Method for constructing a spatially barcoded surface

Abstract

The systems, devices, and methods described herein relate to methods for generating spatially barcoded surfaces using combinatorial synthesis techniques, and their use for analyzing the molecules (particularly, nucleic acid molecules) of biological cells disposed on such surfaces. The methods described herein can further be used to create spatially barcoded nucleic acid molecules. The spatial barcode can be a combination of at least three segments: two segments that identify the position of the array on the surface and a third segment that identifies the position of the barcode oligonucleotide or nucleic acid molecule to which it is attached within the array.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 344,380, filed May 20, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Molecular tagging has a long history in analytical biochemistry and molecular biology. For example, Church U.S. Patent No. 4,942,124; Spitzer et al., Cell, 165(4): 780-791 (2016); Giese, Trends in Analytical Chemistry, 2(7): 166-168 (1983); Hardenbol et al., Nature Biotechnology, 21: 673-678 (2003); Brenner et al., U.S. Patent No. 7,537,897; Fan et al., Science, 347 (6222): 1258367-1 (2015); Macevicz, U.S. Patent Publication US2005 / 0250147; Morris et al., European Patent Publication 0799897A1; Wallace, U.S. Patent No. 5,981,179, etc. In recent years, such techniques have been extended to include the use of spatially distributed oligonucleotide barcodes to identify and study spatial variations in biological processes (e.g., tissue-wide gene expression). For example, Stahl et al., Science, 353(6294): 78-82 (2016); Salmen et al., Nature Protocols, 13: 2501-2534 (2018); Frisen et al., U.S. Patent No. 9,593,365, etc. However, the cost-effective synthesis of spatially barcoded sequences with known sequences, the control of spatial barcode distribution, and the density for resolving cellular and subcellular processes remain difficult. This difficulty has been addressed by a very large number of different approaches, but has only been partially successful.For example, Horgan et al., International Patent Publication WO / 2022 / 013094; Liu et al., Cell, 183: 1665-1681 (2020); Cho et al., bioRxiv (https: / / doi.org / 10.1101 / 2021.01.25.427004); Chen et al. (https: / / doi.org / 10.1101 / 2021.01.17.427807); Delly et al., Scientific Reports, 11: 10857 (2021); Rodriques et al., Science, 363(6434): 1463-1467 (2019), etc. The field of spatial barcode construction has advanced through the availability of cost-effective methods for spatial barcoding.

Prior Art Documents

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Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0005] Abstract The systems, devices, and methods described herein relate to the generation of spatially barcoded surfaces and nucleic acid molecules using combinatorial techniques. In some embodiments, a method of generating a spatially barcoded surface comprises: (a) providing a solid support comprising a surface; (b) synthesizing a plurality of arrays of first oligonucleotides, each array comprising a plurality of discrete reaction sites and each first oligonucleotide coupled to the surface by its 5' end, wherein (i) each first oligonucleotide has a barcode segment comprising a barcode sequence different from those of other first oligonucleotides, (ii) first oligonucleotides having different barcode sequences are coupled at different reaction sites, (iii) the plurality of arrays are arranged in orthogonal rows and columns, and (iv) each first oligonucleotide occupies a reaction site at a known surface position; (c) partitioning the surface into rows by sealingly coupling to the surface a first channel template comprising a plurality of channels; and (d) reacting a second oligonucleotide loaded into each channel of the first channel template with the surface or a previously coupled surface oligonucleotide to form a coupled surface oligonucleotide or a spatially barcoded oligonucleotide, wherein each different channel comprises a second oligonucleotide comprising a second barcode segment comprising a barcode sequence different from those of the second oligonucleotides of the different channels; wherein (A) the rows of the plurality of arrays at least partially coincide with the rows of the first channel template, and (B) each spatially barcoded oligonucleotide uniquely identifies the spatial position of its reaction site on the surface. In some embodiments, in step (d), the second oligonucleotide is reacted directly with the surface, while in other embodiments, in step (d), the second oligonucleotide is reacted with an oligonucleotide already coupled to the surface.In the latter embodiment, the oligonucleotide already attached to the surface is referred to as the "surface-attached oligonucleotide", which can be a continuation of the first, second or third oligonucleotide from the previous step. That is, the surface-attached oligonucleotide is a partially completed oligonucleotide precursor to the spatial barcode oligonucleotide. In some embodiments, step (d) can be the final attachment of the barcode segment (which can be the first, second or third oligonucleotide depending on the embodiment), resulting in the final desired "spatial barcode oligonucleotide".

[0006] In some embodiments, a method of creating a spatially barcoded surface comprises: (a) providing a surface; (b) synthesizing a plurality of arrays of first oligonucleotides, each array comprising a plurality of discrete reaction sites and each first oligonucleotide attached to the surface by its 5' end, wherein (i) each first oligonucleotide has a barcode segment that includes a barcode sequence different from those of the other first oligonucleotides, (ii) first oligonucleotides having different barcode sequences are attached to different reaction sites, (iii) the plurality of arrays are arranged in orthogonal rows and columns, and (iv) each first oligonucleotide occupies a reaction site at a known surface position; (c) partitioning the surface into rows by sealingly attaching to the surface a first channel template that includes a plurality of channels; (d) reacting a second oligonucleotide loaded into each channel of the first channel template with the surface or a preceding attached surface oligonucleotide to form an attached surface oligonucleotide or a spatially barcoded oligonucleotide, wherein each different channel includes a second oligonucleotide that includes a second barcode segment that includes a barcode sequence different from those of the second oligonucleotides of the different channels; (e) partitioning the surface into columns by sealingly attaching to the surface a second channel template that includes a plurality of channels; (f) reacting a third oligonucleotide loaded into each channel of the second channel template with the surface or a preceding attached surface oligonucleotide to form an attached surface oligonucleotide or a spatially barcoded oligonucleotide, wherein each different channel of the second channel template includes a third oligonucleotide that includes a third barcode segment that includes a barcode sequence different from those of the third oligonucleotides of the different channels of the second channel template;Here, (A) the rows of the plurality of arrays at least partially coincide with the rows of the first channel template, and the columns of the plurality of arrays at least partially coincide with the columns of the second channel template, and (B) each spatial barcode oligonucleotide uniquely identifies the spatial position of its reaction site on the surface. As noted above, the term "binding surface oligonucleotide" refers to a partially completed oligonucleotide precursor to a spatial barcode oligonucleotide.;

[0007] In some embodiments, a method for creating spatially barcoded nucleic acid molecules includes: (a) providing a solid support including a surface; (b) capturing a nucleic acid molecule on the surface and transcribing the captured nucleic acid molecule into complementary DNA (cDNA) bound to the surface; (c) synthesizing a plurality of arrays of a first oligonucleotide at different reaction sites on the surface, where (i) the plurality of arrays are arranged in orthogonal rows and columns; (ii) each array includes a plurality of reaction sites, each of which, when located at a different reaction site, always includes a first oligonucleotide including a first barcode segment having a different barcode sequence; (iii) each first oligonucleotide occupies a reaction site at a known surface position; (d) reacting a second oligonucleotide with the surface or an oligonucleotide or cDNA thereon by binding a first channel template including a plurality of channels to the surface in a sealed manner, where each different channel includes a second oligonucleotide including a second barcode segment having a barcode sequence different from those of the second oligonucleotides of the different channels; and (e) reacting a third oligonucleotide with the surface or an oligonucleotide or cDNA thereon by binding a second channel template including a plurality of channels to the surface in a sealed manner, where each different channel of the second channel template includes a third oligonucleotide including a third barcode segment having a barcode sequence different from those of the third oligonucleotides of the different channels; where the channels of the first and second channel templates are aligned with the orthogonal rows and columns of the array, and where the first, second, and third oligonucleotides bound to the cDNA form a spatial barcode that identifies the spatial position of the cDNA on the upper surface.

[0008] In one aspect, a method for generating a spatially barcoded surface, the method comprising: (a) providing a solid support comprising the surface, wherein the surface comprises a plurality of arrays arranged on the surface, wherein each array comprises a plurality of reaction sites, and wherein each reaction site comprises a reaction site oligonucleotide having a barcode sequence specific to the reaction site located within the reaction site; (b) partitioning the surface into one or more channels by coupling a channel template comprising a plurality of channels to the surface; and (c) loading a plurality of channel oligonucleotides into the plurality of channels such that at least one channel oligonucleotide couples to the reaction site oligonucleotide in each array, wherein each channel oligonucleotide comprises a barcode sequence specific to the channel located within the channel.

[0009] In some cases, the method further comprises: (a) partitioning the surface into one or more orthogonal channels by coupling a further channel template comprising a plurality of orthogonal channels to the surface, wherein the one or more orthogonal channels are orthogonal to the one or more channels; and (c) loading a plurality of orthogonal channel oligonucleotides into the plurality of orthogonal channels such that at least one orthogonal channel oligonucleotide couples to the channel oligonucleotide in each array, wherein each orthogonal channel oligonucleotide comprises a barcode sequence specific to the orthogonal channel located within the orthogonal channel.

[0010] In some cases, the plurality of arrays are arranged in rows and columns. In some cases, the one or more channels at least partially coincide with the rows, and the one or more orthogonal channels at least partially coincide with the columns. In some cases, the one or more channels at least partially coincide with the columns, and the one or more orthogonal channels at least partially coincide with the rows.

[0011] In some cases, the step of coupling in (b) includes the step of coupling a channel template including the plurality of channels to the surface so as to seal it. In some cases, the step of coupling in (a) includes the step of coupling the further channel template including the plurality of orthogonal channels to the surface so as to seal it.

[0012] In some cases, each of the plurality of arrays is the same. In some cases, one of the plurality of arrays constitutes a pitch between the reaction sites in the range of 50 to 500 μm, and the reaction sites each have a diameter in the range of 30 to 300 μm. In some cases, one of the plurality of arrays constitutes a density of reaction sites in the range of 50 to 200 reaction sites / mm 2 of the range.

[0013] In some cases, the step of coupling the at least one channel oligonucleotide to the reaction site oligonucleotide of each array in (c) includes the step of extending the at least one channel oligonucleotide using DNA polymerase. In some cases, the step of coupling the at least one channel oligonucleotide to the reaction site oligonucleotide of each array in (c) includes the step of ligating the at least one channel oligonucleotide to the reaction site oligonucleotide in each array.

[0014] In some cases, the surface further comprises capture probes attached thereto, where the method further comprises capturing the sample nucleic acid with the capture probes and extending the capture probes using the captured sample nucleic acid as a template. In some cases, any of the reaction site oligonucleotides, channel oligonucleotides, or orthogonal channel oligonucleotides comprises the capture probe.

[0015] In another aspect, a method of creating a spatially barcoded surface, the method comprising: (a) providing a solid support comprising a surface, where the surface comprises a plurality of arrays, where one of the plurality of arrays comprises at least two reaction sites, where a first reaction site of the at least two reaction sites comprises a first reaction site oligonucleotide comprising a first reaction site barcode sequence, and a second reaction site of the at least two reaction sites comprises a second reaction site oligonucleotide comprising a second reaction site barcode sequence, where the first barcode sequence is different from the second barcode sequence; (b) coupling a channel template comprising a first channel and a second channel to the surface; (c) loading a first channel oligonucleotide into the first channel; and (d) loading a second channel oligonucleotide into the second channel, where subsequent to the loading step of (c), the first channel oligonucleotide couples to the first reaction site oligonucleotide, and subsequent to the loading step of (d), the second channel oligonucleotide couples to the second reaction site oligonucleotide, where the first channel oligonucleotide comprises a third barcode sequence and the second channel oligonucleotide comprises a fourth barcode sequence, where the third barcode sequence is different from the fourth barcode sequence. A method is provided herein that includes these steps.

[0016] In some cases, the method includes: (a) coupling an orthogonal channel template including a first orthogonal channel and a second orthogonal channel to the surface; (b) loading a first orthogonal channel oligonucleotide into the first channel; and (c) loading a second orthogonal channel oligonucleotide into the second channel, wherein following the loading step of (b), the first orthogonal channel oligonucleotide couples to the first channel oligonucleotide, and following the loading step of (c), the second orthogonal channel oligonucleotide couples to the second channel oligonucleotide, and wherein the first orthogonal channel oligonucleotide includes a fifth barcode sequence, the second orthogonal channel oligonucleotide includes a sixth barcode sequence, and wherein the fifth barcode sequence is different from the sixth barcode sequence.

[0017] In some cases, the plurality of arrays are arranged in rows and columns. In some cases, the first channel and the second channel at least partially coincide with the row, and the first orthogonal channel and the second orthogonal channel at least partially coincide with the column. In some cases, the first channel and the second channel at least partially coincide with the column, and the first orthogonal channel and the second orthogonal channel at least partially coincide with the row.

[0018] In some cases, the coupling step in (b) includes coupling the channel template including the first channel and the second channel to the surface so as to seal it. In some cases, the coupling step in (a) includes coupling the orthogonal channel template including the first orthogonal channel and the second orthogonal channel to the surface so as to seal it.

[0019] In some cases, each of the plurality of arrays is the same. In some cases, one of the plurality of arrays constitutes a pitch between the reaction sites in the range of 50 to 500 μm, and the reaction sites each have a diameter in the range of 30 to 300 μm. In some cases, one of the plurality of arrays constitutes a density of reaction sites in the range of 50 to 200 reaction sites / mm 2 of the reaction sites.

[0020] In some cases, the step of coupling the first channel oligonucleotide to the first reaction site oligonucleotide includes the step of extending the first channel oligonucleotide using DNA polymerase. In some cases, the step of coupling the first channel oligonucleotide to the first reaction site oligonucleotide includes the step of ligating the first channel oligonucleotide to the first reaction site oligonucleotide. In some cases, the step of coupling the first orthogonal channel oligonucleotide to the first channel oligonucleotide includes the step of extending the first channel oligonucleotide using DNA polymerase. In some cases, the step of coupling the first orthogonal channel oligonucleotide to the first channel oligonucleotide includes the step of ligating the first orthogonal channel oligonucleotide to the first channel oligonucleotide.

[0021] In some cases, the surface further includes a capture probe bound thereto, and the method further includes the step of capturing the sample nucleic acid with the capture probe and the step of extending the capture probe using the captured sample nucleic acid as a template. In some cases, any one of the first or second reaction site oligonucleotides, the first or second channel oligonucleotides, or the first or second orthogonal channel oligonucleotides includes the capture probe.

[0022] In another aspect, a flow cell comprising one or more arrays, wherein one of the one or more arrays is located at the intersection of rows and columns on the surface of the flow cell, and wherein the array comprises one or more reaction sites, and one of the one or more reaction sites comprises: (a) a first oligonucleotide sequence specific to the spatial position of the reaction site within the array; (b) a second oligonucleotide specific to the row; and (c) a third oligonucleotide specific to the column, is described herein. 59. In some embodiments, the array, the row, the column, or any combination thereof is configured to receive the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, or any combination thereof. In some embodiments, the array constitutes a pitch between the reaction site and a second reaction site in the range of 50 - 500 μm, and the reaction site constitutes a diameter in the range of 30 - 300 μm. In some embodiments, the one or more reaction sites constitute a density in the range of 50 - 200 reaction sites / mm 2 2. In some embodiments, the one or more reaction sites comprise one or more capture probes. In some embodiments, the flow cell further comprises a second array, wherein the second array is located at a second intersection of a second row and a second column, and the second array comprises one or more second reaction sites, and one of the one or more second reaction sites comprises: a fourth oligonucleotide sequence specific to the spatial position of the second reaction site within the second array; a fifth oligonucleotide specific to the second row; and a sixth oligonucleotide specific to the second column. In some embodiments, the spatial position of the second reaction site within the second array corresponds to the spatial position of the reaction site within the array of claim 58, and the first oligonucleotide sequence and the fourth oligonucleotide sequence are the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0023]

Figure 1A

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Figure 1B

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Figure 2A

Figure 2B

Figure 2C

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Figure 3

BEST MODE FOR CARRYING OUT THE INVENTION

[0027] DETAILED DESCRIPTION The practice of the systems and methods described herein may, unless otherwise indicated, use conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such conventional techniques include, but are not limited to, the preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. Specific illustrations of appropriate techniques can be made by reference to the examples below in this specification. However, other equivalent conventional procedures may of course also be used. Such conventional techniques and descriptions can be found in standard experimental manuals such as: Genome Analysis: A Laboratory Manual Series (Volumes I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Renault and Duchateau, eds., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, eds., Protein Engineering Handbook (Wiley-VCH, 2009), etc. Guidance for selecting materials and components for performing specific functions can be found in available papers and references regarding scientific instruments, including but not limited to: Moore et al., Building Scientific Apparatus, 3rd Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013); and similar references.

[0028] The systems and methods described herein relate to creating or generating spatially barcoded surfaces and their use for analyzing molecules of biological cells, particularly nucleic acid molecules, disposed on such surfaces. The systems and methods described herein also relate to spatially barcoding nucleic acid molecules disposed or captured on a surface. The spatial barcode can be combinatorial in the sense that each barcode is a combination of at least three segments: two segments that identify the position of the array on the surface and a third segment that identifies the position of the barcode oligonucleotide or nucleic acid molecule to which it is attached within the array. Further, in some embodiments, the final library of spatial barcodes includes every combination of possible sequences of the first, second, and third barcode segments. Thus, in embodiments using three barcode segments, the number of first oligonucleotides (each including a first barcode segment) in the array, the number of channels for delivering second oligonucleotides (each including a second barcode segment), and the number of channels for delivering third oligonucleotides (each including a third barcode segment) determine the total number of different barcodes on the surface. For example, for an array of 384 first oligonucleotides, with a 50-channel first channel template and a 50-channel second channel template, the surface can have 980,000 (=50×50×384) different barcodes. Channel templates and gaskets for binding the templates to the surface in a sealed manner can be made using fabrication techniques used for microfluidic devices.

[0029] A wide variety of surfaces can be used with the systems and methods described herein. In some embodiments, the surface is a two-dimensional flat surface of a solid support material. Such a solid support material can include a non-porous solid that can be functionalized with conventional functional groups to which oligonucleotides can be attached (e.g., Devor et al., Integrated DNA Technologies (2005), etc.). In some embodiments, such a solid support material can include glass, plastic, silicon, metal oxide, etc. In some embodiments, the surface is a glass support material (e.g., a glass slide).

[0030] In various embodiments of the systems and methods described herein, barcode segments can be attached before and / or after the capture and replication of nucleic acid molecules from a sample. In other words, the order in which the barcode segments and the sample nucleic acid are assembled on the surface can vary such that the order of the cDNA (transcribed from the captured nucleic acid) and the barcode segments that make up the spatial barcode can be selected. In different embodiments, such an ordering (from the above surface) can be as follows: - cDNA - BC1 - BC2 - BC3; BC1 - cDNA - BC2 - BC3; BC1 - BC2 - cDNA - BC3; or BC1 - BC2 - BC3 - cDNA (where BC1, BC2, and BC3 represent the first, second, and third oligonucleotides (including the first, second, and third barcode segments), respectively). The assembly of the first, second, and third oligonucleotides to generate a barcoded surface, or the cDNA to generate a surface with spatially barcoded cDNA, as well as the assembly of the first, second, and third oligonucleotides, is achieved using conventional methods for ligating nucleic acid molecules to each other or to the surface as illustrated for the embodiments described in FIGS. 1A and 1B.

[0031] Embodiments are disclosed that illustrate the formation of spatial barcodes that include two or three barcode segments, although the systems and methods described herein may also include combinatorial spatial barcodes of multiple barcode segments. In some embodiments, the combinatorial spatial barcode includes from 3 to 6 barcode segments; or from 3 to 5 segments; or from 3 to 4 segments. In some embodiments, combinatorial spatial barcodes having more than three barcode segments can be generated by applying additional steps of partitioning (or reusing) and reacting using a channel template loaded with oligonucleotides that include different combinations of barcode arrays.

[0032] In some embodiments, one array of the first oligonucleotide arrays is synthesized (or disposed) on a surface, for example, as shown in FIG. 2A, and then the second and third oligonucleotides are joined by forming orthogonal channels to deliver the oligonucleotides (e.g., as shown in FIGS. 2A-2B). In some embodiments, the surface (202) (see FIG. 2A) may not contain capture oligonucleotides, such that the interstitial space (203) between the arrays (and between the spots or reaction sites within the array) is barcode-free. In other embodiments, the surface (202) may be coated with capture oligonucleotides to capture various barcode oligonucleotides (first, second, or third), followed by either extension or ligation, which can form combinatorial barcodes. In other words, in some embodiments, the surface functional groups can constitute capture oligonucleotides. In such latter embodiments, a barcoded surface can be generated, where the interstitial space (e.g., 203) in one of the plurality of arrays contains one or more segments of barcodes. In some embodiments, the order of channel delivery as well as droplet delivery of the first, second, and third oligonucleotides can be different. In some embodiments, one array of the first oligonucleotide arrays is delivered by droplet, followed by channel delivery of the second and third oligonucleotides. In other embodiments, the first oligonucleotide is delivered by channel, one array of the second oligonucleotide arrays is delivered by droplet, and the third oligonucleotide is delivered by channel. In yet other embodiments, the first oligonucleotide is delivered by channel, the second oligonucleotide is delivered by channel, and one array of the third oligonucleotide arrays is delivered by droplet.

[0033] Figure 1A illustrates one embodiment in which three barcode segments are sequentially linked to form a spatial barcode for the surface, after which sample nucleic acid can be captured (i.e., the above-described fourth format: BC1-BC2-BC3-sample NA). In one embodiment, the first oligonucleotide (102) comprising the first barcode segment (BC1) (106) and the sequence (S1) (104) is attached to the surface (100) by its 5' end by any of a variety of linkages well known to those skilled in the art (e.g., Beaucage, Curr. Med. Chem., 8(10): 1213-1244 (2001); Frydrych-Tomczak et al., BioTechnologia, 95(1): 5-16 (2014); Ratajczak et al., Methods Mol. Biol., 1368: 25-36 (2016); Uszczynska et al., LabChip, 12(6): 1151-1156 (2012), etc.). Such linkages are formed by the reaction of surface functional groups of the attached oligonucleotide and complementary functional groups. In some embodiments, the attached oligonucleotides (e.g., capture oligonucleotides or barcode oligonucleotides) are attached by their 5' ends, such that, for example, their 3' ends remain free for subsequent extension by polymerase.

[0034] According to some embodiments, a first oligonucleotide (102) having different barcode sequences is delivered using a DNA printing device (e.g., a device manufactured by M2 Automation (Berlin, Germany), Scienion (Berlin, Germany), etc.) to separate known positions in an array. In some embodiments, an inkjet delivery system can be used to construct multiple arrays (e.g., Cartesian Technologies (Irvine, CA); Barczak et al., Genome Research, 13: 1775-1785 (2003), etc.). In some embodiments, the first oligonucleotides of the multiple arrays can be synthesized in situ using various array synthesis techniques (e.g., Singh-Gasson et al., Nature Biotechnology, 17: 974-978 (1999); Horgan et al., International Patent Application WO2022 / 013094; Le, Recent Progress in Ink Jet Technologies II, chapter 1 (1999); Hughes et al., Nature Biotechnology, 19: 342-347 (2001), etc.). In some embodiments, such an array includes a spatially small linear or hexagonal array of non-overlapping, i.e., spatially separated, reaction sites substantially uniformly coated with the first oligonucleotide (102). In some embodiments, such an array of reaction sites can have a pitch (center-to-center distance) in the range of 50-500 μm and a diameter in the range of 30-100 μm, but is not limited thereto. Returning to FIG. 1A, the first oligonucleotide (102) bound to the surface (100) can hybridize (or anneal (108)) to a second oligonucleotide (110) comprising a segment S1' (complementary to segment S1 (104)), a second barcode segment BC2, and a segment S2'.Thereafter, a reagent may be introduced to extend the first oligonucleotide (102), such that BC2 and S2’ of the second oligonucleotide (110) are copied to form the first binding surface oligonucleotide. In an alternative embodiment, the second barcode segment (113) may be ligated to the first oligonucleotide (102), for example, by using ligase to ligate the second oligonucleotide, thereby forming the first binding surface oligonucleotide. In some alternative embodiments, consecutive oligonucleotide segments may be joined by ligation using a splint oligonucleotide that forms a duplex with the two oligonucleotides to be ligated and ligase. In a further embodiment, consecutive oligonucleotide segments may be joined by ligation using circligase.

[0035] Methods for delivering a second oligonucleotide (110) and reagents for extending a first oligonucleotide (102) are illustrated in FIGS. 2A-2B. After hybridization and extension (112), the hybridized and copied second oligonucleotide (110) can be melted from the strand (113) (114). The strand (113) (sometimes also referred to herein as the "first binding surface oligonucleotide") can anneal to a third oligonucleotide (118) comprising a segment S2' (complementary to segment S2 of strand (113)), a third barcode segment (BC3), and a segment S3' (116). After extension (120) and washing and melting (122), the result is a spatial barcode (124) comprising barcode segments BC1, BC2, and BC3, the combination of which can be unique for each reaction site in the plurality of arrays. In some embodiments, segment S3 (125) can serve as a capture oligonucleotide. For example, it can be a poly-T sequence for capturing poly-A-tailed messenger RNA from cells of a sample being analyzed on the surface (100). Similarly, in alternative embodiments, the third barcode segment (118) can be ligated to the strand (113).

[0036] Figure 1B illustrates an alternative embodiment that uses tagging to join a third barcode component, thereby forming the above-described third-form barcoded sample nucleic acid, namely: BC1-BC2-sample NA-BC3. A general review of the tagging techniques is shown in Adey, Genome Research, 31: 1693-1705 (2021); U.S. Patent Nos. 9,115,396; 9,085,801; 11,319,534, etc. (which are incorporated herein by reference). The barcode segments, BC1-S1 and BC2-S2 (150), can be assembled as described in Figure 1A. Here segment S2 is a capture probe (e.g., a poly-T segment specific for the poly-A messenger RNA of a biological sample). The biological sample (151) can be contacted with the surface (100), such that the poly-A mRNA contained therein anneals (152) to the capture oligonucleotide (S2, 153). Here the mRNA includes a poly-A segment (154) and a coding segment (156). After extension with reverse transcriptase and optional template switching, a double-stranded structure (159) can be obtained. It is then subjected to tagging (160) to join the final barcode segment, BC3, to give the final sequence (162). Similar procedures can be used to synthesize each of the formats (-sample NA-BC1-BC2-BC3; -BC1-sample NA-BC2-BC3; -BC1-BC2-sample NA-BC3; or -BC1-BC2-BC3-sample NA).

[0037] According to some embodiments described herein, second and third oligonucleotides each including a second and a third barcode segment are delivered to the plurality of arrays by a channel as illustrated in FIGS. 2A-2B. Alternative embodiments for delivery and conjugation to the first oligonucleotide (or a conjugate of the first and second oligonucleotides) can include, for example, the use of a photomask and photoactivated ligation as taught by van Dam, Thesis (California Institute of Technology, 2005). As shown in FIG. 2A, the plurality of arrays (e.g., 204) can be synthesized on a slide, or the surface (202) of a substrate (200). In this illustration, the plurality of arrays is 240 and is arranged in a 24×10 linear format. The spacing between the arrays on the surface (202) is exaggerated for illustrative purposes. The stretch of the arrays (206) shows a 32×24 array of reaction sites (208). In some embodiments, each of the plurality of arrays has the same first oligonucleotide at the same position. Thus, for example, the sequence of the barcode segment of the first oligonucleotide at the 18th row and 11th column of array (205) is the same as those of the first oligonucleotide at the 18th row and 11th column of array (204). That is, in some embodiments, each of the plurality of arrays includes the same first oligonucleotide.

[0038] In this embodiment, the second oligonucleotide and related extension reagents (e.g., DNA polymerase, reaction buffer, dNTP, etc.) can be placed on the plurality of arrays and delivered by channels formed in a layer of material (e.g., elastic plastic, etc.) that forms channel bodies or templates partitioning it into a plurality of rows or columns. Those skilled in the art will understand that the plurality of arrays, rows, columns, first channels, second channels, etc. are independent quantities; that is, the plurality of values for these distinct features need not be the same in any particular embodiment. As illustrated in FIG. 2A, a channel template (210) can be placed (212) on a surface (202) to partition the plurality of arrays into a plurality of 24 rows for each of the 10 arrays. Placing the channel template (210) on the surface (202) can be performed using a simple instrument similar to that illustrated in FIG. 3, sandwiching the channel template (210) between the surface (202) of a substrate (203) and a cover (207). The channel template can vary widely in design and composition depending on the scale and arrangement of the plurality of arrays, the size and arrangement of the arrays of reaction sites, and the method used to couple the first, second, and third oligonucleotides. The channel template can be fabricated from a wide variety of materials well known in the microfluidics field (e.g., silicon, glass, plastic, etc.) (e.g., Ren et al., Acc. Chem. Res., 46(11): 2396-2406 (2013)). In some embodiments, the channel template can include plastics (e.g., polystyrene, polyethylene terephthalate glycol, polyethylene terephthalate, polymethyl methacrylate, polyvinyl chloride, polycarbonate, thermoplastic elastomer, etc.).Guidance in the selection of plastics and fabrication methodologies can be found in the following references: Becker et al., Talanta, 56: 267-287 (2002); Fiorini et al., Biotechniques, 38(3): 429-446 (2005); Bjornson et al., U.S. Patent No. 6,803,019; Soane et al., U.S. Patent No. 6,176,962; Schaevitz et al., U.S. Patent No. 6,908,594; Neyer et al., U.S. Patent No. 6,838,156, etc. (these references are incorporated herein by reference).

[0039] As shown in the cross-sectional view (216), along the median (214) of the channel (213), after the assembly of the substrate (203), the channel template (210) and the cover (207), a dedicated flow path (211) is fabricated for each row of the array. Thus, each array of a given row can receive the same second oligonucleotide. In some embodiments, the sequences of the barcode segments of the second oligonucleotides of different rows are different, such that the sequence of the second barcode segment uniquely identifies the row in which the spatial barcode is located.

[0040] After the second oligonucleotide is delivered and coupled to the first oligonucleotide, the row channel template (210) can be removed. As shown in FIG. 2B, the column channel template (220) can be placed (224) on the surface (202) of the substrate (203) to partition the plurality of arrays into a plurality of 10 columns (e.g., 222) of each of the 24 arrays. Similar to the partitioning into rows, the channel template (220) can create dedicated flow paths for each column, which allows the arrays of each column to be exposed to the same third oligonucleotide. In some embodiments, the sequences of the barcode segments of each third oligonucleotide of different columns are different, such that the sequence of the third barcode segment uniquely identifies the column in which the spatial barcode is located. After the coupling of the third oligonucleotide (226), the spatially barcoded surface can be created with a spatial barcode in the form shown in stretching (228).

[0041] In some embodiments, the number of unique barcodes on the surface can be increased by providing channels that match a subset of the reaction sites in a row or column of the array. FIG. 2C illustrates an example of this embodiment with respect to a column of array (232) shown in an extended view of the array among a plurality of arrays (230). In this embodiment, the width of the channels (e.g., 234a and 234b in the extension) is fabricated such that the channels match half of the spots (or reaction sites) of the array of the column (231, the dark shaded subarray), such that the barcode oligonucleotides can be sequentially coupled (237) to the first half of the reaction sites (e.g., 240), and then can be coupled (238) to the second half of the reaction sites (e.g., 242). This can be achieved by using two different channel templates with the channel position offset by (e.g.) half of the array width, or by moving one channel template by half of the array width. FIG. 2E illustrates the case when two channel templates (262 and 264) (or gasket components of such channel templates) are used with channels offset by a predetermined amount (266) such that different reaction sites are exposed to the reagents delivered by the channels. After such a process, the array among the plurality of arrays (230) has twice the number of different spatial barcodes than in an embodiment where each overall subarray (e.g., 232) matches the channel that delivers the barcode oligonucleotides. In some embodiments as shown in FIG. 2D, the channel template can be fabricated such that half of the subarray matches different channels, where the space (248) is selected such that the walls of the channel template can be adapted without covering or blocking the reaction sites. This configuration increases the speed of fabrication and simplifies the oligonucleotide placement process, such that, for example, different barcode segments are added homogeneously to each half of the reaction sites that give rise to products (258 and 260) in one subarray (256).This embodiment is fabricated by forming a subarray having a gap (255) in a subarray (256) that separates half of it and provides space for the walls of the channel template. In other embodiments, the subarray may be formed with a number of gaps by dividing the reaction sites, for example, into three instead of two, such gaps being formed in both the horizontal and vertical directions, for example, for the attachment of the second and third oligonucleotides, respectively.

[0042] In some embodiments, as illustrated in FIG. 2F, spatial barcode oligonucleotides can be combinatorially formed by delivering their components in channels of different widths. For example, channel (270) can be established by binding a first channel template to a surface containing subarray (268), which reacts with either the surface or the binding surface oligonucleotide to deliver a barcode oligonucleotide that gives a product (e.g., 274) at half of the reaction sites of subarray (268). Subsequently, channel (272) can be established by binding a second channel template, which reacts with either the surface or the binding surface oligonucleotide to deliver a barcode oligonucleotide that gives a product (e.g., 276) at the other half of the reaction sites of subarray (268). Next, channels (282 and 284) are established by binding a third channel template having channels spaced closer together than those delivering reagents (280) to the first and third quadrants of the reaction sites of subarray (268) that give products (290) and (294) after reaction. After step (280), channels (286 and 288) are established by binding a fourth channel template having channels spaced apart that deliver reagents (281) to the second and fourth quadrants of the reaction sites of subarray (268) that give products (292 and 296) after reaction. In such embodiments, four times the number of unique barcodes can be generated compared to embodiments where the channels coincide with the entire subarray, resulting in a barcoded surface.

[0043] As described above, channel templates (210) and (220) can be applied to the surface (202) using an instrument as illustrated in FIG. 3, or a similar device. A substrate (300) including a surface (302) having a plurality of arrays (304) can be placed on a base (306), and a channel template (308) can be placed on top to create dividers for rows (or columns) of the arrays. A manifold (310) can be placed on top of the channel template (308) to provide conduits from a reagent reservoir or plate to the channels created by the channel template (308). Finally, a top plate (not shown) can be aligned by alignment pins (312) and placed on top to complete the assembly.

[0044] The present invention has been described with reference to several specific exemplary embodiments, but those skilled in the art will recognize that many changes can be made to them without departing from the spirit and scope of the present invention. The present invention is applicable to various sensor implementations and other subjects in addition to those considered above.

[0045] Definitions Unless specifically defined otherwise herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow those of standard treatises and texts in the field (e.g., Kornberg and Baker, DNA Replication, 2nd ed. (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immunology, 6th ed. (Saunders, 2007)).

[0046] "Barcode" means a molecular label or identifier. In some embodiments, the barcode is a molecule that can be attached to an analyte or a segment of an analyte (e.g., in the case of a polynucleotide barcode and an analyte) and used to identify that analyte. In some embodiments, the barcode (also referred to herein as a "spatial barcode") is attached to the surface to identify a position on the surface. In some embodiments, a population of identical spatial barcodes can be arranged within a particular region on the surface. The size and shape of such regions can vary widely. In some embodiments, regions having unique spatial barcodes have the same scale and are arranged in a regular pattern on the surface with the density of spatial barcodes per unit area. In some embodiments, the density of such barcodes is 1 barcode / mm 2 ~1000 barcodes / mm 2 or 1 barcode / mm 2 ~500 barcodes / mm 2 or 1 barcode~200 barcodes / mm 2It can vary. In some embodiments, there can be a one-to-one correspondence between different spatial barcodes on the surface and different regions; that is, each different region can have a different and unique barcode. In some embodiments, what the spatial barcode is can be determined, for example, by sequencing when the spatial barcode is a polynucleotide. In some embodiments, the spatial barcode is an oligonucleotide. In some embodiments, the oligonucleotide spatial barcode includes random array oligonucleotides. Random array oligonucleotides are typically synthesized by, for example, the "split and mix synthesis technique" as described in the following references incorporated herein by reference: Church, U.S. Patent No. 4,942,124; Godron et al., International Patent Publication WO2020 / 120442; Seelig et al., U.S. Patent Publication 2016 / 0138086, etc. Sometimes, the random oligonucleotide is represented as "NNN... N". In some embodiments, the term "barcode" includes a composite barcode; that is, an oligonucleotide segment that includes sub-segments that identify different objects. For example, the first segment of the composite barcode can identify a specific region of the surface, and the second segment of the composite barcode can identify a specific molecule (so-called "unique molecular identifier" or UMI).

[0047] "Microfluidics" devices or "nanofluidics" devices are used interchangeably herein and each refers to an integrated system for capturing, moving, mixing, dispensing, or analyzing small volumes of fluid, including, in turn, samples (which may contain or include the cells or molecular analytes of interest), reagents, diluents, buffers, etc. Generally, references to "microfluidics" and "nanofluidics" indicate that they differ in scale in terms of the size of the device and the volume of fluid handled. In some embodiments, microfluidic devices are characterized by having cross-sectional dimensions of less than a few hundred square micrometers and having capillary dimensions, e.g., passages or channels having cross-sectional dimensions from about 1 - 2 mm to about 0.1 μm. In some embodiments, microfluidic devices have capillaries with volumes in the range of 100 μL to a few nL, e.g., 10 - 100 nL, or in the range of 100 μL - 1 μL. The corresponding features, or dimensional sizes, in nanofluidic devices are typically one to three orders of magnitude smaller than those of microfluidic devices. Those skilled in the art know which dimensionality is appropriate from the context of a particular application. In some embodiments, microfluidic or nanofluidic devices have one or more chambers, ports, and channels, which are interconnected and in fluid communication and are designed to perform one or more reactions or processes either alone or in cooperation with instruments or equipment that provide ancillary functions (e.g., sample introduction, fluid and / or reagent propulsion means (e.g., positive or negative pressure, acoustic energy, etc.)), temperature control, detection systems, data collection and / or integration systems, etc. In some embodiments, microfluidic and nanofluidic devices may further include valves, pumps, filters, and special functional coatings on the inner walls for, e.g., preventing adsorption of sample components or reactants and promoting reagent movement by electroosmosis.Such devices can be fabricated as integrated devices in a solid substrate, which can be glass, plastic, or other solid polymeric materials, and can have a planar format, in particular, to facilitate the detection and monitoring of sample and reagent movement via optical or electrochemical means. In some embodiments, such devices can be disposable after single use. In some embodiments, microfluidic and nanofluidic devices include devices that form and control the movement, mixing, dispensing, and analysis of droplets, such as aqueous droplets immersed in immiscible fluids such as light oil. The fabrication and operation of microfluidic and nanofluidic devices are well known in the art, as exemplified by the following references incorporated by reference: Ramsey, U.S. Patent Nos. 6,001,229; 5,858,195; 6,010,607; and 6,033,546; Soane et al., U.S. Patent Nos. 5,126,022 and 6,054,034; Nelson et al., U.S. Patent No. 6,613,525; Maher et al., U.S. Patent No. 6,399,952; Ricco et al., International Patent Publication WO 02 / 24322; Bjornson et al., International Patent Publication WO 99 / 19717; Wilding et al., U.S. Patent Nos. 5,587,128; 5,498,392; Sia et al., Electrophoresis, 24: 3563-3576 (2003); Unger et al., Science, 288: 113-116 (2000); Enzelberger et al., U.S. Patent No. 6,960,437; Cao, “Nanostructures & Nanomaterials: Synthesis, Properties & Applications, (Imperial College Press, London, 2004); Haeberle et al., LabChip, 7: 1094-1110 (2007); Ren et al., Acc. Chem. Res., 46(11): 2396-2406 (2013); Cheng et al., Biochip Technology (CRC Press, 2001), etc.

Claims

Claim 1 A method of creating a spatially barcoded surface, the method comprising: (a) providing a solid support comprising a surface; (b) synthesizing a plurality of arrays of first oligonucleotides, each array comprising a plurality of discrete reaction sites and each first oligonucleotide attached to the surface by its 5' end, wherein (i) each first oligonucleotide has a barcode segment comprising a barcode sequence different from those of the other first oligonucleotides, (ii) first oligonucleotides having different barcode sequences are attached at different reaction sites, (iii) the plurality of arrays are arranged in orthogonal rows and columns; (iv) each first oligonucleotide occupies a reaction site at a known surface position; (c) partitioning the surface into rows by sealingly attaching to the surface a first channel template comprising a plurality of channels; and (d) reacting a second oligonucleotide loaded into each channel of the first channel template with the surface or a preceding bound surface oligonucleotide to form a bound surface oligonucleotide or a spatially barcoded oligonucleotide, wherein each different channel contains a second oligonucleotide comprising a second barcode segment comprising a barcode sequence different from those of the second oligonucleotides of the different channels, comprising: wherein (i) the rows of the plurality of arrays at least partially coincide with the rows of the first channel template, and (ii) each spatially barcoded oligonucleotide uniquely identifies the spatial position of its reaction site on the surface. Claim 2 (a) partitioning the surface into columns by sealingly attaching to the surface a second channel template comprising a plurality of channels; and (b) reacting the third oligonucleotide loaded into each channel of the second channel template with the surface or a preceding bound surface oligonucleotide to form a bound surface oligonucleotide or a spatial barcode oligonucleotide, wherein each different channel of the second channel template contains a third oligonucleotide containing a third barcode segment containing a barcode sequence different from those of the third oligonucleotides of the different channels of the second channel template; further comprising wherein the columns of the plurality of arrays at least partially coincide with the columns of the second channel template, the method according to claim 1. **Claim 3** each of the plurality of arrays is the same, the method according to claim 1. **Claim 4** any row of the orthogonal rows and columns of the array has no array in common with any other row and shares at most a column of any single array, the method according to claim 1. **Claim 5** each of the arrays of the reaction sites has a pitch between the reaction sites in the range of 50 to 500 μm, and the reaction sites each have a diameter in the range of 30 to 300 μm, the method according to claim 1. **Claim 6** Each of the arrays of the reaction sites has a density of reaction sites in the range of 50 to 200 reaction sites / mm ２ The method according to claim 1, wherein the density of the reaction sites is in the range of 50 to 200 reaction sites / mm. **Claim 7** each reaction step is performed by extending the first oligonucleotide, the second oligonucleotide or the third oligonucleotide with DNA polymerase, the method according to claim 1. **Claim 8** each reaction step is performed by ligating the first oligonucleotide to the second oligonucleotide or the third oligonucleotide, or the second oligonucleotide to the first oligonucleotide or the third oligonucleotide, or the third oligonucleotide to the first oligonucleotide or the third oligonucleotide, the method according to claim 1. **Claim 9** the surface further comprises a capture probe bound thereto, and the method further comprises capturing a sample nucleic acid with the capture probe and extending the capture probe using the captured sample nucleic acid as a template, the method according to claim 1. **Claim 10** Any one of the first oligonucleotide, the second oligonucleotide, or the third oligonucleotide includes the capture probe, and the capturing step and the extending step occur before or after any one of the reaction steps. The method according to claim 9.

11. A method for providing spatially barcoded nucleic acid molecules, the method comprising: (a) providing a solid support comprising a surface; (b) capturing a nucleic acid molecule on the surface and transcribing the captured nucleic acid molecule into complementary DNA (cDNA) bound to the surface; synthesizing a plurality of arrays of a first oligonucleotide at different reaction sites on the surface, wherein (i) the plurality of arrays are arranged in orthogonal rows and columns; (ii) each array comprises a plurality of reaction sites, each of which always has a different barcode sequence when located at a different reaction site, and comprises a first oligonucleotide comprising a first barcode segment; (iii) each first oligonucleotide occupies a reaction site at a known surface position; (c) reacting a second oligonucleotide with the surface or an oligonucleotide or cDNA thereon by sealingly binding a first channel template comprising a plurality of channels to the surface, wherein each different channel comprises a second oligonucleotide comprising a second barcode segment having a different barcode sequence from those of the second oligonucleotides of the different channels; and (d) reacting a third oligonucleotide with the surface or an oligonucleotide or cDNA thereon by sealingly binding a second channel template comprising a plurality of channels to the surface, wherein each different channel of the second channel template comprises a third oligonucleotide comprising a third barcode segment having a different barcode sequence from those of the third oligonucleotides of the different channels, comprising wherein the channels of the first and second channel templates are aligned with the orthogonal rows and columns of the array, and wherein the first, second, and third oligonucleotides bound to the cDNA form a spatial barcode that identifies the spatial position of the cDNA on the surface.

12. When the synthesizing step is always performed before the step of capturing or reacting the second or third oligonucleotide, the surface comprises surface functional groups; the first oligonucleotide each comprises a 5'-complementary functional group, such that the surface functional groups react with the 5'-complementary functional groups to form covalent bonds. The method according to claim 11.

13. When the capturing step is always performed before the step of synthesizing or reacting the second or third oligonucleotide, the surface comprises a capture oligonucleotide specific for the nucleic acid molecule. The method according to claim 11.

14. When the capturing step is always performed immediately after the synthesizing step, the first oligonucleotide comprises a capture oligonucleotide. The method according to claim 11.

15. When the capturing step is always performed immediately after the step of reacting the second oligonucleotide, the second oligonucleotide comprises a capture oligonucleotide. The method according to claim 11.

16. When the capturing step is always performed immediately after the step of reacting the third oligonucleotide, the third oligonucleotide comprises a capture oligonucleotide. The method according to claim 11.

17. The nucleic acid molecule is messenger RNA. The method according to claim 1.

18. The messenger RNA is derived from a single cell. The method according to claim 17.

19. A method of creating a spatially barcoded surface, the method comprising: (a) providing a surface comprising surface functional groups; (b) depositing a first oligonucleotide on different barcode sequences at different reaction sites on the surface under conditions such that the surface functional groups react with 5'-complementary functional groups to form covalent bonds, thereby synthesizing a plurality of arrays of first oligonucleotides each comprising a 5'-complementary functional group, wherein (i) the plurality of arrays are arranged in orthogonal rows and columns; (ii) each array comprises a plurality of reaction sites, each of which, when located at a different reaction site, always comprises a first oligonucleotide comprising a first barcode segment having a different barcode sequence; (iii) each first oligonucleotide occupies a reaction site at a known surface position. (c) partitioning the plurality of arrays into rows by sealingly coupling to the surface a first channel template including a plurality of channels aligned respectively with different rows of the array; (d) reacting a first oligonucleotide in each channel with a second oligonucleotide loaded into each channel of the first channel template to form a first binding surface oligonucleotide, wherein each different channel includes a second oligonucleotide including a second barcode segment including a barcode sequence different from those of the second oligonucleotides of the different channels; (e) partitioning the plurality of arrays into columns by sealingly coupling to the surface a second channel template including a plurality of channels aligned respectively with different columns of the array; and (f) reacting a first binding surface oligonucleotide in each channel with a third oligonucleotide loaded into each channel of the second channel template to form a spatial barcode oligonucleotide, comprising wherein (i) each different channel of the second channel template includes a third oligonucleotide including a third barcode segment including a barcode sequence different from that of the third oligonucleotides of the different channels of the second channel body, and (ii) each spatial barcode oligonucleotide uniquely identifies the spatial position of its reaction site on the surface.

20. The method according to claim 19, wherein each of the plurality of arrays is the same.

21. The method according to claim 19, wherein none of the rows of the orthogonal rows and columns of the array have an array in common with any other row and share at most a column of any single array.

22. The method according to claim 19, wherein each of the arrays of the reaction sites has a pitch between the reaction sites in the range of 50 to 500 μm, and the reaction sites each have a diameter in the range of 30 to 300 μm.

23. Each of the arrays of the reaction sites has a density of reaction sites in the range of 50 to 200 reaction sites / mm ２ The method according to claim 19, having a density of reaction sites in the range of

24. The method according to claim 19, wherein each reaction step is performed by extending the first oligonucleotide, the second oligonucleotide or the third oligonucleotide with DNA polymerase.

25. The method according to claim 19, wherein each reaction step is performed by ligating the first oligonucleotide to the second oligonucleotide or the third oligonucleotide, or the second oligonucleotide to the first oligonucleotide or the third oligonucleotide, or the third oligonucleotide to the first oligonucleotide or the second oligonucleotide.

26. The method according to claim 19, wherein the surface further comprises a capture probe bound thereto, and the method further includes a step of capturing a sample nucleic acid with the capture probe and a step of extending the capture probe using the captured sample nucleic acid as a template.

27. The method according to claim 26, wherein any one of the first oligonucleotide, the second oligonucleotide, or the third oligonucleotide comprises the capture probe, and the capturing step and the extending step occur before or after any one of the reaction steps.

28. A method for generating a spatially barcoded surface, the method comprising: (a) providing a solid support comprising a surface, wherein the surface comprises a plurality of arrays arranged on the surface, each array comprising a plurality of reaction sites, each reaction site comprising a reaction site oligonucleotide having a barcode sequence specific to the reaction site located within the reaction site; (b) partitioning the surface into one or more channels by coupling a channel template comprising a plurality of channels to the surface; and (c) loading a plurality of channel oligonucleotides into the plurality of channels, such that at least one channel oligonucleotide couples to the reaction site oligonucleotide in each array, wherein each channel oligonucleotide comprises a barcode sequence specific to the channel located within the channel. A method comprising the above steps.

29. (a) partitioning the surface into one or more sets of orthogonal channels by coupling an additional channel template including a plurality of orthogonal channels to the surface, where the one or more sets of orthogonal channels are orthogonal to the one or more channels; and (b) loading a plurality of orthogonal channel oligonucleotides into the plurality of orthogonal channels, such that at least one orthogonal channel oligonucleotide couples to the channel oligonucleotides in each array, where each orthogonal channel oligonucleotide includes a barcode sequence specific to the orthogonal channel in which it is located, The method of claim 28, further comprising.

30. The method of claim 29, wherein the plurality of arrays are arranged in rows and columns.

31. The method of claim 30, wherein the one or more channels at least partially coincide with the rows, and the one or more sets of orthogonal channels at least partially coincide with the columns.

32. The method of claim 30, wherein the one or more channels at least partially coincide with the columns, and the one or more sets of orthogonal channels at least partially coincide with the rows.

33. The method of claim 28, wherein the coupling step in (b) includes binding the channel template including the plurality of channels to the surface to seal it.

34. The method of claim 29, wherein the coupling step in (a) includes binding the additional channel template including the plurality of orthogonal channels to the surface to seal it.

35. The method of claim 28, wherein each of the plurality of arrays is the same.

36. The method of claim 28, wherein one of the plurality of arrays has a pitch between the reaction sites in the range of 50 to 500 μm, and the reaction sites each have a diameter in the range of 30 to 300 μm.

37. One of the plurality of arrays has a density of reaction sites in the range of 50 to 200 reaction sites / mm ２ The method according to claim 28, wherein the density of reaction sites in the range of

38. The step of coupling the at least one channel oligonucleotide to the reaction site oligonucleotide of each array in (c) includes the step of extending the at least one channel oligonucleotide using DNA polymerase, the method according to claim 28.

39. The step of coupling the at least one channel oligonucleotide to the reaction site oligonucleotide of each array in (c) includes the step of ligating the at least one channel oligonucleotide to the reaction site oligonucleotide of each array, the method according to claim 28.

40. The surface further includes a capture probe bound thereto, and the method further includes the step of capturing the sample nucleic acid with the capture probe and the step of extending the capture probe using the captured sample nucleic acid as a template, the method according to claim 29.

41. Any one of the reaction site oligonucleotide, the channel oligonucleotide, or the orthogonal channel oligonucleotide includes the capture probe, the method according to claim 40.

42. A method of creating a spatially barcoded surface, the method comprising: (a) providing a solid support including a surface, wherein the surface includes a plurality of arrays, wherein one of the plurality of arrays includes at least two reaction sites, wherein a first reaction site of the at least two reaction sites includes a first reaction site oligonucleotide including a first reaction site barcode sequence, and a second reaction site of the at least two reaction sites includes a second reaction site oligonucleotide including a second reaction site barcode sequence, wherein the first barcode sequence is different from the second barcode sequence; (b) coupling a channel template including a first channel and a second channel to the surface; (c) loading a first channel oligonucleotide into the first channel; and (d) loading a second channel oligonucleotide into the second channel, including, wherein subsequent to the loading step of (c), the first channel oligonucleotide couples to the first reaction site oligonucleotide, Here, following the step of loading the (d), the second channel oligonucleotide couples to the second reaction site oligonucleotide, wherein the first channel oligonucleotide includes a third barcode sequence, the second channel oligonucleotide includes a fourth barcode sequence, and wherein the third barcode sequence is different from the fourth barcode sequence, Method. **Claim 43** (a) coupling an orthogonal channel template including a first orthogonal channel and a second orthogonal channel to the surface; (b) loading a first orthogonal channel oligonucleotide into the first channel; and (c) loading a second orthogonal channel oligonucleotide into the second channel, further comprising, where, following the step of loading in (b), the first orthogonal channel oligonucleotide couples to the first channel oligonucleotide, where, following the step of loading in (c), the second orthogonal channel oligonucleotide couples to the second channel oligonucleotide, wherein the first orthogonal channel oligonucleotide includes a fifth barcode sequence, the second orthogonal channel oligonucleotide includes a sixth barcode sequence, and wherein the fifth barcode sequence is different from the sixth barcode sequence, The method according to claim 42. **Claim 44** The method according to claim 43, wherein the plurality of arrays are arranged in rows and columns. **Claim 45** The method according to claim 43, wherein the first channel and the second channel at least partially coincide with the row, and the first orthogonal channel and the second orthogonal channel at least partially coincide with the column. **Claim 46** The method according to claim 43, wherein the first channel and the second channel at least partially coincide with the column, and the first orthogonal channel and the second orthogonal channel at least partially coincide with the row. **Claim 47** The method according to claim 42, wherein the coupling step in (b) includes a step of binding to seal the channel template including the first channel and the second channel to the surface. **Claim 48** The step of coupling in (a) includes the step of coupling to seal the orthogonal channel template including the first orthogonal channel and the second orthogonal channel to the surface, the method according to claim 43.

49. Each of the plurality of arrays is the same, the method according to claim 42.

50. One of the plurality of arrays constitutes a pitch between the reaction sites in the range of 50 to 500 μm, and the reaction sites each have a diameter in the range of 30 to 300 μm, the method according to claim 42.

51. One of the plurality of arrays has a density of reaction sites in the range of 50 to 200 reaction sites / mm ２ The method according to claim 42, comprising a density of reaction sites in the range of

52. The step of coupling the first channel oligonucleotide to the first reaction site oligonucleotide includes the step of extending the first channel oligonucleotide using DNA polymerase, the method according to claim 42.

53. The step of coupling the first channel oligonucleotide to the first reaction site oligonucleotide includes the step of ligating the first channel oligonucleotide to the first reaction site oligonucleotide, the method according to claim 42.

54. The step of coupling the first orthogonal channel oligonucleotide to the first channel oligonucleotide includes the step of extending the first channel oligonucleotide using DNA polymerase, the method according to claim 43.

55. The step of coupling the first orthogonal channel oligonucleotide to the first channel oligonucleotide includes the step of ligating the first orthogonal channel oligonucleotide to the first channel oligonucleotide, the method according to claim 42.

56. The surface further includes a capture probe coupled thereto, and the method further includes the step of capturing a sample nucleic acid with the capture probe and the step of extending the capture probe using the captured sample nucleic acid as a template, the method according to claim 43.

57. Any one of the first or second reaction site oligonucleotides, the first or second channel oligonucleotides, or the first or second orthogonal channel oligonucleotides includes the capture probe, the method according to claim 56.

58. A flow cell comprising one or more arrays, wherein one of the one or more arrays is located at the intersection of rows and columns on the surface of the flow cell, the array comprising one or more reaction sites, and one of the one or more reaction sites is a) a first oligonucleotide sequence specific to the spatial position of the reaction site within the array; b) a second oligonucleotide specific to the row; and c) a third oligonucleotide specific to the column, the flow cell comprising. **Claim 59** The flow cell according to claim 58, wherein the array, the row, the column, or any combination thereof is configured to receive the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, or any combination thereof. **Claim 60** The flow cell according to claim 58, wherein the array constitutes a pitch between the reaction site and a second reaction site in the range of 50 to 500 μm, and the reaction site constitutes a diameter in the range of 30 to 300 μm. **Claim 61** The one or more reaction sites described above have a density in the range of 50 to 200 reaction sites / mm ２ The flow cell according to claim 58, which constitutes a density in the range of **Claim 62** The flow cell according to claim 58, wherein the one or more reaction sites comprise one or more capture probes. **Claim 63** Further comprising a second array, wherein the second array is located at a second intersection of a second row and a second column, the second array comprising one or more second reaction sites, and one of the one or more second reaction sites is a) a fourth oligonucleotide sequence specific to the spatial position of the second reaction site within the second array; b) a fifth oligonucleotide specific to the second row; and c) a sixth oligonucleotide specific to the second column, the flow cell according to claim 58 comprising. **Claim 64** The flow cell according to claim 63, wherein the spatial position of the second reaction site within the second array corresponds to the spatial position of the reaction site within the array of claim 58, and the first oligonucleotide sequence and the fourth oligonucleotide sequence are the same.

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