Method for constructing spatially barcoded surface

Abstract

The systems, devices and methods described herein relate to methods for producing spatially barcoded surfaces using combinatorial synthesis techniques and their use for analyzing molecules of biological cells disposed on such surfaces, particularly nucleic acid molecules. The methods described herein can also be used to create spatially barcoded nucleic acid molecules.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 344,380, filed on May 20, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Molecular tagging has a long history in analytical biochemistry and molecular biology, e.g., Church, U.S. Pat. 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. Pat. No. 7,537,897; Fan et al., Science, 347(6222):1258367-1 (2015); Macevicz, U.S. Pat. No. US2005 / 0250147; Morris et al., European Patent Publication No. 0799897A1; Wallace, U.S. Pat. No. 5,981,179; etc. Recently, such techniques have been extended to include the use of spatially distributed oligonucleotide barcodes to identify and study spatial variations in biological processes, such as tissue-wide gene expression, e.g., Stahl et al., Science, 353(6294):78-82 (2016); Salmen et al., Nature Protocols, 13:2501-2534 (2018); Frisen et al., U.S. Pat. No. 9,593,365; etc. However, the cost-effective synthesis of spatial barcodes with known sequences, control over the distribution of spatial barcodes, and density for resolving cellular and subcellular processes has been a challenge. This challenge has been addressed by a variety of different approaches, with only partial success, such as 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 availability of cost-effective spatial barcoding methods will advance the field of spatial barcode construction. Summary of the invention

[0004] The systems, devices, and methods described herein relate to using combinatorial techniques to make spatially barcoded surfaces and nucleic acid molecules. In some embodiments, a method for making a spatially barcoded surface comprises: (a) providing a solid support comprising a surface; (b) synthesizing multiple arrays of first oligonucleotides, each array comprising multiple discrete reaction sites, and each first oligonucleotide is attached to the surface via its 5'-end, wherein (i) each first oligonucleotide has a barcode segment that comprises a barcode sequence that is different from the barcode sequences of other first oligonucleotides; (ii) first oligonucleotides with different barcode sequences are attached to different reaction sites; (iii) multiple arrays are arranged in orthogonal rows and columns; and (iv) each first oligonucleotide occupies a reaction site at a known surface position. ; (c) dividing the surface into rows by sealingly attaching a first channel template comprising a plurality of channels to the surface; (d) reacting a second oligonucleotide loaded into each channel of the first channel template with the surface or a previously conjugated surface oligonucleotide to form a conjugated surface oligonucleotide or a spatial barcode oligonucleotide, wherein each different channel comprises a second oligonucleotide comprising a second barcode segment, the second barcode segment comprising a barcode sequence different from the barcode sequence of the second oligonucleotide of the different channels; wherein (A) the rows of the plurality of arrays at least partially overlap with the rows of the first channel template, and (B) each spatial barcode oligonucleotide uniquely identifies the spatial position of its reaction site on the surface. In some embodiments, in step (d), the second oligonucleotide reacts directly with the surface, while in other embodiments, in step (d), the second oligonucleotide reacts with an oligonucleotide already attached to the surface. In the latter embodiment, the oligonucleotide already attached to the surface is referred to as a "conjugated surface oligonucleotide", which can be a concatenation of the first oligonucleotide, the second oligonucleotide, or the third oligonucleotide from the previous step. That is, the conjugated surface oligonucleotide is a partially completed oligonucleotide precursor of the spatial barcode oligonucleotide. In some embodiments, step (d) can be the final attachment of the barcode segment (which can be the first oligonucleotide, the second oligonucleotide, or the third oligonucleotide, depending on the embodiment), thereby producing the final desired "spatial barcode oligonucleotide".

[0005] In some embodiments, a method for making a spatially barcoded surface includes: (a) providing a surface; (b) synthesizing multiple arrays of first oligonucleotides, each array comprising multiple discrete reaction sites, and each first oligonucleotide is attached to the surface via its 5'-end, wherein (i) each first oligonucleotide has a barcode segment comprising a barcode sequence different from the barcode sequences of other first oligonucleotides; (ii) first oligonucleotides with different barcode sequences are attached to different reaction sites; (iii) the multiple arrays are arranged in orthogonal rows and columns; and (iv) each first oligonucleotide occupies a reaction site at a known surface position; (c) dividing the surface into rows by sealingly attaching a first channel template comprising multiple channels to the surface; (d) reacting a second oligonucleotide loaded into each channel of the first channel template with the surface or a previously conjugated surface oligonucleotide to form a conjugated surface oligonucleotide or a spatially barcoded oligonucleotide, wherein each Different channels include a second oligonucleotide comprising a second barcode segment, the second barcode segment including a barcode sequence different from the barcode sequence of the second oligonucleotide of the different channels; (e) the surface is divided into columns by sealingly attaching a second channel template including multiple channels to the surface; (f) reacting a third oligonucleotide loaded into each channel of the second channel template with the surface or a previously conjugated surface oligonucleotide to form a conjugated surface oligonucleotide or a spatial barcode oligonucleotide, wherein each different channel of the second channel template includes a third oligonucleotide comprising a third barcode segment, the third barcode segment including a barcode sequence different from the barcode sequence of the third oligonucleotide of the different channels of the second channel template; and wherein (A) the rows of the multiple arrays at least partially overlap with the rows of the first channel template, and the columns of the multiple arrays at least partially overlap 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 described above, the term "conjugated surface oligonucleotide" refers to a partially completed oligonucleotide precursor of a spatial barcode oligonucleotide.

[0006] In some embodiments, a method for making spatially barcoded nucleic acid molecules comprises (a) providing a solid support comprising a surface; (b) capturing nucleic acid molecules on the surface and transcribing the captured nucleic acid molecules into complementary DNA (cDNA) attached to the surface; (c) synthesizing multiple arrays of first oligonucleotides at different reaction sites on the surface, wherein (i) the multiple arrays are arranged in orthogonal rows and columns; (ii) each array comprises multiple reaction sites, each of the multiple reaction sites comprises a first oligonucleotide, the first oligonucleotide comprising a first barcode segment having a different barcode sequence when located at a different reaction site; and (iii) each first oligonucleotide occupies a reaction site at a known surface position; (d) by sealingly attaching a first channel template comprising multiple channels to the surface, allowing a second oligonucleotide to bind to the surface or an oligonucleotide or oligonucleotide on the surface cDNA reaction, wherein each different channel includes a second oligonucleotide comprising a second barcode segment, the second barcode segment comprising a barcode sequence different from the barcode sequence of the second oligonucleotide of the different channels; and (e) reacting a third oligonucleotide with a surface or an oligonucleotide or cDNA on the surface by sealingly attaching a second channel template comprising multiple channels to the surface, wherein each different channel of the second channel template includes a third oligonucleotide comprising a third barcode segment, the third barcode segment comprising a barcode sequence different from the barcode sequence of the third oligonucleotide of the different channels; wherein the channels of the first channel template and the second channel template are aligned with orthogonal rows and columns of the array, and wherein the first oligonucleotide, the second oligonucleotide and the third oligonucleotide attached to the cDNA form a spatial barcode, which identifies the spatial position of the cDNA on the surface.

[0007] In one aspect, provided herein is 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, wherein each array comprises a plurality of reaction sites, wherein each reaction site comprises a reaction site oligonucleotide having a barcode sequence unique to the reaction site in which it is located; (b) dividing 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 is coupled to a reaction site oligonucleotide in each array, wherein each channel oligonucleotide comprises a barcode sequence unique to the channel in which it is located.

[0008] In some cases, the method further includes: (a) dividing the surface into one or more orthogonal lanes by coupling an additional channel template comprising a plurality of orthogonal lanes to the surface, wherein the one or more orthogonal lanes are orthogonal to the one or more channels; and (c) loading a plurality of orthogonal lane oligonucleotides into the plurality of orthogonal lanes such that at least one orthogonal lane oligonucleotide is coupled to a channel oligonucleotide in each array, wherein each orthogonal lane oligonucleotide comprises a barcode sequence unique to the orthogonal lane in which it is located.

[0009] In some cases, wherein the plurality of arrays are arranged in rows and columns. In some cases, one or more channels at least partially coincide with rows, and wherein one or more orthogonal channels at least partially coincide with columns. In some cases, one or more channels at least partially coincide with columns, and wherein one or more orthogonal channels at least partially coincide with rows.

[0010] In some cases, the coupling in (b) comprises sealingly attaching a channel template comprising a plurality of channels to the surface. In some cases, the coupling in (a) comprises sealingly attaching an additional channel template comprising a plurality of orthogonal channels to the surface.

[0011] In some cases, each of the plurality of arrays is identical. In some cases, the spacing between reaction sites of an array in the plurality of arrays is in the range of 50-500 μm, and wherein each of the reaction sites has a diameter in the range of 30-300 μm. In some cases, the density of reaction sites of an array in the plurality of arrays is in the range of 100-1500 μm per mm. 2 The number of reaction sites ranges from 50 to 200.

[0012] In some cases, coupling of at least one channel oligonucleotide in (c) to a reaction site oligonucleotide in each array comprises extending at least one channel oligonucleotide using a DNA polymerase. In some cases, coupling of at least one channel oligonucleotide in (c) to a reaction site oligonucleotide in each array comprises ligating at least one channel oligonucleotide to a reaction site oligonucleotide in each array.

[0013] In some cases, the surface also includes a capture probe attached thereto, and wherein the method further includes capturing the sample nucleic acid with the capture probe, and extending the capture probe using the captured sample nucleic acid as a template. In some cases, any of the reaction site oligonucleotide, channel oligonucleotide, or orthogonal channel oligonucleotide includes the capture probe.

[0014] In another aspect, provided herein is a method of making a spatially barcoded surface, the method comprising: (a) providing a solid support comprising a surface, wherein the surface comprises a plurality of arrays, wherein an array in the plurality of arrays comprises at least two reaction sites, wherein a first reaction site in the at least two reaction sites comprises a first reaction site oligonucleotide, the first reaction site oligonucleotide comprises a first reaction site barcode sequence, and wherein a second reaction site in the at least two reaction sites comprises a second reaction site oligonucleotide, the second reaction site oligonucleotide comprises a second reaction site barcode sequence, and wherein the first barcode sequence is identical to the second barcode sequence; The invention relates to a method for preparing an aqueous solution of at least one molecular structure comprising a first channel and a second channel, wherein the first channel oligonucleotide has a different barcode sequence; (b) coupling a channel template comprising a first channel and a second channel to a surface; (c) loading a first channel oligonucleotide into the first channel; and (d) loading a second channel oligonucleotide into the second channel, wherein after loading in (c), the first channel oligonucleotide is coupled to the first reaction site oligonucleotide, and wherein after loading in (d), the second channel oligonucleotide is coupled to the second reaction site oligonucleotide, and wherein the first channel oligonucleotide comprises a third barcode sequence, and the second channel oligonucleotide comprises a fourth barcode sequence, and wherein the third barcode sequence is different from the fourth barcode sequence.

[0015] In some cases, the method further comprises: (a) coupling an orthogonal channel template comprising a first orthogonal channel and a second orthogonal channel to a 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 after loading in (b), the first orthogonal channel oligonucleotide is coupled to the first channel oligonucleotide, and wherein after loading in (c), the second orthogonal channel oligonucleotide is coupled to the second channel oligonucleotide, and wherein the first orthogonal channel oligonucleotide comprises a fifth barcode sequence, and the second orthogonal channel oligonucleotide comprises a sixth barcode sequence, and wherein the fifth barcode sequence is different from the sixth third barcode sequence.

[0016] 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 rows, and wherein the first orthogonal channel and the second orthogonal channel at least partially coincide with the columns. In some cases, the first channel and the second channel at least partially coincide with the columns, and wherein the first orthogonal channel and the second orthogonal channel at least partially coincide with the rows.

[0017] In some cases, the coupling in (b) comprises sealingly attaching a channel template comprising a first channel and a second channel to a surface. In some cases, the coupling in (a) comprises sealingly attaching an orthogonal channel template comprising a first orthogonal channel and a second orthogonal channel to a surface.

[0018] In some cases, each of the plurality of arrays is identical. In some cases, the spacing between reaction sites of an array in the plurality of arrays is in the range of 50-500 μm, and wherein each of the reaction sites has a diameter in the range of 30-300 μm. In some cases, the density of reaction sites of an array in the plurality of arrays is in the range of 100-1500 μm per mm. 2 The number of reaction sites ranges from 50 to 200.

[0019] In some cases, the first channel oligonucleotide is coupled to the first reaction site oligonucleotide and includes extending the first channel oligonucleotide using a DNA polymerase. In some cases, the first channel oligonucleotide is coupled to the first reaction site oligonucleotide and includes connecting the first channel oligonucleotide to the first reaction site oligonucleotide. In some cases, the first orthogonal channel oligonucleotide is coupled to the first channel oligonucleotide and includes extending the first channel oligonucleotide using a DNA polymerase. In some cases, the first orthogonal channel oligonucleotide is coupled to the first channel oligonucleotide and includes connecting the first orthogonal channel oligonucleotide to the first channel oligonucleotide.

[0020] In some cases, the surface also includes a capture probe attached thereto, and wherein the method also includes capturing the sample nucleic acid with the capture probe, and extending the capture probe using the captured sample nucleic acid as a template. In some cases, any one of the first reaction site oligonucleotide or the second reaction site oligonucleotide, the first channel oligonucleotide or the second channel oligonucleotide, or the first orthogonal channel oligonucleotide or the second orthogonal channel oligonucleotide includes the capture probe.

[0021] In another aspect, a flow cell is provided herein, comprising: one or more arrays, wherein an array in the one or more arrays is located at the intersection of a row and a column on the surface of the flow cell, wherein the array comprises one or more reaction sites, and wherein the reaction sites in the one or more reaction sites comprise: (a) a first oligonucleotide sequence unique to the spatial position of the reaction site within the array, (b) a second oligonucleotide unique to the row, and (c) a third oligonucleotide unique to the column. 59. In some embodiments, the array, row, column, or any combination thereof is configured to receive a first oligonucleotide, a second oligonucleotide, a third oligonucleotide, or any combination thereof. In some embodiments, the spacing between a reaction site of the array and a second reaction site is in the range of 50-500 μm, and wherein the diameter of the reaction site is in the range of 30-300 μm. In some embodiments, the density of one or more reaction sites is per mm 2In some embodiments, the flow cell further comprises a second array, wherein the second array is located at a second intersection of the second row and the second column, wherein the second array comprises one or more second reaction sites, and wherein the second reaction sites in the one or more second reaction sites comprise: a fourth oligonucleotide sequence unique to the spatial position of the second reaction site within the second array; a fifth oligonucleotide unique to the second row; and a sixth oligonucleotide unique 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 according to claim 58, and wherein the first oligonucleotide sequence and the fourth oligonucleotide sequence are identical. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A One format of a combined spatial barcode that may be used with the systems and methods described herein is shown.

[0023] Figure 1B An embodiment of the systems and methods described herein is shown in which the final barcode segments are attached by tagging.

[0024] Figure 2A and Figure 2B An embodiment for producing a spatially barcoded surface is shown.

[0025] Figure 2C-Figure 2E An embodiment for generating a spatially barcoded surface is shown, wherein by adjusting the dot pattern of the array ( Figure 2D ) or multiple channel templates with offset channel positions ( Figure 2E ) to partially overlap the channel with the array of the first oligonucleotides.

[0026] Figure 2F The generation of a combined barcode in one dimension by employing multiple channel templates with different channel widths and offsets is shown.

[0027] Figure 3 Apparatus for creating channels for applying reagents to rows or columns of a dotted array on a surface is shown. DETAILED DESCRIPTION

[0028] Unless otherwise stated, the practice of the systems and methods described herein can adopt the conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant technology), cell biology and biochemistry within the technical scope 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. By referring to the examples below this article, a specific description of suitable technology can be obtained. However, other equivalent routine procedures can also be used, of course. Such conventional techniques and descriptions can be found in standard laboratory 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, ed., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, ed., Protein Engineering Handbook (Wiley-VCH, 2009); and the like. Guidance for selecting materials and components to perform particular functions can be found in available treatises and references on scientific instrumentation, including but not limited to Moore et al., Building Scientific Apparatus, 3rd ed. (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd ed. (Academic Press, 2013); and similar references.

[0029] The systems and methods described herein relate to making or generating a surface barcoded in space, and the use thereof for analyzing the molecules (especially nucleic acid molecules) of biological cells arranged on such surfaces. The systems and methods described herein also relate to making the nucleic acid molecules arranged or captured on the surface barcoded in space. Spatial barcodes can be combined, because each barcode is a combination of at least three segments (two segments of the position of the array on the recognition surface and the third segment of the position of the barcode oligonucleotide or the nucleic acid molecule attached to it in the recognition array). In addition, in some embodiments, the final spatial barcode library includes every combination of the possible sequences of the first barcode segment, the second barcode segment and the third barcode segment. Therefore, in the embodiment using three barcode segments, the number of the first oligonucleotide (each comprising the first barcode segment) in the array, the number of channels for delivering the second oligonucleotide (each comprising the second barcode segment) and the number of channels for delivering the third oligonucleotide (each comprising the third barcode segment) determine the total number of different barcodes on the surface. For example, for an array of 384 first oligonucleotides, a 50-channel first channel template and a 50-channel second channel template, the surface can have 980,000 (=50×50×384) different barcodes. The channel template and the gasket for sealingly attaching the template to the surface can be made using the manufacturing technology used for microfluidic devices.

[0030] A variety of surfaces can be used together with systems and methods described herein. In some embodiments, the surface is a two-dimensional planar surface of a solid support material. Such solid support materials can include non-porous solids, which can be derived with conventional functional groups, and oligonucleotides can be attached by these conventional functional groups (e.g., Devor et al., Integrated DNA Technologies (2005) etc.). In some embodiments, such solid support materials can include glass, plastics, silicon, metal oxides, etc. In some embodiments, the surface is a glass support material, such as a glass slide.

[0031] In various embodiments of the systems and methods described herein, barcode segments can be attached before and / or after capturing and replicating nucleic acid molecules from a sample. In other words, the order in which the barcode segments and sample nucleic acids are assembled on the surface can be different, so that the order of cDNA (transcribed from the captured nucleic acid) and the barcode segments that constitute the spatial barcode can be selected. In different embodiments, such an ordering (starting from the surface) can be as follows: -cDNA-BC1-BC2-BC3; BC1-cDNA-BC2-BC3; BC1-BC2-cDNA-BC3; or BC1-BC2-BC3-cDNA, wherein BC1, BC2, and BC3 represent the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, respectively (comprising the first barcode segment, the second barcode segment, and the third barcode segment). The assembly of the first oligonucleotide, the second oligonucleotide and the third oligonucleotide for producing a barcoded surface, or the assembly of the cDNA and the first oligonucleotide, the second oligonucleotide and the third oligonucleotide for producing a surface having a spatially barcoded cDNA, is accomplished using conventional methods for attaching nucleic acid molecules to each other or to a surface, which is described in Figure 1A and Figure 1B The examples are illustrated in the embodiments described in FIG.

[0032] Although embodiments showing the formation of a spatial barcode comprising two or three barcode segments are disclosed, the systems and methods described herein may also include a combined spatial barcode of multiple barcode segments. In some embodiments, the combined spatial barcode comprises 3 to 6 barcode segments; or 3 to 5 barcode segments; or 3 to 4 barcode segments. In some embodiments, a combined spatial barcode having more than three barcode segments may be generated by applying additional steps of partitioning and reacting using (or reusing) a channel template loaded with oligonucleotides comprising different barcode sequence combinations.

[0033] In some embodiments, the array of the first oligonucleotide array is synthesized (or disposed) on a surface, e.g. Figure 2A As shown, the second and third oligonucleotides are then attached by forming orthogonal channels for delivering the oligonucleotides (e.g., Figure 2A-2B In some embodiments, the surface (202) (see Figure 2A) may not contain capture oligonucleotides, so that the gap space (203) between arrays (and between points or reaction sites within the array) does not contain barcodes. In other embodiments, the surface (202) can be coated with capture oligonucleotides to capture various barcode oligonucleotides (first, second or third), which can then be extended or connected to form a combined barcode. In other words, in some embodiments, the surface functional group can include capture oligonucleotides. In such a latter embodiment, a barcoded surface can be produced, wherein the gap space (e.g., 203) in the arrays in the multiple arrays contains one or more segments of barcodes. In some embodiments, the channel delivery and droplet delivery of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide can be different. In some embodiments, the arrays in the multiple arrays of the first oligonucleotide are delivered by droplets, and then the second oligonucleotide and the third oligonucleotide are delivered by channels. In other embodiments, the first oligonucleotide is delivered by channel, the arrays in the multiple arrays of the second oligonucleotide are delivered by droplets, and the third oligonucleotide is delivered by channel. In yet other embodiments, the first oligonucleotide is delivered via a channel, the second oligonucleotide is delivered via a channel, and an array of a plurality of arrays of third oligonucleotides is delivered via droplets.

[0034] Figure 1A An embodiment is shown for sequentially linking three barcode segments to form a spatial barcode on a surface, after which sample nucleic acid can be captured (ie, the fourth format described above: BC1-BC2-BC3-sample NA). In one embodiment, a first oligonucleotide (102) comprising a first barcode segment (BC1) (106) and a sequence (S1) (104) is attached to a surface (100) via its 5'-end by any of a variety of linkages 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 reaction of surface functional groups with complementary functional groups of the attached oligonucleotide. In some embodiments, an attached oligonucleotide (such as a capture oligonucleotide or a barcode oligonucleotide) is attached via its 5 '-end, for example, leaving its 3 '-end free for subsequent extension by a polymerase.

[0035] According to some embodiments, a DNA printing device (such as a device manufactured by M2 Automation (Berlin, Germany), Scienion (Berlin, Germany), etc.) is used to deliver first oligonucleotides (102) with different barcode sequences to separate known locations in the array. In some embodiments, an inkjet delivery system can be used to construct multiple arrays, for example, Cartesian Technologies (Irvine, California); Barczak et al., Genome Research, 13: 1775-1785 (2003); etc. In some embodiments, a variety of array synthesis techniques can be used to synthesize the first oligonucleotides of multiple arrays in situ, for example, 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 arrays include a spatially compact linear or hexagonal array of non-overlapping (i.e., spatially discrete) reaction sites substantially uniformly coated with the first oligonucleotide (102). In some embodiments, arrays of such reaction sites may have a pitch (center-to-center distance) in the range of (but not limited to) 50-500 μm and a diameter in the range of 30-100 μm. Return Figure 1A , a first oligonucleotide (102) attached to a surface (100) can be hybridized (or annealed (108)) with a second oligonucleotide (110), the second oligonucleotide (110) comprising segment S1' (complementary to segment S1 (104)), a second barcode segment BC2 and segment S2'. Thereafter, a reagent can be introduced to extend the first oligonucleotide (102) so that BC2 and S2' of the second oligonucleotide (110) are replicated to form a first conjugated surface oligonucleotide. In alternative embodiments, the second barcode segment (113) can be attached to the first oligonucleotide (102) by connecting the second oligonucleotide using, for example, a ligase, thereby forming a first conjugated surface oligonucleotide. In some alternative embodiments, continuous oligonucleotide segments can be attached by connecting using a ligase and a splint oligonucleotide, which forms a duplex with the two oligonucleotides to be connected. In further embodiments, continuous oligonucleotide segments can be attached by connecting using a circular ligase.

[0036] Figure 2A-2BA method of delivering a second oligonucleotide (110) and an agent for extending a first oligonucleotide (102) is shown. After hybridization and extension (112), the hybridized and replicated second oligonucleotide (110) can be unzipped (114) from a chain (113). Chain (113) (sometimes referred to herein as a "first conjugated surface oligonucleotide") can be annealed (116) with a third oligonucleotide (118), which includes segment S2' (complementary to segment S2 of chain (113)), a third barcode segment (BC3), and segment S3'. After extension (120) and washing and unzipping (122), the result is a spatial barcode (124) including barcode segments BC1, BC2, and BC3, the combination of which can be unique for each reaction site in a plurality of arrays. In some embodiments, segment S3 (125) can be used as a capture oligonucleotide. For example, it may be a polyT sequence for capturing polyA-tailed messenger RNA from cells of the sample being analyzed on the surface (100).Similar to above, in an alternative embodiment, the third barcode segment (118) may be attached to the strand (113) by ligation.

[0037] Figure 1B An alternative embodiment is shown that uses tagging to attach a third barcode component (thereby forming the barcoded sample nucleic acid of the third format described above, i.e., BC1-BC2-sampleNA-BC3). A review of tagging technology is given in Adey, Genome Research, 31: 1693-1705 (2021); U.S. Patents 9115396, 9085801, 11319534; etc. (which are incorporated herein by reference). The barcode segments BC1-S1 and BC2-S2 (150) can be as follows Figure 1A The biological sample (151) can be assembled as described in, wherein segment S2 is a capture probe (e.g., a polyT segment specific for the polyA messenger RNA of the biological sample). The biological sample (151) can be contacted with the surface (100) so that the polyA mRNA contained therein anneals (152) to capture oligonucleotides (S2, 153), wherein the mRNA includes a polyA segment (154) and a coding segment (156). After extension with a reverse transcriptase and optional template switching, a double-stranded structure (159) can be obtained, which is then labeled (160) to attach the final barcode segment BC3, thereby generating a final sequence (162). Using a similar procedure, each format (-sample NA-BC1-BC2-BC3; -BC1-sample NA-BC2-BC3; -BC1-BC2-sample NA-BC3; or -BC1-BC2-BC3-sample NA) can be synthesized.

[0038] According to some embodiments described herein, a second oligonucleotide and a third oligonucleotide comprising a second barcode segment and a third barcode segment, respectively, are delivered to a plurality of arrays via a channel, such as Figure 2A-2B Alternative embodiments for delivery and conjugation to the first oligonucleotide (or conjugate of the first and second oligonucleotides) may include the use of a photomask and photoactivated ligation, for example, as taught by van Dam (California Institute of Technology, 2005). Figure 2A As shown, multiple arrays (e.g., 204) can be synthesized on a surface (202) of a slide or substrate (200). In this illustration, the multiple arrays are 240 arranged in a 24×10 linear format. For ease of illustration, the spacing of the arrays on the surface (202) is exaggerated. An enlarged view (206) of the array shows a 32×24 array of reaction sites (208). In some embodiments, each of the multiple 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 the 11th column of the array (205) is the same as the sequence of the barcode segment of the first oligonucleotide at the 18th row and the 11th column of the array (204). That is, in some embodiments, each of the multiple arrays includes the same first oligonucleotide.

[0039] In this embodiment, the second oligonucleotide and associated extension reagents (e.g., DNA polymerase, reaction buffer, dNTPs, etc.) are delivered through a channel formed in a material layer (e.g., elastomeric plastic, etc.), forming a channel body or template that can be placed over multiple arrays and divided into multiple rows or columns. One of ordinary skill in the art will understand that multiple arrays, rows, columns, first channels, second channels, etc. are independent quantities; that is, multiple values ​​of these individual features do not have to be the same in any particular embodiment. Figure 2A As shown, the channel template (210) can be placed (212) on the surface (202) to divide the plurality of arrays into a plurality of 24 rows, with 10 arrays in each row. The channel template (210) can be placed on the surface (202) using a method similar to Figure 3The channel template (210) can be realized by a simple device shown in the figure, which sandwiches the channel template (210) between the surface (202) and the cover (207) of the substrate (203). Depending on the scale and arrangement of the multiple arrays, the size and arrangement of the array of reaction sites, and the method for coupling the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, the design and composition of the channel template can vary greatly. The channel template can be made of a variety of materials (such as silicon, glass, plastic, etc.) known in the field of microfluidics, for example, Ren et al., Acc. Chem. Res., 46 (11): 2396-2406 (2013). In some embodiments, the channel template can include plastics, such as polystyrene, polyethylenetetraphthalate glycol, polyethylene terephthalate, polymethyl methacrylate, polyvinyl chloride, polycarbonate, thermoplastic elastomer, etc. Guidance for the selection of plastics and manufacturing methods 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 6,803,019; Soane et al., U.S. Patent 6,176,962; Schaevitz et al., U.S. Patent 6,908,594; Neyer et al., U.S. Patent 6,838,156; etc., these references are incorporated herein by reference.

[0040] As shown in the cross-sectional view (216), along the centerline (214) of the channel (213), after assembling the base (203), the channel template (210) and the cover (207), a dedicated flow path (211) is created for each row of arrays. Thus, each array of a given row can receive the same second oligonucleotide. In some embodiments, the sequence of the barcode segment of each second oligonucleotide in different rows is different, so that the sequence of the second barcode segment uniquely identifies the row where the spatial barcode is located.

[0041] After the second oligonucleotide is delivered and coupled to the first oligonucleotide, the lane template can be removed (210). Figure 2BAs shown, a column channel template (220) can be placed (224) on the surface (202) of the substrate (203) to divide the multiple arrays into a plurality of 10 columns (e.g., 222), with 24 arrays in each column. As with the division into rows, the channel template (220) can create a dedicated flow path for each column, which allows the arrays in each column to be exposed to the same third oligonucleotide. In some embodiments, the sequence of the barcode segment of each third oligonucleotide in different columns is different, so that the sequence of the third barcode segment uniquely identifies the column where the spatial barcode is located. After coupling the third oligonucleotide (226), a spatially barcoded surface having a spatial barcode in the form shown in the enlarged view (228) can be created.

[0042] In some embodiments, the number of unique barcodes on a surface can be increased by providing channels that coincide with subsets of reaction sites in rows or columns of an array. Figure 2C An example of a column of an array (232) shown in an enlarged view relative to an array in a plurality of arrays (230) of this embodiment is shown. In this embodiment, the width of the channel (e.g., 234a and 234b in the enlarged view) is made so that the channel coincides with half of the points (or reaction sites) of the array (231, darker shaded subarray) of the column, so that the barcode oligonucleotide can be attached (237) to the first half reaction site (e.g., 240) and then attached (238) to the second half reaction site (e.g., 242) in sequence. This can be achieved by using two different channel templates in which the channel positions are offset (e.g.) by half the array width or by shifting a single channel template by half the array width. Figure 2E A case is shown where two channel templates (262 and 264) (or a gasket assembly of such channel templates) are used, where the channels are offset by a predetermined amount (266) such that different reaction sites are exposed to reagents delivered through the channels. After these steps, the number of different spatial barcodes of arrays in the plurality of arrays (230) will be twice that of an embodiment where each entire subarray (e.g., 232) coincides with a channel delivering a barcode oligonucleotide. In some embodiments, such as Figure 2DAs shown, the channel template can be manufactured so that the two halves of the subarray overlap with different channels, wherein the space (248) is selected so that the wall of the channel template can be installed without covering or obstructing the reaction site. This configuration improves the manufacturing speed and simplifies the oligonucleotide deposition process, so that, for example, different barcode segments are added to each half of the reaction site at the same time, thereby producing products (258 and 260) in a single subarray (256). This embodiment is realized by forming a subarray with a gap (255) in the subarray (256), and the gap (255) separates the two halves and provides space for the wall of the channel template. In other embodiments, the subarray can be formed with multiple gaps, so that, for example, the reaction site is divided into three regions instead of two regions, and such gaps can be formed in both the horizontal direction and the vertical direction, for example, respectively for attaching the second oligonucleotide and the third oligonucleotide.

[0043] In some embodiments, Figure 2F As shown, spatial barcode oligonucleotides can be formed in combination by delivering their components with channels of different widths. For example, a channel (270) can be established by attaching a first channel template to a surface including a subarray (268), and the channel (270) delivers a barcode oligonucleotide that reacts with a surface or conjugated surface oligonucleotide to produce a product (e.g., 274) in half of the reaction sites of the subarray (268). Subsequently, a channel (272) can be established by attaching a second channel template, and the channel (272) delivers a barcode oligonucleotide that reacts with a surface or conjugated surface oligonucleotide to produce a product (e.g., 276) in the other half of the reaction sites of the subarray (268). Next, a channel (282 and 284) is established by attaching a third channel template with a narrower interval channel, and the channel (282 and 284) delivers a reagent (280) to the reaction sites of the first and third quadrants of the subarray (268), producing products (290) and (294) after the reaction. After step (280), channels (286 and 288) are created by attaching a fourth channel template with spaced channels, which deliver reagents (281) to the reaction sites in the second and fourth quadrants of the subarray (268), producing products (292 and 296) after reaction. In such an embodiment, a barcoded surface can be produced with four times the number of unique barcodes as in an embodiment where the channels coincide with the entire subarray.

[0044] As mentioned above, you can use Figure 3The apparatus or similar device shown applies channel templates (210) and (220) to the surface (202). A substrate (300) including a surface (302) having a plurality of arrays (304) can be placed in a base (306), and a channel template (308) can be placed on top, thereby creating partitions of rows (or columns) of the array. A manifold (310) can be placed on top of the channel template (308) to provide conduits from reagent reservoirs or plates 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.

[0045] Although the invention has been described with reference to several specific example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the invention. The invention is also applicable to various sensor implementations and other subjects in addition to those discussed above.

[0046] definition

[0047] Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow standard treatises and texts in the field, e.g., Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immuology, Sixth Edition (Saunders, 2007).

[0048] "Barcode" refers to a molecular marker or identifier. In some embodiments, a barcode is a molecule attached to an analyte or a segment of an analyte (e.g., in the case of a polynucleotide barcode and an analyte) that can be used to identify the analyte. In some embodiments, barcodes (referred to herein as "spatial barcodes") can be attached to a surface to identify locations on the surface. In some embodiments, populations of identical spatial barcodes can be set within specific areas on a surface. The size and shape of such areas can vary greatly. In some embodiments, areas with unique spatial barcodes are of the same size and are set on the surface in a regular pattern with a spatial barcode density per unit area. In some embodiments, the density of such barcodes can range from 100% per mm2 1 barcode per mm 2 1000 barcodes or more, or from 1000 barcodes per mm 2 1 barcode per mm 2 500 barcodes or more, or from 1 mm 2 1 barcode per mm 2 200 barcodes vary. In some embodiments, there may be a one-to-one correspondence between different spatial barcodes and different areas on the surface; that is, each different area may have a different and unique barcode. In some embodiments, the identity of the spatial barcode is determinable, for example, as long as the spatial barcode is a polynucleotide, it can be determined by sequencing. In some embodiments, the spatial barcode is an oligonucleotide. In some embodiments, the oligonucleotide spatial barcode includes a random sequence oligonucleotide. Random sequence oligonucleotides are typically synthesized by a "split and mix" synthesis technique, for example, as described in the following references incorporated herein by reference: Church, U.S. Patent 4942124; Godron et al., International Patent Publication WO2020 / 120442; Seelig et al., U.S. Patent Publication 2016 / 0138086; etc. Sometimes, random oligonucleotides are represented as "NNN...N". In some embodiments, the term "barcode" includes a composite barcode; that is, an oligonucleotide segment including subsegments that identify different objects. For example, a first segment of a composite barcode may identify a specific area of ​​a surface, and a second segment of the composite barcode may identify a specific molecule (a so-called "unique molecular identifier," or UMI).

[0049] A "microfluidic" device or a "nanofluidic" device, used interchangeably herein, each refers to an integrated system for capturing, moving, mixing, dispensing, or analyzing small volumes of fluids, including samples (which in turn may contain or include cellular or molecular analytes of interest), reagents, diluents, buffers, and the like. In general, references to "microfluidics" and "nanofluidics" indicate different scales of device size and volume of fluid processed. In some embodiments, features of a microfluidic device have cross-sectional dimensions of less than a few hundred square microns, and have passages or channels having capillary dimensions, for example, having cross-sectional dimensions of about 1-2 mm to about 0.1 μm. In some embodiments, the volume capacity of a microfluidic device is in the range of 100 μL to a few nL (e.g., 10-100 nL), or in the range of 100 μL to 1 μL. The dimensions of corresponding features or structures in a nanofluidic device are typically 1 to 3 orders of magnitude smaller than the dimensions of corresponding features or structures of a microfluidic device. One skilled in the art will know which dimensions will be relevant from the context of a particular application. In some embodiments, the microfluidic or nanofluidic device has one or more chambers, ports and channels, which are interconnected and fluidically connected, and are designed to perform one or more reactions or processes alone or in conjunction with an apparatus or instrument that provides a supporting function (such as sample introduction, fluid and / or reagent drive device, such as positive or negative pressure, acoustic energy, etc., temperature control, detection system, data collection and / or integrated system, etc.). In some embodiments, the microfluidic and nanofluidic devices may also include valves, pumps, filters and special functional coatings on the inner wall, such as to prevent adsorption of sample components or reactants, promote reagent movement by electroosmosis, etc. Such devices can be manufactured as integrated devices in solid substrates, which can be glass, plastic or other solid polymer materials, and can have a planar format to facilitate detection and monitoring of sample and reagent movement, especially via optical or electrochemical methods. In some embodiments, such devices are disposable after a single use. In some embodiments, the microfluidic and nanofluidic devices include devices for forming and controlling the movement, mixing, distribution and analysis of droplets (such as aqueous droplets immersed in immiscible fluids (such as light oils)).The manufacture and operation of microfluidic and nanofluidic devices are well known in the art, as exemplified in the following references, which are incorporated by reference: Ramsey, U.S. Pat. Nos. 6,001,229, 5,858,195, 6,010,607, and 6,033,546; Soane et al., U.S. Pat. Nos. 5,126,022 and 6,054,034; Nelson et al., U.S. Pat. No. 6,613,525; Maher et al., U.S. Pat. No. 6,399,952; Ricco et al., International Patent Publication No. WO 02 / 24322; Bjornson et al., International Patent Publication No. WO 99 / 19717; Wilding et al., U.S. Patents 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 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

1. A method for producing 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 being attached to the surface via its 5'-end, wherein (i) each first oligonucleotide has a barcode segment comprising a barcode sequence that is different from the barcode sequences of other first oligonucleotides; and (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 location; (c) dividing the surface into rows by sealingly attaching a first channel template comprising a plurality of channels to the surface; as well as (d) reacting a second oligonucleotide loaded into each channel of the first channel template with the surface or a previously conjugated surface oligonucleotide to form a conjugated surface oligonucleotide or a spatial barcode oligonucleotide, wherein each different channel comprises a second oligonucleotide comprising a second barcode segment, the second barcode segment comprising a barcode sequence different from the barcode sequence of the second oligonucleotide of the different channel, wherein (i) the rows of the plurality of arrays at least partially overlap with the rows of the first channel template, and (ii) each spatial barcode oligonucleotide uniquely identifies the spatial location of its reaction site on the surface.

2. The method according to claim 1, further comprising: (a) dividing the surface into columns by sealingly attaching a second channel template comprising a plurality of channels to the surface; as well as (b) reacting the third oligonucleotide loaded into each channel of the second channel template with the surface or previously conjugated surface oligonucleotide to form a conjugated surface oligonucleotide or a spatial barcode oligonucleotide, wherein each different channel of the second channel template comprises a third oligonucleotide comprising a third barcode segment, the third barcode segment comprising a barcode sequence different from the barcode sequence of the third oligonucleotide of the different channel of the second channel template, Wherein the columns of the plurality of arrays at least partially overlap with the columns of the second channel template. The method of claim 1 , wherein each of the plurality of arrays is identical.

4. The method of claim 1, wherein 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 single array with any column.

5. The method of claim 1, wherein the spacing between the reaction sites of each of the array of reaction sites is in the range of 50-500 μm, and wherein the diameter of each of the reaction sites is in the range of 30-300 μm.

6. The method of claim 1, wherein the density of each reaction site in the array of reaction sites is 2 The number of reaction sites ranges from 50 to 200. The method of claim 1 , wherein each reaction step is performed by extending the first oligonucleotide, the second oligonucleotide or the third oligonucleotide by a DNA polymerase.

8. The method of claim 1, wherein each reaction step is performed by connecting the first oligonucleotide to the second oligonucleotide or the third oligonucleotide, or connecting the second oligonucleotide to the first oligonucleotide or the third oligonucleotide, or connecting the third oligonucleotide to the first oligonucleotide or the third oligonucleotide.

9. The method of claim 1, wherein the surface further comprises a capture probe attached 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.

10. The method of claim 9, wherein any of the first oligonucleotide, the second oligonucleotide, or the third oligonucleotide comprises the capture probe, and wherein the capturing and the extending occur before or after any of the reacting steps.

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

12. A method according to claim 11, wherein whenever a synthesis step is performed before the step of capturing the second oligonucleotide or the third oligonucleotide or reacting the second oligonucleotide or the third oligonucleotide, the surface includes surface functional groups; and the first oligonucleotides each include a 5'-complementary functional group, so that the surface functional group reacts with the 5'-complementary functional group to form a covalent bond.

13. The method of claim 11, wherein whenever a capture step is 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.

14. The method of claim 11, wherein whenever the capture step is performed immediately after the synthesizing step, the first oligonucleotide comprises a capture oligonucleotide.

15. The method of claim 11, wherein whenever the capturing step is performed immediately after the step of reacting the second oligonucleotide, the second oligonucleotide comprises a capture oligonucleotide.

16. The method of claim 11, wherein whenever the capturing step is performed immediately after the step of reacting the third oligonucleotide, the third oligonucleotide comprises a capture oligonucleotide.

17. The method of claim 1, wherein the nucleic acid molecule is messenger RNA.

18. The method of claim 17, wherein the messenger RNA is from a single cell.

19. A method of making a spatially barcoded surface, the method comprising: (a) providing a surface comprising surface functional groups; (b) synthesizing a plurality of arrays of first oligonucleotides each comprising a 5'-complementary functional group by depositing first oligonucleotides having different barcode sequences at different reaction sites on the surface under conditions in which the surface functional groups react with the 5'-complementary functional groups to form covalent bonds, wherein (i) the plurality of arrays are arranged in orthogonal rows and columns; (ii) each array comprises a plurality of reaction sites, the plurality of reaction sites each comprising a first oligonucleotide comprising a first barcode segment having a different barcode sequence whenever located at a different reaction site; and (iii) each first oligonucleotide occupies a reaction site at a known surface location; (c) dividing the plurality of arrays into rows by sealingly attaching to the surface a first channel template comprising a plurality of channels, each of the plurality of channels being aligned with a different row of the array; (d) reacting the first oligonucleotide in each channel with the second oligonucleotide loaded into each channel of the first channel template to form a first conjugated surface oligonucleotide, wherein each different channel includes a second oligonucleotide comprising a second barcode segment, the second barcode segment comprising a barcode sequence different from the barcode sequence of the second oligonucleotide of the different channel; (e) dividing the plurality of arrays into columns by sealingly attaching to the surface a second channel template comprising a plurality of channels, each of the plurality of channels being aligned with a different column of the array; as well as (f) reacting the first conjugated surface oligonucleotide in each channel with the third oligonucleotide loaded into each channel of the second channel template to form a spatial barcode oligonucleotide, wherein (i) each different channel of the second channel template comprises a third oligonucleotide comprising a third barcode segment, wherein the third barcode segment comprises a barcode sequence different from the barcode sequence of the third oligonucleotide 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 of claim 19, wherein each of the plurality of arrays is identical.

21. The method of claim 19, wherein 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 single array with any column.

22. The method of claim 19, wherein the spacing between the reaction sites of each of the array of reaction sites is in the range of 50-500 μm, and wherein the diameter of each of the reaction sites is in the range of 30-300 μm.

23. The method of claim 19, wherein the density of each of the reaction sites in the array of reaction sites is 2 The number of reaction sites ranges from 50 to 200.

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

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

26. The method of claim 19, wherein the surface further comprises a capture probe attached 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.

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

28. A method of generating a spatially barcoded surface, comprising: (a) providing a solid support comprising a surface, wherein the surface comprises a plurality of arrays arranged on the surface, wherein each array comprises a plurality of reaction sites, wherein each reaction site comprises a reaction site oligonucleotide having a barcode sequence unique to the reaction site where the reaction site is located; (b) dividing the surface into one or more channels by coupling a channel template comprising a plurality of channels to the surface; as well as (c) loading a plurality of channel oligonucleotides into the plurality of channels such that at least one channel oligonucleotide is coupled to the reaction site oligonucleotides in each array, wherein each channel oligonucleotide comprises a barcode sequence unique to the channel in which it is located.

29. The method according to claim 28, further comprising: (a) dividing the surface into one or more orthogonal channels by coupling an additional 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; as well as (b) loading a plurality of orthogonal lane oligonucleotides into the plurality of orthogonal lanes such that at least one orthogonal lane oligonucleotide is coupled to the channel oligonucleotide in each array, wherein each orthogonal lane oligonucleotide comprises a barcode sequence unique to the orthogonal lane in which it is located.

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 wherein the one or more 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 wherein the one or more orthogonal channels at least partially coincide with the rows.

33. The method of claim 28, wherein the coupling in (b) comprises sealingly attaching the channel template comprising the plurality of channels to the surface.

34. The method of claim 29, wherein the coupling in (a) comprises sealingly attaching the additional channel template comprising the plurality of orthogonal channels to the surface.

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

36. The method of claim 28, wherein the spacing between the reaction sites of an array in the plurality of arrays is in the range of 50-500 μm, and wherein the diameter of each of the reaction sites is in the range of 30-300 μm.

37. The method of claim 28, wherein the density of reaction sites in the arrays of the plurality of arrays is 2 The number of reaction sites ranges from 50 to 200.

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

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

40. The method of claim 29, wherein the surface further comprises a capture probe attached thereto, and wherein 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.

41. The method of claim 40, wherein any of the reaction site oligonucleotide, the channel oligonucleotide, or the orthogonal channel oligonucleotide comprises the capture probe.

42. A method of making a spatially barcoded surface, the method comprising: (a) providing a solid support comprising a surface, wherein the surface comprises a plurality of arrays, wherein an array in the plurality of arrays comprises at least two reaction sites, wherein a first reaction site in the at least two reaction sites comprises a first reaction site oligonucleotide comprising a first reaction site barcode sequence, and wherein a second reaction site in the at least two reaction sites comprises a second reaction site oligonucleotide comprising a second reaction site barcode sequence, and wherein 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; as well as (d) loading a second channel oligonucleotide into the second channel, wherein after said loading of (c), said first channel oligonucleotide is coupled to said first reaction site oligonucleotide, and wherein after said loading of (d), said second channel oligonucleotide is coupled to said second reaction site oligonucleotide, and Wherein the first channel oligonucleotide comprises a third barcode sequence, and the second channel oligonucleotide comprises a fourth barcode sequence, and wherein the third barcode sequence is different from the fourth third barcode sequence.

43. The method of claim 42, further comprising: (a) coupling an orthogonal channel template comprising a first orthogonal channel and a second orthogonal channel to the surface; (b) loading a first orthogonal channel oligonucleotide into the first channel; as well as (c) loading a second orthogonal channel oligonucleotide into the second channel, wherein after said loading of (b), said first orthogonal channel oligonucleotide is coupled to said first channel oligonucleotide, and wherein after said loading of (c), said second orthogonal channel oligonucleotide is coupled to said second channel oligonucleotide, and Wherein the first orthogonal channel oligonucleotide comprises a fifth barcode sequence, and the second orthogonal channel oligonucleotide comprises a sixth barcode sequence, and wherein the fifth barcode sequence is different from the sixth third barcode sequence.

44. The method of claim 43, wherein the plurality of arrays are arranged in rows and columns.

45. The method of claim 43, wherein the first channels and the second channels at least partially coincide with the rows, and wherein the first orthogonal channels and the second orthogonal channels at least partially coincide with the columns.

46. ​​The method of claim 43, wherein the first channels and the second channels at least partially coincide with the columns, and wherein the first orthogonal channels and the second orthogonal channels at least partially coincide with the rows.

47. The method of claim 42, wherein the coupling in (b) comprises sealingly attaching the channel template comprising the first channel and the second channel to the surface.

48. The method of claim 43, wherein the coupling in (a) comprises sealingly attaching the orthogonal channel template comprising the first orthogonal channel and the second orthogonal channel to the surface.

49. The method of claim 42, wherein each of the plurality of arrays is identical.

50. The method of claim 42, wherein the spacing between the reaction sites of an array in the plurality of arrays is in the range of 50-500 μm, and wherein the diameter of each of the reaction sites is in the range of 30-300 μm.

51. The method of claim 42, wherein the density of reaction sites in the arrays of the plurality of arrays is 2 The number of reaction sites ranges from 50 to 200.

52. The method of claim 42, wherein coupling the first channel oligonucleotide to the first reaction site oligonucleotide comprises extending the first channel oligonucleotide using a DNA polymerase.

53. The method of claim 42, wherein coupling the first channel oligonucleotide to the first reaction site oligonucleotide comprises ligating the first channel oligonucleotide to the first reaction site oligonucleotide.

54. The method of claim 43, wherein coupling the first orthogonal channel oligonucleotide to the first channel oligonucleotide comprises extending the first channel oligonucleotide using a DNA polymerase.

55. The method of claim 42, wherein coupling the first orthogonal channeling oligonucleotide to the first channeling oligonucleotide comprises ligating the first orthogonal channeling oligonucleotide to the first channeling oligonucleotide.

56. The method of claim 43, wherein the surface further comprises a capture probe attached thereto, and wherein 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.

57. The method of claim 56, wherein any one of the first reaction site oligonucleotide or the second reaction site oligonucleotide, the first channel oligonucleotide or the second channel oligonucleotide, or the first or second orthogonal channel oligonucleotide comprises the capture probe.

58. A flow cell comprising: one or more arrays, wherein an array in the one or more arrays is located at an intersection of a row and a column on a surface of the flow cell, wherein the array comprises one or more reaction sites, and wherein a reaction site in the one or more reaction sites comprises: (a) a first oligonucleotide sequence unique to the spatial location of the reaction site within the array; (b) a second oligonucleotide unique to said row; and (c) a third oligonucleotide unique to said column.

59. The flow cell of 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.

60. The flow cell of claim 58, wherein the spacing between the reaction sites of the array and a second reaction site is in the range of 50-500 μm, and wherein the diameter of the reaction sites is in the range of 30-300 μm.

61. The flow cell of claim 58, wherein the density of the one or more reaction sites is 2 The number of reaction sites ranges from 50 to 200.

62. The flow cell of claim 58, wherein the one or more reaction sites comprise one or more capture probes.

63. The flow cell of claim 58, further comprising a second array, wherein the second array is located at a second intersection of a second row and a second column, wherein the second array comprises one or more second reaction sites, and wherein a second reaction site of the one or more second reaction sites comprises: (a) a fourth oligonucleotide sequence unique to the spatial position of the second reaction site within the second array; (b) a fifth oligonucleotide unique to said second row; as well as (c) a sixth oligonucleotide unique to said second column.

64. The flow cell of claim 63, wherein the spatial positions of the second reaction sites within the second array correspond to the spatial positions of the reaction sites within the array of claim 58, and wherein the first oligonucleotide sequence and the fourth oligonucleotide sequence are identical.

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