Generation of large libraries of barcoded beads using a combinatorial divide-and-pool strategy and chemical ligation

Abstract

The present disclosure generally relates to methods for generating highly diverse barcoded bead pools via a combinatorial split-pool strategy.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 477,747, filed December 29, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Spatially resolved genomics and transcriptomics is an area of ​​great interest, but is limited by the challenge of generating diverse bead arrays containing beads with unique oligonucleotide sequences relative to each other. Summary of the Invention

[0003] While bead arrays can be used in spatial genomics and transcriptomics, one drawback is the insufficient number of unique bead types (i.e., beads containing unique nucleotide sequences). Typically, multiple beads in a bead array contain the same nucleotide sequence, resulting in informatics conflicts. To enable the use of bead arrays as a substrate for capturing spatial transcriptomics information of tissues, each bead on the array must have a unique barcode so that the attached oligonucleotides can be identified as unique sequences in the bead array decoding process.

[0004] To be compatible with existing bead array substrate configurations, a large number of bead types (e.g., 50-500 million) are required. To achieve such a large number of bead types, the present disclosure provides a combinatorial split-pool method in which, during each split round, chemical ligation is performed to attach new oligonucleotide sequences, thereby expanding the barcode library.

[0005] Thus, in some aspects, the present disclosure provides a method of generating a bead array, comprising: (i) attaching a first oligonucleotide to a first bead population, a second oligonucleotide to a second bead population, and a third oligonucleotide to a third bead population, wherein the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide comprise a nucleotide sequence that is unique relative to one another; (ii) combining the first bead population, the second bead population, and the third bead population to create a first pool; (iii) dividing the first pool into a first plurality of aliquots; and (iv) chemically ligating, in a first aliquot of the first plurality of aliquots, a fourth oligonucleotide to the first oligonucleotide in the presence of a first splint oligonucleotide, the first splint oligonucleotide hybridizing to both the fourth oligonucleotide and the first oligonucleotide, and a second splint oligonucleotide, the second splint oligonucleotide hybridizing to the fourth oligonucleotide. a second splint oligonucleotide that hybridizes to both the first splint oligonucleotide and the second oligonucleotide, and a third splint oligonucleotide that hybridizes to both the fourth oligonucleotide and the third oligonucleotide, wherein the first splint oligonucleotide, the second splint oligonucleotide, and the third splint oligonucleotide comprise unique nucleotide sequences relative to one another; and in a second aliquot of the first plurality of aliquots, a fifth oligonucleotide that hybridizes to both the first oligonucleotide and the first oligonucleotide, and a fourth splint oligonucleotide that hybridizes to both the fifth oligonucleotide and the second ... fifth oligonucleotide is chemically ligated to the first oligonucleotide in the presence of a fourth splint oligonucleotide that hybridizes to both the fifth oligonucleotide and the first oligonucleotide, and a fifth splint oligonucleotide that hybridizes to both the fifth oligonucleotide and the second oligonucleotide.a fourth splint oligonucleotide, a fifth splint oligonucleotide, and a sixth splint oligonucleotide that hybridizes to both the fifth and third oligonucleotides; wherein the fourth splint oligonucleotide, the fifth splint oligonucleotide, and the sixth splint oligonucleotide comprise unique nucleotide sequences relative to one another; and in a third aliquot of the first plurality of aliquots, the sixth oligonucleotide is chemically ligated to the first oligonucleotide in the presence of a seventh splint oligonucleotide that hybridizes to both the sixth and first oligonucleotides; and a eighth splint oligonucleotide that hybridizes to both the sixth and second oligonucleotides. (v) combining a first aliquot of the first plurality of aliquots, a second aliquot of the first plurality of aliquots, and a third aliquot of the first plurality of aliquots to create a second pool; (vi) dividing the second pool into a second plurality of aliquots; (vii) chemically ligating, in the first aliquot of the second plurality of aliquots, the seventh oligonucleotide to the third oligonucleotide in the presence of a tenth splint oligonucleotide, wherein the tenth splint oligonucleotide hybridizes to both the seventh oligonucleotide and the fourth oligonucleotide;and in a second aliquot of the second plurality of aliquots, chemically ligating the ninth oligonucleotide to the sixth oligonucleotide in the presence of a twelfth splint oligonucleotide that hybridizes to both the ninth oligonucleotide and the sixth oligonucleotide, wherein the tenth splint oligonucleotide, the eleventh splint oligonucleotide, and the twelfth splint oligonucleotide comprise unique nucleotide sequences relative to one another. In some embodiments, the method includes (viii) combining a first aliquot of the second plurality of aliquots, a second aliquot of the second plurality of aliquots, and a third aliquot of the second plurality of aliquots to create a third pool; (ix) dividing the third pool into a third plurality of aliquots; and (x) chemically ligating, in the first aliquot of the third plurality of aliquots, the tenth oligonucleotide to a thirteenth splint oligonucleotide, wherein the tenth oligonucleotide is hybridized to both the tenth oligonucleotide and the seventh oligonucleotide. in a second aliquot of the third plurality of aliquots, the eleventh oligonucleotide is chemically ligated to the eighth oligonucleotide in the presence of a fourteenth splint oligonucleotide, wherein the eighth oligonucleotide hybridizes to both the eleventh oligonucleotide and the eighth oligonucleotide; and in a third aliquot of the third plurality of aliquots, the twelfth oligonucleotide is chemically ligated to a fifteenth splint oligonucleotide, wherein the fifteenth splint oligonucleotide hybridizes to both the eleventh oligonucleotide and the eighth oligonucleotide.The method further includes chemically ligating the ninth oligonucleotide in the presence of a fifteenth splint oligonucleotide that hybridizes to both the twelfth oligonucleotide and the ninth oligonucleotide, wherein the thirteenth splint oligonucleotide, the fourteenth splint oligonucleotide, and the fifteenth splint oligonucleotide comprise unique nucleotide sequences relative to each other. In a further aspect, the disclosure provides a method of generating a bead array, comprising: (i) attaching a first oligonucleotide to one or more beads in a first bead population, a second oligonucleotide to one or more beads in a second bead population, and a third oligonucleotide to one or more beads in a third bead population, wherein the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide comprise unique nucleotide sequences relative to one another; (ii) combining the first bead population, the second bead population, and the third bead population to create a first pool; (iii) dividing the first pool into a first plurality of aliquots; and (iv) chemically ligating a fourth oligonucleotide to the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide in a first aliquot of the first plurality of aliquots. (v) chemically ligating a first aliquot of the first plurality of aliquots, a second aliquot of the first plurality of aliquots, and a third aliquot of the first plurality of aliquots to create a second pool; (vi) dividing the second pool into a second plurality of aliquots; (vii) chemically ligating a second aliquot of the first plurality of aliquots to a third aliquot of the first plurality of aliquots; (viii) chemically ligating a third aliquot of the first plurality of aliquots to a fourth aliquot of the first plurality of aliquots; (viii) chemically ligating a fifth aliquot of the first plurality of aliquots to a fifth aliquot of the first plurality of aliquots; and (viii) chemically ligating a sixth aliquot of the first plurality of aliquots to a third aliquot of the first plurality of aliquots;In a first aliquot of the second plurality of aliquots, a seventh oligonucleotide is chemically ligated to the fourth oligonucleotide, in a second aliquot of the second plurality of aliquots, an eighth oligonucleotide is chemically ligated to the fifth oligonucleotide, and in a third aliquot of the second plurality of aliquots, a ninth oligonucleotide is chemically ligated to the sixth oligonucleotide. In some embodiments, the method further includes (viii) combining a first aliquot of the second plurality of aliquots, a second aliquot of the second plurality of aliquots, and a third aliquot of the second plurality of aliquots to create a third pool, (ix) dividing the third pool into a third plurality of aliquots, and (x) chemically ligating, wherein in a first aliquot of the third plurality of aliquots, the tenth oligonucleotide is chemically ligated to the seventh oligonucleotide, in a second aliquot of the second plurality of aliquots, the eleventh oligonucleotide is chemically ligated to the eighth oligonucleotide, and in a third aliquot of the second plurality of aliquots, the twelfth oligonucleotide is chemically ligated to the ninth oligonucleotide. In some embodiments, the method further includes chemically ligating at least about 1 x 10 attached to a bead array. 6 In a further embodiment, the method results in a bead array comprising at least about 1 x 10 unique oligonucleotide sequences attached to the bead array. 7 In yet a further embodiment, the method results in a bead array comprising at least about 1 x 10 unique oligonucleotide sequences attached to the bead array. 8In some embodiments, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, and twelfth splint oligonucleotides have the same nucleotide sequence. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and fifteenth splint oligonucleotides have the same nucleotide sequence. In various embodiments, the oligonucleotides are selected from the group consisting of (i) a poly(T) nucleotide sequence or a poly(A) nucleotide sequence, (ii) one or more gene-specific capture sequences, (iii) one or more universal capture sequences (e.g., random or semi-random nucleotide sequences), or a combination thereof. and is chemically ligated to the end of each oligonucleotide on each bead in the bead array. In some embodiments, (i) comprises about 100 bead populations, each bead population comprising an oligonucleotide attached thereto that is unique relative to all other bead populations. In further embodiments, (i) comprises about 13,600 bead populations, each bead population comprising an oligonucleotide attached thereto that is unique relative to all other bead populations. In some embodiments, the first oligonucleotide, the second oligonucleotide, and / or the third oligonucleotide are attached to one or more beads via a cleavable linker. In various embodiments, a 3'-modified nucleotide is incorporated using terminal deoxynucleotidyl transferase (TdT) enzyme prior to chemical ligation. In some embodiments, one or more of the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, the seventh oligonucleotide, the eighth oligonucleotide, or the ninth oligonucleotide is a barcode. In further embodiments, each of the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, the seventh oligonucleotide, the eighth oligonucleotide, and the ninth oligonucleotide is a barcode. In some embodiments, one or more of the tenth oligonucleotide, the eleventh oligonucleotide, or the twelfth oligonucleotide is a barcode. In further embodiments, each of the tenth oligonucleotide, the eleventh oligonucleotide, and the twelfth oligonucleotide is a barcode. In further embodiments, the barcode is a spatial barcode. [Brief explanation of the drawings]

[0006] [Figure 1]1 shows a general schematic of an exemplary combinatorial split-and-pool method of the present disclosure for generating bead pools. At each split-pool step, oligonucleotides are ligated (e.g., chemically ligated) to the ends of growing barcode strands. [Figure 2] Various non-limiting examples of unnatural backbone linkages that can be used in library synthesis are shown (1-7: triazole, 8: squaramide, 9: urea). [Figure 3] Non-limiting examples of a) phosphate or phosphate derivative linkages for chemical ligation, b) types of chemistries that can be used to generate phosphate-derived backbones in chemical ligation are shown. [Figure 4] The structures of various 3' and 5' modifications that can be used on the non-natural but biocompatible scaffold shown in Figure 2 are shown. [Figure 5] 1 shows an exemplary workflow for barcode generation using TdT enzyme to incorporate modified nucleotides that allow for chemical ligation. SM = surface modification. CL = cleavable linker. The "N" in the phrase "N no. of sequences for a unique barcode on bead" refers to the number of times chemical ligation is performed. [Figure 6] 1 shows an exemplary workflow for barcode generation using only chemical ligation without TdT. SM = surface modification. CL = cleavable linker. The "N" in the phrase "Nth sequence for a unique barcode on a bead" refers to the number of times chemical ligation is performed. [Figure 7] An exemplary workflow involving protection of one reactive group (two protected chemical reaction handles) is shown. [Figure 8] An exemplary workflow involving two pairs of orthogonal reactive groups (four chemical reaction handles) is shown. [Figure 9]Schematic diagram of ligation with splint oligonucleotides. R1 and R2 are functional groups for chemical ligation. [Figure 10] 1 shows non-limiting examples of chemically cleavable attachments that can be used for barcode attachment to beads and optional subsequent cleavage from the beads for sequencing. [Figure 11] The following shows the types of splint oligonucleotides contemplated in this disclosure: a) Universal Bridge Sequence. Nucleotide sequences A' and B' are universal sequences that hybridize with nucleotide sequences A and B of the universal bridge sequence. Nucleotide sequence C is a unique oligo sequence 1 sequence. Nucleotide sequence D is a unique oligo sequence 2 sequence. Nucleotide sequence E is a unique oligo sequence 3 sequence. b) Unique Bridge Sequence. Nucleotide sequences A' and B' are unique sequences that hybridize with nucleotide sequences A and B of a first unique bridge sequence. Similarly, nucleotide sequences G' and H' are unique nucleotide sequences that hybridize with nucleotide sequences G and H of a second unique bridge sequence. Nucleotide sequence A', nucleotide sequence B', nucleotide sequence G', nucleotide sequence H', nucleotide sequence F', and nucleotide sequence I' are each unique nucleotide sequences relative to each other. Nucleotide sequence C is a unique oligo sequence 1 sequence. Nucleotide sequence D is a unique oligo sequence 2 sequence. Nucleotide sequence E is a unique oligo sequence 3 sequence. R1 and R2 are functional groups for chemical ligation. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present disclosure generally relates to a method for generating a bead pool via a combinatorial split-pool strategy. Oligonucleotides on a bead array are typically fully synthesized before attachment to the beads. However, generating a large pool of unique oligonucleotide barcoded beads may require synthesizing a wide variety of longer oligonucleotide sequences. The synthesis of large oligonucleotides poses significant challenges and often results in reduced yields. Therefore, the present disclosure provides a method for generating a large pool of unique oligonucleotide sequences using combinatorial split-pool and ligation (e.g., chemical ligation). A general, non-limiting schematic diagram of the disclosed method is shown in Figure 1.

[0008] term As used in this specification and the recited paragraphs herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0009] "About" and "approximately" generally refer to an acceptable degree of error for the quantity measured, given the nature or precision of the measurement. Exemplary degrees of error are within 20-25 percent (%), e.g., within 20 percent, 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range of values.

[0010] The term "bead" refers to a small object made of a rigid or semi-rigid material. The object can have a shape characterized as, for example, a sphere, an ellipsoid, a microsphere, or other recognized particle shape, whether with regular or irregular dimensions. Examples of materials useful for beads include glass; plastics, such as acrylic, polystyrene, or copolymers of styrene with another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (TEFLON® from Chemours); polysaccharides or cross-linked polysaccharides, such as agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metals; inorganic glass; fiber optic bundles; or various other polymers. Examples of beads include, but are not limited to, controlled pore glass beads, paramagnetic beads, triazoles, Sepharose beads, nanocrystals, and others known in the art, such as those described in the Microsphere Detection Guide from Bangs Laboratories, Fishers Ind. The beads can also be coated with a polymer that has functional groups to which oligonucleotides can attach.

[0011] As used herein, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquids. The substrate can be non-porous or porous. The substrate can optionally incorporate liquid (e.g., through porosity), but will typically be sufficiently rigid so that it does not significantly swell when incorporating liquid and does not significantly shrink when the liquid is removed by drying. Non-porous solid supports are generally impermeable to liquids or gases. Exemplary solid support materials include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials, including silicon and modified silicon, carbon, metals, inorganic glasses, fiber optic bundles, and polymers. A particularly useful material is glass. Other suitable substrate materials can include polymeric materials, plastics, silicon, quartz (fused silica), borofloat glass, silica, silica-based materials, carbon, metals including gold, optical fibers or fiber optic bundles, sapphire, or plastic materials such as COC and epoxy. A particular material can be selected based on properties desired for a particular use. For example, a material that is transparent to radiation of a desired wavelength is useful for analytical techniques that will utilize radiation of a desired wavelength, such as one or more of the techniques described herein. Conversely, it may be desirable to select a material that does not transmit radiation of a certain wavelength (e.g., opaque, absorbing, or reflective). This can be useful for forming masks used during the fabrication of structured substrates or for chemical reactions or analytical detection performed using the structured substrates. Other properties of materials that can be utilized include inertness or reactivity to certain reagents used in downstream processes, or ease or low cost of manipulation during manufacturing processes. Further examples of materials that may be used in the structured substrates or methods of the present disclosure are described in U.S. Patent Application Publication Nos. 2012 / 0316086(A1) and 2013 / 0116153, the entire contents of each of which are incorporated herein by reference.In some embodiments, the solid support is a flow cell, as described herein below.

[0012] In any of the aspects or embodiments of the present disclosure, the solid support can comprise a collection of beads or other particles. The particles can be suspended in a solution or disposed on the surface of a substrate. Examples of arrays with beads disposed on the surface include those in which beads are disposed in wells, such as the BeadChip array (Illumina Inc., San Diego, Calif.), the substrate used in the sequencing platform from 454 LifeSciences (a subsidiary of Roche, Basel, Switzerland), or the substrate used in the sequencing platform from Ion Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.). Other solid supports having beads disposed on their surfaces are described in U.S. Patent Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570, 6,210,891, 6,258,568, or 6,274,320, U.S. Patent Application Publication Nos. 2009 / 0026082A1, 2009 / 0127589A1, 2010 / 0137143A1, or 2010 / 0282617A1, or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Some of the above references describe methods for attaching nucleic acids to beads prior to loading the beads into or onto the solid support. However, it will be understood that ligated oligonucleotides can be first generated, then attached to beads, which can then be loaded onto an array and used in the methods described herein. In some embodiments, the ligated oligonucleotides are released from the beads and attached to a solid support (for example, but not limited to, a flow cell). Thus, the present disclosure also contemplates the use of a flow cell. As used herein, the term "flow cell" is intended to mean a container having a chamber in which a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some embodiments, the chamber is configured to detect a reaction occurring within the chamber.For example, the chamber may include one or more transparent surfaces to allow optical detection of biological specimens, optically labeled molecules, etc. within the chamber. Exemplary flow cells include, but are not limited to, those used in nucleic acid sequencing devices, such as flow cells for the Genome Analyzer®, MiSeq®, NextSeq®, or HiSeq® platforms marketed by Illumina, Inc. (San Diego, CA), or flow cells for the SOLiD™ or Ion Torrent™ sequencing platforms marketed by Life Technologies (Carlsbad, CA). Exemplary flow cells and methods for their manufacture and use are also described, for example, in International Publication No. WO 2014 / 142841 A1, U.S. Patent Application Publication No. 2010 / 0111768 A1, and U.S. Patent No. 8,951,781, each of which is incorporated herein by reference.

[0013] In some embodiments, the solid support typically used in bead arrays is used without beads. For example, nucleic acids, such as the ligated oligonucleotides described herein, can be directly attached to the well or the gel material in the well. Thus, the above references are examples of materials, compositions, or devices that can be modified for use in the methods and compositions described herein.

[0014] The solid support used in the methods described herein can include an array of beads, with different ligated oligonucleotides attached to different beads in the array. In various embodiments, each bead can be attached with a different ligated oligonucleotide, and the beads can be randomly distributed on the solid support to effectively attach different nucleic acid probes to the solid support.

[0015] Optionally, the solid support may include wells sized to accommodate one or fewer beads. In such a configuration, the beads may be attached to the wells due to forces resulting from the fit of the beads in the wells. It is also possible to use adhesive chemicals or glues to hold the beads in the wells.

[0016] As described herein, the ligated oligonucleotides attached to the beads can include or consist of barcode sequences. According to the methods provided herein, a population of beads can be configured such that each bead is attached with only one type of ligated oligonucleotide, including multiple barcodes, and there are many different beads (each with a different ligated oligonucleotide) in the population.

[0017] As used herein, the term "different" when used in reference to nucleic acids means that the nucleic acids have nucleotide sequences that are not the same as each other.Two or more nucleic acids can have different nucleotide sequences along their entire length.Alternatively, two or more nucleic acids can have different nucleotide sequences along a substantial portion of their length.For example, two or more nucleic acids can have different target nucleotide sequence portions for two or more molecules, but also have universal sequence portions that are the same for two or more molecules.This term can also be applied to proteins that can be distinguished as different from each other based on differences in amino acid sequence.

[0018] "Complementary" means that the oligonucleotide contains a sequence of nucleotides that can form a double-stranded structure by base pairing with another oligonucleotide or portion thereof. "Substantially complementary" means that the oligonucleotide has at least 85%, 90%, 95%, 98%, 99%, or 100% overall sequence identity with the complementary sequence.

[0019] Bead arrays containing oligonucleotide barcodes can also be used in sequencing procedures, such as sequencing-by-synthesis (SBS) techniques. Briefly, SBS can be initiated by contacting the barcode with one or more labeled nucleotides, DNA polymerase, or the like. These features, where a primer is extended using the barcode-containing sequence as a template, can incorporate a detectable labeled nucleotide. Optionally, the labeled nucleotide can further include a reversible termination feature that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the primer so that further extension does not occur until a deblocking agent is delivered to remove the moiety. Thus, in embodiments using reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between various delivery steps. The cycle is then repeated n times to extend the primer with n nucleotides, thereby allowing a sequence of length n to be detected. Exemplary SBS procedures, fluidic systems, and detection platforms that can be readily adapted for use with arrays generated by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, WO 91 / 06678, WO 07 / 123744, U.S. Patent Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, or 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082A1, each of which is incorporated herein by reference.

[0020] As used herein, a "primer" is a nucleic acid molecule capable of hybridizing to a target sequence, such as an adapter attached to a library fragment. As an example, an amplification primer can serve as a starting point for template amplification and cluster generation. In another example, a synthesized nucleic acid (template) strand can contain a site to which a primer (e.g., a sequencing primer) can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid strand. Any primer can contain any combination of nucleotides or their analogs. In some examples, a primer is a single-stranded oligonucleotide or polynucleotide. The primer can be any number of bases long and can contain a variety of non-naturally occurring nucleotides. In various embodiments, sequencing primers are short strands ranging from 5-60 bases, 10-60 bases, 10-20 bases, 10-30 bases, 10-40 bases, 10-50 bases, or 20-40 bases.

[0021] As used herein, the term "unique molecular identifier" or "UMI" refers to a molecular tag that can be attached to a nucleic acid, either randomly, non-randomly, or semi-randomly. When incorporated into a nucleic acid, the unique molecular identifier (UMI) can be used to correct for subsequent amplification bias by directly counting the UMI after amplification and sequencing. The UMI can be attached to a similar nucleic acid, such as an adapter, making each nucleic acid unique.

[0022] As used herein, the term "adapter" generally refers to any linear nucleic acid molecule that can be ligated to the oligonucleotides of the present disclosure. In some embodiments, the adapter comprises two reverse-complementary oligonucleotides that form a double-stranded structure. In some embodiments, the adapter comprises two oligonucleotides that are complementary in one portion and mismatched in another portion, forming a Y-shaped or forked adapter that is double-stranded in the complementary portion and has two floppy overhangs in the mismatched portion.

[0023] As used herein, the term "barcode" is intended to refer to a sequence of nucleotides in an oligonucleotide that can be used to identify an oligonucleotide, its spatial address on a surface, a feature of the oligonucleotide, or an operation performed on the oligonucleotide. A barcode can be a naturally occurring nucleotide sequence or a nucleotide sequence that does not naturally occur in the organism from which the barcoded nucleic acid was obtained. A barcode sequence can be unique to a single nucleic acid species in a population, or the barcode sequence can be shared by multiple different nucleic acid species in a population. For example, each nucleic acid capture probe in a population on a substrate for spatial capture of nucleic acids in a biological sample, such as a permeabilized tissue sample or cell suspension, can contain a barcode sequence that is different from all other nucleic acid capture probes in the population. Alternatively, each nucleic acid probe in the population can contain a barcode sequence that is different from some or most other nucleic acid capture probes in the population. For example, each capture probe in a population can have a barcode that is present for multiple different capture probes in the population, even if capture probes with a common barcode differ from each other in other sequence regions along their length. In various embodiments, one or more barcode sequences used in biological tissue analysis are not present in the genome, transcriptome, or other nucleic acids of the biological sample. For example, the barcode sequence can have less than 80%, 70%, 60%, 50%, or 40% sequence identity with a nucleic acid sequence in a particular biological tissue.

[0024] As used herein, the term "array" refers to a collection of sites that can be distinguished from one another according to their relative positions. Different molecules at different sites of an array can be distinguished from one another according to the site's position within the array. Each site of an array can contain one or more molecules of a particular type. For example, a site can contain a single nucleic acid molecule having a particular sequence, or a site can contain several nucleic acid molecules having the same sequence (and / or its complementary sequence). The sites of an array can be different features located on the same substrate. Exemplary features include, but are not limited to, wells in a substrate, beads (or other particles) in or on a substrate, droplets, wells in a substrate, protrusions from a substrate, ridges on a substrate, or channels in a substrate. The sites of an array can be separate substrates, each with a different molecule. The different molecules attached to the separate substrates can be identified according to the position of the substrate on a surface to which the substrates are associated, or according to the position of the substrate within a liquid or gel. An exemplary array in which separate substrates are located on a surface includes, but is not limited to, beads in wells.

[0025] As used herein, the term "tissue" is intended to mean a collection of cells and, optionally, intercellular material. Typically, the cells in a tissue are not free-floating in solution but are attached to each other to form a multicellular structure. Exemplary tissue types include muscle, nerve, epidermis, and connective tissue.

[0026] The terms "P5" and "P7" may be used when referring to example adapters. The terms "P5'" (P5 prime) and "P7'" (P7 prime) refer to the complements of P5 and P7, respectively. It will be understood that any suitable adapter can be used in the methods presented herein, and the use of P5 and P7 is only an exemplary embodiment. The use of adapters such as P5 and P7 or their complements on flow cells is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, each of which is incorporated herein by reference. For example, any suitable forward amplification primer, whether immobilized or in solution, can be useful in the methods provided herein for amplifying complementary sequences and sequences. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be useful in the methods provided herein for amplifying complementary sequences and sequences. Those skilled in the art will understand how to design and use suitable primer sequences for capturing and / or amplifying nucleic acids as provided herein.

[0027] As used herein, the terms "ligate," "ligation," and derivatives thereof generally refer to the process of covalently linking two or more molecules to one another, e.g., covalently linking two or more nucleic acid molecules to one another. In some embodiments, ligation involves joining nicks between adjacent nucleotides of a nucleic acid. In some embodiments, ligation involves forming a covalent bond between the end of a first oligonucleotide and the end of a second oligonucleotide. In some embodiments, ligation can involve forming a covalent bond between the 5' phosphate group of one oligonucleotide and the 3' hydroxyl group of a second oligonucleotide, thereby forming a ligated nucleic acid molecule. As used herein, a "ligated oligonucleotide" refers to an oligonucleotide produced after two or more rounds of the combinatorial split-pool method of the present disclosure. Chemical ligation of oligonucleotides is further described herein.

[0028] As used herein, "hybridize" refers to the non-covalent binding of a first oligonucleotide to a second oligonucleotide along the length of the polymer to form a double-stranded "duplex." For example, two DNA oligonucleotide strands can associate through complementary base pairing. The strength of association between a first and a second oligonucleotide increases with the complementarity between the nucleotide sequences within the oligonucleotides. The strength of hybridization between oligonucleotides can be characterized by the melting temperature (Tm) at which 50% of the duplex has oligonucleotide strands dissociated from each other. Oligonucleotides that are "partially" hybridized to each other mean that they have complementary sequences, but such sequences hybridize to each other along only a portion of their length to form a partial duplex. Oligonucleotides that are "incapable" of hybridizing include those that are physically separated from each other with insufficient bases that can contact each other in a manner that allows them to hybridize.

[0029] As used herein, the term "plurality" is intended to mean a population of two or more members, which may all be the same or in which two or more members are different. Pluralities can range in size from small, medium, large, to very large. A small-sized plurality can range, for example, from a few members to tens of members. A medium-sized plurality can range, for example, from tens of members to about 100 or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1,000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, pluralities can range in size from 2 to well over 100 million members, as well as all sizes measured by number of members and ranges larger than the exemplary ranges listed above. Therefore, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the plurality value can be set, for example, by the theoretical diversity of bead types in an array.

[0030] As used herein, the term "attached" refers to the state in which two things are joined, fastened, adhered, connected, or bonded to one another. For example, an oligonucleotide may be attached to a material such as a bead by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a chemical bond that does not involve the sharing of electron pairs, and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, and hydrophobic interactions.

[0031] As used herein, a "semi-random" nucleotide sequence comprises or consists of a partially predetermined nucleotide sequence combined with random nucleotide sequences.

[0032] Oligonucleotides Oligonucleotide is a polymer composed of nucleotides.The oligonucleotide of the present disclosure can be of any length, and in various embodiments, comprises DNA oligonucleotide, RNA oligonucleotide, their analog, or a combination thereof.In any aspect or embodiment described herein, the oligonucleotide is single-stranded, double-stranded, or partially double-stranded.

[0033] Nucleotides can include naturally occurring nucleotides and their functional analogs. Examples of functional analogs are those that can hybridize to nucleic acids in a sequence-specific manner or can be used as templates for replicating a specific nucleotide sequence. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone linkages, including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). Analog structures can have alternative sugar moieties, including any of a variety known in the art. Nucleotides can include natural or unnatural bases. Natural DNA can include one or more of adenine, thymine, cytosine, and / or guanine, while natural RNA can include one or more of adenine, uracil, cytosine, and / or guanine. Any unnatural base can be used, such as locked nucleic acids (LNA) and bridged nucleic acids (BNA). Exemplary modified nucleotides include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azo- ... Examples of amino acids that can be used include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.As is known in the art, certain nucleotide analogs cannot become incorporated into polynucleotides, e.g., nucleotide analogs such as adenosine 5'-phosphosulfate. The nucleotide can contain any suitable number of phosphates, e.g., 3, 4, 5, 6, or more than 6 phosphates.

[0034] Oligonucleotides contemplated by the present disclosure also include those having at least one modified internucleoside linkage. In some embodiments, the oligonucleotide is entirely or partially a peptide nucleic acid. Other modified internucleoside linkages include at least one phosphorothioate linkage. Still other modified oligonucleotides include those containing one or more universal bases. A "universal base" refers to a molecule that can bind to and replace any one of A, C, G, T, and U in a nucleic acid by forming a hydrogen bond without significantly destabilizing the structure. Examples of universal bases include, but are not limited to, 5'-nitroindole-2'-deoxyriboside, 3-nitropyrrole, inosine, and hypoxanthine.

[0035] method The present disclosure generally relates to a method for generating a large library of barcoded beads using a combinatorial split-pool strategy and ligation (e.g., chemical ligation). Generally, the method involves successive rounds of adding oligonucleotides (e.g., barcodes) to a growing oligonucleotide chain. The successive rounds of adding oligonucleotides to a growing oligonucleotide chain can be performed in solution, or the successive rounds of adding oligonucleotides to a growing oligonucleotide chain can be performed, for example, directly on beads. Thus, during each "split" round, ligation (e.g., chemical ligation) is performed to add new oligonucleotide sequences (e.g., barcode sequences), thus expanding the library (e.g., barcode library). Thus, the disclosed method includes at least one "split and pool" step that collects pooled oligonucleotides (e.g., barcodes), distributes them into aliquots, and adds additional oligonucleotides (e.g., barcodes), with the number of "split and pool" steps determining the number of different oligonucleotides (e.g., barcodes) added to the growing oligonucleotides. This process generates unique oligonucleotide (e.g., barcode) combinations. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more rounds of splitting and pooling are performed. In various embodiments, the methods of the present disclosure generate approximately 1×10 2 , about 1×10 3 , about 1×10 4 , about 1×10 5 , about 1×10 6 , about 1×10 7 , about 1×10 8 The method provides a bead array comprising, or at least about, or less than about, or more than about, those numbers of unique oligonucleotide sequences attached to the bead array.

[0036] Thus, in some aspects, the present disclosure provides a method of generating a bead array, comprising: (i) attaching a first oligonucleotide to a first bead population, a second oligonucleotide to a second bead population, and a third oligonucleotide to a third bead population, wherein the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide comprise a nucleotide sequence that is unique relative to one another; (ii) combining the first bead population, the second bead population, and the third bead population to create a first pool; (iii) dividing the first pool into a first plurality of aliquots; and (iv) chemically ligating, in a first aliquot of the first plurality of aliquots, a fourth oligonucleotide to the first oligonucleotide in the presence of a first splint oligonucleotide, the first splint oligonucleotide hybridizing to both the fourth oligonucleotide and the first oligonucleotide, and a second splint oligonucleotide, the second splint oligonucleotide hybridizing to the fourth oligonucleotide. a second splint oligonucleotide that hybridizes to both the first splint oligonucleotide and the second oligonucleotide, and a third splint oligonucleotide that hybridizes to both the fourth oligonucleotide and the third oligonucleotide, wherein the first splint oligonucleotide, the second splint oligonucleotide, and the third splint oligonucleotide comprise unique nucleotide sequences relative to one another; and in a second aliquot of the first plurality of aliquots, a fifth oligonucleotide that hybridizes to both the first oligonucleotide and the first oligonucleotide, and a fourth splint oligonucleotide that hybridizes to both the fifth oligonucleotide and the second ... fifth oligonucleotide is chemically ligated to the first oligonucleotide in the presence of a fourth splint oligonucleotide that hybridizes to both the fifth oligonucleotide and the first oligonucleotide, and a fifth splint oligonucleotide that hybridizes to both the fifth oligonucleotide and the second oligonucleotide.a fourth splint oligonucleotide, a fifth splint oligonucleotide, and a sixth splint oligonucleotide that hybridizes to both the fifth and third oligonucleotides; wherein the fourth splint oligonucleotide, the fifth splint oligonucleotide, and the sixth splint oligonucleotide comprise unique nucleotide sequences relative to one another; and in a third aliquot of the first plurality of aliquots, the sixth oligonucleotide is chemically ligated to the first oligonucleotide in the presence of a seventh splint oligonucleotide that hybridizes to both the sixth and first oligonucleotides; and a eighth splint oligonucleotide that hybridizes to both the sixth and second oligonucleotides. (v) combining a first aliquot of the first plurality of aliquots, a second aliquot of the first plurality of aliquots, and a third aliquot of the first plurality of aliquots to create a second pool; (vi) dividing the second pool into a second plurality of aliquots; (vii) chemically ligating, in the first aliquot of the second plurality of aliquots, the seventh oligonucleotide to the third oligonucleotide in the presence of a tenth splint oligonucleotide, wherein the tenth splint oligonucleotide hybridizes to both the seventh oligonucleotide and the fourth oligonucleotide;and in a second aliquot of the second plurality of aliquots, chemically ligating the eighth oligonucleotide to the fifth oligonucleotide in the presence of an eleventh splint oligonucleotide, wherein the eleventh splint oligonucleotide hybridizes to both the eighth oligonucleotide and the fifth oligonucleotide; and in a third aliquot of the second plurality of aliquots, chemically ligating the ninth oligonucleotide to the sixth oligonucleotide in the presence of a twelfth splint oligonucleotide, wherein the twelfth splint oligonucleotide hybridizes to both the ninth oligonucleotide and the sixth oligonucleotide. In various embodiments, step (i) is carried out at a concentration of about 10, about 20, about 50, about 100, about 200, about 500, about 1,000, about 2,000, about 5,000, about 10,000, about 13,600, about 15,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, about 700,000, about 1 x 10, 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , or at least about those numbers, or less than about those numbers, or more bead populations, each bead population having attached thereto an oligonucleotide that is unique relative to all other bead populations.

[0037] In a further aspect, the disclosure provides a method of generating a bead array, comprising: (i) attaching a first oligonucleotide to one or more beads in a first bead population, a second oligonucleotide to one or more beads in a second bead population, and a third oligonucleotide to one or more beads in a third bead population, wherein the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide comprise a unique nucleotide sequence relative to one another; (ii) combining the first bead population, the second bead population, and the third bead population to create a first pool; (iii) dividing the first pool into a first plurality of aliquots; and (iv) chemically ligating a fourth oligonucleotide to the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide in a first aliquot of the first plurality of aliquots; (v) combining the first aliquot of the first plurality of aliquots, the second aliquot of the first plurality of aliquots, and the third aliquot of the first plurality of aliquots to create a second pool; (vi) dividing the second pool into a second plurality of aliquots; (vii) chemically ligating the seventh oligonucleotide to the fourth oligonucleotide in the first aliquot of the second plurality of aliquots, the eighth oligonucleotide to the fifth oligonucleotide in the second aliquot of the second plurality of aliquots, and the ninth oligonucleotide to the sixth oligonucleotide in the third aliquot of the second plurality of aliquots.In various embodiments, step (i) comprises chemically ligating about 10, about 20, about 50, about 100, about 200, about 500, about 1,000, about 2,000, about 5,000, about 10,000, about 13,600, about 15,000, about 20,000, about 50,000, about 100,000, about 200,000, about 500,000, about 700,000, about 1 x 10, 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×10 6 , about 8×10 6 , or at least about those numbers, or less than about those numbers, or more bead populations, each bead population having attached thereto an oligonucleotide that is unique relative to all other bead populations.

[0038] In any of the aspects or embodiments of the present disclosure, the final oligonucleotide sequence (e.g., barcode sequence) added to the growing oligonucleotide chain comprises a unique molecular identifier (UMI). In any of the aspects or embodiments of the present disclosure, the final oligonucleotide sequence (e.g., barcode sequence) added to the growing oligonucleotide chain comprises a poly(T) or poly(A) nucleotide sequence. In some embodiments, the final oligonucleotide sequence (e.g., barcode sequence) added to the growing oligonucleotide chain (e.g., a reverse transcription primer) is cleaved from the bead surface for sequencing. Thus, in some embodiments, the present disclosure contemplates the use of a cleavable linkage between the bead and the barcoded primer. Cleavable linkers for use in such methods include, but are not limited to, the cleavable linkers described in Bioorg. Med. Chem. 2012, 20, 571-582 and the cleavable linker shown in FIG. 10.

[0039] Oligonucleotides for use in the methods described herein generally range in length from about 5 to about 100 nucleotides. In various embodiments, oligonucleotides of the disclosure range in length from about 5 to about 100 nucleotides, about 5 to about 90 nucleotides, about 5 to about 80 nucleotides, about 5 to about 70 nucleotides, about 5 to about 60 nucleotides, about 5 to about 50 nucleotides, about 5 to about 40 nucleotides, about 5 to about 30 nucleotides, about 5 to about 20 nucleotides, about 5 to about 10 nucleotides, about 10 to about 100 nucleotides, about 10 to about 90 nucleotides, about 10 to about 80 nucleotides, about 10 to about 70 nucleotides, about 10 to about 60 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 20 nucleotides. In further embodiments, oligonucleotides of the disclosure are, about, at least about, or less than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.

[0040] Ligation Any method for ligating oligonucleotides can be used in the methods of the present disclosure. In any of the aspects or embodiments of the present disclosure, the oligonucleotides are ligated using chemical ligation. In some embodiments, the oligonucleotides are ligated using enzymatic ligation. In any of the aspects or embodiments of the present disclosure, a splint oligonucleotide is used during ligation of the oligonucleotides. A "splint oligonucleotide" is an oligonucleotide that improves the efficiency of ligation of oligonucleotides. For example, when ligating two oligonucleotides together, a splint oligonucleotide hybridizes to the end of each oligonucleotide, and the ends are then ligated. See, e.g., Figures 3a and 3b. In any of the aspects or embodiments of the present disclosure, a splint oligonucleotide is used for each ligation in the method. In some embodiments, each splint oligonucleotide contains a unique nucleotide sequence relative to all other splint oligonucleotides. See, e.g., Figure 11b. In some embodiments, each splint oligonucleotide contains a semi-random nucleotide sequence. In some embodiments, each oligonucleotide (e.g., barcode) used in the methods of the present disclosure comprises a nucleotide sequence such that a universal splint oligonucleotide may be used. As used herein, a "universal splint oligonucleotide" refers to a splint oligonucleotide in a population of splint oligonucleotides, where each splint oligonucleotide in the population comprises or consists of the same nucleotide sequence. See, e.g., Figure 11a.

[0041] In some embodiments, the oligonucleotide (e.g., barcode) comprises a modified backbone, such as a phosphorothiolate backbone. In various embodiments, reactions that can be performed to obtain a phosphate derivative backbone are shown in Figure 3b, and the phosphate derivative group can be present at either the 3' or 5' end of either fragment.

[0042] In some embodiments, methods using enzymatic ligation include the use of splint oligonucleotides. In some embodiments, methods using chemical ligation include the use of splint oligonucleotides. In some embodiments, methods using chemical ligation do not include the use of splint oligonucleotides. In some embodiments, variables that can increase ligation efficiency include, but are not limited to, temperature, buffers, divalent metals [Sci. Rep. 2014, 4, 4595], and the use of double-stranded DNA interfering substances [(a) Nat. Commun. 2016, 6, 7304; (b) PNAS 2010, 107, 5288]. In various embodiments, terminal deoxynucleotidyl transferase (TdT) enzymes are used to incorporate 3'-modified nucleotides prior to chemical ligation.

[0043] In some embodiments, chemically ligating oligonucleotides in the methods disclosed herein involves the synthesis of a 3'-5' phosphodiester bond. Phosphodiester bonds can be synthesized by methods known in the art. For example, phosphodiester bonds can be synthesized by reacting a phosphate- or thiophosphate-terminated nucleoside with a hydroxyl-containing nucleoside. The reaction can be facilitated by a suitable reagent, such as 1-fluoro-2,4-dinitrobenzene (DNFB) or 1-cyanoimidazole. In some embodiments, chemically ligating oligonucleotides in the methods disclosed herein involves the synthesis of a non-natural backbone bond between two nucleosides. A "non-natural backbone bond" is a bond between two nucleotides other than the naturally occurring 3'-5' phosphodiester bond. Non-limiting examples of non-natural backbone bonds include phosphoramidate, phosphorothiolate, triazole, squaramide, and urea. Non-natural backbone bonds can be synthesized by methods known in the art. For example, a phosphoramidate bond can be formed by reacting a terminal thiophosphate-modified nucleoside with an amine-modified nucleoside. The reaction can be promoted by a suitable reagent, such as 1-fluoro-2,4-dinitrobenzene (DNFB). A phosphorothiolate bond can be formed by reacting a terminal thiophosphate-modified nucleoside with a hydroxyl-containing nucleoside. The reaction can be promoted by a suitable reagent, such as dabsyl chloride. A triazole bond can be formed via an azide-alkyne cycloaddition reaction (e.g., a "click reaction"). The azide-alkyne cycloaddition reaction can be catalyzed (e.g., by Cu(I)) or uncatalyzed. A squaramide bond can be formed by reacting an amine-modified nucleoside with squaric acid or its nucleophilic derivative. Squaramide formation may be facilitated (eg, using carbodiimide-mediated reaction conditions, such as using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)) or unfacilitated.Non-limiting examples of methods for synthesizing urea linkages include reacting an amine-modified nucleoside with an activating reagent (such as 1,1'-carbonyldiimidazole (CDI)), reacting an amine-modified nucleoside with an isocyanate-modified nucleoside, or reacting an amine-modified nucleoside with a carbamate-modified nucleoside.

[0044] Exemplary Ligation Synthesis Workflow As described herein, in some embodiments, the oligonucleotide comprises a modified backbone. To utilize a modified backbone, the method requires modified nucleotide bases at the 5' and 3' ends of each oligonucleotide sequence. There are several ways to include modified nucleotide bases at the ends of each oligonucleotide sequence.

[0045] In some embodiments, modified nucleotide bases are added to the ends of oligonucleotide sequences using terminal deoxynucleotide transferase (TdT) enzyme. In some embodiments, the general steps of the ligation synthesis workflow, as shown in Figure 5, are: 1. conjugating a first oligonucleotide to a solid phase particle that allows for oligonucleotide synthesis (e.g., any particle that can be separated from other particles and combined as a pool), such as, but not limited to, controlled pore glass (CPG) or beads; 2. Incorporating a 3'-modified nucleotide into the first oligonucleotide using TdT; 3. Chemically ligating a second oligonucleotide with the 5'-orthogonal chemistry and 3'-orthogonal chemistry of the first oligonucleotide; 4. Capping any unreacted 3'-modified nucleotides that failed to ligate; 5. Repeating steps 2-4 up to n rounds, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; optionally, 6. Cleavage of the oligonucleotide (e.g., barcode) from the solid-phase particle and optionally purifying it; optionally, 7. Conjugating the oligonucleotide (e.g., barcode) to a second solid phase particle or solid support.

[0046] Before each new ligation in the aforementioned workflow (i.e., before each step (5) is performed), the pool is split (see Figure 1). One unique oligonucleotide is then conjugated to each split-pool, increasing the diversity of oligonucleotide sequences and depending on the number of rounds of split-pooling performed. In some embodiments, a wash step to wash away unbound material and / or excess reagents is performed after step (1), after step (2), after step (3), after step (4), after step (5), and / or after step (7).

[0047] In some embodiments, modified nucleotide bases are added to the ends of oligonucleotide sequences in the absence of terminal deoxynucleotidyl transferase (TdT) enzyme. In some embodiments, the general steps of the ligation synthesis workflow, as shown in Figure 6, are: 1. conjugating a first oligonucleotide to a solid phase particle that allows for oligonucleotide synthesis (e.g., any particle that can be separated from other particles and combined as a pool), such as, but not limited to, controlled pore glass (CPG) or beads; 2. Chemically ligating a second oligonucleotide with the 5'-orthogonal chemistry and 3'-orthogonal chemistry of the first oligonucleotide; 3. Capping any unreacted 3'-modified nucleotides that failed to ligate; 4. Repeating steps 2-3 up to n rounds, where n is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more; optionally, 5. Cleavage of the oligonucleotide (e.g., barcode) from the solid-phase particle and optionally purifying it; optionally, 6. Conjugating the oligonucleotide (e.g., barcode) to a second solid phase particle or solid support.

[0048] Before each new ligation in the aforementioned workflow (i.e., before each step (3) is performed), the pool is split (see Figure 1). One unique oligonucleotide is then conjugated to each split-pool, increasing the diversity of oligonucleotide sequences and depending on the number of split-pool rounds performed. In some embodiments, a wash step to wash away unbound material and / or excess reagents is performed after step (1), after step (2), after step (3), after step (4), and / or after step (6).

[0049] For any of the ligation workflows described herein, there are optional additional steps that can be performed to improve conjugation efficiency and yield. In some embodiments, when R1 and R2 (e.g., as shown in Figures 5 and 6) are the only two reactive groups used in the workflow, R1 on the second oligonucleotide sequence can be capped with a protecting group, while R2 of the second oligonucleotide sequence is conjugated to R1 of the first oligonucleotide sequence. Once conjugation is complete, R1 of the second oligonucleotide sequence is then deprotected, allowing for conjugation of a third oligonucleotide sequence, and the cycle continues. (See Figure 7.)

[0050] Alternatively, in some embodiments, the number of reactive groups is expanded to four (i.e., R1, R2, R3, R4). For example, a first oligonucleotide may have R1 and R2 reactive handles, where R1 is either capped or conjugated to a solid support. A second oligonucleotide may have R3 and R4 reactive handles. R2 of the first oligonucleotide can then be conjugated to R3 of the second oligonucleotide. Subsequently, R4 of the second oligonucleotide can then be conjugated to R1 of a third oligonucleotide having the same reactive entity as the first oligonucleotide, where R2 + R3 are orthogonal to R1 + R4. (See Figure 8).

[0051] To minimize undesired barcodes, in some embodiments, a capping step is included to terminate failed ligation synthesis for any of the workflows of the present disclosure. While the workflows described herein focus on chemical ligation of solid supports, it is further contemplated that ligation synthesis can also be performed in solution prior to bead attachment. Additionally, while the workflows described herein focus on synthesis prior to bead attachment, in some embodiments, ligation synthesis can also be performed directly on beads.

[0052] To further illustrate the present disclosure, examples are presented herein. It should be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure. [Example]

[0053] Example 1 Synthesis of exemplary modified nucleotides for ligation.

[0054] Synthesis of 3'-alkyne dT triphosphate:

[0055] [ka]

[0056] Synthesis of 3'-alkyne dA triphosphate:

[0057] [ka]

[0058] In this example, two 3'-alkyne modified deoxyribonucleotides useful in the chemical ligation methods disclosed herein were synthesized. These modified deoxyribonucleotides can be used to generate triazole linkages (i.e., non-natural backbone linkages) between nucleotides in the chemical ligation methods disclosed herein.

[0059] Synthesis of 3'-alkyne dT triphosphate: Briefly, deoxythymidine was treated with tert-butyldimethylsilyl chloride (TBDMSCl) in pyridine / DMF to protect the 5' hydroxyl group. The silylated deoxythymidine was then treated with propargyl bromide and sodium hydride in anhydrous THF to give 3'-alkyne deoxythymidine. Desilylation with tetrabutylammonium fluoride (TBAF) in THF, followed by stepwise treatment with phosphoryl chloride / trimethylphosphite (POCl3 / PO(OMe)3), tributylammonium pyrophosphate ((Bu3NH)4PO7), and Bu3N / TEAB, gave the desired 3'-alkyne dT triphosphate.

[0060] Synthesis of 3'-alkyne dA triphosphate: Briefly, deoxyadenosine was treated with N,N-dimethylformamide dimethyl acetal to give 10-N,N-dimethylformamidine-protected deoxyadenosine. The protected dA was treated with tert-butyldimethylsilyl chloride (TBDMSCl) in pyridine / DMF to protect the 5' hydroxyl group. The silylated deoxyadenosine was then treated with propargyl bromide and sodium hydride in anhydrous THF to give the 3'-alkyne deoxyadenosine. Desilylation with tetrabutylammonium fluoride (TBAF) in THF, followed by stepwise treatment with phosphoryl chloride / trimethylphosphite (POCl3 / PO(OMe)3), tributylammonium pyrophosphate ((Bu3NH)4PO7), and Bu3N / TEAB, gave the desired 3'-alkyne dA triphosphate.

[0061] Example 2 In this example, an array of the present disclosure was produced. Array production begins by synthesizing a first set of 100 unique oligonucleotide beads. Next, another 100 unique oligonucleotide barcodes were chemically ligated in each split-pool step. After three rounds of split-pool and ligation, a pool of 100,000,000 barcoded beads was produced. Chemical ligation can occur on beads or in solution. If in solution, the resulting oligonucleotides are chemically attached to the beads after all ligation cycles are completed.

[0062] Three unique oligonucleotide sequences (Figure 10) with 5'-terminal amines or hydrazines and cleavable linkers were first synthesized and purified on an oligo synthesizer. Each of these unique oligonucleotide sequences was then chemically attached via its 5' end to three pools of aldehyde-functionalized beads, i.e., one unique oligonucleotide sequence was attached to one of the three pools of beads. Sodium cyanoborohydride was then added to reduce the imine bond formed between the 5'-terminal hydrazine and the aldehyde-functionalized beads. After oligonucleotide attachment, excess aldehydes on the beads were capped using any small amine and further reduced with sodium cyanoborohydride. The beads from the three pools were then combined. TdT enzyme and 3'-alkyne-modified nucleotides were then added to the combined bead pool. After one 3'-alkyne-modified nucleotide was added to the 3'-OH terminus of the attached oligonucleotide, the TdT enzyme and 3'-alkyne-modified nucleotide were washed away using a buffer solution. This combined pool was then randomly split into three new pools. A unique oligonucleotide sequence bearing a 5'-azide was added to each of the three new pools of beads. Click ligation was performed with or without a splint. Once ligation was complete, the beads were washed multiple times with urea in buffer and buffer to dehybridize any splints and remove unreacted oligonucleotides. Unreacted 3'-alkynes were capped using small azides and washed. Another round of TdT enzyme and 3'-alkyne-modified nucleotides was then added to allow ligation of a third oligonucleotide sequence to the 3' OH terminus of the second oligonucleotide sequence. The process of TdT enzyme and 3'-alkyne-modified nucleotide addition, washing, and oligonucleotide ligation, along with the capping step, was repeated until the desired oligonucleotide barcode length was obtained.

[0063] Example 3 In this example, the disclosed method is used for spatial applications. Barcodes are generated on beads and either (1) directly immobilized on a solid support (such as a flow cell) (e.g., via hybridization with a surface primer), (2) oligonucleotides are released from the beads, purified, and then attached to new beads, which are then immobilized on a solid support (e.g., via hybridization with a surface primer), or (3) oligonucleotides are released from the beads and immobilized on a solid support (e.g., via hybridization with a surface primer). Tissues are then placed on the solid support, and nucleic acids from the tissue are captured with oligonucleotides (e.g., mRNA capture with a poly(T) capture sequence). The captured nucleic acids can be sequenced in situ or ex situ. For mRNA, cDNA was generated on the solid support.

Claims

1. 1. A method for generating a bead array, comprising: (i) attaching a first oligonucleotide to a first population of beads, a second oligonucleotide to a second population of beads, and a third oligonucleotide to a third population of beads, wherein the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide comprise unique nucleotide sequences relative to one another; (ii) combining the first bead population, the second bead population, and the third bead population to create a first pool; (iii) dividing the first pool into a first plurality of aliquots; (iv) chemically ligating, in a first aliquot of the first plurality of aliquots, a fourth oligonucleotide to chemically ligating the first oligonucleotide in the presence of a first splint oligonucleotide, wherein the first splint oligonucleotide hybridizes to both the fourth oligonucleotide and the first oligonucleotide; chemically ligating to the second oligonucleotide in the presence of a second splint oligonucleotide, wherein the second splint oligonucleotide hybridizes to both the fourth oligonucleotide and the second oligonucleotide; and chemically ligating the fourth oligonucleotide to the third oligonucleotide in the presence of a third splint oligonucleotide, the third splint oligonucleotide hybridizing to both the fourth oligonucleotide and the third oligonucleotide; the first splint oligonucleotide, the second splint oligonucleotide, and the third splint oligonucleotide comprise unique nucleotide sequences relative to one another; In a second aliquot of said first plurality of aliquots, a fifth oligonucleotide is chemically ligating the first oligonucleotide in the presence of a fourth splint oligonucleotide, wherein the fourth splint oligonucleotide hybridizes to both the fifth oligonucleotide and the first oligonucleotide; chemically ligating to the second oligonucleotide in the presence of a fifth splint oligonucleotide, wherein the fifth splint oligonucleotide hybridizes to both the fifth oligonucleotide and the second oligonucleotide; and chemically ligating the third oligonucleotide in the presence of a sixth splint oligonucleotide, wherein the sixth splint oligonucleotide hybridizes to both the fifth oligonucleotide and the third oligonucleotide; the fourth splint oligonucleotide, the fifth splint oligonucleotide, and the sixth splint oligonucleotide comprise unique nucleotide sequences relative to one another; and In a third aliquot of the first plurality of aliquots, a sixth oligonucleotide is chemically ligating the first oligonucleotide in the presence of a seventh splint oligonucleotide, wherein the seventh splint oligonucleotide hybridizes to both the sixth oligonucleotide and the first oligonucleotide; chemically ligating to the second oligonucleotide in the presence of an eighth splint oligonucleotide, wherein the eighth splint oligonucleotide hybridizes to both the sixth oligonucleotide and the second oligonucleotide; and chemically ligating to the third oligonucleotide in the presence of a ninth splint oligonucleotide, wherein the ninth splint oligonucleotide hybridizes to both the sixth oligonucleotide and the third oligonucleotide; chemically ligating the seventh splint oligonucleotide, the eighth splint oligonucleotide, and the ninth splint oligonucleotide, wherein the seventh splint oligonucleotide, the eighth splint oligonucleotide, and the ninth splint oligonucleotide comprise unique nucleotide sequences relative to one another; (v) combining the first aliquot of the first plurality of aliquots, the second aliquot of the first plurality of aliquots, and the third aliquot of the first plurality of aliquots to create a second pool; (vi) dividing the second pool into a second plurality of aliquots; (vii) chemically ligating, in a first aliquot of the second plurality of aliquots, a seventh oligonucleotide to the fourth oligonucleotide in the presence of a tenth splint oligonucleotide, wherein the tenth splint oligonucleotide hybridizes to both the seventh oligonucleotide and the fourth oligonucleotide; in a second aliquot of the second plurality of aliquots, chemically ligating an eighth oligonucleotide to the fifth oligonucleotide in the presence of an eleventh splint oligonucleotide, wherein the eleventh splint oligonucleotide hybridizes to both the eighth oligonucleotide and the fifth oligonucleotide; and In a third aliquot of the second plurality of aliquots, a ninth oligonucleotide is chemically ligated to the sixth oligonucleotide in the presence of a twelfth splint oligonucleotide, wherein the twelfth splint oligonucleotide hybridizes to both the ninth oligonucleotide and the sixth oligonucleotide, and the tenth splint oligonucleotide, the eleventh splint oligonucleotide, and the twelfth splint oligonucleotide comprise unique nucleotide sequences relative to one another.

2. 1. A method for generating a bead array, comprising: (i) attaching a first oligonucleotide to one or more beads in a first population of beads, a second oligonucleotide to one or more beads in a second population of beads, and a third oligonucleotide to one or more beads in a third population of beads, wherein the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide comprise unique nucleotide sequences relative to one another; (ii) combining the first bead population, the second bead population, and the third bead population to create a first pool; (iii) dividing the first pool into a first plurality of aliquots; (iv) chemically ligating, in a first aliquot of the first plurality of aliquots, a fourth oligonucleotide is chemically ligated to the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, in a second aliquot of the first plurality of aliquots, a fifth oligonucleotide is chemically ligated to the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, and in a third aliquot of the first plurality of aliquots, a sixth oligonucleotide is chemically ligated to the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide; (v) combining the first aliquot of the first plurality of aliquots, the second aliquot of the first plurality of aliquots, and the third aliquot of the first plurality of aliquots to create a second pool; (vi) dividing the second pool into a second plurality of aliquots; (vii) chemically ligating, wherein in a first aliquot of the second plurality of aliquots, a seventh oligonucleotide is chemically ligated to the fourth oligonucleotide, in a second aliquot of the second plurality of aliquots, an eighth oligonucleotide is chemically ligated to the fifth oligonucleotide, and in a third aliquot of the second plurality of aliquots, a ninth oligonucleotide is chemically ligated to the sixth oligonucleotide.

3. (viii) combining the first aliquot of the second plurality of aliquots, the second aliquot of the second plurality of aliquots, and the third aliquot of the second plurality of aliquots to create a third pool; (ix) dividing the third pool into a third plurality of aliquots; (x) chemically ligating, in a first aliquot of the third plurality of aliquots, a tenth oligonucleotide to the seventh oligonucleotide in the presence of a thirteenth splint oligonucleotide, wherein the thirteenth splint oligonucleotide hybridizes to both the tenth oligonucleotide and the seventh oligonucleotide; in a second aliquot of the third plurality of aliquots, chemically ligating an eleventh oligonucleotide to the eighth oligonucleotide in the presence of a fourteenth splint oligonucleotide, wherein the fourteenth splint oligonucleotide hybridizes to both the eleventh oligonucleotide and the eighth oligonucleotide; 2. The method of claim 1, further comprising chemically ligating, in a third aliquot of the third plurality of aliquots, a 12th oligonucleotide to the ninth oligonucleotide in the presence of a 15th splint oligonucleotide, wherein the 15th splint oligonucleotide hybridizes to both the 12th oligonucleotide and the ninth oligonucleotide, and the 13th splint oligonucleotide, the 14th splint oligonucleotide, and the 15th splint oligonucleotide comprise unique nucleotide sequences relative to one another.

4. (viii) combining the first aliquot of the second plurality of aliquots, the second aliquot of the second plurality of aliquots, and the third aliquot of the second plurality of aliquots to create a third pool; (ix) dividing the third pool into a third plurality of aliquots; 3. The method of claim 2, further comprising: (x) chemically ligating a tenth oligonucleotide to the seventh oligonucleotide in a first aliquot of the third plurality of aliquots; chemically ligating an eleventh oligonucleotide to the eighth oligonucleotide in a second aliquot of the second plurality of aliquots; and chemically ligating a twelfth oligonucleotide to the ninth oligonucleotide in a third aliquot of the second plurality of aliquots.

5. The method comprises the steps of: providing an array of beads and at least about 1×10 ６ 5. The method of claim 1, wherein the bead array comprises a plurality of unique oligonucleotide sequences.

6. The method comprises the steps of: providing an array of beads and at least about 1×10 ７ 6. The method of claim 5, wherein the bead array comprises a plurality of unique oligonucleotide sequences.

7. The method comprises the steps of: providing an array of beads and at least about 1×10 ８ 7. The method of claim 5 or 6, wherein the bead array comprises a plurality of unique oligonucleotide sequences.

8. 4. The method of claim 3, wherein the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, and fifteenth splint oligonucleotides have the same nucleotide sequence.

9. 9. The method of any one of claims 1 to 8, wherein the oligonucleotides comprise (i) a poly(T) nucleotide sequence or a poly(A) nucleotide sequence, (ii) one or more gene-specific capture sequences, (iii) one or more universal capture sequences (e.g., random or semi-random nucleotide sequences), or a combination thereof, and are chemically ligated to the end of each oligonucleotide on each bead in the bead array.

10. 10. The method of any one of claims 1 to 9, wherein (i) comprises about 100 bead populations, each bead population having attached thereto an oligonucleotide that is unique relative to all other bead populations.

11. 11. The method of any one of claims 1 to 10, wherein (i) comprises about 13,600 bead populations, each bead population comprising attached thereto an oligonucleotide that is unique relative to all other bead populations.

12. 12. The method of any one of claims 1 to 11, wherein the first oligonucleotide, the second oligonucleotide, and / or the third oligonucleotide are attached to the one or more beads via a cleavable linker.

13. The method of any one of claims 1 to 12, wherein a 3'-modified nucleotide is incorporated using terminal deoxynucleotidyl transferase (TdT) enzyme prior to said chemical ligation.

14. 13. The method of any one of claims 1 to 12, wherein one or more of the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, the seventh oligonucleotide, the eighth oligonucleotide, or the ninth oligonucleotide is a barcode.

15. 15. The method of any one of claims 1 to 14, wherein each of the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, the seventh oligonucleotide, the eighth oligonucleotide, and the ninth oligonucleotide is a barcode.

16. 16. The method of any one of claims 3 to 15, wherein one or more of the tenth oligonucleotide, the eleventh oligonucleotide, or the twelfth oligonucleotide is a barcode.

17. The method of any one of claims 3 to 15, wherein each of the tenth oligonucleotide, the eleventh oligonucleotide, and the twelfth oligonucleotide is a barcode.

18. The method according to any one of claims 14 to 17, wherein the barcode is a spatial barcode.

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