on RNA sequences is shown. A cDNA strand extension associated with the node barcode strand is generated from reverse transcription of the 5N3G domain. Fig.28DA node barcode chain comprising a PCR primer, a unique modifier and a node barcode and a random sequence (5N3G) for priming on an RNA sequence is shown. A cDNA chain extension associated with a node barcode chain is generated from the reverse transcription of the 5N3G domain. In some embodiments, the primer sequence is random or specific. In some embodiments, the primer sequence comprises TTTTTTTTTT, TTTTTTTTTTTTTTTTTTTT, TTTTTTTTTTTTTTTTTTTTTTT, TTTTTTTTTTTTTTTTTTTTTTVN, NNNNNNN, NNNNNNNN, NNNNGGG, NNNNNCCC, NNNNNTTT or any combination thereof. In some embodiments, the priming sequence is specific to the RNA of interest. In some embodiments, the primer sequence is a CRISPR guide RNA sequence, an mRNA therapeutic sequence, a T or B cell receptor sequence or any combination thereof. In some embodiments, the second chain synthesis is applied to the reverse transcription product. In some embodiments, a template switching oligonucleotide (TSO) is used for the second chain synthesis. In some embodiments, the second chain synthesis generates a chain comprising a PCR primer flanked by one or more barcodes and a cDNA sequence.

[0328] In some embodiments, a node barcode strand described herein is used as a primer and also comprises a docking sequence. Figure 29A-Figure 29D An exemplary embodiment of using a node barcode strand as a primer for reverse transcribing a target mRNA is shown, wherein the node barcode strand also comprises a docking sequence. Fig.29A A node barcode strand comprising a docking sequence and a 3' poly-T domain hybridized to a poly-A tail on an mRNA target is shown. A cDNA strand extension associated with the node barcode strand is generated from reverse transcription of the poly-T domain. A barcode strand comprising a PCR primer, a unique identifier, and an additional barcode is photocrosslinked to the docking sequence. Fig.29B A node barcode strand comprising a docking sequence, a unique modifier, and a 3' poly-T domain hybridized to a poly-A tail on an mRNA target is shown. A cDNA strand extension associated with the node barcode strand is generated from reverse transcription of the poly-T domain. A barcode strand comprising a PCR primer, a unique identifier, and an additional barcode is photocrosslinked to the docking sequence. Fig.29C A node barcode strand comprising a docking sequence and a node barcode with a random sequence (5N3G) for priming on an RNA sequence is shown. A cDNA strand extension associated with the node barcode strand is generated from reverse transcription of the 5N3G domain. The barcode strand comprising a PCR primer, a unique identifier, and an additional barcode is photocrosslinked to the docking sequence. Fig.29DA node barcode strand comprising a docking sequence, a unique modifier, and a node barcode with a random sequence (5N3G) for priming on an RNA sequence is shown. A cDNA strand extension associated with the node barcode strand is generated from reverse transcription of the 5N3G domain. The barcode strand comprising a PCR primer, a unique identifier, and an additional barcode is photocrosslinked to the docking sequence.

[0329] Reconstructing spatial landscape from diffuse clouds

[0330] The frequency and combination of barcodes in concatemers provide information for calculating spatial proximity information. Sequencing of node barcode sequences from different node positions provides handshake or triangulation information about the relative position of the node. In some embodiments, the target nucleic acid is combined with the node nucleic acid in the concatemer. Sequencing these concatemers provides specific position information of the target nucleic acid of the subject. The node barcode sequences generated from one or more node positions can be connected in series. In some embodiments, the series sequence contains part or all of 2 or more node barcode sequences. The series sequence can contain additional sequences, including unique modifiers and other additional sequences of 1-50nt or longer. These series of node barcode chains are called node barcode concatemers. The node barcode chain can contain additional sequences introduced or eliminated during the series connection. The node barcode chain is connected in series as a pair or multiple node barcode concatemers can indicate the proximity of the node position at a specific or non-specific time through direct interaction (such as through base pairing) or indirect interaction (such as enzyme-mediated or splint-mediated combination). The node barcode concatemer can be sequenced. In some embodiments, the sequence is read using next generation sequencing, ISH, microarray, or any variation or combination thereof. In some embodiments, the sequencing results are used to reconstruct which node barcode chains are connected in series. The association of node barcode sequences within or excluded from the chain connected in series with their node positions (e.g., derived from a single or more biomolecules) can enable reconstruction of the spatial topology of the sample.

[0331] In some embodiments, the node barcode strands are derived from one or more strands, wherein one or both strands can be fixed on a substrate, in a substrate, or in a fixed or living sample. In some embodiments, concatenating node barcode strands includes, but is not limited to, ligation, splinting, cross-linking synthesis, ligation and cross-linking synthesis, cross-linking and nicking synthesis, and paired hybridization and copying (see Fig.18 Alternatively, PER, asymmetric PCR, Gibson assembly, transposase-based tandem assembly (e.g., Tn5 transposition for RNA-DNA hybrids), or any other strategy for assembling sequences may be used.

[0332] In some embodiments, a concatenated node barcode strand is amplified and prepared for sequencing, node barcode-specific readout (eg, ISH), or other methods to identify the concatenated original node barcode.

[0333] The spatial relationship between node positions can be deconvoluted based on the co-occurrence characteristics of node barcode sequences within the concatemer and the associated unique identifiers, barcodes and / or additional information collected from the same tissue / cell / matrix. In some embodiments, the association of node barcodes and target barcodes is used to reconstruct the positions of node positions relative to each other in the sample. This additional information can be related to protein or RNA detection, morphological imaging, spatial delivery of barcode sequences, or other sample information. The interaction of node barcode chains from node positions can be controlled by titrating the diffusion rate within the sample, which can change the incidence of series connection. In some embodiments, one or more of the imaging data of nodes, node concatemers, diffusion clouds, light-directed ROI sizes, or light-directed ROI shapes are used in any combination to help calculate deconvolution.

[0334] Layering process

[0335] Methods described herein can be used alone or in combination. In some embodiments, other levels of space and expression information are advantageous. The embodiments described include sequencing from different diffusion clouds, overlapping diffusion clouds, light-directed barcoding, fractional light-directed barcoding or any combination thereof. In various embodiments, the selection of methods as described herein or combinations is guided by the ability of each method.

[0336] Mapping using different diffusion clouds provides a relative position mapping of the node position to the target. Using different diffusion clouds from the node position allows subcellular resolution of the entire sample without special equipment. The integrity of the tissue sample remains intact. Additionally, the reaction is completed without having to perform multiple washing cycles, in other words, it is a "one pot" reaction.

[0337] In some embodiments, the diffusion of the generated node barcode nucleic acid chains from more than one node position is adjusted so that the diffusion clouds from the individual node positions overlap. The node barcode nucleic acids from the individual node positions are combined by hybridization, extension, connection, splint connection, cross-linking, connection and cross-chain crosslinking (ICL) synthesis, cross-ICL and nick synthesis, paired copies or any combination thereof to form a concatemer of node barcode nucleic acids. The concatenated node barcode nucleic acid chains from overlapping diffusion clouds allow the spatial map of the entire sample to be fully reconstructed. This method is also a "one pot" reaction.

[0338] Mapping samples using light-directed barcoding of cells allows for position mapping in complete sample tissue. A light mask during light-directed barcoding allows sequencing of only cells or regions of interest. As described herein, fractional barcoding provides identification of cells or regions of interest using light-directed barcoding, wherein the fraction of the barcode spans a population of reads from the same region. Fractional barcoding allows for position mapping with minimal fluid exchange sequences. Fractional barcoding can include any other fractional percentage of binary, ternary, quaternary or one or more barcodes.

[0339] In some embodiments, the barcoding method is optionally combined with a diffusion cloud labeling method. The combination of methods allows for position mapping at subcellular resolution with minimal fluid exchange. The combined method allows mapping only in cells or regions of interest. Additionally, the sample remains intact after barcoding. In some embodiments, spatial analysis is performed using light-directed barcoding. In some embodiments, spatial analysis is performed using one or more types of fractional barcoding. In some embodiments, spatial analysis is performed using light-directed barcoding and one or more types of fractional barcoding. In some embodiments, spatial analysis is performed using different diffusion clouds. In some embodiments, spatial analysis is performed using overlapping diffusion clouds. In some embodiments, spatial analysis is performed using light-directed barcoding and different diffusion clouds. In some embodiments, spatial analysis is performed using light-directed barcoding and overlapping diffusion clouds. In some embodiments, spatial analysis is performed using one or more types of fractional barcoding and different diffusion clouds. In some embodiments, spatial analysis is performed using one or more types of fractional barcoding and overlapping diffusion clouds. In some embodiments, spatial analysis is performed using any combination of light-directed barcoding, one or more types of fractional barcoding, distinct diffusion clouds, and overlapping diffusion clouds.

[0340] filter

[0341] Also provided herein is a method for screening a region of interest affected by intervention compared to an unaffected region of interest. In some embodiments, the method includes screening one or more regions of interest in a sample. In some embodiments, the method includes screening one or more regions of interest across one or more samples. In some embodiments, one or more regions of interest are affected by intervention before screening, after screening, or between multiple screenings. In some embodiments, the impact includes applying small molecules, peptides, antibodies, cells, proteins, energy, or any combination thereof. In some embodiments, energy includes thermal energy, radiation energy, chemical energy, nuclear energy, electrical energy, kinetic energy, acoustic energy, or any combination thereof.

[0342] Nucleic acid-based information storage

[0343] Provided herein are compositions, methods and systems utilizing the above innovations in the context of writing and reading DNA for storing digital information. In short, utilizing DNA "write" technology described herein, optionally with "reading" combination by methods such as fluorescence microscopy or next generation sequencing, allows efficient and proportional data storage. In some embodiments, digital information is converted into nucleic acid information. In some embodiments, nucleic acid information includes nucleic acid sequence. In some embodiments, digital information items are converted into nucleic acid sequence. In some embodiments, information items are converted into nucleic acid sequence of one or more nucleic acids. In some embodiments, information items are converted into sequences of about 2, about 5, about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900 or about 1000 kinds of nucleic acids. In some embodiments, barcodes as described herein each include information items.

[0344] An exemplary depiction of data being written to DNA and subsequently read is depicted in Fig.33 The digital information is converted into nucleic acid information. The nucleic acid information is partitioned into barcodes. Based on the generated information, the barcodes containing the partition information are assembled into nucleic acid chains on a glass disk similar to a Blu-ray disc using the light-directed barcoding method described herein. A representative disk for sequence assembly is shown in Fig.34 The disk includes an input port and an output port, with a width of approximately 113 cm 2 Reading the generated barcode strands can be performed using imaging or next generation sequencing techniques. The digital data can then be reconstructed using information from the molecular reading step.

[0345] Combinatorial libraries of sequences are assembled using light-directed oligonucleotide-by-oligonucleotide synthesis. An exemplary depiction of the magnitude of information that can be stored using this approach is shown in Fig.35 Providing m barcodes at n index positions provides m*n rounds of patterning. This results in m^n possible combinations, or log_2(m^n) bits of possible information. Fig.35 As shown in the center of the figure, each pixel on the substrate contains multiple copies of the same "metapolymer" sequence. Continuing to the right side of the figure, a representative metapolymer from each pixel is shown, showing covalently linked DNA chains. These chains are generated using an ultrafast scanner or a digital micromirror device using a light-directed assembly as described herein.

[0346] In some embodiments, the barcoded information is deposited on the surface of the node position. In some embodiments, multiple copies of the barcoded information are provided and diffused from the node position. In some embodiments, copies of the barcoded information are generated and diffused from the node position. The series connection of the barcodes from the node position is assembled and translated according to the methods described herein.

[0347] It is possible to extract the stored information as digital information by different methods. In some embodiments, sequencing methods are applied to access the nucleic acid information for conversion into digital information. In some embodiments, fluorescent labeling methods are applied to the sample to translate the stored information. Two exemplary strategies for extracting molecularly encoded information are described below. In one embodiment, as Fig.36A As shown in , a one-pot cross-linking reaction is used to copy the information into a continuous chain that can be amplified by PCR and sequenced using next generation sequencing. In a second embodiment, as Fig.36B As shown in , the identity of the molecule is read out by fluorescence, such as by the same ultrafast scanning device used to write the information.

[0348] Table 1 lists exemplary write and read strategies.

[0349] Table 1

[0350]

[0351] Estimation of capacity, cost and time

[0352] General estimates include storage capacity, read time, and approximate cost. With a pixel size of 2 μm x 2 μm, it is estimated that 10^6 sequences can be assembled per field of view (FOV), and up to ~3 billion sequences can be assembled on a glass substrate approximately the size of a Blu-ray disc.

[0353] If each pixel uses m = 8 barcodes and n = 8 index positions for data encoding, this would correspond to 24 bits of information per pixel, or 8.5 GB per glass disk. The sequence length is flexible depending on the design of the library. With a barcode sequence of about 20 nucleotides to accommodate error correction + imaging, the estimated assembled sequence length is about 300 nucleotides, or up to twice that, depending on the indexing strategy used.

[0354] Early studies have shown that exposure times as low as 100 microseconds result in barcoding, and it is expected that the use of powerful lasers can significantly reduce this time, allowing the development of ultrafast scanners that minimize the movement time to reach the speed of a 16× Blu-ray drive (such as by adopting a spinning disk format), and the hybridization and washing steps are approximately 2 minutes per round. It is speculated that the time to write each 8.5GB disc may be as low as 10 minutes per round or 10.7 hours per disc. For imaging-based readout, the same scanning system can be used to read out the fluorescent signal. With such a scanner, it is expected that the time to read each 8.5GB disc by imaging can be comparable (10.7 hours per disc).

[0355] The cost and time of sequencing-based reading of stored data is expected to approach similar levels as writing. Additionally, sequencing-based reading allows molecular copies to be separated from the substrate and stored in an ultra-dense format.

[0356] If sequencing-based readout is not to be used, single bit value writing and imaging-based reading can adopt a spatial encoding strategy even more similar to that of Blu-ray, with expected resolution, read and write times comparable to Blu-ray, while the total cost of writing and reading is about 100 times lower. The single bit value strategy is also compatible with more complex molecular encryption chemistries that will increase data security.

[0357] Oligonucleotide library generation

[0358] In some embodiments, the tandem method based on light described herein is used to generate an oligonucleotide library. In some embodiments, the sequence of oligonucleotide uses a computational generation method to generate. In other embodiments, the oligonucleotide sequence is synthesized from the surface according to the method described herein. The synthetic oligonucleotide is cracked from the support. Optionally carry out other steps, including removing blocking groups, purification or separation.

[0359] Surface material

[0360] In some embodiments, the surface of nucleic acid storage provided herein comprises flexible or rigid materials. Exemplary rigid materials include, but are not limited to, glass; fused silica; silicon such as silicon dioxide or silicon nitride; metals such as gold or platinum; plastics, such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate and combinations thereof. Rigid surfaces can be made of materials selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS) and glass or a combination thereof. Flexible materials are materials that can be bent, folded or similarly operated without breaking. In some cases, the flexible material is bent at least 30 degrees around the roller. In some cases, the flexible material is bent at least 180 degrees around the roller. In some cases, the flexible surface is bent at least 270 degrees around the roller. In some cases, the flexible material is bent about 360 degrees around the roller. In some cases, the radius of the roller is less than about 10cm, 5cm, 3cm, 2cm or 1cm. Exemplary flexible materials include, but are not limited to, nylon (unmodified nylon, modified nylon, clear nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic acid, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resins, transparent PVC foil, transparent foil for printers, poly (methyl methacrylate) (PMMA), methacrylate copolymers, styrene polymers, high refractive index polymers, fluoropolymers, polyethersulfones, polyimides containing alicyclic structures, rubber, fabrics, metal foils, and any combination thereof. In some cases, the nylon and PMAA surfaces herein are provided as sheets, or alternatively provided as layers coated on another material (such as silicon). Various plasticizers and modifiers can be used together with polymer substrate materials to achieve selected flexible properties. In some cases, the flexible material is a hydrogel, such as agarose or polyacrylamide.

[0361] In some embodiments, the nucleic acid chain is covalently or non-covalently attached to the surface. In some embodiments, the surface is first coated with biotin and then with streptavidin. In other embodiments, the biotinylated nucleic acid is attached to the coated surface. In some embodiments, the surface is coated with a copolymer of N,N-dimethylacrylamide. In some embodiments, the nucleic acid is baked onto a glass surface using drying and UV light. In some embodiments, the nucleic acid chain contains acrylamide modification and is covalently attached to a hydrogel. In some embodiments, the nucleic acid chain is attached to a bead fixed on the surface. In other embodiments, the fixed chain is UV-cleavable from the bead. In some embodiments, the bead is a hydrogel bead, a metal bead, a streptavidin bead, a magnetic bead, a magnetic streptavidin bead, a gold nanoparticle, a metal nanoparticle, or any combination thereof. In some embodiments, the nucleic acid chain is encapsulated in a biological sample by a bead, LNP, AAV, a virus, an exosome. In some embodiments, the biological sample is a fixed tissue or cell. In some embodiments, the biological sample is a living cell.

[0362] In some embodiments, the surface is silanized. In other embodiments, the nucleic acid chain comprises a base or other reactive groups modified by amination, and is covalently bound to the silanized surface. In some embodiments, it is directly attached to the silanized surface by reacting with an epoxy group. In some embodiments, a cross-linking agent such as 1,4-phenylene diisothiocyanate (PDC) is used to indirectly attach to the silanized surface. In some embodiments, the nucleic acid chain is covalently bound to the surface using click chemistry, wherein the chain is modified with an azide or alkyne reactive group, and the azide or alkyne reactive group reacts with an alkyne or azide group on the surface, for example, in the presence of copper.

[0363] Exemplary embodiments

[0364] The present invention provides a method for generating biological information, which includes providing a node factory at a node position on a biological sample, wherein the node factory contains one or more node nucleic acids, and wherein each node nucleic acid contains: a node barcode region, one or more flanking node barcode hybridization regions and optionally a node primer region; amplifying one or more node nucleic acids to generate multiple concatemers of node nucleic acids, wherein the multiple concatemers diffuse away from the node position over time; cleaving the multiple concatemers into more than one node nucleic acid; attaching two or more node nucleic acids generated by the node factory to generate multiple node nucleic acids; and analyzing the association frequency of the two or more node nucleic acids, wherein the association frequency provides information for spatial mapping of the biological sample. The present invention also provides a method, wherein the node factory also comprises one or more factory target nucleic acids, wherein each factory target nucleic acid comprises: a target binding region; a target barcode region; one or more flanking target binding hybridization regions; and optionally, a target primer region; wherein each factory target nucleic acid is adjacent to a node nucleic acid, and wherein amplification generates a concatemer comprising repeats of a node nucleic acid and a factory target nucleic acid, and cleavage generates more than one node nucleic acid and a factory target nucleic acid; and wherein the method further comprises: attaching the factory target nucleic acid to a target sequence; and measuring the association frequency of the factory target nucleic acid and the target sequence, wherein the association frequency provides information for spatial mapping of the biological sample. Also provided herein are methods, wherein amplification comprises rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branched amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER), or primer exchange reaction (PER). Also provided herein are methods, wherein each node factory comprises a padlock probe comprising a node nucleic acid and a node nucleic acid restriction site, the method further comprising: hybridizing the padlock probe to a nucleic acid splint, wherein the nucleic acid splint comprises a region complementary to a region at the 3' end and the 5' end of the padlock probe; applying a ligase to the sample, wherein the ligase ligates the 3' end and the 5' end of the padlock probe; applying a rolling circle polymerase to the ligated padlock probe; and generating concatemers comprising a node nucleic acid and a node nucleic acid restriction site by RCA. Also provided herein are methods, wherein the nucleic acid splint comprises at least a portion of an mRNA sequence in a biological sample. Also provided herein are methods, wherein the nucleic acid splint is conjugated to a surface, a protein, a small molecule, a nucleic acid, a lipid, or any combination thereof. Also provided herein are methods, wherein the protein comprises a receptor, a ligand, an antibody, or any functional fragment thereof. Also provided herein are methods, wherein the biological sample further comprises a feature, wherein the feature in the biological sample comprises a cell structure, a receptor, a scaffold, a matrix, a nucleic acid, a tissue, or an organelle.Also provided herein are methods, the methods further comprising depositing a restriction enzyme or nickase on the sample, wherein the restriction enzyme or nickase recognizes the node nucleic acid restriction site, thereby cleaving the concatemer to provide more than one node nucleic acid. Also provided herein are methods, the methods further comprising depositing a restriction enzyme or nickase on the sample, wherein the concatemer forms a hairpin comprising a node nucleic acid restriction site. Also provided herein are methods, wherein the restriction enzyme performs double-stranded cleavage at the node nucleic acid restriction site. Also provided herein are methods, wherein the nickase performs single-stranded cleavage at the node nucleic acid restriction site. Also provided herein are methods, the methods further comprising: depositing an oligonucleotide on the sample, wherein the oligonucleotide hybridizes to a region comprising a node nucleic acid restriction site to form a double-stranded restriction region, depositing a double-stranded specific restriction enzyme on the sample, wherein the restriction enzyme recognizes the node nucleic acid restriction site, thereby cleaving the concatemer to provide more than one node nucleic acid. Also provided herein are methods, wherein more than one node nucleic acid is generated separately. Also provided herein is a method, wherein the node factory also comprises one or more factory target nucleic acids, wherein each factory target nucleic acid comprises: a target binding region; a target barcode region; one or more flanking target binding hybridization regions; and optionally, a target primer region; wherein each factory target nucleic acid is adjacent to the node nucleic acid, and wherein amplification generates a concatemer comprising a repeat of the node nucleic acid and the factory target nucleic acid, and cleavage generates more than one node nucleic acid and the factory target nucleic acid; and wherein the method further comprises: attaching the factory target nucleic acid to the target sequence; and measuring the association frequency of the factory target nucleic acid and the target sequence, wherein the association frequency provides information for spatial mapping of the biological sample. Also provided herein is a method, wherein the generated concatemer comprises alternating node nucleic acids, node nucleic acid restriction sites, factory target nucleic acids, and factory target nucleic acid restriction sites. Also provided herein is a method, the method further comprising depositing a restriction enzyme or nicking enzyme on the sample, wherein the restriction enzyme or nicking enzyme recognizes the node nucleic acid restriction site and the factory target nucleic acid restriction site. Also provided herein is a method, the method further comprising depositing a first restriction enzyme on the sample, wherein the first restriction enzyme recognizes a factory target nucleic acid restriction site and does not recognize a node nucleic acid restriction site, thereby generating more than one nucleic acid comprising a node nucleic acid and a factory target nucleic acid. Also provided herein is a method, the method further comprising depositing a second restriction enzyme on the sample after depositing the first restriction enzyme, wherein the second restriction enzyme recognizes a node nucleic acid restriction site, thereby generating more than one node nucleic acid and a factory target nucleic acid. Also provided herein is a method, wherein one or more flanking node barcode hybridization regions or one or more flanking target binding hybridization regions are located on the flanks of the node barcode region or the target binding region. Also provided herein is a method, wherein providing includes affinity reaction, conjugation, incorporation into a hydrogel, cross-linking or photo-cross-linking. Also provided herein is a method, wherein the template node nucleic acid is conjugated to an antibody, an antibody fragment, a protein, a nanobody, a small molecule, a nucleic acid therapeutic agent, a lipid, a nanoparticle, a lipid nanoparticle or other affinity agent.Also provided herein is a method, wherein attachment includes hybridization, extension, connection, splint connection, cross-linking synthesis, connection and cross-linking (ICL) synthesis, cross-ICL and nick synthesis, paired copies or any combination thereof. Also provided herein is a method, wherein spatial mapping includes sequencing concatemers. Also provided herein is a method, wherein sequencing includes chain termination sequencing, nanopore sequencing, connection sequencing, combined probe anchor synthesis, synthetic sequencing, pyrophosphate sequencing, ion semiconductor or single molecule real-time sequencing. Also provided herein is a method, wherein the node barcode includes one or more fluorophore labels. Also provided herein is a method, wherein spatial mapping includes imaging concatemers. Also provided herein is a method, wherein one or more fluorophore labels are attached to the node barcode by synthesis or hybridization. Also provided herein is a method, wherein the node position includes a pattern. Also provided herein is a method, wherein the node position is preselected. Also provided herein is a method, wherein the biological sample includes cells in a synthetic matrix. Also provided herein is a method, and the method also includes imaging the biological sample. Also provided herein is a method, wherein the biological sample includes a colony of cells, a tissue sample or a protein. Also provided herein are methods wherein the node barcode region is about 3 to about 30 nucleotides. Also provided herein are methods wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000 or more than 1000 nucleotides. Also provided herein are methods wherein the biological sample is placed on a microscope slide or cover slip. Also provided herein are methods wherein the diffused node nucleic acid comprises a gradient in which the concentration decreases as the distance from the node position increases. Also provided herein are methods wherein the diffused node nucleic acids from different node positions do not overlap. Also provided herein are methods wherein the node nucleic acids from different node positions overlap. Also provided herein are methods wherein the node nucleic acids from two or more node positions are combined by hybridization, extension, ligation, splint ligation, cross-linking synthesis, ligation and cross-linked interchain crosslinking (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof. Also provided herein are methods, the method further comprising depositing a polymerase on the node nucleic acid. Also provided herein are methods, wherein the polymerase is a DNA polymerase or an RNA polymerase. Also provided herein are methods, wherein the DNA polymerase comprises Phi29, Pol I, Pol II, Pol III, Pol IV, Pol V, T4, Bsm, Bst, a reverse transcriptase, or any combination thereof. Also provided herein are methods, wherein the RNA polymerase comprises T7, T3, or SP6. Also provided herein are methods, the method further comprising depositing a nickase, wherein the node nucleic acid comprises a nicking site that can be targeted by the nickase.Also provided herein are methods, wherein the node nucleic acid further comprises a unique identifier sequence, the unique identifier sequence comprising one or more atypical bases, universal bases, other bases that the polymerase will randomly or incorrectly pair, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof. Also provided herein are methods, wherein the method further comprises controlling a diffusion factor, wherein the diffusion factor comprises viscosity, time, temperature, the presence of a crowding agent, pH, an electric field, a physical feature, or any combination thereof. Also provided herein are methods, wherein the viscosity is greater than 1 cP. Also provided herein are methods, wherein the biological sample medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP. Also provided herein are methods, wherein the deposition is at random locations throughout the biological sample. Also provided herein are methods, wherein the deposition is at a specific target in the biological sample. Also provided herein are methods, the method also comprising generating a second more than one node nucleic acid from the node nucleic acid.

[0365] Provided herein is a method for generating biological information, the method comprising: depositing a template nucleic acid at a node position on a biological sample, wherein the template nucleic acid comprises: a node barcode region; and one or more flanking node barcode hybridization regions; amplifying the template nucleic acid to generate a node nucleic acid, wherein the node nucleic acid diffuses away from the node position over time; depositing a factory target nucleic acid onto the biological sample, wherein the factory target nucleic acid comprises: a target binding region; and one or more flanking target binding hybridization regions; and attaching at least one of the node nucleic acids to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node nucleic acid and the factory target nucleic acid provides biological information for spatial mapping of the biological sample. Also provided herein is a method, wherein the factory target nucleic acid also comprises a target recognition region. Also provided herein is a method, wherein one or more flanking node barcode hybridization regions or one or more flanking target binding hybridization regions are located on the flanks of the node barcode region or the target binding region. Also provided herein is a method, wherein providing includes affinity reaction, conjugation, incorporation into a hydrogel, cross-linking or photo-cross-linking. Also provided herein is a method, wherein template node nucleic acid is conjugated to an antibody, an antibody fragment, an albumen, a nanobody, a small molecule, a nucleic acid therapeutic agent, a lipid, a nanoparticle, a lipid nanoparticle or other affinity reagent. Also provided herein is a method, wherein amplification includes rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branch amplification method (RAM), amplification based on nucleic acid sequence (NASBA), signal amplification (SABER) or primer exchange reaction (PER) by exchange reaction. Also provided herein is a method, wherein attachment includes hybridization, extension, connection, splint connection, cross-combination, connection and cross-chain cross-linking (ICL) synthesis, cross ICL and nick synthesis, paired copies or any combination thereof. Also provided herein is a method, wherein spatial mapping includes sequencing concatemers. Also provided herein is a method, wherein sequencing includes chain termination sequencing, nanopore sequencing, ligation sequencing, combined probe anchor synthesis, synthetic sequencing, pyrophosphate sequencing, ion semiconductor or single molecule real-time sequencing. Also provided herein is a method, wherein the node barcode comprises one or more fluorophore labels. Also provided herein is a method, wherein spatial mapping comprises imaging of concatemers. Also provided herein is a method, wherein one or more fluorophore labels are attached to the node barcode by synthesis or hybridization. Also provided herein is a method, wherein the node position comprises a pattern. Also provided herein is a method, wherein the node position is preselected. Also provided herein is a method, wherein the biological sample comprises cells in a synthetic matrix. Also provided herein is a method, and the method further comprises imaging the biological sample. Also provided herein is a method, wherein the biological sample comprises a colony of cells, a tissue sample or a protein.Also provided herein are methods wherein the node barcode region is about 3 to about 30 nucleotides. Also provided herein are methods wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000 or more than 1000 nucleotides. Also provided herein are methods wherein the biological sample is placed on a microscope slide or cover slip. Also provided herein are methods wherein the diffused node nucleic acid comprises a gradient in which the concentration decreases as the distance from the node position increases. Also provided herein are methods wherein the diffused node nucleic acids from different node positions do not overlap. Also provided herein are methods wherein the node nucleic acids from different node positions overlap. Also provided herein are methods wherein the node nucleic acids from two or more node positions are combined by hybridization, extension, ligation, splint ligation, cross-linking synthesis, ligation and cross-linked interchain crosslinking (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof. Also provided herein are methods, the method further comprising depositing a polymerase on the node nucleic acid. Also provided herein are methods, wherein the polymerase is a DNA polymerase or an RNA polymerase. Also provided herein are methods, wherein the DNA polymerase comprises Phi29, Pol I, Pol II, Pol III, Pol IV, Pol V, T4, Bsm, Bst, a reverse transcriptase, or any combination thereof. Also provided herein are methods, wherein the RNA polymerase comprises T7, T3, or SP6. Also provided herein are methods, the method further comprising depositing a nicking enzyme, wherein the node nucleic acid comprises a nicking site that can be targeted by the nicking enzyme. Also provided herein are methods, wherein the node nucleic acid further comprises a unique identifier sequence, the unique identifier sequence comprising one or more atypical bases, universal bases, other bases that the polymerase will randomly or incorrectly pair, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof. Methods are also provided herein, wherein the diffusion factor includes viscosity, time, temperature, the presence of crowding agents, pH, electric field, physical characteristics or any combination thereof. Methods are also provided herein, wherein the viscosity is greater than 1 cP. Methods are also provided herein, wherein the biological sample medium includes a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP. Methods are also provided herein, wherein the deposition is at a random position throughout the biological sample. Methods are also provided herein, wherein the deposition is at a specific target in the biological sample.Also provided herein are methods that further include generating a second one or more node nucleic acids from the node nucleic acid.

[0366] Provided herein is a method for generating biological information, the method comprising: depositing a node nucleic acid at a node position on a biological sample, wherein the node nucleic acid comprises: a node barcode region; and one or more flanking node barcode hybridization regions; amplifying the node nucleic acid to generate a node amplification product, wherein the node amplification product diffuses away from the node position; depositing a factory target nucleic acid onto the biological sample, wherein the factory target nucleic acid comprises: a target binding region; and one or more flanking target binding hybridization regions; and attaching at least one of the node amplification products to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node amplification product and the factory target nucleic acid provides biological information for spatial mapping of the biological sample. Also provided herein is a method, wherein the factory target nucleic acid also comprises a target recognition region. Also provided herein is a method, wherein one or more flanking node barcode hybridization regions or one or more flanking target binding hybridization regions are located flanking the node barcode region or the target binding region. Also provided herein is a method, wherein the deposition comprises an affinity reaction, conjugation, incorporation into a hydrogel, cross-linking, or photo-cross-linking. Also provided herein is a method, wherein the node nucleic acid is attached to another molecule. Methods are also provided herein, wherein another molecule includes an antibody, an antibody fragment, an albumen, a nano antibody, a small molecule, a nucleic acid therapeutic agent, a lipid, a nanoparticle, a lipid nanoparticle or other affinity reagents. Methods are also provided herein, wherein amplification includes rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branch amplification method (RAM), amplification based on nucleic acid sequence (NASBA), signal amplification (SABER) or primer exchange reaction (PER) by exchange reaction. Methods are also provided herein, wherein attachment includes being synthesized by hybridization, connection, splint connection, cross-linking, connection and cross-chain cross-linking (ICL) synthesis, cross ICL and nick synthesis, paired copies or any combination thereof. Methods are also provided herein, wherein spatial mapping includes sequencing concatemers. Also provided herein is a method, wherein sequencing is chain termination sequencing, nanopore sequencing, ligation sequencing, combined probe anchor synthesis, synthetic sequencing, pyrophosphate sequencing, ion semiconductor or single molecule real-time sequencing. Also provided herein is a method, wherein the node barcode comprises one or more fluorophore labels. Also provided herein is a method, wherein spatial mapping comprises imaging of concatemers. Also provided herein is a method, wherein one or more fluorophore labels are attached to the node barcode by synthesis or hybridization. Also provided herein is a method, wherein the node position comprises a pattern. Also provided herein is a method, wherein the node position is preselected. Also provided herein is a method, wherein the biological sample comprises cells in a synthetic matrix. Also provided herein is a method, and the method further comprises imaging the biological sample. Also provided herein is a method, wherein the biological sample comprises a colony of cells, a tissue sample or a protein.Also provided herein are methods, wherein the node barcode region is about 3 to about 30 nucleotides. Also provided herein are methods, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000 or more than 1000 nucleotides. Also provided herein are methods, wherein the biological sample is placed on a microscope slide or cover slip. Also provided herein are methods, wherein the diffused node amplification products are in a gradient where the concentration decreases as the distance from the node increases. Also provided herein are methods, wherein the node amplification products from different node positions do not overlap. Also provided herein are methods, wherein the node amplification products from different node positions overlap. Also provided herein are methods, wherein the node amplification products from two or more node positions are combined by hybridization, ligation, splint ligation, cross-linking synthesis, ligation and cross-linking (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof. Also provided herein are methods, the method further comprising depositing a polymerase on the node nucleic acid. Also provided herein are methods, wherein the polymerase is a DNA polymerase or an RNA polymerase. Also provided herein are methods, wherein the DNA polymerase is Pol I, PolII, Pol III, Pol IV, Pol V, or any combination thereof. Also provided herein are methods, wherein the RNA polymerase is T7, T3, or a reverse transcriptase. Also provided herein are methods, the method further comprising a nicking enzyme, wherein the node nucleic acid comprises a nicking site that can be targeted by the nicking enzyme. Also provided herein are methods, wherein the node nucleic acid further comprises a unique identifier sequence, the unique identifier sequence comprising one or more atypical bases, universal bases, other bases that the polymerase will randomly or incorrectly pair, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof. Also provided herein are methods, wherein the biological sample further comprises a biological sample medium, and wherein the biological sample medium comprises a viscosity greater than 1 cP. Also provided herein are methods, wherein the biological sample medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP. Also provided herein are methods, wherein the deposition is at random locations throughout the biological sample. Also provided herein are methods, wherein the deposition is at a specific target in the biological sample.Also provided herein is a method, the method further comprising: depositing a DNA barcode onto a biological sample, wherein the DNA barcode comprises a region complementary to a region of a concatemer, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; selectively irradiating the biological sample to form a covalently linked DNA barcode-concatemer complex; washing the biological sample to remove DNA barcodes that are not complexed with the concatemer; and repeating the deposition, selective irradiation, and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence than the DNA barcode in the second deposition step, and the selective irradiation occurs at different locations for the first selective irradiation step and the second selective irradiation step. Also provided herein is a method, the method further comprising synthesizing a continuous DNA chain, wherein the synthesis comprises contacting the DNA barcode-concatemer complex with a polymerase. Also provided herein is a method, wherein the synthesis comprises crosslinking. Also provided herein is a method, the method further comprising displacing a continuous DNA chain from a biological sample. Also provided herein is a method, wherein the synthesis and displacing occur simultaneously. Also provided herein is a method, wherein the displaced continuous DNA chains are pooled. Also provided herein is a method, wherein the pooled continuous DNA chains are amplified. Also provided herein is a method in which the continuous DNA strands collected are sequenced. Also provided herein is a method in which the DNA barcode further comprises a promoter region, a primer region, or any combination thereof. Also provided herein is a method in which the primer is one or more random primers or in which the primer is a non-random primer. Also provided herein is a method in which the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Also provided herein is a method in which selective radiation is performed using a photomask. Also provided herein is a method in which the photomask is different in different selective radiation steps. Also provided herein is a method in which the photomask is manually generated. Also provided herein is a method in which the photomask is generated by a machine. Also provided herein is a method in which the selective radiation step comprises applying radiation at a wavelength of about 300nm to up to 450nm. Also provided herein is a method in which the selective radiation step comprises applying radiation at a wavelength of about 350nm to up to 420nm. Also provided herein is a method in which the selective radiation step comprises applying radiation at a wavelength of about 365nm to up to 405nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation to a preselected region of interest (ROI). Also provided herein are methods, wherein the ROI is cell-specific or subcellular-specific. Also provided herein are methods, wherein the ROI is based on morphology. Also provided herein are methods, wherein deposition is performed manually, automatically, by a robot, or any combination thereof. Also provided herein are methods, wherein deposition is performed with a modified inkjet printer. Also provided herein are methods, wherein deposition or washing is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers, inkjet printers, automatic liquid handlers, or any combination thereof.Also provided herein are methods wherein one or more DNA barcodes are combined with one or more node barcode strands, wherein the combination is performed by hybridization, extension, ligation, splinting, crossover synthesis, ligation and crossover interstrand crosslink (ICL) synthesis, crossover ICL and nick synthesis, paired copying, or any combination thereof.

[0367] The present invention provides a method for information storage, which includes: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a template nucleic acid at a node position on a surface, wherein the template nucleic acid comprises: a node barcode region, each corresponding to one of more than one barcode; and one or more flanking node barcode hybridization regions; amplifying the template nucleic acid to generate a node nucleic acid; depositing a factory target nucleic acid onto the surface, wherein the factory target nucleic acid comprises: a target binding region; one or more flanking target binding hybridization regions; optionally, a target primer region; and attaching at least one of the node nucleic acids to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node nucleic acid and the factory target nucleic acid provides the stored information. Also provided herein is a method, wherein amplification includes rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branch amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER) or primer exchange reaction (PER). Also provided herein is a method, wherein attachment includes synthesis by hybridization, extension, connection, splint connection, cross-linking, connection and cross-linking (ICL) synthesis, cross-ICL and nick synthesis, paired copies or any combination thereof. Also provided herein is a method, and the method also includes converting nucleic acid information into digital information, wherein conversion includes sequencing concatemers. Also provided herein is a method, wherein sequencing is chain termination sequencing, nanopore sequencing, ligation sequencing, combined probe anchor synthesis, synthesis sequencing, pyrophosphate sequencing, ion semiconductor or single molecule real-time sequencing. Also provided herein is a method, wherein concatemers include one or more fluorophore labels. Also provided herein is a method, wherein spatial mapping comprises imaging a concatemer. Also provided herein is a method, wherein one or more fluorophore labels are attached to a concatemer by synthesis or hybridization. Also provided herein is a method, wherein the node position comprises a pattern. Also provided herein is a method, wherein the node position is preselected. Also provided herein is a method, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000 or more than 1000 nucleotides. Also provided herein is a method, wherein the diffused node nucleic acid comprises a gradient whose concentration decreases as the distance from the node position increases. Also provided herein is a method, wherein the node nucleic acids from different node positions do not overlap. Also provided herein is a method, wherein the node nucleic acids from different node positions overlap.Also provided herein are methods, wherein node nucleic acids from two or more node positions are combined by hybridization, extension, ligation, splinting, cross-linking, ligation and cross-linking (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof. Also provided herein are methods, the method further comprising depositing a polymerase on the node nucleic acid. Also provided herein are methods, wherein the polymerase is a DNA polymerase or an RNA polymerase. Also provided herein are methods, wherein the node nucleic acid further comprises a unique identifier sequence, the unique identifier sequence comprising one or more atypical bases, universal bases, other bases that the polymerase will randomly or incorrectly pair, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof. Also provided herein are methods, wherein the diffusion factor comprises viscosity, time, temperature, the presence of a crowding agent, pH, an electric field, a physical feature, or any combination thereof. Also provided herein are methods, wherein the viscosity is greater than 1 cP. Also provided herein are methods wherein the medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP.

[0368] The present invention provides a method for information storage, which includes: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a node nucleic acid at a node position on a surface, wherein the node nucleic acid comprises: a node barcode region, each corresponding to one of more than one barcode; and one or more flanking node barcode hybridization regions; amplifying the node nucleic acid to generate a node amplification product, wherein the node amplification product diffuses away from the node position; depositing a factory target nucleic acid onto the surface, wherein the factory target nucleic acid comprises: a target binding region; and one or more flanking target binding hybridization regions; and attaching at least one of the node amplification products to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node amplification product and the factory target nucleic acid provides information for spatially mapping the node position. Also provided herein is a method, wherein amplification includes rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branch amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER) or primer exchange reaction (PER). Also provided herein is a method, wherein attachment includes synthesis by hybridization, connection, splint connection, cross-linking, connection and cross-linking (ICL) synthesis, cross ICL and nick synthesis, paired copies or any combination thereof. Also provided herein is a method, wherein spatial mapping includes sequencing concatemers. Also provided herein is a method, wherein sequencing is chain termination sequencing, nanopore sequencing, connection sequencing, combined probe anchor synthesis, synthetic sequencing, pyrophosphate sequencing, ion semiconductor or single molecule real-time sequencing. Also provided herein is a method, wherein concatemers include one or more fluorophore labels. Also provided herein is a method, wherein spatial mapping includes imaging the concatemer. Also provided herein is a method, wherein one or more fluorophore labels are attached to the concatemer by synthesis or hybridization. Also provided herein is a method, wherein the node position includes a pattern. Also provided herein is a method, wherein the node position is preselected. Also provided herein is a method, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000 or more than 1000 nucleotides. Also provided herein is a method, wherein the diffused node amplification product is in a gradient in which the concentration decreases as the distance from the node increases. Also provided herein is a method, wherein the node amplification products from different node positions do not overlap. Also provided herein is a method, wherein the node amplification products from different node positions overlap.Also provided herein are methods, wherein node amplification products from two or more node positions are combined by hybridization, ligation, splinting, cross-linking synthesis, ligation and cross-linking interstrand crosslink (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof. Also provided herein are methods, the method further comprising depositing a polymerase on the node nucleic acid. Also provided herein are methods, wherein the polymerase is a DNA polymerase or an RNA polymerase. Also provided herein are methods, wherein the node nucleic acid further comprises a unique identifier sequence, the unique identifier sequence comprising one or more atypical bases, universal bases, other bases that the polymerase will randomly or incorrectly pair, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid is double-stranded, is single-stranded, comprises a hairpin structure, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a medium, and wherein the medium comprises a viscosity greater than 1 cP. Also provided herein are methods wherein the medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP.

[0369] Provided herein is a method for information storage, the method comprising: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a first oligonucleotide onto a surface, wherein the first oligonucleotide comprises: optionally, a surface binding region; a first barcode region corresponding to a barcode in the more than one barcode; and one or more flanking first oligonucleotide hybridization regions, wherein the surface binding region and the one or more flanking second oligonucleotide hybridization regions optionally comprise a photoreactive nucleobase; optionally selectively irradiating the surface to form covalently linked oligonucleotides; depositing a second oligonucleotide onto the surface, wherein the second oligonucleotide comprises: a second barcode region corresponding to a barcode in the more than one barcode; and one or more flanking second oligonucleotide hybridization regions, wherein the one or more flanking second oligonucleotide hybridization regions comprise a photoreactive nucleobase, and at least one of the one or more flanking first oligonucleotide hybridization regions is complementary to at least one of the one or more flanking second oligonucleotide hybridization regions; and selectively irradiating the surface to covalently link the second oligonucleotide to the first oligonucleotide; and optionally repeating the second depositing step and the irradiating step one or more times, thereby generating a concatemer. Also provided herein is a method, wherein the surface comprises an oligonucleotide, the oligonucleotide comprising a photocleavable nucleobase and a region complementary to at least one of the one or more flanking first oligonucleotide hybridization regions. Also provided herein is a method, wherein the surface comprises a reactive group capable of binding to a nucleoside. Also provided herein is a method, wherein the radiation comprises applying light at a wavelength of about 365nm to about 405nm. Also provided herein is a method, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Also provided herein is a method, wherein the selective radiation comprises applying radiation to a preselected region of interest (ROI). Also provided herein is a method, wherein the ROI is described with a photomask. Also provided herein is a method, wherein the photomask is different in each selective radiation step. Also provided herein is a method, wherein the photomask is manually generated. Also provided herein is a method, wherein the photomask is generated by a machine. Also provided herein is a method, wherein the selective radiation step comprises applying radiation at a wavelength of about 300nm to up to 450nm. Also provided herein is a method, wherein the selective radiation step comprises applying radiation at a wavelength of about 365nm to up to 405nm. Methods are also provided herein, and the methods also include washing to remove unbound oligonucleotides after each selective radiation step. Methods are also provided herein, wherein the concatemers also include a primer region, a promoter region, or any combination thereof. Methods are also provided herein, and the methods also include depositing a polymerase on the concatemers. Methods are also provided herein, wherein the continuous complementary nucleic acid chains of the concatemers are generated by cross-linking, hybridization, extension, connection, splinting, connection and cross-linking (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof.Also provided herein is a method in which a continuous complementary nucleic acid chain is amplified and sequenced to generate sequencing data. Also provided herein is a method in which sequencing data allows nucleic acid information to be translated into digital information. Also provided herein is a method in which a concatemer is contacted with a sequence of a fluorophore-labeled concatemer complementary to the barcode region of an oligonucleotide to generate a fluorophore-concatemer complex. Also provided herein is a method in which the fluorophore-concatemer complex is imaged. Also provided herein is a method in which imaging includes applying light at a wavelength of about 365nm to about 405nm. Also provided herein is a method in which imaging allows nucleic acid information to be translated into digital information.

[0370] Provided herein is a method for bioinformation generation, the method comprising depositing a node factory comprising a matrix at a node location on a biological sample, wherein each node factory comprises more than one node nucleic acid, and wherein each node nucleic acid comprises: a node barcode region; one or more flanking node barcode hybridization regions; optionally, a node primer region, a target binding region, a target barcode region; one or more flanking target binding hybridization regions; and optionally, a target primer region, which releases at least one node nucleic acid from the node factory, wherein the release provides bioinformation for spatial mapping of the biological sample. Also provided herein is a method, wherein the node factory comprises more than one node nucleic acid reversibly fixed to a carrier. Also provided herein is a method, wherein the carrier comprises a liposome, a droplet, a bead, an LNP, an AAV, an exosome, a lentivirus, or a protein. Also provided herein is a method, wherein the carrier comprises a bead. Also provided herein is a method, the method further comprising releasing the node nucleic acid from the carrier. Also provided herein is a method, wherein each node nucleic acid further comprises a restriction site, and wherein more than one node nucleic acid is provided in concatemer form. Also provided herein are methods, further comprising depositing a restriction enzyme on the sample, wherein the restriction enzyme recognizes the restriction site, thereby cleaving the concatemer to provide more than one node nucleic acid. Also provided herein are methods, further comprising depositing an oligonucleotide on the sample, wherein the oligonucleotide hybridizes to a region comprising a restriction site on the node nucleic acid, and wherein the restriction enzyme recognizes the double-stranded cleavage site on the sample, thereby cleaving the concatemer to provide more than one node nucleic acid.

[0371] The present invention provides a method for generating bioinformatics, which includes: depositing a node template nucleic acid at a node position on a sample, wherein the node nucleic acid comprises: a node barcode region, and a flanking hybridization region; depositing a factory target nucleic acid onto a population of cells, wherein the factory target nucleic acid comprises: a target recognition region, a target binding region, and a flanking hybridization region; and hybridizing at least one of the node nucleic acids with at least one of the factory target nucleic acids.

[0372] The present invention provides a method for generating bioinformation, comprising: depositing a factory target nucleic acid at a node position on a sample that contains a node nucleic acid; performing more than one polymerase-based amplification to generate an amplification product, wherein the amplification product contains at least one node nucleic acid and more than one factory target nucleic acid; wherein the amplification product provides information for spatially mapping a population of cells based on the frequency of the factory target nucleic acid and the node nucleic acid present in the amplification product.

[0373] Provided herein is a method for generating bioinformatics, the method comprising: depositing node nucleic acids onto node locations in a sample, wherein the locations comprise a pattern, wherein the tissue sample comprises cells in a synthetic matrix, and wherein the node nucleic acids comprise: a node barcode region, and flanking hybridization regions; depositing factory target nucleic acids onto a population of cells, wherein the factory target nucleic acids comprise: a target recognition region; a target binding region; and flanking hybridization regions; and hybridizing at least one of the node nucleic acids with at least one of the factory target nucleic acids, wherein the density of the synthetic matrix regulates the hybridization rate.

[0374] The present invention provides a method for generating bioinformatics, which includes: depositing a node nucleic acid to a preselected node position on a sample, wherein the node nucleic acid comprises: a node barcode region, and a flanking hybridization region; depositing a factory target nucleic acid onto a population of cells, wherein the factory target nucleic acid comprises: a target recognition region, a target binding region, and a flanking hybridization region; performing polymerase-based amplification to generate amplification products, wherein the amplification products comprise at least one node nucleic acid and more than one factory target nucleic acid; sequencing the amplification products to generate sequencing data; and generating a spatial map based on the sequencing data.

[0375] Also provided herein are methods as described herein, wherein the sample comprises a population of cells, a tissue sample, or a protein. Also provided herein are methods, wherein the node barcode region is about 3 to about 30 nucleotides. Also provided herein are methods, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000 or more than 1000 nucleotides. Also provided herein are methods, the methods further comprising amplifying the node nucleic acid, wherein the amplification product diffuses away from the node. Also provided herein are methods, wherein the diffused amplification product is in a gradient in which the concentration decreases as the distance from the node increases. Also provided herein are methods, wherein the amplification products from different node positions do not overlap. Also provided herein are methods, wherein the amplification products from different node positions overlap. Also provided herein are methods, wherein the node barcode chains from two or more node positions are combined by hybridization, connection, splint connection, cross-linking synthesis, connection and cross-linking inter-chain crosslinking (ICL) synthesis, cross-ICL and nicking synthesis, paired copies, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof. Also provided herein are methods, the method further comprising depositing a polymerase on the node nucleic acid. Also provided herein are methods, wherein the polymerase is a DNA polymerase or an RNA polymerase. Also provided herein are methods, wherein the DNA polymerase is Pol I, Pol II, Pol III, Pol IV, Pol V, or any combination thereof. Also provided herein are methods, wherein the RNA polymerase is T7, T3, or a reverse transcriptase. Also provided herein are methods, the method further comprising a nicking enzyme, wherein the node barcode chain comprises a nicking site that can be targeted by the nicking enzyme. Also provided herein are methods, wherein the node nucleic acid further comprises a unique identifier sequence, the unique identifier sequence comprising one or more atypical bases, universal bases, other bases that the polymerase will randomly or incorrectly pair, or any combination thereof. Also provided herein are methods, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof. Also provided herein are methods, wherein the sample medium comprises a viscosity greater than 1 cP. Also provided herein are methods, wherein the sample medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP. Also provided herein are methods, wherein the deposition is at random locations throughout the sample. Also provided herein are methods, wherein the deposition is at a specific target in the sample.

[0376] Provided herein is a method as described herein, further comprising: contacting a population of cells with a nucleic acid polymerase and a primer to generate an amplification product; depositing a DNA barcode onto the population of cells, wherein the DNA barcode comprises a region complementary to a region of the amplification product, and wherein the DNA barcode comprises a photoreactive nucleobase capable of crosslinking with another nucleobase; selectively irradiating the population of cells to form a covalently linked DNA barcode-amplification product complex; washing the population of cells to remove DNA barcodes that are not complexed with the amplification product; and repeating the deposition, selective irradiation and washing steps, wherein: the DNA barcode in the first deposition step comprises a different sequence from the DNA barcode in the second deposition step, and the selective irradiation occurs at different locations for the first selective irradiation step and the second selective irradiation step. Provided herein is a method, the method further comprising synthesizing a continuous DNA chain, wherein the synthesis comprises contacting the DNA barcode-amplification product complex with a polymerase. Provided herein is a method, wherein the synthesis comprises cross-linking. Provided herein is a method, the method further comprising replacing a continuous DNA chain from a population of cells. Provided herein is a method, wherein the synthesis and replacement occur simultaneously. Provided herein is a method, wherein the replaced continuous DNA chains are pooled. Also provided herein is a method in which the continuous DNA chains collected are amplified. Also provided herein is a method in which the continuous DNA chains collected are sequenced. Also provided herein is a method in which the primer is one or more random primers. Also provided herein is a method in which the primer is a non-random primer. Also provided herein is a method in which the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK). Also provided herein is a method in which the selective radiation is carried out using a photomask. Also provided herein is a method in which the photomask is different in different selective radiation steps. Also provided herein is a method in which the photomask is manually generated. Also provided herein is a method in which the photomask is generated by a machine. Also provided herein is a method in which the selective radiation step includes applying radiation at a wavelength of about 300nm to up to 450nm. Also provided herein is a method in which the selective radiation step includes applying radiation at a wavelength of about 350nm to up to 420nm. Also provided herein is a method in which the selective radiation step includes applying radiation at a wavelength of about 365nm to up to 405nm. Also provided herein are methods, wherein the selective radiation step comprises applying radiation to a preselected region of interest (ROI). Also provided herein are methods, wherein the ROI is cell-specific or subcellular-specific. Also provided herein are methods, wherein the ROI is based on morphology. Also provided herein are methods, wherein deposition is performed manually, automatically, by a robot, or any combination thereof. Also provided herein are methods, wherein deposition is performed with a modified inkjet printer. Also provided herein are methods, wherein deposition or washing is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, acoustic liquid handlers, inkjet printers, automatic liquid handlers, or any combination thereof.Also provided herein are methods wherein one or more DNA barcodes are combined with one or more node barcode strands, wherein the combination is performed using ligation, splint ligation, crossover synthesis, ligation and crossover interstrand crosslink (ICL) synthesis, crossover ICL and nick synthesis, paired copies, or any combination thereof. Example

[0377] The following examples further illustrate the described embodiments but are not intended to limit the scope of the disclosure.

[0378] Example 1: Fixation of samples

[0379] MRC-5 (human, ATCC CCL-171) and HEK293T cells (human, ATCC CRL-1573) were grown in Dulbecco's Modified Eagle's Medium (Gibco #10564) supplemented with 10% (vol / vol) serum (Gibco #10437 for MRC-5 and Peak Serum PS-FB2 for HEK293T), 50 U / mL penicillin and 50 μg / mL streptomycin (Gibco #15070). EY.T 4 embryonic fibroblasts (mouse) 49 were grown in Dulbecco's Modified Eagle's Medium (Gibco #10564) supplemented with 15% (vol / vol) serum, 50 U / mL penicillin and 50 μg / mL streptomycin. Cells were cultured at 37°C in the presence of 5% CO2.

[0380] The cells were washed with phosphate buffered saline (PBS), 0.1% 80, 0.1% polysorbate 20 in PBS ( 20) or Hank's balanced salt solution (HBSS). The samples were fixed in 1%-10% formaldehyde and then washed with PBS or HBSS. Optionally, 0%-2.5% Triton X-100 + 0.1% 20 to permeabilize the cells. Fixed samples can be maintained at a temperature of 4°C to 45°C. Prior to barcode hybridization, samples are fixed at room temperature (20°C to 25°C) for approximately 5 to 20 minutes. Alternatively, samples are fixed at a lower temperature of 4°C and stored for up to two weeks prior to barcoding. After fixation, samples are washed with PBS or 0.1% 20 washes. Bovine serum albumin (BSA) is used as a blocking agent to reduce nonspecific detection of target nucleic acids and proteins. Other blocking agents or detergents can be used. When blocking, cells are incubated in BSA or detergent at room temperature.

[0381] Example 2: Barcode hybridization and imaging.

[0382] For fixed tissue samples, such as those generated in Example 1, barcodes are hybridized to target nucleic acids using the reagents listed in Table 2. Reagent conditions include ranges that allow hybridization of the DNA barcodes to the target.

[0383] Table 2. Barcode hybridization reagents

[0384]

[0385]

[0386] The barcoding solution consisting of 250 nM barcode 1 strand was mixed in PBS and 20 (PBST) mixed with 2mg / ml sheared salmon sperm DNA (Invitrogen AM9680), 10% dextran sulfate (wt / vol, Sigma Aldrich S4030) and 500mM NaCl, applied to fixed tissue samples for 20min to 1 hour. The samples were washed 3 times with PBST containing 1M NaCl, each for about 1min. Before light stimulation, the fixed samples can be maintained in the barcoding solution at a temperature of about 20°C to up to 60°C.

[0387] Using microscopy, the desired area on the tissue sample is visually identified in a brightfield or fluorescent image. A hand-drawn mask is set to the photostimulation area. In each barcode wheel, a high-power liquid light guide-coupled LED source is applied at 365 nm to the selected area of ​​interest on the sample. The microscope focus is set to the desired layer on the sample and the photomask area is manually selected. The sample is photostimulated, imaged, and then treated with approximately 0.1×-1× PBS; 25% to 80% formamide; and 0% to up to 1% 20 Washing.

[0388] After finding the same cell region in the fixed sample, additional barcode strands can be added to the barcoding solution. After barcoding, the sample is optionally treated with a cross-linking solution consisting of RNase H and reverse transcription buffer. cDNA can be extracted for amplification and / or next generation sequencing by washing the cells with PBST and collecting the supernatant in a tube. cDNA can be pooled together or separately pooled for sequencing analysis in a continuous barcoding step.

[0389] Example 3: Binary encoding.

[0390] The sample was spatially defined with 16 regions of interest (ROIs). Four unique barcodes were applied in four rounds of crosslinking iterations. In each of the four rounds of crosslinking, light was applied to half of the ROIs. The fraction of any illumination in each round contained about 1 / 4 (25%) saturation or less, providing 2^4 = 16 unique combinations of the 4 barcode strands being crosslinked or not crosslinked in each region. This Fig.30 , where the filled squares represent some fraction of reads in the region containing the barcode corresponding to that column, and the empty white squares represent no illumination during that barcoding round. On the right, the binary codes of the four barcodes can be assigned to each of these combinations.

[0391] Example 4: quaternary encoding.

[0392] The sample was spatially defined with 16 regions of interest (ROIs). Two unique barcodes were applied in two rounds of crosslinking iterations. In each of the two crosslinking rounds, light was applied to 75% of the ROIs at one of three different levels, so that the fraction of maximum illumination in each round included crosslink saturation of approximately 1 / 2 (50%) or less of the sites. This provided 4^2 = 16 unique combinations of the crosslink fractions of the 2 barcode strands. Fig.31 The combinations are illustrated in , where the hierarchy of filled squares represents the different fractions of reads in the region containing the barcode corresponding to that column, and the empty white squares represent the absence of illumination during that barcoding round. On the right, the quaternary encodings of the two barcodes can be assigned to each of these cases.

[0393] Example 5: Fractional encoding.

[0394] The sample was spatially defined with 11 regions of interest (ROIs). Two unique barcodes were applied in two rounds of crosslinking iterations. In each of the crosslinking rounds, light was applied to each ROI at 10 different levels such that there were unique ratios of barcode fractions. Light was applied at levels that provided 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% barcode fractions. These 11 unique ratios of the two barcode strands were Fig.32 , where a hierarchy of filled squares represents different fractions of reads in the region containing the barcode corresponding to that column, and empty white squares represent no illumination during that barcoding pass. On the right, ratio-based encodings of the two barcodes can be assigned to each of these cases.

[0395] Example 6: Node chain generation

[0396] Diffused strands from single-stranded rolling circle amplification (RCA) repeats (concatemers) in cells are generated by hybridizing short oligonucleotides to create double-stranded restriction enzyme (RE) sites that can be cleaved to release the strands. An exemplary schematic of the workflow is shown in Fig.37A middle.

[0397] Cell seeding and fixation

[0398] 3T3 cells are inoculated and grown overnight in a glass bottom chamber on a slide. The next day, the culture medium is removed, and the cells are washed with preheated DPBS (calcium-free, magnesium-free), then fixed with a 4% paraformaldehyde solution (4% PFA) in 1×PBS. The cells are incubated at room temperature for 5 minutes in a fixative, then washed twice in 1×PBS (PBST) containing 0.1% Tween-20 for 2 minutes.

[0399] The cells were then permeabilized with 1×PBS containing 0.25% Triton-X-100 (PBSTx) for 10 minutes and then washed twice in 1×PBS for 1 min. Finally, the cells were washed twice with PBST for 2 minutes.

[0400] The blocking solution contained 1×Ampligase buffer (ThermoFisher), 0.05M KCl, 20% formamide, 0.1 μM blocking oligonucleotide, 0.2 μg / mL BSA, 1 U / μL RiboLock (ThermoFisher), 0.2 μg / μL yeast tRNA (ThermoFisher). The blocking solution was incubated with the sample at room temperature for 30 minutes, and then washed twice with PBST for 1 minute.

[0401] Clamp connection and RCA

[0402] Padlock probe hybridization generation Fig.37F was performed using a padlock probe at a concentration of 0.05 μM in blocking solution ( Fig.37B ), without adding blocking oligonucleotide. Three padlock probes ( Fig.37C ) is hybridized with the sample to generate Figure 37G , three padlock probes were incubated at a final incubation concentration of 0.02 μM each during padlock hybridization in blocking solution, without adding blocking oligonucleotide. Cells were incubated at 55°C for 15 minutes for padlock hybridization, followed by 45°C for 120 minutes. Slides were then washed three times at room temperature with wash buffer (10% formamide in 2×SSC) for 5 minutes, followed by three washes in PBST for 1 minute.

[0403] The padlock that generates the node chain is shown in Fig.37B and Fig.37CIn situ hybridization with nucleic acid sequences and splint ligation to form circular constructs. The padlock probe was incubated in a 1× T4 RNA ligase buffer containing 0.01 mM ATP, 0.2 μg / μl BSA, 0.005 to 0.5 U / μL SplintR ( Fig.37E , use 0.5U / μl, Fig.37F , 0.005U / μl) and 1U / μl RiboLock ligation solution were used for ligation. The incubation time was overnight at room temperature, followed by two 1-minute washes with PBST.

[0404] Rolling circle amplification was then performed in a solution of 1X Phi29 buffer, 5% glycerol, 0.2 μg / μl BSA, 0.25 mM dNTP mix, 0.1 μM RCA initiator primer and 0.5 U / μl phi29 polymerase using a primer complementary to the padlock with a phosphorothioate bond on the 3' nucleotide (RCA initiator primer).

[0405] The chamber slides were incubated at 30°C for 2 hours and washed twice for 1 minute with PBST. The slides were then fixed with 4% PFA in 1×PBS for 5 minutes at room temperature, followed by 3 washes for 1 minute with PBST, then 3 washes for 5 minutes with 65% formamide at 30°C, then 1 minute washes twice with PBST. The slides were stored in PBST at 4°C overnight.

[0406] Cleavage of RCA products

[0407] Hybridization of the cleavage oligonucleotide to the RCA product was then performed, followed by treatment with a restriction enzyme to double-strand the RCA product at the restriction enzyme site. Fig.37D and Fig.37E The light grey fork (harpoon) in the double helix differs in the length and number of bases between the recognition site of the enzyme and the end of the oligonucleotide (two restriction enzymes with recognition sites in the duplex were also tested). The same cleavage oligonucleotide was used for both TsprI (restriction site: NNCASTGNN) and BstI-v2 (restriction site: GCAGTGNN) because their restriction enzyme recognition sites overlap (where N is any nucleotide and S is G or C). The slides were first treated with 10mM EDTA aqueous solution and incubated at 65°C for 5 minutes, followed by three washes of 1×PBS for 1 minute. The cleavage oligonucleotides were hybridized in 1×SSC, 5% formamide, 0.06μM cleavage oligonucleotides and 0.1μg / mL BSA. The slides were incubated at 33°C for 20 minutes for cleavage oligonucleotide hybridization, followed by three washes of 5 minutes at 33°C in 10% formamide in 2×SSC, followed by three washes of 1 minute with 6×SSC.

[0408] For the "Before cutting", "After cutting, before cleaning", and "After cleaning" conditions ( Figure 37G ), then the cleavage oligonucleotide and fluorescent probe were hybridized to the sample in a single solution containing 1×SSC, 5% formamide, 0.02 μM of each probe (one RCA product for detecting each padlock) and 0.1 μg / mL BSA, 0.06 μM of the cleavage oligonucleotide. The samples were incubated at 33°C for 30 minutes and then washed 3 times with 10% formamide in 2×SSC for 5 min, followed by 3 washes with 6×SSC for 1 min. The "before cleavage" samples were imaged with a 20× spinning disk confocal after hybridization and before washing.

[0409] Restriction enzymes (BtsI-v2 or TsprI) were then added in 1× CutSmart buffer at 55°C for 1 hour. The samples were then washed three times with PBST for one minute ( Fig.37F ) or lysis was performed by incubation at 50°C for 30 min with 0.2 U / μl BtsI-v2 (+BtsI) or water (-BtsI condition) in 1× CutSmart buffer. The “post-cut, pre-wash” samples were then imaged ( Figure 37G ). The samples were then washed three times with 6× SSC. The “after wash” condition was then imaged using a 20× spinning disk confocal.

[0410] The "no Og" control (no oligonucleotide control) was treated with the same protocol, except that water was used to replace the cleavage oligonucleotide. The "no RE" control (no restriction enzyme control) followed the same protocol similarly, but water was used to replace the restriction enzyme during the cleavage step. The "re-probe" sample was subjected to fluorescent probe hybridization after cleavage and washing. The fluorescent probe was incubated in 1 × SSC, 5% formamide, 0.02 μM of each fluorescent probe and 0.1 μg / mL BSA. The incubation occurred at 33 ° C for 30 minutes, followed by washing three times for 5 minutes at 33 ° C in 10% formamide in 2 × SSC, and washing three times for 1 minute at room temperature with 6 × SSC. The "re-probe" sample was then imaged with 20 × spinning disk confocal imaging.

[0411] For all samples, images were acquired with equivalent acquisition parameters and maximum intensity projections of z-stack images are shown in the accompanying figures.

[0412] Detection and imaging of RCA products

[0413] Detection of the cleaved products is performed by hybridization of fluorescent oligonucleotides to target sequences within the RCA products ( Fig.37F and Figure 37G). The 3'Cy3-labeled probe oligonucleotide was incubated in a hybridization solution containing 1× SSC, 5% formamide, 0.06 μM fluorescent probe and 0.1 μg / mL BSA at room temperature in the dark for 20 minutes. The slides were then washed three times for 5 minutes at 33°C with 10% formamide in 2× SSC, followed by three washes for 1 minute at room temperature with 6× SSC. The slides were then imaged at 20× with a spinning disk confocal imager to visualize the RCA products after cleavage.

[0414] The maximum intensity projections of the acquired z-stack images were used to quantify the mean fluorescent spot intensity and area and are shown in Fig.37H In. Use the Laplacian of Gaussian method to segment individual spots and use Otsu thresholding to define spot image pixels. Calculate the total spot area and the average intensity of all segmented spot pixels for each spot.

[0415] To generate a line graph ( Fig.37H ), the average spot intensity of the "after cleavage, before washing", "after washing" and "re-probing" samples was divided by the average spot intensity of the "after cleavage, before w...

Claims

1. A method for generating biological information, the method comprising: A node factory is provided at a node location on a biological sample, wherein the node factory comprises one or more node nucleic acids, and wherein each node nucleic acid comprises: Node barcode region; one or more flanking node barcode hybridization regions; and Optionally, a node primer region; amplifying the one or more node nucleic acids to generate concatemers of node nucleic acids, wherein the concatemers diffuse away from the node location over time; cleaving the concatemer into more than one node nucleic acid; attaching two or more node nucleic acids produced by the node factory to produce a multi-node nucleic acid; as well as The association frequencies of the two or more node nucleic acids are analyzed, wherein the association frequencies provide information for spatially mapping the biological sample.

2. The method of claim 1, wherein the node factory further comprises one or more factory target nucleic acids, wherein each factory target nucleic acid comprises: target binding region; target barcode region; one or more flanking target binding hybridization regions; and Optionally, a target primer region; wherein each factory target nucleic acid is contiguous with a node nucleic acid, and wherein said amplifying generates concatemers comprising repeats of a node nucleic acid and a factory target nucleic acid, and wherein said cleavage generates more than one node nucleic acid and factory target nucleic acid; and The method further comprises: attaching the factory target nucleic acid to a target sequence; as well as The association frequency of the factory target nucleic acid and the target sequence is measured, wherein the association frequency provides information for spatially mapping the biological sample.

3. The method of claim 1, wherein the amplification comprises rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branched amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER) or primer exchange reaction (PER).

4. The method of claim 1, wherein each node factory comprises a padlock probe, the padlock probe comprising the node nucleic acid and a node nucleic acid restriction site, the method further comprising: hybridizing the padlock probe to a nucleic acid splint, wherein the nucleic acid splint comprises regions complementary to regions at the 3' end and the 5' end of the padlock probe; applying a ligase to the sample, wherein the ligase ligates the 3' end and the 5' end of the padlock probe; applying a rolling circle polymerase to the attached padlock probe; as well as Concatemers containing the node nucleic acid and the node nucleic acid restriction site are generated by RCA. The method of claim 4 , wherein the nucleic acid splint comprises at least a portion of an mRNA sequence in the biological sample.

6. The method of claim 4, wherein the nucleic acid splint is conjugated to a surface, a protein, a small molecule, a nucleic acid, a lipid, or any combination thereof.

7. The method of claim 6, wherein the protein comprises a receptor, a ligand, an antibody or any functional fragment thereof.

8. The method of claim 6, wherein the biological sample further comprises a feature, wherein the feature in the biological sample comprises a cell structure, a receptor, a scaffold, a matrix, a nucleic acid, a tissue, or an organelle.

9. The method of claim 4, further comprising depositing a restriction enzyme or nicking enzyme onto the sample, wherein the restriction enzyme or nicking enzyme recognizes the node nucleic acid restriction site, thereby cleaving the concatemer to provide more than one node nucleic acid.

10. The method of claim 4, further comprising depositing a restriction enzyme or nicking enzyme onto the sample, wherein the concatemer forms a hairpin comprising the node nucleic acid restriction site.

11. The method of claim 10, wherein the restriction endonuclease performs double-stranded cleavage at the node nucleic acid restriction site.

12. The method of claim 10, wherein the nicking enzyme performs single-stranded cleavage at the node nucleic acid restriction site.

13. The method according to claim 4, further comprising: depositing an oligonucleotide onto the sample, wherein the oligonucleotide hybridizes to a region comprising the node nucleic acid restriction site to form a double-stranded restriction region, A double-strand specific restriction enzyme is deposited onto the sample, wherein the restriction enzyme recognizes the node nucleic acid restriction site, thereby cleaving the concatemer to provide more than one node nucleic acid.

14. The method of claim 1, wherein the more than one node nucleic acid is generated separately.

15. The method according to any one of claims 3 to 14, wherein the node factory further comprises one or more factory target nucleic acids, wherein each factory target nucleic acid comprises: target binding region; target barcode region; one or more flanking target binding hybridization regions; and Optionally, a target primer region; wherein each factory target nucleic acid is contiguous with a node nucleic acid, and wherein said amplifying generates concatemers comprising repeats of a node nucleic acid and a factory target nucleic acid, and wherein said cleavage generates more than one node nucleic acid and factory target nucleic acid; and The method further comprises: attaching the factory target nucleic acid to a target sequence; as well as The association frequency of the factory target nucleic acid and the target sequence is measured, wherein the association frequency provides information for spatially mapping the biological sample.

16. The method of claim 15, wherein the generated concatemers comprise alternating node nucleic acids, node nucleic acid restriction sites, factory target nucleic acids, and factory target nucleic acid restriction sites.

17. The method of claim 16, further comprising depositing a restriction enzyme or nicking enzyme on the sample, wherein the restriction enzyme or nicking enzyme recognizes the node nucleic acid restriction site and the factory target nucleic acid restriction site.

18. The method according to claim 17 further includes depositing a first restriction enzyme on the sample, wherein the first restriction enzyme recognizes the factory target nucleic acid restriction site but not the node nucleic acid restriction site, thereby generating more than one nucleic acid comprising the node nucleic acid and the factory target nucleic acid.

19. The method of claim 18, further comprising depositing a second restriction enzyme on the sample after depositing the first restriction enzyme, wherein the second restriction enzyme recognizes the node nucleic acid restriction site, thereby generating more than one node nucleic acid and factory target nucleic acid.

20. The method of claim 1, wherein the one or more flanking node barcode hybridization regions or the one or more flanking target binding hybridization regions flank the node barcode region or the target binding region.

21. The method of claim 1, wherein the providing comprises affinity reaction, conjugation, incorporation into a hydrogel, cross-linking, or photo-cross-linking.

22. The method of claim 21, wherein the template node nucleic acid is conjugated to an antibody, antibody fragment, protein, nanobody, small molecule, nucleic acid therapeutic, lipid, nanoparticle, lipid nanoparticle or other affinity reagent.

23. The method of claim 1, wherein the attachment comprises hybridization, extension, ligation, splinting, cross-linking synthesis, ligation and cross-linked interstrand crosslink (ICL) synthesis, cross-ICL and nick synthesis, paired copies, or any combination thereof.

24. The method of claim 1, wherein the spatial mapping comprises sequencing the concatemers.

25. The method of claim 25, wherein the sequencing comprises chain termination sequencing, nanopore sequencing, ligation sequencing, combinatorial probe anchor synthesis, synthesis sequencing, pyrosequencing, ion semiconductor or single molecule real-time sequencing.

26. The method of claim 1, wherein the node barcode comprises one or more fluorophore labels.

27. The method of claim 26, wherein the spatial mapping comprises imaging the concatemer.

28. The method of claim 27, wherein the one or more fluorophore labels are attached to the node barcode by synthesis or hybridization.

29. The method of claim 1, wherein the node positions comprise a pattern.

30. The method of claim 1, wherein the node locations are pre-selected.

31. The method of claim 1, wherein the biological sample comprises cells in a synthetic matrix.

32. The method of claim 1, further comprising imaging the biological sample.

33. The method of claim 1, wherein the biological sample comprises a population of cells, a tissue sample, or a protein.

34. The method of claim 1, wherein the node barcode region is about 3 to about 30 nucleotides.

35. The method of claim 1, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000, or more than 1000 nucleotides.

36. The method of claim 1, wherein the biological sample is placed on a microscope slide or a cover slip.

37. The method of claim 1, wherein the diffused node nucleic acid comprises a gradient of decreasing concentration with increasing distance from the node location.

38. The method of claim 1, wherein diffused node nucleic acids from different node locations do not overlap.

39. The method of claim 1, wherein the node nucleic acids from different node positions overlap.

40. The method of claim 39, wherein the node nucleic acids from two or more node positions are combined by hybridization, extension, ligation, splint ligation, crossover synthesis, ligation and crossover interstrand crosslink (ICL) synthesis, crossover ICL and nick synthesis, paired copies, or any combination thereof.

41. The method of claim 1, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof.

42. The method of claim 41, further comprising depositing a polymerase on the node nucleic acid.

43. The method of claim 42, wherein the polymerase is a DNA polymerase or an RNA polymerase.

44. The method of claim 43, wherein the DNA polymerase comprises Phi29, Pol I, Pol II, Pol III, Pol IV, Pol V, T4, Bsm, Bst, reverse transcriptase, or any combination thereof.

45. The method of claim 43, wherein the RNA polymerase comprises T7, T3, or SP6.

46. ​​The method of claim 42, further comprising depositing a nicking enzyme, wherein the node nucleic acid comprises a nicking site that is targetable by the nicking enzyme.

47. The method of claim 1, wherein the node nucleic acid further comprises a unique identifier sequence comprising one or more atypical bases, universal bases, other bases that a polymerase will randomly or incorrectly pair with, or any combination thereof.

48. The method of claim 1, wherein the node nucleic acid is double-stranded, is single-stranded, comprises a hairpin structure, or any combination thereof.

49. The method of claim 1, further comprising controlling a diffusion factor, wherein the diffusion factor comprises viscosity, time, temperature, the presence of a crowding agent, pH, an electric field, a physical characteristic, or any combination thereof.

50. The method of claim 49, wherein the viscosity is greater than 1 cP.

51. The method of claim 50, wherein the biological sample medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP.

52. The method of claim 1, wherein the depositing is at random locations throughout the biological sample.

53. The method of claim 1, wherein the depositing is at a specific target in the biological sample.

54. The method of claim 1 further comprises generating a second more than one node nucleic acid from the node nucleic acid.

55. A method according to claim 2, wherein the cleavage generates more than one nucleic acid, and the more than one nucleic acid together comprises the node nucleic acid and the factory nucleic acid.

56. A method for generating biological information, the method comprising: Depositing a template nucleic acid at a node location on a biological sample, wherein the template nucleic acid comprises: Node barcode region; and one or more flanking node barcode hybridization regions; amplifying the template nucleic acid to generate a node nucleic acid, wherein the node nucleic acid diffuses away from the node location over time; depositing a factory target nucleic acid onto the biological sample, wherein the factory target nucleic acid comprises: target binding region; and one or more flanking target binding hybridization regions; and At least one of the node nucleic acids is attached to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node nucleic acid and the factory target nucleic acid provides biological information for spatial mapping of the biological sample.

57. The method of claim 56, wherein the factory target nucleic acid further comprises a target recognition region.

58. The method of claim 56, wherein the one or more flanking node barcode hybridization regions or one or more flanking target binding hybridization regions flank the node barcode region or the target binding region.

59. The method of claim 56, wherein the providing comprises affinity reaction, conjugation, incorporation into a hydrogel, cross-linking, or photo-cross-linking.

60. The method of claim 59, wherein the template node nucleic acid is conjugated to an antibody, antibody fragment, protein, nanobody, small molecule, nucleic acid therapeutic, lipid, nanoparticle, lipid nanoparticle or other affinity reagent.

61. The method of claim 56, wherein the amplification comprises rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), exponential amplification reaction (EXPAR), enzymatic oligonucleotide synthesis via internal inosine modification and exonuclease V, nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branched amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER), or primer exchange reaction (PER).

62. The method of claim 56, wherein the attachment comprises hybridization, extension, ligation, splinting, cross-linking synthesis, ligation and cross-linked interstrand crosslink (ICL) synthesis, cross-ICL and nick synthesis, paired copies, or any combination thereof.

63. The method of claim 56, wherein the spatial mapping comprises sequencing the concatemers.

64. The method of claim 63, wherein the sequencing comprises chain termination sequencing, nanopore sequencing, ligation sequencing, combinatorial probe anchor synthesis, sequencing by synthesis, pyrosequencing, ion semiconductor, or single molecule real-time sequencing.

65. The method of claim 56, wherein the node barcode comprises one or more fluorophore labels.

66. The method of claim 65, wherein the spatial mapping comprises imaging the concatemer.

67. The method of claim 65, wherein the one or more fluorophore labels are attached to the node barcode by synthesis or hybridization.

68. The method of claim 56, wherein the node positions include a pattern.

69. The method of claim 56, wherein the node locations are preselected.

70. The method of claim 56, wherein the biological sample comprises cells in a synthetic matrix.

71. The method of claim 56, further comprising imaging the biological sample.

72. The method of claim 56, wherein the biological sample comprises a population of cells, a tissue sample, or a protein.

73. The method of claim 56, wherein the node barcode region is about 3 to about 30 nucleotides.

74. The method of claim 56, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000, or more than 1000 nucleotides.

75. The method of claim 56, wherein the biological sample is placed on a microscope slide or a coverslip.

76. The method of claim 56, wherein the diffused node nucleic acid comprises a gradient of decreasing concentration with increasing distance from the node location.

77. A method according to claim 56, wherein diffused node nucleic acids from different node locations do not overlap.

78. The method of claim 56, wherein the node nucleic acids from different node positions overlap.

79. A method according to claim 78, wherein the node nucleic acids from two or more node positions are combined by hybridization, extension, ligation, splint ligation, cross-linking synthesis, ligation and cross-link interchain crosslink (ICL) synthesis, cross-ICL and nick synthesis, paired copies or any combination thereof.

80. The method of claim 56, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof.

81. The method of claim 80, further comprising depositing a polymerase on the node nucleic acid.

82. The method of claim 81, wherein the polymerase is a DNA polymerase or an RNA polymerase.

83. The method of claim 82, wherein the DNA polymerase comprises Phi29, Pol I, Pol II, PolIII, Pol IV, Pol V, T4, Bsm, Bst, reverse transcriptase, or any combination thereof.

84. The method of claim 82, wherein the RNA polymerase comprises T7, T3, or SP6.

85. The method of claim 81 further comprising depositing a nicking enzyme, wherein the node nucleic acid comprises a nicking site that can be targeted by the nicking enzyme.

86. The method of claim 56, wherein the node nucleic acid further comprises a unique identifier sequence comprising one or more atypical bases, universal bases, other bases that a polymerase will randomly or incorrectly pair with, or any combination thereof.

87. The method of claim 56, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof.

88. The method of claim 56, wherein the diffusion factor comprises viscosity, time, temperature, the presence of a crowding agent, pH, an electric field, a physical characteristic, or any combination thereof.

89. The method of claim 88, wherein the viscosity is greater than 1 cP.

90. The method of claim 89, wherein the biological sample medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP.

91. The method of claim 56, wherein the depositing is at random locations throughout the biological sample.

92. The method of claim 56, wherein the deposition is at a specific target in the biological sample.

93. The method of claim 56, further comprising generating a second more than one node nucleic acid from the node nucleic acid.

94. The method of claim 56, further comprising: depositing a DNA barcode onto the biological sample, wherein the DNA barcode comprises a region complementary to a region of the concatemer, and wherein the DNA barcode comprises a photoreactive nucleobase capable of cross-linking to another nucleobase; selectively irradiating the biological sample to form covalently linked DNA barcode-concatemer complexes; washing the biological sample to remove DNA barcodes that are not complexed with concatemers; as well as The steps of deposition, selective irradiation and washing are repeated, wherein: The DNA barcode in the first deposition step comprises a different sequence than the DNA barcode in the second deposition step, and For the first selective irradiation step and the second selective irradiation step, the selective irradiation occurs at different locations.

95. The method of claim 94, further comprising synthesizing a continuous DNA strand, wherein the synthesizing comprises contacting the DNA barcode-concatemer complex with a polymerase.

96. The method of claim 95, wherein the synthesizing comprises cross-linking synthesis.

97. The method of claim 95, further comprising replacing the continuous DNA strand from the biological sample.

98. The method of claim 97, wherein the synthesizing and replacing occur simultaneously.

99. The method of claim 97, wherein the displaced continuous DNA strands are pooled.

100. The method of claim 99, wherein the pooled continuous DNA strands are amplified.

101. The method of claim 100, wherein the pooled continuous DNA strands are sequenced.

102. The method of any one of claims 94 to 101, wherein the DNA barcode further comprises a promoter region, a primer region, or any combination thereof.

103. The method of claim 102, wherein the primers are one or more random primers, or wherein the primers are non-random primers.

104. The method of any one of claims 94 to 103, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK).

105. The method of any one of claims 94 to 104, wherein the selective irradiation is performed using a photomask.

106. The method of claim 105, wherein the photomask is different in different selective irradiation steps.

107. The method of claim 105, wherein the photomask is generated manually.

108. The method of claim 105, wherein the photomask is generated by a machine.

109. The method of any one of claims 943 to 108, wherein the selectively irradiating step comprises applying radiation at a wavelength of about 300 nm up to 450 nm.

110. The method of claim 109, wherein the selectively irradiating step comprises administering radiation at a wavelength of about 350 nm up to 420 nm.

111. The method of claim 110, wherein the selectively irradiating step comprises administering radiation at a wavelength of about 365 nm up to 405 nm.

112. The method of any one of claims 94 to 111, wherein the selectively irradiating step comprises applying radiation to a preselected region of interest (ROI).

113. The method of claim 112, wherein the ROI is cell-specific or subcellular-specific.

114. The method of claim 112, wherein the ROI is based on morphology.

115. The method of any one of claims 56 to 114, wherein the depositing is performed manually, automatically, by robot, or any combination thereof.

116. The method of claim 114, wherein the depositing is performed using a modified inkjet printer.

117. The method of any one of claims 56 to 116, wherein the depositing or washing is performed using microfluidics, passive diffusion, electrophoresis, digital microfluidics, an acoustic liquid processor, an inkjet printer, an automated liquid processor, or any combination thereof.

118. The method of any one of claims 56 to 117, wherein one or more DNA barcodes are combined with one or more node barcode strands, wherein the combination is performed by hybridization, extension, ligation, splinting, crossover synthesis, ligation and crossover interstrand crosslink (ICL) synthesis, crossover ICL and nick synthesis, paired copying, or any combination thereof.

119. A method for storing information, the method comprising: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a template nucleic acid at a node location on a surface, wherein the template nucleic acid comprises: node barcode regions, each corresponding to one of the more than one barcodes; and one or more flanking node barcode hybridization regions; amplifying the template nucleic acid to generate a node nucleic acid; depositing a factory target nucleic acid onto the surface, wherein the factory target nucleic acid comprises: target binding region; one or more flanking target binding hybridization regions; Optionally, a target primer region; and At least one of the node nucleic acids is attached to at least one of the factory target nucleic acids to form a concatemer, wherein the combination of the node nucleic acid and the factory target nucleic acid provides storage information.

120. The method of claim 119, wherein the amplification comprises rolling circle amplification (RCA), strand displacement amplification (SDA), nickase amplification reaction (NEAR), nick translation, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), ligase chain reaction (LCR), recombinase polymerase amplification (RPA), branched amplification method (RAM), nucleic acid sequence-based amplification (NASBA), signal amplification by exchange reaction (SABER) or primer exchange reaction (PER).

121. The method of claim 119, wherein the attachment comprises synthesis by hybridization, extension, ligation, splinting, cross-linking, ligation and cross-linked inter-strand crosslink (ICL) synthesis, cross-ICL and nick synthesis, paired copies, or any combination thereof.

122. The method of claim 119, further comprising converting the nucleic acid information into digital information, wherein the converting comprises sequencing the concatemers.

123. The method of claim 122, wherein the sequencing is chain termination sequencing, nanopore sequencing, ligation sequencing, combinatorial probe anchor synthesis, synthesis sequencing, pyrosequencing, ion semiconductor, or single molecule real-time sequencing.

124. The method of claim 119, wherein the concatemer comprises one or more fluorophore labels.

125. The method of claim 124, wherein the spatial mapping comprises imaging the multimer.

126. The method of claim 124, wherein the one or more fluorophore labels are attached to the concatemer by synthesis or hybridization.

127. The method of claim 119, wherein the node positions comprise a pattern.

128. The method of claim 119, wherein the node locations are preselected.

129. The method of claim 119, wherein the node nucleic acid is about 20 to about 50, about 50 to about 100, about 100 to about 200, about 200 to about 500, about 500 to about 1000, or more than 1000 nucleotides.

130. A method according to claim 119, wherein the diffused node nucleic acid includes a gradient in which the concentration decreases with increasing distance from the node location.

131. A method according to claim 119, wherein the node nucleic acids from different node positions do not overlap.

132. A method according to claim 119, wherein the node nucleic acids from different node positions overlap.

133. A method according to claim 132, wherein the node nucleic acids from two or more node positions are combined by hybridization, extension, ligation, splint ligation, cross-linking synthesis, ligation and cross-link interchain crosslink (ICL) synthesis, cross-ICL and nick synthesis, paired copies or any combination thereof.

134. The method of claim 119, wherein the node nucleic acid further comprises a promoter region, a primer region, or any combination thereof.

135. The method of claim 134, further comprising depositing a polymerase on the node nucleic acid.

136. The method of claim 135, wherein the polymerase is a DNA polymerase or an RNA polymerase.

137. The method of claim 119, wherein the node nucleic acid further comprises a unique identifier sequence comprising one or more atypical bases, universal bases, other bases that a polymerase will randomly or incorrectly pair with, or any combination thereof.

138. The method of claim 119, wherein the node nucleic acid is double-stranded, single-stranded, comprises a hairpin structure, or any combination thereof.

139. The method of claim 119, wherein the diffusion factor comprises viscosity, time, temperature, the presence of a crowding agent, pH, an electric field, a physical characteristic, or any combination thereof.

140. The method of claim 139, wherein the viscosity is greater than 1 cP.

141. A method according to claim 140, wherein the medium comprises a viscosity of about 1 to about 10 cP, about 10 to about 20 cP, about 20 to about 30 cP, about 30 to about 40 cP, about 40 to about 50 cP, about 50 to about 60 cP, about 60 to about 70 cP, about 70 to about 80 cP, about 80 to about 90 cP, about 100 to about 150 cP, about 150 to about 300 cP, about 300 to about 500 cP, about 500 to about 1000 cP, about 1000 to about 2000 cP, about 2000 to about 3000 cP.

142. A method for information storage, the method comprising: receiving digital information; converting the digital information into nucleic acid information in the form of more than one nucleic acid barcode; depositing a first oligonucleotide onto a surface, wherein the first oligonucleotide comprises: Optionally, a surface binding region; a first barcode region corresponding to a barcode of the more than one barcodes; and one or more flanking first oligonucleotide hybridization regions, wherein the surface binding region and the one or more flanking hybridization regions optionally comprise photoreactive nucleobases; optionally selectively irradiating the surface to form covalently attached oligonucleotides; depositing a second oligonucleotide on the surface, wherein the second oligonucleotide comprises: a second barcode region corresponding to a barcode of the more than one barcodes; and one or more flanking second oligonucleotide hybridization regions, wherein the one or more flanking second oligonucleotide hybridization regions comprise a photoreactive nucleobase, and wherein at least one of the one or more flanking first oligonucleotide hybridization regions is complementary to at least one of the one or more flanking second oligonucleotide hybridization regions; and selectively irradiating the surface to covalently attach the second oligonucleotide to the first oligonucleotide; and The second depositing step and irradiating step are optionally repeated one or more times to form concatemers.

143. The method of claim 142, wherein the surface comprises oligonucleotides comprising a photocleavable nucleobase and a region complementary to at least one of the one or more flanking first oligonucleotide hybridization regions.

144. The method of claim 142, wherein the surface comprises reactive groups capable of binding to nucleosides.

145. The method of claim 142, wherein the irradiating comprises applying light at a wavelength of about 365 nm to about 405 nm.

146. The method of claim 142, wherein the photoreactive nucleobase is 3-cyanovinylcarbazole phosphoramidite (CNVK).

147. The method of claim 142, wherein the selectively irradiating comprises applying radiation to a preselected region of interest (ROI).

148. The method of claim 147, wherein the ROI is described by a photomask.

149. The method of claim 148, wherein the photomask is different in each selective irradiation step.

150. The method of claim 148, wherein the photomask is generated manually.

151. The method of claim 148, wherein the photomask is generated by a machine.

152. The method of claim 151, wherein the selectively irradiating step comprises administering radiation at a wavelength of about 300 nm up to 450 nm.

153. The method of claim 151, wherein the selectively irradiating step comprises administering radiation at a wavelength of about 365 nm up to 405 nm.

154. The method of claim 151 further comprising washing after each selective irradiation step to remove unbound oligonucleotides.

155. The method of claim 151, wherein the concatemer further comprises a primer region, a promoter region, or any combination thereof.

156. The method of claim 155, further comprising depositing a polymerase on the concatemer.

157. The method of claim 156, wherein the concatemer continuous complementary nucleic acid chains are generated by cross-linking, hybridization, extension, ligation, splinting, ligation and cross-link interstrand cross-link (ICL) synthesis, cross-ICL and nick synthesis, paired copying, or any combination thereof.

158. A method according to claim 157, wherein the continuous complementary nucleic acid chain is amplified and sequenced to generate sequencing data.

159. A method according to claim 158, wherein the sequencing data allows translation of the nucleic acid information into the digital information.

160. The method of claim 142, wherein the concatemer is contacted with a fluorophore-labeled sequence complementary to a barcode region of the oligonucleotide to generate a fluorophore-concatemer complex.

161. The method of claim 160, wherein the fluorophore-concatemer complex is imaged.

162. The method of claim 161, wherein the imaging comprises applying light at a wavelength of about 365 nm to about 405 nm.

163. The method of claim 162, wherein the imaging allows translation of the nucleic acid information into the digital information.

164. A method for generating biological information, the method comprising: Depositing a node factory comprising a matrix at a node location on a biological sample, wherein each node factory comprises more than one node nucleic acid, and wherein each node nucleic acid comprises: Node barcode region; one or more flanking node barcode hybridization regions; Optionally, a node primer region, target binding region; target barcode region; one or more flanking target binding hybridization regions; and Optionally, the target primer region, At least one of the node nucleic acids is released from the node factory, wherein the release provides biological information for spatially mapping the biological sample.

165. A method according to claim 164, wherein the node factory comprises more than one node nucleic acid reversibly fixed to a carrier.

166. The method of claim 165, wherein the carrier comprises a liposome, a droplet, a bead, a LNP, an AAV, an exosome, a lentivirus, or a protein.

167. The method of claim 166, wherein the carrier comprises beads.

168. The method of claim 165, further comprising releasing the node nucleic acid from the vector.

169. The method of claim 164, wherein each node nucleic acid further comprises a restriction site, and wherein the more than one node nucleic acid is provided as a concatemer.

170. The method of claim 164, further comprising depositing a restriction enzyme on the sample, wherein the restriction enzyme recognizes the restriction site, thereby cleaving the concatemer to provide more than one node nucleic acid.

171. The method of claim 164, further comprising depositing an oligonucleotide on the sample, wherein the oligonucleotide hybridizes to a region comprising a restriction site on the node nucleic acid, and wherein the restriction enzyme recognizes a double-stranded cleavage site on the sample, thereby cleaving the concatemer to provide more than one node nucleic acid.

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