Spatial barcodes by hydrogel lithography

Abstract

The invention relates to a method and a system for producing a surface coated with a plurality of spatial barcodes. In one aspect, beads carrying barcode oligonucleotides are disposed on a surface, after which a hydrogel chamber is synthesized around each bead, and barcode oligonucleotides are released to be captured on the surface of the interior of the chamber. The captured oligonucleotide barcodes are amplified and optionally sequenced to form a coating of spatial barcodes.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 329,049, filed on April 8, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. Background Art

[0005] Molecular tagging has a long history in analytical biochemistry and molecular biology, such as Church's 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; and so on. Recently, such techniques have been expanded 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. Patent 9593365; etc. However, cost-effective synthesis of spatial barcodes with known sequences, control over their distribution, and density for resolving cellular and subcellular processes has been a challenge that has been addressed by a large number of different approaches with only partial success, e.g., Horgan et al., Intl. 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 that allow flexible synthesis and deposition as well as control over the barcoded area will advance the field of spatial barcode construction. Summary of the invention

[0006] The present invention relates to a method for coating a surface with a spatial barcode that allows a high degree of control over barcode density and geometry. In one aspect, a hydrogel barrier is used to control the geometry and pattern of the spatial barcode in a process sometimes referred to herein as "hydrogel lithography."

[0007] In one aspect, provided herein is a method for making a spatially barcoded surface, the method comprising: (a) arranging particles on the surface of a channel, wherein the surface is coated with primers for solid phase amplification and wherein each particle comprises a cleavably attached oligonucleotide comprising a barcode sequence; (b) synthesizing hydrogel chambers on the surface such that each hydrogel chamber encapsulates a particle; and (c) cleaving the oligonucleotide from the particle so that the oligonucleotide is released into the chamber such that at least a portion of the oligonucleotide is captured by the primer, thereby obtaining a captured oligonucleotide.

[0008] In some cases, the method also includes replicating the captured oligonucleotide by using the captured oligonucleotide as a template to extend a primer, thereby obtaining a copy of the captured oligonucleotide. In some cases, the method also includes amplifying a copy of the captured oligonucleotide to form a cluster. In some cases, the method also includes sequencing the barcode sequence of the copy of the captured oligonucleotide of the cluster.

[0009] In some cases, the copies of the captured oligonucleotide each include a sequencing primer binding site adjacent to the barcode sequence, and wherein the method further comprises determining the barcode sequence by a sequencing-by-synthesis method. In some cases, the particles are randomly arranged on the surface. In some cases, the particles are arranged on the surface in a regular pattern. In some cases, the particles are arranged on the surface in a linear pattern or a hexagonal pattern.

[0010] In one aspect, a method for making a spatially barcoded surface is provided herein, the method comprising: (a) providing a fluidic device comprising: (i) a channel comprising one or more polymer precursors and a surface comprising a capture element and a plurality of particles arranged on the surface, the particles having oligonucleotides cleavably attached thereto, the oligonucleotides comprising a barcode; (ii) a spatial energy modulation element optically connected to the surface; and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element; (b) detecting each of the plurality of particles using the detector and identifying the plurality of particles. The invention relates to a method for synthesizing a plurality of chambers in a channel, wherein the one or more chambers encapsulate one or more of the plurality of particles, wherein the polymer matrix wall is substantially impermeable to the oligonucleotide, and wherein the position of the synthesized chamber is determined by the position of the particle encapsulated by the synthesized chamber as identified by a detector; and (d) cutting the oligonucleotide from the particle so that the oligonucleotide is released into the one or more chambers and captured by the capture element, thereby obtaining a captured oligonucleotide.

[0011] In some cases, the method further comprises replicating the captured oligonucleotide by extending the capture element using the captured oligonucleotide as a template, thereby obtaining a copy of the captured oligonucleotide. In some cases, the method further comprises performing solid phase amplification on the copy of the captured oligonucleotide on the surface. In some cases, the method further comprises degrading the polymer wall of one or more chambers.

[0012] In another aspect, provided herein is a method for making a spatially barcoded surface, the method comprising one or more cycles of the following operations: (a) arranging one or more particles on the surface of a channel, wherein the surface is coated with primers for solid phase amplification and wherein each of the one or more particles comprises one or more cleavably attached oligonucleotides, each of which comprises a barcode sequence; (b) synthesizing hydrogel chambers on the surface such that each hydrogel chamber defines a subregion of the surface and encapsulates a particle in the one or more particles; (c) cleaving one or more oligonucleotides from the one or more particles so that the one or more oligonucleotides are released into the hydrogel chamber such that at least a portion of the one or more oligonucleotides are captured by the primers and replicated, thereby obtaining one or more replicated oligonucleotides; and (d) degrading the hydrogel chambers, thereby leaving subregions, each of which has a replicated oligonucleotide with a different barcode in the one or more replicated oligonucleotides attached thereto.

[0013] In some cases, the sub-regions do not overlap each other. In some cases, the method also includes amplifying the replicated oligonucleotides to obtain amplified oligonucleotides. In some cases, the barcode sequence is determined by sequencing the amplified oligonucleotides. In some cases, the sum of the areas of the sub-regions accounts for at least 70% of the area of ​​the surface. In some cases, the sub-regions form a fractal barcode array. In some cases, each of the sub-regions overlaps with at least one other sub-region to form an overlapping region. In some cases, the surface in the overlapping region includes at least one oligonucleotide including two or more barcode sequences. In some cases, the relative position of the sub-region on the surface is determined by the oligonucleotides including two or more barcode sequences in the overlapping region. In some cases, one or more cycles are multiple cycles.

[0014] In another aspect, provided herein is a composition comprising a substrate having a planar surface comprising a fractal barcode array. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A-1E An embodiment of the invention is shown which employs a random arrangement of beads on a surface.

[0016] Figure 2 An embodiment for generating spatially barcoded capture probes is shown.

[0017] Figure 3A and Figure 3B Another embodiment of the invention is shown using beads arranged regularly on a surface.

[0018] Figure 4A-4D An embodiment of non-overlapping spatial barcode regions is diagrammatically shown.

[0019] Figure 5A-5E An embodiment employing overlapping spatial barcode regions is shown.

[0020] Fig. 6A The distribution of primary and secondary barcodes in each chamber footprint in a set of overlapping chamber footprints is shown.

[0021] Figure 6B is a flow chart of an algorithm for assigning bar codes to chamber footprints and their overlaps.

[0022] Fig. 7A and Figure 7B An embodiment of a system for carrying out the method of the present invention is schematically shown.

[0023] Fig. 8A and Figure 8B One embodiment comprising a flow cell comprising a plurality of channels is shown.

[0024] Fig. 9 Shown are the steps for preparing cDNA from a single cell target template, such as mRNA, and its sequencing in an external sequencing instrument after elution from the channel.

[0025] Figure 10A-10C Shown is the pattern of a spatial barcode generated by the method of the present invention. DETAILED DESCRIPTION

[0026] Unless otherwise stated, the practice of the present invention may adopt 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, preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. Suitable techniques may be specifically described by reference to the examples below this article. However, other equivalent routine procedures may also be used, of course. Such conventional techniques and descriptions may 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, Third Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, Third Edition (Academic Press, 2013); and similar references.

[0027] The present invention relates to a method for producing a surface that is barcoded in space; the surface that is barcoded in space is also such a surface: it has an attached molecular indicator (indicator), from which the surface position can be determined or indicated. In some embodiments, such molecular indicators include surface position information encoded in a polymer sequence (such as an oligonucleotide sequence). In some embodiments, multiple sub-regions of a surface can be formed, in which the same unique molecular indicator or barcode is present. In other words, in some embodiments, among multiple sub-regions, each different sub-region has a different molecular indicator. In some embodiments, the surface that is barcoded in space can have sub-regions, each of which has a unique barcode, but its sequence is not known until it is used for certain processes or determinations (such as determining the spatial distribution of gene expression in tissues). In other embodiments, the surface that is barcoded in space can have sub-regions, each of which has a unique barcode, and its sequence is determined before use, so that there is a map of barcode sequence and surface position before use. The present invention covers these two embodiments. The latter embodiment is advantageous for many applications because it allows associating molecular processes and / or species with locations and features of the image of the surface.

[0028] Figure 1A and Figure 1BAn embodiment of the method described herein is shown. A fluidic device can be provided, which has: a channel (100), the channel (100) having a surface (102); a detector (104), the detector (104) is optically connected to the surface (102); and a spatial energy modulation element (106), which can be, for example, a digital micromirror device (DMD), the spatial energy modulation element (106) is also optically connected to the surface (102) and is operatively associated with the detector (104). A reaction mixture including one or more polymer precursors and beads or particles (e.g., 101) can be loaded into the channel (100). The beads or particles can have a cleavably attached molecular indicator (such as an oligonucleotide having at least one segment having a unique nucleotide sequence) or a barcode. The terms "beads" and "particles" are used interchangeably to represent discrete solid supports that are typically used or manipulated in a group but can be separated from each other. In some embodiments, the term "bead" refers to a population of monodisperse, approximately spherical particles whose diameters typically have a coefficient of variation of less than 25%. After loading, the position of the particles on the surface (102) can be determined based on an optical signal collected by a detector (104), which optical signal can include an image of all or part of the surface (102). As part of the operational association between the detector (104) and the spatial energy modulation element (106), the particle position information determined based on the optical signal collected by the detector (104) can be converted into instructions transmitted to the spatial energy modulation element (106) to generate a light beam and direct it into the channel (100), thereby causing the gel barrier or polymer matrix wall to perform photoenergy synthesis (108). The gel barrier or polymer matrix wall can have a predetermined size and shape to form a chamber (e.g., 109) around each particle on the surface (102).

[0029] The enlarged view (110) shows that the solid appearance structure (112, 113 and 114) can have an interior (111) and a wall (121) including a predetermined thickness (116). Similarly, the hydrogel chamber can have a predetermined shape (e.g., a circle (or ring) having a diameter and a predetermined area enclosed. In the accompanying drawings, for convenience, the chamber is shown as being isolated and not connected to adjacent chambers, and is shown as having a cylindrical or ring-like shape; however, the spatial energy modulation element can synthesize chambers of different shapes and sizes, as can be used for specific applications. In some embodiments of the proliferation assay, each synthesized hydrogel chamber can have the same shape and area, for example, having a diameter selected from 0.001 to 0.01 mm 2 The inner area of ​​the range is annular.

[0030] As used herein, "channel" means a container capable of accommodating a fluid (which may be static or mobile) and having at least one surface on which beads may be arranged and a synthetic chamber may be formed. In some embodiments, a channel may have a first surface and / or a second surface on which a synthetic chamber may be formed and / or on which beads or particles may be arranged. As used herein, reference to "surface" without reference to "first" or "second" is intended to include a first surface or a second surface (if two surfaces are present in a fluid device (such as a flow cell)). In some embodiments, a channel may limit the flow of a fluid passing therethrough from an inlet to an outlet. In other embodiments, a channel may contain a non-flowable volume of fluid that may be removed, substituted, or added through an opening or an inlet; that is, in some embodiments, a channel of the present invention may be a hole or a pore-like structure.

[0031] return Figure 1A, after the beads on the surface (102) are encapsulated by the chamber (117), the oligonucleotides attached to the beads can be cleaved and captured by the capture elements on the surface (102) within the corresponding chambers of the beads. The porosity of the polymer matrix walls of the chambers can be selected so that the oligonucleotides released from the beads are effectively confined to the interior of the chamber into which they are released. The capture elements on the surface (102) can include a uniform coating (or substantially uniform coating) of surface-attached oligonucleotides that serve as primers or hybridization binding sites for desired polynucleotides (such as oligonucleotides released from the beads). For example, the surface (102) can include a coating of P5 primers and P7 primers used for cluster formation in Illumina, Inc. (San Diego) DNA sequencing instruments. In some embodiments, the released oligonucleotides are captured (130) on a portion (132) of the surface (102) within the chamber that encapsulates the beads. After capturing the released oligonucleotide, the captured oligonucleotide can be extended (134) using the captured oligonucleotide as a template so that the barcode carried by the released oligonucleotide is replicated along with other segments such as primer binding sites for sequencing the barcode and segments (e.g., polyT regions) for capturing molecules of interest such as messenger RNA. The cleavable linkages tethering the oligonucleotide to the particle or bead can vary widely. Such linkages can include, but are not limited to, cleavable chemical linkages (e.g., base or acid labile), enzymatically cleavable linkages, hybridization (which can be cleaved by heating or treatment with a chaotropic agent such as formamide), photochemical cleavage, and the like. In some embodiments, after capture, the hydrogel chamber can be degraded, synthetic reagents loaded, and the captured oligonucleotide replicated. In other embodiments, after capture, synthetic reagents are loaded and replicated before the chamber is degraded and the beads are removed. After degradation of the hydrogel chamber and removal of the beads, the pattern of sub-regions (136) can remain on the surface, each such sub-region seeded with an identical copy of the barcoded oligonucleotide. The seeded copies can then be amplified using conventional surface amplification techniques (such as bridge amplification, etc.) to produce a large number of surface-bound copies, for example, U.S. Patents 6090592, 6060288, 6787308, 9057097, 9169513, 9476080, 9476080; Adessi et al., Nucleic Acids Research, 28(20): e87 (2000); etc. In some embodiments, clusters can be formed from the seeded copies by bridge amplification. Such embodiments can also include cutting the end of the amplified sequence away from the surface to expose the extendable capture sequence.

[0032] Figure 1C-1E Include further description Figure 1A-1B. In the figure, an enlarged portion (146) of the surface (102) of the channel (100) shows two types of exemplary capture oligonucleotides (P7' (140) and P5' (142)), which in this embodiment can be used as surface binding primers for cluster formation achieved by bridge PCR. The capture oligonucleotides can be attached via their 5' ends so that whenever the oligonucleotides released from the beads are captured (e.g., 144), their 3' ends can be extended by a polymerase. The released oligonucleotides having a P5 sequence at one end can also hybridize and be captured by a P5' complementary primer (142) (not shown in the figure), so that the capture oligonucleotides (e.g., 312 of FIG. 3) and other segments are in opposite directions relative to the surface (102). In some embodiments, the P5' primer can be attached to the surface (102) via a cleavable linker to remove such sequences (including their extension products) prior to capturing the polynucleotide of interest (e.g., mRNA), for example as taught by Cho et al. (cited above). After the surface-bound primer is extended to form the extension product (146), the captured chain can be melted (148), leaving behind the covalently attached seed oligonucleotide (147), from which clusters (150) are formed. After cluster formation, the amplified chains in the undesirable direction can be cut or digested as described above. Among the chains in the desired direction, the sequences away from the surface (102) required for cluster formation can be cut to release the capture oligonucleotide for capture and extension (e.g., see Figure 3).

[0033] Figure 2An example of the structure of an oligonucleotide attached to a bead or particle for use in the methods of the present invention is shown. A bead-oligonucleotide conjugate (200) can include a bead (201) and an oligonucleotide (202) cleavably attached to the bead (201), for example, the oligonucleotide (202) is cleavably attached to the bead (201) by a cleavable linkage (204) or by hybridization with an oligonucleotide attached to the bead (201). In some embodiments, the oligonucleotide (202) can include multiple segments, including but not limited to segments at either end for surface amplification (shown as P7 (206) and P5 (216)), a segment with one or more barcodes (208), a primer binding segment (210) for sequencing all or part of the barcode (208), a capture segment (212), and a cleavage site (214) for removing an amplification segment (e.g., P5), so that the end of the capture segment (212) can be extended. The beads or particles (201) can vary widely in size, composition, and loading of oligonucleotides (202). In some embodiments, the size of the beads or particles can be in the range of 2-500 μm in diameter, or in the range of 4-200 μm, or in the range of 4-100 μm. In some embodiments, the amount (or loading) of oligonucleotides (202) on the beads (201) is sufficient to provide 100% oligonucleotides (202) per 1 μm. 2 Oligonucleotides (202) are arranged on the surface encapsulated by the chamber at a density in the range of 0.5 to 2 oligonucleotides. As briefly described above, the chamber can be synthesized around the bead (218), the oligonucleotide (202) can be released by cutting the cleavable linkage (204), the oligonucleotide (202) can be captured on the surface (222) by a complementary surface-bound primer, such primer is an extended replication oligonucleotide (202), and the extension product (219) can be amplified to form a cluster (221). After the cluster of double-stranded oligonucleotides is formed, the segment (220) containing the P5 primer binding site can be cut. For example, whenever the capture oligonucleotide (212) is a polyT segment for capturing mRNA, the cleavage site (214) can be a DraI endonuclease recognition site.

[0034] In some embodiments, the beads arranged on the channel surface can be arranged into a regular pattern by modifying the channel surface to have discrete sites for which the beads have an affinity. This affinity can be established by a variety of methods, including but not limited to providing a pattern of hydrophilic sites surrounded by hydrophobic interstitial spaces, hybridization of complementary oligonucleotides, covalent bonding, etc., such as Gopinath et al., Nature, 535: 401-405 (2016) and the like. Figure 3A-3BAn embodiment of a regular array comprising beads is shown. In some embodiments, as used herein, "regular array" refers to a non-random array. In some embodiments, the regular array can be a linear array, a hexagonal array, etc. Figure 3A The surface (300) of the channel is shown to have a linear array of sites (302) to which beads (e.g., 306) have affinity. When beads are loaded into the channel (304), the beads can be attracted to the sites (302) and occupy the sites (302). In some embodiments, this occupation can be promoted by shaking or vibrating the channel. After the beads are loaded and placed on the affinity sites in this way, a hydrogel chamber (310) can be synthesized (308) around each bead. The polymer matrix wall of this chamber can be selected to be (i) substantially impermeable to the oligonucleotide once it is cut and released from the beads, and (ii) degradable so that they can be removed after capturing the released oligonucleotide. The captured oligonucleotide can be treated (312) as described above to produce a surface coated (314) with a unique barcode in each chamber. After the barcode is attached to the "bottom" of the chamber, the polymer matrix wall can be degraded (316) to produce an array (318) with a spatial barcode pattern. In some embodiments, the diameter of such affinity sites or dots is in the range of 5-500 μm or in the range of 10-1000 μm. In some embodiments, such dots or affinity sites are arranged in a linear array, or in a hexagonal array. In some embodiments, the density of such arrays of such dots or reaction sites ranges from 10 to 2500 sites / mm 2 , or 10 to 1000 sites / mm 2 or 10 to 500 sites / mm 2 or 10 to 100 sites / mm 2 .

[0035] In some embodiments, the methods described herein can be implemented in the following manner: (a) particles are arranged on the surface of a channel, wherein the surface is coated with primers for solid phase amplification, and wherein each particle includes an oligonucleotide attached to a cleavable surface, the oligonucleotide including a barcode sequence; (b) hydrogel chambers are synthesized on the surface so that each hydrogel chamber encapsulates the particle; and (c) oligonucleotides are cut from the particles so that the oligonucleotides are released into the chamber so that a portion of the oligonucleotides are captured by the primers. In some embodiments, the method also includes replicating the captured oligonucleotides by extending the primers on the surface using the captured oligonucleotides as templates. In some embodiments, the method also includes amplifying copies of the captured oligonucleotides by solid phase amplification or surface amplification.

[0036] In some embodiments, synthesizing hydrogel chambers is performed by: (a) providing a fluidic device comprising: (i) a channel comprising one or more polymer precursors, a surface comprising a capture element, and particles arranged on the surface, the particles having oligonucleotides cleavably attached, the oligonucleotides comprising a barcode; (ii) a spatial energy modulation element optically connected to the surface; and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element, the detector detecting each of a plurality of particles and determining a position of each of the plurality of particles on the surface; and (b) synthesizing one or more chambers in the channel by projecting light into the channel using the spatial energy modulation element such that the projected light causes one or more polymer precursors to cross-link to form a polymer matrix wall of the chamber, wherein each chamber encapsulates a particle, wherein the polymer matrix wall is substantially impermeable to the oligonucleotides, and wherein the position of the synthesized chamber is determined by the position of the particle encapsulated by the synthesized chamber as identified by the detector.

[0037] Exemplary solid phase amplification techniques include, but are not limited to, bridge polymerase chain reaction (bPCR), recombinase-polymerase solid phase amplification (RPA), kinetic exclusion amplification, and the like. Solid phase amplification techniques are disclosed in the following references (which are incorporated by reference): Adams, U.S. Pat. No. 5,641,658; Boles, U.S. Pat. No. 6,300,070; Mayer, U.S. Pat. Nos. 7,790,418, 7,985,565, 8,652,810, 9,593,328, 9,902,951 and International Patent Publication No. WO 1998 / 44151; Ronaghi, U.S. Pat. Nos. 9,777,3268, 9,416,415; U.S. Pat. Nos. 7,763,427, 8,426,134, 7,666,598, 9,309,558; or U.S. Pat. Nos. 6,090,592, 6,060,288, 6,787,308, 9,057,097, 9,169,513, 9,476,080; 9,476,080; Adessi et al., Nucleic Acids Research, 28(20):e87(2000); etc.

[0038] Continuous cycles of bead loading and chamber synthesis

[0039] By sequentially arranging beads on a surface and then performing chamber synthesis, it is possible to substantially cover the entire surface with a spatial barcode. To this end, chambers of the same or different sizes and / or shapes may be used. In some embodiments, where beads are randomly arranged on a surface, chambers of the same shape (e.g., annular) but of successively smaller areas may be used, such as Figure 4A-4C shown. Figure 4AThe figure above shows a random arrangement of beads (e.g., 402) on a surface (400). Chambers (e.g., 406) are synthesized (404) around each bead. Similarly, the chambers can have various shapes. For simplicity, a circular (or annular) shape is shown in the figure. In some embodiments, the position of the chamber around a specific bead can be selected to promote more uniform coverage of the surface, promote useful overlap of the chamber coverage area in continuous cycles, etc. After the chamber is synthesized, the oligonucleotide containing the barcode can be released from the bead, and then a portion (via the capture oligonucleotide on the surface (400)) is captured in the area defined by the chamber (i.e., the chamber coverage area). In this example, the capture oligonucleotide is covalently attached to the surface (400) via its 5' end, and its 3'-OH end is free so that the captured chain can be used as a template to extend the 3'-OH end with a polymerase. Finally, the gel wall of the chamber can be degraded and the beads removed (408), leaving a surface (400) having regions or zones or chamber footprints (these terms are used interchangeably) (e.g., (410)) each having an attached oligonucleotide that includes the same barcode within such region or zone or chamber footprint. Such attached oligonucleotides can also include additional elements, such as capture sequences, primer binding sites, etc., such as Figure 2 and Figure 5C-5D As shown. Figure 4B As shown, in some embodiments, within the chamber footprint, the surface (400) includes primers (412) for solid phase amplification (e.g., bridge PCR) and oligonucleotide chains (414) for amplification. In some embodiments, especially when the beads are randomly arranged on the surface (400) during loading, continuous loading of beads can be performed so that the beads are arranged in a better position to be encapsulated in the chamber. Similarly, the concentration of beads can be varied in such continuous loading so that beads are obtained in a desired position. In some embodiments, beads can be repositioned in various ways to promote the arrangement of beads that is more conducive to encapsulation in the chamber. For example, magnetic beads can be used, or beads can be moved by optical tweezers, such as Hui, Masters Thesis, MIT, Dept. Eng. Comp. Sci. (2001); Grier, Nature, 424: 810-816 (2003), etc.

[0040] like Figure 4B As shown, the chamber footprints including the spatial barcodes are randomly distributed and do not cover the entirety of the surface (400). Further coverage of the surface can be obtained by performing successive cycles of bead deposition and chamber synthesis. In some embodiments, chambers with smaller surface areas can be used so that more of the barcode-free interstitial spaces between the current chamber footprints can be filled with additional spatial barcodes. Such embodiments are Figure 4C and Figure 4D. A second arrangement of beads (e.g., 430) is performed on a surface (400) comprising a shadowed chamber footprint (e.g., 410), after which a further chamber is synthesized around the selected beads, the chamber having the same area (e.g., 433) or a smaller area (e.g., 432) than the pre-existing chamber footprint (e.g., 410). Beads (e.g., 435) not encapsulated by a chamber (e.g., 437 or 439) are washed away (418), after which oligonucleotides including additional barcodes are released, captured, and replicated (420). After degradation of the gel chamber, Figure 4C The lower figure shows a surface (400) having an additional area covered by a chamber including an additional spatial barcode. Figure 4D An embodiment of the above process referred to herein as a "fractal barcode array" is shown. In some embodiments, the fractal barcode array comprises a surface comprising regions or zones of successive smaller areas, wherein each zone comprises a spatial barcode comprising the same unique nucleotide sequence, and wherein the total area of ​​the zones accounts for at least 70% or at least 80% or at least 90% or at least 95% of the area of ​​the surface. In some embodiments, the fractal barcode array comprises at least three distinct regions of spatial barcode zones, e.g., Figure 4D In some embodiments, the fractal barcode array includes spatial barcode regions having the same shape. In some embodiments, the shape of the spatial barcode region is circular or polygonal.

[0041] Overlapping chamber footprint with dual barcodes

[0042] In some embodiments, continuous cycles of bead loading and chamber synthesis can result in overlapping spatial barcode regions or zones, such as Figure 5A In the above figure, the surface (501) includes a shaded space barcode containing area (e.g., 503), such as Figure 5B These can be the result of a first cycle of bead placement, chamber synthesis, release, capture, and replication. For such a surface, a second cycle of bead placement, chamber synthesis, release, capture, and replication can be applied, where at least a portion of the beads are selected to be enclosed in a chamber because the resulting chamber overlaps an area of ​​one or more previously made chamber footprints, e.g. Figure 5AAs shown in the chamber (504) of the above figure. In the figure, the position of the chamber (504) of the second cycle can be selected to overlap with the chamber coverage areas (509) and (511) of the first cycle and is selected to encapsulate the beads (507). The overlapping areas (506) and (508) can each include a dual barcode sequence, which in turn includes in a single oligonucleotide chain (i) for (506), a barcode sequence from the bead (507) in the chamber coverage area (509) and a barcode sequence already attached to the surface (501), and (ii) for (508), a barcode sequence from the bead (507) in the chamber coverage area (511) and a barcode sequence already attached to the surface (501). The structures of the various barcoded chains attached to the surface (501) and how they are formed are described above in conjunction with Figure 2 And the following combination Figure 5C and Figure 5D The region (513) enclosed by the chamber (504) and located between the first cycle chamber footprint (509) and (511) may include oligonucleotide chains attached to the surface (501), each of which includes only the barcode sequence from the bead (507). After chamber synthesis, the unencapsulated beads (e.g., 502) may be removed (510) from the surface (501), as Figure 5A The barcode containing oligonucleotides can then be released from the encapsulated beads and the released sequences captured by capture oligonucleotides attached to the surface (501) and replicated (512), or they are linked to the previously formed first cycle oligonucleotide chain in the overlap region to form a double barcoded oligonucleotide chain, after which the second cycle chamber is degraded. The result is an arrangement of the overlap chamber footprint shown in Figure 5A In the figure below.

[0043] Figure 5B and Figure 5C The formation of single barcode oligonucleotide chains and double barcode oligonucleotide chains on a surface (501) is shown. Figure 5B Describes in a slightly different form the Figure 1C-1E The process described in . Briefly, a surface (501) can have attached to it a "lawn" (530) of two primers, designated P5 and P7 (i.e., capture oligonucleotides), which are attached to the surface (501) via their 5' ends. The barcode oligonucleotides (534) can be released or cleaved (532) from the beads and captured by the P5 capture oligonucleotide, after which they can be replicated by extending the P5 primer (in the presence of a suitable DNA polymerase and dNTPs). After such replication, the extension products can be surface amplified to obtain clusters (536).

[0044] Dual barcode oligonucleotides can be used to determine the relative position of the chamber coverage area on the surface. This is advantageous because the barcode only needs to be sequenced once (e.g., after integration into the cDNA), rather than twice as many current spatial barcode methods, such as Chen et al., Cell, 185: 1777-1792 (2022); Cho et al., bioRxiv (https: / / boi.org / 10.1101 / 2021.01.25.427807); Fu et al., Cell, 185: 4621-4633 (2022); Rodriques et al., Science, 363 (6434): 1463-1467 (2019); etc. In some embodiments, these conditions are achieved by delivering one or more splint oligonucleotides and ligase activity to a surface for reacting with the barcode oligonucleotide. A portion of the barcode oligonucleotide released from each bead may include a 5' phosphate group, which forms a phosphodiester bond that covalently links the barcode oligonucleotide whenever it is juxtaposed with the 3' hydroxyl of a barcode oligonucleotide attached to a surface, for example, by hybridization with a splint oligonucleotide. In some cases, self-ligation or linkage with other released barcode oligonucleotides may occur, but the products of such side reactions may be removed by washing and will not affect the method. In some embodiments, for example, the formation of such side products can be adjusted by adjusting the portion of the barcode oligonucleotide that includes a 5' phosphate group. In some embodiments, the portion of the barcode oligonucleotide that includes a 5'-phosphate group is in the range of 0.5% to 10%; in other embodiments, the portion of the barcode oligonucleotide that includes a 5'-phosphate group is in the range of 1% to 5%. These percentages can be conveniently obtained when using the nitrobenzyl photocleavable linker of Urdea et al. (cited above) because the position of the nitrobenzyl moiety in the linker can be selected to produce a 5'-phosphate or a 5'-hydroxyl. Therefore, the two forms of adapters can be used in a ratio corresponding to the desired fraction of barcode oligonucleotides having a 5'-phosphate group. Figure 5C The implementation plan is to Figure 5BThe embodiment of the present invention begins with the same surface (501) and capture oligonucleotide plateau. The barcode oligonucleotide (544) (a portion of which includes a 5'-phosphate group (542)) is released (540) in the presence of a splint oligonucleotide (546) on or near the surface (501), so that at least some of the barcode oligonucleotides from the adjacent chamber coverage area are connected to form a dual barcode oligonucleotide (548). In some embodiments, the splint oligonucleotide can be rendered non-connectable by the presence of a 3' blocking group or dideoxynucleotide at its 3' end, i.e., preventing the formation of a dimer of the splint oligonucleotide and the phosphorylated barcode oligonucleotide. In some cases, the dual barcode oligonucleotide can be captured by its internal primer binding site (555), but as with the circularized barcode oligonucleotide, such byproducts will not be significant because the desired capture configuration can be increased or decreased by adjusting the reaction conditions (such as Figure 5C After capturing and extending the barcode oligonucleotide (544) and any tandem barcode oligonucleotides formed therefrom, clusters of clonal oligonucleotides comprising the barcode sequence can be formed by surface amplification (550). In the overlapping region of adjacent chamber footprints on the surface (501), clusters comprising three barcode sequences are formed, an exemplary embodiment of which is shown in FIG. Figure 5C and Figure 5D For example, Figure 5C As shown, some clusters may include dual barcode sequences (552), such as b2 and b8, while other clusters may include single barcode sequences (e.g., (554) and (556)), which include only b8 and only b2, respectively.

[0045] Figure 5DAn example of three different oligonucleotide chains that can be attached to a surface (501) in an overlapping region is shown: a first cycle barcode chain (560), a second cycle dual barcode chain (562), and a second cycle barcode chain (564) (where "cycle" refers to the step of using a chamber to define the area where the barcode is attached to the surface). In some embodiments, as described above, the oligonucleotide chain containing the barcode sequence includes a primer (565) attached to the surface (501) via its 5' end and a primer (566) having a free 3' hydroxyl group to allow solid phase amplification, a barcode sequence BC1 (567), a capture sequence (568) (which can be a polyT segment for transcriptome analysis) and a restriction endonuclease site RE (569) (which is used to cut the distal portion of the oligonucleotide chain to produce a free 3' end of the capture oligonucleotide). Whenever restriction digestion is used to generate a capture sequence that can be extended, the second (or subsequent) cycle oligonucleotide chain can also include a primer binding site (570). The restriction sites of the first cycle oligonucleotide and the second cycle oligonucleotide can be the same or different. In some embodiments, the first cycle (or any cycle before the last cycle) can include a restriction site for a methylation-sensitive endonuclease (i.e., an endonuclease that prevents cleavage whenever its recognition site is methylated in a specific manner). With such an arrangement, cleavage can be accomplished only at the distal restriction site of the dual barcode strand (562), such as Figure 5E As shown. A portion of the sequence of primer (572) is complementary to the recognition site (569) so that when annealed with the double barcode oligonucleotide after amplification, a non-methylated restriction site is formed. Then, primer (572) can be extended with a polymerase in the presence of four kinds of deoxyribonucleoside triphosphates (dNTPs), wherein one of the dNTPs is replaced by an appropriately methylated analog. Restriction sites (such as restriction sites (571)) in the direction from restriction site (569) to surface (501) will be methylated. In some embodiments, type IIs endonuclease AwlI (New England Biolabs) can be used to achieve this purpose. In this case, primer (572) will be extended with dATP substituted with the analog N6-methyldeoxyadenosine 5'-triphosphate (which can be prepared as described in Mace, J. Biol. Chem. 259(6):3616-3669 (1984); and / or Jones et al., J. Am. Chem. Soc., 85(2):193-201 (1963)). After this extension and digestion, the extension products and cleaved distal segment can be washed away to obtain a dual barcode sequence (576) with the distal capture sequence (573) exposed.

[0046] The relative positions of the chamber footprint and overlap regions on the surface are known and can be determined as Fig. 6A . (The chamber coverage areas are arbitrarily labeled 1 to 33). Each chamber can be associated with a primary barcode, which corresponds to the barcode sequence released from the beads encapsulated by the chamber. The problem is to determine which primary barcode is associated with which chamber. Whenever there is an overlap, each chamber with an overlap is also associated with one or more secondary barcodes (i.e., a barcode that is part of a dual barcode oligonucleotide and is derived from the primary barcode of an adjacent and overlapping chamber). The solution to the above problem is to determine which of the 33 primary barcodes is associated with which chamber in a manner consistent with the type and number of single barcode sequences and dual barcode sequences. Fig. 6A Table 1 in provides an example of such data from an inspection of chambers 1 to 7 of the surface represented in the upper diagram of the figure. Figure 6B The algorithm represented by the flowchart is to systematically use the tabular data to find an ordering of primary barcodes in chambers 1-33, which is a solution as described above. The chambers are assigned numbers K=1 to N, and the primary barcodes are assigned numbers J=1 to N. According to steps (602)-(606), starting with chamber K=1, primary barcodes can be selected for chambers K and K+1 (selected from the primary barcode set without replacement), and then the type and number of secondary barcodes are checked to determine whether they are consistent with the currently selected primary barcode. If they are consistent, consideration will be given to moving to the next chamber (K+2) and selecting the next primary barcode from the remaining primary barcodes. This can continue until no primary barcode selection is consistent, in which case the process restarts (608) and a different primary barcode is selected in chamber 1. The process continues until an ordering of primary barcodes that meets the solution conditions is found.

[0047] Systems and Instruments

[0048] exist Fig. 7A An example of a system for performing the above method is shown in . A flow cell (700) can be a component of a fluidic device that provides one or more channels and liquid handling components under programmable control to deliver beads and reagents to the channels. In this illustration, four channels (702, 704, 706, and 708) are shown, and an enlarged view (712) of a segment (710) of channel 2 (704) is shown below. Fig. 7AIn the abstract view of the flow cell (700), the inlet, outlet and other features of the channel are not shown. On the first surface (714) of channel 2 (704), a plurality of beads (e.g., (718)) can each be encapsulated by a hydrogel chamber (e.g., (716)). In some embodiments, the porosity of the polymer matrix wall of the hydrogel chamber is selected to be impermeable to the beads, but permeable to reagents used to form the spatial barcode. Thus, reagents can be introduced into the interior of the hydrogel chamber and removed from the interior of the hydrogel chamber by flowing (720) the reagents through the channel, but the beads remain inside. The enlarged view (712) below of the channel segment (710) shows an optical system (721) for causing the hydrogel chamber to perform photosynthesis at the location of the beads in the channel. One of ordinary skill in the art will recognize that a device having the same structure as the hydrogel chamber can be used. Fig. 7A and 7B Optical systems of different configurations can perform these functions. In some embodiments, one or more DMD-objective subsystems for synthesizing hydrogel structures can be used to increase the synthesis speed by synthesizing multiple structures simultaneously.

[0049] return Fig. 7A , for making the hydrogel chamber perform photosynthesis, the light source (722) can generate a beam (723) of light of appropriate wavelength (e.g., UV light), which passes through an appropriate photomask or beam shaping or beam pointing (Galvo) system to shape the beam so as to synthesize the desired one or more structures in the channel. In some embodiments, a digital micromirror device (DMD) (724) is used, and in other embodiments, a physical photomask can be used. The chamber position, shape, and polymer matrix wall thickness can be determined at least in part based on the bead position information determined by the image collected by the detector (732). The reflected light from the DMD (724) can be shaped using conventional optical devices (e.g., collimating optical devices (728)) and is guided through the objective lens system (734) into the channel 2 section (710). The objective lens (734) and the flow cell (700) can be moved relative to each other in the xy direction (736) to make the chamber perform photosynthesis at any position in any channel. In some embodiments, the flow cell (700) moves while the optical system (721) is stationary. In some embodiments, the objective lens (734) can also direct the light beam (727) from the light source (729) to the target (such as cells) on the first surface (714) and collect the light signal (such as fluorescent signal) from the measurement performed on the first surface (714). Alternatively, the objective lens (734) can be used as Figure 7B The light signal is collected by the independent objective lens shown. Figure 7BThe information collected by the counterparts in the embodiments, in particular the cell positions in their corresponding channels, can be used by a computer (738) and / or an auxiliary controller to guide the DMD (724) and a translation device that controls the relative positions of the objective lens (734) and the flow cell (700) to synthesize hydrogel chambers of appropriate shapes and sizes at appropriate locations.

[0050] Figure 7B An alternative optical system is shown in which the detection portion (750) of the optical system moves (772) independently of the movement (768) of the combining portion (752) of the optical system. In some cases, the detection portion (750) of the optical system includes a detector (756), an objective lens (758), a light source (760), and interconnected optical elements, such as a dichroic mirror (762). Fig. 7A As in the embodiment of the present invention, the detector (756) can be operatively associated with the computer (764) and the synthesis portion (752) of the optical system to provide bead position information to the synthesis portion (752). The computer (764) and (738) can also be operatively associated with a platform and / or motor that controls the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the synthesis portion (752) of the optical system is located on the side of the first surface (564) opposite to the detection portion (750). Fig. 7A Like the embodiment of the present invention, it includes conventional components: objective lens (774), reflector (776), collimating optics (780), DMD (782) and light source (778).

[0051] In some embodiments, beads (e.g. Fig. 7A The beads are randomly arranged on the first surface (714) in (718). In alternative embodiments, the first surface (714) may include regularly spaced locations or features for capturing beads, such that the beads are arranged substantially only on such locations or features on the first surface. For example, in some embodiments, such locations or features may be a linear or hexagonal array of dots.

[0052] In some embodiments, the system described herein includes: (a) a channel including a first surface, a plurality of beads arranged on the first surface, and one or more polymer precursors; (b) a spatial energy modulation element optically connected to the first surface; (c) a detector optically connected to the first surface and operably associated with the spatial energy modulation element, the detector detecting each of the plurality of cells and determining its position on the first surface; and (d) a plurality of gel chambers, each gel chamber encapsulating a single bead from the plurality of beads, wherein the gel chamber is synthesized by projecting light into the channel using the spatial energy modulation element so that the projected light causes one or more polymer precursors to crosslink to form a polymer matrix wall of the chamber, wherein the position of the synthesized chamber is determined by the position of the bead encapsulated by the synthesized chamber as identified by the detector. It should be understood that the term "detector" as used herein may include, but is not limited to: a microscope element that collects and optionally amplifies an image of a portion of the channel; and an image analysis element that includes information for identifying beads and associated position information. The computer element uses such information generated by the detectors, as well as user input, to generate commands to other elements (such as spatial energy modulation elements) to perform a variety of functions including, but not limited to, synthesis chambers, "on demand" degradation of chambers, selective photodegradation chambers, and the like.

[0053] The configuration of such an embodiment is described above Figure 7A-7B In some embodiments, the channel of the fluidic device further comprises a second surface, wherein the first surface and the second surface are arranged relative to each other across the channel, and wherein the polymer matrix wall of the chamber extends from the first surface to the second surface to form a chamber each having an interior. In some embodiments, the chambers in the channel each encapsulate a single cell. In some embodiments, the first wall and the second wall are both made of a light-transmitting material (such as glass, plastic, etc.), and are positioned so that the first surface and the second surface are substantially parallel to each other. The vertical distance between the first surface and the second surface can be in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm.

[0054] In some embodiments, the cells can be randomly arranged on the first surface. In some embodiments, the beads are randomly arranged on the first surface in a Poisson distribution. In some embodiments, in this Poisson distribution, the beads have a nearest neighbor distance equal to or greater than 10 μm, equal to or greater than 20 μm, equal to or greater than 30 μm, equal to or greater than 40 μm, equal to or greater than 50 μm, or equal to or greater than 100 μm. In some embodiments, a subset of the beads of this Poisson distribution is each encapsulated by a ring-shaped chamber with a diameter in the range of 10-500 μm. In some embodiments, the beads are randomly arranged on the first surface in a Poisson distribution with a density of 10 to 2500 beads / mm 2, or 10 to 1000 beads / mm 2 , or 10 to 500 beads / mm 2 , or 10 to 100 beads / mm 2 within the range.

[0055] In some embodiments, multiple channels can be Figure 8A-8B 1 to 6, or 808 to 802 to 804 to 806 to 802 to 804 to 806 to 802 to 806 ... In some embodiments, the thickness of the spacer (804) can be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm, which determines the internal height of the channel. The top wall (802) includes an inlet (814) and an outlet (816), which are used to load and remove reagents and beads from channel 1-channel 6 individually or collectively. In some embodiments, at least one of the walls (802) and (806) is made of a light-transmitting material (such as glass, plastic, etc.). The flow cell (800) can be operationally associated with a fluid device that delivers reagents and beads to any one of channel 1-channel 6 under program control. Guidelines for specific designs (including fluid handling and valve adjustment of such fluid systems) can be found in U.S. Patents 8921073, 8173080 and 8900828, etc., which are incorporated herein by reference. Figure 8B A channel of a flow cell (800) is shown having randomly distributed (not to scale) hydrogel chambers having a quasi-ring-shaped cross-section, such as (820), on a first surface thereof.

[0056] As described above, any of the first surface, second surface, or polymer matrix wall of the chamber may include capture elements and other functional groups for performing various operations including, but not limited to, capturing beads, capturing analytes (such as mRNA, secreted proteins, intracellular proteins, or genomic sequences), capturing components of analytical reagents (such as oligonucleotide tags from antibodies), etc. Derivatized surfaces for such purposes are well known to those skilled in the art, as demonstrated by the following exemplary references: Integrated DNA Technologies brochure (cited above); Hermanson (cited above); etc.

[0057] As described above, in some embodiments, the fluid device of the method includes a detector or is operationally associated with a detector, which can share the optical path of the spatial energy modulation element, or can be arranged near the second wall or arranged near the first wall opposite to the spatial energy modulation element in an embodiment (such as a groove) having only a first wall and a first surface. The detector is positioned so that it can detect light signals from cells in the channel or detect light signals near cells in the channel, for example, the cells are distributed on the first surface in the chamber. In some embodiments, the first wall and the second wall each include a light-transmitting material, for example, so that the spatial energy modulation element can project light energy into the interior of the channel, and so that the detector can detect light signals (such as fluorescence emission or reflected light from biological components). In some embodiments, the projected energy from the spatial energy modulation element is light energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulation element can have a complex cross-section, thereby allowing (in various embodiments) to synthesize multiple chambers simultaneously. Light-transmitting materials include but are not limited to glass, quartz, plastics and similar materials.

[0058] The spatial energy modulation element for achieving polymerization using light energy may include a physical photomask or a virtual photomask, such as a digital micromirror device (DMD). The following references are incorporated by reference to provide guidance in selecting and operating a DMD for achieving photopolymerization of gels: Chung et al., U.S. Patent 10464307; Hribar et al., U.S. Patent 10351819; Das et al., U.S. Patent 9561622; Huang et al., Biomicrofluidics, 5:034109 (2011); etc.

[0059] application

[0060] According to the present invention, after establishing a spatial barcode on the surface, oligonucleotide markers, barcodes, genomic fragments, messenger RNA and similar polynucleotide targets can be sequenced by the method and system of the present invention. In some embodiments, the capture element for this purpose includes an oligonucleotide attached to the surface in the channel, wherein such oligonucleotide includes a sequence segment complementary to the sequence segment of the nucleic acid to be captured, and the sequence segment can be a polyA segment of mRNA or any "handle" sequence region adjacent to the barcode or oligonucleotide marker. The spatial barcode can provide channel position information and allow the sequence determined externally to be associated with each chamber. In some embodiments, the spatial barcode exists at a sufficiently high density so that each chamber covers the first surface area uniquely associated with one or more spatial barcodes (and typically a single spatial barcode). In some embodiments, the preparation of polynucleotides for sequencing operations occurs after the target template (e.g., oligonucleotide markers, mRNA, genomic fragments) is released from the cell and captured by the complementary sequence in the capture element. The release step depends on the properties of the target template. For example, an oligonucleotide marker attached to an antibody by a disulfide bond can be released by a reducing agent (which can be the same as a cleavage agent). mRNA can be released by treating cells with conventional lysis reagents. Releasing genomic fragments may require lysis and pre-amplification steps. Lysis conditions may vary greatly and may be based on the effects of heat, detergents, proteases, alkalis, or a combination of such factors. The following references provide guidance for selecting lysis reagents or lysis buffers for single cell lysis conditions of mRNA and / or genomic DNA: Thronhill et al., Prenatal Diagnosis, 21: 490-497 (2001); Kim et al., Fertility and Sterility, 92: 814-818 (2009); Spencer et al., ISME Journal, 10: 427-436 (2016); Tamminen et al., Frontiers Microbiol. Methods, 6: article 195 (2015); etc.Exemplary lysis conditions include the following: 1) cells in H2O at 96°C for 15 min, then at 10°C for 15 min; 2) 200 mM KOH, 50 mM dithiothreitol, heated to 65°C for 10 min; 3) for 4 μL of protease-based lysis buffer: 1 μL of 17 μM SDS combined with 3 μL of 125 μg / mL proteinase K, then incubated at 37°C for 60 min, then incubated at 95°C for 15 min (to inactivate proteinase K); 4) for 10 μL of detergent-based lysis buffer: 2 μL of H2O, 2 μL of 10 mM EDTA, 2 μL of 250 mM dithiothreitol, 2 μL of 0.5% N-lauryl sarcosinate solution; 5) 200 mM Tris pH 7.5, 20 mM EDTA, 2% sarcoyl, 6% Ficoll.

[0061] Fig. 9A process for capturing a target template and preparing cDNA for external sequencing is shown. Those skilled in the art will recognize that the details of the following examples of target template capture and cDNA synthesis may vary greatly depending on the sequencing system employed. In some embodiments, the preparation of cDNA includes a tagging step. Guidance for specific embodiments can be found in the following references: Picelli et al., Genome Research, 24: 2033-2040 (2014); Bose et al., Genome Biology, 16: 120 (2015); Hashimshony et al., Genome Biology, 17: 77 (2016); Yuan et al., Scientific Reports, 6: 33883 (2016); and similar references. Attached to the surface (901) by its 5' end is an oligonucleotide having the following components: a primer binding site P7 (for Illumina sequencers) (902), an optional primer binding site R1 (for Illumina paired end sequencing), a barcode oligonucleotide (906) (which can be or include a spatial barcode), an optional unique molecular identifier (908), and a capture oligonucleotide (910), which can be a polyT segment whenever mRNA is to be captured. The target template (912) is captured by hybridization of a polyA segment or sequence handle (914) to the capture oligonucleotide (910). After capture, the capture oligonucleotide (910) and the polyA segment (914) are extended by a polymerase (e.g., Moloney murine leukemia virus (MMLV) reverse transcriptase), leaving a single-stranded polyC tail (916). In some embodiments, a template switching oligonucleotide (918) is hybridized thereto and the polyC tail is further extended, as shown in (930), for example, Zhu et al., Biotechniques, 30:892-897 (2001). The unattached strand is melted, and the attached strand is amplified, for example, by PCR and eluted for external sequencing (932).

[0062] Hydrogel chamber

[0063] Function A variety of photosynthesizable gels can be used in conjunction with the present invention. In some embodiments, hydrogels are used with the present invention, particularly because they are compatible with living cells and are widely used in formulating gels with desired properties, including but not limited to porosity (which largely determines the contents and passage of materials through the gel (or polymer matrix) wall), degradability, mechanical strength, ease and speed of synthesis, etc.

[0064] Porosity. In some embodiments, the hydrogel porosity is selected to allow the selected reagent to pass through while preventing other reagents or objects (such as cells) from passing through. In some embodiments, the hydrogel porosity is selected to prevent biological cells from passing through, but allows reagents (including proteins, such as polymerases) to pass through. In some embodiments, such reagents that can penetrate the polymer matrix wall include lysozyme, proteinase K, random hexamer, polymerase, transposase, ligase, deoxynucleotide triphosphate, buffer, cell culture medium or divalent cations. In some embodiments, at least one polymer matrix includes a pore, which is sized to allow the reagent to diffuse through at least one polymer matrix, but is too small to allow the DNA or RNA for analysis to cross the pore (size greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs). In some embodiments, the polymer chains of the cross-linked hydrogel structure form a hydrogel matrix (i.e., a porous hydrogel matrix) with pores. In some versions, the size of the pores in the hydrogel structure can be regulated or tuned, and can be formulated to encapsulate sufficiently large genetic material (such as cells or nucleic acids) (e.g., genetic material greater than about 300 base pairs), but allow smaller substances (such as reagents) or nucleic acids of smaller size (e.g., less than about 50 base pairs) (such as primers) to pass through the pores, thereby entering and exiting the hydrogel structure. In some embodiments, the hydrogel can have a diameter sufficient to allow the reagents listed above to diffuse through the structure while retaining any pore size of nucleic acid molecules greater than 500 nucleotides or base pairs in length. In some embodiments, when the hydrogel is hydrated, the hydrogel structure can expand. Then, the size of the pores can be changed depending on the water content in the hydrogel of the hydrogel structure. In some embodiments, the diameter of the pores is from about 10nm to about 100nm. In some embodiments, the pore size of the hydrogel structure is tuned by routine experiments by changing the ratio of the concentration of the polymer precursor to the concentration of the cross-linking agent, changing pH, salt concentration, temperature, light intensity, etc. In some embodiments, the average diameter of the pores of the polymer matrix wall prevents the passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater, a molecular weight of 50 kDa or greater, a molecular weight of 75 kDa or greater, a molecular weight of 100 kDa or greater, or a molecular weight of 150 kDa or greater.

[0065] In some embodiments, the length of the DNA or RNA retained can be sequenced using conventional synthetic sequencing techniques. For example, such DNA or RNA comprises at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the average diameter of the pore can be 5nm to 100nm. In some embodiments, the average diameter of the pore can be 5nm to 10nm, 10nm to 20nm, 20nm to 30nm, 30nm to 40nm, 50nm to 60nm, 60nm to 70nm, 70nm to 80nm, 80nm to 90nm, 90nm to 100nm. In some embodiments, the average diameter of the pore can be greater than 100nm. In some embodiments, the average diameter of the pore can be less than 5nm. Reagents can include enzymes or primers with a size less than 50 base pairs (bp). Primers can include single-stranded DNA (ssDNA). In some embodiments, the size of primers can be 5bp to 50bp. In some embodiments, the size of the primer can be 5bp to 10bp, 10bp to 20bp, 20bp to 30bp, 30bp to 40bp or 40bp to 50bp. In some embodiments, the size of the primer can exceed 50bp. In some cases, the size of the primer can be less than 5bp. In some embodiments, the diameter of the pore can be 5nm to 100nm. In some embodiments, the diameter of the pore can be 5nm to 10nm, 10nm to 20nm, 20nm to 30nm, 30nm to 40nm, 50nm to 60nm, 60nm to 70nm, 70nm to 80nm, 80nm to 90nm, 90nm to 100nm. In some embodiments, the diameter of the pore can be greater than 100nm. In some embodiments, the average diameter of the pore can be less than 5nm. The pore size of the polymer matrix can be from about 5 nanometers (nm) to about 100nm.The pore size of the polymer matrix can be from about 5 nm to about 10 nm, from about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, from about 5 nm to about 100 nm, from about 5 nm to about 110 nm, from about 10 nm to about 20 nm, from about 10 nm to about 30 nm, from about 10 nm to about 40 nm, from about 10 nm to about 50 nm, from about 10 nm to about 60 nm, from about 10 nm to about 70 nm, nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 10 nm to about 100 nm, about 10 nm to about 110 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm about 70nm, about 30nm to about 80nm, about 30nm to about 90nm, about 30nm to about 100nm, about 30nm to about 110nm, about 40nm to about 50nm, about 40nm to about 60nm, about 40nm to about 70nm, about 40nm to about 80nm, about 40nm to about 90nm, about 40nm to about 100nm, about 40nm to about 110nm, about 50nm to about 60nm, about 50nm to about 70nm, about 50nm to about 80nm, about 50nm to about 90nm, about 50nm to about 100nm The pore size of the polymer matrix can be about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 60nm to about 110nm, about 70nm to about 80nm, about 70nm to about 90nm, about 70nm to about 100nm, about 70nm to about 110nm, about 80nm to about 90nm, about 80nm to about 100nm, about 80nm to about 110nm, about 90nm to about 100nm, about 90nm to about 100nm, or about 100nm to about 110nm. The pore size of the polymer matrix can be about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm or about 110nm. The pore size of the polymer matrix may be at least about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, or less.The pore size of the polymer matrix may be up to about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or more.

[0066] Modulation of porosity . The pore size in the polymer matrix can be adjusted using chemical reagents or by applying heat, electric field, light or another suitable stimulus. In other words, the polymer matrix can include tunable properties (e.g., pore size). In some cases, the polymer matrix can include thermoresponsive or temperature responsive polymers. Thermoresponsive polymers (e.g., poly (N-isopropylacrylamide) (NIPAAM)) may phase separate with the solution after heating or after cooling (e.g., polymers showing lower critical solution temperature (LCST) or upper critical solution temperature (UCST)). The polymer matrix can include polymers that can collapse at high temperatures, such as to control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that can be used to form a hydrogel / polymer matrix with tunable properties can include poly (N-vinyl caprolactam), poly (N-ethyl oxazoline), poly (methyl vinyl ether), poly (acrylic acid-acrylamide) copolymers or combinations thereof. Changes in temperature can enlarge or shrink the average pore size in the polymer matrix to allow selected molecules such as nucleic acid molecules, proteins or any biomolecules or molecules smaller than the adjusted pore size to be released from the hydrogel chamber.

[0067] Size and shape of hydrogel chambers. In some embodiments, the polymer matrix wall of the chamber inhibits the passage of predetermined components, such as mammalian cells, genomic DNA, larger polynucleotides (e.g., mRNA greater than 200 ribonucleotides, greater than 300 ribonucleotides, or 500 ribonucleotides), etc. In some embodiments, the polymer matrix wall extends from the first surface to the second surface (parallel to the first surface) to form a chamber in the channel. In some embodiments, the chamber has a polymer matrix wall and an interior. In some embodiments, the size of the interior of the chamber is designed to encapsulate cells. For example, such chambers may include a cylindrical shell or a polygonal shell, which includes an interior space or an interior and a polymer matrix wall. In some embodiments, such chambers have a cross-section of a ring-like shape. As used herein, the term "ring-like cross-section" means a cross-section that is topologically equivalent to a ring. In some embodiments, the interior space or interior of the chamber has an inner diameter of 1 μm to 500 μm and a volume ranging from 1 picoliter to 200 nanoliters or 100 picoliters to 100 nanoliters or 100 picoliters to 10 nanoliters. In some embodiments, the thickness of the polymer matrix wall is at least 1 μm (micrometer). In some embodiments, the value of the height of the chamber with a quasi-annular cross section ranges from 10 μm to 500 μm or ranges from 50 μm to 250 μm. In some embodiments, the aspect ratio (i.e., height / width) of the polymer matrix wall with a quasi-annular cross section is 1 or less. In some embodiments, the aspect ratio and polymer matrix wall thickness are selected to maximize chamber stability of resistance (such as reagent flow through the channel), washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix is ​​degradable. In some embodiments, the degradation of at least one polymer matrix is ​​"as needed". In some embodiments, the chamber in the channel is discontinuous. In some embodiments, the chamber in the channel can be adjacent to an adjacent chamber. In some embodiments, the chamber can share a polymer matrix wall with each other. In some embodiments, the chamber can be synthetic, and its slit or other orifice is large enough to allow certain components (such as beads) to pass through, but small enough to prevent other components (such as cells) from passing through.

[0068] Hydrogel composition. In some embodiments, the channel of the fluid device of the system of the present invention comprises one or more polymer precursors for forming a chamber. In some embodiments, the one or more polymer precursors comprise a hydrogel precursor. Such precursors can be selected from a variety of compounds, including but not limited to polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PV In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEG thiol / PEG-acrylate, acrylamide / N, N'-bis (acryloyl) cystamine (BACy) or PEG / PPO. In some embodiments, the following precursors and cross-linking agents can be used to form a chamber with a degradable polymer matrix (hydrogel) wall. The polymer precursor can be formed by using any of the hydrogel precursors and crosslinking agents (columns 1 and 3, respectively) of Table 2. The resulting polymer matrix can be degraded using a degradation agent as indicated in Table 2 (column 4).

[0069] Table 2

[0070]

[0071] Table 3

[0072]

[0073] Hydrogel degradation. In some embodiments, the hydrogel chambers of the present invention are generally degradable or depolymerizable in the channel or "as needed" in the channel. Generally, the degradable hydrogel chambers are degraded by treatment with a degradation agent or a depolymerizing agent that is equivalently exposed to all chambers in the channel. Exemplary depolymerizing agents include, but are not limited to, heat, light, and / or chemical depolymerizing agents (sometimes also referred to as cutting agents or degradation agents). In some embodiments, polymer precursors that allow photocrosslinking and photodegradation can be used according to the degradation required, for example, different wavelengths are used for crosslinking and degradation to achieve. For example, Eosin Y can be used for free radical polymerization at a defined area using a 500nm wavelength, after which 380nm irradiation can be used to cut the crosslinker. In other embodiments, a light cage hydrogel cutting agent can be included in the formation of the polymer matrix wall. For example, an acid-labile crosslinker (such as an ester, etc.) can be used to produce a hydrogel, and then UV light can be used to generate local acidic conditions, which in turn degrade the hydrogel. In some embodiments, at least one polymer matrix can be degraded by at least one of the following: (i) contacting at least one polymer matrix with a cleavage agent; (ii) heating at least one polymer matrix to at least 90°C; or (iii) exposing at least one polymer matrix to a certain wavelength of light that cuts a photo-cleavable cross-linking agent, which cross-links the polymers of at least one polymer matrix. In some embodiments, at least one polymer matrix comprises a hydrogel. In some embodiments, the cleavage agent degrades the hydrogel. In some embodiments, the cleavage agent includes a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage agent, or a combination thereof. In some embodiments, the cleavage agent includes dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), tris (3-hydroxypropyl) phosphine (THP) or a combination thereof. In some embodiments, the surface of the polymer matrix or hydrogel can be functionalized by coupling functional groups to the polymer matrix or hydrogel. Some non-limiting examples of functional groups can include capture reagents (e.g., picolinaldehyde (PCA)), acrylamide, agarose, biotin, streptavidin, strep-tag II, linkers, functional groups comprising aldehydes, phosphates, silicates, esters, acids, amides, aldehyde dithiolanes, PEGs, thiols, olefins, alkynes, azides, or combinations thereof. In some cases, the functionalized polymer matrix can be used to capture biomolecules within the polymer matrix compartments formed adjacent to (e.g., around or on) the biological component. The biomolecules can be produced by the biological component (e.g., from the secretome of the cell). The functionalized surface of the polymer matrix within the compartment can be used to capture reagents or molecules from outside the compartment. The functionalized surface can increase the surface area covered by reagents, molecular sensors, or any molecules of interest (e.g., antibodies).

[0074] Partial degradationIn some embodiments, the existing polymer matrix wall can be partially degraded, for example to change the porosity. In some embodiments, the polymer precursor can include degradable beads that form part of the polymer matrix wall and are embedded therein when synthesized and can then be degraded on demand or routinely, resulting in an increase in porosity.

[0075] Photosynthesis . In some embodiments, generating a polymer matrix in the fluid device includes exposing one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light of 350nm to 800nm. In some embodiments, the light generating device generates light of 350nm to 600nm. In some embodiments, the light generating device generates light of 350nm to 450nm. In some embodiments, the light generating device generates UV light. In some embodiments, generating a polymer matrix in the fluid device is performed using a spatial light modulator (SLM) (i.e., a spatial energy modulation element capable of spatially generating a desired light intensity pattern). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam manipulated using a galvanometer. In some embodiments, the SLM is based on liquid crystal.

[0076] Example

[0077] Spatial barcodes constructed from randomly arranged beads on a surface

[0078] In this embodiment, each of them carries Fig. 10A 10 μm beads (streptavidin coated, Bangs Lab Inc., UMC0102) of oligonucleotides of the structure shown are loaded into a flow cell channel whose surface includes a "plateau" of P7 primers and P5 primers (Illumina sequencing flow cell). After loading the beads with polymer precursors, an annular (or ring-shaped) hydrogel chamber is synthesized around each bead using the above-mentioned equipment. After the oligonucleotides are released from the beads, captured and amplified, the portion of the barcode sequence is determined by fluorescence-based sequencing by synthesis to confirm the distribution and identity of the barcode in the space within the chamber. Oligonucleotides (1000, Fig. 10A) is attached to the bead (1002) by forming a duplex with a shorter biotinylated oligonucleotide (not shown) attached to the streptavidin on the bead (1002). From 5' to 3', the oligonucleotide (1000) includes a P7 primer binding site (1004), a composite barcode segment (1006) comprising three barcodes separated by a spacer segment, a spacer "TTCGAG", a random sequence segment (1008), an R2 primer binding site (1010) for sequencing the captured and amplified oligonucleotide, a capture oligonucleotide ("T30", 1012) designed to capture mRNA, a cleavage site (1014) for removing the distal segment of the oligonucleotide (1000) so that the sequence captured by segment (1012) can be extended, and a P5 primer binding site (1016).

[0079] The above steps were performed as follows: a hydrogel precursor solution (40% acrylamide gel with 30 mg / mL photoinitiator (3X lithium phenyl-2,4,6-trimethylbenzoylphosphonate)) containing 10 μm beads with barcode oligonucleotides attached by hybridization was loaded into the flow cell channel. Photosynthesis of the chambers was performed using a DMD programmed to project a ring beam with a radius of 200 uM around each bead. After the gel structure was formed, the excess precursor solution was washed with 600 ul of 1X PBS. The barcode oligonucleotides were released (or melted) from the beads by loading the flow cell with a release solution consisting of 50% formamide in a 350 mM NaCl solution, followed by heating the flow cell to 70°C. The barcode oligonucleotide from each bead was confined to its corresponding hydrogel chamber and captured by the anchored P7 primer. The Bst polymerase mix is ​​then loaded into the flow cell and the captured barcode oligonucleotide is used as a template to extend the P7 primer, followed by cluster formation from the extended sequence via bridge amplification. The sequencing primer anneals to the amplified sequence and recognizes the barcode sequence. Fig. 10B On the left is shown an enlarged section of a flow cell channel (1018) where a single bead has been encapsulated by a hydrogel chamber (e.g., (1020)). On the right is an enlarged section (1022) of the flow channel (1018) after incorporation of fluorescently labeled nucleotides in sequencing of the barcode segment of an amplified oligonucleotide. The fluorescence distribution in the chamber (e.g., 1024) shows uniform coating of the spatial barcode inside the chamber. Fig. 10C A larger section of a flow cell channel (1026) is shown with a spatial barcode distribution.

[0080] 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 applicable to various sensor embodiments and other subject matter in addition to those discussed above.

[0081] definition

[0082] 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, 2nd edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd edition (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immuology, 6th edition (Saunders, 2007).

[0083] In some embodiments, "determination" refers to a process for detecting or measuring a cell property or property of a single cell or a cell group. Typically, the process steps of the determination include chemical, biochemical or molecular reactions (such as the cutting of a bond, specific binding of complementary components, enzyme reactions, dissolution of complementary components, etc.) or changes in physical state (such as the increase or decrease of temperature, changes in energy levels, etc.), and result in the generation of a signal (or multiple signals), from which the presence, absence or size of a quantity associated with the cell can be inferred. The properties of the signal generated by the determination may vary greatly, and may include but are not limited to electrical signals, optical signals, chemical signals or material signals. Material signals include the generation of materials containing information that can be extracted. For example, a material signal can be an amplification of a polynucleotide whose length, quantity, composition or nucleotide sequence indicates a cell property. For example, a barcode oligonucleotide can be a material signal. The characteristics or properties of cells detected or measured may vary widely and include, but are not limited to, cytotoxicity, viability, ability to proliferate under selected conditions, size, shape, motility, types and profiles of cell surface or membrane proteins, types and profiles of secreted proteins, production of metabolites, transcriptome, gene copy number, gene or allele identity, chromatin accessibility profile, vector copy number of engineered or infected cells, etc. Assays of particular interest for cell-based therapies include, but are not limited to, cytotoxicity, viability, activation, ability to proliferate under selected conditions, chromatin accessibility profile, types and profiles of cell surface or membrane proteins, types and profiles of secreted proteins, intracellular proteins, transcriptome, vector copy number, etc.

[0084] "Barcode" means 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), which can be used to identify an analyte. In some embodiments, a barcode (referred to herein as a "spatial barcode") can be attached to a surface to identify the location on the surface. In some embodiments, a population of the same spatial barcode can be arranged in a specific area on the surface. 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 has 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 comprises 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, which are incorporated herein by reference: Church, U.S. Pat. No. 4,942,124; Godron et al., International Patent Publication No. WO2020 / 120442; Seelig et al., U.S. Patent Publication No. 2016 / 0138086, etc. Sometimes, random oligonucleotides are represented as "NNN...N". In some embodiments, the term "barcode" includes composite barcodes; that is, oligonucleotide segments containing subsegments that identify different objects. For example, the first segment of a composite barcode can identify a specific area of ​​a surface, and the second segment of the composite barcode can identify a specific molecule (a so-called "unique molecule identifier" or UMI).

[0085] "Cell" refers to a biological cell that can be determined by the method and system of the present invention, including but not limited to vertebrate cells, invertebrate cells, eukaryotic cells, mammalian cells, microbial cells, protozoan cells, prokaryotic cells, bacterial cells, insect cells or fungal cells. In some embodiments, mammalian cells are determined by the method and system of the present invention. Specifically, any mammalian cell that can or has been genetically altered to be used in medicine, industry, environment or treatment can be analyzed by the method and system of the present invention. In some embodiments, "cell" as used herein includes genetically modified mammalian cells. In some embodiments, "cell" includes stem cells. In some embodiments, "cell" refers to cells modified by CRISPR Cas9 technology. In some embodiments, "cell" refers to cells of the immune system, including but not limited to cytotoxic T lymphocytes, regulatory T cells, CD4+T cells, CD8+T cells, natural killer cells, antigen presenting cells or dendritic cells. Particularly interesting are cytotoxic T lymphocytes engineered for therapeutic applications (such as cancer therapy).

[0086] "Cleavable linkage" or "cleavable nucleotide" means any of a variety of cleavable linkages, or more specifically, cleavable nucleotides can be used with embodiments of the present invention. As used herein, the term "cleavable site" refers to a nucleotide or backbone linkage of a single-stranded nucleic acid sequence that can be excised or cut under predetermined conditions, thereby dividing the single-stranded nucleic acid sequence into two parts. In some embodiments, the step of cutting a cleavable nucleotide or a cleavable linkage leaves a free 3'-hydroxyl on the cut chain, thereby, for example, allowing the cut chain to be extended by a polymerase. The cutting step can be performed by chemical, thermal, enzymatic or by light-based cutting. Sometimes, the term "release" can be used to refer to cutting an oligonucleotide tag, for example, by releasing an agent or a releasing agent, which can be one or more of those listed above. In some embodiments, a cleavable nucleotide can be a nucleotide analog (such as deoxyuridine or 8-oxodeoxyguanosine), which is recognized by a specific glycosylase (e.g., uracil deoxyglycosylase, followed by endonuclease VIII and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage by glycosylases and / or endonucleases may require a double-stranded DNA substrate. Methods for synthesizing and cleaving nucleic acids containing chemically cleavable, thermally cleavable, and photolabile groups are described in, for example, U.S. Patent No. 5,700,642, which is incorporated herein by reference. Further cleavable linkages are disclosed in the following references: Pon, R., Methods Mol. Biol. 20: 465-496 (1993); Verma et al., Ann. Rev. Biochem. 67: 99-134 (1998); U.S. Patent Nos. 5,739,386, 5,700,642, and 5,830,655; and U.S. Patent Publication Nos. 2003 / 0186226 and 2004 / 0106728; Urdea et al., U.S. Patent No. 5367066, which are incorporated herein by reference. The synthesis and cleavage conditions of chemically cleavable oligonucleotides are described in U.S. Patent Nos. 5,700,642 and 5,830,655. Phosphorothioate internucleotide linkages can be selectively cleaved under mild oxidative conditions. Selective cleavage of phosphoramide bonds can be performed under mild acidic conditions (such as 80% acetic acid). Selective cleavage of ribose can be performed by treatment with dilute ammonium hydroxide. In another embodiment, the cleavable linking portion can be an amino linker. The resulting oligonucleotide bound to the linker via a phosphoramidite linkage can be cleaved with 80% acetic acid to produce a 3'-phosphorylated oligonucleotide, which can be removed (if necessary) by a phosphatase. In some embodiments, the cleavable linking portion can be a photocleavable linker, such as an o-nitrobenzyl photocleavable linker.For example, in Venkatesan et al., J.Org.Chem.61:525-529 (1996), Kahl et al., J.Org.Chem.64:507-510 (1999), Kahl et al., J.Org.Chem.63:4870-4871 (1998), Greenberg et al., J.Org.Chem.59:746-753 (1994), Holmes et al., J.Org.Chem.62:2370-2380 (1997) and U.S. Patent No. 5,739,386, the synthesis and cleavage conditions of light-labile oligonucleotides on solid supports are described. Linkers based on o-nitrobenzyl (such as hydroxymethyl, hydroxyethyl and Fmoc-aminoethyl carboxylic acid linkers) can also be obtained commercially. In some embodiments, ribonucleotides can be used as cleavable nucleotides, wherein the cleavage step can be achieved using ribonucleases (such as RNase H). In other embodiments, the cleavage step can be performed by treatment with a nicking enzyme.

[0087] "Cluster" means an amplicon or clonal population of a single polynucleotide amplified by a surface amplification technique such as bridge PCR. In some embodiments, the term "cluster" includes amplicons generated by rolling circle amplification.

[0088] "Hydrogel" means a gel comprising a cross-linked hydrophilic polymer network that can absorb and retain large amounts of water (e.g., 60% to 90% water, or 70% to 80%) without dissolving due to the establishment of physical or chemical bonds (which can be covalent, ionic or hydrogen bonds) between polymer chains. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications. Hydrogels can comprise natural or synthetic polymers and can be reversible (i.e., degradable or depolymerizable) or irreversible. Exemplary synthetic hydrogel polymers include polyethylene glycol (PEG), poly (2-hydroxyethyl methacrylate) and poly (vinyl alcohol). Exemplary natural hydrogel polymers include alginate, hyaluronic acid and collagen. The following references describe hydrogels and their biomedical uses: Drury et al., Biomaterials, 24:4337-4351 (2003); Garagorri et al., Acta Biomatter, 4(5):1139-1147 (2008); Caliari et al., Nature Methods, 13(5):405-414 (2016); Bowman et al., U.S. Patent 9631092; Koh et al., Langmuir, 18(7):2459-2462 (2002).

[0089] The term "as needed" means that operations can be directed to individual, discrete, selected locations (e.g., spatial location of a polymer precursor solution; or a selected polymer matrix chamber). Such selection can be based on manual observation of optical signals or data collected by a detector, or such selection can be based on a computer algorithm that operates on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include real-time detection or detection of a certain period of time before modulating unit energy to polymerize a polymer precursor or degrading a chamber. For example, a subset of chambers (all formed with photodegradable polymer matrix walls) can be preselected based on position information and the value of the optical signal from the analytical determination performed in the chamber for release and removal of its contents. The preselected chamber can be photodegraded by selectively projecting a beam of light with appropriate wavelength characteristics (e.g., with a spatial energy modulation element) to degrade the polymer matrix wall of the preselected chamber. In another example, multiple chambers can be observed in real time (e.g., via fluorescence microscopy) to detect an analyte of interest, and one or more chambers in multiple chambers can be selected in real time after detecting the analyte of interest for degradation.

[0090] A "physical photomask" generally refers to a physical structure having a plurality of openings or holes through which light can be projected. A physical photomask can be used to produce a hydrogel matrix as described herein by polymerizing a polymer precursor solution and forming a three-dimensional structure corresponding to the pattern on the photomask. A physical photomask can be patterned with a specific layout or geometric pattern. A physical photomask can be adhered to the upper surface of a flow cell.

[0091] "Polymerase chain reaction" or "PCR" means a reaction in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary DNA strands. In other words, PCR is a reaction for preparing multiple copies or replications of a target nucleic acid flanked by primer binding sites, such a reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing the primer to the primer binding site, and (iii) extending the primer by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled in a thermal cycler at different temperatures optimized for each step. The specific temperature, duration of each step, and rate of change between steps depend on many factors well known to those of ordinary skill in the art, such as those illustrated in the following references: McPherson et al., eds., PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in conventional PCR using Taq DNA polymerase, double-stranded target nucleic acid can be denatured at a temperature of>90°C, primers are annealed at a temperature in the range of 50-75°C, and primers are extended at a temperature in the range of 72-78°C. The term "PCR" covers derivative forms of reactions, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, bridge PCR, etc. The reaction volume ranges from several hundred nanoliters (e.g., 200 nL) to several hundred microliters (e.g., 200 μL). "Reverse transcription PCR" or "RT-PCR" means PCR performed before the reverse transcription reaction, which converts the target RNA into complementary single-stranded DNA, which is then amplified, such as Tecott et al., U.S. Patent No. 5,168,038, which is incorporated herein by reference. "Real-time PCR" or "quantitative PCR" means PCR that monitors the amount of the reaction product (i.e., amplicon) as the reaction proceeds. There are many forms of real-time PCR, which differ primarily in the detection chemistry used to monitor the reaction products, such as Gelfand et al., U.S. Pat. No. 5,210,015 ("taqman"); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons); these patents are incorporated herein by reference. Detection chemistry for real-time PCR is reviewed in Mackay et al., Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporated herein by reference.

[0092] "Polymer matrix" generally refers to a phase material (e.g., continuous phase material) comprising at least one polymer. In some embodiments, polymer matrix refers to at least one polymer and interstitial space not occupied by polymer. Polymer matrix can be composed of one or more types of polymers. Polymer matrix can include straight chain, branched and cross-linked polymer units. Polymer matrix can also include non-polymer species inserted into the interstitial space not occupied by polymer chains. The inserted species can be solid, liquid or gas species. For example, the term "polymer matrix" can encompass dry hydrogels, hydrated hydrogels and hydrogels containing glass fibers. Polymer matrix can include polymer precursors, which generally refer to one or more molecules that can trigger or initiate polymerization when activated. Polymer precursors can be activated by electrochemical energy, photochemical energy, photons, magnetic energy or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which are polymerized to produce polymer matrix) and cross-linked compounds, which can include photoinitiators, other compounds necessary or useful for generating polymer matrix (especially the polymer matrix of hydrogel), etc.

[0093] "Polynucleotide" and "oligonucleotide" are used interchangeably, and each means a linear polymer of nucleotide monomers. The monomers that make up polynucleotides and oligonucleotides can specifically bind to natural polynucleotides through regular patterns of monomer-monomer interactions, such as base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, etc. Such monomers and their internucleoside linkages can be naturally occurring or can be analogs thereof, such as naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs can include PNA, phosphorothioate internucleoside linkages, bases containing linking groups that allow attachment of labels (such as fluorophores) or haptens, etc. Whenever the use of oligonucleotides or polynucleotides requires enzymatic treatment, such as extension by polymerase, connection by ligase, etc., it will be understood by ordinary technicians that oligonucleotides or polynucleotides in these cases will not contain certain analogs of internucleoside linkages, sugar moieties, or bases at any or certain positions. The size range of polynucleotides is generally several monomer units, such as 5-40 (when they are generally referred to as "oligonucleotides") to several thousand monomer units. Unless otherwise indicated or apparent from the context, whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (upper or lower case), such as "ATGCCTG", it is understood that the nucleotides are in 5'→3' order from left to right, and "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents thymidine, "I" represents deoxyinosine, and "U" represents uridine. Unless otherwise indicated, the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Typically, polynucleotides contain four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine of DNA or their ribose counterparts of RNA) linked by phosphodiester linkages; however, they may also contain non-natural nucleotide analogs, for example, including modified bases, sugars, or internucleoside linkages. It is clear to those skilled in the art that when the enzyme has specific oligonucleotide or polynucleotide substrate activity requirements, such as single-stranded DNA, RNA / DNA duplexes, etc., then selecting the appropriate composition for the oligonucleotide or polynucleotide substrate is within the knowledge of those skilled in the art, especially with the guidance of papers (such as Sambrook et al., Molecular Cloning, 2nd Edition (Cold Spring Harbor Laboratory, New York, 1989)) and similar references.

[0094] "Primer" means such a natural or synthetic oligonucleotide, which can serve as the starting point of nucleic acid synthesis after forming a duplex with a polynucleotide template and extend from its 3' end along the template to form an extended duplex. The extension of the primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of the nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Usually the primer is extended by a DNA polymerase. The length of the primer is usually in the range of 14 to 40 nucleotides or in the range of 18 to 36 nucleotides. The primer is used for a variety of nucleic acid amplification reactions, for example, a linear amplification reaction using a single primer or a polymerase chain reaction using two or more primers. The guidance of the length and sequence of the primer selected for a specific application is well known to those of ordinary skill in the art, as demonstrated by the following reference, which is incorporated by reference: Dieffenbach, ed., PCR Primer: A Laboratory Manual, 2nd edition (Cold Spring Harbor Press, New York, 2003).

Claims

1. A method for producing a spatially barcoded surface, the method comprising: (a) arranging particles on a surface of a channel, wherein the surface is coated with primers for solid phase amplification and wherein each particle comprises a cleavably attached oligonucleotide comprising a barcode sequence; (b) synthesizing hydrogel chambers on the surface such that each hydrogel chamber encapsulates a particle; as well as (c) cleaving the oligonucleotide from the particle so that the oligonucleotide is released into the chamber, causing at least a portion of the oligonucleotide to be captured by the primer, thereby obtaining a captured oligonucleotide.

2. The method of claim 1, further comprising replicating the captured oligonucleotide by extending the primer using the captured oligonucleotide as a template, thereby obtaining a copy of the captured oligonucleotide.

3. The method of claim 2, further comprising amplifying the copies of the captured oligonucleotide to form clusters.

4. The method of claim 3, further comprising sequencing the barcode sequence of the copies of the captured oligonucleotides of the cluster.

5. The method of claim 4, wherein the copies of the captured oligonucleotide each include a sequencing primer binding site adjacent to the barcode sequence, and wherein the method further comprises determining the barcode sequence by a sequencing-by-synthesis method. The method of claim 1 , wherein the particles are randomly arranged on the surface.

7. The method of claim 1, wherein the particles are arranged on the surface in a regular pattern.

8. The method of claim 7, wherein the particles are arranged on the surface in a linear pattern or a hexagonal pattern.

9. A method of making a spatially barcoded surface, the method comprising: (a) providing a fluidic device comprising: (i) a channel comprising one or more polymer precursors and a surface comprising a capture element and a plurality of particles disposed on the surface, the particles having cleavably attached oligonucleotides, the oligonucleotides comprising barcodes; (ii) a spatial energy modulation element, the spatial energy modulation element being in optical communication with the surface; and (iii) a detector, the detector being in optical communication with the surface and operably associated with the spatial energy modulation element; (b) detecting each of the plurality of particles using the detector and identifying a location of each of the plurality of particles on the surface; (c) synthesizing the one or more chambers in the channel by projecting light into the channel using the spatial energy modulation element such that the projected light causes the one or more polymer precursors to cross-link to form polymer matrix walls of the one or more chambers, each chamber encapsulating one or more of the plurality of particles, wherein the polymer matrix walls are substantially impermeable to the oligonucleotides, and wherein the position of the synthesized chamber is determined by the position of the particle encapsulated by the synthesized chamber as identified by the detector; and (d) cleaving the oligonucleotide from the particle such that the oligonucleotide is released into the one or more chambers and captured by the capture element, thereby obtaining a captured oligonucleotide.

10. The method of claim 9, further comprising replicating the captured oligonucleotide by extending the capture element using the captured oligonucleotide as a template, thereby obtaining copies of the captured oligonucleotide.

11. The method of claim 10, further comprising performing solid phase amplification of the copies of the captured oligonucleotide on the surface.

12. The method of claim 11, further comprising degrading the polymer walls of the one or more chambers.

13. A method of making a spatially barcoded surface, the method comprising one or more cycles of the following operations: (a) disposing one or more particles on a surface of a channel, wherein the surface is coated with primers for solid phase amplification, and wherein each of the one or more particles comprises one or more cleavably attached oligonucleotides, each of the oligonucleotides comprising a barcode sequence; (b) synthesizing hydrogel chambers on the surface such that each hydrogel chamber defines a subregion of the surface and encapsulates a particle of the one or more particles; (c) cleaving the one or more oligonucleotides from the one or more particles so that the one or more oligonucleotides are released into the hydrogel chamber, such that at least a portion of the one or more oligonucleotides are captured by the primer and replicated, thereby obtaining one or more replicated oligonucleotides; as well as (d) degrading the hydrogel chamber, thereby leaving the sub-regions, each of which has a replicated oligonucleotide with a different barcode among the one or more replicated oligonucleotides attached thereto. The method of claim 13 , wherein the sub-regions do not overlap with each other.

15. The method of claim 14, further comprising amplifying the replicated oligonucleotides to obtain amplified oligonucleotides.

16. The method of claim 15, wherein the barcode sequence is determined by sequencing the amplified oligonucleotides.

17. The method of claim 15, wherein the sum of the areas of the sub-regions accounts for at least 70% of the area of ​​the surface.

18. The method of claim 17, wherein the sub-regions form a fractal barcode array. The method of claim 13 , wherein each of the sub-regions overlaps with at least one other sub-region to form an overlapping region.

20. The method of claim 18, wherein the surface in the overlapping region comprises at least one oligonucleotide comprising two or more barcode sequences.

21. The method of claim 19, wherein the relative positions of the sub-regions on the surface are determined by the oligonucleotides comprising two or more barcode sequences in the overlapping region.

22. The method of claim 13, wherein the one or more cycles is a plurality of cycles.

23. A composition comprising a substrate having a planar surface comprising an array of fractal barcodes.

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