Application for peptide nucleic acid (PNA) blockers

Abstract

Disclosed herein include systems, methods, compositions, and kits for preventing antibody oligonucleotide extension. In some embodiments, blocking oligonucleotides are provided. In some embodiments, the blocking oligonucleotide comprises a binding region capable of hybridizing to the barcoded oligonucleotide to form a protected duplex. The blocking oligonucleotide may 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'terminal of the blocking oligonucleotide cannot be extended by a reverse transcriptase or polymerase, In some embodiments, the blocking oligonucleotide of the protected duplex cannot be removed by the strand displacement activity of the reverse transcriptase or polymerase.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 421,744, filed on November 2, 2022, under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] background

[0004] field

[0005] The present disclosure relates generally to the field of molecular biology, such as determining gene expression using molecular barcoding.

[0006] Description of the Prior Art

[0007] Current technology allows for the measurement of gene expression of single cells in a massively parallel manner (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to multiple (A) mRNA molecules from individual cells when each cell is co-localized with a barcoding reagent bead in a compartment. Gene expression can affect protein expression. Protein-protein interactions can affect gene expression and protein expression. There is a need for systems and methods that can quantitatively analyze protein expression in cells and simultaneously measure protein expression and gene expression in cells. In addition, existing protein spectrum analysis methods include methods in which antibody oligonucleotides can hybridize with barcode oligonucleotides and can be used as templates for barcoding reactions and can extend their own 3' ends. There is a need for compositions, methods, systems, and kits that reduce or prevent the extension of antibody oligonucleotides hybridized with barcoded oligonucleotides.

[0008] Overview

[0009] The disclosure herein includes blocking oligonucleotides. In some embodiments, the blocking oligonucleotide comprises: a binding region capable of hybridizing to each of more than one barcoded oligonucleotide 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.

[0010] The disclosure herein includes protected duplexes. In some embodiments, the protected duplex comprises: a blocking oligonucleotide that hybridizes to a barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide comprises a binding region that is capable of hybridizing to a barcoded oligonucleotide. In some embodiments, the barcoded oligonucleotide comprises: a 3' target binding region that is 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.

[0011] In some embodiments, the blocking oligonucleotide cannot be used as a primer for a reverse transcriptase or polymerase. In some embodiments, the 3' end of the blocking oligonucleotide cannot be extended by a reverse transcriptase or polymerase. In some embodiments, the blocking oligonucleotide of the protected duplex cannot be removed by the strand displacement activity of a reverse transcriptase or polymerase. In some embodiments, the blocking oligonucleotide is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. In some embodiments, the blocking oligonucleotide has a T of at least 50°C, at least 60°C, or at least 70°C. m .

[0012] 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 not capable of binding to the barcoded oligonucleotide. In some embodiments, the 3' non-annealing region is 1 nt to 100 nt long, 1 nt to 50 nt long, 1 nt to 21 nt long, 1 nt to 10 nt long, or about 5 nt long. In some embodiments, the non-complementarity between the 3' non-annealing region and the 5' adjacent region of the sequence in 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 blocking oligonucleotide and / or the barcoded oligonucleotide is a single-stranded oligonucleotide.

[0013] In some embodiments, the barcoded oligonucleotide comprises a blocker region, and wherein: 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 bound by the blocking oligonucleotide 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 with 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.

[0014] In some embodiments, the protected duplex comprises a double-stranded nucleic acid flanked by single-stranded overhangs. In some embodiments, when the protected duplex is contacted with a nucleic acid target, a polymerase or reverse transcriptase is capable of extending the 3' end of a barcoded oligonucleotide hybridized with the nucleic acid target to generate an extended protected duplex, wherein the extended protected duplex comprises a blocking oligonucleotide hybridized with 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. In some embodiments, the blocking oligonucleotide is capable of stopping the polymerase extension and / or reverse transcriptase extension of the 3' end of the hybridized nucleic acid target to the 5' end of the barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide is capable of stopping the polymerase extension 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 can hybridize with an adapter oligonucleotide to form a triplex. In some embodiments, the adapter oligonucleotide comprises a second universal sequence capable of binding to an oligonucleotide barcode, and wherein the triplex can bind 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 can be ligated together by a ligase.

[0015] In some embodiments, in the absence of a blocking oligonucleotide, the 3' end of the hybridized nucleic acid target extends to the 5' end of the barcoding oligonucleotide to form an undesired duplex. In some embodiments, the undesired duplex is double-stranded and / or incapable of hybridizing with an adapter oligonucleotide. In some embodiments, the blocking oligonucleotide can reduce the generation 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%.

[0016] In some embodiments, the barcode comprises a first cell marker. In some embodiments, the oligonucleotide barcode comprises a second cell marker. In some embodiments, more than one barcoded oligonucleotide comprises at least 100, at least 1000, or at least 10,000 different barcode sequences. In some embodiments, more than one barcoded oligonucleotide each comprises the same barcode sequence. In some embodiments, the barcoded oligonucleotide is 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 are 1 nt to 100 nt long, 1 nt to 50 nt long, 1 nt to 21 nt long, or about 12 nt long.

[0017] In some embodiments, the nucleic acid target is a nucleic acid target molecule selected from a group comprising 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 cell component binding reagent specific oligonucleotide, and wherein the cell component binding reagent specific oligonucleotide is associated with the cell component binding reagent. In some embodiments, the cell component binding reagent is an antibody or a fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, or any combination thereof. In some embodiments, the cell component binding reagent specific oligonucleotide comprises a unique identifier sequence for the cell 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 cell component binding reagent specific oligonucleotide comprises a sequence complementary to the capture sequence, and the capture sequence is configured to capture the cell component binding reagent specific oligonucleotide. In some embodiments, the sequence complementary to the capture sequence comprises a poly (dA) region.

[0018] In some embodiments, the cell component binding reagent can be specifically bound to at least one of more than one cell component target of the cell. In some embodiments, the cell component binding reagent specific oligonucleotide comprises a third universal sequence. In some embodiments, the cell component binding reagent specific oligonucleotide comprises a molecular marker. In some embodiments, at least 10 of more than one cell component binding reagent specific oligonucleotides comprise different molecular marker sequences. In some embodiments, the cell component binding reagent specific oligonucleotide comprises multiple (dA) districts. In some embodiments, the cell component binding reagent specific oligonucleotide comprises an alignment sequence adjacent to multiple (dA) districts. In some embodiments, the cell component binding reagent specific oligonucleotide is associated with the cell component binding reagent by a joint. In some embodiments, the cell component binding reagent specific oligonucleotide is configured to be able to detach from the cell component binding reagent. In some embodiments, the length of the alignment sequence is one or more nucleotides, or a length of two or more nucleotides. In some embodiments, (a) the alignment sequence comprises 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 located 5' of the poly(dA) region. In some embodiments, the linker comprises a carbon chain. In some embodiments, the carbon chain comprises 2-30 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 cell component target comprises a protein target. In some embodiments, the cell component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell 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. In some embodiments, the cell component target is on the 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).

[0019] The disclosure herein includes a reaction mixture. In some embodiments, the reaction mixture comprises: more than one blocking oligonucleotide provided herein; more than one protected duplex provided herein; more than one protected duplex; more than one barcoded oligonucleotide; more than one cellular component binding reagent; ligase; dNTP; polymerase; reverse transcriptase and / or more than one oligonucleotide barcode.

[0020] The disclosure herein includes kits. In some embodiments, the kit comprises: more than one blocking oligonucleotide provided herein; more than one protected duplex provided herein; more than one protected duplex; more than one barcoded oligonucleotide; more than one cellular component binding reagent; ligase; dNTP; polymerase; reverse transcriptase and / or more than one oligonucleotide barcode.

[0021] In some embodiments, more than one oligonucleotide barcode is fixed on a substrate. In some embodiments, the substrate is a particle (e.g., a bead). In some embodiments, more than one oligonucleotide barcode comprises at least 100 different molecular marker sequences or at least 100 different molecular marker sequences. In some embodiments, more than one oligonucleotide barcode comprises the same cell marker sequence. In some embodiments, each of more than one oligonucleotide barcode comprises a cell marker sequence, a sample marker sequence, a position marker sequence, a binding site for a universal primer, or a combination thereof. In some embodiments, more than one oligonucleotide barcode comprises at least 100, at least 1000, or at least 10000 different molecular marker sequences. In some embodiments, more than one oligonucleotide barcode comprises the same cell marker sequence. In some embodiments, more than one oligonucleotide barcode is associated with a particle. In some embodiments, the oligonucleotide barcode is fixed on a particle, partially fixed on a particle, embedded in a particle, partially embedded in a particle, or a combination thereof. In some embodiments, a particle is a bead. In some embodiments, the beads comprise a material of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or a combination thereof. In some embodiments, the beads are hydrogel beads or magnetic beads. In some embodiments, the beads are destructible. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Non-limiting exemplary bar codes are illustrated.

[0024] Figure 2 A non-limiting exemplary workflow for barcoding and digital counting is shown.

[0025] Figure 3 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of targets barcoded at the 3' end from more than one target.

[0026] Figure 4A-4B Depicted is a non-limiting exemplary schematic depicting capture of molecules barcoding cDNA by Rhapsody beads and attachment of the barcoded cDNA to the beads ( Figure 4A), and the lack of capture of molecular barcoded AbSeq oligonucleotides due to double-stranded extension ( Figure 4B ).

[0027] Figure 5A-Figure 5B Depicted is a non-limiting exemplary schematic showing that polymerase extension of both barcoded oligonucleotides and antibody oligonucleotides can occur ( Figure 5A ), which is disadvantageous in some scenarios ( Figure 5B ).

[0028] Figure 6A-6B Depicted is a non-limiting exemplary schematic diagram showing a PNA blocking oligonucleotide provided herein hybridized to a barcoded primer ( Fig. 6A ) and subsequently preventing extension of the 3' end of the antibody oligonucleotide hybridized thereto (eg, by blocking the strand displacement activity of the reverse transcriptase) ( Figure 6B ).

[0029] Details

[0030] Reference is made to the accompanying drawings forming a part of this document in the following detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context otherwise indicates. The illustrative 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 accompanying drawings can be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and constitute a part of the present disclosure.

[0031] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

[0032] Quantification of small amounts of nucleic acids, such as messenger ribonucleotide (mRNA) molecules, is clinically important for determining genes expressed in cells, for example, at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules, such as mRNA molecules, can also be very challenging, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is the digital polymerase chain reaction (PCR). Ideally, PCR produces identical copies of molecules in each cycle. However, PCR can have the disadvantage that each molecule is replicated with a random probability, and this probability varies depending on the PCR cycle and the gene sequence, which leads to amplification bias and inaccurate gene expression measurements. Random barcodes with unique molecular labels (molecular labels, also called molecular indexes (MI)) can be used to count the number of molecules and correct for amplification bias. Random barcoding, such as Precise TM Assay (Cellular Research, Inc. (Palo Alto, CA)) and Rhapsody TM The PCR amplification assay (Becton, Dickinson and Company (Franklin Lakes, NJ)) can correct for bias induced by PCR and library preparation steps by labeling mRNA during reverse transcription (RT) using a molecular marker (ML).

[0033] Precise TM The assay can utilize a non-depleting pool of random barcodes with a large number (e.g., 6561 to 65536) of unique molecular marker sequences on poly (T) oligonucleotides to hybridize with all poly (A)-mRNAs in the sample during the RT step. The random barcode can include a universal PCR priming site. During RT, the target gene molecule reacts randomly with the random barcode. Each target molecule can hybridize with the random barcode, resulting in the generation of randomly barcoded complementary ribonucleotides (cDNA) molecules. After labeling, the randomly barcoded cDNA molecules from the microwells of the microplate can be pooled into a single tube for PCR amplification and sequencing. The original sequencing data can be analyzed to generate the number of reads, the number of random barcodes with unique molecular marker sequences, and the number of mRNA molecules.

[0034] The disclosure herein includes blocking oligonucleotides. In some embodiments, the blocking oligonucleotide comprises: a binding region capable of hybridizing to each of more than one barcoded oligonucleotide 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.

[0035] The disclosure herein includes protected duplexes. In some embodiments, the protected duplex comprises: a blocking oligonucleotide that hybridizes to a barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide comprises a binding region that is capable of hybridizing to a barcoded oligonucleotide. In some embodiments, the barcoded oligonucleotide comprises: a 3' target binding region that is 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.

[0036] The disclosure herein includes a reaction mixture. In some embodiments, the reaction mixture comprises: more than one blocking oligonucleotide provided herein; more than one protected duplex provided herein; more than one protected duplex; more than one barcoded oligonucleotide; more than one cellular component binding reagent; ligase; dNTP; polymerase; reverse transcriptase and / or more than one oligonucleotide barcode.

[0037] The disclosure herein includes kits. In some embodiments, the kit comprises: more than one blocking oligonucleotide provided herein; more than one protected duplex provided herein; more than one protected duplex; more than one barcoded oligonucleotide; more than one cellular component binding reagent; ligase; dNTP; polymerase; reverse transcriptase and / or more than one oligonucleotide barcode.

[0038] definition

[0039] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present 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 the purposes of the present disclosure, the following terms are defined below.

[0040] As used herein, the term "adapter" may mean a sequence that promotes the 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 marker, a target marker, a sample marker, an index marker, or a barcode sequence (e.g., a molecular marker). The adaptor may be linear. The adaptor may be a pre-adenylated adaptor. The adaptor may be double-stranded or single-stranded. One or more adaptors may be located at the 5' end or 3' end of the nucleic acid. When the adaptor comprises a known sequence at the 5' end and the 3' end, the known sequence may be the same or different sequence. The adaptor located at the 5' end and / or the 3' end of the polynucleotide may be able to hybridize with one or more oligonucleotides fixed on the surface. In some embodiments, the adaptor may include a universal sequence. The universal sequence may be a region of a nucleotide sequence common to two or more nucleic acid molecules. Two or more nucleic acid molecules may also have regions of different sequences. Therefore, for example, the 5' adaptor may include the same and / or universal nucleic acid sequence, and the 3' adaptor may include the same and / or universal sequence. Universal sequences that may be present in different members of more than one nucleic acid molecule may allow the use of a single universal primer complementary to the universal sequence to replicate or amplify more than one different sequence. Similarly, at least one, two (e.g., a pair) or more universal sequences that may be present in different members of a set of nucleic acid molecules may allow the use of at least one, two (e.g., a pair) or more single universal primers complementary to the universal sequence to replicate or amplify more than one different sequence. Therefore, universal primers include sequences that can hybridize with such universal sequences. Molecules with target nucleic acid sequences may be modified to attach universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of different target nucleic acid sequences. One or more universal primers attached to target nucleic acid may provide a site for universal primer hybridization. One or more universal primers attached to target nucleic acid may be identical or different from each other.

[0041] As used herein, the term "association" or "associated with..." can mean that two or more substances can be identified as being co-located at a certain point in time. Association can mean that two or more substances are or have been in similar containers. Association can be an informatics association. For example, digital information about two or more substances can be stored and can be used to determine that one or more substances are co-located at a certain point in time. Association can also be a physical association. In some embodiments, two or more associated substances are "tethered", "attached" or "fixed" to each other or to a common solid or semi-solid surface. Association can refer to a covalent or non-covalent means for attaching a label to a solid or semi-solid support (such as a bead). Association can be a covalent bond between a target and a label. Association can include hybridization between two molecules (such as a target molecule and a label).

[0042] As used herein, the term "complementary" can refer to the ability of accurate pairing between two nucleotides. For example, if the nucleotides of a nucleic acid at a given position can hydrogen bond with the nucleotides of another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules can be "partial", in which only some nucleotides bind, or it can be complete when there is full complementarity between single-stranded molecules. If a first nucleotide sequence is complementary to a second nucleotide sequence, the first nucleotide sequence can be referred to as the "complement" of the second sequence. If a first nucleotide sequence is complementary to a sequence opposite to the second sequence (i.e., the nucleotide order is opposite), the first nucleotide sequence can be referred to as the "reverse complement" of the second sequence. As used herein, a "complementary" sequence can refer to the "complement" or "reverse complement" of a sequence. It is understood from the present disclosure that if a molecule can hybridize with another molecule, it can be complementary or partially complementary to the molecule it hybridizes with.

[0043] As used herein, the term "digital counting" may refer to a method for estimating the number of target molecules in a sample. Digital counting may include a step of determining the number of unique markers that have been associated with a target in a sample. This method, which may be stochastic in nature, converts the problem of counting molecules from one of localization and identification of identical molecules to a series of yes / no digital questions about detecting a set of predefined markers.

[0044] As used herein, the term "a label" or "more than one labels" can refer to a nucleic acid code associated with a target in a sample. A label can be, for example, a nucleic acid label. A label can be a fully or partially amplifiable label. A label can be a fully or partially sequenceable label. A label can be a part of a natural nucleic acid that can be identified as being distinguished. A label can be a known sequence. A label can include a junction of a nucleic acid sequence, such as a junction of a natural and a non-natural sequence. As used herein, the term "label" can be used interchangeably with the term "index," "tag," or "label-tag." A label can convey information. For example, in various embodiments, a label can be used to determine the identity of a sample, the source of a sample, the identity of a cell, and / or a target.

[0045] As used herein, the term "non-depleting reservoir" may refer to a pool of barcodes (e.g., random barcodes) composed of many different markers. A non-depleting reservoir may include a large number of different barcodes such that when the non-depleting reservoir is associated with a pool of targets, each target may be associated with a unique barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random selection and depends on the number of copies of the same target molecule in the set compared to the diversity of the markers. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and then analysis of the number of detected barcodes allows calculation of the number of target molecules present in the original set or sample. When the ratio of the number of copies of the target molecules present to the number of unique barcodes is low, the labeled target molecules are highly unique (i.e., the probability that more than one target molecule is labeled by a given marker is very low).

[0046] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. Nucleic acid may include nucleotides. Nucleic acid may be exogenous or endogenous to a cell. Nucleic acid may be present in a cell-free environment. Nucleic acid may be a gene or a fragment thereof. Nucleic acid may be DNA. Nucleic acid may be RNA. Nucleic acid may include one or more analogs (e.g., altered backbones, sugars or nucleobases). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xenonucleic acid, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescent flavin linked to sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" are used interchangeably.

[0047] Nucleic acids can include one or more modifications (e.g., base modifications, backbone modifications) to provide new or enhanced features (e.g., improved stability) to nucleic acids. Nucleic acids can include nucleic acid affinity tags. Nucleosides can be base-sugar combinations. The base portion of a nucleoside can be a heterocyclic base. The two most common categories of such heterocyclic bases are purine and pyrimidine. Nucleotides can be nucleosides that also include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides including furanose, the phosphate group can be linked to the 2', 3' or 5' hydroxyl portion of the sugar. When forming nucleic acids, the phosphate group can covalently link adjacent nucleosides to each other to form a linear polymer compound. Subsequently, each end of this linear polymer compound can be further connected to form a cyclic compound; however, linear compounds are generally suitable. In addition, linear compounds can have internal nucleotide base complementarity, and can therefore be folded in a manner that produces a completely or partially double-stranded compound. In nucleic acids, phosphate groups can generally be referred to as the internucleoside backbone that forms nucleic acids. Linkages or backbones can be 3' to 5' phosphodiester linkages.

[0048] Nucleic acids can include modified backbones and / or modified internucleoside linkages. Modified backbones can include those that retain phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone. Suitable modified nucleic acid backbones containing phosphorus atoms therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3'-alkylenephosphonates, 5'-alkylenephosphonates, chiral phosphonates, phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates), thioalkylphosphonates, thioalkylphosphotriesters, selenophosphoros and boranophosphates, analogs with normal 3'-5' linkages, 2'-5' linkages, and analogs with reversed polarity (wherein one or more of the internucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' linkages).

[0049] Nucleic acids can include polynucleotide backbones formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms, and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatomic or heterocyclic nucleoside linkages. These can include those with morpholino linkages (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboseacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and those with mixed N, O, S and CH2 component parts.

[0050] Nucleic acid can include nucleic acid mimics.The term "simulations" may be intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide bond are replaced by non-furanose groups, and the replacement of only the furanose ring may also be referred to as a sugar substitute (surrogate). The heterocyclic base moiety or the modified heterocyclic base moiety may be maintained to hybridize with an appropriate target nucleic acid. Such a nucleic acid may be a peptide nucleic acid (PNA). In PNA, the sugar backbone of the polynucleotide may be replaced by an amide-containing backbone, particularly by an aminoethylglycine backbone. Nucleotides may be retained and directly or indirectly combined with the aza nitrogen atom of the amide moiety of the backbone. The backbone in the PNA compound may include two or more aminoethylglycine units connected, which allows the PNA to have an amide-containing backbone. The heterocyclic base moiety may be directly or indirectly combined with the aza nitrogen atom of the amide moiety of the backbone.

[0051] Nucleic acid can include morpholino backbone structure.For example, nucleic acid can include 6-membered morpholino rings replacing ribose rings.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages can replace phosphodiester linkages.

[0052] Nucleic acid can include morpholino units (e.g., morpholino nucleic acids) having a connection to a heterocyclic base attached to a morpholino ring. A linking group can connect the morpholino monomer units in morpholino nucleic acids. Oligomeric compounds based on nonionic morpholinos can have less undesirable interactions with cellular proteins. Polynucleotides based on morpholinos can be nonionic mimics of nucleic acids. Various compounds within the morpholino category can be connected using different linking groups. Polynucleotide mimics of other categories can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose rings commonly present in nucleic acid molecules can be replaced by cyclohexenyl rings. Phosphoramidite monomers protected by CeNA DMT can be prepared using phosphoramidite chemistry and are used for oligomeric compound synthesis. CeNA monomers are incorporated into nucleic acid chains to increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements, with stability similar to natural complexes. Additional modifications may include locked nucleic acids (LNA) in which the 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 may be a methylene (-CH2-) bridging the 2' oxygen atom and the 4' carbon atom. n A group wherein n is 1 or 2. LNA and LNA analogs can show very high duplex thermal stability (Tm = +3°C to +10°C) with complementary nucleic acids, stability to 3'-exonuclease degradation and good solubility.

[0053] Nucleic acids can also include modifications or substitutions of nucleobases (often referred to as "bases" for short). 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 may include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives 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 pyrimidine bases. Alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, especially 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 may 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 cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido (5,4-(b) (1,4) benzoxazin-2 (3H) -one), phenoxazine cytidine (1H-pyrimido (5,4-b) (1,4) benzothiazin-2 (3H) -one), Thiazide 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 cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0054] As used herein, the term "sample" may refer to a composition comprising a target. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.

[0055] As used herein, the term "sampling device" or "device" may refer to a device that can take a slice of a sample and / or place the slice on a substrate. The sampling 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 an ultramicrotome.

[0056] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which more than one barcode (e.g., a random barcode) may be attached. A solid support may include any type of solid, porous or hollow sphere, ball, bearing, cylinder, or other similar configuration comprising plastic, ceramic, metal, or polymeric material (e.g., hydrogel) on which nucleic acids may be fixed (e.g., covalently or non-covalently). A solid support may include discrete particles that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as a cubic, rectangular, conical, cylindrical, conical, elliptical, or disc-shaped, etc. The shape of a bead may be non-spherical. More than one solid support spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead".

[0057] As used herein, the term "random barcode" may refer to a polynucleotide sequence comprising a tag of the present disclosure. A random barcode may be a polynucleotide sequence that can be used for random barcoding. A random barcode may be used to quantify a target in a sample. A random barcode may be used to control errors that may occur after a tag is associated with a target. For example, a random barcode may be used to assess amplification or sequencing errors. A random barcode associated with a target may be referred to as a random barcode-target or a random barcode-tag-target.

[0058] As used herein, the term "gene-specific random barcode" may refer to a polynucleotide sequence comprising a marker and a gene-specific target binding region. A random barcode may be a polynucleotide sequence that can be used for random barcoding. A random barcode may be used to quantify a target in a sample. A random barcode may be used to control errors that may occur after a marker is associated with a target. For example, a random barcode may be used to assess amplification or sequencing errors. A random barcode associated with a target may be referred to as a random barcode-target or a random barcode-tag-target.

[0059] As used herein, the term "random barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Random barcoding can utilize a recursive Poisson strategy to associate and quantify labels associated with a target. As used herein, the term "random barcoding" can be used interchangeably with "random labeling."

[0060] As used herein, the term "target" may refer to a composition that can be associated with a barcode (e.g., a random barcode). Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, etc. The target may be single-stranded or double-stranded. In some embodiments, the target may be a protein, peptide, or polypeptide. In some embodiments, the target is a lipid. As used herein, "target" may be used interchangeably with "species".

[0061] As used herein, the term "reverse transcriptase" may refer to a group of enzymes having reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Typically, such enzymes include, but are not limited to, reverse transcriptases derived from retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retrotranscriptases, bacterial reverse transcriptases, and reverse transcriptases derived from group II introns and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retrotranscriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include Lactococcus lactis LI.LtrB intron reverse transcriptases, Thermosynechococcus elongatus TeI4c intron reverse transcriptases, or Geobacillus stearothermophilus GsI-IIC intron reverse transcriptases. Other classes of reverse transcriptases may include many types of non-retroviral reverse transcriptases (ie, retrovirals, group II introns, and diversity-generating retroelements, among others).

[0062] The terms "universal adapter primer," "universal primer adapter," or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can be used to hybridize with a barcode (e.g., a random barcode) to generate a gene-specific barcode. The universal adapter sequence can be, for example, a known sequence that is common to all barcodes used in the methods of the present disclosure. For example, when more than one target is labeled using the methods disclosed herein, each target-specific sequence can be connected to the same universal adapter sequence. In some embodiments, more than one universal adapter sequence can be used in the methods disclosed herein. For example, when more than one target is labeled using the methods disclosed herein, at least two target-specific sequences are connected to different universal adapter sequences. The universal adapter primer and its complement can be included in two oligonucleotides, one of which contains a target-specific sequence and the other oligonucleotide contains a barcode. For example, the universal adapter sequence can be a portion of an oligonucleotide containing a target-specific sequence to generate a nucleotide sequence complementary to a target nucleic acid. A second oligonucleotide containing a barcode and a complementary sequence to the universal adapter sequence can hybridize with the nucleotide sequence and generate a target-specific barcode (e.g., a target-specific random barcode). In some embodiments, the universal adapter primer has a different sequence than the universal PCR primer used in the methods of the disclosure.

[0063] Barcode

[0064] Barcoding, such as random barcoding, has been described in, for example, Fu et al., Proc Natl Acad Sci U.S.A., May 31, 2011, 108(22):9026-31; US2011 / 0160078; Fan et al., Science, February 6, 2015, 347(6222):1258367; US2015 / 0299784; and WO2015 / 031691; the contents of each of these, including any supporting or supplementary information or materials, are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein can be random barcodes, which can be polynucleotide sequences that can be used to randomly label (e.g., barcode, tag) a target. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences of a stochastic barcode to the number of occurrences of any target to be labeled can be or can 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 can be an mRNA species including mRNA molecules having the same or nearly the same sequence. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences of a stochastic barcode to the number of occurrences of any target 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, or 100: 1. The barcode sequences of a stochastic barcode can be referred to as molecular markers.

[0065] The barcode (e.g., random barcode) may include one or more labels. Exemplary labels may include universal labels, cell labels, barcode sequences (e.g., molecular labels), sample labels, plate labels, spatial labels, and / or pre-spatial labels. Figure 1An exemplary barcode 104 with spatial markers is shown. The barcode 104 can include a 5' amine that can link the barcode to the solid support 105. The barcode can include universal markers, dimensional markers, spatial markers, cellular markers, and / or molecular markers. The order of the different markers (including but not limited to universal markers, dimensional markers, spatial markers, cellular markers, and molecular markers) in the barcode can vary. For example, Figure 1 As shown in , the universal label can be the label of the most 5' side (5'-most label), and the molecular label can be the label of the most 3' side (3'-most label). Spatial label, dimensional label and cell label can be in any order. In some embodiments, universal label, spatial label, dimensional label, cell label and molecular label are in any order. Barcode can include target binding region. Target binding region can interact with the target (for example, target nucleic acid, RNA, mRNA, DNA) in sample. For example, the target binding region can include oligo (dT) sequence that can interact with the poly (A) tail of mRNA. In some cases, the label of barcode (for example, universal label, dimensional label, spatial label, cell label and barcode sequence) can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides.

[0066] Mark (such as cell marker) can comprise a group of unique defined length nucleic acid subsequences, for example, each seven nucleotides (equivalent to the number of bits used in some Hamming error correction codes), which can be designed to provide error correction capabilities. The error correction subsequence group comprising seven nucleotide sequences can be designed so that any paired combination of the sequences in the group exhibits a defined "genetic distance" (or mismatched base number), for example, a group of error correction subsequences can be designed to exhibit a genetic distance of three nucleotides. In this case, the review of the error correction sequence in the sequence data group of the target nucleic acid molecule of the mark (described in more detail below) can allow people to detect or correct amplification errors or sequencing errors. In some embodiments, the length of the nucleic acid subsequence for generating the error correction code can vary, for example, their length can be following or can be about following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 or the nucleotides of the number or range between any two of these values. In some embodiments, nucleic acid subsequences of other lengths can be used to generate error correction codes.

[0067] The barcode may include a target binding region. The target binding region may interact with a target in a sample. The target may be or include: ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each containing a poly (A) tail, or any combination thereof. In some embodiments, more than one target may include deoxyribonucleic acid (DNA).

[0068] In some embodiments, the target binding region may include an oligo (dT) sequence that can interact with the poly (A) tail of the mRNA. One or more markers of the barcode (e.g., universal markers, dimensional markers, spatial markers, cell markers, and barcode sequences (e.g., molecular markers)) may be separated from another or two remaining markers 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 or 20 or more nucleotides. In some embodiments, none of the markers of the barcode are separated by a spacer.

[0069] General Tags

[0070] The barcode may include one or more universal markers. In some embodiments, one or more universal markers may be the same for all barcodes in the barcode group attached to a given solid support. In some embodiments, one or more universal markers may be the same for all barcodes attached to more than one bead. In some embodiments, the universal marker may include a nucleic acid sequence that can be hybridized with a sequencing primer. A sequencing primer may be used to sequence the barcode including the universal marker. A 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 marker may include a nucleic acid sequence that can be hybridized with a PCR primer. In some embodiments, the universal marker may include a nucleic acid sequence that can be hybridized with a sequencing primer and a PCR primer. The nucleic acid sequence of the universal marker that can be hybridized with a sequencing primer or a PCR primer may be referred to as a primer binding site. The universal marker may include a sequence that can be used to initiate transcription of a barcode. The universal marker may include a sequence that can be used to extend a barcode or a region within a barcode. The length of the universal tag can be or can 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. For example, the universal tag can include at least about 10 nucleotides. The length of the universal tag can be at least or can be at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal tag sequence to enable the barcode to be cleaved from the support.

[0071] Dimension tagging

[0072] The barcode may include one or more dimensional markers. In some embodiments, the dimensional marker may include a nucleic acid sequence that provides information about the dimension in which the marker (e.g., random marker) occurs. For example, the dimensional marker may provide information about the time when the target is barcoded. The dimensional marker may be associated with the time of barcoding (e.g., random barcoding) in the sample. The dimensional marker may be activated at the time of the marker. Different dimensional markers may be activated at different times. The dimensional marker provides information about the order in which the target, the target group, and / or the sample are barcoded. For example, a population of cells may be barcoded in the G0 phase of the cell cycle. In the G1 phase of the cell cycle, the cell may be pulsed again with a barcode (e.g., a random barcode). In the S phase of the cell cycle, the cell may be pulsed again with a barcode, and so on. The barcode at each pulse (e.g., each period of the cell cycle) may include different dimensional markers. In this way, the dimensional marker provides information about which targets are labeled in which period of the cell cycle. Dimensional markers can interrogate many different biological times. Exemplary biological events may 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 population of cells) may be labeled before and / or after treatment with a drug and / or therapy. Changes in the copy number of different targets may indicate the response of a sample to a drug and / or therapy.

[0073] Dimensional markers can be activatable. Activatable dimensional markers can be activated at a specific time point. Activatable markers can be, for example, constitutively activated (for example, not closed). Activatable dimensional markers can be, for example, reversibly activated (for example, activatable dimensional markers can be turned on and off). Dimensional markers can be, for example, reversibly activated at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times. Dimensional markers can be reversibly activated, for example, at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times. In some embodiments, the dimensional markers can be activated by fluorescence, light, chemical events (for example, cleavage, connecting another molecule, adding modifications (for example, pegylation, ubiquitination (sumoylate), acetylation, methylation, deacetylation, demethylation), photochemical events (for example, photocaging) and the introduction of non-natural nucleotides.

[0074] In some embodiments, the dimension mark can be the same for all bar codes (e.g., random bar codes) attached to a given solid support (e.g., beads), but 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 bar codes on the same solid support can include the same dimension mark. In some embodiments, at least 60% of the bar codes on the same solid support can include the same dimension mark. In some embodiments, at least 95% of the bar codes on the same solid support can include the same dimension mark.

[0075] Up to 10 may be present on more than one solid support (e.g., beads). 6 The length of the dimension marker can be as follows or can be about as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any two of these values ​​between the number or range of nucleotides. The length of the dimension marker can be at least as follows or can be at most as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. The dimension marker can include about 5 to about 200 nucleotides. The dimension marker can include about 10 to about 150 nucleotides. The dimension marker can include a length of about 20 to about 125 nucleotides.

[0076] Space Marking

[0077] The barcode may include one or more spatial markers. In some embodiments, the spatial marker may include a nucleic acid sequence that provides information about the spatial orientation of the target molecule associated with the barcode. The spatial marker may be associated with a coordinate in the sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed with reference to a substrate. The spatial marker may be referenced to a two-dimensional or three-dimensional grid. The coordinate may be fixed with reference to a landmark. A landmark may be identified in space. A landmark may be a structure that may be imaged. A landmark may be a biological structure, such as an anatomical landmark. A landmark may be a cell landmark, such as an organelle. A landmark may be a non-natural landmark, such as a structure with an identifiable identifier (such as a color code, a barcode, a magnetic property, fluorescence, radioactivity, or a unique size or shape). The spatial marker may be associated with a physical partition (e.g., a hole, a container, or a droplet). In some embodiments, more than one spatial marker may be used together to encode one or more positions in space.

[0078] The spatial tag can be the same for all barcodes attached to a given solid support (e.g., beads), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same spatial tag on the same solid support can be the following or can be about the following: 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 spatial tag on the same solid support can be at least the following or at most the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. In some embodiments, at least 60% of the barcodes on the same solid support can contain the same spatial tag. In some embodiments, at least 95% of the barcodes on the same solid support can contain the same spatial tag.

[0079] Up to 10 may be present on more than one solid support (e.g., beads). 6 The length of the spatial marker can be as follows or can be about: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or any two of these values ​​between the nucleotide of the number or range. The length of the spatial marker can be at least below or at most below: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. The spatial marker can include about 5 to about 200 nucleotides. The spatial marker can include about 10 to about 150 nucleotides. The spatial marker can include a length of about 20 to about 125 nucleotides.

[0080] Cell labeling

[0081] Barcodes (e.g., random barcodes) may include one or more cell markers. In some embodiments, cell markers may include nucleic acid sequences that provide information for determining which target nucleic acid is derived from which cell. In some embodiments, cell markers are identical for all barcodes attached to a given solid support (e.g., beads), but are different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support may be as follows or may be approximately as follows: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100% or any two of these values. In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support may be as follows or may be approximately as follows: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. For example, at least 60% of the barcodes on the same solid support may include the same cell marker. As another example, at least 95% of the barcodes on the same solid support can comprise the same cellular marker.

[0082] Up to 10 may be present on more than one solid support (e.g., beads). 6 In some embodiments, the cell marker can be a nucleotide sequence having at least one ...

[0083] Barcode sequence

[0084] The barcode can comprise one or more barcode sequences. In some embodiments, the barcode sequence can comprise a nucleic acid sequence that provides identification information for a particular type of target nucleic acid species that hybridizes to the barcode. The barcode sequence can comprise a nucleic acid sequence that provides a counter (e.g., provides a rough estimate) for a particular occurrence of a target nucleic acid species that hybridizes to a barcode (e.g., a target binding region).

[0085] In some embodiments, a set of diverse barcode sequences are attached to a given solid support (e.g., a bead). In some embodiments, there may be the following, or there may be about the following unique molecular marker sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 or a number or range between any two of these values. For example, more than one barcode may include about 6561 barcode sequences with different sequences. As another example, more than one barcode may include about 65536 barcode sequences with different sequences. In some embodiments, there may be at least the following, or at most the following unique barcode sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Species or 10 9 The unique molecular marker sequence can be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular marker sequence is partially or completely contained by the particle (e.g., a hydrogel bead).

[0086] In different embodiments, the length of the barcode can be different. For example, the length of the barcode can be or can 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. As another example, the length of the barcode can be at least or can be at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0087] Molecular markers

[0088] The barcode (e.g., a random barcode) can include one or more molecular markers. The molecular marker can include a barcode sequence. In some embodiments, the molecular marker can include a nucleic acid sequence that provides identification information for a specific type of target nucleic acid species that hybridizes to the barcode. The molecular marker can include a nucleic acid sequence that provides a counter for a specific occurrence of a target nucleic acid species that hybridizes to a barcode (e.g., a target binding region).

[0089] In some embodiments, a set of distinct molecular markers are attached to a given solid support (e.g., a bead). In some embodiments, there may be the following, or there may be about the following unique molecular marker sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 or a number or range between any two of these values. For example, more than one barcode may include about 6561 molecular markers with different sequences. As another example, more than one barcode may include about 65536 molecular markers with different sequences. In some embodiments, there may be at least the following, or at most the following unique molecular marker sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Species or 10 9 A barcode having a unique molecular marker sequence can be attached to a given solid support (e.g., a bead).

[0090] For barcoding using more than one random barcode (e.g., random barcoding), the ratio of the number of different molecular marker sequences to the number of occurrences of any target can be or can 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 can be an mRNA species including mRNA molecules having the same or nearly the same sequence. In some embodiments, the ratio of the number of different molecular marker sequences to the number of occurrences of any target 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.

[0091] The length of a molecular marker can be or can be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range of nucleotides between any two of these values. The length of a molecular marker can be at least or can be at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0092] Target binding region

[0093] The barcode may include one or more target binding regions, such as capture probes. In some embodiments, the target binding region may hybridize with a target of interest. In some embodiments, the target binding region may include a nucleic acid sequence that specifically hybridizes with a target (e.g., a target nucleic acid, a target molecule, e.g., a cell nucleic acid to be analyzed) (e.g., specifically hybridizes with a specific gene sequence). In some embodiments, the target binding region may include a nucleic acid sequence that can be attached (e.g., hybridized) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region may include a nucleic acid sequence that can specifically hybridize with a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode may then be connected to any nucleic acid molecule comprising a sequence complementary to a restriction site overhang.

[0094] In some embodiments, the target binding region may include a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence may refer to a sequence that can bind to more than one target nucleic acid independently of a specific sequence of the target nucleic acid. For example, the target binding region may include a random polymer 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 hybridized with a poly (A) tail on an mRNA molecule. A random polymer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or a higher polymer sequence of any length. In some embodiments, the target binding region is identical for all barcodes attached to a given bead. In some embodiments, for more than one barcode attached to a given bead, the target binding region may include two or more different target binding sequences. The length of the target binding region can be or can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or a number or range between any two of these values. The length of the target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. For example, a reverse transcriptase such as Moloney murine leukemia virus (MMLV) reverse transcriptase can be used to reverse transcribe an mRNA molecule to produce a cDNA molecule with a poly (dC) tail. The barcode can include a target binding region with a poly (dG) tail. After base pairing between the poly (dG) tail of the barcode and the poly (dC) tail of the cDNA molecule, the reverse transcriptase converts the template strand from the cellular RNA molecule to the barcode and continues to replicate to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains a barcode sequence (such as a molecular tag) at the 3' end of the cDNA molecule.

[0095] In some embodiments, the target binding region can include oligo(dT), which can hybridize to an mRNA comprising a polyadenylated end. The target binding region can be gene specific. For example, the target binding region can be configured to hybridize to a specific region of the target. The length of the target binding region can be or can be about 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 of nucleotides between any two of these values. The length of the target binding region can be at least or can be 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. The length of the target binding region can be about 5-30 nucleotides. When the barcode comprises a gene-specific target binding region, the barcode can be referred to herein as a gene-specific barcode.

[0096] Orientation Property

[0097] Random barcodes (e.g., random barcodes) can include one or more directional properties that can be used to orient (e.g., align) the barcodes. The barcodes can include portions for isoelectric focusing. Different barcodes can include different isoelectric focusing points. When these barcodes are introduced into a sample, the sample can undergo isoelectric focusing to orient the barcodes in a known manner. In this way, the directional properties can be used to develop known mappings of barcodes in a sample. Exemplary directional 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 a directional property of self-assembly can self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0098] Affinity Property

[0099] Barcodes (e.g., random barcodes) can include one or more affinity properties. For example, spatial labels can include affinity properties. Affinity properties can include chemical and / or biological parts that can promote the binding of barcodes to another entity (e.g., cell receptors). For example, affinity properties can include antibodies, for example, antibodies specific to specific parts (e.g., receptors) on a sample. In some embodiments, antibodies can guide barcodes to specific cell types or molecules. Targets at and / or near specific cell types or molecules can be marked (e.g., randomly marked). In some embodiments, affinity properties can provide spatial information beyond the nucleotide sequence of the spatial label because antibodies can guide barcodes to specific locations. The antibody can be a therapeutic antibody, such as a monoclonal antibody or a polyclonal antibody. The antibody can be humanized or chimeric. The antibody can be a naked antibody or a fusion antibody.

[0100] Antibodies can be full-length (ie, naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) immunoglobulin molecules (eg, IgG antibodies) or immunologically active (ie, specifically binding) portions of immunoglobulin molecules (eg, antibody fragments).

[0101] The antibody fragment can be, for example, a part of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include a separated fragment consisting of the variable region of the antibody, such as a "Fv" fragment consisting of the variable region of the heavy chain and the light chain, and a recombinant single-chain polypeptide molecule ("scFv protein") in which the light chain and the heavy chain variable region are connected by a peptide linker. Exemplary antibodies can include, but are not limited to, cancer cell antibodies, virus antibodies, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0102] Universal adapter primer

[0103] The barcode may comprise one or more universal adapter primers. For example, a gene-specific barcode (such as a gene-specific random barcode) may comprise a universal adapter primer. A universal adapter primer may refer to a universal nucleotide sequence across all barcodes. A universal adapter primer may be used to construct a gene-specific barcode. The length of a universal adapter primer may be or may be about 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 of nucleotides between any two of these values. The length of the universal adapter primer can be at least or can be 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. The length of the universal adapter primer can be 5-30 nucleotides.

[0104] Connectors

[0105] When the barcode comprises more than one type of marker (e.g., more than one cell marker or more than one barcode sequence, such as a molecular marker), the markers may be interspersed with adapter marker sequences. The length of the adapter marker sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. The length of the adapter marker sequence may be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. In some cases, the length of the adapter marker sequence is 12 nucleotides. The adapter marker sequence can be used to facilitate the synthesis of the barcode. The adapter marker may include an error correction (e.g., Hamming) code.

[0106] Solid support

[0107] In some embodiments, the barcodes disclosed herein (such as random barcodes) can be associated with solid supports. The solid support can be, for example, synthetic particles. In some embodiments, some or all barcode sequences (such as, molecular markers of random barcodes (e.g., first barcode sequences)) of more than one barcode on a solid support differ by at least one nucleotide. The cell markers of the barcodes on the same solid support can be the same. The cell markers of the barcodes on different solid supports can differ by at least one nucleotide. For example, the first cell marker of the first more than one barcode on the first solid support can have the same sequence, and the second cell marker of the second more than one barcode on the second solid support can have the same sequence. The first cell marker of the first more than one barcode on the first solid support and the second cell marker of the second more than one barcode on the second solid support can differ by at least one nucleotide. The cell marker can be, for example, about 5-20 nucleotides long. The barcode sequence can be, for example, about 5-20 nucleotides long. The synthetic particles can be, for example, beads.

[0108] The beads can be, for example, silica beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / agarose 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 substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or any combination thereof.

[0109] In some embodiments, the beads can be polymer beads (e.g., deformable beads or gel beads) functionalized with barcodes or random barcodes (such as gel beads from 10X Genomics (San Francisco, CA)). In some embodiments, the gel beads can include a polymer-based gel. The gel beads can be produced, for example, by encapsulating one or more polymer precursors into droplets. The gel beads can be produced after exposing the polymer precursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0110] In some embodiments, the particles can be destructible (e.g., soluble, degradable). For example, the polymer beads can dissolve, melt or degrade, for example, under desired conditions. The desired conditions can include environmental conditions. The desired conditions can cause the polymer beads to dissolve, melt or degrade in a controlled manner. The gel beads can dissolve, melt or degrade due to chemical stimulation, physical stimulation, biological stimulation, thermal stimulation, magnetic stimulation, electrical stimulation, light stimulation or any combination thereof.

[0111] For example, analyte and / or reagent (such as oligonucleotide barcode) can be coupled / fixed to the inner surface of gel beads (for example, via the diffusion of oligonucleotide barcode and / or the material for producing oligonucleotide barcode) and / or the outer surface of gel beads or any other microcapsule described herein. Coupling / fixation can be via any form of chemical bonding (for example, covalent bond, ionic bond) or physical phenomenon (for example, van der Waals force, dipole-dipole interaction, etc.). In some embodiments, the coupling / fixation of reagent described herein with gel beads or any other microcapsule can be reversible, such as, for example, via unstable part (for example, via chemical crosslinking, including chemical crosslinking described herein). After applying stimulation, the unstable part can be cleaved and release the fixed reagent. In some embodiments, the unstable part is a disulfide bond. For example, in the case where the oligonucleotide barcode is fixed to the gel beads via a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the beads. The labile moiety can be included as part of a gel bead or microcapsule, as part of a chemical linker that connects a reagent or analyte to a gel bead or microcapsule, and / or as part of a reagent or analyte. In some embodiments, at least one of the more than one barcodes can be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof.

[0112] In some embodiments, the gel beads may include a wide range of different polymers, including but not limited to: polymers, thermosensitive polymers, photosensitive polymers, magnetic polymers, pH sensitive polymers, salt sensitive polymers, chemical sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins and / or plastics. The polymer may include, but is not limited to, materials such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(bisallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrrole) (poly(pyrolle), PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(o-phthalaldehyde) (PTHF), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic-co-glycolic acid) (PLGA).

[0113] Many chemical stimuli can be used to trigger the destruction, dissolution or degradation of beads. Examples of these chemical changes can include, but are not limited to, pH-mediated changes in the bead wall, disintegration of the bead wall via chemical cleavage of cross-links, triggered depolymerization of the bead wall, and bead wall switching reactions. Bulk changes can also be used to trigger the destruction of beads.

[0114] Bulk or physical alteration of microcapsules by various stimuli also provides many advantages in designing capsules to release agents. Bulk or physical alteration occurs on a macroscopic scale, where bead rupture is the result of mechanical-physical forces caused by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in the porosity of the bead wall.

[0115] Biostimulation can also be used to trigger the destruction, dissolution or degradation of beads. Generally, biological triggers are similar to chemical triggers, but many examples use biomolecules or molecules common in living systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, etc. For example, beads can include polymers with peptide crosslinks that are sensitive to cleavage by specific proteases. More specifically, one example can include microcapsules containing GFLGK peptide crosslinks. Upon addition of a biological trigger (such as the protease cathepsin B), the peptide crosslinks of the shell wall are cleaved and the contents of the beads are released. In other cases, the protease can be heat-activated. In another example, the beads include a shell wall that includes cellulose. The addition of chitosan hydrolase serves as a biological trigger for cleavage of cellulose bonds, depolymerization of the shell wall and release of its internal contents.

[0116] The beads can also be induced to release their contents upon application of a thermal stimulus. Changes in temperature can cause various changes in the beads. Changes in heat can cause the beads to melt, causing the bead wall to disintegrate. In other cases, heat can increase the internal pressure of the internal components of the beads, causing the beads to rupture or explode. In still other cases, heat can cause the beads to transform into a shrunken, dehydrated state. Heat can also act on the thermosensitive polymers within the bead wall, causing the destruction of the beads.

[0117] Including magnetic nanoparticles in the bead wall of the microcapsule can allow for triggered rupture of the beads and directing the beads into an array. The device of the present disclosure may include magnetic beads for any purpose. In one example, Fe3O4 nanoparticles are incorporated into beads containing polyelectrolytes to trigger rupture in the presence of an oscillating magnetic field stimulus.

[0118] The beads can also be destroyed, dissolved or degraded as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, the electrosensitive beads can allow for triggered rupture of the beads as well as other functions such as alignment in an electric field, conductivity or redox reactions. In one example, beads containing electrosensitive materials align in an electric field so that the release of an internal agent can be controlled. In other examples, the electric field can induce a redox reaction within the bead wall itself, which can increase porosity.

[0119] Light stimulation can also be used to destroy the beads. Many light triggers are possible and can include systems using various molecules such as nanoparticles and chromophores that can absorb photons of a specific wavelength range. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of polyelectrolyte capsules coated with SiO2 can lead to the disintegration of the bead wall. In yet another example, light-switchable materials such as azobenzene groups can be incorporated into the bead wall. Upon application of UV or visible light, chemicals such as these undergo reversible cis-to-trans isomerization upon absorption of photons. In this regard, the incorporation of a photon switch produces a bead wall that can disintegrate or become more porous upon application of a light trigger.

[0120] For example, in Figure 2 In the non-limiting example of barcoding (e.g., random barcoding) illustrated in FIG, after introducing cells such as single cells into more than one microwell of a microwell array at block 208, beads may be introduced into more than one microwell of a microwell array at block 212. Each microwell may contain one bead. The beads may contain more than one barcode. The barcode may contain a 5' amine region attached to the bead. The barcode may contain a universal tag, a barcode sequence (e.g., a molecular tag), a target binding region, or any combination thereof.

[0121] The barcodes disclosed herein can be associated (e.g., attached) with a solid support (e.g., a bead). The barcodes associated with a solid support can each include a barcode sequence selected from the following group, the group including at least 100 or 1000 barcode sequences with unique sequences. In some embodiments, different barcodes associated with a solid support can include barcodes with different sequences. In some embodiments, a certain percentage of the barcodes associated with a solid support include the same cell marker. For example, the percentage can be the following or can be about the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100% or a number or range between any two of these values. As another example, the percentage can be at least the following or can be at most the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. In some embodiments, the barcodes associated with a solid support can have the same cell marker. The barcodes associated with different solid supports can have different cell markers selected from the group consisting of at least 100 or 1000 cell markers having unique sequences.

[0122] The barcodes disclosed herein can be associated (e.g., attached) with a solid support (e.g., a bead). In some embodiments, more than one target in a sample can be barcoded with a solid support including more than one synthetic particle associated with more than one barcode. In some embodiments, a solid support can include more than one synthetic particle associated with more than one barcode. The spatial markings of more than one barcode on different solid supports can differ by at least one nucleotide. The solid support can include more than one barcode, for example, in two or three dimensions. The synthetic particle can be a bead. The bead can be a silica bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a Sephadex / agarose gel bead, a cellulose bead, a polystyrene bead, or any combination thereof. The solid support can include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support can float freely. In some embodiments, the solid support can be embedded in a semi-solid or solid array. The barcode may not be associated with the solid support. The barcode can be a single nucleotide. The barcode can be associated with a substrate.

[0123] As used herein, the terms "tethered," "attached," and "fixed" can be 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 as a solid support for attaching pre-synthesized barcodes or for in situ solid phase synthesis of barcodes.

[0124] In some embodiments, the solid support is a bead. The bead may include one or more types of solid, porous or hollow spheres, balls, sockets, cylinders, or other similar configurations that can fix nucleic acids (e.g., covalently or non-covalently). The bead may comprise, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. The bead may be or include spherical (e.g., microspheres) or discrete particles having a non-spherical or irregular shape, such as a cubic, rectangular, conical, cylindrical, conical, elliptical, or disc-shaped, etc. In some embodiments, the shape of the bead may be non-spherical.

[0125] The beads can include 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), ceramics, plastics, glass, polystyrene, silica, methyl styrene, acrylic polymers, titanium, latex, agarose gel, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof.

[0126] In some embodiments, the beads (e.g., beads to which labels are attached) are hydrogel beads. In some embodiments, the beads include a hydrogel.

[0127] Some embodiments disclosed herein include one or more particles (e.g., beads). Each particle can contain more than one oligonucleotide (e.g., barcode). Each of more than one oligonucleotide can contain a barcode sequence (e.g., a molecular marker sequence), a cell marker, and a target binding region (e.g., an oligo (dT) sequence, a gene-specific sequence, a random polymer, or a combination thereof). The cell marker sequence of each of more than one oligonucleotide can be the same. The cell marker sequences of the oligonucleotides on different particles can be different, so that the oligonucleotides on different particles can be identified. In different embodiments, the number of different cell marker sequences can be different. In some embodiments, the number of cell marker sequences may be or may be about the following: 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, 10 ... 6 , 10 7 , 10 8 , 10 9, a number or range between any two of these values ​​or more. In some embodiments, the number of cell marker sequences can be at least the following or at most the following: 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, 10 ... 6 , 10 7 , 10 8 or 10 9 In 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 particles in more than one particle include oligonucleotides with the same cell sequence. In some embodiments, more than one particle including oligonucleotides with the same cell sequence can be at most 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. In some embodiments, all of the more than one particles do not have the same cell marker sequence.

[0128] More than one oligonucleotide on each particle can comprise different barcode sequences (e.g., molecular markers). In some embodiments, the number of barcode sequences can be or can be about the following: 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, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of barcode sequences can be at least the following or can be at most the following: 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, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 or 10 9 For example, at least 100 of the more than one 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 more than one oligonucleotides comprise different barcode sequences. Some embodiments provide more than one particle comprising a barcode. In some embodiments, the ratio of the occurrence (or copies or number) of the target to be labeled and the different 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 more than one oligonucleotides further comprises a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or a microparticle.

[0129] The size of the beads can be different. For example, the diameter of the beads can range from 0.1 micron to 50 microns. In some embodiments, the diameter of the beads can be or can be about 0.1 micron, 0.5 micron, 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, or a number or range between any two of these values.

[0130] The diameter of the bead can be related to the diameter of the hole of the substrate. In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the hole or about less than: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or a number or range between any two of these values. The diameter of the bead can be related to the diameter of the cell (e.g., a single cell captured by the hole of the substrate). In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the hole by at least less than or at most less than: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The diameter of the bead can be related to the diameter of the cell (e.g., a single cell captured by the hole of the substrate). In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the cell by less than or about less than 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. In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the cell by at least less than or at most less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300%.

[0131] The beads can be attached to a substrate and / or embedded in a substrate. The beads can be attached to a gel, a hydrogel, a polymer and / or a matrix and / or embedded in a gel, a hydrogel, a polymer and / or a matrix. The spatial position of the beads in a substrate (e.g., a gel, a matrix, a support or a polymer) can be identified using a spatial marker present on a barcode on the beads, which can be used as a positional address.

[0132] Examples of beads may include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, 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 TM Carboxyl-terminated magnetic beads.

[0133] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluoresce in one fluorescent optical channel or more than one optical channel. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be identifiable. For example, a camera can be used to image the beads. The beads can have a detectable code associated with the beads. For example, the beads can contain 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.

[0134] The solid support (e.g., bead) can be visualized. The solid support can include a visualization label (e.g., a fluorescent dye). The solid support (e.g., bead) can be etched with an identifier (e.g., a number). The identifier can be visualized by imaging the bead.

[0135] The solid support may include soluble, semi-soluble or insoluble materials. When the solid support includes a linker, a scaffold, a building block or other reactive moiety attached thereto, the solid support may be referred to as "functionalized", while when the solid support lacks such reactive moieties attached thereto, the solid support may be referred to as "non-functionalized". The solid support may be free in solution, such as in a microtiter well; in a flow-through form, such as in a column; or used as a dipstick.

[0136] Solid supports can include films, paper, plastics, coated surfaces, flat surfaces, glass, slides, chips or any combination thereof. Solid supports can take the form of resins, gels, microspheres or other geometric configurations. Solid supports can include silicon dioxide chips, micron particles, nanoparticles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, films, microplates, slides, plastic materials including porous plates or films (e.g., formed by polyethylene, polypropylene, polyamide, polyvinylidene fluoride), and / or wafers, combs, needles or pinheads (e.g., suitable for combined synthesis or analysis of needle arrays) or beads, flat surfaces such as recesses or nanoliter well arrays of wafers (e.g., silicon wafers), wafers with recesses (with or without filter bottoms).

[0137] The solid support may include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix may be capable of permeating intracellular spaces (e.g., around organelles). The polymer matrix may be capable of being pumped throughout the circulatory system.

[0138] Substrates and microwell arrays

[0139] As used herein, substrate can refer to a solid support type. Substrate can refer to a solid support that can contain a barcode or a random barcode of the present disclosure. Substrate can, for example, include more than one microwell. Substrate can, for example, be a hole array including two or more microwells. In some embodiments, microwells can include a small reaction chamber of a defined volume. In some embodiments, microwells can capture one or more cells. In some embodiments, microwells can only capture one cell. In some embodiments, microwells can capture one or more solid supports. In some embodiments, microwells can only capture one solid support. In some embodiments, microwells capture single cells and single solid supports (e.g., beads). Microwells can contain barcode reagents of the present disclosure.

[0140] Barcoding method

[0141] The present disclosure provides methods for estimating the number of different targets at different locations in a body sample (e.g., tissue, organ, tumor, cell). The method may include placing a barcode (e.g., a random barcode) in close proximity to the sample, lysing the sample, associating different targets with the barcode, amplifying the target and / or digitally counting the target. The method may also include analyzing and / or visualizing the information obtained from the spatial markers on the barcode. In some embodiments, the method includes visualizing more than one target in the sample. Mapping more than one target to a map of the sample may include generating a two-dimensional map or a three-dimensional map of the sample. Two-dimensional maps and three-dimensional maps may be generated before or after barcoding more than one target in the sample (e.g., random barcoding). Visualizing more than one target in the sample may include mapping more than one target to a map of the sample. Mapping more than one target to a map of the sample may include generating a two-dimensional map or a three-dimensional map of the sample. Two-dimensional maps and three-dimensional maps may be generated before or after barcoding more than one target in the sample. In some embodiments, the two-dimensional map and the three-dimensional map can be generated before or after the sample is lysed. Lysing the sample before or after generating the two-dimensional map or the three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0142] In some embodiments, barcoding more than one target comprises hybridizing more than one barcode to more than one target to generate barcoded targets (e.g., randomly barcoded targets). Barcoding more than one target can comprise generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets can be performed with a solid support comprising more than one barcode (e.g., random barcodes).

[0143] Bring the sample and barcode into contact

[0144] The present disclosure provides methods for contacting a sample (e.g., a cell) with a substrate of the present disclosure. Samples including, for example, thin sections of cells, organs, or tissues can be contacted with a barcode (e.g., a random barcode). Cells can be contacted, for example, by gravity flow, wherein the cells can be sedimented and a monolayer can be produced. The sample can be a thin section of tissue. The thin section can be placed on a substrate. The sample can be one-dimensional (e.g., forming a flat surface). The sample (e.g., a cell) can be dispersed throughout a substrate, for example, by growing / culturing cells on a substrate.

[0145] 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-exhaustive ratio so that each different target can be associated with a different barcode of the present disclosure. To ensure effective association between the target and the barcode, the target and the barcode can be cross-linked.

[0146] Cell lysis

[0147] After the distribution of cells and barcodes, the cells can be lysed to release the target molecules. Cell lysis can be accomplished by any of a variety of means, such as by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis or optical lysis. Cells can be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, lithium 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. In order to increase the association of the target with the barcode, the diffusion rate of the target molecule can be changed by, for example, reducing the temperature of the lysate and / or increasing the viscosity of the lysate.

[0148] In some embodiments, filter paper can be used to crack the sample. The filter paper can be soaked with lysis buffer on the top of the filter paper. The filter paper can be applied to the sample with pressure, which can promote the cracking of the sample and the hybridization of the target of the sample with the substrate.

[0149] 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. Cleavage can be performed by adding lysis buffer to the substrate. Lysis buffer can include Tris HCl. Lysis buffer can include at least about 0.01M, 0.05M, 0.1M, 0.5M or 1M or more Tris HCl. Lysis buffer can include up to about 0.01M, 0.05M, 0.1M, 0.5M or 1M or more Tris HCl. Lysis buffer can include about 0.1M Tris HCl. The pH of lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. The pH of lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. In some embodiments, the pH of lysis buffer is about 7.5. Lysis buffer can include salt (e.g., LiCl). The salt concentration in the lysis buffer can be at least about 0.1M, 0.5M, or 1M or more. The salt concentration in the lysis buffer can be up to about 0.1M, 0.5M, or 1M or more. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5M. The lysis buffer can contain a detergent (e.g., SDS, lithium dodecyl sulfate, triton X, Tween, NP-40). The detergent concentration in the lysis buffer can 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 more. The detergent concentration in the lysis buffer can 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 more. In some embodiments, the detergent concentration in the lysis buffer is about 1% lithium dodecyl sulfate. The time used in the lysis method can depend on the amount of detergent used. In some embodiments, the more detergent is used, the less time is required for lysis. The lysis buffer can contain a chelating agent (e.g., EDTA, EGTA). The chelating agent concentration in the lysis buffer can be at least about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM or 30mM or more. The chelating agent concentration in the lysis buffer can be at most about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM or 30mM or higher. In some embodiments, the chelating agent concentration in the lysis buffer is about 10mM. The lysis buffer can contain a reducing agent (e.g., β-mercaptoethanol, DTT). The reducing agent concentration in the lysis buffer can be at least about 1mM, 5mM, 10mM, 15mM or 20mM or higher.The reducing agent concentration in the lysis buffer can be up to about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or higher. In some embodiments, the reducing agent concentration in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer can 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.

[0150] The lysis can be carried out at a temperature of about 4°C, 10°C, 15°C, 20°C, 25°C or 30°C. The lysis can be carried out for about 1 minute, 5 minutes, 10 minutes, 15 minutes or 20 minutes or more minutes. The lysis cells can include at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000 or 700,000 or more target nucleic acid molecules. The lysis cells can include at most about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000 or 700,000 or more target nucleic acid molecules.

[0151] Attaching barcodes to target nucleic acid molecules

[0152] After cell lysis and nucleic acid molecules are released from the cells, the nucleic acid molecules can be randomly associated with the barcodes of the co-located solid support. The association can include hybridizing the target recognition region of the barcode with the complementary portion of the target nucleic acid molecule (for example, the oligo (dT) of the barcode can interact with the poly (A) tail of the target). The assay conditions for hybridization (for example, buffer pH, ionic strength, temperature, etc.) can be selected to promote the formation of specific stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with more than one probe on the substrate (for example, hybridized with the probe on the substrate). When the probe contains oligo (dT), the mRNA molecule can be hybridized with the probe and reverse transcribed. The oligo (dT) portion of the oligonucleotide can act as a primer for the first chain synthesis of the cDNA molecule. For example, in Figure 2 In the non-limiting example of barcoding illustrated in FIG, at block 216, the mRNA molecule can be hybridized to the barcode on the bead. For example, a single-stranded nucleotide fragment can be hybridized to the target binding region of the barcode.

[0153] Attachment can also include connecting the target recognition region of the barcode to a portion of the target nucleic acid molecule. For example, the target binding region can include a nucleic acid sequence that can be specifically hybridized with a restriction site overhang (e.g., EcoRI sticky end overhang). The assay procedure can also include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to produce a restriction site overhang. The barcode can then be connected to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang. Ligase (e.g., T4 DNA ligase) can be used to connect two fragments.

[0154] For example, in Figure 2 In the non-limiting example of barcoding illustrated in FIG, labeled targets (e.g., target-barcode molecules) from more than one cell (or more than one sample) can then be pooled, for example, into a tube at block 220. The labeled targets can be pooled by, for example, retrieving barcodes and / or beads to which target-barcode molecules are attached.

[0155] Recovery of a solid support-based collection of attached target-barcode molecules can be achieved by using magnetic beads and an externally applied magnetic field. After pooling the target-barcode molecules, all further processing can be performed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions and / or nucleic acid extension reactions. Further processing reactions can be performed within the microwells, i.e., without first pooling the labeled target nucleic acid molecules from more than one cell.

[0156] Reverse transcription or nucleic acid extension

[0157] The present disclosure provides methods for using reverse transcription (e.g., in Figure 2 The method of producing a target-barcode conjugate by a barcode or nucleic acid extension. The target-barcode conjugate may include a barcode and a complementary sequence of all or a portion of a target nucleic acid (i.e., a barcoded cDNA molecule, such as a randomly barcoded cDNA molecule). Reverse transcription of the associated RNA molecule may occur by adding a reverse transcription primer together with a reverse transcriptase. The reverse transcription primer may be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The length of the oligo(dT) primer may be 12-18 nucleotides or may be about 12-18 nucleotides and is bound to an endogenous poly(A) tail at the 3' end of a mammalian mRNA. The random hexanucleotide primer may bind to the mRNA at each complementary site. The target-specific oligonucleotide primer typically selectively triggers the mRNA of interest.

[0158] In some embodiments, the reverse transcription of mRNA molecules to the RNA molecules of the mark can occur by adding 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, the length of the oligo (dT) primer is 12-18 nucleotides, and is combined with the endogenous poly (A) tail at the 3' end of mammalian mRNA. Random hexanucleotide primers can be combined with mRNA at each complementary site. Target-specific oligonucleotide primers selectively trigger mRNA of interest usually.

[0159] In some embodiments, the target is a cDNA molecule. For example, a reverse transcriptase such as Moloney murine leukemia virus (MMLV) reverse transcriptase can be used to reverse transcribe an mRNA molecule to produce a cDNA molecule with a poly (dC) tail. The barcode can include a target binding region with a poly (dG) tail. After base pairing between the poly (dG) tail of the barcode and the poly (dC) tail of the cDNA molecule, the reverse transcriptase converts the template strand from the cellular RNA molecule to the barcode and continues to replicate to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains a barcode sequence (such as a molecular marker) at the 3' end of the cDNA molecule.

[0160] Reverse transcription can occur repeatedly to produce more than one labeled cDNA molecule. 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 reverse transcription reactions. The methods may 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.

[0161] Amplification

[0162] One or more nucleic acid amplification reactions can be performed (e.g., Figure 2The amplification reaction may be performed in a multiplexed manner, wherein more than one target nucleic acid sequence is amplified simultaneously. The amplification reaction may be used to add sequencing adapters to the nucleic acid molecules. The amplification reaction may include amplifying at least a portion of the sample label (if present). The amplification reaction may include amplifying at least a portion of a cell label and / or a barcode sequence (e.g., a molecular marker). The amplification reaction may include amplifying at least a portion of a sample label, a cell label, a spatial label, a barcode sequence (e.g., a molecular marker), a target nucleic acid, or a combination thereof. The amplification reaction can 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 any range or number between two of these values ​​of more than one nucleic acid. The method can also include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of a target-barcode molecule comprising a sample marker, a cell marker, a spatial marker and / or a barcode sequence (e.g., a molecular marker).

[0163] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for simultaneously extending a primer of a complementary strand of DNA to amplify a specific DNA sequence in vitro. As used herein, PCR can encompass derivative forms of reactions, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR.

[0164] The amplification of the nucleic acid of labeling can include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), amplification based on nucleic acid sequences (NASBA), chain displacement amplification (SDA), real-time SDA, rolling circle amplification, or ring-to-ring amplification. Other non-PCR-based amplification methods include more than one cycle of DNA synthesis and transcription of DNA-dependent RNA polymerase-driven transcription amplification or RNA-guided DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) methods, the use of palindromic probes, chain displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, primers hybridized to nucleic acid sequences and the resulting duplexes cleaved before extension reactions and amplification, chain displacement amplification, rolling circle amplification, and branch extension amplification (RAM) using nucleic acid polymerases lacking 5' exonuclease activity. In some embodiments, amplification does not produce circularized transcripts.

[0165] In some embodiments, the method disclosed herein also includes carrying out polymerase chain reaction to the labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to produce labeled amplicon (e.g., randomly labeled amplicon). The labeled amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid of the present disclosure can include a synthetic or altered nucleic acid.

[0166] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable or triggerable nucleotides. Examples of non-natural nucleotides can include but are not limited to peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA) and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of the amplified reaction. Adding non-natural nucleotides can be used to identify the product of a specific cycle or time point in the amplified reaction.

[0167] Carrying out one or more amplification reactions can include using one or more primers.One or more primers can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides.One or more primers can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides.One or more primers can include less than 12-15 nucleotides.One or more primers can anneal to at least a portion of a target (for example, a randomly labeled target) of more than one mark.One or more primers can anneal to 3' ends or 5' ends of a target of more than one mark.One or more primers can anneal to the internal region of a target of more than one mark. 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, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910 In some embodiments, the present invention provides at least one or more primers of the present invention. 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. One or more primers can include a group of fixed primers. One or more primers can include at least one or more custom primers. One or more primers can include at least one or more control primers. One or more primers can include at least one or more gene specific primers.

[0168] One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers may anneal to a first sample marker, a second sample marker, a spatial marker, a cell marker, a barcode sequence (e.g., a molecular marker), a target, or any combination thereof. One or more primers may include universal primers and custom primers. Custom primers may be designed to amplify one or more targets. The target may include a subset of total nucleic acid in one or more samples. The target may include a subset of total marker targets in one or more samples. One or more primers may include at least 96 or more custom primers. One or more primers may include at least 960 or more custom primers. One or more primers may include at least 9600 or more custom primers. One or more custom primers may anneal to two or more different labeled nucleic acids. Two or more different labeled nucleic acids may correspond to one or more genes.

[0169] Any amplification scheme can be used in the methods of the present disclosure. For example, in one approach, the first round of PCR can use gene-specific primers and primers for universal Illumina sequencing primer 1 sequences to amplify molecules attached to beads. The second round of PCR can use nested gene-specific primers flanked by Illumina sequencing primer 2 sequences and primers for universal Illumina sequencing primer 1 sequences to amplify the first PCR product. The third round of PCR adds P5 and P7 and a sample index to turn the PCR product into an Illumina sequencing library. Sequencing using 150bp×2 sequencing can reveal cell markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and sample indexes on index 1 reads.

[0170] In some embodiments, chemical cleavage can be used to remove nucleic acid from substrate. For example, chemical groups or modified bases present in nucleic acid can be used to promote the removal of nucleic acid from solid support. For example, enzymes can be used to remove nucleic acid from substrate. For example, nucleic acid can be removed from substrate by restriction endonuclease digestion. For example, nucleic acid containing dUTP or ddUTP can be treated with uracil-d-glycosidase (UDG) to remove nucleic acid from substrate. For example, nucleic acid can be removed from substrate using enzymes (such as base excision repair enzymes, such as apurinic / apyrimidinic (ap) endonucleases) that perform nucleotide excision. In some embodiments, photocleavable groups and light can be used to remove nucleic acid from substrate. In some embodiments, cleavable joints can be used to remove nucleic acid from substrate. For example, cleavable joints can include at least one of the following: biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig protein A, light-labile joints, acid or base-labile joint groups or adapters.

[0171] When the probe is gene specific, the molecule can be hybridized with the probe and reverse transcribed and / or amplified. In some embodiments, after the nucleic acid has been synthesized (e.g., reverse transcribed), the nucleic acid can be amplified. Amplification can be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acid.

[0172] In some embodiments, amplification can be performed on substrates, for example, with bridging amplification.cDNA can be added with homopolymer tails to produce compatible ends for bridging amplification using oligo (dT) probes on substrates.In bridging amplification, the primer complementary to the 3' end of the template nucleic acid can be the first primer in each pair of primers covalently attached to solid particles.When the sample containing the template nucleic acid contacts the particle and performs a single thermal cycle, the template molecule can be annealed to the first primer, and the first primer is extended forward by adding nucleotides to form a duplex molecule, which is composed of the template molecule and the newly formed DNA chain complementary to the template.In the heating step of the next cycle, the duplex molecule can be denatured, release the template molecule from the particle and leave the complementary DNA chain attached to the particle by the first primer.In the annealing stage of the subsequent annealing and extension step, the complementary chain can be hybridized with the second primer, and the second primer is complementary to the segment of the complementary chain at the position removed from the first primer. This hybridization can cause complementary strands to form a bridge between the first primer and the second primer, connecting the first primer by a covalent bond and connecting the second primer by hybridization. In the extension phase, by adding nucleotides in the same reaction mixture, the second primer can be extended in the reverse direction, thereby converting the bridge into a double-stranded bridge. Then start the next cycle, and the double-stranded bridge can be denatured to produce two single-stranded nucleic acid molecules, each of which has an end that is attached to the particle surface via the first primer and the second primer, respectively, wherein the other end of each single-stranded nucleic acid molecule is unattached. In the annealing and extension steps of this second cycle, each chain can hybridize with other complementary primers that were not previously used on the same particle to form a new single-stranded bridge. Now the two previously unused primers that hybridize extend so that two new bridges are converted into double-stranded bridges.

[0173] 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 more than one nucleic acid.

[0174] The amplification of the labeled nucleic acid may include a PCR-based method or a non-PCR-based method. The amplification of the labeled nucleic acid may include an exponential amplification of the labeled nucleic acid. The amplification of the labeled nucleic acid may include a linear amplification of the labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for simultaneously extending the primers of the complementary strands of DNA to amplify a specific DNA sequence in vitro. PCR may encompass derivative forms of reactions, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, inhibition PCR, semi-inhibition PCR, and assembly PCR.

[0175] In some embodiments, the amplification of the labeled nucleic acid includes 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), chain displacement amplification (SDA), real-time SDA, rolling circle amplification, or ring-to-ring amplification. Other non-PCR-based amplification methods include more than one cycle of DNA synthesis and transcription of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-guided DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ) method, use of palindromic probes, chain displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, primers hybridized to nucleic acid sequences and the resulting duplexes cleaved before extension reactions and amplification, chain displacement amplification using nucleic acid polymerases lacking 5' exonuclease activity, rolling circle amplification, and / or branch extension amplification (RAM).

[0176] The methods disclosed herein can also include performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample label or a molecular identifier tag. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid of the present invention can include a synthetic or altered nucleic acid.

[0177] In some embodiments, the method includes repeatedly amplifying the labeled nucleic acid to produce more than one amplicon. The method 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 amplification reactions. Alternatively, the method includes performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0178] Amplification can also include adding one or more control nucleic acids to one or more samples comprising more than one nucleic acid. Amplification can also include adding one or more control nucleic acids to more than one nucleic acid. The control nucleic acid can include a control label.

[0179] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable and / or triggerable nucleotides. Examples of non-natural nucleotides include but are not limited to peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA) and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of amplified reactions. Adding non-natural nucleotides can be used to identify the product of a specific cycle or time point in an amplified reaction.

[0180] Carrying out one or more amplification reactions can include using one or more primers. One or more primers can include one or more oligonucleotides. One or more oligonucleotides can contain at least about 7-9 nucleotides. One or more oligonucleotides can contain less than 12-15 nucleotides. One or more primers can anneal to at least a portion of more than one labeled nucleic acid. One or more primers can anneal to the 3' end and / or 5' end of more than one labeled nucleic acid. One or more primers can anneal to the internal region of more than one labeled nucleic acid. 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, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 , 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. One or more primers may include a set of fixed primers. One or more primers may include at least one or more custom primers. One or more primers may include at least one or more control primers. One or more primers may include at least one or more housekeeping gene primers. One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers can anneal to the first sample tag, the second sample tag, a molecular identifier label, a nucleic acid or its product. One or more primers can include universal primers and custom primers. Custom primers can be designed to amplify one or more target nucleic acids. Target nucleic acids can include a subset of total nucleic acids in one or more samples. In some embodiments, primers are probes attached to an array of the present disclosure.

[0181] In some embodiments, barcoding more than one target in a sample (e.g., random barcoding) further comprises generating an index library of barcoded targets (e.g., randomly barcoded targets) or barcoded fragments of targets. The barcode sequences of different barcodes (e.g., molecular markers of different random barcodes) can be different from each other. Generating an index library of barcoded targets comprises generating more than one index polynucleotide from more than one target in a sample. For example, for an index library of barcoded targets comprising a first index target and a second index target, the marker region of the first index polynucleotide can differ from the marker region of the second index polynucleotide by less than, about less than, at least less than, or at most less than: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or a number or range of nucleotides between any two of these values. In some embodiments, generating an index library of barcoded targets includes contacting more than one target (e.g., mRNA molecules) with more than one oligonucleotide comprising a poly (T) region and a tag region; and performing first-strand synthesis using a reverse transcriptase to generate single-stranded labeled cDNA molecules (each comprising a cDNA region and a tag region), wherein the more than one target includes at least two mRNA molecules of different sequences, and the more than one oligonucleotide includes at least two oligonucleotides of different sequences. Generating an index library of barcoded targets may also include amplifying single-stranded labeled cDNA molecules to generate double-stranded labeled cDNA molecules; and performing nested PCR on double-stranded labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may include generating adapter-tagged amplicons.

[0182] Barcoding (e.g., random barcoding) can include the use of nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it includes adding DNA barcodes or tags to cDNA molecules when generating cDNA molecules from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters used for sequencing (e.g., next generation sequencing (NGS)) can be added. For example, in Figure 2 At block 232 of the method, the sequencing results can be used to determine the sequence of cellular markers, molecular markers, and nucleotide fragments of one or more copies of the target.

[0183] Figure 3is a schematic diagram showing a non-limiting exemplary process for generating an index library of barcoded targets (e.g., random barcoded targets), such as an index library of barcoded mRNAs or fragments thereof. As shown in step 1, the reverse transcription process can encode each mRNA molecule with a unique molecular marker sequence, a cell marker sequence, and a universal PCR site. Specifically, by hybridizing (e.g., randomly hybridizing) a set of barcodes (e.g., random barcodes) 310 with a poly (A) tail region 308 of an RNA molecule 302, an RNA molecule 302 can be reverse transcribed to produce a labeled cDNA molecule 304 (including a cDNA region 306). Each of the barcodes 310 can include a target binding region, such as a poly (dT) region 312, a marker region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0184] In some embodiments, the cell marker sequence may comprise 3 to 20 nucleotides. In some embodiments, the molecular marker sequence may comprise 3 to 20 nucleotides. In some embodiments, each of more than one random barcode further comprises one or more of a universal marker and a cell marker, wherein the universal marker is the same for more than one random barcode on the solid support, and the cell marker is the same for more than one random barcode on the solid support. In some embodiments, the universal marker may comprise 3 to 20 nucleotides. In some embodiments, the cell marker comprises 3 to 20 nucleotides.

[0185] In some embodiments, the label region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, the label region 314 may include one or more of a universal label, a dimensional label, and a cell label. The length of the barcode sequence or molecular label 318 may be less than, may be about less than, may be at least less than, or may be at most less than: 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 between any two of these values. The length of the cell label 320 can be, can be about, can be at least, or can be 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 between any two of these values. The length of the universal label can be, can be about, can be at least, or can be 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 between any two of these values. The universal label can be the same for more than one random barcode on the solid support, and the cell label is the same for more than one random barcode on the solid support. The length of a dimensional marker can be less than, can be about less than, can be at least less than, or can be at most less than: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range between any two of these values.

[0186] In some embodiments, the marker area 314 may include the following, may include about the following, may include at least the following, or may include at most the following: 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 two of these values ​​of different markers, such as barcode sequences or molecular markers 318 and cell markers 320. The length of each marker can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values. A set of barcodes or random barcodes 310 can include, can include about, can include at least, or can include at most 10, 20, 40, 50, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values. 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 20 The barcodes or random barcodes 310 may be a number or range of barcodes or random barcodes 310 of any type or number between any two of these values. And the group of barcodes or random barcodes 310 may, for example, each contain a unique tag region 314. The labeled cDNA molecules 304 may be purified to remove excess barcodes or random barcodes 310. Purification may include Ampure bead purification.

[0187] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled into 1 tube and PCR amplified using the 1st PCR primer pool and the 1st universal PCR primer. Pooling is possible because of the unique tag region 314. In particular, the labeled cDNA molecules 304 can be amplified to produce nested PCR labeled amplicons 322. Amplification can include multiplex PCR amplification. Amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, in a single reaction volume, the multiplex PCR amplification can utilize the following, utilize about the following, utilize at least the following, or utilize at most the following: 10, 20, 40, 50, 70, 80, 90, 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 20 Amplification can include using a first PCR primer pool 324 including custom primers 326A-C targeting a specific gene and a universal primer 328. Custom primers 326 can hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. Universal primers 328 can hybridize to universal PCR region 316 of the labeled cDNA molecule 304.

[0188] like Figure 3As shown in step 3 of , the product from the PCR amplification in step 2 can be amplified with a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the amplicon 322 of the nested PCR marker can be further amplified by nested PCR. Nested PCR can include multiple PCR performed in a single reaction volume using a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328'. The nested PCR primer pool 328 can include the following, can include about the following, can include at least the following, or can include at most the following: 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 two of these values ​​of different nested PCR primers 330. Nested PCR primer 332 may include adapter 334 and hybridize to a region within cDNA portion 306″ of labeled amplicon 322. Universal primer 328′ may include adapter 336 and hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 produces adapter-tagged amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328′ may not include adapter 334 and adapter 336. Instead, adapter 334 and adapter 336 may be ligated to the product of nested PCR to produce adapter-tagged amplicon 338.

[0189] As shown in step 4, the PCR product from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, one or more additional assays can be performed on the adapter-tagged amplicon 338 using adapter 334 and adapter 336. Adapter 334 and adapter 336 can hybridize with primer 340 and primer 342. One or more primers 340 and primer 342 can be PCR amplification primers. One or more primers 340 and primer 342 can be sequencing primers. One or more adapters 334 and adapter 336 can be used for further amplification of adapter-tagged amplicon 338. One or more adapters 334 and adapter 336 can be used for sequencing adapter-tagged amplicon 338. Primer 342 can include a plate index 344 so that the amplicons generated using the same set of barcodes or random barcodes 310 can be sequenced in one sequencing reaction using next generation sequencing (NGS).

[0190] PNA Blockers

[0191] In some embodiments provided herein, peptide nucleic acid (PNA) blockers for selective amplification of libraries are provided. The disclosure herein includes methods for selectively blocking reverse transcription and / or PCR amplification for single-cell multi-omics library generation (e.g., Rhapsody) using peptide nucleic acids (PNA). In some embodiments, blockers provided herein can be used to block the extension of AbSeq oligonucleotides from AbSeq oligonucleotides, but allow other extension directions of molecular barcoding methods. PNA can bind to DNA through sequence complementarity, and it can have a stronger affinity due to the neutral charge of the peptide backbone. Due to the precise distance between each base, PNA can be highly specific. Therefore, PNA provided herein can selectively block PCR amplification or reverse transcription. Reverse transcriptase has a strong chain displacement activity, which can remove conventional DNA blockers, and in some embodiments provided herein, PNA will not be removed due to the higher affinity of PNA. PNA is a DNA mimic that has an uncharged backbone instead of an entire negatively charged sugar-phosphate backbone. Via a peptide bond, PNA can be easily conjugated to a peptide or a fluorescent dye. Due to the correct intramolecular spacing and hybridization between complementary nucleic acids, PNA can show high sequence selectivity and affinity for DNA molecules or RNA molecules. Due to the uncharged main chain, PNA can bind to negatively charged DNA or RNA without any electrostatic repulsion, and therefore has a much higher binding affinity. In some embodiments, the melting temperature is about 1°C higher per base pair than that of DNA / DNA molecules or DNA / RNA molecules, and this higher thermal stability and improved hybridization properties can exist independently of salt concentration.

[0192] In some embodiments, a method including superloading is provided (e.g., as described in Cao et al., Science 2017, which is incorporated herein by reference in its entirety). In some embodiments, adding a barcode during the RT step before entering a single-cell workflow (e.g., Rhapsody) will achieve superloading because each cDNA will have an additional unique barcode independent of the bead barcode. There is a demand for an in situ indexing solution for a higher throughput single-cell workflow (e.g., Rhapsody method). In order to achieve high throughput of in situ indexing methods using RNA and AbSeq workflows, it is necessary to capture AbSeq extensions in Rhapsody beads and connect to them. In order to be able to produce single-stranded oligonucleotides for capture and connection, blocking of AbSeq oligonucleotide extension is required in some embodiments. Both methods can be improved by the PNA blocking system provided herein. In some embodiments, the blocking oligonucleotides provided herein are included in the in situ cell indexing adapter (e.g., barcoded oligonucleotides) that can be provided in a high-throughput solution kit. Figure 4A-4BDepicted is a non-limiting exemplary schematic depicting capture of molecules barcoding cDNA by Rhapsody beads and attachment of the barcoded cDNA to the beads ( Figure 4A ), and the lack of capture of molecular barcoded AbSeq oligonucleotides due to double-stranded extension ( Figure 4B ). Figure 5A-Figure 5B Depicted is a non-limiting exemplary schematic showing that polymerase extension of both barcoded oligonucleotides and antibody oligonucleotides can occur ( Figure 5A ), which is disadvantageous in some scenarios ( Figure 5B ). Figure 6A-6B Depicted is a non-limiting exemplary schematic depicting a PNA blocking oligonucleotide provided herein hybridized to a barcoded primer ( Fig. 6A ) and subsequent prevention of extension of the 3' end of the antibody oligonucleotide hybridized thereto (eg, by blocking the strand displacement activity of the reverse transcriptase).

[0193] Cellular component binding reagents (such as barcoded antibodies) and their uses (such as sample indexing of cells) have been described in US2018 / 0088112 and US2018 / 0346970; The contents of each of these are incorporated herein by reference in their entirety. In some embodiments, the systems, methods, compositions and kits provided herein can be used in coordination with the systems, methods, compositions and kits described in US2020 / 0232032, the contents of which are incorporated herein by reference in their entirety. In some embodiments of the methods and compositions provided herein, DNA cell component binding reagent specific oligonucleotides (e.g., antibody oligonucleotides) are hybridized with oligonucleotide barcodes, and extended to realize separate but parallel workflows for protein quantification and mRNA quantification from the same beads, as described in US20210214784A1, the contents of which are incorporated herein by reference in their entirety. In some embodiments of the methods and compositions provided herein, the oligonucleotide barcode comprises a cleavage region (comprising, for example, one or more cleavage sites, such as atypical nucleotides (e.g., deoxyuridine) or a restriction enzyme recognition sequence), as described in US20210214770A1, the contents of which are incorporated herein by reference in their entirety.

[0194] In some embodiments, a blocking oligonucleotide is provided. In some embodiments, the blocking oligonucleotide comprises: a binding region capable of hybridizing to each of more than one barcoded oligonucleotide 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.

[0195] In some embodiments, a protected duplex is provided. In some embodiments, the protected duplex comprises: a blocking oligonucleotide that hybridizes to a barcoded oligonucleotide. In some embodiments, the blocking oligonucleotide comprises a binding region that can hybridize to a barcoded oligonucleotide. In some embodiments, the barcoded oligonucleotide comprises: a 3' target binding region that can hybridize to a nucleic acid target, a 5' first universal sequence, and a barcode located between the target binding region and the first universal sequence.

[0196] In some embodiments, the blocking oligonucleotide cannot be used as a primer for a reverse transcriptase or polymerase. In some embodiments, the 3' end of the blocking oligonucleotide cannot be extended by a reverse transcriptase or polymerase. In some embodiments, the blocking oligonucleotide of the protected duplex cannot be removed by the strand displacement activity of a reverse transcriptase or polymerase. The blocking oligonucleotide can be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. The blocking oligonucleotide can have a T of at least 50°C, at least 60°C, at least 70°C, or a number or range between any two of these values. m .

[0197] In some embodiments, the blocking oligonucleotide does not comprise non-natural nucleotides. In some embodiments, the blocking oligonucleotide comprises a 3' non-annealing region that is incapable of binding to a barcoded oligonucleotide. In some embodiments, the 3' non-annealing region is 1 nt to 100 nt long, 1 nt to 50 nt long, 1 nt to 21 nt long, 1 nt to 10 nt long, about 5 nt long, 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 5' adjacent region of the sequence in 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%, or a number or range between any two of these values.

[0198] The blocking oligonucleotide and / or the barcoded oligonucleotide can be a single-stranded oligonucleotide. The barcoded oligonucleotide can 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 bound by the blocking oligonucleotide 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 can be capable of hybridizing with 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 can include a double-stranded nucleic acid flanked by single-stranded overhangs.

[0199] After the protected duplex is contacted with the nucleic acid target, a polymerase or reverse transcriptase may be able to extend the 3' end of the barcoded oligonucleotide hybridized with the nucleic acid target to produce an extended protected duplex. In some embodiments, the extended protected duplex comprises a blocking oligonucleotide hybridized with 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 stop the polymerase extension and / or reverse transcriptase extension of the 3' end of the hybridized nucleic acid target to the 5' end of the barcoded oligonucleotide. The blocking oligonucleotide may be able to stop the polymerase extension 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-stranded 5' first universal sequence of the extended protected duplex may be able to hybridize with the adapter oligonucleotide to form a triplex. The adapter oligonucleotide may comprise a second universal sequence capable of binding to the 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 can be ligated together by a ligase.

[0200] In the absence of a blocking oligonucleotide, the 3' end of the hybridized nucleic acid target can extend to the 5' end of the barcoded oligonucleotide to form an undesirable duplex. The undesirable duplex can be double-stranded and / or can be incapable of hybridizing with a linker oligonucleotide. The blocking oligonucleotide can be capable of reducing the generation of undesirable 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 can comprise a first cell marker. The oligonucleotide barcode can comprise a second cell marker. More than one barcoded oligonucleotide can comprise at least 100, at least 1000, or at least 10,000 different barcode sequences. More than one barcoded oligonucleotide can each comprise the same barcode sequence. The barcoded oligonucleotide can be an in situ cell index adapter. In some embodiments, the blocking oligonucleotide, barcoded oligonucleotide, barcode, target binding region, first universal sequence, binding region and / or blocker region is 1 nt to 100 nt long, 1 nt to 50 nt long, 1 nt to 21 nt long, about 12 nt long, or a number or range between any two of these values.

[0201] The nucleic acid target can be a nucleic acid target molecule selected from a group comprising 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. The nucleic acid target can be a cell component binding reagent specific oligonucleotide, and the cell component binding reagent specific oligonucleotide can be associated with the cell component binding reagent. The cell component binding reagent can be an antibody or a fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, or any combination thereof. The cell component binding reagent specific oligonucleotide can include a unique identifier sequence for the cell component binding reagent. The target binding region can include a capture sequence. The target binding region can include a poly (dT) region. The cell component binding reagent specific oligonucleotide can include a sequence complementary to the capture sequence, and the capture sequence is configured to capture the cell component binding reagent specific oligonucleotide. The sequence complementary to the capture sequence can include a poly (dA) region. The cell component binding reagent can be capable of specifically binding to at least one of more than one cell component targets of the cell. The cell component binding reagent specific oligonucleotide can include a third universal sequence. The cell component binding reagent specific oligonucleotide can include a molecular tag. At least 10 of the more than one cellular component binding agent-specific oligonucleotides may comprise different molecular marker sequences.

[0202] Cell component binding reagent specific oligonucleotides may include poly (dA) regions. Cell component binding reagent specific oligonucleotides may include alignment sequences adjacent to poly (dA) regions. Cell component binding reagent specific oligonucleotides may be associated with cell component binding reagents via joints. Cell component binding reagent specific oligonucleotides may be configured to be able to detach from cell component binding reagents. The length of the alignment sequence may be one or more nucleotides, or the length may be two or more nucleotides. In some embodiments, (a) the alignment sequence includes guanine, cytosine, thymine, uracil, or a combination thereof; (b) the alignment sequence includes poly (dT) sequences, poly (dG) sequences, poly (dC) sequences, poly (dU) sequences, or a combination thereof; and / or (c) the alignment sequence is located at the 5' of poly (dA) regions. The joint may include a carbon chain. The carbon chain may include 2-30 carbon atoms (e.g., 12 carbon atoms). The joint may include 5' amino modifier C12 (5AmMC12) or a derivative thereof. The cell component target may include a protein target. The cellular component target can include a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell 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. The cellular component target can be on the cell surface. The DNA polymerase can include a Klenow fragment. The reverse transcriptase can include a viral reverse transcriptase (e.g., a murine leukemia virus (MLV) reverse transcriptase and / or a Moloney murine leukemia virus (MMLV) reverse transcriptase).

[0203] In some embodiments, a reaction mixture is provided. In some embodiments, the reaction mixture comprises: more than one blocking oligonucleotide provided herein; more than one protected duplex provided herein; more than one protected duplex; more than one barcoded oligonucleotide; more than one cellular component binding reagent; ligase; dNTP; polymerase; reverse transcriptase and / or more than one oligonucleotide barcode.

[0204] In some embodiments, a kit is provided. In some embodiments, the kit comprises: more than one blocking oligonucleotide provided herein; more than one protected duplex provided herein; more than one protected duplex; more than one barcoded oligonucleotide; more than one cellular component binding reagent; ligase; dNTP; polymerase; reverse transcriptase and / or more than one oligonucleotide barcode.

[0205] More than one oligonucleotide barcode can be fixed on a substrate. The substrate can be a particle (e.g., a bead). More than one oligonucleotide barcode can contain at least 100 different molecular marker sequences or at least 100 different molecular marker sequences. More than one oligonucleotide barcode can contain the same cell marker sequence. Each of more than one oligonucleotide barcode can contain a cell marker sequence, a sample marker sequence, a position marker sequence, a binding site for a universal primer, or a combination thereof. More than one oligonucleotide barcode can contain at least 100, at least 1000, or at least 10000 different molecular marker sequences. More than one oligonucleotide barcode can contain the same cell marker sequence. More than one oligonucleotide barcode can be associated with a particle. The oligonucleotide barcode is fixed on a particle, partially fixed on a particle, embedded in a particle, partially embedded in a particle, or a combination thereof. The particle can be a bead. Beads can include materials of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or a combination thereof. Beads can be hydrogel beads or magnetic beads. Beads can be destructible. In some embodiments, the systems, methods, compositions, and kits provided herein can be used in conjunction with the systems, methods, compositions, and kits described in provisional patent application serial number 63 / 421,759, entitled "POLYMERASE MEDIATED END MODIFICATION OF ABSEQ", filed on November 2, 2022, the contents of which are incorporated herein by reference in their entirety.

[0206] the term

[0207] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0208] Those skilled in the art will appreciate that for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in a different order. In addition, the steps and operations outlined are provided only as examples, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without departing from the essence of the disclosed embodiments.

[0209] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art may convert from the plural to the singular and / or from the singular to the plural where appropriate for the context and / or application. For clarity, various singular / plural arrangements may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Unless otherwise indicated, any reference to "or" herein is intended to encompass "and / or".

[0210] Those skilled in the art will understand that, in general, the terms used herein, and especially in the appended claims (e.g., the bodies of the appended claims), are generally intended as "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.). Those skilled in the art will further understand that if a specific number of an introduced claim recitation is intended, such intent will be explicitly stated in the claim, and in the absence of such a statement, no such intent is present. For example, as an aid to understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such words should not be interpreted as meaning that introduction of a claim recitation by the indefinite article "a" or "an" will limit any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory words "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim recitations. In addition, even if a particular number of introduced claim recitations is explicitly stated, one skilled in the art will recognize that such statement should be interpreted to mean at least the stated number (e.g., merely stating "two statements" without other modifiers means at least two statements or two or more statements). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, such syntactic structure is generally intended so that those skilled in the art will understand the meaning of the convention (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, a system having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally such syntactic structure is intended so that those skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further understand that, in practice, any disjunctive words and / or phrases presenting 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 term, or both terms. For example, the phrase "A or B" should be understood to include the possibility of "A" or "B" or "A and B."

[0211] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0212] As will be understood by those skilled in the art, for any and all purposes, such as in providing written description, all scopes disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges of the scope.Any listed scope can be easily identified as fully describing and enabling the same scope to be decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As non-limiting examples, each scope discussed herein can be easily decomposed into lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all languages, such as "up to", "at least", "greater than", "less than", etc. include stated numbers, and refer to the scope that can be subsequently decomposed into sub-ranges as discussed above.Finally, as will be understood by those skilled in the art, scope includes each individual member.Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles.Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0213] From the foregoing, it should be understood that various embodiments of the present disclosure have been described herein for illustrative purposes, and various modifications may be made without departing from the scope and spirit of the present disclosure. Therefore, the various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. A blocking oligonucleotide comprising a binding region capable of hybridizing to each of more than one barcoded oligonucleotide to form a protected duplex, wherein the barcoded oligonucleotide comprises: a 3' target binding region capable of hybridizing to a nucleic acid target, 5' first universal sequence, and A barcode is located between the target binding region and the first universal sequence.

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

3. The blocking oligonucleotide or protected duplex according to any one of claims 1 to 2, wherein the blocking oligonucleotide cannot serve as a primer for a reverse transcriptase or a polymerase.

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

5. The blocking oligonucleotide or protected duplex according to any one of claims 1 to 4, wherein the blocking oligonucleotide of the protected duplex cannot be removed by the strand displacement activity of a reverse transcriptase or a polymerase.

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

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

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

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 the barcoding oligonucleotide.

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

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

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

13. The blocking oligonucleotide or protected duplex of any one of claims 1 to 12, wherein the barcoded oligonucleotide comprises a blocker region, and wherein: 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.

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

15. The blocking oligonucleotide or protected duplex according to any one of claims 1 to 14, wherein the binding region is capable of hybridizing with 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 the protected duplex.

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 by single-stranded overhangs.

17. The blocking oligonucleotide or protected duplex according to any one of claims 1 to 16, wherein after contacting the protected duplex with a nucleic acid target, A polymerase or reverse transcriptase is capable of extending the 3' end of the barcoded oligonucleotide hybridized to the nucleic acid target to generate an extended protected duplex, wherein the extended protected duplex comprises the 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) the target binding region, (iii) the first universal sequence, and (iv) the barcode.

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

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

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

21. The blocking oligonucleotide or protected duplex according to any one of claims 1 to 20, wherein the single-stranded 5' first universal sequence of the extended protected duplex is capable of hybridizing with an adaptor oligonucleotide to form a triplex.

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

23. The blocking oligonucleotide or protected duplex of any one of claims 1-22, wherein the 5' end of the extended barcoded oligonucleotide and the 3' end of the oligonucleotide barcode are capable of being ligated together by a ligase.

24. The blocking oligonucleotide or protected duplex according to 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 barcoding oligonucleotide to form an undesired duplex.

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

26. The blocking oligonucleotide or protected duplex of any one of claims 1-25, wherein the blocking oligonucleotide is capable of reducing the generation 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%.

27. The blocking oligonucleotide or protected duplex of any one of claims 1-26, wherein the barcode comprises a first cellular marker, optionally the oligonucleotide barcode comprises a second cellular marker.

28. The blocking oligonucleotide or protected duplex of any one of claims 1-27, wherein the more than one barcoded oligonucleotide comprises at least 100, at least 1000, or at least 10000 different barcode sequences.

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

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

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

32. The blocking oligonucleotide or protected duplex according to any one of claims 1-31, 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, an mtDNA, an siRNA molecule or any combination thereof.

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

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

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

36. A blocking oligonucleotide or protected duplex according to any one of claims 1-35, wherein the cellular component binding reagent specific oligonucleotide comprises a sequence complementary to a capture sequence, and the capture sequence is configured to capture the cellular component binding reagent specific oligonucleotide, and optionally the sequence complementary to the capture sequence comprises a poly (dA) region.

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

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

39. The blocking oligonucleotide or protected duplex according to any one of claims 1-38, wherein the cellular component binding agent specific oligonucleotide comprises a molecular tag, and optionally, at least 10 of the more than one cellular component binding agent specific oligonucleotides comprise different molecular tag sequences.

40. The blocking oligonucleotide or protected duplex according to any one of claims 1-39, wherein the cellular component binding agent specific oligonucleotide comprises a poly (dA) region, optionally the cellular component binding agent specific oligonucleotide comprises an alignment sequence adjacent to the poly (dA) region.

41. The blocking oligonucleotide or protected duplex according to any one of claims 1-40, wherein the cellular component binding agent specific oligonucleotide is associated with the cellular component binding agent via a linker.

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

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

44. The blocking oligonucleotide or protected duplex of any one of claims 1 to 43, wherein: (a) the aligned sequences contain guanine, cytosine, thymine, uracil or a combination thereof; (b) the aligned sequences comprise poly(dT) sequences, poly(dG) sequences, poly(dC) sequences, poly(dU) sequences or a combination thereof; and / or (c) The aligned sequences are located 5' to the poly(dA) region.

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

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

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

48. The blocking oligonucleotide or protected duplex of any one of claims 1-47, wherein the cellular component target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell 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. The blocking oligonucleotide or protected duplex of any one of claims 1-48, wherein the cellular component target is on the surface of a cell.

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

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

52. A reaction mixture comprising: More than one blocking oligonucleotide according to any one of claims 1-51; More than one protected duplex according to any one of claims 1 to 51; More than one protected duplex; more than one barcoded oligonucleotide; More than one cellular component binding agent; Ligase; dNTPs; polymerase; Reverse transcriptase, and / or More than one oligonucleotide barcode.

53. A kit, comprising: More than one blocking oligonucleotide according to any one of claims 1-51; More than one protected duplex according to any one of claims 1 to 51; More than one protected duplex; more than one barcoded oligonucleotide; More than one cellular component binding agent; Ligase; dNTPs; polymerase; Reverse transcriptase, and / or More than one oligonucleotide barcode.

54. The reaction mixture or kit of any one of claims 52-53, wherein the more than one oligonucleotide barcode is immobilized on a substrate.

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

56. The reaction mixture or kit of any one of claims 52-55, wherein the more than one oligonucleotide barcode comprises at least 100 different molecular marker sequences or at least 100 different molecular marker sequences.

57. The reaction mixture or kit of any one of claims 52-56, wherein the more than one oligonucleotide barcodes comprise the same cellular marker sequence.

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

59. The reaction mixture or kit of any one of claims 52-58, wherein the more than one oligonucleotide barcode comprises at least 100, at least 1000, or at least 10,000 different molecular marker sequences.

60. The reaction mixture or kit of any one of claims 52-59, wherein the more than one oligonucleotide barcodes comprise the same cellular marker sequence.

61. A reaction mixture or kit according to any one of claims 52-60, wherein the more than one oligonucleotide barcode is associated with a particle, optionally wherein the oligonucleotide barcode is fixed on the particle, partially fixed on the particle, embedded in the particle, partially embedded in the particle, or a combination thereof.

62. The reaction mixture or kit of any one of claims 52-61, wherein the particles are beads, optionally wherein the beads comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or combinations thereof.

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

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

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