Application of Peptide Nucleic Acid (PNA) Blockers

Abstract

The present disclosure includes systems, methods, compositions, and kits for preventing antibody oligonucleotide extension. In some embodiments, a blocking oligonucleotide is provided. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing to a barcoded oligonucleotide to form a protected duplex. The blocking oligonucleotide can be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. In some embodiments, the 3' end of the blocking oligonucleotide is not extendible by a reverse transcriptase or a polymerase. In some embodiments, the blocking oligonucleotide of the protected duplex is not removable by the strand displacement activity of a reverse transcriptase or a polymerase.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 USC §119(e) of U.S. Provisional Patent Application No. 63 / 421,744, filed November 2, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] The present disclosure relates generally to the field of molecular biology, for example, determining gene expression using molecular barcoding. Current technology allows for the attachment of cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells, allowing each cell to colocalize with a barcoded reagent bead in a compartment, thereby enabling the measurement of gene expression in a single cell in a massively parallel manner (e.g., more than 10,000 cells). Gene expression can affect protein expression. Protein-protein interactions can affect gene and protein expression. There is a need for systems and methods that can quantitatively analyze protein expression in cells and simultaneously measure protein and gene expression in cells. Furthermore, existing methods of protein profiling include methods in which antibody oligonucleotides can hybridize to barcode oligonucleotides and serve as templates for the barcoding reaction, as well as be extended at their own 3' ends. There is a need for compositions, methods, systems, and kits that reduce or prevent the extension of antibody oligonucleotides hybridized to barcoded oligonucleotides. Summary of the Invention

[0003] The present disclosure includes a blocking oligonucleotide. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing to each of a plurality of barcoded oligonucleotides to form a protected duplex. In some embodiments, the barcoded oligonucleotide comprises a 3' target binding region capable of hybridizing to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence. The present disclosure includes protected duplexes. In some embodiments, the protected duplexes include a blocking oligonucleotide hybridized to a barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide includes a binding region capable of hybridizing to the barcoded oligonucleotide. In some embodiments, the barcoded oligonucleotide includes a 3' target binding region capable of hybridizing to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence.

[0004] In some embodiments, the blocking oligonucleotide cannot function as a primer for reverse transcriptase or polymerase. In some embodiments, the 3' end of the blocking oligonucleotide is not extendable by reverse transcriptase or polymerase. In some embodiments, the protected duplex of the blocking oligonucleotide cannot be removed by the strand displacement activity of reverse transcriptase or polymerase. In some embodiments, the blocking oligonucleotide is a locked nucleic acid (LNA), peptide nucleic acid (PNA), DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. In some embodiments, the T of the blocking oligonucleotide m is at least 50°C, at least 60°C, or at least 70°C.

[0005] In some embodiments, the blocking oligonucleotide does not contain non-natural nucleotides. In some embodiments, the blocking oligonucleotide comprises a 3' non-annealing region that is incapable of binding to the barcoded oligonucleotide. In some embodiments, the 3' non-annealing region is 1 to 100 nucleotides in length, 1 to 50 nucleotides in length, 1 to 21 nucleotides in length, 1 to 10 nucleotides in length, or about 5 nucleotides in length. In some embodiments, the non-complementarity between the 3' non-annealing region and the region of the barcoded oligonucleotide adjacent to the 5' side of the sequence to which the blocking oligonucleotide binds is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%. In some embodiments, the blocking oligonucleotide and / or the barcoded oligonucleotide are single-stranded oligonucleotides.

[0006] In some embodiments, the barcoded oligonucleotide comprises a blocker region, the blocker region being located between the barcode and the target binding region; or the blocker region being located between the barcode and the first universal sequence. In some embodiments, the complementarity between the binding region of the barcoded oligonucleotide and the sequence of the barcoded oligonucleotide to which the blocking oligonucleotide binds is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%. In some embodiments, the binding region is capable of hybridizing to at least a portion of one or more of the target binding region, the first universal sequence, the barcode, and the blocker region to form a protected duplex.

[0007] In some embodiments, the protected duplex comprises a double-stranded nucleic acid flanked by single-stranded overhangs. In some embodiments, upon contact of the protected duplex with a nucleic acid target, a polymerase or reverse transcriptase can extend the 3' end of the barcoded oligonucleotide hybridized to the nucleic acid target to generate an extended protected duplex, the extended protected duplex comprising a blocking oligonucleotide hybridized to the extended barcoded oligonucleotide, the blocking oligonucleotide comprising (i) a sequence complementary to at least a portion of the nucleic acid target, (ii) a target binding region, (iii) a first universal sequence, and (iv) a barcode. In some embodiments, the blocking oligonucleotide can terminate polymerase and / or reverse transcriptase extension of the 3' end of the hybridized nucleic acid target beyond the 5' end of the barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide can terminate polymerase and / or reverse transcriptase extension of the 3' end of the hybridized nucleic acid target beyond the barcoded oligonucleotide. In some embodiments, the extended protected duplex comprises a single-stranded 5' first universal sequence. In some embodiments, the single-stranded 5' first universal sequence of the extended protected duplex is capable of hybridizing to a linker oligonucleotide to form a triplex. In some embodiments, the linker oligonucleotide comprises a second universal sequence capable of binding to an oligonucleotide barcode, and the triplex is capable of binding to the oligonucleotide barcode via the second universal sequence to form a quadruplex. In some embodiments, the 5' end of the extended barcoded oligonucleotide and the 3' end of the oligonucleotide barcode are ligated to each other by a ligase. It is capable of being ligated.

[0008] In some embodiments, in the absence of the blocking oligonucleotide, the 3' end of the hybridized nucleic acid target extends to the 5' end of the barcoded oligonucleotide, forming an undesired duplex. In some embodiments, the undesired duplex is double-stranded and / or unable to hybridize to the linker oligonucleotide. In some embodiments, the blocking oligonucleotide can reduce the formation of undesired duplexes by at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0009] In some embodiments, the barcode comprises a first cell label. In some embodiments, the oligonucleotide barcode comprises a second cell label. In some embodiments, the plurality of barcoded oligonucleotides comprises at least 100, at least 1,000, or at least 10,000 different barcode sequences. In some embodiments, each of the plurality of barcoded oligonucleotides comprises the same barcode sequence. In some embodiments, the barcoded oligonucleotide is an in situ cell indexing adaptor. In some embodiments, the blocking oligonucleotide, barcoded oligonucleotide, barcode, target binding region, first universal sequence, binding region, and / or blocker region is between 1 and 100 nucleotides in length, between 1 and 50 nucleotides in length, between 1 and 21 nucleotides in length, or about 12 nucleotides in length.

[0010] In some embodiments, the nucleic acid target is a nucleic acid target molecule selected from the group including a DNA molecule, an RNA molecule, a genomic DNA molecule, a cDNA molecule, an mRNA molecule, an rRNA molecule, an mtDNA, an siRNA molecule, or any combination thereof. In some embodiments, the nucleic acid target is a cellular component binding reagent-specific oligonucleotide, and the cellular component binding reagent-specific oligonucleotide is associated with the cellular component binding reagent. In some embodiments, the cellular component binding reagent is an antibody or fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, or any combination thereof. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a unique identifier sequence for the cellular component binding reagent. In some embodiments, the target binding region comprises a capture sequence. In some embodiments, the target binding region comprises a poly(dT) region. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the cellular component binding reagent-specific oligonucleotide. In some embodiments, the sequence complementary to the capture sequence comprises a poly(dA) region.

[0011] In some embodiments, the cellular component binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets of a cell. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a third universal sequence. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a molecular label. In some embodiments, at least 10 of the plurality of cellular component binding reagent-specific oligonucleotides comprise different molecular label sequences. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a poly(dA) tract. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises an alignment sequence adjacent to the poly(dA) tract. In some embodiments, the cellular component binding reagent-specific oligonucleotide is associated with the cellular component binding reagent through a linker. In some embodiments, the cellular component binding reagent-specific oligonucleotide is configured to be detachable from the cellular component binding reagent. In some embodiments, the alignment sequence is one or more nucleotides in length, or two or more nucleotides in length. In some embodiments, (a) the alignment sequence comprises guanine, cytosine, thymine, uracil, or a combination thereof; (b) the alignment sequence comprises a poly(dT), poly(dG), poly(dC), poly(dU), or a combination thereof; and / or (c) the alignment sequence is 5' to the poly(dA) region. In some embodiments, the linker comprises a carbon chain. In some embodiments, the carbon chain comprises 2 to 30 carbons (e.g., 12 carbons). In some embodiments, the linker comprises a 5' amino modifier C12 (5AmMC12) or a derivative thereof. In some embodiments, the cellular component target comprises a protein target. In some embodiments, the cellular component target comprises a carbohydrate, lipid, protein, extracellular protein, cell surface protein, cell marker, B cell receptor, T cell receptor, major histocompatibility complex, tumor antigen, receptor, intracellular protein, or any combination thereof. In some embodiments, the cellular component target is present on a cell surface. In some embodiments, the DNA polymerase comprises a Klenow fragment.In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase (eg, murine leukemia virus (MLV) reverse transcriptase and / or Moloney murine leukemia virus (MMLV) reverse transcriptase).

[0012] The present disclosure includes a reaction mixture, in some embodiments, the reaction mixture comprises a plurality of blocking oligonucleotides provided herein, a plurality of protected duplexes provided herein, a plurality of protected duplexes, a plurality of barcoded oligonucleotides, a plurality of cellular component binding reagents, a ligase, dNTPs, a polymerase, a reverse transcriptase, and / or a plurality of oligonucleotide barcodes.

[0013] The present disclosure includes kits, in some embodiments, the kits include a plurality of blocking oligonucleotides provided herein, a plurality of protected duplexes provided herein, a plurality of protected duplexes, a plurality of barcoded oligonucleotides, a plurality of cellular component binding reagents, a ligase, a dNTP, a polymerase, a reverse transcriptase, and / or a plurality of oligonucleotide barcodes.

[0014] In some embodiments, the plurality of oligonucleotide barcodes are immobilized on a substrate. In some embodiments, the substrate is a particle (e.g., a bead). In some embodiments, the plurality of oligonucleotide barcodes comprises at least 100 different molecular label sequences or at least 100 different molecular label sequences. In some embodiments, the plurality of oligonucleotide barcodes comprises the same cell label sequence. In some embodiments, each of the plurality of oligonucleotide barcodes comprises a cell label sequence, a sample label sequence, a location label sequence, a binding site for a universal primer, or a combination thereof. In some embodiments, the plurality of oligonucleotide barcodes comprises at least 100, at least 1,000, or at least 10,000 different molecular label sequences. In some embodiments, the plurality of oligonucleotide barcodes comprises the same cell label sequence. In some embodiments, the plurality of oligonucleotide barcodes are associated with particles. In some embodiments, the oligonucleotide barcodes are immobilized on the particle, partially immobilized on the particle, embedded in the particle, partially embedded in the particle, or a combination thereof. In some embodiments, the particles are beads. In some embodiments, the beads comprise polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or a combination thereof. In some embodiments, the beads are hydrogel beads or magnetic beads. In some embodiments, the beads are disintegratable. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 illustrates a non-limiting exemplary barcode. [Figure 2] FIG. 1 illustrates a non-limiting exemplary workflow for barcoding and electronic counting. [Figure 3]1 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of 3′-barcoded targets from multiple targets. [Figure 4A] 4A-4B show non-limiting exemplary schematics illustrating the capture of molecularly barcoded cDNA by Rhapsody beads and ligation of the barcoded cDNA to the beads (FIG. 4A), and the lack of capture of molecularly barcoded AbSeq oligonucleotides due to double-stranded extension (FIG. 4B). [Figure 4B] 4A-4B show non-limiting exemplary schematics illustrating the capture of molecularly barcoded cDNA by Rhapsody beads and ligation of the barcoded cDNA to the beads (FIG. 4A), and the lack of capture of molecularly barcoded AbSeq oligonucleotides due to double-stranded extension (FIG. 4B). [Figure 5A] A non-limiting exemplary schematic diagram is shown showing that polymerase extension of both barcoded and antibody oligonucleotides can occur (FIG. 5A), but is not preferred in some protocols (FIG. 5B). [Figure 5B] A non-limiting exemplary schematic diagram is shown showing that polymerase extension of both barcoded and antibody oligonucleotides can occur (FIG. 5A), but is not preferred in some protocols (FIG. 5B). [Figure 6A] 6A-6B show non-limiting exemplary schematic diagrams illustrating that a PNA blocking oligonucleotide provided herein hybridizes to a barcoded primer (FIG. 6A), subsequently preventing extension of the 3' end of an antibody oligonucleotide hybridized thereto (e.g., by blocking the strand displacement activity of reverse transcriptase) (FIG. 6B). [Figure 6B]6A-6B show non-limiting exemplary schematic diagrams illustrating that a PNA blocking oligonucleotide provided herein hybridizes to a barcoded primer (FIG. 6A), subsequently preventing extension of the 3' end of an antibody oligonucleotide hybridized thereto (e.g., by blocking the strand displacement activity of reverse transcriptase) (FIG. 6B). DETAILED DESCRIPTION OF THE INVENTION

[0016] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and make part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety for relevant art.

[0017] Quantifying a small number of nucleic acids, such as messenger ribonucleotide acid (mRNA) molecules, is clinically important, for example, to determine the genes expressed in cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can be very difficult, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR produces identical molecular copies in each cycle. However, PCR can have drawbacks because each molecule replicates with a stochastic probability, which varies depending on the PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Stochastic barcodes with unique molecular labels (also called molecular indexes (MIs)) can be used to count the number of molecules and correct for amplification bias. Stochastic barcoding, such as the Precise™ assay (Cellular Research, Inc., Palo Alto, CA) and the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ), can correct for biases induced by the library preparation step by using molecular beacons (MLs) to label mRNA during PCR and reverse transcription (RT).

[0018] The Precise™ assay utilizes a non-depleting pool of stochastic barcodes with a large number of unique molecular tag sequences, e.g., 6561-65536, on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step. The stochastic barcodes may contain universal PCR priming sites. During RT, target gene molecules react randomly with the stochastic barcodes. Each target molecule hybridizes to a stochastic barcode, resulting in the generation of a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules from the microwells of a microwell plate may be pooled into a single tube for PCR amplification and sequencing. Raw sequencing data can be analyzed to obtain the number of reads, the number of stochastic barcodes with unique molecular tag sequences, and the number of mRNA molecules.

[0019] The present disclosure includes a blocking oligonucleotide. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing to each of a plurality of barcoded oligonucleotides to form a protected duplex. In some embodiments, the barcoded oligonucleotide comprises a 3' target binding region capable of hybridizing to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence. The present disclosure includes protected duplexes. In some embodiments, the protected duplexes include a blocking oligonucleotide hybridized to a barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide includes a binding region capable of hybridizing to the barcoded oligonucleotide. In some embodiments, the barcoded oligonucleotide includes a 3' target binding region capable of hybridizing to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence.

[0020] The present disclosure includes a reaction mixture, in some embodiments, the reaction mixture comprises a plurality of blocking oligonucleotides provided herein, a plurality of protected duplexes provided herein, a plurality of protected duplexes, a plurality of barcoded oligonucleotides, a plurality of cellular component binding reagents, a ligase, dNTPs, a polymerase, a reverse transcriptase, and / or a plurality of oligonucleotide barcodes. The present disclosure includes kits, in some embodiments, the kits include a plurality of blocking oligonucleotides provided herein, a plurality of protected duplexes provided herein, a plurality of protected duplexes, a plurality of barcoded oligonucleotides, a plurality of cellular component binding reagents, a ligase, a dNTP, a polymerase, a reverse transcriptase, and / or a plurality of oligonucleotide barcodes.

[0021] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0022] As used herein, the term "adapter" may refer to a sequence for facilitating amplification or sequencing of an associated nucleic acid. The associated nucleic acid may include a target nucleic acid. The associated nucleic acid may include one or more of a spatial label, a target label, a sample label, an indexing label, or a barcode sequence (e.g., a molecular label). The adapter may be linear. The adapter may be a pre-adenylated adapter. The adapter may be double-stranded or single-stranded. One or more adapters may be positioned at the 5' or 3' end of a nucleic acid. When an adapter includes known sequences at the 5' and 3' ends, the known sequences may be the same or different sequences. The adapters positioned at the 5' and / or 3' ends of a polynucleotide may be capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, the adapter may include a universal sequence. A universal sequence may be a region of nucleotide sequence that is common to two or more nucleic acid molecules. The two or more nucleic acid molecules may also have regions of different sequences. Thus, for example, the 5' adapters can contain identical and / or universal nucleic acid sequences, and the 3' adapters can contain identical and / or universal sequences. The presence of a universal sequence in different members of a plurality of nucleic acid molecules can allow for the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair), or more universal sequences can be present in different members of a collection of nucleic acid molecules, allowing for the replication or amplification of multiple different sequences using at least one, two (e.g., a pair), or more single universal primers that are complementary to the universal sequence. Thus, a universal primer includes a sequence that can hybridize to such a universal sequence. A molecule having a target nucleic acid sequence can be modified to add universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of the different target nucleic acid sequences.The one or more universal primers bound to the target nucleic acid may provide a site for hybridization of the universal primer. The one or more universal primers bound to the target nucleic acid may be the same or different from each other.

[0023] As used herein, the terms "associated" or "associated with" can mean that two or more species are identifiable as being located together at a time. Association can mean that two or more species are or were located in similar containers. Association can also be an informational association. For example, digital information about two or more species can be stored and used to determine that one or more of the species were located together at a time. Association can also be a physical association. In some embodiments, two or more associated species are "tethered," "bound," or "immobilized" to each other or to a common solid or semi-solid surface. Association can refer to covalent or non-covalent means for attaching a label to a solid or semi-solid support, such as a bead. Association can also be a covalent bond between a target and a label. Association can include hybridization between two molecules (e.g., a target molecule and a label).

[0024] As used herein, the term "complementary" may refer to the ability for precise pairing between two nucleotides. For example, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules may be "partial," in which only some of the nucleotides bind, or may be complete, in which total complementarity exists between the single-stranded molecules. A first nucleotide sequence may be referred to as the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence may be referred to as the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, a "complementary" sequence may refer to the "complement" or "reverse complement" of a sequence. It is understood from this disclosure that when a molecule is capable of hybridizing to another molecule, it may be complementary or partially complementary to the hybridizing molecule.

[0025] As used herein, the term "digital counting" can refer to a method for estimating the number of target molecules in a sample. Digital counting can include determining the number of unique labels associated with targets in a sample. This methodology, which can be probabilistic in nature, transforms the molecular counting problem into one of locating and identifying identical molecules into a series of yes / no digital questions regarding the detection of a predefined set of labels.

[0026] As used herein, the term "label" or "labels" can refer to a nucleic acid code associated with a target in a sample. The label can be, for example, a nucleic acid label. The label can be a wholly or partially amplifiable label. The label can be a wholly or partially sequenceable label. The label can be a portion of a naturally occurring nucleic acid that can be identified as distinct. The label can be a known sequence. The label can include a junction of a nucleic acid sequence, e.g., a junction of a naturally occurring and non-naturally occurring sequence. As used herein, the term "label" can be used interchangeably with the terms "index," "tag," or "label tag." The label can carry information. For example, in various embodiments, the label can be used to determine the identity of the sample, the source of the sample, the identity of the cell, and / or the target.

[0027] As used herein, the term "non-depletion reservoir" can refer to a pool of barcodes (e.g., stochastic barcodes) composed of many different labels. The non-depletion reservoir can contain many different barcodes so that when the non-depletion reservoir is associated with a pool of targets, each target is likely to associate with a unique barcode. The uniqueness of each labeled target molecule can be determined by random selection statistics and depends on the copy number of identical target molecules in the population compared to the diversity of the labels. The size of the resulting labeled target molecule can be determined by the stochastic nature of the barcoding process, and analysis of the number of detected barcodes then allows for calculation of the number of target molecules present in the original population or sample. If the ratio of the number of target molecules present to the number of unique barcodes is low, the labeled target molecule is highly unique (i.e., the probability that more than one target molecule will be labeled with a given label is very low).

[0028] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid may comprise nucleotides. A nucleic acid may be exogenous or endogenous to a cell. A nucleic acid may exist in a cell-free environment. A nucleic acid may be a gene or a fragment thereof. A nucleic acid may be DNA. A nucleic acid may be RNA. A nucleic acid may comprise one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0029] Nucleic acids may contain one or more modifications (e.g., base modifications, backbone modifications) to result in nucleic acids with new or enhanced properties (e.g., improved stability). Nucleic acids may also contain nucleic acid affinity tags. A nucleoside may be a base-sugar combination. The base portion of a nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide may be a nucleoside further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides containing a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of this linear polymeric compound may then be further joined to form a circular compound, although linear compounds are generally preferred. In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold in such a manner as to yield fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups may commonly be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone may be a 3' and 5' phosphodiester linkage.

[0030] The nucleic acids can contain modified backbones and / or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs, and those with reverse polarity, wherein one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0031] Nucleic acids can contain polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH moieties.

[0032] Nucleic acids may include nucleic acid mimetics. The term "mimetics" is intended to include polynucleotides in which only the furanose ring, or both the furanose ring and the internucleotide linkage, are replaced with non-furanose groups; replacement of only the furanose ring may also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide may be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides may be retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The backbone in a PNA compound may contain two or more linked aminoethylglycine units, thereby providing the PNA with an amide-containing backbone. The heterocyclic base moiety may be directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The nucleic acid may include a morpholino backbone structure. For example, the nucleic acid may include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidates or other non-phosphodiester internucleoside linkages may replace the phosphodiester linkages.

[0033] Nucleic acids can contain linked morpholino units (e.g., morpholino nucleic acids) with heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds may have fewer undesirable interactions with intracellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be attached using different linking groups. A further class of polynucleotide mimics can be called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used to synthesize oligomeric compounds using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of native complexes. Further modifications include locked nucleic acids (LNAs), in which a 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and LNA analogs can exhibit very high duplex thermal stability with complementary nucleic acids (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility characteristics.

[0034] Nucleic acids can also include nucleobase (often simply referred to as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases can include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkyl derivatives of the pyrimidine base, 6-azouracil, cytosine ... Mention may be made of tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidines (2H-pyrimido(4,5-b)indol-2-one), and pyridoindole cytidines (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0035] As used herein, the term "sample" can refer to a composition that contains a target. Samples suitable for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms. As used herein, the terms "sample collection device" or "device" may refer to a device capable of collecting a section of a sample and / or placing the section on a substrate. A sample device may refer to, for example, a fluorescence activated cell sorter (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a microtome.

[0036] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which multiple barcodes (e.g., stochastic barcodes) can be attached. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar structure composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) to which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support may comprise discrete particles that may be spherical (e.g., microspheres) or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. A bead may be non-spherical in shape. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead."

[0037] As used herein, the term "stochastic barcode" may refer to a polynucleotide sequence comprising a label of the present disclosure. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target. As used herein, the term "gene-specific stochastic barcode" may refer to a polynucleotide sequence comprising a label and a gene-specific target-binding region. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0038] As used herein, the term "stochastic barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a Poisson recursion strategy to associate labels and quantify the labels associated with targets. As used herein, the term "stochastic barcoding" can be used interchangeably with "stochastic labeling." As used herein, the term "target" may refer to a composition that can be associated with a barcode (e.g., a stochastic barcode). Exemplary targets suitable for analysis by the methods, devices, and systems of the present disclosure include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. Targets may be single-stranded or double-stranded. In some embodiments, targets may be proteins, peptides, or polypeptides. In some embodiments, targets are lipids. As used herein, "target" may be used interchangeably with "species."

[0039] As used herein, the term "reverse transcriptase" can refer to a group of enzymes that have reverse transcriptase activity (i.e., that catalyze the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include the Lactococcus lactis LI.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases include the numerous classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity-generating retroelements, among others).

[0040] The terms "universal adapter primer," "universal primer adapter," or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can hybridize to a barcode (e.g., a stochastic barcode) and be used to generate a gene-specific barcode. A universal adapter sequence can be, for example, a known sequence that is universal across all barcodes used in the methods of the present disclosure. For example, when multiple targets are labeled using the methods disclosed herein, each of the target-specific sequences can be ligated to the same universal adapter sequence. In some embodiments, two or more universal adapter sequences can be used in the methods disclosed herein. For example, when multiple targets are labeled using the methods disclosed herein, at least two of the target-specific sequences are ligated to different universal adapter sequences. The universal adapter primer and its complement can be included in two oligonucleotides, one of which contains the target-specific sequence and the other of which contains the barcode. For example, a universal adapter sequence can be part of an oligonucleotide that contains a target-specific sequence to generate a nucleotide sequence that is complementary to a target nucleic acid. A second oligonucleotide comprising the complement of the barcode and universal adapter sequence can hybridize to the nucleotide sequence to generate a target-specific barcode (target-specific stochastic barcode). In some embodiments, the universal adapter primer has a different sequence than the universal PCR primer used in the disclosed methods.

[0041] Barcode Barcoding, e.g., probabilistic barcoding, is described, for example, in Fu et al., Proc Natl Acad Sci USA, 2011 May 31, 108(22):9026-31; U.S. Patent Application Publication No. 2011 / 0160078; Fan et al., Science, 2015 February 6, 347(6222):1258367; U.S. Patent Application Publication No. 2015 / 0299784; and International Publication No. WO2015 / 031691, the contents of each of which, including any supplementary or additional information or materials, are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein may be probabilistic barcodes, which may be polynucleotide sequences that can be used to stochastically label (e.g., barcode, tag) targets. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences in the stochastic barcode to the number of occurrences of any of the targets to be labeled can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, or can be approximately these values ​​or such numbers or ranges. The targets may be mRNA species that include mRNA molecules with identical or nearly identical sequences. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences of the stochastic barcode to the number of occurrences of any of the targets to be labeled is at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1. The barcode sequences of a stochastic barcode may be referred to as molecular labels.

[0042] A barcode, e.g., a stochastic barcode, can include one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. FIG. 1 shows an exemplary barcode 104 having a spatial label. The barcode 104 can include a 5' amine that can link the barcode to a solid support 105. The barcode can include a universal label, a dimensional label, a spatial label, a cell label, and / or a molecular label. The order of different labels (including, but not limited to, the universal label, the dimensional label, the spatial label, the cell label, and the molecular label) within the barcode can vary. For example, as shown in FIG. 1, the universal label can be the 5'-most label and the molecular label can be the 3'-most label. The spatial label, the dimensional label, and the cell label can be in any order. In some embodiments, the universal label, the spatial label, the dimensional label, the cell label, and the molecular label are in any order. The barcode can include a target binding region. The target binding region can interact with a target (e.g., a target nucleic acid, RNA, mRNA, DNA) in a sample. For example, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of an mRNA. In some cases, the labels of a barcode (e.g., a universal label, a dimensional label, a spatial label, a cellular label, and a barcode sequence) can be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides apart.

[0043] Labels, e.g., cellular labels, can include a set of unique nucleic acid subsequences of defined length, e.g., seven nucleotides each (equivalent to the number of bits used in some Hamming error-correcting codes), that can be designed to provide error-correcting capabilities. An error-correcting subsequence set including seven-nucleotide sequences can be designed so that any pairwise combination of sequences in the set exhibits a defined "genetic distance" (or number of mismatched bases); for example, an error-correcting subsequence set can be designed to exhibit a genetic distance of three nucleotides. In this case, consideration of the error-correcting sequences in a sequence dataset of a labeled target nucleic acid molecule (described in more detail below) can enable detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequences used to create the error-correcting code can vary, e.g., be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or a number or range between or about any two of these values. In some embodiments, nucleic acid subsequences of other lengths may be used to create error-correcting codes.

[0044] The barcode may include a target binding region. The target binding region may interact with a target in the sample. The target may be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNAs each including a poly(A) tail, or any combination thereof. In some embodiments, the multiple targets may include deoxyribonucleic acid (DNA).

[0045] In some embodiments, the target binding region may include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more of the labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence (e.g., molecular label)) may be separated from another one or two of the remaining labels of the barcode by a spacer. The spacer may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, none of the labels of the barcode are separated by a spacer.

[0046] Universal Signage A barcode may include one or more universal labels. In some embodiments, the one or more universal labels may be the same for all barcodes in a set of barcodes bound to a given solid support. In some embodiments, the one or more universal labels may be the same for all barcodes bound to a plurality of beads. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer. The sequencing primer can be used to sequence barcodes that include a universal label. The sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a PCR primer. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer and a PCR primer. The nucleic acid sequence of a universal label that can hybridize to a sequencing primer or a PCR primer may be referred to as a primer binding site. The universal label may include a sequence that can be used to initiate transcription of the barcode. A universal label can include a sequence that can be used to extend a barcode or a region within the barcode. A universal label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between or about any two of these values. For example, a universal label can include at least about 10 nucleotides. A universal label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or modified nucleotides can be part of the universal label sequence to allow the barcode to be cleaved from the support.

[0047] dimensional indicator A barcode may include one or more dimensional labels. In some embodiments, a dimensional label may include a nucleic acid sequence that provides information about the dimension in which labeling (e.g., stochastic labeling) occurred. For example, a dimensional label can provide information about the time a target was barcoded. A dimensional label may be associated with the time of barcoding (e.g., stochastic barcoding) in a sample. A dimensional label may be activated at the time of labeling. Different dimensional labels may be activated at different time points. A dimensional label provides information about the order in which a target, a group of targets, and / or a sample was barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. Cells may be pulsed again with a barcode (e.g., a stochastic barcode) in the G1 phase of the cell cycle. Cells may be pulsed again with a barcode in the S phase of the cell cycle, and so on. The barcode in each pulse (e.g., each stage of the cell cycle) may include a different dimensional label. In this way, the dimensional label provides information about which targets were labeled at which stage of the cell cycle. Dimensional labeling can examine many different biological time periods. Exemplary biological time periods can include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a cell population) can be stochastically labeled before and / or after treatment with a drug and / or therapy. Changes in copy number of distinct targets can indicate the sample's response to the drug and / or therapy.

[0048] Dimensional labels may be activatable. Activatable dimensional labels can be activated at a specific time. Activatable labels can, for example, be continuously activated (e.g., not turned off). Activatable dimensional labels can, for example, be reversibly activatable (e.g., they can be turned on and turned off). Dimensional labels can be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Dimensional labels can be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, dimensional labels can be activated by fluorescence, light, chemical events (e.g., cleavage, ligation of another molecule, addition of a modification (e.g., pegylation, sumoylation, acetylation, deacetylation, demethylation), photochemical events (e.g., photocaging), and introduction of unnatural nucleotides.

[0049] In some embodiments, the dimension labels may be the same for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., a bead), but may be different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 60% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 95% of the barcodes on the same solid support may comprise the same dimension label. On multiple solid supports (e.g., beads), 6Many unique dimension label sequences, even more than 10, may be presented. Dimension labels can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Dimension labels can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Dimension labels can comprise from about 5 to about 200 nucleotides. Dimension labels can comprise from about 10 to about 150 nucleotides. Dimension labels can comprise from about 20 to about 125 nucleotides in length.

[0050] spatial sign A barcode may include one or more spatial labels. In some embodiments, a spatial label may include a nucleic acid sequence that provides information about the spatial orientation of a target molecule associated with the barcode. A spatial label may be associated with a coordinate in a sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed relative to a substrate. A spatial label may reference a two-dimensional or three-dimensional grid. The coordinate may be fixed relative to a landmark. The landmark may be identifiable in space. The landmark may be a structure that can be imaged. The landmark may be a biological structure, e.g., an anatomical landmark. The landmark may be a cellular landmark, e.g., an organelle. The landmark may be a non-natural landmark, e.g., an identifiable identifier, e.g., a color code, a barcode, a magnetic property, fluorescence, radioactivity, or a structure with a unique size or shape. A spatial label may be associated with a physical compartment (e.g., a well, a container, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more locations in space.

[0051] Spatial labels may be the same for all barcodes attached to a given solid support (e.g., beads), but may be different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values ​​or such numbers or ranges. In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support may contain the same spatial label. In some embodiments, at least 95% of the barcodes on the same solid support may contain the same spatial label.

[0052] On multiple solid supports (e.g., beads), 6 Many unique spatial marker sequences, even more than 10, may be presented. Spatial markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Spatial markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Spatial markers can comprise between about 5 and about 200 nucleotides. Spatial markers can comprise between about 10 and about 150 nucleotides. Spatial markers can comprise between about 20 and about 125 nucleotides in length.

[0053] cell labeling A barcode (e.g., a stochastic barcode) may include one or more cell labels. In some embodiments, the cell label may include a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell. In some embodiments, the cell label is the same for all barcodes attached to a given solid support (e.g., a bead) but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of the barcodes on the same solid support may contain the same cell label. As another example, at least 95% of the barcodes on the same solid support may contain the same cell label. On multiple solid supports (e.g., beads), 6 Many unique cell marker sequences, even more than 10, may be presented. Cell markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Cell markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. For example, a cell marker can comprise between about 5 and about 200 nucleotides. As another example, a cell marker can comprise between about 10 and about 150 nucleotides. As yet another example, a cell marker can comprise between about 20 and about 125 nucleotides in length.

[0054] Barcode sequence The barcode may include one or more barcode sequences. In some embodiments, the barcode sequence may include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. The barcode sequence includes a nucleic acid sequence that provides a counter (e.g., provides a rough approximation) for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target binding region).

[0055] In some embodiments, a diverse set of barcode sequences is attached to a given solid support (e.g., a bead). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 There may be at least 10, or a number or range between, or about, any two of these values, or such number or range of unique molecular label sequences. For example, the plurality of barcodes may include about 6561 barcode sequences having distinct sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences having distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There may be multiple unique barcode sequences. The unique molecular beacon sequences may be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular beacon sequences are partially or entirely encompassed by a particle (e.g., a hydrogel bead).

[0056] The length of the barcode may vary in different implementations. For example, the barcode may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, or may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, in length. As another example, the barcode may be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0057] molecular label A barcode (e.g., a stochastic barcode) can include one or more molecular labels. A molecular label can include a barcode sequence. In some embodiments, a molecular label can include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. A molecular label includes a nucleic acid sequence that provides a counter for the specific occurrence of a target nucleic acid species hybridized to the barcode (e.g., a target binding region). In some embodiments, a diverse set of molecular labels is attached to a given solid support (e.g., beads). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 or a number or range between any two of these values, or about 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9There may be at least 10 unique molecular label sequences, or a number or range between any two of these values. For example, the plurality of barcodes may include about 6561 molecular labels with distinct sequences. As another example, the plurality of barcodes may include about 65536 molecular labels with distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There can be a number of unique molecular beacon sequences. A barcode with a unique molecular beacon sequence can be attached to a given solid support (e.g., a bead).

[0058] For barcoding using multiple stochastic barcodes (e.g., stochastic barcoding), the ratio of the number of distinct molecular label sequences to the number of occurrences of any of the targets can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, or may be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target may be an mRNA species that includes mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular beacon sequences to the number of occurrences of any of the targets is at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0059] A molecular label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or a number or range of nucleotides approximately equal to or equal to these values ​​or such number or range. A molecular label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0060] Target binding region The barcode may comprise one or more target binding regions, e.g., capture probes. In some embodiments, the target binding region may hybridize with a target of interest. In some embodiments, the target binding region may comprise a nucleic acid sequence that specifically hybridizes to a target (e.g., a target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), e.g., a specific gene sequence. In some embodiments, the target binding region may comprise a nucleic acid sequence that can bind (e.g., hybridize) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region may comprise a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang.

[0061] In some embodiments, the target binding region may comprise a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence may refer to a sequence that can bind to multiple target nucleic acids independently of the specific sequence of the target nucleic acid. For example, the target binding region may comprise a random multimer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, or a combination thereof. For example, the target binding region may be an oligo(dT) sequence that hybridizes to a poly(A) tail on an mRNA molecule. The random multimer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or any longer multimer sequence of any length. In some embodiments, the target binding region is the same for all barcodes bound to a given bead. In some embodiments, the target binding regions of multiple barcodes bound to a given bead may comprise two or more different target binding sequences. The target binding region can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. The target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or more. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail. The barcode can include a target binding region with a poly(dG) tail. Upon base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues replication to the 5' end of the barcode. In doing so, the resulting cDNA molecules contain a barcode sequence (eg, a molecular tag) on ​​the 3' end of the cDNA molecule.

[0062] In some embodiments, the target binding region may comprise an oligo(dT) capable of hybridizing to an mRNA containing a polyadenylated end. The target binding region may be gene-specific. For example, the target binding region may be configured to hybridize to a specific region of the target. The target binding region may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a number or range between any two of these values, or approximately these values ​​or such number or range, in length. The target binding region can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The target binding region can be about 5-30 nucleotides in length. When a barcode includes a gene-specific target binding region, the barcode may be referred to herein as a gene-specific barcode.

[0063] Orientation Characteristics A stochastic barcode (e.g., a stochastic barcode) may include one or more orientation properties that can be used to orient (e.g., align) the barcode. The barcode may include a moiety for isoelectric focusing. Different barcodes may include different isoelectric focusing points. When these barcodes are introduced into a sample, the sample may undergo isoelectric focusing to orient the barcodes in a known manner. In this manner, the orientation properties can be used to develop a known map of the barcodes in the sample. Exemplary orientation properties can include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, a barcode with an orientation property for self-assembly may self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0064] affinity properties A barcode (e.g., a stochastic barcode) can include one or more affinity features. For example, a spatial label can include an affinity feature. Affinity features can include chemical and / or biological moieties that can facilitate binding of the barcode to another entity (e.g., a cellular receptor). For example, an affinity feature can include an antibody, e.g., an antibody specific to a particular moiety (e.g., a receptor) on a sample. In some embodiments, the antibody can direct the barcode to a particular cell type or molecule. Targets on and / or near a particular cell type or molecule can be labeled (e.g., stochastically labeled). In some embodiments, the affinity feature can provide spatial information in addition to the nucleotide sequence of the spatial label, as the antibody can direct the barcode to a specific location. The antibody can be a therapeutic antibody, e.g., a monoclonal or polyclonal antibody. The antibody can be humanized or chimeric. The antibody can be a naked antibody or a fusion antibody.

[0065] An antibody can be a full-length (i.e., naturally occurring or formed by conventional immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., an IgG antibody), or an immunologically active (i.e., specific binding) portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment can be, for example, a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, an antibody fragment can bind to the same antigen recognized by the full-length antibody. Antibody fragments can include isolated fragments consisting of the variable regions of an antibody, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are connected by a peptide linker ("scFv protein"). Exemplary antibodies can include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0066] Universal Adapter Primer A barcode may include one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, may include a universal adapter primer. A universal adapter primer may refer to a nucleotide sequence that is universal across all barcodes. A universal adapter primer can be used to construct a gene-specific barcode. A universal adapter primer may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides in length, or a number or range between any two of these, or approximately these values ​​or such number or range of nucleotides. The universal adapter primer can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The universal adapter primer can be 5 to 30 nucleotides in length.

[0067] Linker When a barcode includes more than one type of label (e.g., more than one cellular label or more than one barcode sequence, e.g., one molecular label), the labels may be interspersed with linker label sequences. The linker label sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. The linker label sequence may be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. In some cases, the linker label sequence is 12 nucleotides in length. The linker label sequence may be used to facilitate synthesis of the barcode. The linker label may include an error-correcting (e.g., Hamming) code.

[0068] solid support In some embodiments, the barcodes disclosed herein, e.g., stochastic barcodes, may be associated with a solid support. The solid support may be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences, e.g., molecular labels of stochastic barcodes (e.g., first barcode sequences) of a plurality of barcodes (e.g., a first plurality of barcodes) on a solid support, differ by at least one nucleotide. Cell labels of barcodes on the same solid support may be the same. Cell labels of barcodes on different solid supports may differ by at least one nucleotide. For example, a first cell label of a first plurality of barcodes on a first solid support may have the same sequence, and a second cell label of a second plurality of barcodes on a second solid support may have the same sequence. A first cell label of a first plurality of barcodes on a first solid support and a second cell label of a second plurality of barcodes on a second solid support may differ by at least one nucleotide. Cell labels may be, for example, about 5 to 20 nucleotides in length. The barcode sequence can be, for example, about 5-20 nucleotides in length. The synthetic particle can be, for example, a bead.

[0069] The beads can be, for example, silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic materials, ceramic, plastic, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof. In some embodiments, the beads can be polymer beads, such as deformable beads or gel beads (e.g., gel beads from 10X Genomics (San Francisco, CA)) functionalized with barcodes or stochastic barcodes. In some implementations, the gel beads can comprise a polymer-based gel. Gel beads can be generated, for example, by encapsulating one or more polymer precursors into droplets. Gel beads can be generated when the polymer precursors are exposed to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0070] In some embodiments, the particles may be disintegrable (e.g., dissolvable, degradable). For example, polymer beads may dissolve, melt, or decompose under desired conditions. The desired conditions may include environmental conditions. The desired conditions may result in the dissolution, melting, or decomposition of the polymer beads in a controlled manner. Gel beads may dissolve, melt, or decompose due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electrical stimulus, a light stimulus, or any combination thereof.

[0071] Analytes and / or reagents, e.g., oligonucleotide barcodes, may be linked / immobilized, for example, to the interior surface of a gel bead (e.g., the interior accessible through diffusion of the oligonucleotide barcodes and / or the material used to generate the oligonucleotide barcodes) and / or to the exterior surface of a gel bead or any other microcapsule described herein. Linkage / immobilization may be via any form of chemical bond (e.g., covalent bond, ionic bond) or physical phenomenon (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the linkage / immobilization of reagents to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical crosslinker, including those described herein). Upon application of a stimulus, the labile moiety can be cleaved, releasing the immobilized reagent. In some embodiments, the labile moiety is a disulfide bond. For example, in cases where an oligonucleotide barcode is immobilized to a gel bead via a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the bead. The labile moiety may be included as part of the gel bead or microcapsule, as part of a chemical linker connecting the reagent or analyte to the gel bead or microcapsule, and / or as part of the reagent or analyte. In some embodiments, at least one barcode of the plurality of barcodes may be immobilized to the particle, partially immobilized to the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof.

[0072] In some embodiments, the gel beads may comprise a wide variety of different polymers, including, but not limited to, polymers, thermosensitive polymers, light-sensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. Polymers can include, but are not limited to, materials such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethylammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthalaldehyde) (PTHF), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic-co-glycolic acid) (PLGA).

[0073] A number of chemical stimuli can be used to trigger bead collapse, dissolution, or degradation. Examples of these chemical changes include, but are not limited to, pH-mediated changes to the bead wall, bead wall collapse via chemical cleavage of cross-links, triggering bead wall depolymerization, and bead wall switching reactions. Bulk changes can also be used to trigger bead collapse. Bulk or physical changes to microcapsules through various stimuli also offer many advantages in designing capsules for releasing reagents. Bulk or physical changes occur on a macroscopic scale, with bead rupture being the result of mechanical-physical forces induced by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in bead wall porosity.

[0074] Biological stimuli can also be used to trigger bead disintegration, dissolution, or degradation. Generally, biological triggers resemble chemical triggers, but many examples use biomolecules, or molecules commonly found in biological systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, and the like. For example, beads can contain polymers with peptide crosslinks that are susceptible to cleavage by specific proteases. More specifically, one example can include microcapsules containing GFLGK peptide crosslinks. Addition of a biological trigger, such as the protease cathepsin B, cleaves the peptide crosslinks in the shell wall, releasing the contents of the bead. In other cases, the protease can be heat-activated. In another example, beads contain a shell wall containing cellulose. Addition of the hydrolytic enzyme chitosan serves as a biological trigger for cleavage of the cellulose bonds, depolymerization of the shell wall, and release of its contents.

[0075] Beads can also be induced to release their contents upon application of a thermal stimulus. A change in temperature can cause various changes in the beads. A change in heat can cause the beads to melt, causing the bead walls to collapse. In other cases, heat can increase the internal pressure of the beads' internal components, causing the beads to collapse or explode. In still other cases, heat can transform the beads into a compressed, dehydrated state. Heat can also act on heat-sensitive polymers within the bead walls, causing the beads to collapse. The inclusion of magnetic nanoparticles in the bead walls of microcapsules can trigger the collapse of the beads as well as guide the beads in an array. The devices of the present disclosure can include magnetic beads for either purpose. In one example, the incorporation of Fe3O4 nanoparticles into polyelectrolyte-containing beads triggers collapse in the presence of an oscillating magnetic field stimulus.

[0076] Beads can also be disintegrated, dissolved, or decomposed as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrically sensitive beads can trigger both bead disintegration and other functions, such as alignment in an electric field, electrical conduction, or redox reactions. In one example, beads containing electrically sensitive materials are aligned in an electric field to control the release of internal reagents. In another example, an electric field can induce redox reactions within the bead wall itself, which can increase porosity. Light stimulation can also be used to disrupt the beads. Numerous optical triggers are possible, including systems using various molecules such as nanoparticles and chromophores that can absorb photons of specific wavelengths. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of SiO2-coated polyelectrolyte capsules can result in the collapse of the bead wall. In yet another example, photoswitchable materials, such as azobenzene groups, can be incorporated into the bead wall. Upon application of UV or visible light, chemicals such as these absorb photons and undergo reversible cis-to-trans isomerization. In this embodiment, the incorporation of a photoswitch results in a bead wall that can collapse or become more porous upon application of a light trigger.

[0077] For example, in a non-limiting example of barcoding (e.g., stochastic barcoding) shown in FIG. 2, after cells, e.g., single cells, are introduced into multiple microwells of a microwell array in block 208, beads can be introduced into multiple microwells of the microwell array in block 212. Each microwell can contain one bead. The beads can contain multiple barcodes. The barcodes can include 5' amine regions attached to the beads. The barcodes can include a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0078] The barcodes disclosed herein may be associated with (e.g., attached to) a solid support (e.g., a bead). The barcodes associated with the solid support may comprise a barcode sequence selected from a group comprising at least 100 or 1000 barcode sequences, each having a unique sequence. In some embodiments, different barcodes associated with a solid support may comprise barcodes with different sequences. In some embodiments, a percentage of the barcodes associated with a solid support comprise the same cell label. For example, the percentage may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values ​​or such a number or range. As another example, the percentage may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support may have the same cell label. Barcodes associated with different solid supports may have different cell labels selected from a group comprising at least 100 or 1000 cell labels having unique sequences.

[0079] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a bead). In some embodiments, barcoding a plurality of labels in a sample can be performed using a solid support comprising a plurality of synthetic particles associated with a plurality of barcodes. In some embodiments, the solid support may comprise a plurality of synthetic particles associated with a plurality of barcodes. The spatial labeling of the plurality of barcodes on different solid supports may differ by at least one nucleotide. The solid support may comprise a plurality of barcodes, for example, in two or three dimensions. The synthetic particles may be beads. The beads may be silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support may include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support may be free-floating. In some embodiments, the solid support may be embedded in a semi-solid or solid array. The barcodes may not be associated with a solid support. The barcodes may be individual nucleotides. The barcode may be associated with the substrate.

[0080] As used herein, the terms "tethered," "attached," and "immobilized" are used interchangeably and can refer to covalent or non-covalent means for attaching a barcode to a solid support. Any of a variety of different solid supports can be used to attach pre-synthesized barcodes or as a solid support for in situ solid phase synthesis of barcodes. In some embodiments, the solid support is a bead. Beads may include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures to which nucleic acids can be immobilized (e.g., covalently or non-covalently). Beads may be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. Beads may be or include discrete particles that are spherical (e.g., microspheres), or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. In some embodiments, beads may be non-spherical in shape.

[0081] The beads may comprise a variety of materials, including, but not limited to, paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe3O4, magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof. In some embodiments, the beads (e.g., the beads to which the label is attached) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0082] Some embodiments disclosed herein include one or more particles (e.g., beads). Each of the particles may include a plurality of oligonucleotides (e.g., barcodes). Each of the plurality of oligonucleotides may include a barcode sequence (e.g., a molecular label sequence), a cell label, and a target binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequence of each of the plurality of oligonucleotides may be the same. The cell label sequences of oligonucleotides on different particles may be different so that the oligonucleotides on different particles can be identified. The number of different cell label sequences may vary in different implementations. In some embodiments, the number of cell labeling sequences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 In some embodiments, the number of cell labeling sequences may be at least or up to 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the plurality of particles comprise oligonucleotides having the same cellular sequence. In some embodiments, up to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of the plurality of particles comprise oligonucleotides with the same cellular sequence, hi some embodiments, none of the plurality of particles have the same cellular targeting sequence.

[0083] The multiple oligonucleotides on each particle can include different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9In some embodiments, the number of barcode sequences may be at least or at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 For example, at least 100 of the plurality of oligonucleotides comprise different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or range between any two of these values, or more of the plurality of oligonucleotides comprise different barcode sequences. Some embodiments provide a plurality of particles comprising barcodes. In some embodiments, the ratio of occurrences (or copies or numbers) of targets to be labeled to distinct barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particles can be, for example, nanoparticles or microparticles.

[0084] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or range between any two of these values, or can be approximately these values ​​or such numbers or ranges. The diameter of the beads may be related to the diameter of the wells of the substrate. In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, longer or shorter than the diameter of the wells, or may be approximately these values ​​or any such number or range, longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or range between any two of these values, longer or shorter than the diameter of the cells, or may be approximately these values ​​or any such number or range, longer or shorter than the diameter of the cells. In some embodiments, the diameter of the beads may be at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the cells.

[0085] The beads may be bound and / or embedded in a substrate. The beads may be bound and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial location of the beads within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial labels present in barcodes on the beads, which can serve as locational addresses. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag™ carboxyl-terminated magnetic beads.

[0086] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one fluorescent optical channel or multiple optical channels. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be identifiable. For example, the beads can be imaged using a camera. The beads can have a detectable code associated with them. For example, the beads can include a barcode. The beads can change size, for example, due to swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible. The solid support (e.g., beads) can be visualized. The solid support can include a visualization tag (e.g., a fluorescent dye). The solid support (e.g., beads) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging of the beads.

[0087] A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as "functionalized" if it includes a linker, scaffold, building block, or other reactive moiety attached thereto, while a solid support can be "non-functionalized" if it lacks such reactive moieties attached thereto. A solid support can be freely used in solution, e.g., in a microtiter well format, in a flow-through format, e.g., in a column, or in a dipstick.

[0088] The solid support may comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. The solid support may take the form of a resin, gel, microsphere, or other geometric configuration. The solid support may comprise a silica chip, microparticle, nanoparticle, plate, array, caliper, flat support, such as a glass fiber filter, glass surface, metal surface, metal surface (steel, gold silver, aluminum, silicone, and copper), glass support, plastic support, silicone support, chip, filter, membrane, microwell plate, slide, multiwell plate, or plastic material (e.g., formed from polyethylene, polypropylene, polyamide, polyvinylidene difluoride), including membrane, and / or wafer, comb, pin, or needle (e.g., an array of pins suitable for combinatorial synthesis or analysis), or an array of holes or nanoliter wells on a flat surface, such as a wafer (e.g., a silicone wafer), a wafer with holes or without a filter bottom, or beads. The solid support may comprise a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix may be capable of penetrating intracellular spaces (e.g., around organelles). The polymer matrix may be capable of being pumped through the circulatory system.

[0089] Substrates and Microwell Arrays As used herein, a substrate may refer to a type of solid support. A substrate may refer to a solid support that may include a barcode or stochastic barcode of the present disclosure. A substrate may include, for example, a plurality of microwells. For example, a substrate may be a well array including two or more microwells. In some embodiments, a microwell may include a small reaction chamber having a defined volume. In some embodiments, a microwell may incorporate one or more cells. In some embodiments, a microwell may incorporate only one cell. In some embodiments, a microwell may incorporate one or more solid supports. In some embodiments, a microwell may incorporate only one solid support. In some embodiments, a microwell incorporates a single cell and a single solid support (e.g., a bead). A microwell may include a barcode reagent of the present disclosure.

[0090] Barcoding methods The present disclosure provides methods for estimating the number of distinct targets in distinct locations of a body sample (e.g., tissue, organ, tumor, cell). The method may include placing a barcode (e.g., a stochastic barcode) in proximity to the sample, lysing the sample, associating distinct targets with the barcode, amplifying the targets, and / or digitally counting the targets. The method may further include analyzing and / or visualizing information obtained from the spatial labeling of the barcode. In some embodiments, the method includes visualizing a plurality of targets in the sample. Mapping the plurality of targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps may be created before or after barcoding (e.g., stochastically barcoding) the plurality of targets in the sample. Visualizing a plurality of targets in the sample may include mapping the plurality of targets to a map of the sample. Mapping the plurality of targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps can be generated before or after barcoding multiple targets in a sample. In some embodiments, the two-dimensional and three-dimensional maps can be generated before or after lysing the sample. Lysing the sample before or after generating the two-dimensional or three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0091] In some embodiments, barcoding the plurality of targets comprises hybridizing a plurality of barcodes to the plurality of targets to create barcoded targets (e.g., stochastically barcoded targets). Barcoding the plurality of targets may comprise generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets may be performed using a solid support comprising a plurality of barcodes (e.g., stochastic barcodes).

[0092] Contacting the sample with the barcode The present disclosure provides methods for contacting a sample (e.g., cells) with a substrate of the present disclosure. For example, a sample including cells, an organ, or a thin section of tissue can be contacted with a barcode (e.g., a stochastic barcode). For example, the cells can be contacted by gravity flow, where they can settle and form a monolayer. The sample can be a tissue slice. The slice can be disposed on a substrate. The sample can be one-dimensional (e.g., forming a planar surface). For example, the sample (e.g., cells) can be spread across the substrate by growing / culturing the cells on the substrate. When the barcode is in close proximity to the target, the target can hybridize to the barcode. The barcodes can be contacted in a non-depleting ratio so that each distinct target can associate with a distinct barcode of the present disclosure. To ensure efficient association between the target and the barcode, the target can be cross-linked to the barcode.

[0093] Cell lysis After partitioning the cells and barcodes, the cells can be lysed to liberate the target molecules. Cell lysis can be achieved by any of a variety of means, such as chemical or biochemical means, osmotic shock, or thermal, mechanical, or optical lysis. Cells can also be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, Li-dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of the target with the barcode, the diffusion rate of the target molecule can be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0094] In some embodiments, the sample may be lysed using filter paper, which can be soaked with a lysis buffer over the filter paper, and pressure can be applied to the sample, which can promote lysis of the sample and hybridization of the sample's targets to the substrate. In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris-HCl. The lysis buffer can include at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include up to about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include about 0.1 M Tris-HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may include a salt (e.g., LiCl). The salt concentration in the lysis buffer may be at least about 0.1, 0.5, or 1 M or higher. The salt concentration in the lysis buffer may be up to about 0.1, 0.5, or 1 M or higher. In some embodiments, the salt concentration in the lysis buffer is about 0.5 M. The lysis buffer may include a detergent (e.g., SDS, Li-dodecyl sulfate, triton X, tween, NP-40). The concentration of the detergent in the lysis buffer may be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. The concentration of detergent in the lysis buffer may be up to about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. In some embodiments, the concentration of detergent in the lysis buffer is about 1% Li-dodecyl sulfate. The time used in the lysis method may depend on the amount of detergent used. In some embodiments, the more detergent used, the less time is required for lysis.The lysis buffer may include a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of the chelating agent in the lysis buffer may be up to about 1, 5, 10, 15, 20, 25, or 30 mM or more. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer may include a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer may be up to about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of the reducing reagent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may comprise about 0.1 M Tris-HCl (about pH 7.5), about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0095] Lysing can be performed at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysing can be performed for about 1, 5, 10, 15, 20 minutes, or longer. Lysed cells may contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules. Lysed cells may contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules.

[0096] Attaching the barcode to the target nucleic acid molecule After cell lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with the barcodes on the co-localized solid support. Association may involve hybridization of the target recognition region of the barcode to a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode may interact with the poly(A) tail of the target). Assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) used for hybridization may be selected to promote the formation of specific, stable hybrids. In some embodiments, nucleic acid molecules released from lysed cells may associate with multiple probes on a substrate (e.g., hybridize to the probes on a substrate). If the probes contain oligo(dT), mRNA molecules may hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide may act as a primer for first-strand synthesis of cDNA molecules. For example, in the non-limiting example of barcoding shown in block 216 of FIG. 2, mRNA molecules may hybridize to barcodes on beads. For example, a single-stranded nucleotide fragment can hybridize to the target binding region of the barcode.

[0097] The binding may further include ligating the target recognition region of the barcode with a portion of the target nucleic acid molecule. For example, the target binding region may include a nucleic acid sequence capable of specific hybridization to a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure may further include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to generate a restriction site overhang. The barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to connect the two fragments.

[0098] For example, in a non-limiting example of barcoding shown in block 220 of Figure 2, labeled targets (e.g., target-barcode molecules) from multiple cells (or multiple samples) can then be pooled, e.g., in a tube. For example, the labeled targets can be pooled by collecting beads to which barcodes and / or target-barcode molecules are bound. Solid support-based collection recovery of bound target-barcode molecules can be achieved through the use of magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can proceed within a single reaction vessel. Further processing can include, for example, reverse transcription, amplification, cleavage, dissociation, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling labeled target nucleic acid molecules from multiple cells.

[0099] Reverse transcription or nucleic acid extension The present disclosure provides methods for generating target-barcode conjugates using reverse transcription (e.g., block 224 of Figure 2) or nucleic acid extension. The target-barcode conjugates can include a barcode and a complementary sequence of all or a portion of a target nucleic acid (i.e., a barcoded cDNA molecule, e.g., a stochastically barcoded cDNA molecule). Reverse transcription of the associated RNA molecule can occur by adding a reverse transcription primer along with a reverse transcriptase. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo(dT) primer can be 12-18 nucleotides in length, or about 12-18 nucleotides in length, and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexanucleotide primer can bind to the mRNA at various complementary sites. The target-specific oligonucleotide primer typically selectively primes the mRNA of interest.

[0100] In some embodiments, reverse transcription of mRNA molecules into labeled RNA molecules can occur by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Typically, oligo(dT) primers are 12-18 nucleotides in length and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNAs. Random hexanucleotide primers can bind to mRNAs at various complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

[0101] In some embodiments, the target is a cDNA molecule. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase such as Moloney murine leukemia virus (MMLV) reverse transcriptase to generate a cDNA molecule with a poly(dC) tail. The barcode can include a target binding region with a poly(dG) tail. When base pairing occurs between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues replication to the 5' end of the barcode. In this way, the resulting cDNA molecule contains the barcode sequence (e.g., a molecular tag) on ​​the 3' end of the cDNA molecule. Reverse transcription can occur repeatedly to generate multiple labeled cDNA molecules. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methods can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0102] amplification One or more nucleic acid amplification reactions (e.g., block 228 of FIG. 2 ) can be performed to generate multiple copies of the labeled target nucleic acid molecule. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to add sequencing adapters to the nucleic acid molecule. The amplification reaction can include amplifying at least a portion of the sample label, if present. The amplification reaction can include amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction can include amplifying at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or a combination thereof. The amplification reaction may include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a range or number between any two of these values ​​of the plurality of nucleic acids. The method may further include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of the target-barcode molecule comprising the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0103] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for amplifying specific DNA sequences in vitro by simultaneous primer extension of complementary strands of DNA. As used herein, PCR can encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, and assembly PCR.

[0104] Amplification of labeled nucleic acids may include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiplex displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) methods, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and branched extension amplification (RAM). In some embodiments, amplification does not produce circularized transcripts.

[0105] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on the labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to generate labeled amplicons (e.g., stochastically labeled amplicons). The labeled amplicons may be double-stranded molecules. The double-stranded molecules may comprise double-stranded RNA molecules, double-stranded DNA molecules, or RNA molecules hybridized to DNA molecules. One or both strands of the double-stranded molecules may comprise a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicons may be single-stranded molecules. The single-stranded molecules may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure may include synthetic or modified nucleic acids.

[0106] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0107] Performing one or more amplification reactions may include the use of one or more primers. The one or more primers may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the multiple labeled targets (e.g., stochastically labeled targets). The one or more primers may anneal to the 3' or 5' ends of the multiple labeled targets. The one or more primers may anneal to an internal region of the multiple labeled targets. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the multiple labeled targets. The one or more primers may comprise a constant panel of primers. The one or more primers may include at least one or more custom primers, the one or more primers may include at least one or more control primers, or the one or more primers may include at least one or more gene-specific primers.

[0108] The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. The one or more primers may include a universal primer and a custom primer. The custom primers may be designed to amplify one or more targets. The targets may comprise a subset of all nucleic acids in one or more samples. The targets may comprise a subset of all labeled targets in one or more samples. The one or more primers may include at least 96 or more custom primers. The one or more primers may include at least 960 or more custom primers. The one or more primers may include at least 9600 or more custom primers. The one or more custom primers may anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids may correspond to one or more genes.

[0109] Any amplification scheme can be used in the disclosed methods. For example, in one scheme, a first PCR can amplify the molecules bound to the beads using a gene-specific primer and a primer for the sequence of universal Illumina sequencing primer 1. A second PCR can amplify the first PCR product using a nested gene-specific primer adjacent to the sequence of Illumina sequencing primer 2 and a primer for the sequence of universal Illumina sequencing primer 1. A third PCR adds P5 and P7 and a sample index to place the PCR product into an Illumina sequencing library. Sequencing using 150 bp x 2 sequencing can reveal cell label and barcode sequences (e.g., molecular labels) on read 1, genes on read 2, and a sample index on index 1 read.

[0110] In some embodiments, chemical cleavage can be used to remove nucleic acids from a substrate. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from a solid support. For example, enzymes can be used to remove nucleic acids from a substrate. For example, nucleic acids can be removed from a substrate by restriction endonuclease digestion. For example, nucleic acids containing dUTP or ddUTP can be removed from a substrate using uracil-d-glycosylase (UDG) treatment. For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, for example, apurinic / apyrimidinic (AP) endonucleases, can be used to remove nucleic acids from a substrate. In some embodiments, photocleavable groups and light can be used to remove nucleic acids from a substrate. In some embodiments, a cleavable linker can be used to remove nucleic acids from a substrate. For example, the cleavable linker can comprise at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-Protein A, a photolabile linker, an acid or base labile linker group, or an aptamer.

[0111] If the probe is gene-specific, the molecule can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, the nucleic acid can be amplified after it is synthesized (e.g., reverse transcribed). Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acid.

[0112] In some embodiments, amplification can be performed on the substrate using, for example, bridge amplification. Homopolymer tails can be added to cDNA to generate ends compatible with bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, a primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to solid particles. When a sample containing the template nucleic acid is contacted with the particles and a single thermal cycle is performed, the template molecule anneals to the first primer, and the first primer can be extended in the forward direction by adding nucleotides to form a double-stranded molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing stage of the subsequent annealing and extension step, the complementary strand can hybridize to a second primer complementary to the segment of the complementary strand at the position removed from the first primer. Through this hybridization, the complementary strand can form a bridge between the first and second primers, immobilized by covalent bonding to the first primer and by hybridization to the second primer. In the extension step, the second primer can be extended in the opposite direction by adding nucleotides to the same reaction mixture, thereby converting the bridge into a double-stranded bridge. The next cycle then begins, and the double-stranded bridge is denatured to yield two single-stranded nucleic acid molecules, each with one end bound to the particle surface via the first and second primers, and the other end unbound. In the annealing and extension step of this second cycle, each strand can hybridize to a previously unused additional complementary primer on the same particle to form a new single-stranded bridge. The two previously unused hybridized primers then extend, converting the two new bridges into double-stranded bridges.

[0113] The amplification reaction can include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids. Amplification of the labeled nucleic acid may include PCR-based or non-PCR-based methods. Amplification of the labeled nucleic acid may include exponential amplification of the labeled nucleic acid. Amplification of the labeled nucleic acid may include linear amplification of the labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, suppression PCR, semi-suppressive PCR, and assembly PCR.

[0114] In some embodiments, amplification of the labeled nucleic acid comprises a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ) method, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and / or branched extension amplification (RAM).

[0115] The methods disclosed herein may further include performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon may be a double-stranded molecule. The double-stranded molecule may comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule may comprise a sample tag or molecular identifier label. Alternatively, the amplicon may be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present invention may include synthetic or modified nucleic acids. In some embodiments, the methods include repeatedly amplifying a labeled nucleic acid to generate multiple amplicons. The methods disclosed herein may include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the methods include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0116] The amplification may further include adding one or more control nucleic acids to one or more samples containing the plurality of nucleic acids. The amplification may further include adding one or more control nucleic acids to the plurality of nucleic acids. The control nucleic acids may include a control label. Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile and / or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0117] Performing one or more amplification reactions may involve the use of one or more primers. The one or more primers may comprise one or more oligonucleotides. The one or more oligonucleotides may comprise at least about 7 to 9 nucleotides. The one or more oligonucleotides may comprise fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the plurality of labeled nucleic acids. The one or more primers may anneal to the 3' and / or 5' ends of the plurality of labeled nucleic acids. The one or more primers may anneal to an internal region of the plurality of labeled nucleic acids. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the plurality of labeled nucleic acids. The one or more primers can comprise a fixed panel of primers. The one or more primers may include at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more housekeeping gene primers. The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. The one or more primers may include a universal primer and a custom primer. The custom primer may be designed to amplify one or more target nucleic acids. The target nucleic acids may comprise a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are probes bound to an array of the present disclosure.

[0118] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further includes generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of those targets. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) may differ from each other. Generating an indexed library of barcoded targets includes generating a plurality of indexed polynucleotides from the plurality of targets in the sample. For example, for an indexed library of barcoded targets including a first indexed target and a second indexed target, the labeled region of the first indexed polynucleotide may differ from the labeled region of the second indexed polynucleotide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range between, or about, or at least, or at most, any two of these values. In some embodiments, generating an indexed library of barcoded targets includes contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides comprising a poly(T) region and a label region, and performing first-strand synthesis using a reverse transcriptase to generate single-stranded, labeled cDNA molecules, each comprising a cDNA region and a label region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an indexed library of barcoded targets may further include amplifying the single-stranded, labeled cDNA molecules to generate double-stranded, labeled cDNA molecules, and performing nested PCR on the double-stranded, labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may include generating adapter-labeled amplicons.

[0119] Barcoding (e.g., stochastic barcoding) can include labeling individual nucleic acid (e.g., DNA or RNA) molecules with nucleic acid barcodes or tags. In some embodiments, this includes adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can minimize PCR amplification bias. Adapters can be added for sequencing, e.g., using next-generation sequencing (NGS). For example, sequencing results can be used to determine the sequence of cellular labels, molecular labels, and nucleotide fragments of one or more copies of the target in block 232 of FIG. 2.

[0120] 3 is a schematic diagram illustrating a non-limiting, exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), e.g., barcoded mRNAs or fragments thereof. As shown in step 1, a reverse transcription process can encode each mRNA molecule containing a unique molecular label sequence, a cellular label sequence, and a universal PCR site. In particular, the RNA molecule 302 can be reverse transcribed to generate labeled cDNA molecules 304 containing cDNA regions 306 by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to a poly(A) tail region 308 of the RNA molecule 302. Each of the barcodes 310 can include a target binding region, e.g., a poly(dT) region 312, a label region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0121] In some embodiments, the cell label sequence may comprise 3 to 20 nucleotides. In some embodiments, the molecular label sequence may comprise 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cell label, wherein the universal label is the same for the plurality of stochastic barcodes on the solid support and the cell label is the same for the plurality of stochastic barcodes on the solid support. In some embodiments, the universal label may comprise 3 to 20 nucleotides. In some embodiments, the cell label comprises 3 to 20 nucleotides.

[0122] In some embodiments, label region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, label region 314 may include one or more of a universal label, a dimensional label, and a cell label. Barcode sequence or molecular label 318 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between any of these values, in length, or may be approximately, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in length between any of these values. A cell label 320 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between, or may be approximately, or may be at least, or may be up to, these values ​​or such number or range of nucleotides in length. A universal label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between, or may be approximately, or may be at least, or may be up to, these values ​​or such number or range of nucleotides in length. The universal label may be the same for multiple stochastic barcodes on a solid support, and the cell label may be the same for multiple stochastic barcodes on a solid support.A dimension marker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between any of these values, in length, or may be approximately, at least, or at most, these values ​​or numbers or ranges of nucleotides in length.

[0123] In some embodiments, label region 314 may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include approximately these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include at least these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include up to these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320. Each label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range between any of these values, in length, or may be approximately, at least, or at most these values ​​or number or range of nucleotides in length. A set of barcodes or stochastic barcodes 310 may be 10, 20, 40, 50, 70, 80, 90, 100, or a number or range between any of these values. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 , or a number or range of barcodes or stochastic barcodes 310 between any of these values, or about these values ​​or such number or range of barcodes or stochastic barcodes 310, or at least these values ​​or such number or range of barcodes or stochastic barcodes 310, or at most these values ​​or such number or range of barcodes or stochastic barcodes 310. The set of barcodes or stochastic barcodes 310 may also each contain, for example, a unique labeled region 314. The labeled cDNA molecules 304 may be purified to remove excess barcodes or stochastic barcodes 310. Purification may include Ampure bead purification.

[0124] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled in one tube and PCR amplified using a first pool of PCR primers and a first universal PCR primer. Pooling is possible due to the unique label region 314. In particular, the labeled cDNA molecules 304 can be amplified to generate nested PCR-labeled amplicons 322. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, the multiplex PCR amplification can be performed using 10, 20, 40, 50, 70, 80, 90, 10, 25, 30, 45, 50, 60, 75, 80, 90, 100, 150, 250, 300, 450, 500, 600, 750, 800, 900, 1500, 1500, 2500, 3000, 4500, 5000, 6000, 15000, 25000, 30000, 45000, 50000, 50000, 60000, 75000, 8000, 9000, 15000, 15000, 15000, 25000, 30000, 45000, 50000, 15000, 25000, 30000, 45000, 50000, 60000, 150000, 250000, 300000, 450000, 50000, 60000, 150000, 25000 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 1013 , 10 14 , 10 15 , 10 20 The amplification may utilize a number or range of multiplex primers between, or between, or at about, or at least, or at most. The amplification may include a first PCR primer pool 324 including custom primers 326A-C targeting specific genes and a universal primer 328. The custom primer 326 may hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. The universal primer 328 may hybridize to the universal PCR region 316 of the labeled cDNA molecule 304.

[0125] As shown in step 3 of Figure 3, the product from the PCR amplification in step 2 can be amplified using a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, nested PCR-labeled amplicons 322 can be further amplified by nested PCR. Nested PCR can include multiplex PCR including a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328' in a single reaction volume. The nested PCR primer pool 328 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, of different nested PCR primers 330, or may contain about, or at least, or at most, of different nested PCR primers 330. The nested PCR primers 332 may contain an adaptor 334 and hybridize to a region within the cDNA portion 306" of the labeled amplicon 322. Universal primer 328' contains adapter 336 and can hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 generates adapter-labeled amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328' may not contain adapters 334 and 336. Instead, adapters 334 and 336 can ligate to the product of the nested PCR to generate adapter-labeled amplicon 338.

[0126] As shown in step 4, the PCR products from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 334 and 336 can be used to perform one or more additional assays on adapter-labeled amplicons 338. Adapters 334 and 336 can be hybridized with primers 340 and 342. One or more of primers 340 and 342 can be PCR amplification primers. One or more of primers 340 and 342 can be sequencing primers. One or more of adapters 334 and 336 can be used for further amplification of adapter-labeled amplicons 338. One or more of adapters 334 and 336 can be used for sequencing of adapter-labeled amplicons 338. Primer 342 can contain a plate index 344, which allows amplicons generated using the same set of barcodes or stochastic barcodes 310 to be sequenced in a single sequencing reaction using next-generation sequencing (NGS).

[0127] PNA blockers Some embodiments provided herein provide peptide nucleic acid (PNA) blockers for selective library amplification. The disclosure herein includes methods for selectively blocking reverse transcription and / or PCR amplification using peptide nucleic acids (PNAs) in single-cell multi-omics library generation (e.g., Rhapsody). In some embodiments, the blockers provided herein can be used to block AbSeq oligo extension from AbSeq oligos, while allowing extension in other directions in molecular barcoding approaches. PNAs can bind to DNA through sequence complementarity and may have stronger affinity due to the neutral charge of the peptide backbone. PNAs can be highly specific due to the precise distance between each base. Therefore, the PNAs provided herein can selectively block PCR amplification or reverse transcription. Reverse transcriptase has strong strand displacement activity, which can remove conventional DNA blockers; in some embodiments provided herein, PNAs are not removed due to their higher affinity. PNAs are DNA mimics with an uncharged backbone rather than a completely negatively charged sugar-phosphate backbone. PNAs can be easily conjugated to peptides or fluorescent dyes via peptide bonds. Due to precise intramolecular spacing and hybridization between complementary nucleic acids, PNAs can exhibit high sequence selectivity and affinity for DNA or RNA molecules. Because PNAs have an uncharged backbone, they can bind to negatively charged DNA or RNA without any electrostatic repulsion, thus exhibiting much higher binding affinity. In some embodiments, the melting temperature is approximately 1°C higher per base pair compared to DNA / DNA or DNA / RNA molecules, and this higher thermal stability and improved hybridization properties can be independent of the salt concentration present.

[0128] In some embodiments, methods are provided that include superloading (e.g., as described in Cao et al., Science 2017, incorporated herein by reference in its entirety). In some embodiments, adding barcodes during the RT step before proceeding to a single-cell workflow (e.g., Rhapsody) enables superloading because each cDNA has an additional unique barcode independent of the bead barcode. There is a need for an in situ indexing solution for higher-throughput single-cell workflows (e.g., the Rhapsody approach). To enable high throughput using an in situ indexing approach for RNA and an AbSeq workflow, there is a need to capture AbSeq extensions on Rhapsody beads and ligate them onto Rhapsody beads. In some embodiments, it is necessary to block AbSeq oligo extensions to enable generation of single-stranded oligos for capture and ligation. Both approaches can be improved by the PNA blocking system provided herein. In some embodiments, the blocking oligonucleotides provided herein are included in in situ cell indexing adapters (e.g., barcoded oligonucleotides), which may be provided as high-throughput solution kits. Figures 4A-4B show non-limiting, exemplary schematic diagrams illustrating the capture of molecularly barcoded cDNAs by Rhapsody beads and ligation of the barcoded cDNAs to the beads (Figure 4A), as well as the lack of capture of molecularly barcoded AbSeq oligonucleotides due to double-stranded extension (Figure 4B). Figures 5A-5B show non-limiting, exemplary schematic diagrams illustrating that polymerase-mediated extension of both barcoded and antibody oligonucleotides can occur (Figure 5A), which may be undesirable in some protocols (Figure 5B).6A-6B show non-limiting exemplary schematic diagrams illustrating that a PNA blocking oligonucleotide provided herein hybridizes to a barcoded primer (FIG. 6A), subsequently preventing extension of the 3' end of an antibody oligonucleotide hybridized thereto (e.g., by blocking the strand displacement activity of reverse transcriptase).

[0129] Cellular component binding reagents (e.g., barcoded antibodies, etc.) and their uses (e.g., cellular sample indexing) are described in U.S. Patent Application Publication Nos. 2018 / 0088112 and 2018 / 0346970, the contents of each of which are incorporated herein by reference in their entireties. The systems, methods, compositions, and kits provided herein can, in some embodiments, be used with the systems, methods, compositions, and kits described in U.S. Patent Application Publication No. 2020 / 0232032, the contents of which are incorporated herein by reference in their entireties. In some embodiments of the methods and compositions provided herein, DNA cellular component binding reagent-specific oligonucleotides (e.g., antibody oligonucleotides) are hybridized to oligonucleotide barcodes and extended, as described in U.S. Patent Application Publication No. 20210214784, the contents of which are incorporated herein by reference in their entireties, enabling separate but parallel workflows for protein quantification and mRNA quantification from the same beads. In some embodiments of the methods and compositions provided herein, the oligonucleotide barcode comprises a cleavage region (e.g., comprising one or more cleavage sites, such as non-canonical nucleotides (e.g., deoxyuridine) or restriction enzyme recognition sequences), as described in U.S. Patent Application Publication No. 20210214770, the contents of which are incorporated herein by reference in their entirety.

[0130] In some embodiments, a blocking oligonucleotide is provided. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing to each of a plurality of barcoded oligonucleotides to form a protected duplex. In some embodiments, the barcoded oligonucleotide comprises a 3' target binding region capable of hybridizing to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence.

[0131] In some embodiments, a protected duplex is provided. In some embodiments, the protected duplex comprises a blocking oligonucleotide hybridized to a barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing to the barcoded oligonucleotide. In some embodiments, the barcoded oligonucleotide comprises a 3' target binding region capable of hybridizing to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence.

[0132] In some embodiments, the blocking oligonucleotide cannot function as a primer for reverse transcriptase or polymerase. In some embodiments, the 3' end of the blocking oligonucleotide cannot be extended by reverse transcriptase or polymerase. In some embodiments, the protected duplex blocking oligonucleotide cannot be removed by the strand displacement activity of reverse transcriptase or polymerase. The blocking oligonucleotide can be a locked nucleic acid (LNA), peptide nucleic acid (PNA), DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. The T of the blocking oligonucleotide m may be at least 50°C, at least 60°C, at least 70°C, or a number or range between any two of these values.

[0133] In some embodiments, the blocking oligonucleotide does not contain non-naturally occurring nucleotides. In some embodiments, the blocking oligonucleotide comprises a 3' non-annealing region that is incapable of binding to the barcoded oligonucleotide. In some embodiments, the 3' non-annealing region is 1 to 100 nucleotides in length, 1 to 50 nucleotides in length, 1 to 21 nucleotides in length, 1 to 10 nucleotides in length, about 5 nucleotides in length, or a number or range between any two of these values. In some embodiments, the non-complementarity between the 3' non-annealing region and the region of the barcoded oligonucleotide adjacent to the 5' side of the sequence to which the blocking oligonucleotide binds is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, about 100%, or a number or range between any two of these values.

[0134] The blocking oligonucleotide and / or barcoded oligonucleotide may be a single-stranded oligonucleotide. The barcoded oligonucleotide may include a blocker region. In some embodiments, the blocker region is located between the barcode and the target binding region, or the blocker region is located between the barcode and the first universal sequence. In some embodiments, the complementarity between the binding region of the barcoded oligonucleotide and the sequence of the barcoded oligonucleotide to which the blocking oligonucleotide binds is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, about 100%, or a number or range between any two of these values. The binding region is capable of hybridizing to at least a portion of one or more of the target binding region, the first universal sequence, the barcode, and the blocker region to form a protected duplex. The protected duplex may comprise a double-stranded nucleic acid flanked on both sides by single-stranded overhangs.

[0135] When the protected duplex contacts the nucleic acid target, a polymerase or reverse transcriptase may be able to extend the 3' end of the barcoded oligonucleotide hybridized to the nucleic acid target to form an extended protected duplex. In some embodiments, the extended protected duplex comprises a blocking oligonucleotide hybridized to the extended barcoded oligonucleotide, the extended barcoded oligonucleotide comprising (i) a sequence complementary to at least a portion of the nucleic acid target, (ii) a target binding region, (iii) a first universal sequence, and (iv) a barcode. The blocking oligonucleotide may be able to terminate polymerase and / or transcriptase extension of the 3' end of the hybridized nucleic acid target up to the 5' end of the barcoded oligonucleotide. The blocking oligonucleotide may be able to terminate polymerase and / or reverse transcriptase extension of the 3' end of the hybridized nucleic acid target beyond the barcoded oligonucleotide. The extended protected duplex may comprise a single-stranded 5' first universal sequence. The single strand 5' of the extended protected duplex may be capable of hybridizing to a linker oligonucleotide to form a triplex. The linker oligonucleotide may include a second universal sequence capable of binding to an oligonucleotide barcode, and the triplex may be capable of binding to the oligonucleotide barcode via the second universal sequence to form a quadruplex. In some embodiments, the 5' end of the extended barcoded oligonucleotide and the 3' end of the oligonucleotide barcode may be ligated to each other by a ligase.

[0136] In the absence of a blocking oligonucleotide, the 3' end of the hybridized nucleic acid target may extend to the 5' end of the barcoded oligonucleotide, forming an undesired duplex. The undesired duplex may be double-stranded and / or may be capable of hybridizing to a linker oligonucleotide. The blocking oligonucleotide may be capable of reducing the formation of undesired duplexes by at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, at least 95%, at least 99%, or a number or range between any two of these values. The barcode may include a first cell marker. The oligonucleotide barcode may include a second cell marker. The plurality of barcoded oligonucleotides may include at least 100, at least 1,000, or at least 10,000 different barcode sequences. Each of the plurality of barcoded oligonucleotides may include the same barcode sequence. The barcoded oligonucleotide may be an in situ cell indexing adapter. In some embodiments, the blocking oligonucleotide, barcoded oligonucleotide, barcode, target binding region, first universal sequence, binding region, and / or blocker region is between 1 and 100 nucleotides in length, between 1 and 50 nucleotides in length, between 1 and 21 nucleotides in length, about 12 nucleotides in length, or a number or range of nucleotides in length between any two of these values.

[0137] The nucleic acid target may be a nucleic acid target molecule selected from the group including DNA molecules, RNA molecules, genomic DNA molecules, cDNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof. The nucleic acid target may be a cellular component binding reagent-specific oligonucleotide, and the cellular component binding reagent-specific oligonucleotide may be associated with the cellular component binding reagent. The cellular component binding reagent may be an antibody or fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, or any combination thereof. The cellular component binding reagent-specific oligonucleotide may include a unique identifier sequence for the cellular component binding reagent. The target binding region may include a capture sequence. The target binding region may include a poly(dT) region. The cellular component binding reagent-specific oligonucleotide may include a sequence complementary to a capture sequence configured to capture the cellular component binding reagent-specific oligonucleotide. The sequence complementary to the capture sequence may include a poly(dA) region. The cellular component binding reagent may be capable of specifically binding to at least one of multiple cellular component targets of a cell. The cellular component binding reagent-specific oligonucleotide may include a third universal sequence. The cellular component binding reagent-specific oligonucleotides may comprise a molecular beacon. At least 10 of the plurality of cellular component binding reagent-specific oligonucleotides may comprise different molecular beacon sequences.

[0138] The cellular component binding reagent-specific oligonucleotide may include a poly(dA) region. The cellular component binding reagent-specific oligonucleotide may include an alignment sequence adjacent to the poly(dA) region. The cellular component binding reagent-specific oligonucleotide may be associated with the cellular component binding reagent via a linker. The cellular component binding reagent-specific oligonucleotide may be configured to be detachable from the cellular component binding reagent. The alignment sequence may be one or more nucleotides in length, or two or more nucleotides in length. In some embodiments, (a) the alignment sequence includes guanine, cytosine, thymine, uracil, or a combination thereof; (b) the alignment sequence includes a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof; and / or (c) the alignment sequence is located 5' to the poly(dA) region. The linker may include a carbon chain. The carbon chain may include 2 to 30 carbons (e.g., 12 carbons). The linker may comprise a 5' amino modifier C12 (5AmMC12) or a derivative thereof. The subcellular target may comprise a protein target. The subcellular target may comprise a carbohydrate, lipid, protein, extracellular protein, cell surface protein, cell marker, B cell receptor, T cell receptor, major histocompatibility complex, tumor antigen, receptor, intracellular protein, or any combination thereof. The subcellular target may be present on a cell surface. The DNA polymerase may comprise a Klenow fragment. The reverse transcriptase may comprise a viral reverse transcriptase (e.g., murine leukemia virus (MLV) reverse transcriptase and / or Moloney murine leukemia virus (MMLV) reverse transcriptase).

[0139] In some embodiments, a reaction mixture is provided, which includes a plurality of blocking oligonucleotides provided herein, a plurality of protected duplexes provided herein, a plurality of protected duplexes, a plurality of barcoded oligonucleotides, a plurality of cellular component binding reagents, a ligase, dNTPs, a polymerase, a reverse transcriptase, and / or a plurality of oligonucleotide barcodes. In some embodiments, a kit is provided, which includes a plurality of blocking oligonucleotides provided herein, a plurality of protected duplexes provided herein, a plurality of protected duplexes, a plurality of barcoded oligonucleotides, a plurality of cellular component binding reagents, a ligase, a dNTP, a polymerase, a reverse transcriptase, and / or a plurality of oligonucleotide barcodes.

[0140] A plurality of oligonucleotide barcodes can be immobilized on a substrate. The substrate can be a particle (e.g., a bead). The plurality of oligonucleotide barcodes can include at least 100 different molecular label sequences or at least 100 different molecular label sequences. The plurality of oligonucleotide barcodes can include the same cell label sequence. Each of the plurality of oligonucleotide barcodes can include a cell label sequence, a sample label sequence, a location label sequence, a binding site for a universal primer, or a combination thereof. The plurality of oligonucleotide barcodes can include at least 100, at least 1,000, or at least 10,000 different molecular label sequences. The plurality of oligonucleotide barcodes can include the same cell label sequence. The plurality of oligonucleotide barcodes can be associated with particles. The oligonucleotide barcodes can be immobilized on particles, partially immobilized on particles, embedded in particles, partially embedded in particles, or a combination thereof. The particles can be beads. The beads may comprise polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or a combination thereof. The beads may be hydrogel beads or magnetic beads. The beads may be disintegrable. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in Provisional Patent Application No. 63 / 421,759, filed November 2, 2022, entitled "POLYMERASE-MEDIATED END MODIFICATION OF ABSEQ," the contents of which are incorporated herein by reference in their entirety.

[0141] term In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, except where such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter as defined by the appended claims. Those skilled in the art will recognize that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing order. Furthermore, the outlined steps and operations are provided only as examples, and some of the steps and operations may be arbitrarily combined into fewer steps and operations or expanded into additional steps and operations without diminishing the essential elements of the embodiments of the present disclosure.

[0142] In connection with the use of virtually any plural and / or singular term herein, those of skill in the art can convert from plural to singular and / or from singular to plural where appropriate in context and / or application. Various singular / plural permutations may be expressly stated herein for clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to encompass "and / or" unless specified otherwise.

[0143] In general, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Furthermore, where a specific number of introduced claim recitations is intended, such intention will be explicitly set forth in the claim; it will be understood by those skilled in the art that, in the absence of such a recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means that any particular claim containing such introduced claim recitation is limited to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), nor should the use of definite articles used to introduce claim recitations.Furthermore, even if a particular number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., an unqualified recitation such as "two recitations" without other modifiers means at least two recitations, or more than two recitations). Furthermore, when a convention similar to "such as at least one of A, B, and C" is used, it is generally intended that such a configuration be understood by one of skill in the art (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). When a convention similar to "such as at least one of A, B, or C" is used, it is generally intended that such construction have the meaning that one of ordinary skill in the art would understand that convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Furthermore, it will be understood by those skilled in the art that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0144] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will recognize that the present disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0145] As will be understood by those skilled in the art, for all purposes, e.g., with respect to the provision of a specification, all ranges disclosed herein encompass all possible subranges and combinations of such subranges. Any recited range is readily recognizable as being fully descriptive and that the range can be divided into at least 2, 3, 4, 5, 10, etc. equal parts. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. Similarly, as will be understood by those skilled in the art, terms such as "up to," "at least," "greater than," "less than," and the like all refer to ranges that are inclusive of the recited numbers and that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4, or 5 items, and so forth.

[0146] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. a blocking oligonucleotide comprising a binding region capable of hybridizing to each of a plurality of barcoded oligonucleotides to form a protected duplex, The barcoded oligonucleotide is a 3' target binding region capable of hybridizing to a nucleic acid target; 5' a first universal sequence, and A barcode located between the target binding region and the first universal sequence. A blocking oligonucleotide comprising:

2. A protected duplex comprising a blocking oligonucleotide hybridized to a barcoded oligonucleotide, the blocking oligonucleotide comprises a binding region capable of hybridizing to the barcoded oligonucleotide; The barcoded oligonucleotide is a 3' target binding region capable of hybridizing to a nucleic acid target; 5' a first universal sequence, and A barcode located between the target binding region and the first universal sequence. a protected duplex comprising:

3. 3. The blocking oligonucleotide or protected duplex of any one of claims 1 to 2, wherein the blocking oligonucleotide is incapable of functioning as a primer for a reverse transcriptase or a polymerase.

4. 4. The blocking oligonucleotide or protected duplex of any one of claims 1 to 3, wherein the 3' end of the blocking oligonucleotide is not extendable by reverse transcriptase or polymerase.

5. 5. The blocking oligonucleotide or protected duplex of any one of claims 1 to 4, wherein the blocking oligonucleotide of the protected duplex is not removable by the strand displacement activity of a reverse transcriptase or a polymerase.

6. 6. The blocking oligonucleotide or protected duplex of any one of claims 1 to 5, wherein the blocking oligonucleotide is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera.

7. T of blocking oligonucleotide m 7. The blocking oligonucleotide or protected duplex of any one of claims 1 to 6, wherein the temperature is at least 50°C, at least 60°C, or at least 70°C.

8. The blocking oligonucleotide or protected duplex of any one of claims 1 to 7, wherein the blocking oligonucleotide does not contain any non-naturally occurring nucleotides.

9. 9. The blocking oligonucleotide or protected duplex of any one of claims 1 to 8, wherein the blocking oligonucleotide comprises a 3' non-annealing region that is incapable of binding to a barcoded oligonucleotide.

10. 10. The blocking oligonucleotide or protected duplex of any one of claims 1 to 9, wherein the 3' non-annealing region is 1 to 100 nucleotides in length, 1 to 50 nucleotides in length, 1 to 21 nucleotides in length, 1 to 10 nucleotides in length, or about 5 nucleotides in length.

11. 11. The blocking oligonucleotide or protected duplex of any one of claims 1 to 10, wherein the non-complementarity between the 3' non-annealing region and the region of the barcoded oligonucleotide adjacent to the 5' side of the sequence to which the blocking oligonucleotide binds is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%.

12. 12. The blocking oligonucleotide or protected duplex of any one of claims 1 to 11, wherein the blocking oligonucleotide and / or barcoded oligonucleotide is a single-stranded oligonucleotide.

13. the barcoded oligonucleotide comprises a blocker region; a blocker region located between the barcode and the target binding region; or a blocker region located between the barcode and the first universal sequence; A blocking oligonucleotide or protected duplex according to any one of claims 1 to 12.

14. 14. The blocking oligonucleotide or protected duplex of any one of claims 1 to 13, wherein the complementarity between the binding region of the barcoded oligonucleotide and the sequence of the barcoded oligonucleotide to which the blocking oligonucleotide binds is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or about 100%.

15. 15. The blocking oligonucleotide or protected duplex of any one of claims 1 to 14, wherein the binding region is capable of hybridizing to at least a portion of one or more of the target binding region, the first universal sequence, the barcode, and the blocker region to form a protected duplex.

16. 16. The blocking oligonucleotide or protected duplex of any one of claims 1 to 15, wherein the protected duplex comprises a double-stranded nucleic acid flanked on both sides by single-stranded overhangs.

17. When the protected duplex comes into contact with the nucleic acid target, 17. The blocking oligonucleotide or protected duplex of any one of claims 1 to 16, wherein a polymerase or reverse transcriptase is capable of extending a 3' end of a barcoded oligonucleotide hybridized to a nucleic acid target to generate an extended protected duplex, the extended protected duplex comprising a blocking oligonucleotide hybridized to the extended barcoded oligonucleotide, the extended barcoded oligonucleotide comprising: (i) a sequence complementary to at least a portion of the nucleic acid target; (ii) a target binding region; (iii) a first universal sequence; and (iv) a barcode.

18. 18. The blocking oligonucleotide or protected duplex of any one of claims 1 to 17, wherein the blocking oligonucleotide is capable of terminating polymerase-mediated extension and / or reverse transcriptase-mediated extension of the 3'-end of a hybridized nucleic acid target towards the 5'-end of the barcoded oligonucleotide.

19. 19. The blocking oligonucleotide or protected duplex of any one of claims 1 to 18, wherein the blocking oligonucleotide is capable of terminating polymerase extension and / or reverse transcriptase extension of the 3' end of a hybridized nucleic acid target beyond the barcoded oligonucleotide.

20. 20. The blocking oligonucleotide or protected duplex of any one of claims 1 to 19, wherein the extended protected duplex comprises a single-stranded 5' first universal sequence.

21. 21. The blocking oligonucleotide or protected duplex of any one of claims 1 to 20, wherein a single strand 5' first universal sequence of the extended protected duplex is capable of hybridizing to a linker oligonucleotide to form a triplex.

22. 22. The blocking oligonucleotide or protected duplex of any one of claims 1 to 21, wherein the linker oligonucleotide comprises a second universal sequence capable of binding to the oligonucleotide barcode, and the triplex is capable of binding to the oligonucleotide barcode via the second universal sequence to form a quadruplex.

23. 23. The blocking oligonucleotide or protected duplex of any one of claims 1 to 22, wherein the 5' end of the extended barcoded oligonucleotide and the 3' end of the oligonucleotide barcode are capable of ligating to each other by a ligase.

24. 24. The blocking oligonucleotide or protected duplex of any one of claims 1 to 23, wherein in the absence of the blocking oligonucleotide, the 3' end of the hybridized nucleic acid target extends to the 5' end of the barcoded oligonucleotide, forming an undesired duplex.

25. 25. The blocking oligonucleotide or protected duplex of any one of claims 1 to 24, wherein the undesired duplex is double-stranded and / or incapable of hybridizing to the linker oligonucleotide.

26. 26. The blocking oligonucleotide or protected duplex of any one of claims 1 to 25, wherein the blocking oligonucleotide is capable of reducing the formation of unwanted duplexes by at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, at least 95%, or at least 99%.

27. 27. The blocking oligonucleotide or protected duplex of any one of claims 1 to 26, wherein the barcode comprises a first cell label and the oligonucleotide barcode optionally comprises a second cell label.

28. 28. The blocking oligonucleotide or protected duplex of any one of claims 1 to 27, wherein the plurality of barcoded oligonucleotides comprises at least 100, at least 1,000, or at least 10,000 different barcode sequences.

29. 29. The blocking oligonucleotide or protected duplex of any one of claims 1 to 28, wherein each of the plurality of barcoded oligonucleotides comprises the same barcode sequence.

30. 30. The blocking oligonucleotide or protected duplex of any one of claims 1 to 29, wherein the barcoded oligonucleotide is an in situ cell indexing adaptor.

31. 31. The blocking oligonucleotide or protected duplex of any one of claims 1 to 30, wherein the blocking oligonucleotide, barcoded oligonucleotide, barcode, target binding region, first universal sequence, binding region, and / or blocker region is 1 to 100 nucleotides in length, 1 to 50 nucleotides in length, 1 to 21 nucleotides in length, or about 12 nucleotides in length.

32. 32. The blocking oligonucleotide or protected duplex of any one of claims 1 to 31, wherein the nucleic acid target is a nucleic acid target molecule selected from the group comprising a DNA molecule, an RNA molecule, a genomic DNA molecule, a cDNA molecule, an mRNA molecule, an rRNA molecule, mtDNA, an siRNA molecule, or any combination thereof.

33. 33. The blocking oligonucleotide or protected duplex of any one of claims 1 to 32, wherein the nucleic acid target is a cellular component binding reagent-specific oligonucleotide, the cellular component binding reagent-specific oligonucleotide is associated with a cellular component binding reagent, and the cellular component binding reagent may be an antibody or fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, or any combination thereof.

34. 34. The blocking oligonucleotide or protected duplex of any one of claims 1 to 33, wherein the cellular component binding reagent-specific oligonucleotide comprises a unique identifier sequence for the cellular component binding reagent.

35. 35. The blocking oligonucleotide or protected duplex of any one of claims 1 to 34, wherein the target binding region comprises a capture sequence, and the target binding region optionally comprises a poly(dT) region.

36. The blocking oligonucleotide or protected duplex described in any one of claims 1 to 35, wherein the cell component binding reagent-specific oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the cell component binding reagent-specific oligonucleotide, and the sequence complementary to the capture sequence may comprise a poly(dA) region.

37. 37. The blocking oligonucleotide or protected duplex of any one of claims 1 to 36, wherein the cellular component binding reagent is capable of specifically binding to at least one of multiple cellular component targets of a cell.

38. 38. The blocking oligonucleotide or protected duplex of any one of claims 1 to 37, wherein the cell component binding reagent-specific oligonucleotide comprises a third universal sequence.

39. The blocking oligonucleotide or protected duplex of any one of claims 1 to 38, wherein the cellular component binding reagent-specific oligonucleotide comprises a molecular beacon, and at least 10 of the plurality of cellular component binding reagent-specific oligonucleotides may comprise different molecular beacon sequences.

40. The blocking oligonucleotide or protected duplex described in any one of claims 1 to 39, wherein the cell component binding reagent-specific oligonucleotide comprises a poly(dA) region, and the cell component binding reagent-specific oligonucleotide may comprise an alignment sequence adjacent to the poly(dA) region.

41. 41. The blocking oligonucleotide or protected duplex of any one of claims 1 to 40, wherein the cell component binding reagent-specific oligonucleotide is associated with the cell component binding reagent through a linker.

42. A blocking oligonucleotide or protected duplex according to any one of claims 1 to 41, wherein the cell component binding reagent-specific oligonucleotide is configured to be detachable from the cell component binding reagent.

43. 43. The blocking oligonucleotide or protected duplex of any one of claims 1 to 42, wherein the alignment sequence is one or more nucleotides in length, or two or more nucleotides in length.

44. (a) the alignment sequence contains guanine, cytosine, thymine, uracil, or a combination thereof; (b) the alignment sequence comprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof; and / or (c) the alignment sequence is 5' to the poly(dA) region; 44. A blocking oligonucleotide or protected duplex according to any one of claims 1 to 43.

45. 45. The blocking oligonucleotide or protected duplex of any one of claims 1 to 44, wherein the linker comprises a carbon chain, and optionally the carbon chain comprises 2 to 30 carbons, and further optionally the carbon chain comprises 12 carbons.

46. 46. ​​The blocking oligonucleotide or protected duplex of any one of claims 1 to 45, wherein the linker comprises a 5' amino modifier C12 (5AmMC12) or a derivative thereof.

47. 47. The blocking oligonucleotide or protected duplex of any one of claims 1 to 46, wherein the cellular component target comprises a protein target.

48. 48. The blocking oligonucleotide or protected duplex of any one of claims 1 to 47, wherein the cellular component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cellular marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

49. 49. The blocking oligonucleotide or protected duplex of any one of claims 1 to 48, wherein the cellular component target is present on the cell surface.

50. 50. The blocking oligonucleotide or protected duplex of any one of claims 1 to 49, wherein the DNA polymerase comprises a Klenow fragment.

51. 51. The blocking oligonucleotide or protected duplex of any one of claims 1 to 50, wherein the reverse transcriptase comprises a viral reverse transcriptase, which may be murine leukemia virus (MLV) reverse transcriptase and / or Moloney murine leukemia virus (MMLV) reverse transcriptase.

52. A plurality of blocking oligonucleotides according to any one of claims 1 to 51; A plurality of protected duplexes according to any one of claims 1 to 51; Multiple protected duplexes, a plurality of barcoded oligonucleotides; a plurality of cellular component binding reagents; ligase, dNTPs, polymerase, reverse transcriptase, and / or Multiple oligonucleotide barcodes A reaction mixture comprising:

53. A plurality of blocking oligonucleotides according to any one of claims 1 to 51; A plurality of protected duplexes according to any one of claims 1 to 51; Multiple protected duplexes, a plurality of barcoded oligonucleotides; a plurality of cellular component binding reagents; ligase, dNTPs, polymerase, reverse transcriptase, and / or Multiple oligonucleotide barcodes Kit including:

54. 54. The reaction mixture or kit of any one of claims 52 to 53, wherein a plurality of oligonucleotide barcodes are immobilized on a substrate.

55. 55. The reaction mixture or kit of any one of claims 52 to 54, wherein the substrate is a particle, optionally a bead.

56. 56. The reaction mixture or kit of any one of claims 52 to 55, wherein the plurality of oligonucleotide barcodes comprises at least 100 different molecular beacon sequences or at least 100 different molecular beacon sequences.

57. 57. The reaction mixture or kit of any one of claims 52 to 56, wherein multiple oligonucleotide barcodes comprise the same cell labeling sequence.

58. 58. The reaction mixture or kit of any one of claims 52 to 57, wherein each of the plurality of oligonucleotide barcodes comprises a cell labeling sequence, a sample labeling sequence, a position labeling sequence, a binding site for a universal primer, or a combination thereof.

59. 59. The reaction mixture or kit of any one of claims 52 to 58, wherein the plurality of oligonucleotide barcodes comprises at least 100, at least 1000, or at least 10000 different molecular label sequences.

60. 60. The reaction mixture or kit of any one of claims 52 to 59, wherein multiple oligonucleotide barcodes comprise the same cell targeting sequence.

61. 61. The reaction mixture or kit of any one of claims 52 to 60, wherein a plurality of oligonucleotide barcodes are associated with the particles, and the oligonucleotide barcodes may be immobilized on the particles, partially immobilized on the particles, embedded in the particles, partially embedded in the particles, or combinations thereof.

62. 62. The reaction mixture or kit of any one of claims 52 to 61, wherein the particles are beads, and the beads may comprise polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, or a combination of these materials.

63. 63. The reaction mixture or kit according to any one of claims 52 to 62, wherein the beads are hydrogel beads or magnetic beads.

64. 64. The reaction mixture or kit of any one of claims 52 to 63, wherein the beads are disintegrable.

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