Methods of constructing spatially barcoded surfaces

Abstract

A method of making a barcoded surface includes synthesizing a plurality of arrays arranged in rows and columns. Each array can include a plurality of discrete reaction sites having a first oligonucleotide comprising a first unique array barcode sequence. The first unique array barcode sequence of the first oligonucleotide corresponds to a mapped discrete reaction site location within the array. A second oligonucleotide can be coupled to the first oligonucleotide comprising a second unique barcode sequence. The first unique barcode sequence of the second oligonucleotide corresponds to a mapped first subset location within the plurality of arrays.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application No. 63 / 602,180, filed November 22, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0002] Molecular tagging has a long history in analytical biochemistry and molecular biology, as evidenced by Church, U.S. Patent 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 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 5,981,179; and so on. 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, as exemplified by Stahl et al., Science, 353(6294): 78-82 (2016); Salmen et al., Nature Protocols, 13:2501-2534 (2018); Frisen et al., U.S. Patent 9,593,365; and so on. However, the cost-effective synthesis of spatial barcodes with known sequences, the control over the distribution of spatial barcodes, and their density for resolving cellular and subcellular processes have remained challenges. This challenge has been addressed through various approaches, but with only partial success, as seen in publications 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

[0003] The systems, apparatus, and methods described herein relate to the use of combinatorial techniques to fabricate spatially barcoded surfaces and nucleic acid molecules. One method for fabricating a barcoded surface includes synthesizing a plurality of arrays. Each array includes a plurality of discrete reaction sites. Each discrete reaction site includes a first oligonucleotide coupled at its 5' end to a surface of a solid support. The surface includes uncoated regions between adjacent arrays. Uncoated regions include regions where the first oligonucleotide is not coupled to the surface. The first oligonucleotide includes a first unique array barcode sequence for each discrete reaction site location within the array. Each of the first unique array barcode sequences of the first oligonucleotide corresponds to a mapped discrete reaction site location within the array. A liquid containing a second oligonucleotide is disposed on each of the plurality of arrays, wherein portions of the uncoated regions between adjacent arrays are not covered by the liquid containing the second oligonucleotide. The second oligonucleotide is coupled to the first oligonucleotide to form a first surface oligonucleotide complex. The second oligonucleotide includes a second unique barcode sequence. The second unique barcode sequence includes identical sequences within a first subgroup of the plurality of arrays and distinct sequences between the respective first subgroups of the plurality of arrays. Each of the second unique barcode sequences of the second oligonucleotide corresponds to the first subgroup position of the mapping within multiple arrays.

[0004] Regarding various methods, the method may further include applying another liquid containing a third oligonucleotide to each of a plurality of arrays, wherein portions of uncoated regions between adjacent arrays are not covered by the other liquid containing the third oligonucleotide. The third oligonucleotide is coupled to a second oligonucleotide to form a second surface oligonucleotide complex. The third oligonucleotide includes a third unique barcode sequence. The third unique barcode sequence includes identical sequences within second subgroups of the plurality of arrays and distinct sequences between each of the second subgroups of the plurality of arrays. Each of the third unique barcode sequences of the third oligonucleotide corresponds to a mapped second subgroup position within the plurality of arrays.

[0005] Regarding various methods, the second oligonucleotide may further include a random barcode sequence, which is at least 99% unique in the formed second surface oligonucleotide complex.

[0006] Regarding various methods, setting the liquid containing the second oligonucleotide on each of multiple arrays may include setting liquid pools (puddles) on each array, wherein each pool does not contact adjacent pools.

[0007] Regarding the various methods, each liquid pool can be a single, unified entity of liquid.

[0008] Regarding various methods, a liquid pool is formed by setting the liquid as multiple distributed droplets that are merged together.

[0009] Regarding various methods, multiple dispensed droplets have a spacing ranging from about 1 micrometer to about 40 micrometers along the x-axis and a spacing ranging from about 1 micrometer to about 40 micrometers along the y-axis.

[0010] Regarding various methods, droplets are formed from inkjet nozzles or piezoelectric pumps.

[0011] Regarding the various methods, the droplets can each have a diameter ranging from about 80 micrometers to about 140 micrometers.

[0012] Regarding the various methods, the droplets can each have a volume ranging from about 200 picoliters to about 350 picoliters.

[0013] Regarding various methods, setting a liquid containing a second oligonucleotide on each of a plurality of arrays may include setting the liquid as a plurality of droplets, wherein each droplet at least partially covers each discrete reaction site. In various methods, each droplet is a single droplet that at least partially covers each discrete reaction site.

[0014] Regarding various methods, setting a liquid containing a second oligonucleotide on each of multiple arrays may include setting the liquid as multiple droplets, wherein each droplet covers each discrete reaction site. In various methods, each droplet is a single droplet covering each discrete reaction site.

[0015] Regarding various methods, synthesizing multiple arrays may include coupling the amine portion of a first oligonucleotide to a surface via a coupling layer, wherein the amine reactive portion of the coupling layer binds to the amine portion.

[0016] Regarding the various methods, the surface may include a glass surface, and the coupling layer may include silane bonding to the glass surface, and the amine reactive portion may include an N-hydroxysuccinimide portion.

[0017] Regarding various methods, further examples include adding a ligase to extend the first oligonucleotide after coupling the second oligonucleotide to the first oligonucleotide to form a first surface oligonucleotide complex, so that a portion of the second oligonucleotide is replicated.

[0018] Regarding various methods, multiple arrays are arranged in rows and columns.

[0019] In various methods, the rows and columns of multiple arrays are arranged in an orthogonal relationship.

[0020] Regarding various methods, each discrete reaction site of the array is formed from a droplet containing a single set of first oligonucleotides.

[0021] Regarding various methods, each of the multiple arrays may have the same patterned position of the first unique array barcode sequence of the first oligonucleotide for each discrete reaction site of the array.

[0022] Regarding various methods, each discrete reaction site in the array can have a spacing between reaction sites in the range of 50-500 micrometers, and each discrete reaction site can have a diameter in the range of 30-300 micrometers.

[0023] Regarding various methods, each discrete reaction site in the array can have [number] sites per mm. 2 Reaction site density in the range of 50 to 200 reaction sites.

[0024] Regarding the various methods, the first subgroup can be an array category selected from the following: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, first chessboard pattern, second chessboard pattern, and combinations thereof.

[0025] Regarding the various methods, the second subgroup is selected from the following array categories: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, first chessboard pattern, second chessboard pattern, and combinations thereof, wherein the array categories of the first subgroup and the second subgroup are different.

[0026] Regarding various methods, the first subgroup can be rows, where the second unique barcode sequence includes the same sequence within rows of multiple arrays and different sequences between each row of multiple arrays.

[0027] Regarding various methods, the second subgroup can be columns, where the third unique barcode sequence includes the same sequence within columns of multiple arrays and different sequences between each column of multiple arrays.

[0028] A flow cell configured to capture cells from a biological sample in a hydrogel cage includes: (a) an inlet configured to receive liquid; (b) an outlet configured to discharge liquid; (c) a top layer; (d) a spacer layer having cut-out portions; and (e) a bottom layer. The top and bottom layers are relative to the spacer layer located between the top and bottom layers. The top, bottom, and cut-out portions cooperate to form channels. The inlet and outlet are in liquid communication with the channels, thereby allowing liquid to flow through the channels. The bottom layer comprises a plurality of arrays as described herein.

[0029] For various flow pools, multiple arrays can be arranged in rows and columns.

[0030] Regarding various flow cells, each array may include multiple discrete sites, wherein conjugated surface oligonucleotides are coupled to the surface of the underlying layer at each discrete site. The conjugated surface oligonucleotides include a first oligonucleotide and a second oligonucleotide. The surface may include uncoated regions between adjacent arrays, comprising regions where the conjugated surface oligonucleotides are not coupled to the surface. The first oligonucleotide may include a first unique array barcode sequence for each discrete site location within the array. Each first unique array barcode sequence of the first oligonucleotide may correspond to a mapped discrete site location within the array. The second oligonucleotide may include a second unique barcode sequence. The second unique barcode sequence may include identical sequences within first subgroups of multiple arrays and different sequences between the first subgroups of multiple arrays. Each second unique barcode sequence of the second oligonucleotide may correspond to a mapped first subgroup location within multiple arrays.

[0031] For various flow cells, the second oligonucleotide may further include a random barcode sequence, which is at least 99% unique of the conjugated surface oligonucleotides.

[0032] Regarding various flow pools, the first subgroup can be an array category selected from the following: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, first chessboard pattern, second chessboard pattern, and combinations thereof.

[0033] Regarding the various flow pools, the second subgroup is selected from the following array categories: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, first chessboard pattern, second chessboard pattern, and combinations thereof, wherein the array categories of the first subgroup and the second subgroup are different.

[0034] Regarding various flow pools, the first subgroup can be rows, where the second unique barcode sequence includes the same sequence within rows of multiple arrays and different sequences between each row of multiple arrays.

[0035] Regarding various flow pools, the second subgroup can be a column, where the third unique barcode sequence includes the same sequence within columns of multiple arrays and different sequences between each column of multiple arrays.

[0036] In various embodiments, the first surface oligonucleotide complex can be a concatenation of a first oligonucleotide and a second oligonucleotide, and the second surface oligonucleotide complex can be a concatenation of the first, second, and third oligonucleotides. That is, the surface oligonucleotide complex can be a partially completed oligonucleotide precursor of a spatially barcoded oligonucleotide. In some embodiments, a ligase can be used to extend either the first or second surface oligonucleotide complex to produce a "spatially barcoded oligonucleotide."

[0037] As mentioned above, the terms first conjugated surface oligonucleotide or second conjugated surface oligonucleotide can refer to a partially completed oligonucleotide precursor of a spatial barcode oligonucleotide.

[0038] In some cases, each of the multiple arrays is identical. In other cases, the spacing between the reaction sites of the multiple arrays is in the range of 50-500 micrometers, and the diameter of each reaction site is in the range of 30-300 micrometers. In still other cases, the arrays of the multiple arrays have a spacing of 1 mm per... 2 Reaction site density in the range of 50 to 200 reaction sites.

[0039] In various embodiments, the first oligonucleotide may include a first barcode segment (BC1 or 106) and a first sequence (S1). The first barcode segment may include a unique array barcode sequence. In various embodiments, the second oligonucleotide may include a second barcode segment (BC2) and a second sequence (S2). The second barcode segment may include a second unique barcode sequence. In various embodiments, the third oligonucleotide may include a third barcode segment (BC3) and a third sequence (S3). The third barcode segment may include a third unique barcode sequence. Attached Figure Description

[0040] Figure 1A This paper illustrates one format of combined spatial barcodes that can be used with the systems and methods described herein.

[0041] Figure 1B An implementation scheme of the system and method described herein is shown, wherein the final barcode segment is attached via labeling.

[0042] Figure 2A and Figure 2B An implementation scheme for generating a surface that is barcode-like in space is shown.

[0043] Figures 2C-2E An embodiment for generating a surface that is spatially barcode-like is shown, wherein the dot pattern of the array is adjusted ( Figure 2D ), using multiple channel templates with offset channel positions ( Figure 2E This allows the channel to partially overlap with the array of the first oligonucleotides.

[0044] Figure 2F This demonstrates how to generate a combined barcode in one dimension by using multiple channel templates with different channel widths and offsets.

[0045] Figure 3 An apparatus is shown for creating channels to apply reagents to rows or columns of a dotted array on a surface.

[0046] Figure 4A A top view of the bottom layer of oligonucleotides in the flow cell without any spatial barcoding is shown.

[0047] Figure 4B A top view shows a spacer layer with cut-out areas suitable for use as a flow pool.

[0048] Figure 4C A top view of the top layer of the flow cell is shown, with inlet and outlet openings.

[0049] Figure 5 A top view is shown of the bottom layer and spacer layers stacked together along multiple arrays containing barcoded oligonucleotides.

[0050] Figure 6 An exploded top view of a single array of multiple overlapping droplets that partially extend beyond the boundaries of the single array is shown. Detailed Implementation

[0051] Unless otherwise stated, the systems and methods described herein can be practiced using conventional techniques and descriptions within the scope of the art in organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry. 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 descriptions of suitable techniques can be obtained by referring to the examples below. However, other equivalent conventional procedures may of course be used. 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); Site-directed Insertion of Transgenes, edited by Renault and Duchateau (Springer, Heidelberg, 2013); Protein Engineering Handbook, edited by Lutz and Bornscheuer (Wiley-VCH, 2009); and so on. Guidelines for selecting materials and components to perform specific functions can be found in available papers and references on 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.

[0052] The systems and methods described herein relate to the fabrication or generation of spatially barcoded surfaces and their use for analyzing molecules (especially nucleic acid molecules) of biological cells disposed on such surfaces. The systems and methods described herein also relate to spatially barcoding nucleic acid molecules disposed on or captured on a surface. Spatial barcodes can be combined, as each barcode is a combination of at least three segments: two segments identifying the location of an array on the surface and a third segment identifying the location of a barcode oligonucleotide within the array or a nucleic acid molecule to which it is attached. Furthermore, in some embodiments, the final spatial barcode library includes every possible combination of the sequences of the first, second, and third barcode segments. Thus, in embodiments employing three barcode segments, the number of first oligonucleotides (each containing a first barcode segment) in the array, the number of channels for delivering second oligonucleotides (each containing a second barcode segment), and the number of channels for delivering third oligonucleotides (each containing a third barcode segment) determine the total number of different barcodes on the surface. For example, for an array of 384 first oligonucleotides, a first channel template with 50 channels, and a second channel template with 50 channels, the surface can have 980,000 (=50×50×384) different barcodes. The channel templates and the gaskets for hermetically attaching the templates to the surface can be fabricated using the manufacturing techniques employed in microfluidic devices.

[0053] Various surfaces can be used with the 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 may include non-porous solids that may be derived with conventional functional groups through which oligonucleotides can be attached (e.g., Devor et al., Integrated DNA Technologies (2005), etc.). In some embodiments, such solid support materials may include glass, plastics, silicon, metal oxides, etc. In some embodiments, the surface is a glass support material, such as a glass slide.

[0054] 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 vary, allowing selection of the order of cDNA (transcribed from the captured nucleic acid) and the barcode segments constituting the spatial barcode. In different embodiments, such an order (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, where BC1, BC2, and BC3 represent the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide (containing the first barcode segment, the second barcode segment, and the third barcode segment, respectively). The assembly of the first, second, and third oligonucleotides for producing barcoded surfaces, or the assembly of cDNA and the first, second, and third oligonucleotides for producing surfaces with spatially barcoded cDNA, is accomplished using conventional methods for linking nucleic acid molecules to each other or to surfaces. Figure 1A and Figure 1B The implementation scheme described herein is illustrated with examples.

[0055] While embodiments illustrating the formation of spatial barcodes comprising two or three barcode segments have been disclosed, the systems and methods described herein may also include combined spatial barcodes comprising 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, combined spatial barcodes having more than three barcode segments can be generated by applying additional partitioning and reaction steps using (or reusing) a channel template loaded with oligonucleotides comprising different combinations of barcode sequences.

[0056] In some implementations, the array in the first oligonucleotide array is synthesized (or set) on the surface, for example, as Figure 2A As shown, the second and third oligonucleotides are then attached by forming a positive communication channel for delivery of the oligonucleotide (e.g., as shown). Figures 2A-2B As shown). In some embodiments, surface (202) (see...) Figure 2AThe surface (202) may be free of capturing oligonucleotides, such that the gap spaces (203) between arrays (and between points or reaction sites within an array) are barcode-free. In other embodiments, the surface (202) may be coated with capturing oligonucleotides to capture various barcode oligonucleotides (first, second, or third), which may then be extended or joined to form a combined barcode. In other words, in some embodiments, surface functional groups may include capturing oligonucleotides. In such a latter embodiment, a barcode-encoded surface may be produced, wherein the gap spaces (e.g., 203) between arrays of multiple arrays contain one or more segments of barcode. In some embodiments, the order of channel delivery and droplet delivery of the first, second, and third oligonucleotides may differ. In some embodiments, arrays of the first oligonucleotide are delivered by droplet delivery, followed by channel delivery of the second and third oligonucleotides. In other embodiments, the first oligonucleotide is delivered by channel, arrays of the second oligonucleotide are delivered by droplet delivery, and the third oligonucleotide is delivered by channel delivery. In some other embodiments, the first oligonucleotide is delivered via a channel, the second oligonucleotide is delivered via a channel, and an array of multiple arrays of the third oligonucleotide is delivered via droplets.

[0057] Figure 1A An embodiment is shown for sequentially connecting three barcode segments to form a spatial barcode on a surface, which can then capture sample nucleic acid (i.e., 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, for example, 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 with complementary functional groups of the attached oligonucleotide. In some implementations, the attached oligonucleotide (such as a capture oligonucleotide or a barcode oligonucleotide) is attached via its 5' end, for example, leaving its 3' end free so that it can be subsequently extended by a polymerase.

[0058] According to some embodiments, a DNA printing device (such as those manufactured by M2 Automation (Berlin, Germany), Scienion (Berlin, Germany), etc.) is used to deliver a first oligonucleotide (102) with a different barcode sequence to a separate, known location in an array. In some embodiments, an inkjet delivery system can be used to construct multiple arrays, e.g., Cartesian Technologies (Irvine, California); Barczak et al., Genome Research, 13:1775-1785 (2003); etc. In some embodiments, multiple arrays of the first oligonucleotide 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 arrays comprise 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, such arrays of reaction sites may have (but are not limited to) 50-500 Spacing within a range of m (center-to-center distance) and 30-100 Diameters within a range of m. Return Figure 1A The first oligonucleotide (102) attached to the surface (100) can hybridize (or anneal (108)) with the second oligonucleotide (110), which includes segment S1' (complementary to segment S1 (104)), a second barcode segment BC2, and segment S2'. A reagent can then be introduced to extend the first oligonucleotide (102) such that BC2 and S2' of the second oligonucleotide (110) are replicated to form the first conjugated surface oligonucleotide. In an alternative embodiment, the second barcode segment (113) can be attached to the first oligonucleotide (102) by ligating the second oligonucleotide using, for example, a ligase, to form the first conjugated surface oligonucleotide. In some alternative embodiments, consecutive oligonucleotide segments can be attached by ligation using a ligase and a splint oligonucleotide, which forms a double strand with the two oligonucleotides to be ligated. In a further embodiment, consecutive oligonucleotide segments can be attached by ligation using a cyclic ligase.

[0059] Figures 2A-2BA method for delivering a second oligonucleotide (110) and reagents for extending a first oligonucleotide (102) is illustrated. Following hybridization and extension (112), the hybridized and replicated second oligonucleotide (110) can be unwound from the strand (113) (114). The strand (113) (sometimes referred to herein as the “first conjugated surface oligonucleotide”) can be annealed (116) with a third oligonucleotide (118) comprising segment S2' (complementary to segment S2 of the strand (113)), a third barcode segment (BC3), and segment S3'. Following extension (120) and washing and unwinding (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 multiple arrays. In some embodiments, segment S3 (125) can be used as a trapping oligonucleotide. For example, it could be a polyT sequence used to capture messenger RNA with a polyA tail from cells of a sample being analyzed on the surface (100). Similarly, in an alternative embodiment, the third barcode segment (118) can be attached to the strand (113) by linking.

[0060] Figure 1B An alternative implementation is shown, which employs tagging to attach a third barcode component (thus forming a barcode of sample nucleic acid in the aforementioned third format, namely: BC1-BC2-sample NA-BC3). A review of tagging techniques is given in Adey, Genome Research, 31: 1693-1705 (2021); U.S. Patents 9,115,396, 9,085,801, 11,319,534; etc. (which are incorporated herein by reference). Barcode segments BC1-S1 and BC2-S2 (150) can be as follows Figure 1A Assembled as described above, where segment S2 is a capture probe (e.g., a polyT segment specific to polyA messenger RNA of the biological sample). The biological sample (151) can be contacted with the surface (100) such that the polyA mRNA contained therein anneals (152) to capture oligonucleotides (S2, 153), wherein the mRNA comprises a polyA segment (154) and a coding segment (156). After extension with reverse transcriptase and optional template switching, a double-stranded structure (159) can be obtained, which is then tagged (160) to attach the final barcode segment BC3, thereby producing the final sequence (162). Using a similar procedure, each format (-sampleNA-BC1-BC2-BC3; -BC1-sampleNA-BC2-BC3; -BC1-BC2-sampleNA-BC3; or -BC1-BC2-BC3-sampleNA) can be synthesized.

[0061] According to some embodiments described herein, second and third oligonucleotides, including second and third barcode segments respectively, are delivered to multiple arrays via channels, such as... Figures 2A-2B As shown. Alternative embodiments for delivery and conjugation to the first oligonucleotide (or a conjugate of the first and second oligonucleotides) may include the use of a photomask and photoactivated linkage, for example, as taught in the paper by van Dam (California Institute of Technology, 2005). Figure 2A As shown, multiple arrays (e.g., 204) can be synthesized on the surface (202) of a glass slide or substrate (200). In this illustration, the multiple arrays are 240 arrays arranged in a 24 × 10 linear format. For ease of illustration, the spacing of the arrays on the surface (202) is exaggerated. A magnified view (206) of the arrays 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 row 18 and column 11 of array (205) is the same as the sequence of the barcode segment of the first oligonucleotide at row 18 and column 11 of array (204). That is, in some embodiments, each of the multiple arrays comprises the same first oligonucleotide.

[0062] In this embodiment, the second oligonucleotide and associated extending agents (e.g., DNA polymerase, reaction buffer, dNTPs, etc.) are delivered through channels formed in a material layer (e.g., elastomeric plastic, etc.) to form a channel body or template that can be placed over multiple arrays and divided into multiple rows or columns. Those skilled in the art will understand that multiple arrays, rows, columns, first channels, second channels, etc., are independent quantities; that is, the multiple values ​​of these individual characteristics need not be the same in any particular embodiment. Figure 2A As shown, a channel template (210) can be placed (212) on a surface (202) to divide multiple arrays into multiple 24 rows, with 10 arrays per row. Placing the channel template (210) on the surface (202) can be done using methods similar to Figure 3The simple apparatus shown sandwiches the channel template (210) between the surface (202) and the cap (207) of the substrate (203). The design and composition of the channel template can vary considerably depending on the size and arrangement of the multiple arrays, the size and arrangement of the array of reaction sites, and the method used for coupling the first, second, and third oligonucleotides. The channel template can be made from a variety of materials known in the field of microfluidics, such as silicon, glass, plastics, etc., for example, Ren et al., Acc. Chem. Res., 46(11): 2396-2406 (2013). In some embodiments, the channel template may include plastics such as polystyrene, polyethylene tetraphthalate glycol, polyethylene terephthalate, polymethyl methacrylate, polyvinyl chloride, polycarbonate, thermoplastic elastomers, etc. Guidelines on 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., which are incorporated herein by reference.

[0063] As shown in the cross-sectional view (216), along the midline (214) of the channel (213), after assembling the substrate (203), channel template (210), and cap (207), a dedicated flow path (211) is created for each row of the array. Therefore, each array in a given row can receive the same second oligonucleotide. In some embodiments, the barcode segment sequence of each second oligonucleotide in different rows is different, such that the sequence of the second barcode segment uniquely identifies the row where the spatial barcode is located.

[0064] After the second oligonucleotide is delivered and coupled to the first oligonucleotide, the line channel template (210) can be removed. For example... Figure 2BAs shown, a column channel template (220) can be placed (224) on the surface (202) of the substrate (203) to divide multiple arrays into multiple 10 columns (e.g., 222), with 24 arrays in each column. Similar to dividing into rows, the channel template (220) can create a dedicated flow path for each column, which allows arrays in each column to be exposed to the same third oligonucleotide. In some embodiments, the barcode segment sequence of each third oligonucleotide in different columns is different, such that the sequence of the third barcode segment uniquely identifies the column in which the spatial barcode is located. After coupling the third oligonucleotide (226), a spatially barcoded surface with the form of the spatial barcode shown in the enlarged view (228) can be created.

[0065] In some implementations, the number of unique barcodes on a surface can be increased by providing channels that overlap with a subset of reaction sites in the rows or columns of the array. Figure 2C An example of a column of array (232) of this embodiment is shown in a magnified view relative to the arrays in a plurality of arrays (230). In this embodiment, the width of the channels (e.g., 234a and 234b in the magnified view) is fabricated such that the channels coincide halfway with the points (or reaction sites) of the array (231, the darker shaded subarray) of the column, so that the barcode oligonucleotide can be sequentially attached (237) to the first half-reaction site (e.g., 240) and then (238) to the second half-reaction site (e.g., 242). 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 moving a single channel template by half the array width. Figure 2E The illustration shows a case where two channel templates (262 and 264) (or a liner assembly of such channel templates) are used, with the channel offset predetermined (266) such that different reaction sites are exposed to the reagent delivered through the channel. After these steps, the number of different spatial barcodes in the arrays (230) of multiple arrays will be twice that of an embodiment where each entire subarray (e.g., 232) coincides with the channel delivering the barcode oligonucleotide. In some embodiments, such as Figure 2DAs shown, the channel template can be fabricated such that the two halves of the subarray coincide with different channels, wherein the selection space (248) allows the walls of the channel template to be mounted without covering or obstructing the reaction site. This configuration increases fabrication speed and simplifies the oligonucleotide deposition process, thus, for example, different barcode segments are simultaneously added to each half of the reaction site, thereby producing products (258 and 260) in a single subarray (256). This embodiment is achieved by forming a subarray with gaps (255) in the subarray (256), which separate the two halves and provide space for the walls of the channel template. In other embodiments, the subarray can be formed with multiple gaps, thereby dividing the reaction site into three regions instead of two regions, for example, and such gaps can be formed in both the horizontal and vertical directions for, for example, to attach a second oligonucleotide and a third oligonucleotide, respectively.

[0066] In some implementation schemes, such as Figure 2F As shown, spatially barcoded oligonucleotides can be configured to deliver their components using channels of varying widths. For example, a channel (270) can be constructed by attaching a first channel template to a surface comprising a subarray (268), which delivers a barcoded oligonucleotide that reacts with the surface or conjugated surface oligonucleotide to produce a product (e.g., 274) in one half of the reaction sites of the subarray (268). Subsequently, a channel (272) can be constructed by attaching a second channel template, which delivers a barcoded oligonucleotide that reacts with the 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, channels (282 and 284) are constructed by attaching a third channel template with narrower spacing channels, which deliver reagents (280) to the reaction sites in the first and third quadrants of the subarray (268), producing products (290) and (294) after the reaction. Following step (280), channels (286 and 288) are established by attaching a fourth channel template with spaced channels, which deliver reagents (281) to reaction sites in the second and fourth quadrants of the subarray (268), producing products (292 and 296) after the reaction. In such embodiments, the number of unique barcodes that can be generated is four times that of embodiments where the channels overlap the entire subarray.

[0067] As mentioned above, it is possible to use, such as Figure 3The apparatus or similar device shown applies channel templates (210) and (220) to the surface (202). A substrate (300) comprising a surface (302) having multiple arrays (304) can be placed in a base (306), and the channel template (308) can be placed on top to create partitions of 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, the top plate can be aligned and placed on top using alignment pins (312) to complete the assembly.

[0068] As described herein, sample fluids containing cells can be analyzed in a flow cell comprising a bottom layer (400), a spacer layer (402), and a top layer (404) (see each for details). Figure 4A , Figure 4B and Figure 4C The bottom layer (400) and top layer (404) can both be made of glass or plastic. The spacer layer (402) can be a double-sided pressure-sensitive adhesive with cutouts (406). (Return to Reference) Figure 4B The peripheral portion (407) provides the boundary of the cutout portion (406). In various embodiments, the spacer layer (402) may include a core PET layer having a pressure-sensitive adhesive coating on both sides of the PET layer. The top layer (404) includes an inlet (408) configured to receive liquid and an outlet (410) configured to discharge liquid. In various embodiments, both the inlet (408) and the outlet (410) may be presented as through holes in the top layer (404).

[0069] In various embodiments, rows may be three or more arrays arranged along a generally linear line (e.g., horizontal) extending substantially perpendicular to the liquid flow in the channel. Columns may be three or more arrays arranged along a generally linear line (e.g., vertical) extending substantially parallel to the liquid flow in the channel. In various embodiments, the generally linear line of the rows is approximately orthogonal to the generally linear line of the columns.

[0070] To form a flow pool, a top layer (404) and a bottom layer (400) are positioned relative to a spacer layer (402) between the top layer (404) and the bottom layer (400). The top layer (404), the bottom layer (400), and the cutout portion (406) cooperate to form a channel, wherein an inlet (408) and an outlet (410) are in liquid communication with the channel, thereby allowing liquid to flow through the channel. The channel may have a height defined by the height of the spacer layer (402) and an area defined by the cutout portion (406). The outer portion (407) of the spacer layer (402) forms the wall of the channel. The bottom layer (400) forms the bottom of the channel, and the top layer (404) forms the top of the channel. Figure 4BThe spacer layer (402) includes eight cut sections (406). Figure 4C The top layer (404) includes eight inlets and eight outlets arranged at opposite ends of the cut portion (406) when laminated.

[0071] In various implementations, the substrate (400) may have a spatial barcode surface. Cells may be captured by hydrogel cages while encapsulating portions of the substrate having discrete regions containing specific spatial barcodes. This specific spatial barcode may contain information such as the location of the cells captured in the hydrogel cages within the flow cell. The spatial barcode may be referred to as a surface oligonucleotide complex, which includes a first oligonucleotide, a second oligonucleotide, and / or a third oligonucleotide. The various oligonucleotides of the spatial barcode may be coupled together as a complex oligonucleotide.

[0072] In various implementations, the underlying layer can be composited into a surface with barcodes arranged in multiple arrays. After composited, spacer layers can be laminated onto the underlying layer with the barcode-like surface, such as... Figure 5 As shown in the diagram. The laminated bottom layer and spacer layer form a composite partial assembly (500), as shown. Figure 5 As shown in the image. Furthermore... Figure 5 Multiple arrays (204) are shown arranged such that the columns of the arrays coincide with the channels defined by the cutout portions (406). In various embodiments, the area of ​​the arrays can be approximately 5 mm. 2 Approximately 200 mm 2 Range. For example, each array could have a size of 5 mm x 8 mm or 40 mm. 2 The area.

[0073] In an alternative implementation, the second and / or third oligonucleotides may be coupled to the first oligonucleotide without using a channel template (e.g., 210 or 220). For example, instead of having the liquid containing the second oligonucleotide flow through a temporary flow channel, a liquid may be placed along the row on each array. Figure 6 An example is shown of a single array (204) having multiple overlapping droplets 602 that partially extend beyond the boundary of a single array. The overlapping droplets 602 can converge to form a single liquid pool 504, the boundary of which is shown by the rectangular dotted dashed line and is larger than the boundary of the array (204) shown by the smaller rectangular solid line. However, the single liquid pool does not contact another liquid pool in an adjacent array, thus creating uncoated or gap regions 506, such as... Figure 5 As shown in the image.

[0074] Methods for creating barcode-like surfaces include synthesizing multiple arrays (204) arranged in rows (e.g., 24) and columns (e.g., 10), such as Figure 2AAs shown in the diagram. In various implementations, the rows and columns of multiple arrays can be arranged in an orthogonal relationship, such as... Figure 2A As shown in the diagram. Each array may include multiple discrete reaction sites (208), as... Figure 2A The magnified portion (206) is shown. For example, the array can be formed by a 24 x 32 discrete matrix of reaction sites, wherein each reaction site has a first oligonucleotide having a unique array barcode corresponding to its position within the array. In various embodiments, each discrete reaction site of the array is formed by a set droplet containing a first oligonucleotide comprising a unique array barcode sequence.

[0075] In various embodiments, the unique array barcode sequence of the first oligonucleotide represents a series of nucleotides having codes mapped to specific discrete reaction sites within the array. During the fabrication of the discrete reaction sites, separate liquid solutions can be used to prepare each unique array barcode for the oligonucleotide. For example, a 24 x 32 discrete reaction site matrix can have 768 separate solutions for preparing the first oligonucleotide, each solution having a unique array barcode mapped to a specific discrete reaction site. In various embodiments, each discrete reaction site can have a spacing between reaction sites in the range of 50-500 micrometers, and each discrete reaction site can have a diameter in the range of 30-300 micrometers. In various embodiments, each discrete reaction site in the array can have a diameter per mm. 2 Reaction site density in the range of 50 to 200 discrete reaction sites.

[0076] The surface of the solid support may include a coupling layer coupled to the surface. The coupling layer may include a silane reactive portion and an amine reactive portion bound to silanol groups on the surface. The first oligonucleotide may be derived to have an amine group adjacent to its 5' end. In various embodiments, synthesizing multiple arrays includes coupling the amine portion of the first oligonucleotide to the surface via the coupling layer, wherein the coupling layer includes the amine reactive portion. In various embodiments, each discrete reactive site includes a first oligonucleotide (102) coupled at the 5' end of the first oligonucleotide to the surface (100) of the solid support (see [link to documentation]). Figure 1AIt is noteworthy that discrete reaction sites are configured to react or bind with another oligonucleotide in a subsequent process. In various embodiments, the surface includes a glass surface, and the coupling layer includes silane bonding to the glass surface, and the amine reactive portion includes an N-hydroxysuccinimide portion. In various embodiments, the coupling layer may be attached to the surface and has reactive groups such as an alkyne portion, an azide portion, and / or an amine portion. The alkyne portion of the coupling layer may react with the azide portion coupled to the first oligonucleotide. The azide portion of the coupling layer may react with the alkyne portion of the (dibenzocyclooctyne)DBCO portion coupled to the first oligonucleotide. The amine portion of the coupling layer may react with the N-hydroxysuccinimide portion coupled to the first oligonucleotide.

[0077] Multiple arrays can be constructed such that the surface includes uncoated regions (506) between adjacent arrays (204), as... Figure 5 As shown in the diagram. The uncoated region (506) includes the area where the first oligonucleotide is not coupled to the surface. In various embodiments, such as Figure 2A or Figure 5 The arrays (204) shown can be identical, where each array has the same patterned position of a unique array barcode sequence of the first oligonucleotide for each discrete reaction site of the array. Therefore, the deposition process for the discrete reaction sites can be identical for each array (204), where the arrays can be formed in a 12-row x 8-column format, as shown below. Figure 5 As shown in the diagram. In various embodiments, the unique array barcode sequence of the first oligonucleotide provides the location mapped within the array, rather than the row and / or column positions of multiple arrays.

[0078] Now that the first oligonucleotide (102) has been attached to the surface to form multiple arrays, the second oligonucleotide can be coupled to the first oligonucleotide (110) through an annealing step (108), such as Figure 1A As shown in the diagram. A liquid containing a second oligonucleotide can be disposed on each of a plurality of arrays (204), wherein portions of uncoated areas between adjacent arrays are not covered by the liquid containing the second oligonucleotide. Figure 5 An example of a liquid pool 504 containing a second oligonucleotide is shown, coated with an array (204). Dashed lines are used to show... Figure 5 Boundary of intermediate liquid tank 504.

[0079] In various embodiments, a liquid containing a second oligonucleotide can be disposed on each array, resulting in multiple liquid pools, but in a manner where adjacent liquid pools do not contact each other. Each liquid pool can be in the form of a single small liquid pool. In various embodiments, the liquid pools can be formed by arranging the liquid into multiple dispensed droplets that are merged together. For example, droplets can be formed from an inkjet nozzle or a piezoelectric pump. The multiple dispensed droplets can have a spacing ranging from about 1 micrometer to about 40 micrometers along the x-axis and a spacing ranging from about 1 micrometer to about 40 micrometers along the y-axis. The droplets can have a diameter ranging from about 80 micrometers to about 140 micrometers and a volume ranging from about 200 picoliters to about 350 picoliters. In various embodiments, the piezoelectric pump can dispense droplets at a rate of 1 to about 500 droplets / second, preferably from 2 to about 50 droplets / second. The dispensing pipette of the piezoelectric pump can travel at a rate of 10,000 to 500,000 micrometers / sec, preferably from 100,000 to 300,000 micrometers / sec while dispensing liquid.

[0080] Multiple liquid pools can be incubated on the surface for a predetermined period ranging from 10 minutes to 24 hours, preferably from 10 minutes to 3 hours. During this incubation, humidity can be controlled between approximately 50% RH and approximately 90% RH. After incubation with the second oligonucleotide, the liquid containing the second oligonucleotide can be washed away. It should be noted that all gap areas between the arrays are not coated, and a lower volume of liquid containing the second oligonucleotide is used for deposition compared to the process using a channel template. In various cases, it is advantageous to reduce the volume of liquid containing the customized oligonucleotide, thereby reducing production costs. The instrument used for liquid deposition may require approximately 20 to 30 microliters of reagent to cover 8 arrays in a single row, which is about 1 / 10 of the liquid volume required to flow through a row channel template. Furthermore, the time required to coat multiple rows of arrays using liquid pools is approximately 5 to 7 minutes, significantly less than the time required to coat multiple rows of arrays using a row channel template.

[0081] Each liquid pool can be in the form of a single small liquid pool. In various embodiments, the liquid pool can be formed by setting the liquid as multiple distributed droplets merged together. For example, the droplets can be formed from an inkjet nozzle or a piezoelectric pump. The multiple distributed droplets can have a spacing ranging from about 1 micrometer to about 40 micrometers along the x-axis and a spacing ranging from about 1 micrometer to about 40 micrometers along the y-axis. The droplets can have a diameter ranging from about 80 micrometers to about 140 micrometers and a volume ranging from about 200 picoliters to about 350 picoliters.

[0082] In various embodiments, the liquid containing the second oligonucleotide can be disposed as a single droplet at each discrete reaction site of the array, wherein each droplet at least partially or completely covers each discrete reaction site. The droplets can be dispensed in a manner that neighboring droplets adjacent to the discrete reaction sites do not contact each other.

[0083] In another embodiment, the liquid containing the second oligonucleotide can be disposed as a single droplet on each discrete reaction site of the array, wherein each droplet completely covers each discrete reaction site and partially covers a portion of the gap region between the discrete reaction sites. However, droplets that over-coat the discrete reaction sites are distributed in a manner that neighboring droplets adjacent to the discrete reaction sites do not contact each other.

[0084] In various implementations, the second oligonucleotide is coupled to the first oligonucleotide to form a first surface oligonucleotide complex. The process of forming the first surface oligonucleotide complex may also be referred to as hybridization or annealing. The second oligonucleotide comprises a unique row barcode sequence having identical sequences within rows and distinct sequences between each row of multiple arrays. Each unique row barcode sequence of the second oligonucleotide corresponds to a mapped or known row position within the multiple arrays. The first oligonucleotide complex (e.g., the output of annealing (108)) can be extended, such as... Figure 1A As shown in the extension step (112), the BC2 and S2' of the second oligonucleotide (110) are replicated to extend the first oligonucleotide moiety. After extension, the output of the extension step (112) can be washed and unstretched (114) to form a single-stranded oligonucleotide comprising the first and second oligonucleotides. In various embodiments, a ligase can be used to extend the first oligonucleotide, thereby replicating the second oligonucleotide moiety.

[0085] Now that the second oligonucleotide has been coupled to the surface, the third oligonucleotide can be coupled to the surface via an annealing step (116), as follows: Figure 1A As shown in the diagram. Another liquid containing a third oligonucleotide can be applied to each of the multiple arrays, wherein portions of uncoated areas between adjacent arrays are not covered by the liquid containing the third oligonucleotide. The third oligonucleotide (118) can be coupled to a second oligonucleotide to form a second surface oligonucleotide complex, as shown by the output of the annealing step (116). The third oligonucleotide may include a unique column barcode sequence (i.e., the same oligonucleotide sequence within a column) and different sequences between each column of the multiple arrays. The unique column barcode sequence of the second oligonucleotide each corresponds to a mapped or known column position within the multiple arrays.

[0086] In various embodiments, the second oligonucleotide further includes a random barcode sequence, which is at least 99% unique in the formed second surface oligonucleotide complex. The random barcode sequence may be referred to as a UMI or unique molecular identifier.

[0087] While the invention has been described with reference to several specific exemplary embodiments, those skilled in the art will recognize that many changes can be made therein without departing from the spirit and scope of the invention. In addition to those discussed above, the invention is applicable to various sensor implementations and other subjects.

[0088] definition Unless otherwise explicitly defined herein, the terms and symbols used in this paper for nucleic acid chemistry, biochemistry, genetics, and molecular biology follow the standard papers and texts in the field, such as Kornberg and Baker, DNA Replication, 2nd ed. (WH 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 Immuology, 6th ed. (Saunders, 2007).

[0089] A "barcode" refers to a molecular marker or identifier. In some embodiments, a barcode is a molecule or segment of an analyte (e.g., in the case of a polynucleotide barcode and the analyte) attached to it, which can be used to identify the analyte. In some embodiments, a barcode (referred to herein as a "spatial barcode") can be attached to a surface to identify its location on the surface. In some embodiments, a group of identical spatial barcodes can be set in specific areas on the surface. The size and shape of such areas can vary considerably. In some embodiments, areas with unique spatial barcodes are of the same size and are set on the surface in a regular pattern at a spatial barcode density per unit area. In some embodiments, the density of such barcodes can range from per mm... 2 One barcode per mm 2 The number of barcodes varies, ranging from 1000 to... (in mm). 2 One barcode per mm 2 The number of barcodes varies, ranging from 500 to 1 mm. 2 One barcode per mm 2There are a range of 200 barcodes. In some embodiments, there may be a one-to-one correspondence between different spatial barcodes and different regions on the surface; that is, each different region may have a different and unique barcode. In some embodiments, the identity of the spatial barcode is determinable, for example, it can be determined by sequencing as long as the spatial barcode is a polynucleotide. In some embodiments, the spatial barcode is an oligonucleotide. In some embodiments, the oligonucleotide spatial barcode includes random sequence oligonucleotides. Random sequence oligonucleotides are typically synthesized using a "split and mix" synthesis technique, for example, as described by reference in the following references incorporated herein by reference: Church, U.S. Patent 4,942,124; 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 composite barcodes; that is, oligonucleotide segments that include segments that identify different objects. For example, the first segment of a composite barcode can identify a specific area on the surface, and the second segment of the composite barcode can identify a specific molecule (the so-called "unique molecular identifier" or UMI).

[0090] The terms "microfluidic" and "nanofluidic" are used interchangeably herein, each referring to an integrated system for capturing, moving, mixing, dispensing, or analyzing small volumes of fluids, including samples (which may in turn contain or include analytes of interest), reagents, diluents, buffers, etc. Generally, references to "microfluidic" and "nanofluidic" indicate different scales in device size and the volume of fluid handled. In some embodiments, microfluidic devices are characterized by cross-sectional dimensions less than several hundred square micrometers and have channels or passages having capillary dimensions, for example, having a cross-sectional dimension of about 1-2 mm to about 0.1 μm. In some embodiments, the volumetric capacity of microfluidic devices ranges from 100 μL to several nL (e.g., 10-100 nL), or from 100 μL to 1 μL. The dimensions of corresponding features or structures in nanofluidic devices are typically 1 to 3 orders of magnitude smaller than the dimensions of corresponding features or structures in microfluidic devices. Those skilled in the art will know which dimensions will be relevant from the context of a particular application. In some embodiments, microfluidic or nanofluidic devices have one or more interconnected and fluidly communicated chambers and channels, and are designed to perform one or more reactions or processes, either alone or in conjunction with supporting appliances or instruments, such as sample introduction, fluid and / or reagent driving devices (e.g., positive or negative pressure, acoustic energy, temperature control, detection systems, data collection, and / or integrated systems). In some embodiments, microfluidic and nanofluidic devices may also include valves, pumps, filters, and specialized functional coatings on the inner walls to, for example, prevent adsorption of sample components or reactants, facilitate reagent movement via electroosmosis, etc. Such devices can be fabricated as integrated devices in a solid substrate, which may be glass, plastic, or other solid polymer materials, and may have a planar format to facilitate the detection and monitoring of sample and reagent movement, particularly via optical or electrochemical methods. In some embodiments, such devices are disposable after a single use. In some embodiments, microfluidic and nanofluidic devices include means for forming and controlling the movement, mixing, dispensing, and analysis of droplets, such as aqueous droplets immersed in an immiscible fluid (e.g., light oil).The fabrication and operation of microfluidic and nanofluidic devices are well known in the art, as illustrated by examples in the following references, which are incorporated herein by reference: Ramsey, U.S. Patents 6,001,229, 5,858,195, 6,010,607, and 6,033,546; Soane et al., U.S. Patents 5,126,022 and 6,054,034; Nelson et al., U.S. Patent 6,613,525; Maher et al., U.S. Patent 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. Patents 5,587,128 and 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 creating a barcode-enabled surface, the method comprising: Synthesize multiple arrays, where: (i) Each array includes multiple discrete reaction sites. (ii) Each discrete reaction site includes a first oligonucleotide coupled to the surface of a solid support at its 5' end. (iii) The surface includes uncoated regions between adjacent arrays, the uncoated regions including areas where the first oligonucleotide is not coupled to the surface. (iv) The first oligonucleotide comprises a first unique array barcode sequence for each discrete reaction site location within the array. (v) Each of the first unique array barcode sequences of the first oligonucleotide corresponds to a discrete reaction site location mapped within the array; and A liquid containing a second oligonucleotide is disposed on each of the plurality of arrays, wherein portions of the uncoated areas between adjacent arrays are not covered by the liquid containing the second oligonucleotide, wherein: (vi) The second oligonucleotide is coupled to the first oligonucleotide to form a first surface oligonucleotide complex. (vii) The second oligonucleotide includes a second unique barcode sequence, which includes identical sequences within the first subgroups of the plurality of arrays and distinct sequences between the respective first subgroups of the plurality of arrays. (viii) Each of the second unique barcode sequences of the second oligonucleotide corresponds to the first subgroup position of the mapping within the plurality of arrays.

2. The method according to claim 1, further comprising: Another liquid containing a third oligonucleotide is disposed on each of the plurality of arrays, wherein portions of the uncoated areas between the adjacent arrays are not covered by the other liquid containing the third oligonucleotide, wherein: (ix) The third oligonucleotide is coupled to the second oligonucleotide to form a second surface oligonucleotide complex. (x) The third oligonucleotide comprises a third unique barcode sequence, which includes identical sequences within the second subgroups of the plurality of arrays and distinct sequences between each of the second subgroups. (xi) Each of the third unique barcode sequences of the third oligonucleotide corresponds to a second subgroup position of the mapping within the plurality of arrays.

3. The method of claim 1, wherein the second oligonucleotide further comprises a random barcode sequence, wherein the random barcode sequence is a unique sequence in at least 99% of the formed second surface oligonucleotide complex.

4. The method of claim 1, wherein disposing of the liquid containing the second oligonucleotide on each of the plurality of arrays comprises: The liquid pools are arranged on each array, wherein each liquid pool does not contact the adjacent liquid pools.

5. The method of claim 4, wherein each liquid pool is a single unit of the liquid.

6. The method of claim 4, wherein the liquid pool is formed by configuring the liquid as a plurality of distributed droplets merged together.

7. The method of claim 6, wherein the plurality of dispensed droplets have: The spacing along the x-axis, ranging from approximately 1 micrometer to approximately 40 micrometers, and The spacing ranges from approximately 1 micrometer to approximately 40 micrometers along the y-axis.

8. The method of claim 6, wherein the plurality of dispensed droplets are formed from a piezoelectric pump.

9. The method of claim 6, wherein each of the plurality of dispensed droplets has a diameter ranging from about 80 micrometers to about 140 micrometers.

10. The method of claim 6, wherein each of the plurality of dispensed droplets has a volume ranging from about 200 picoliters to about 350 picoliters.

11. The method of claim 1, wherein disposing of the liquid containing the second oligonucleotide on each of the plurality of arrays comprises: The liquid is configured as a plurality of droplets, wherein each droplet at least partially covers each of the discrete reaction sites.

12. The method of claim 1, wherein disposing the liquid containing the second oligonucleotide on each of the plurality of arrays comprises: The liquid is configured as multiple droplets, wherein each droplet covers each of the discrete reaction sites.

13. The method of claim 1, wherein the synthesis of the plurality of arrays comprises coupling the amine portion of the first oligonucleotide to the surface via a coupling layer, wherein the amine reactive portion of the coupling layer binds to the amine portion.

14. The method of claim 13, wherein the surface comprises a glass surface, the coupling layer comprises a silane bonded to the glass surface, and the amine reactive portion comprises an N-hydroxysuccinimide portion.

15. The method of claim 1, further comprising, after coupling the second oligonucleotide to the first oligonucleotide to form the first surface oligonucleotide complex, adding a ligase to extend the first oligonucleotide such that a portion of the second oligonucleotide is replicated.

16. The method of claim 1, wherein the plurality of arrays are arranged in rows and columns.

17. The method of claim 16, wherein the rows and columns of the plurality of arrays are arranged in an orthogonal relationship.

18. The method of claim 1, wherein each of the discrete reaction sites of the array is formed from a single droplet containing the first oligonucleotide.

19. The method of claim 1, wherein each of the plurality of arrays has the same patterned position of the first unique array barcode sequence of the first oligonucleotide for each of the discrete reaction sites of the array.

20. The method of claim 1, wherein each of the discrete reaction sites in the array has a spacing between the reaction sites in the range of 50-500 micrometers, and wherein each of the discrete reaction sites has a diameter in the range of 30-300 micrometers.

21. The method of claim 1, wherein each of the discrete reaction sites in the array has a density per mm. 2 Reaction site density in the range of 50 to 200 reaction sites.

22. The method of claim 1, wherein the first subgroup is an array category selected from the following: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, a first chessboard pattern, a second chessboard pattern, and combinations thereof.

23. The method of claim 2, wherein the second subgroup is an array category selected from the following: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, a first chessboard pattern, a second chessboard pattern, and combinations thereof, wherein the array categories of the first subgroup and the second subgroup are different.

24. The method of claim 1, wherein the first subgroup comprises the rows, wherein the second unique barcode sequence comprises identical sequences within the rows of the plurality of arrays and distinct sequences between each row of the plurality of arrays.

25. The method of claim 2, wherein the second subgroup comprises the columns, wherein the third unique barcode sequence comprises identical sequences within the columns of the plurality of arrays and distinct sequences between each column of the plurality of arrays.

26. A flow cell configured to capture cells from a biological sample in a hydrogel cage, comprising: (a) Configure an inlet for receiving liquid; (b) Configure an outlet for discharging the liquid; (c) Top layer; (d) A spacer layer with a cut portion; and (e) A bottom layer, wherein the top layer and the bottom layer are relative to the spacer layer between the top layer and the bottom layer, wherein the top layer, the bottom layer, and the cutout portion cooperate to form a channel, wherein the inlet and the outlet are in liquid communication with the channel, thereby allowing liquid to flow through the channel, the bottom layer comprising a plurality of arrays, wherein (i) Each array includes multiple discrete sites, wherein a spatially barcoded oligonucleotide is coupled to the surface of the underlying layer at each of the discrete sites, wherein the spatially barcoded oligonucleotide comprises: a first oligonucleotide and a second oligonucleotide. (ii) The surface includes uncoated regions between adjacent arrays, the uncoated regions including areas where spatial barcode oligonucleotides are not coupled to the surface. (iii) The first oligonucleotide comprises a first unique array barcode sequence for each discrete site location within the array. (iv) The first unique array barcode sequence of the first oligonucleotide corresponds to a discrete site location mapped within the array; (v) The second oligonucleotide includes a second unique barcode sequence, the second unique barcode sequence comprising identical sequences within the first subgroups of the plurality of arrays and distinct sequences between each of the first subgroups of the plurality of arrays, and (vi) The second unique barcode sequence of the second oligonucleotide corresponds to the first subgroup position of the mapping within the plurality of arrays.

27. The flow cell of claim 26, wherein the second oligonucleotide further comprises a random barcode sequence, wherein the random barcode sequence is a unique sequence in at least 99% of the spatial barcode oligonucleotides.

28. The flow pool of claim 26, wherein the first subgroup is an array category selected from the following: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, a first checkerboard pattern, a second checkerboard pattern, and combinations thereof.

29. The flow pool of claim 28, wherein the first subgroup comprises the rows, wherein the second unique barcode sequence comprises the same sequence within the rows of the plurality of arrays and different sequences between each row of the plurality of arrays.

30. The flow cell according to claim 26, further comprising: A third oligonucleotide is coupled to the second oligonucleotide to form a second surface oligonucleotide complex, wherein the third oligonucleotide includes a third unique barcode sequence.

31. The flow cell of claim 31, wherein the third unique barcode sequence comprises identical sequences within the second subgroups of the plurality of arrays and distinct sequences between each of the second subgroups, and wherein each of the third unique barcode sequences of the third oligonucleotide corresponds to a mapped second subgroup position within the plurality of arrays.

32. The flow pool of claim 32, wherein the second subgroup is an array category selected from the following: rows, columns, diagonals, even rows, odd rows, even columns, odd columns, a first checkerboard pattern, a second checkerboard pattern, and combinations thereof, wherein the array categories of the first subgroup and the second subgroup are different.

33. The flow pool of claim 33, wherein the second subgroup comprises the column, wherein the third unique barcode sequence comprises the same sequence within the column of the plurality of arrays and different sequences between each column of the plurality of arrays.

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