RNA probing and amplification methods for single-cell analysis in fixed cells

Abstract

The disclosures herein include systems, methods, compositions, and kits for determining the spatial location and / or copy number of a nucleic acid target in an immobilized sample. In some embodiments, a probing oligonucleotide is provided comprising a coupling sequence, a probe sequence configured to hybridize with the nucleic acid target, and / or a predetermined spatial label. The method may include the step of contacting each of two or more spatial locations in a sample containing a copy of the nucleic acid target with the probing oligonucleotide. The method may include the step of extending the probing oligonucleotide hybridized to the copy of the nucleic acid target with insights to produce a plurality of extended probing oligonucleotides. The method may include the step of barcoding the plurality of extended probing oligonucleotides using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 503,856, filed May 23, 2023, under 35 U.S.C. § 119(e), and the contents of this related application are hereby incorporated by reference in their entirety for all purposes. The present disclosure generally relates to the field of molecular biology, for example, the determination of profiles of secreted analytes of cells using molecular barcoding. The present disclosure generally relates to the field of molecular biology, for example, the determination of gene expression using molecular barcoding.

Background Art

[0002] Many cell samples, including patient samples that need to be stored in fixatives, contain fixed cells. However, for fixed samples, mRNA is cross-linked and unavailable for capture by conventional mRNA capture methods (e.g., by polyA / dT capture), so current gene expression profiling compositions and methods, including single-cell RNA sequencing workflows and platforms, are not compatible. Although mRNA in fixed samples has been visualized using in situ hybridization and FISH methods, the number of fluorescence detectors is limited, and thus the number of probes that can be used to detect mRNA is limited due to the difficulty of analyzing at the transcriptome level. There is a need for compositions, methods, systems, and kits capable of analyzing gene expression in fixed samples. Furthermore, there is a need for compositions, methods, systems, and kits for spatial gene expression analysis in fixed samples.

Summary of the Invention

[0003] The disclosure herein includes methods for labeling nucleic acid targets in a sample. In some embodiments, the method includes contacting a sample containing a copy of a nucleic acid target with a plurality of probing oligonucleotides, each of which includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target. The method may include extending the plurality of probing oligonucleotides hybridized to the copy of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include barcoding the plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides, each of which includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular label sequence and a partial sequence of a nucleic acid target. The method may also include the step of determining the copy number of a nucleic acid target in a sample based on the number of molecular labels associated with the multiple barcoded probing oligonucleotides or their product.

[0004] The disclosure herein includes a method for determining the copy number of a nucleic acid target in a sample. In some embodiments, the method includes contacting a sample containing a copy of the nucleic acid target with a plurality of probing oligonucleotides, each of which includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target. The method may include extending the plurality of probing oligonucleotides hybridized to a copy of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include barcoding the plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides, each of which includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular label sequence and a partial sequence of a nucleic acid target. The method may also include the step of determining the copy number of a nucleic acid target in a sample based on the number of molecular labels associated with the multiple barcoded probing oligonucleotides or their product.

[0005] The disclosure herein includes methods for determining the spatial location and copy number of a nucleic acid target in a sample. In some embodiments, the method includes contacting each of two or more spatial locations in a sample containing copies of the nucleic acid target with a plurality of probing oligonucleotides, each of which comprises a coupling sequence, a probe sequence configured to hybridize with the nucleic acid target, and a predetermined spatial label. In some embodiments, probing oligonucleotides contacted with the same spatial location contain the same spatial label sequence, while probing oligonucleotides contacted with separate spatial locations in the sample contain different spatial label sequences. The method may include extending the plurality of probing oligonucleotides hybridized with copies of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of generating multiple barcoded probing oligonucleotides by barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes, wherein each oligonucleotide barcode of the multiple oligonucleotide barcodes includes a molecular label, and each of the multiple barcoded probing oligonucleotides includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may also include the step of obtaining sequencing data including multiple sequencing reads of the barcoded probing oligonucleotides or their products, wherein each of the multiple sequencing reads includes a spatial label sequence, a molecular label sequence, and a partial sequence of the nucleic acid target. The method may also include the step of determining the copy number of the nucleic acid target at each spatial location of the sample by counting the number of molecular labels having a distinct sequence associated with the nucleic acid target for each unique spatial label sequence associated with a distinct spatial location of the sample.

[0006] In some embodiments, the step of barcoding a plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes includes the steps of preparing coupling oligonucleotides comprising a 5' coupling sequence complement and a 3' capture sequence complement; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5' coupling sequence complement of the coupling oligonucleotide; hybridizing the 3' capture sequence complement of the coupling oligonucleotide with the capture sequence of an oligonucleotide barcode among the plurality of oligonucleotide barcodes; and / or ligating the extended probing oligonucleotide to the hybridized oligonucleotide barcode. The method may include, prior to the step of ligating the extended probing oligonucleotide to the oligonucleotide barcode, a step of filling the gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode using a DNA polymerase lacking at least one of 5'-to-3' exonuclease activity and 3'-to-5' exonuclease activity. In some embodiments, the step of ligating the extended probing oligonucleotide to the hybridized oligonucleotide barcode is carried out using DNA ligase. In some embodiments, the coupling oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the coupling oligonucleotide contains at least six nucleotides. In some embodiments, the coupling sequence contains at least four nucleotides. In some embodiments, the 5' end of each probing oligonucleotide is phosphorylated. In some embodiments, the probing oligonucleotide is capable of entering the cells and / or nuclei of the sample (e.g., permeabilized cells and / or permeabilized nuclei of the sample).The method may include, after the step of contacting the probing oligonucleotides with the sample, a step of removing one or more probing oligonucleotides from a plurality of probing oligonucleotides that have not come into contact with the sample, and the step of removing one or more probing oligonucleotides that have not come into contact with the sample may include a step of removing one or more probing oligonucleotides that have not entered the cells of the sample. In some embodiments, the contact step includes bringing the sample into contact with a device configured to place the probing oligonucleotides (e.g., an inkjet device). In some embodiments, the device is a needle, a needle array, a tube, an aspiration device, an injection device, an electroporation device, a fluorescence-activated cell sorter device, an inkjet device, a microfluidic device, or any combination thereof. In some embodiments, the device brings separate spatial locations of the sample into contact at a specific speed. In some embodiments, the spatial label is 6 to 60 nucleotides long. In some embodiments, the two or more spatial locations include at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 separate spatial locations of the sample. In some embodiments, the spatial locations of the sample correspond to regions containing about 50 cells or less, about 45 cells or less, about 40 cells or less, about 35 cells or less, about 30 cells or less, about 25 cells or less, about 20 cells or less, about 15 cells or less, about 10 cells or less, about 9 cells or less, about 8 cells or less, about 7 cells or less, about 6 cells or less, about 5 cells or less, about 4 cells or less, about 3 cells or less, about 2 cells or less, or about 1 cell or less.

[0007] The method may include a step of contacting the sample with an elongation reagent. In some embodiments, at least a portion of the contact step is performed in the presence of the elongation reagent. In some embodiments, the entire contact step is performed in the presence of the elongation reagent. In some embodiments, the contact step and the elongation step are simultaneous. In some embodiments, elongation is performed with insights. In some embodiments, the elongation includes insights reverse transcription. In some embodiments, the cells of the sample remain intact during the elongation step. In some embodiments, the elongation reagent includes a reverse transcription reagent. In some embodiments, the reverse transcription reagent includes reverse transcriptase and dNTPs. In some embodiments, the reverse transcriptase includes viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is mouse leukemia virus (MLV) reverse transcriptase or Moloney's mouse leukemia virus (MMLV) reverse transcriptase.

[0008] In some embodiments, the sample is physically separated or intact during the contact step. In some embodiments, the sample comprises a single cell. In some embodiments, the sample comprises multiple single cells. In some embodiments, the sample comprises multiple cells, and the method may include a step of dissociating the sample to produce multiple single cells, wherein the dissociation step may include chemical dissociation, enzymatic dissociation, and / or mechanical dissociation, and the dissociation step may use one or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin. The method may include a step of distributing the multiple single cells into multiple compartments prior to the barcoding step, wherein one of the compartments comprises a single cell from the multiple single cells, and in the compartment containing the single cells, an extended probing oligonucleotide may be brought into contact with the multiple oligonucleotide barcodes. In some embodiments, in the compartment containing the single cells, the single cells are brought into contact with a lysis buffer at 15-65°C to lyse the single cells. In some embodiments, the lysis buffer contains an active substance capable of dissociating protein-nucleic acid complexes.

[0009] In some embodiments, each oligonucleotide barcode of a plurality of oligonucleotide barcodes includes a first universal sequence. In some embodiments, the step of obtaining sequencing data includes amplifying a plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or its complement, and an amplification primer capable of hybridizing to a nucleic acid target or its complement, thereby generating a plurality of amplified barcoded probing oligonucleotides, and the step of obtaining sequencing data includes obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides or their products. In some embodiments, the step of obtaining sequencing data includes binding sites of sequencing primers and / or sequencing adapters to the plurality of barcoded probing oligonucleotides or their products. In some embodiments, the amplification primer includes a second universal sequence, and / or the first primer includes a third universal sequence. In some embodiments, the first universal sequence, the second universal sequence, and / or the third universal sequence are the same. In some embodiments, the first universal sequence, the second universal sequence, and / or the third universal sequence differ. In some embodiments, the first universal sequence, the second universal sequence, and / or the third universal sequence include binding sites of the sequencing primer and / or sequencing adapter, their complementary sequences, and / or portions thereof. In some embodiments, the sequencing adapter includes a P5 sequence, a P7 sequence, their complementary sequences, and / or portions thereof. In some embodiments, the sequencing primer includes a lead 1 sequencing primer, a lead 2 sequencing primer, their complementary sequences, and / or portions thereof.

[0010] In some embodiments, the sample includes a target panel of multiple nucleic acid targets, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or about 500 distinct nucleic acid targets. In some embodiments, two or more nucleic acid targets in the target panel are biomarkers. In some embodiments, the biomarker is a biomarker for a disease or condition. In some embodiments, the disease or condition is cancer, an infectious disease, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof. In some embodiments, the contact step includes contacting the sample with a panel of probing oligonucleotides comprising two or more plurality of probing oligonucleotides, each plurality comprising a probe sequence configured to hybridize with a nucleic acid target among a plurality of nucleic acid targets. In some embodiments, the step of determining the copy number of nucleic acid targets in the sample includes determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels having distinct sequences associated with a plurality of barcoded probing oligonucleotides or their products, each comprising the respective sequences of the plurality of nucleic acid targets. The method may include determining the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample by counting the number of molecular labels having distinct sequences associated with each of the plurality of nucleic acid targets for each unique spatial label sequence associated with a distinct spatial location of the sample.In some embodiments, the amplification primers include a panel of amplification primers configured to hybridize with a plurality of nucleic acid targets or their complements, for example, a panel of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 distinct amplification primers. In some embodiments, the nucleic acid target includes a nucleic acid molecule. In some embodiments, the nucleic acid molecule includes ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, sample-indexed oligonucleotides, cell component-binding reagent-specific oligonucleotides, or any combination thereof.

[0011] In some embodiments, the plurality of cells comprises one or more cell types. In some embodiments, the one or more cell types are selected from the group consisting of brain cells, cardiac cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, and circulating cells, or any combination thereof. In some embodiments, the sample comprises a biological sample, clinical sample, environmental sample, biological fluid, tissue, tissue section, or any combination thereof derived from the subject. In some embodiments, the subject is a human, mouse, dog, rat, or vertebrate. The method may include a step of determining the genotype, phenotype, or one or more gene mutations of the subject based on the spatial location of nucleic acid targets in the sample. The method may include a step of predicting the subject's susceptibility to one or more diseases, such as cancer or a genetic disease. The method may include a step of determining the cell types of the plurality of cells in the sample. In some embodiments, a drug is selected based on the predicted responsiveness of the cell types of the plurality of cells in the sample. The method may include a step of imaging the sample, optionally an image of the sample before and / or after the contact step, and the imaging step may generate imaging data. In some embodiments, the image of the sample step includes a step of staining the sample using staining, the staining being fluorescence staining, negative staining, antibody staining, or any combination thereof. In some embodiments, the staining step includes immunocytochemistry (ICC), immunohistochemical testing (IHC), immunofluorescence (IF), or any combination thereof. In some embodiments, the imaging step includes microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multiphoton microscopy, quantitative phase microscopy, surface-enhanced Raman spectroscopy, video recording, manual visual analysis, automated visual analysis, or any combination thereof. The method may include a step of correlating imaging data of one or more spatial locations of the sample with sequencing data. The method may include correlation analysis of spatial location imaging data with sequencing data.In some embodiments, correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and / or cellular targets of candidate therapeutic agents. In some embodiments, the imaging step creates images used to construct a map that physically represents the sample. In some embodiments, the map is two-dimensional or three-dimensional. The method may include the step of mapping nucleic acid targets and / or cellular component targets onto the map of the sample. The method may include the step of mapping one or more single cells from a plurality of cells onto the map of the sample.

[0012] In some embodiments, the sample has been in contact with one or more fixatives and / or permeabilizing agents. In some embodiments, the sample includes tissue, cell monolayers, fixed cells, tissue sections, or any combination thereof. In some embodiments, the sample includes fresh tissue sections, frozen tissue sections, fixed tissue sections, formalin-fixed tissue sections, formalin-fixed paraffin-embedded (FFPE) tissue sections, acetone-fixed tissue sections, paraformaldehyde (PFA)-fixed tissue sections, and / or methanol-fixed tissue sections. In some embodiments, the sample includes nuclear suspensions, such as fixed nuclear suspensions and / or permeabilized nuclear suspensions. In some embodiments, the sample includes cells, such as fresh cells, frozen cells, fixed cells, formalin-fixed cells, formalin-fixed paraffin-embedded (FFPE) cells, acetone-fixed cells, paraformaldehyde (PFA)-fixed cells, and / or methanol-fixed cells. The method may include the step of permeabilizing the sample and / or fixing the sample. In some embodiments, the step of fixing the sample includes bringing the sample into contact with a fixative. In some embodiments, the fixative includes a non-crosslinking fixative (e.g., methanol). In some embodiments, the fixative includes a crosslinking agent. In some embodiments, the crosslinking agent includes a cleavable crosslinking agent.In some embodiments, the cleavable crosslinking agents are dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3,3'-dithiobispropionimidate (DTBP), 3-(2-pyridyldithio)propionate succinimidyl (SPDP), 6-(3(2-pyridyldithio)propionate The cleavable crosslinking agent includes or is derived from succinimidyl lysyldithio)propionamide)hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene (SMPT), 3-(2-pyridyldithio)propionylhydrazide (PDPH), 2-((4,4'-adipentanamide)ethyl)-1,3'-dithiopropionate succinimidyl (SDAD, NHS-SS-diaziline), or any combination thereof. In some embodiments, the cleavable crosslinking agent includes cleavable linkages selected from the group consisting of chemically cleavable linkages, photocleavable linkages, acid-unstable linkers, heat-sensitive linkages, enzymatically cleavable linkages, and combinations thereof. In some embodiments, the cleavable crosslinking agent is a thiol-cleavable crosslinking agent or includes a disulfide linker. In some embodiments, the fixative includes paraformaldehyde (PFA), succinimidyl dithiobis(propionate) (DSP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), CellCover, or a combination thereof. In some embodiments, the steps of fixing the sample and permeabilizing the sample are performed simultaneously. In some embodiments, the steps of fixing the sample and permeabilizing the sample are performed in the presence of a dual-function substance capable of fixing and permeabilizing the sample. In some embodiments, the dual-function substance is methanol.

[0013] In some embodiments, the step of permeabilizing the sample includes the step of contacting the sample with a permeabilizing agent. The method may include the step of contacting the sample with a plurality of probing oligonucleotides or a plurality of cell component binding reagents, followed by the step of removing the permeabilizing agent from the sample. In some embodiments, the permeabilizing agent can (i) permeabilize the cell membrane of a cell, or (ii) make the cell membrane permeable to the probing oligonucleotide, the cell component binding reagent, or both. In some embodiments, the permeabilizing agent includes (i) a solvent, surfactant, or surfactant, (ii) BD Cytoperm, (iii) saponin or a derivative thereof, (iv) Triton X-100, (v) methanol or a derivative thereof, and / or (vi) digitonin or a derivative thereof. In some embodiments, the active agent capable of dissociating protein-nucleic acid complexes includes a serine protease with broad substrate specificity. In some embodiments, the serine protease with broad substrate specificity is proteinase K. In some embodiments, the lysis buffer includes a fixation release agent. In some embodiments, the defixation agent includes thiols, hydroxylamines, periodic acid, bases, or any combination thereof. In some embodiments, the lysis buffer includes DTT. The method may include a step of reversing the fixation of the sample and / or single cells. In some embodiments, the step of reversing the fixation of the sample and / or single cells includes UV cutting, chemical treatment, heating, enzymatic treatment, or any combination thereof.

[0014] The sample may contain multiple cell component targets, and the method further includes the steps of: contacting a sample with multiple cell component binding reagents, each of which contains a cell component binding reagent-specific oligonucleotide containing a unique identifier sequence for the cell component binding reagent, and enabling the cell component binding reagent to specifically bind to at least one of the multiple cell component targets; barcoding the cell component binding reagent-specific oligonucleotides to generate a plurality of barcoded cell component binding reagent-specific oligonucleotides, each containing a sequence complementary to at least a portion of the unique identifier sequence and a molecular label sequence; and obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cell component binding reagent-specific oligonucleotides or their products, each of which contains at least a portion of the molecular label sequence and the unique identifier sequence. In some embodiments, the step of obtaining sequencing data includes binding the binding site of a sequencing primer and / or sequencing adapter to the barcoded cell component binding reagent-specific oligonucleotide or its product.

[0015] This method may include a step of contacting a sample with a plurality of cell component binding reagents, followed by a step of removing one or more cell component binding reagents that have not come into contact with the sample, and the step of removing one or more cell component binding reagents that have not come into contact with the sample may include a step of removing one or more cell component binding reagents that have not come into contact with at least one of the plurality of cell component targets. In some embodiments, the cell component targets include intracellular proteins, carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, intracellular proteins, or any combination thereof. In some embodiments, the cell component binding reagent-specific oligonucleotides include a second molecular label, and at least 10 of the plurality of cell component binding reagent-specific oligonucleotides may include different second molecular label sequences. In some embodiments, the second molecular label sequences of at least two cell component binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two cell component binding reagent-specific oligonucleotides are identical. In some embodiments, the second molecular labeling sequences of at least two cell component binding reagent-specific oligonucleotides differ, and the unique identifier sequences of at least two cell component binding reagent-specific oligonucleotides differ. In some embodiments, the number of unique molecular labeling sequences in the sequencing data associated with a unique identifier sequence for a cell component binding reagent capable of specifically binding to at least one cell component target indicates the copy number of at least one cell component target in the sample. In some embodiments, the number of unique second molecular labeling sequences in the sequencing data associated with a unique identifier sequence for a cell component binding reagent capable of specifically binding to at least one cell component target indicates the copy number of at least one cell component target in the sample.

[0016] The method may include the step of contacting a sample with a blocking reagent, one or more decoy oligonucleotides, and / or one or more blocking oligonucleotides, prior to the steps of contacting a plurality of cell component binding reagents with the sample and / or contacting a plurality of probing oligonucleotides with the sample. In some embodiments, the step of contacting a plurality of cell component binding reagents with the sample is performed in the presence of a blocking reagent. In some embodiments, the blocking reagent comprises a plurality of oligonucleotides complementary to at least a portion of the cell component binding reagent-specific oligonucleotides. In some embodiments, the blocking reagent comprises an antibody or fragment thereof derived from a first species, and the blocking reagent comprises serum derived from the first species. In some embodiments, the sample comprises one or more non-target nucleic acids, and the blocking reagent comprises a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids. In some embodiments, each of the plurality of decoy oligonucleotides is capable of hybridizing to at least a portion of the non-target nucleic acids.In some embodiments, the decoy oligonucleotide contains a sequence complementary to at least a portion of the non-target nucleic acid; the decoy oligonucleotide contains a sequence identical or substantially similar to the sequence of the cell component binding reagent-specific oligonucleotide, and the sequence may be 3 to 40 nucleotides in length; the decoy oligonucleotide has up to 50% sequence identity with respect to the cell component binding reagent-specific oligonucleotide; the decoy oligonucleotide does not contain UMI; the decoy oligonucleotide contains a random sequence, and the random sequence may be approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length; The nucleotides do not contain any sequence having five or more, six or more, seven or more, or eight or more consecutive T or A sequences; the decoy oligonucleotides contain at least one G or C for every four, five, six or seven consecutive nucleotides; the decoy oligonucleotides contain one or more modified nucleotides; the decoy oligonucleotides contain a 5' modification, the 5' modification may include a 5' amino-modification factor C12 modification (5AmMC12); the decoy oligonucleotides contain a 3' modification, the 3' modification may include a 3' dideoxy C modification (ddC); and / or the decoy oligonucleotides are 30 to 65 nucleotides in length. In some embodiments, the sample contains one or more undesirable nucleic acid species, and the method includes the step of contacting the sample with a blocking oligonucleotide, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species, thereby reducing the reverse transcription of at least one of the one or more undesirable nucleic acid species by the blocking oligonucleotide. In some embodiments, the blocking oligonucleotide is brought into contact with the sample before the multiple probing oligonucleotides are brought into contact with the sample, the blocking oligonucleotide is brought into contact with the sample after the multiple probing oligonucleotides are brought into contact with the sample, and / or the blocking oligonucleotide is brought into contact with the sample at the same time as the multiple probing oligonucleotides are brought into contact with the sample.This method may include the step of preparing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species in a sample, optionally at least 10 or at least 100 undesirable nucleic acid species. In some embodiments, the blocking oligonucleotides are locked nucleic acid (LNA), peptide nucleic acid (PNA), DNA, LNA / PNA chimeras, LNA / DNA chimeras, or PNA / DNA chimeras; the blocking oligonucleotides specifically bind to within 100 nucleotides, 50 nucleotides, or 25 nucleotides from the 3' end of one or more undesirable nucleic acid species; the blocking oligonucleotides specifically bind to within 100 nucleotides from the 5' end of one or more undesirable nucleic acid species; or the blocking oligonucleotides specifically bind to within 100 nucleotides from the central part of one or more undesirable nucleic acid species. The blocking oligonucleotide, which specifically binds within 10 nucleotides from the 3' poly(A) tail of the 3'

[0017] In some embodiments, each molecular label of a plurality of oligonucleotide barcodes contains at least six nucleotides. In some embodiments, each capture sequence of a plurality of oligonucleotide barcodes contains at least four nucleotides. In some embodiments, a plurality of oligonucleotide barcodes are associated with a solid support, and one of the compartments contains a single solid support. In some embodiments, each of the plurality of oligonucleotide barcodes contains a cell label. In some embodiments, each cell label of a plurality of oligonucleotide barcodes contains at least six nucleotides. In some embodiments, oligonucleotide barcodes associated with the same solid support contain the same cell label. In some embodiments, oligonucleotide barcodes associated with different solid supports contain different cell labels. In some embodiments, the solid support includes synthetic particles, a planar surface, or a combination thereof. The method may include the step of associating synthetic particles containing a plurality of oligonucleotide barcodes with cells in a compartment. The method may include the step of lysing the cells after the step of associating the synthetic particles with cells. In some embodiments, the step of lysing the cells includes the steps of heating the cells, contacting the cells with a surfactant, changing the pH of the cells, or any combination thereof. In some embodiments, the synthetic particles and single cells are in the same compartment, which may be a well or a droplet. In some embodiments, at least one of a plurality of oligonucleotide barcodes is immobilized or partially immobilized on the synthetic particle, or at least one of a plurality of oligonucleotide barcodes is encapsulated or partially encapsulated within the synthetic particle. In some embodiments, the synthetic particles are disintegrable (e.g., disintegrable hydrogel particles). In some embodiments, the synthetic particles include beads.In some embodiments, the beads include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, antibiotin microbeads, antifluorescent dye microbeads, or any combination thereof. In some embodiments, the synthetic particles include materials selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. In some embodiments, each oligonucleotide barcode in a plurality of oligonucleotide barcodes includes a linker functional group. In some embodiments, the synthetic particles include a solid support functional group. In some embodiments, the support functional group and the linker functional group are associated with each other, and the linker functional group and the support functional group may be selected from the group consisting of C6, biotin, streptavidin, primary amines, aldehydes, ketones, and any combination thereof.

[0018] The disclosure herein includes compositions (e.g., kits). In some embodiments, the kit includes: a plurality of probing oligonucleotides, each of which comprises a coupling sequence and a probe sequence configured to hybridize with a nucleic acid target, and the probing oligonucleotides may also include a predetermined spatial label; a coupling oligonucleotide comprising a 5' coupling sequence complement and a 3' capture sequence complement; a plurality of oligonucleotide barcodes, where the 3' end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, and the 5' end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; and capable of hybridizing to a first universal sequence, and further, the A first primer which may contain a universal sequence 3; an amplification primer which may hybridize to a nucleic acid target or its complement and which may further contain a second universal sequence; a DNA ligase; an extension reagent, optionally a reverse transcription reagent, and optionally a reverse transcriptase and dNTPs; one or more fixatives; one or more permeabilizing agents; a crosslinking agent; a defixation agent; a lysis buffer; a plurality of cell component binding reagents, each of which contains a cell component binding reagent-specific oligonucleotide containing a unique identifier sequence for the cell component binding reagent, and which allows the cell component binding reagent to specifically bind to at least one of the plurality of cell component targets; a blocking reagent; one or more decoy oligonucleotides; and / or one or more blocking oligonucleotides.

[0019] In some embodiments, the multiple probing oligonucleotides include a panel of probing oligonucleotides comprising two or more multiple probing oligonucleotides, each multiple comprising multiple nucleic acid targets, optionally at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, and 5. The probe sequence includes a target from a target panel of 0, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 125, approximately 150, approximately 175, approximately 200, approximately 225, approximately 250, approximately 275, approximately 300, approximately 325, approximately 350, approximately 375, approximately 400, approximately 425, approximately 450, approximately 475, or approximately 500 distinct nucleic acid targets. In some embodiments, the amplification primers include a panel of amplification primers configured to hybridize with a plurality of nucleic acid targets or their complements, optionally comprising at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 separate amplification primer panels. [Brief explanation of the drawing]

[0020] [Figure 1] This shows a non-exclusive, illustrative barcode. [Figure 2] This document presents a non-limiting, illustrative workflow for barcoding and electronic counting. [Figure 3] This is a schematic diagram illustrating a non-restrictive, exemplary process for generating an indexed library of targets barcoded at their 3' ends from multiple targets. [Figure 4A] A schematic diagram of a non-limiting, illustrative workflow for gene expression analysis in fixed cells is shown. [Figure 4B]A schematic diagram of a non-limiting, illustrative workflow for gene expression analysis in fixed cells is shown. [Figure 4C] A schematic diagram of a non-limiting, illustrative workflow for gene expression analysis in fixed cells is shown. [Figure 4D] A schematic diagram of a non-limiting, illustrative workflow for gene expression analysis in fixed cells is shown. [Modes for carrying out the invention]

[0021] In the following detailed description, references are made to the accompanying drawings which form part of this specification. In the drawings, similar symbols typically identify similar components unless otherwise indicated by context. The exemplary embodiments described in the detailed description, drawings and claims are not intended to limit. Other embodiments may be used and other modifications may be made without departing from the spirit or scope of the subject matter presented herein. Generally, the aspects of this disclosure described herein and illustrated in the drawings may be arranged, replaced, combined, separated and designed in a wide range of different configurations, all of which are expressly intended herein and form part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank, as well as other databases referenced herein, are incorporated by reference in their entirety with respect to the relevant technology.

[0022] Quantifying a small number of nucleic acids, such as messenger ribonucleotide (mRNA) molecules, is clinically important, for example, to determine the genes expressed in cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can be very difficult, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR yields identical molecular copies in each cycle. However, PCR has drawbacks because each molecule replicates with a probabilistic probability, which varies depending on the PCR cycle and gene sequence, leading to amplification bias and inaccurate gene expression measurements. Probabilistic barcodes with unique molecular labels (also called molecular indices (MIs)) can be used to count molecules and correct for amplification bias. Probabilistic barcoding, such as the Precise® assay (Cellular Research, Inc. (Palo Alto, CA)) and the Rhapsody® assay (Becton, Dickinson and Company (Franklin Lakes, NJ)), can correct for biases induced by the library preparation step by using molecular labeling (ML) to label mRNA between PCR and reverse transcription (RT).

[0023] The Precise® assay allows poly(T) oligonucleotides to hybridize to all poly(A)-mRNA in a sample during the RT step by utilizing an inexhaustible pool of probabilistic barcodes, each containing a large number of unique molecular labeling sequences, e.g., 6561–65536. The probabilistic barcodes may include universal PCR priming sites. During RT, target gene molecules react randomly with the probabilistic barcodes. Each target molecule hybridizes to a probabilistic barcode, potentially generating probabilistically barcoded complementary ribonucleotide acid (cDNA) molecules. After labeling, the probabilistically barcoded cDNA molecules obtained from the microwells of a microwell plate may be pooled in a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to obtain the number of reads, the number of probabilistic barcodes with unique molecular labeling sequences, and the number of mRNA molecules.

[0024] The disclosure herein includes methods for labeling nucleic acid targets in a sample. In some embodiments, the method includes contacting a sample containing a copy of a nucleic acid target with a plurality of probing oligonucleotides, each of which includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target. The method may include extending the plurality of probing oligonucleotides hybridized to the copy of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include barcoding the plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides, each of which includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular label sequence and a partial sequence of a nucleic acid target. The method may also include the step of determining the copy number of a nucleic acid target in a sample based on the number of molecular labels associated with the multiple barcoded probing oligonucleotides or their product.

[0025] The disclosure herein includes a method for determining the copy number of a nucleic acid target in a sample. In some embodiments, the method includes contacting a sample containing a copy of the nucleic acid target with a plurality of probing oligonucleotides, each of which includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target. The method may include extending the plurality of probing oligonucleotides hybridized to a copy of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include barcoding the plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides, each of which includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular label sequence and a partial sequence of a nucleic acid target. The method may also include the step of determining the copy number of a nucleic acid target in a sample based on the number of molecular labels associated with the multiple barcoded probing oligonucleotides or their product.

[0026] The disclosure herein includes methods for determining the spatial location and copy number of a nucleic acid target in a sample. In some embodiments, the method includes contacting each of two or more spatial locations in a sample containing copies of the nucleic acid target with a plurality of probing oligonucleotides, each of which comprises a coupling sequence, a probe sequence configured to hybridize with the nucleic acid target, and a predetermined spatial label. In some embodiments, probing oligonucleotides contacted with the same spatial location contain the same spatial label sequence, while probing oligonucleotides contacted with separate spatial locations in the sample contain different spatial label sequences. The method may include extending the plurality of probing oligonucleotides hybridized with copies of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of generating multiple barcoded probing oligonucleotides by barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes, wherein each oligonucleotide barcode of the multiple oligonucleotide barcodes includes a molecular label, and each of the multiple barcoded probing oligonucleotides includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may also include the step of obtaining sequencing data including multiple sequencing reads of the barcoded probing oligonucleotides or their products, wherein each of the multiple sequencing reads includes a spatial label sequence, a molecular label sequence, and a partial sequence of the nucleic acid target. The method may also include the step of determining the copy number of the nucleic acid target at each spatial location of the sample by counting the number of molecular labels having a distinct sequence associated with the nucleic acid target for each unique spatial label sequence associated with a distinct spatial location of the sample.

[0027] definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those widely understood by those skilled in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994) and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below:

[0028] As used herein, the term “adapter” may mean a sequence for facilitating 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 the following: spatial labels, target labels, sample labels, indexing labels, or barcode sequences (e.g., molecular labels). The adapter may be linear. The adapter may be adenylated. The adapter may be double-stranded or single-stranded. One or more adapters may be positioned at the 5' or 3' end of a nucleic acid. If the adapter includes known sequences at the 5' and 3' ends, the known sequences may be the same or different sequences. Adapters positioned at the 5' and / or 3' ends of a polynucleotide may be hybridized to one or more oligonucleotides immobilized on the surface. In some embodiments, the adapter may include a universal sequence. The universal sequence may be a region of nucleotide sequences common to two or more nucleic acid molecules. The two or more nucleic acid molecules may also have regions of different sequences. Therefore, for example, a 5' adapter may contain the same and / or universal nucleic acid sequence, and a 3' adapter may contain the same and / or universal sequence. The presence of universal sequences in different members of multiple nucleic acid molecules may enable replication or amplification of multiple different sequences using a single universal primer complementary to the universal sequence. Similarly, the presence of at least one, two (e.g., one pair), or more universal sequences in different members of an aggregate of nucleic acid molecules may enable replication or amplification of multiple different sequences using at least one, two (e.g., one pair), or more single universal primers complementary to the universal sequence. Therefore, universal primers include sequences that can hybridize to such universal sequences. A molecule having a target nucleic acid sequence may be modified to have universal adapters (e.g., non-target nucleic acid sequences) attached to one or both ends of different target nucleic acid sequences.One or more universal primers bound to the target nucleic acid may provide sites for hybridization of the universal primers. The one or more universal primers bound to the target nucleic acid may be the same or different from one another.

[0029] As used herein, the terms “associated” or “associated with” may mean that two or more species are identifiable as being located together at a given time. An association may mean that two or more species are in or have been in similar containers. An association may be an informational association. For example, digital information about two or more species may be stored and used to determine that one or more of the species were located together at a given time. An association may also be a physical association. In some embodiments, two or more associated species are “anchored,” “bonded,” or “immobilized” to each other or to a common solid or semi-solid surface. An association may refer to covalent or non-covalent means for bonding a label to a solid or semi-solid support, such as beads. An association may be a covalent bond between a target and a label. An association may include hybridization between two molecules (e.g., a target molecule and a label).

[0030] As used herein, the term “complementary” may refer to the ability of two nucleotides to precisely pair. For example, two nucleic acids are considered complementary at a given position if a nucleotide at a given position in one nucleic acid can form a hydrogen bond with a nucleotide in another nucleic acid. The complementarity between two single-stranded nucleic acid molecules may be “partial,” where only some of the nucleotides are bonded, or it may be complete, where total complementarity exists between the single-stranded molecules. A first nucleotide sequence may be referred to as the “complement” of a second nucleotide sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence may be referred to as the “reverse complement” of a second nucleotide sequence if the first nucleotide sequence is complementary to a sequence that is an inverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, “complementary” sequence may refer to the “complementary” or “reverse complement” of a sequence. It is understood from this disclosure that when one molecule can hybridize with another molecule, it may be complementary to or partially complementary to the molecule it is hybridizing with.

[0031] 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 the step of determining the number of unique labels associated with the target in the sample. This methodology, which can be inherently probabilistic, transforms the problem of molecular counting into a series of yes / no digital questions concerning the detection of a predefined set of labels, one of which is the location and identification of identical molecules. As used herein, the terms “label” or “multiple labels” may refer to a nucleic acid code associated with a target in a sample. A label may, for example, be a nucleic acid label. A label may be a label that is entirely or partially amplified. A label may be a label that is entirely or partially sequenceable. A label may be a portion of a native nucleic acid that can be identified as distinct. A label may be a known sequence. A label may include a junction of nucleic acid sequences, for example, a junction of native and unnatural sequences. As used herein, the term “label” may be used interchangeably with the terms “index,” “tag,” or “labeled tag.” A label can carry information. For example, in various embodiments, a label may be used to determine the identity of a sample, the source of a sample, the identity of cells, and / or targets.

[0032] As used herein, the term “non-exhaustive reservoir” may refer to a pool of barcodes (e.g., probabilistic barcodes) composed of a large number of different labels. A non-exhaustive reservoir may contain a large number of different barcodes such that, when the non-exhaustive reservoir is associated with a pool of targets, each target is likely to 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 identical target molecules in the population, compared to the diversity of labels. The size of the resulting labeled target molecule can be determined by the probabilistic nature of the barcoding process, and then, by analyzing the number of barcodes detected, it is possible to calculate the number of target molecules present in the original population or sample. If the ratio of the number of present target molecules 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 will be labeled with a given label is very low).

[0033] As used herein, the term “nucleic acid” refers to a polynucleotide sequence or fragment thereof. Nucleic acids may include nucleotides. Nucleic acids may be exogenous or endogenous to cells. Nucleic acids may exist in a cell-free environment. Nucleic acids may be genes or fragments thereof. Nucleic acids may be DNA. Nucleic acids may be RNA. Nucleic acids may include one or more analogs (e.g., modified skeletons, sugars, or nucleic acid bases). Some non-exclusive examples of analogs include 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholino, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, quosin, and waiosin. "Nucleic acid," "polynucleotide," "targeted polynucleotide," and "targeted nucleic acid" can be used interchangeably.

[0034] Nucleic acids may include one or more modifications (e.g., base modifications, skeletal modifications) to result in nucleic acids having novel or enhanced properties (e.g., improved stability). Nucleic acids may also include nucleic acid affinity tags. Nucleosides can be base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide may be a nucleoside further containing a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides containing pentofuranosyl sugars, the phosphate group may 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. The ends of this linear polymer compound can then be further bonded to form a cyclic compound, but linear compounds are generally preferred. In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold in a manner that results in fully or partially double-stranded compounds. Within nucleic acids, phosphate groups can generally be said to form the internucleoside skeleton of the nucleic acid. The linkage or skeleton may be a 3'-5' phosphodiester linkage.

[0035] Nucleic acids may include modified skeletons and / or modified nucleoside linkages. Modified skeletons may include those that retain phosphorus atoms and those that do not. Suitable modified nucleic acid skeletons containing a phosphorus atom include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, such as 3'-aminophosphoramidates and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates having a normal 3'-5' linkage, 2'-5' linked analogs, and those having reverse polarity, where one or more internucleotide linkages are 3'-to-3', 5'-to-5', or 2'-to-2' linkages.

[0036] Nucleic acids may include polynucleotide skeletons formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more heteroatoms or heterocyclic nucleoside linkages. These include those having morpholino linkages (partially formed from the sugar portion of a nucleoside), siloxane skeletons, sulfides, sulfoxides, and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, riboacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others having mixed N, O, S, and CH2 component portions.

[0037] Nucleic acids may include nucleic acid mimes. The term “mimetic” may refer to polynucleotides in which only the furanose ring, or both the furanose ring and the internucleotide linkages, are replaced with non-furanose groups, and substitution of only the furanose ring may also be referred to as a sugar substitute. Heterocyclic base moieties or modified heterocyclic base moieties may be retained for hybridization with a suitable target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In PNAs, the sugar backbone of the polynucleotide may be replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleotide may be retained and directly or indirectly bonded to the aza nitrogen atom of the amide moiety of the backbone. The backbone in a PNA compound may contain two or more linked aminoethylglycine units, thereby providing the amide-containing backbone to the PNA. Heterocyclic base moieties may be directly or indirectly bonded to the aza nitrogen atom of the amide moiety of the backbone. Nucleic acids may contain a morpholino backbone structure. For example, a nucleic acid may contain a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, the phosphodiester linkage can be replaced by a phosphorodiamidate or other non-phosphodiester nucleoside linkage.

[0038] Nucleic acids may contain linked morpholino units (e.g., morpholino nucleic acids) having heterocyclic bases bonded to a morpholino ring. Linking groups can link morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds may have fewer undesirable interactions with intracellular proteins. Morpholino-based polynucleotides can be nonionic mimetic forms of nucleic acids. Various compounds within the morpholino class can be linked using different linking groups. A further class of polynucleotide mimetic forms may be called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used in the synthesis of oligomeric compounds using phosphoramidite chemistry. Incorporating CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of natural complexes. Further modifications include locked nucleic acids (LNAs) in which a 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, which in turn forms a bicyclic sugar moiety. The linkage can be a methylene (-CH2) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and LNA analogs can exhibit very high double-chain thermal stability with complementary nucleic acids (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility.

[0039] Nucleic acids may also include modifications or substitutions of nucleic acid bases (often simply referred to as "bases"). As used herein, "unmodified" or "natural" nucleic acid bases include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, pyrimidine bases 5-propynyl(-C=C-CH3)uracil and cytosine, and other alkyl derivatives, 6-azouracil, cytosine Examples include tosine and thymine, 5-uracil (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosine, 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 nucleic acid bases include tricyclic pyrimidines, e.g., phenoxazinecytidine (1H-pyrimido(5,4-b)(1,4)benzoxazine-2(3H)-one), phenothiazinecytidine (1H-pyrimido(5,4-b)(1,4)benzothiadin-2(3H)-one), G-clamps, e.g., substituted phenoxazinecytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazine-2(3H)-one), phenothiazinecytidine Examples include (1H-pyrimido(5,4-b)(1,4)benzothiadin-2(3H)-one), G-clamps, for example, substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazine-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indole-2-one), and pyridoindole cytidine (H-pyrimido(3',2':4,5)pyrrolo[2,3-d]pyrimidine-2-one).

[0040] As used herein, the term “sample” may refer to a composition containing a target. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms. As used herein, the terms “sample collection device” or “device” may refer to a device capable of collecting and / or placing sections of a sample onto a substrate. Sample devices may refer to, for example, fluorescence-activated cell sorting (FACS) machines, cell sorters, biopsy needles, biopsy devices, tissue sectioning devices, microfluidic devices, blade grids, and / or microtomes.

[0041] As used herein, the term “solid support” may refer to separate solid or semi-solid surfaces to which multiple barcodes (e.g., probabilistic barcodes) can be bound. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar structure composed of plastic, ceramic, metal, or polymer material (e.g., hydrogel) to which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support may contain separate particles that are spherical (e.g., microspheres) or have non-spherical or irregular shapes, such as cubes, cuboidal, pyramidal, cylindrical, conical, rectangular, or disc-shaped. Beads may have a non-spherical shape. Multiple solid supports spaced apart in an array may not contain a substrate. The term “solid support” may be used interchangeably with the term “beads.”

[0042] As used herein, the term “probabilistic barcode” may refer to a polynucleotide sequence containing the label of this disclosure. A probabilistic barcode may be a polynucleotide sequence that can be used for probabilistic barcoding. A probabilistic barcode can be used to quantify a target in a sample. A probabilistic barcode can be used to control for errors that may occur after the label has been associated with the target. For example, a probabilistic barcode can be used to evaluate amplification or sequencing errors. A probabilistic barcode associated with a target may be referred to as a probabilistic barcode-target or a probabilistic barcode-tag-target. As used herein, the term “gene-specific stochastic barcode” may refer to a polynucleotide sequence containing a label and a gene-specific target-binding region. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode can be used to quantify a target in a sample. A stochastic barcode can be used to control for errors that may occur after the label has been associated with the target. For example, a stochastic barcode can be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0043] As used herein, the term “probabilistic barcoding” may refer to the random labeling (e.g., barcoding) of nucleic acids. Probabilistic barcoding can utilize a Poisson recursive strategy to associate labels and quantify the labels associated with a target. As used herein, the term “probabilistic barcoding” may be used interchangeably with “probabilistic labeling.” As used herein, the term “target” may refer to a composition that can be associated with a barcode (e.g., a probabilistic barcode). Suitable exemplary targets for analysis by the methods, devices, and systems of this disclosure include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. 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 may be a lipid. As used herein, “target” may be used interchangeably with “species.”

[0044] As used herein, the term “reverse transcriptase” may refer to a group of enzymes that have reverse transcriptase activity (i.e., catalyze the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retroron reverse transcriptases, bacterial reverse transcriptases, group II intron reverse transcriptases, and their variants, variants, or derivatives. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retroron reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include Lactococcus lactis LI.LtrB intron reverse transcriptase, Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases include numerous classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversification-generating retroelements, among others).

[0045] The terms “universal adapter primer,” “universal primer adapter,” or “universal adapter sequence” are used interchangeably to refer to nucleotide sequences that can be hybridized to a barcode (e.g., a probabilistic barcode) and used to generate a gene-specific barcode. A universal adapter sequence may be, for example, a known sequence that is universal across all barcodes used in the methods of this disclosure. For example, if multiple targets are labeled using the methods disclosed herein, each of the target-specific sequences may be ligated to the same universal adapter sequence. In some embodiments, two or more universal adapter sequences may be used in the methods disclosed herein. For example, if multiple targets are labeled using the methods disclosed herein, at least two of the target-specific sequences may be ligated to different universal adapter sequences. A universal adapter primer and its complement may be contained in two oligonucleotides, one of which contains the target-specific sequence and the other contains the barcode. For example, a universal adapter sequence may be part of an oligonucleotide containing a target-specific sequence to generate a nucleotide sequence complementary to the target nucleic acid. A second oligonucleotide containing complementary sequences of the barcode and universal adapter sequences can hybridize with the nucleotide sequence to generate a target-specific barcode (target-specific probabilistic barcode). In some embodiments, the universal adapter primer has a different sequence from the universal PCR primer used in the method of this disclosure.

[0046] barcode Barcoding, such as probabilistic barcoding, is described, for example, in Fu et al., Proc Natl Acad Sci USA, 2011 May 31, 108(22):9026-31; U.S. Patent Application Publication US2011 / 0160078; Fan et al., Science, 2015 February 6, 347(6222):1258367; U.S. Patent Application Publication US2015 / 0299784; and PCT Application Publication WO2015 / 031691, the contents of each of these, including any supplementary or additional information or materials, are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein may be probabilistic barcodes, which may be polynucleotide sequences that can be used to probabilistically label (e.g., barcode, tag) a target. A barcode may be called a probabilistic barcode if the ratio of the number of different barcode sequences in the probabilistic barcode to the number of occurrences of any of the targets to be labeled is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, or is approximately one of these values ​​or such a number or range. The targets may be mRNA species containing mRNA molecules with identical or nearly identical sequences. A barcode can be called a probabilistic barcode if the ratio of the number of different barcode sequences of the probabilistic barcode to the number of occurrences of any of the targets to be labeled is at least 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 sequence of a probabilistic barcode can be called a molecular label.

[0047] A barcode, such as a probabilistic barcode, may contain one or more labels. Exemplary labels include universal labels, cellular labels, barcode sequences (e.g., molecular labels), sample labels, plate labels, spatial labels, and / or pre-spatial labels. Figure 1 shows an exemplary barcode 104 having a spatial label. Barcode 104 may contain a 5' amine that can be linked to a solid support 105. The barcode may include a universal label, a dimension label, a spatial label, a cellular label, and / or a molecular label. The order of different labels within the barcode (including, but not limited to, universal labels, dimension labels, spatial labels, cellular labels, and molecular labels) may vary. For example, as shown in Figure 1, the universal label may be the 5'-side label, and the molecular label may be the 3'-side label. The spatial label, dimension label, and cellular label may be in any order. In some embodiments, the universal label, spatial label, dimension label, cellular label, and molecular label are in any order. The barcode may include a target binding region. The target-binding region may interact with a target in the sample (e.g., target nucleic acid, RNA, mRNA, DNA). For example, the target-binding region may include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. In some cases, the barcode labels (e.g., universal labels, dimensional labels, spatial labels, cellular labels, and barcode sequences) may be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides apart.

[0048] A label, such as a cell label, may include a set of unique nucleic acid subsequences of a defined length, e.g., each containing 7 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 set containing 7 nucleotide sequences can be designed such that any combination of pairs of sequences within the set exhibits a defined "gene distance" (or mismatch number of bases). For example, the error correction subsequence set may be designed to exhibit a gene distance of 3 nucleotides. In this case, consideration of the error correction sequences in the sequence dataset of the labeled target nucleic acid molecule (described in more detail below) may enable the detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequence used to create the error correction code may vary, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or a number or range between any two of these values, or approximately these values ​​or such a number or range of nucleotides. In some embodiments, nucleic acid subsequences of other lengths may be used to create error correction codes.

[0049] The barcode may include a target-binding region. The target-binding region may interact with a target in the sample. The target may be 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, or may include these. In some embodiments, multiple targets may include deoxyribonucleic acid (DNA).

[0050] In some embodiments, the target binding region may include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more of the barcode labels (e.g., universal labels, dimensional labels, spatial labels, cellular labels, and barcode sequences (e.g., molecular labels)) may be spaced apart by spacers from one or two of the remaining barcode labels. The spacers may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, or more. In some embodiments, none of the barcode labels are spaced apart by spacers.

[0051] Universal Sign A barcode may include one or more universal labels. In some embodiments, one or more universal labels may be the same for all barcodes in a set of barcodes bound to a given solid support. In some embodiments, one or more universal labels may be the same for all barcodes bound to multiple beads. In some embodiments, a universal label may include a nucleic acid sequence that can hybridize to a sequencing primer. A sequencing primer can be used to sequence a barcode containing a universal label. A sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, a universal label may include a nucleic acid sequence that can hybridize to a PCR primer. In some embodiments, a universal label may include a nucleic acid sequence that can hybridize to both a sequencing primer and a PCR primer. The nucleic acid sequence of a universal label that can hybridize to a sequencing primer or a PCR primer may be referred to as a primer binding site. A universal label may include a sequence that can be used to initiate the transcription of a barcode. A universal label may include a sequence that can be used for extending a barcode or a region within a barcode. The universal label may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values, or approximately 100, 200, or 300 nucleotides in length. For example, a universal label may include at least about 10 nucleotides. The universal label may be, for example, at least, or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or modified nucleotide may be part of the universal label sequence to allow the barcode to be cleaved from the support.

[0052] dimensional indicator A barcode may include one or more dimensional labels. In some embodiments, a dimensional label may include a nucleic acid sequence that provides information about the dimension in which labeling (e.g., stochastic labeling) occurred. For example, a dimensional label may provide information about the time when a target was barcoded. A dimensional label may be associated with the time of barcoding (e.g., stochastic barcoding) in the sample. A dimensional label may be activated at the time of labeling. Different dimensional labels may be activated at different times. A dimensional label provides information about the target, the group of targets, and / or the order in which the sample was barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. Cells may be pulsed again with a barcode (e.g., stochastic barcode) in the G1 phase of the cell cycle. Cells may be pulsed again with a barcode in the S phase of the cell cycle, and so on. The barcode in each pulse (e.g., each stage of the cell cycle) may include a different dimensional label. In this way, the dimensional label provides information about which target was labeled at which stage of the cell cycle. Dimensional labeling can investigate many different biological time periods. Exemplary biological time periods include, but are not limited to, the cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., cells, cell populations) may be probabilistically labeled before and / or after treatment with a drug and / or therapeutic agent. Changes in the copy number of distinct targets can indicate the sample's response to the drug and / or therapeutic agent.

[0053] Dimensional labels may be activatable. Activatable dimensional labels can be activated at a specific point in time. Activatable labels can, for example, be continuously activated (e.g., not deactivated). Activatable dimensional labels may, for example, be reversibly activatable (e.g., activatable dimensional labels can be activated and deactivated). Dimensional labels may be reversibly activatable, for example, at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. Dimensional labels may be reversibly activatable, for example, at least once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. In some embodiments, dimensional labels can be activated by fluorescence, light, chemical events (e.g., cleavage, ligation of another molecule, addition of modifications (e.g., pegylation, SUMOylation, acetylation, deacetylation, demethylation)), photochemical events (e.g., photocaging), and introduction of unnatural nucleotides.

[0054] In some embodiments, the dimensional identifier may be the same for all barcodes (e.g., probabilistic barcodes) bound to a given solid support (e.g., beads), but may be different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support may include the same dimensional identifier. In some embodiments, at least 60% of the barcodes on the same solid support may include the same dimensional identifier. In some embodiments, at least 95% of the barcodes on the same solid support may include the same dimensional identifier. Multiple solid supports (e.g., beads), 10 6Many unique dimensional label sequences, more than one, may be presented. A dimensional label may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values, or approximately the length of these values ​​or such a number or range of nucleotides. A dimensional label may be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. A dimensional label may contain about 5 to about 200 nucleotides. A dimensional label may contain about 10 to about 150 nucleotides. A dimensional label may contain about 20 to about 125 nucleotides in length.

[0055] spatial sign A 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 a target molecule associated with the barcode. The spatial marker may be associated with coordinates in a sample. The coordinates may be fixed coordinates. For example, the coordinates may be fixed in relation to a substrate. The spatial marker may refer to a two-dimensional or three-dimensional grid. The coordinates may be fixed to a landmark. The landmark may be identifiable in space. The landmark may be a structure that can be imaged. The landmark may be a biological structure, e.g., an anatomical landmark. The landmark may be a cellular landmark, e.g., an organelle. The landmark may be a non-natural landmark, e.g., an identifiable identifier, e.g., a color code, barcode, magnetic properties, fluorescence, radioactivity, or a structure with a unique size or shape. The spatial marker may be associated with a physical compartment (e.g., a well, container, or droplet). In some embodiments, multiple spatial markers are used together to code one or more locations in space.

[0056] The spatial identifier may be the same for all barcodes bound to a given solid support (e.g., beads), but may be different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes on the same solid support containing the same spatial identifier may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or approximately these values ​​or such a number or range. In some embodiments, the percentage of barcodes on the same solid support containing the same spatial identifier may be at least, or at most, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support may contain the same spatial identifier. In some embodiments, at least 95% of the barcodes on the same solid support may contain the same spatial identifier.

[0057] Multiple solid supports (e.g., beads), 10 6 Many unique spatial label sequences, more than one, may be presented. The spatial label may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values, or approximately the length of these values ​​or such a number or range of nucleotides. For example, the spatial label may be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. The spatial label may contain between about 5 and about 200 nucleotides. The spatial label may contain between about 10 and about 150 nucleotides. The spatial label may contain between about 20 and about 125 nucleotides in length.

[0058] cell labeling A barcode (e.g., a probabilistic barcode) may include one or more cell labels. In some embodiments, the cell labels may include nucleic acid sequences that provide information for determining which target nucleic acids originate from which cells. In some embodiments, the cell labels are identical for all barcodes bound 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 cell label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or approximately these values ​​or such a number or range. For example, at least 60% of barcodes on the same solid support may contain the same cell marker. As another example, at least 95% of barcodes on the same solid support may contain the same cell marker. Multiple solid supports (e.g., beads), 10 6 Many unique cell-labeling sequences, more than one, may be presented. The cell label may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values, or approximately these values ​​or such a number or range of nucleotides in length. For example, the cell label may be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. For example, the cell label may contain between approximately 5 and approximately 200 nucleotides. As another example, the cell label may contain between approximately 10 and approximately 150 nucleotides. As yet another example, the cell label may contain between approximately 20 and approximately 125 nucleotides in length.

[0059] barcode array A barcode can include one or more barcode arrays. In some embodiments, a barcode array can include a nucleic acid sequence that provides information regarding a particular type of target nucleic acid species hybridized to the barcode. A barcode array can include a nucleic acid sequence that provides a counter (e.g., provides an approximate value) regarding the occurrence of a particular target nucleic acid species hybridized to the barcode (e.g., the target binding region).

[0060] In some embodiments, a diverse set of barcode arrays is attached to a given solid support (e.g., beads). In some embodiments, 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, or approximately these values or a unique molecular label sequence of such a number or range can be present. For example, a plurality of barcodes can include approximately 6561 barcode arrays having distinct sequences. As another example, a plurality of barcodes can include approximately 65536 barcode arrays having distinct sequences. In some embodiments, at least, or at most 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 unique barcode arrays can be present. The unique molecular label sequence can be attached to a given solid support (e.g., beads). In some embodiments, the unique molecular label sequence is partially or wholly encapsulated by a particle (e.g., a hydrogel bead).

[0061] The length of a barcode may vary in different implementations. For example, a barcode may have a nucleotide length of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, or approximately 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, a barcode may have a length of at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0062] molecular label A barcode (e.g., a probabilistic barcode) may include one or more molecular labels. The molecular labels may include barcode sequences. In some embodiments, the molecular labels may include nucleic acid sequences that provide information about specific types of target nucleic acid species hybridized to the barcode. The molecular labels may include nucleic acid sequences that provide a counter for specific occurrences of target nucleic acid species hybridized to the barcode (e.g., a target binding region). In some embodiments, a diverse set of molecular labels is bonded to a given solid support (e.g., beads). In some embodiments, 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 A unique molecular label sequence of one, or a number or range between any two of these values, or about 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9There may be unique molecular label sequences of 1, or a number or range between any two of these values. For example, multiple barcodes may contain about 6561 molecular labels having distinct sequences. As another example, multiple barcodes may contain about 65536 molecular labels having distinct sequences. In some embodiments, there may be at least, or up to 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 10 9 A unique molecular label sequence may exist. A barcode having a unique molecular label sequence may be bound to a given solid support (e.g., beads).

[0063] For barcoding using multiple probabilistic barcodes (e.g., probabilistic barcoding), the ratio of the number of different molecular label sequences to the number of occurrences of any of the targets is 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, Alternatively, it may be a number or range between any two of these values, or approximately 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target may be an mRNA species containing mRNA molecules having the same or nearly identical sequence. In some embodiments, the ratio of the number of different molecularly labeled sequences to the number of occurrences of any of the targets is at least, or at most, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0064] The molecular label may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values, or approximately the length of these values ​​or such a number or range of nucleotides. For example, the molecular label may be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0065] Target binding region The barcode may include one or more target-binding regions, e.g., capture probes. In some embodiments, the target-binding region may hybridize with a target of interest. In some embodiments, the target-binding region may include a nucleic acid sequence that specifically hybridizes with a target (e.g., a target nucleic acid, a target molecule, e.g., a cellular nucleic acid to be analyzed), e.g., a specific gene sequence. In some embodiments, the target-binding region may include a nucleic acid sequence that can bind (e.g., hybridize) to a specific location on a particular target nucleic acid. In some embodiments, the target-binding region may include a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be ligated to any nucleic acid molecule containing a sequence complementary to the restriction site overhang.

[0066] 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 multiple target nucleic acids independently of a specific sequence of the target nucleic acid. For example, the target binding region may include a random multimer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, or a combination thereof. For example, the target binding region may be an oligo(dT) sequence that hybridizes to the poly(A) tail on an mRNA molecule. A random multimer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, noumer, decamer, or a multimer sequence of any length or more. In some embodiments, the target binding region is the same for all barcodes attached to a given bead. In some embodiments, the target binding regions of multiple barcodes attached to a given bead may include two or more different target binding sequences. The target-binding region may be 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or a number or range between any two of these values, or approximately the length of these values ​​or such a number or range of nucleotides. The target-binding region may be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or more. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase such as Moloney's mouse leukemia virus (MMLV) reverse transcriptase to produce a cDNA molecule with a poly(dC) tail. The barcode may contain a target-binding region with a poly(dG) tail. When base pairing occurs between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues replication to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains a barcode sequence (e.g., molecular label) on the 3' end of the cDNA molecule.

[0067] In some embodiments, the target-binding region may include an oligo(dT) that can hybridize with mRNA containing a polyadenylated end. The target-binding region may be gene-specific. For example, the target-binding region may be configured to hybridize to a specific region of the target. The target-binding region may be a number or range between 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 any two of these values, or an approximate length of nucleotides of these values ​​or such a number or range. The target-binding region may be at least, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The target-binding region may be approximately 5 to 30 nucleotides in length. If a barcode contains a gene-specific target-binding region, the barcode may be referred to herein as a gene-specific barcode.

[0068] Orientation characteristics A probabilistic barcode (e.g., a stochastic barcode) may include one or more orientation properties that can be used to orient the barcode (e.g., alignment). The barcode may include portions for isoelectric focusing. Different barcodes may include different isoelectric focusing points. When these barcodes are introduced into a sample, the sample may undergo isoelectric focusing to orient the barcodes in known forms. In this way, the orientation properties can be used to unfold a known map of the barcode in the sample. Exemplary orientation properties include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric focus, spin, conductivity, and / or self-assembly. For example, a barcode with a self-assembly orientation property may, upon activation, self-assemble into a specific orientation (e.g., a nucleic acid nanostructure).

[0069] affinity properties A barcode (e.g., a probabilistic barcode) may include one or more affinity properties. For example, spatial labeling may include affinity properties. Affinity properties may include chemical and / or biological parts that can facilitate the binding of the barcode to another entity (e.g., a cell receptor). For example, affinity properties may include antibodies, e.g., antibodies specific to a particular part of a sample (e.g., a receptor). In some embodiments, the antibody may guide the barcode to a specific cell type or molecule. Targets on and / or near a specific cell type or molecule can be labeled (e.g., probabilistically labeled). In some embodiments, affinity properties can provide spatial information in addition to the nucleotide sequence of spatial labeling, because the antibody may guide the barcode to a specific location. The antibody may be a therapeutic antibody, e.g., a monoclonal antibody or a polyclonal antibody. The antibody may be humanized or a chimeric antibody. The antibody may be a naked antibody or a fusion antibody.

[0070] Antibodies may be full-length immunoglobulin molecules (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) (e.g., IgG antibodies), or immunologically active (i.e., specifically binding) portions of immunoglobulin molecules, such as antibody fragments. Antibody fragments may be, for example, a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, or sFv. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. Examples of antibody fragments include isolated fragments consisting of the variable region of an antibody, such as "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules ("scFv proteins") in which the variable regions of the light and heavy chains are linked by a peptide linker. Exemplary antibodies include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0071] Universal Adapter Primer A barcode may contain one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, may contain a universal adapter primer. The universal adapter primer may point to a nucleotide sequence that is universal across all barcodes. The universal adapter primer can be used to construct a gene-specific barcode. The universal adapter primer may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, or a number or range between any two of these, or an approximate length of these values ​​or such a number or range of nucleotides. The universal adapter primer may be at least, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The universal adapter primer may be 5 to 30 nucleotides in length.

[0072] Linker If a barcode contains more than one type of label (e.g., more than one cellular label or more than one barcode sequence, e.g., one molecular label), the label may contain interspersed linker label sequences. Linker label sequences may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or longer. Linker label sequences may be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or longer. In some cases, linker label sequences are 12 nucleotides long. Linker label sequences can be used to facilitate the synthesis of barcodes. Linker labels may include error correction (e.g., Hamming) codes.

[0073] solid support The barcodes disclosed herein, for example, stochastic barcodes, may, in some embodiments, be associated with a solid support. The solid support may be, for example, synthetic particles. In some embodiments, the molecular labels of some or all of the barcode sequences, for example, the stochastic barcodes (e.g., the first barcode sequence) of a plurality of barcodes (e.g., the first plurality of barcodes) on a solid support, differ by at least one nucleotide. The cellular labels of barcodes on the same solid support may be the same. The cellular labels of barcodes on different solid supports may differ by at least one nucleotide. For example, the first cellular labels of the first plurality of barcodes on the first solid support may have the same sequence, and the second cellular labels of the second plurality of barcodes on the second solid support may have the same sequence. The first cellular labels of the first plurality of barcodes on the first solid support and the second cellular labels of the second plurality of barcodes on the second solid support may differ by at least one nucleotide. The cellular labels may be, for example, about 5 to 20 nucleotides long. The barcode sequence may be, for example, about 5 to 20 nucleotides long. The synthetic particles may be, for example, beads.

[0074] The beads may be, for example, silica gel beads, pore-controlled glass beads, magnetic beads, Dynabead, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads may contain materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sephadex, cellulose, nylon, silicone, or any combination thereof. In some embodiments, the beads may be polymer beads, such as deformable beads or gel beads functionalized with barcodes or probabilistic barcodes (e.g., gel beads from 10X Genomics (San Francisco, CA)). In some implementations, the gel beads may comprise polymer-based gels. Gel beads can be produced, for example, by encapsulating one or more polymer precursors in droplets. Exposure of the polymer precursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)) may produce gel beads.

[0075] In some embodiments, the particles may be disintegrable (e.g., soluble, decomposable). For example, polymer beads may dissolve, melt, or decompose under desired conditions. Desired conditions may include environmental conditions. Desired conditions may result in the dissolution, melting, or decomposition of polymer beads in a controlled manner. Gel beads may dissolve, melt, or decompose due to chemical, physical, biological, thermal, magnetic, electrical, or optical stimuli, or any combination thereof.

[0076] The analyte and / or reagent, e.g., oligonucleotide barcodes, may be ligated / immobilized, for example, to the inner surface of gel beads (e.g., the interior accessible through diffusion of the material used to generate the oligonucleotide barcodes and / or oligonucleotide barcodes), and / or to the outer surface of gel beads or any other microcapsules described herein. Ligation / immobilization may be via any form of chemical bonding (e.g., covalent bonding, ionic bonding) or physical phenomena (e.g., van der Waals forces, dipole interactions, etc.). In some embodiments, the ligation / immobilization of the reagent to the gel beads or any other microcapsules described herein may be reversible, for example, via an unstable moiety (e.g., via a chemical crosslinker, including a chemical crosslinker described herein). Upon application of a stimulus, the unstable moiety may be cleaved, and the immobilized reagent may be released. In some embodiments, the unstable moiety is a disulfide bond. For example, in cases where oligonucleotide barcodes are immobilized to gel beads via a disulfide bond, exposure of the disulfide bond to a reducing agent may cleave the disulfide bond and release the oligonucleotide barcode from the beads. The unstable portion may be included as part of the gel beads or microcapsules, as part of the chemical linker that links the reagent or analyte to the gel beads or microcapsules, and / or as part of the reagent or analyte. In some embodiments, at least one of the multiple barcodes may be immobilized on the particle, partially immobilized on the particle, encapsulated on the particle, partially encapsulated on the particle, or any combination thereof.

[0077] In some embodiments, the gel beads may comprise a wide range of different polymers, including but not limited to polymers, heat-sensitive polymers, photosensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemical-sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. Examples of polymers include, but are not limited to, poly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethylammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthalaldehyde) (PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic acid-coglycolic acid) (PLGA).

[0078] Numerous chemical stimuli can be used to trigger the collapse, dissolution, or decomposition of beads. Examples of these chemical changes include, but are not limited to, pH-mediated changes to the bead wall, collapse of the bead wall via chemical cleavage of crosslinking bonds, triggering the depolymerization of the bead wall, and switching reactions of the bead wall. Bulk changes can also be used to trigger the collapse of beads. Bulk or physical changes to microcapsules induced by various stimuli also offer many advantages in the design of capsules for reagent release. These bulk or physical changes occur on a macroscopic scale, and bead rupture is a result of mechanical-physical forces induced by the stimulus. These processes include, but are not limited to, pressure-induced rupture, melting of the bead wall, or changes in the porosity of the bead wall.

[0079] Biological stimuli can also be used to trigger the collapse, dissolution, or degradation of beads. Generally, biological triggers are similar to chemical triggers, but in many cases, they use biomolecules or molecules commonly found in biological systems, such as enzymes, peptides, sugars, fatty acids, and nucleic acids. For example, beads may contain polymers with peptide crosslinks that are sensitive to cleavage by specific proteases. More specifically, one example may contain microcapsules containing GFLGK peptide crosslinks. Adding a biological trigger such as protease cathepsin B cleaves the peptide crosslinks in the shell wall, releasing the contents of the bead. In other cases, the protease may be thermally activated. In another example, beads may contain a shell wall containing cellulose. Adding chitosan, a hydrolytic enzyme, acts as a biological trigger for cleavage of the cellulose bonds, depolymerization of the shell wall, and release of its internal contents.

[0080] Beads can also be induced to release their contents when heat is applied. Temperature changes can cause various changes in beads. A change in temperature can cause the beads to melt, causing the bead walls to collapse. In other cases, heat can increase the internal pressure of the bead's internal components, causing the bead to collapse or explode. In yet another case, heat can convert the bead into a compressed and dehydrated state. Heat can also act on the heat-sensitive polymer within the bead walls, causing the bead to collapse. By incorporating magnetic nanoparticles into the bead walls of microcapsules, the collapse of the beads can be triggered, as well as the beads can be guided in an array. The devices of this disclosure may include magnetic beads for either purpose. In one example, Fe3O4 nanoparticles are incorporated into polymer electrolyte-containing beads, thereby triggering collapse in the presence of an oscillating magnetic field stimulus.

[0081] Beads can also disintegrate, dissolve, or decompose as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrosensitive beads can be made capable of triggering both bead disintegration and other functions such as alignment, electrical conduction, or redox reactions in an electric field. In one example, beads containing an electrosensitive material are aligned in an electric field so that the release of internal reagents can be controlled. In another example, an electric field may induce redox reactions within the bead walls themselves, which may increase porosity. Light stimulation can also be used to cause the beads to disintegrate. Numerous phototriggers are possible, and these may include systems using various molecules such as nanoparticles and chromophores that can absorb photons within a specific range of wavelengths. For example, a metal oxide coating can be used as a capsule trigger. UV irradiation of a polymer electrolyte capsule coated with SiO2 can cause the bead wall to disintegrate. In yet another example, a photoswitchable material, such as an azobenzene group, may be incorporated into the bead wall. When UV or visible light is applied, these chemicals absorb photons and undergo reversible cis-to-trans isomerization. In this embodiment, the incorporation of a photoswitch provides a bead wall that can disintegrate or become more porous when a phototrigger is applied. For example, in a non-limiting example of barcoding (e.g., probabilistic barcoding) shown in Figure 2, in block 208, cells, e.g., single cells, can be introduced into multiple microwells of a microwell array, and then in block 212, beads can be introduced into multiple microwells of the microwell array. Each microwell may contain one bead. The beads may contain multiple barcodes. The barcodes may contain a 5' amine region bound to the bead. The barcodes may contain a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0082] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., beads). Each barcode associated with a solid support may include a barcode sequence selected from the group including at least 100 or 1000 barcode sequences, each having a unique sequence. In some embodiments, different barcodes associated with a solid support may include barcodes having different sequences. In some embodiments, a certain percentage of barcodes associated with a solid support may include the same cell label. For example, the percentage may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or approximately these values ​​or such a number or range. As another example, the percentage may be at least, or at most, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support may have the same cell markers. Barcodes associated with different solid supports may have different cell markers, selected from the group comprising at least 100 or 1000 cell markers having unique sequences.

[0083] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., beads). In some embodiments, the step of barcoding multiple labels in a sample can be performed using a solid support comprising multiple synthetic particles associated with multiple barcodes. In some embodiments, the solid support may comprise multiple synthetic particles associated with multiple barcodes. Spatial labeling of multiple barcodes on different solid supports may differ by at least one nucleotide. The solid support may comprise multiple barcodes in two or three dimensions, for example. The synthetic particles may be beads. The beads may be silica gel beads, pore-controlled glass beads, magnetic beads, Dynabead, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. Examples of solid supports include polymers, matrices, hydrogels, needle array devices, antibodies, or any combination thereof. In some embodiments, the solid support may be freely floating. In some embodiments, the solid support may be embedded in a semi-solid or solid array. The barcodes may not be associated with the solid support. The barcodes may be individual nucleotides. The barcode may be associated with the substrate.

[0084] As used herein, the terms “moored,” “bonded,” and “immobilized” are interchangeable and may refer to covalent or non-covalent means for bonding a barcode to a solid support. Any of a variety of different solid supports can be used as a solid support for bonding pre-synthesized barcodes or for solid-phase synthesis of barcode insights. In some embodiments, the solid support is a bead. The bead may include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures on which nucleic acids can be immobilized (e.g., covalently or non-covalently). The bead may be composed of, for example, plastic, ceramic, metal, polymer material, or any combination thereof. The bead may be a separate particle that is spherical (e.g., a microsphere), or may have a non-spherical or irregular shape, such as a cube, cuboidal, pyramidal, cylindrical, conical, rectangular, or disc-shaped. In some embodiments, the bead may be non-spherical in shape.

[0085] The beads may contain 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, methylstyrene, acrylic polymers, titanium, latex, Sepharose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof. In some embodiments, the beads (e.g., the beads to which the label is attached) are hydrogel beads. In some embodiments, the beads contain a hydrogel.

[0086] Some embodiments disclosed herein include one or more particles (e.g., beads). Each particle may contain multiple oligonucleotides (e.g., barcodes). Each of the multiple oligonucleotides may contain a barcode sequence (e.g., a molecular label sequence), a cell label, and a target binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequences of each of the multiple oligonucleotides may be the same. The cell label sequences of oligonucleotides on different particles may be different so as to be able to identify oligonucleotides on different particles. The number of different cell label sequences may vary in different implementations. In some embodiments, the number of cell label essences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 The number of cell-labeled sequences may be at least 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, or 10 9There may be 1 or fewer particles. In some embodiments, one or fewer, two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, twenty or fewer, thirty or fewer, forty or fewer, fifty or fewer, sixty or fewer, seventy or fewer, eighty or fewer, ninety or fewer, one hundred or fewer, two hundred or fewer, three hundred or fewer, four hundred or fewer, five hundred or fewer, six hundred or fewer, seventy or fewer, eighty or fewer, ninety or fewer, one thousand or fewer, or not exceeding that number, contain oligonucleotides having the same cell sequence. In some embodiments, the number of particles containing oligonucleotides having the same cell sequence may be up to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none of the particles have the same cell-labeling sequence.

[0087] Multiple oligonucleotides on each particle may contain different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences can be 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9The number of barcode sequences may be 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, or 10 9 There may be several such particles. For example, at least 100 of the oligonucleotides may contain different barcode sequences. Another example is a single particle in which at least 100, 500, 1,000, 5,000, 10,000, 15,000, 20,000, 50,000, a number or range between any two of these values, or more, of the oligonucleotides may contain different barcode sequences. Some embodiments provide multiple particles containing barcodes. In some embodiments, the ratio of the occurrence (or copies or number) of the target to be labeled to a different barcode sequence may be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, each of the plurality of oligonucleotides further includes sample labeling, universal labeling, or both. The particles may be, for example, nanoparticles or microparticles.

[0088] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads may be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or range between any two of these values, or approximately these values ​​or such a number or range. The diameter of the beads may be related to the diameter of the substrate wells. In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of the cells (e.g., single cells surrounded by the substrate wells). In some embodiments, the diameter of the beads may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of the cells (e.g., single cells surrounded by the substrate wells). In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% longer or shorter than the diameter of the cell, or approximately longer or shorter than these values ​​or such a number or range. In some embodiments, the diameter of the beads may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the cell.

[0089] The beads may be bound to and / or embedded in a substrate. The beads may be bound to and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial position of the beads within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using a spatial label present on a barcode on the bead, which can function as a positional address. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabead®, MACS® microbeads, antibody conjugate beads (e.g., anti-immunoglobulin microbeads), protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag® carboxyl-terminated magnetic beads.

[0090] Beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one or more optical channels. Beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. Beads may be identifiable. For example, beads can be imaged using a camera. Beads may have a detectable code associated with them. For example, beads may contain a barcode. Beads may change size due to expansion in organic or inorganic solutions, for example. Beads may be hydrophobic. Beads may be hydrophilic. Beads may be biocompatible. A solid support (e.g., beads) can be visualized. The solid support may contain a visualization tag (e.g., a fluorescent dye). The solid support (e.g., beads) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging of the beads.

[0091] Solid supports may contain insoluble, semi-soluble, or insoluble materials. A solid support may be described as "functionalized" if it contains bound linkers, scaffolds, building blocks, or other reactive parts, but it may be "non-functionalized" if it lacks such bound reactive parts. Solid supports can be freely used in solution, for example in microtiter wells, in flow-through configurations, for example in columns, or in dipsticks.

[0092] Solid supports may include films, paper, plastics, coated surfaces, flat surfaces, glass, slides, chips, or any combination thereof. Solid supports may take the form of resins, gels, microspheres, or other geometric configurations. Solid supports may include silica chips, microparticles, nanoparticles, plates, arrays, calipers, flat supports, e.g., glass fiber filters, glass surfaces, metal surfaces, metal surfaces (steel, gold / silver, aluminum, silicone, and copper), glass supports, plastic supports, silicone supports, chips, filters, films, microwell plates, slides, multiwell plates, or plastic materials (e.g., polyethylene, polypropylene, polyamide, polyvinylidene difluoride, etc.) formed from films, and / or wafers, combs, pins, or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis), or arrays of holes or nanoliter wells in flat surfaces, e.g., wafers (e.g., silicone wafers), beads in wafers having holes with or without a filter at the bottom. The solid support may include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix may be able to penetrate into the intracellular space (e.g., around organelles). The polymer matrix may be able to be pumped out through the circulatory system.

[0093] Substrates and microwell arrays As used herein, a substrate may refer to a certain type of solid support. A substrate may refer to a solid support that may contain the barcode or probabilistic barcode of the Disclosure. A substrate may, for example, comprise a plurality of microwells. For example, a substrate may be a well array comprising two or more microwells. In some embodiments, a microwell may comprise a small reaction chamber with a defined volume. In some embodiments, a microwell may take up one or more cells. In some embodiments, a microwell may take up only one cell. In some embodiments, a microwell may take up one or more solid supports. In some embodiments, a microwell may take up only one solid support. In some embodiments, a microwell takes up a single cell and a single solid support (e.g., a bead). A microwell may comprise the barcode reagent of the Disclosure.

[0094] Barcode creation method This disclosure provides a method for estimating the number of distinct targets at distinct locations in a body sample (e.g., tissue, organ, tumor, cell). The method may include the steps of: positioning a barcode (e.g., a probabilistic barcode) in close proximity to the sample; dissolving the sample; associating the distinct targets with the barcode; amplifying the targets; and / or digitally counting the targets. The method may further include the steps of analyzing and / or visualizing information obtained from the spatial labeling of the barcode. In some embodiments, the method includes the step of visualizing a plurality of targets in the sample. The step of mapping a plurality of targets to a map of the sample may include the step of creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps may be created before or after barcoding (e.g., probabilistic barcoding) the plurality of targets in the sample. The step of visualizing a plurality of targets in the sample may include the step of mapping a plurality of targets to a map of the sample. The step of mapping a plurality of targets to a map of the sample may include the step of creating a two-dimensional or three-dimensional map of the sample. Two-dimensional and three-dimensional maps may be created before or after barcoding multiple targets in the sample. In some embodiments, two-dimensional and three-dimensional maps may be created before or after the step of dissolving the sample. The step of dissolving the sample before or after creating the two-dimensional or three-dimensional map may include heating the sample, contacting the sample with a surfactant, changing the pH of the sample, or any combination thereof.

[0095] In some embodiments, the step of barcoding multiple targets includes the step of hybridizing multiple barcodes to multiple targets to produce barcoded targets (e.g., probabilistically barcoded targets). The step of barcoding multiple targets may include the step of generating an indexed library of barcoded targets. The step of generating an indexed library of barcoded targets may be carried out using a solid support containing multiple barcodes (e.g., probabilistic barcodes).

[0096] Step of bringing the sample into contact with the barcode. This disclosure provides a method for bringing a sample (e.g., cells) into contact with a substrate of this disclosure. For example, a sample comprising thin sections of cells, organs, or tissues can be brought into contact with a barcode (e.g., a probabilistic barcode). For example, cells can be brought into contact by gravity flow, in which case the cells may settle and form a monolayer. The sample may also be a tissue section. The section can be placed on a substrate. The sample may be one-dimensional (e.g., it may form a planar surface). For example, the sample (e.g., cells) can be spread across the entire substrate by growing / culturing cells on the substrate. If a barcode is located in close proximity to a target, the target may hybridize with the barcode. Barcodes can be brought into contact at a non-depleting ratio so that each separate target may associate with a separate barcode of this disclosure. To ensure efficient association between the target and the barcode, the target may be bridged with the barcode.

[0097] Cell lysis After the distribution of cells and barcodes, the cells can be lysed to release the target molecules. Cell lysis can be achieved by any of the following means, for example, by chemical or biochemical means, by osmotic shock, or by thermal lysis, mechanical lysis, or optical lysis. Cells may be lysed by adding a cell lysis buffer containing a surfactant (e.g., SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of the target with the barcodes, the diffusion rate of the target molecules may be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0098] In some embodiments, the sample may be dissolved by using filter paper. The filter paper can be immersed in a lysis buffer. The sample can be applied at a pressure that promotes the dissolution of the sample and the hybridization of the sample's target into the substrate. In some embodiments, dissolution can be carried out by mechanical dissolution, thermal dissolution, optical dissolution, and / or chemical dissolution. Chemical dissolution may include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Dissolution can be carried out by adding a dissolution buffer to the substrate. The dissolution buffer may contain Tris-HCl. The dissolution buffer may contain at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more of Tris-HCl. The dissolution buffer may contain up to about 0.01, 0.05, 0.1, 0.5, or 1 M or more of Tris-HCl. The dissolution buffer may contain about 0.1 M of Tris-HCl. The pH of the dissolution buffer may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher. The pH of the dissolution buffer may be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may contain a salt (e.g., LiCl). The salt concentration in the lysis buffer may be at least about 0.1, 0.5, or 1 M or higher. The salt concentration in the lysis buffer may be up to about 0.1, 0.5, or 1 M or higher. In some embodiments, the salt concentration in the lysis buffer is about 0.5 M. The lysis buffer may contain a surfactant (e.g., SDS, Li dodecyl sulfate, Triton X, Tween, NP-40). The concentration of the surfactant in the lysis buffer may be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. The concentration of the surfactant in the dissolution buffer may be up to approximately 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. In some embodiments, the concentration of the surfactant in the dissolution buffer is approximately 1% Li dodecyl sulfate. The time used in the dissolution method may depend on the amount of surfactant used. In some embodiments, the more surfactant used, the less time is required for dissolution.The lysis buffer may contain a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1, 5, 10, 15, 20, 25, or 30 mM or higher. The concentration of the chelating agent in the lysis buffer may be up to about 1, 5, 10, 15, 20, 25, or 30 mM or higher. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer may contain a reducing agent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing agent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM or higher. The concentration of the reducing agent in the lysis buffer may be up to about 1, 5, 10, 15, or 20 mM or higher. In some embodiments, the concentration of the reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may contain 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.

[0099] Lysis can be carried out at a temperature of approximately 4, 10, 15, 20, 25, or 30°C. Lysis can be carried out for approximately 1, 5, 10, 15, 20 minutes, or longer. Lysified cells may contain at least approximately 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules. Lysified cells may contain up to approximately 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules.

[0100] Binding barcodes to target nucleic acid molecules Following cell lysis and the release of nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with barcodes on a co-localized solid support. Association may involve hybridization of the target recognition region of the barcode to a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode may interact with the poly(A) tail of the target). Assay conditions used for hybridization (e.g., buffer pH, ionic strength, temperature) can be selected to facilitate the formation of specific stable hybrids. In some embodiments, nucleic acid molecules released from lysed cells may associate with multiple probes on a substrate (e.g., hybridize with probes on the substrate). If the probes contain oligo(dT), the mRNA molecule may hybridize to the probe and be reverse transcribed. The oligo(dT) portion of the oligonucleotide may act as a primer for the first strand synthesis of the cDNA molecule. For example, in a non-limiting example of barcoding shown in block 216 of Figure 2, the mRNA molecule may hybridize to a barcode on a bead. For example, a single-stranded nucleotide fragment can hybridize to the target binding region of a barcode.

[0101] The binding may further involve ligation between the target recognition region of the barcode and a portion of the target nucleic acid molecule. For example, the target binding region may contain a nucleic acid sequence that can be specifically hybridized to a restriction site overhang (e.g., an EcoRI attachment end overhang). The assay procedure may further involve treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to generate a restriction site overhang. The barcode can then be ligated to any nucleic acid molecule containing a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to join the two fragments.

[0102] For example, in a non-limiting example of barcoding shown in block 220 of Figure 2, labeled targets (e.g., target-barcode molecules) derived from multiple cells (or multiple samples) can then be pooled, for example, in a tube. The labeled targets can be pooled, for example, by recovering beads to which the barcode and / or target-barcode molecules are bound. The recovery of a solid support-based collection of bound target-barcode molecules can be achieved using magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can be carried out in a single reaction vessel. Further processing may include, for example, reverse transcription, amplification, cleavage, dissociation, and / or nucleic acid extension. These further processing reactions can be carried out in microwells, i.e., without first pooling labeled target nucleic acid molecules from multiple cells.

[0103] Reverse transcription or nucleic acid elongation This disclosure provides a method for constructing a target-barcode conjugate using reverse transcription (e.g., block 224 in Figure 2) or nucleic acid elongation. The target-barcode conjugate may comprise a barcode and a complementary sequence of all or part of the target nucleic acid (i.e., a barcoded cDNA molecule, e.g., a stochastically barcoded cDNA molecule). Reverse transcription of the associated RNA molecule may occur by adding a reverse transcription primer together with reverse transcriptase. The reverse transcription primer may be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Oligo(dT) primers may be 12–18 nucleotides long, or approximately 12–18 nucleotides long, and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNA. Random hexanucleotide primers may bind to mRNA at various complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

[0104] In some embodiments, reverse transcription of an mRNA molecule into a labeled RNA molecule 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. Generally, oligo(dT) primers are 12-18 nucleotides long and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at various complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest. In some embodiments, the target is a cDNA molecule. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase such as Moloney's mouse leukemia virus (MMLV) reverse transcriptase to produce a cDNA molecule with a poly(dC) tail. The barcode may include a target-binding region with a poly(dG) tail. When base pairing occurs between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues replication to the 5' end of the barcode. In this way, the resulting cDNA molecule contains the barcode sequence (e.g., molecular label) on the 3' end of the cDNA molecule. Reverse transcription can occur repeatedly, potentially producing multiple labeled cDNA molecules. The methods disclosed herein may include steps of 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 steps of performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0105] amplification One or more nucleic acid amplification reactions (e.g., block 228 in Figure 2) can be performed to produce multiple copies of a labeled target nucleic acid molecule. Amplification can be performed in a multiplexing manner, in which multiple target nucleic acid sequences are amplified simultaneously. A sequencing adapter can be attached to the nucleic acid molecule using the amplification reaction. The amplification reaction may include amplifying at least a portion of the sample label, if present. The amplification reaction may include a step of amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction may include amplifying at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or a combination thereof. The amplification reaction may involve amplifying a range or number of multiple nucleic acids to 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 two of these values. The method may further include the step of performing one or more cDNA synthesis reactions to produce one or more cDNA copies of a target-barcode molecule containing a sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0106] In some embodiments, amplification can be carried out using polymerase chain reaction (PCR). As used herein, PCR may refer to a reaction for amplifying a specific DNA sequence in vitro by simultaneous primer extension of complementary strands of DNA. As used herein, PCR may encompass derivatives of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, and assembly PCR.

[0107] Amplification of labeled nucleic acids may include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription for amplifying DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) methods, the use of palindromic probes, strand substitution amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which primers are hybridized to nucleic acid sequences and the resulting double strands are cleaved before the extension reaction and amplification, strand substitution amplification using nucleic acid polymerases lacking 5' exonuclease activity, rolling circle amplification, and branched extension amplification (RAM). In some embodiments, amplification does not produce a cyclized transcript.

[0108] In some embodiments, the methods disclosed herein further include the step of performing a polymerase chain reaction on a labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to produce a labeled amplicon (e.g., a stochastic labeled amplicon). The labeled amplicon may be a double-stranded molecule. The double-stranded molecule may include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule may include a sample label, spatial label, cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon may be a single-stranded molecule. The single-stranded molecule may include DNA, RNA, or a combination thereof. The nucleic acids of this disclosure may include synthetic nucleic acids or modified nucleic acids.

[0109] Amplification may involve the use of one or more non-natural nucleotides. Non-natural nucleotides may include photounstable or triggering nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNA), morpholino nucleic acids and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify the product as a specific cycle or point in time of the amplification reaction.

[0110] Performing one or more amplification reactions may involve the use of one or more primers. One or more primers may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. One or more primers may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. One or more primers may contain less than 12 to 15 nucleotides. One or more primers may anneal to at least a portion of a plurality of labeled targets (e.g., stochastic labeled targets). One or more primers may anneal to the 3' or 5' ends of a plurality of labeled targets. One or more primers may anneal to the internal regions of a plurality of labeled targets. The internal region may consist of at least approximately 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of multiple labeled targets. One or more primers may comprise a constant panel of 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 gene-specific primers.

[0111] One or more primers may include a universal primer. A universal primer may anneal to a universal primer binding site. One or more custom primers may anneal to a first sample label, a second sample label, a spatial label, a cellular label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. One or more primers may include a universal primer and custom primers. Custom primers may be designed to amplify one or more targets. Targets may include a subset of all nucleic acids in one or more samples. Targets may include a subset of all labeled 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.

[0112] Any amplification scheme can be used in the method of this disclosure. For example, in one scheme, the molecule bound to the beads can be amplified by a first PCR using a gene-specific primer and a primer for the sequence of universal Illumina sequencing primer 1. In a second PCR, the first PCR product can be amplified using a nested gene-specific primer adjacent to the sequence of Illumina sequencing primer 2 and a primer for the sequence of universal Illumina sequencing primer 1. In a third PCR, P5 and P7 and the sample index are added, and the PCR product is placed into an Illumina sequencing library. Sequencing using 150 bp × 2 sequencing can reveal cell labels and barcode sequences (e.g., molecular labels) on read 1, genes on read 2, and the sample index on index 1 read.

[0113] In some embodiments, nucleic acids can be removed from a substrate using chemical cleavage. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from a solid support. For example, enzymes can be used to remove nucleic acids from a substrate. For example, nucleic acids can be removed from a substrate by restriction endonuclease digestion. For example, nucleic acids can be removed from a substrate by treatment of dUTP or nucleic acids containing ddUTP with uracil-d-glycosylase (UDG). For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, such as depurine / depyrimidine (AP) endonucleases, can be used to remove nucleic acids from a substrate. In some embodiments, photocleavable groups and light can be used to remove nucleic acids from a substrate. In some embodiments, cleavable linkers can be used to remove nucleic acids from a substrate. For example, a cleavable linker may include at least one of the following: biotin / avidin, biotin / streptavidin, biotin / neutraavidin, Ig-protein A, a photounstable linker, an acid or base unstable linker group, or an aptamer.

[0114] If the probe is gene-specific, the molecule can hybridize to the probe and be reverse transcribed and / or amplified. In some embodiments, amplification may occur after the nucleic acid has been synthesized (e.g., reverse transcribed). Amplification can be carried out in a multiplexing manner in which multiple target nucleic acid sequences are amplified simultaneously. Amplification may add a sequencing adapter to the nucleic acid.

[0115] In some embodiments, amplification can be carried out on a substrate, for example, using bridge amplification. A homopolymer tail can be added to the cDNA to generate ends suitable for bridge amplification using an oligo(dT) probe on the substrate. In bridge amplification, the primer complementary to the 3' end of the template nucleic acid may be each pair of first primers covalently bound to the solid particle. When a sample containing the template nucleic acid is in contact with the particle and one thermal cycle is performed, the template molecule may be annealed to the first primer, and the first primer may be extended forward by the addition of nucleotides to form a double-stranded molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule may be denatured, releasing the template molecule from the particle and leaving behind the complementary DNA strand bound to the particle through the first primer. In the annealing step of the subsequent annealing and extension step, the complementary strand may hybridize to a second primer complementary to the segment of the complementary strand at the position removed from the first primer. This hybridization allows the complementary strand to form a bridge between the first and second primers, covalently bonded to the first primer and hybridized to the second primer. In the extension step, the second primer can be extended in the opposite direction by adding nucleotides to the same reaction mixture, thereby converting the bridge into a double-stranded bridge. The next cycle then begins, and the double-stranded bridge is denatured to obtain two single-stranded nucleic acid molecules, each having one end bound to the particle surface via the first and second primers, and the other end unbound. In the annealing and extension steps of this second cycle, each strand can hybridize to further previously unused complementary primers on the same particle to form a new single-stranded bridge. The two previously unused primers hybridized at this point extend to convert the two new bridges into double-stranded bridges.

[0116] The amplification reaction may involve 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 multiple nucleic acids. Amplification of labeled nucleic acids may include PCR-based or non-PCR-based methods. Amplification of labeled nucleic acids may include exponential amplification of labeled nucleic acids. Amplification of labeled nucleic acids may include linear amplification of labeled nucleic acids. Amplification can be carried out by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivatives of the reaction, including, but is not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, suppression PCR, semi-suppressive PCR, and assembly PCR.

[0117] In some embodiments, amplification of labeled nucleic acids includes non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription for amplifying DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ), use of palindromic probes, strand substitution amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which primers are hybridized to nucleic acid sequences and the resulting double strands are cleaved before extension and amplification, strand substitution amplification using nucleic acid polymerases lacking 5' exonuclease activity, rolling circle amplification, and / or branched extension amplification (RAM).

[0118] In some embodiments, the methods disclosed herein further include the step of performing a nested polymerase chain reaction on an amplified amplicon (e.g., a target). The amplicon may be a double-stranded molecule. The double-stranded molecule may include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule may contain a sample tag or molecular identifier label. Alternatively, the amplicon may be a single-stranded molecule. The single-stranded molecule may include DNA, RNA, or a combination thereof. The nucleic acids of the present invention may include synthetic nucleic acids or modified nucleic acids. In some embodiments, the method includes a step of repeatedly amplifying a labeled nucleic acid to generate a large number of amplicons. The methods disclosed herein may include a step of 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 may include a step of performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0119] Amplification may further involve adding one or more control nucleic acids to one or more samples containing multiple nucleic acids. Amplification may further involve adding one or more control nucleic acids to multiple nucleic acids. The control nucleic acids may include control labels. Amplification may involve the use of one or more non-natural nucleotides. Non-natural nucleotides may include photounstable and / or triggering nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNA), morpholino nucleic acids and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify the product as a specific cycle or point in time of the amplification reaction.

[0120] Performing the amplification reaction one or more times may involve the use of one or more primers. One or more primers may contain one or more oligonucleotides. One or more oligonucleotides may contain at least about 7 to 9 nucleotides. One or more oligonucleotides may contain less than 12 to 15 nucleotides. One or more primers may anneal to at least a portion of the multiple labeled nucleic acids. One or more primers may anneal to the 3' and / or 5' ends of the multiple labeled nucleic acids. One or more primers may anneal to the internal regions of the multiple labeled nucleic acids. The internal region may consist of at least approximately 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of multiple labeled nucleic acids. One or more primers may comprise a constant panel of 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 a universal primer. A universal primer may anneal to a universal primer binding site. One or more custom primers may anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. One or more primers may include a universal primer and a custom primer. A custom primer may be designed to amplify one or more target nucleic acids. The target nucleic acids may include a subset of all nucleic acids in one or more samples. In some embodiments, the primers are probes bound to the array of this disclosure.

[0121] In some embodiments, the step of barcoding multiple targets in a sample (e.g., a step of probabilistic barcoding) further includes the step of generating an indexed library of barcoded targets (e.g., probabilistically barcoded targets) or barcoded fragments of those targets. The barcode sequences of different barcodes (e.g., molecular labels of different probabilistic barcodes) may be different from one another. The step of generating an indexed library of barcoded targets includes the step of generating multiple indexed polynucleotides from multiple targets in a sample. For example, for an indexed library of barcoded targets including a first indexed target and a second indexed target, the labeling region of the first indexed polynucleotide may differ from the labeling region of the second indexed polynucleotide by a number or range between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or any two of these values, or approximately these values ​​or such a number or range, or at least these values ​​or such a number or range, or at most these values ​​or such a number or range. In some embodiments, the step of generating an indexed library of barcoded targets includes contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides, each containing a poly(T) region and a labeling region, and performing a first-strand synthesis using reverse transcriptase to generate single-stranded labeled cDNA molecules, each containing a cDNA region and a labeling region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. The step of generating an indexed library of barcoded targets may further include amplifying the single-stranded labeled cDNA molecules to generate double-stranded labeled cDNA molecules, and performing nested PCR on the double-stranded labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may include a step of generating adapter-labeled amplicons.

[0122] The barcoding step (e.g., a probabilistic barcoding step) may include the step of labeling individual nucleic acid (e.g., DNA or RNA) molecules using nucleic acid barcodes or tags. In some embodiments, this includes the step of attaching DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can perform the minimization of PCR amplification bias. Adapters can be attached for sequencing using next-generation sequencing (NGS), for example. For example, sequencing results can be used to determine the cellular labeling, molecular labeling, and sequencing of nucleotide fragments of one or more copies of the target in block 232 of Figure 2.

[0123] Figure 3 is a schematic diagram illustrating a non-limiting, exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), such as barcoded mRNA or fragments thereof. As shown in Step 1, the reverse transcription process may encode each mRNA molecule, including a unique molecular labeling sequence, a cell labeling sequence, and a universal PCR site. In particular, the RNA molecule 302 can be reverse transcribed by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to the poly(A) tail region 308 of the RNA molecule 302 to generate a labeled cDNA molecule 304 containing a cDNA region 306. Each of the barcodes 310 may include a target-binding region, e.g., a poly(dT) region 312, a labeling region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0124] In some embodiments, the cell labeling sequence may consist of 3 to 20 nucleotides. In some embodiments, the molecular labeling sequence may consist of 3 to 20 nucleotides. In some embodiments, each of a plurality of probabilistic barcodes further comprises one or more of a universal label and a cell label, the universal label being the same for a plurality of probabilistic barcodes on a solid support, and the cell label being the same for a plurality of probabilistic barcodes on a solid support. In some embodiments, the universal label may consist of 3 to 20 nucleotides. In some embodiments, the cell label consists of 3 to 20 nucleotides.

[0125] In some embodiments, the labeling region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, the labeling region 314 may include one or more of a universal label, a dimensional label, and a cell label. The barcode sequence or molecular label 318 may have a nucleotide length of 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 any of these values, or may be approximately one of these values ​​or such a number or range of nucleotide lengths, or may be at least one of these values ​​or such a number or range of nucleotide lengths, or may be at most one of these values ​​or such a number or range of nucleotide lengths. The cell label 320 may have a nucleotide length of 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 any of these values, or it may be approximately one of these values ​​or such a number or range of nucleotide lengths, or it may be at least one of these values ​​or such a number or range of nucleotide lengths, or it may be at most one of these values ​​or such a number or range of nucleotide lengths. The universal label may have a nucleotide length of 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 any of these values, or it may be approximately one of these values ​​or such a number or range of nucleotide lengths, or it may be at least one of these values ​​or such a number or range of nucleotide lengths, or it may be at most one of these values ​​or such a number or range of nucleotide lengths. The universal label may be the same for multiple probabilistic barcodes on a solid support, and the cell label may be the same for multiple probabilistic barcodes on a solid support.The dimensional label may be a nucleotide length of 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 any of these values, or approximately a nucleotide length of these values ​​or such a number or range, or at least a nucleotide length of these values ​​or such a number or range, or at most a nucleotide length of these values ​​or such a number or range.

[0126] In some embodiments, the labeling region 314 may include a number or range of different labels between 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 any of these values, such as a barcode sequence or molecular label 318 and a cell label 320, or approximately these values ​​or a number or range of different labels, such as a barcode sequence or molecular label 318 and a cell label 320, or at least these values ​​or a number or range of different labels, such as a barcode sequence or molecular label 318 and a cell label 320, or up to these values ​​or a number or range of different labels, such as a barcode sequence or molecular label 318 and a cell label 320. Each label may have a nucleotide length of 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 any of these values, or approximately, or at least, or at most, these values ​​or a range of such values. The set of barcodes or probabilistic barcodes 310 may be 10, 20, 40, 50, 70, 80, 90, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 The barcode or probabilistic barcode 310 may contain a number or range of values ​​between any of these values, or approximately a number or range of such values, or at least a number or range of such values, or at most a number or range of such values. Furthermore, each set of barcodes or probabilistic barcodes 310 may contain, for example, a specific labeling region 314. To remove excess barcodes or probabilistic barcodes 310, the labeled cDNA molecule 304 may be purified. Purification may include Ampure bead purification.

[0127] As shown in Step 2, the product from the reverse transcription process in Step 1 is pooled in one tube and can be PCR amplified using a first PCR primer pool and a first universal PCR primer. Pooling is made possible by a specific labeling region 314. In particular, the labeled cDNA molecule 304 can be amplified to produce a nested PCR-labeled amplicon 322. Amplification may include multiplex PCR amplification. Amplification may include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, multiplex PCR amplification may be performed in a single reaction volume of 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 , 1013 , 10 14 , 10 15 , 10 20 Multiple primers of any number or range between any of these values ​​may be used, or approximately these values ​​or such a number or range of multiple primers may be used, or at least these values ​​or such a number or range of multiple primers may be used, or at most these values ​​or such a number or range of multiple primers may be used. Amplification may include a first PCR primer pool 324 containing custom primers 326A-C that target specific genes and universal primers 328. Custom primer 326 may hybridize with a region within the cDNA portion 306' of labeled cDNA molecule 304. Universal primer 328 may hybridize with the universal PCR region 316 of labeled cDNA molecule 304.

[0128] As shown in step 3 of Figure 3, the product from the PCR amplification in step 2 can be amplified using a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the nested PCR-labeled amplicon 322 can be further amplified by nested PCR. Nested PCR may include a multiplex PCR with a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328' in a single reaction volume. The nested PCR primer pool 328 may contain nested PCR primers 330 of different numbers or ranges between 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 any of these values, or may contain nested PCR primers 330 of approximately these values ​​or such numbers or ranges, or may contain at least these values ​​or such numbers or ranges, or may contain up to these values ​​or such numbers or ranges. The nested PCR primer 332 contains an adapter 334 and may hybridize with a region within the cDNA portion 306'' of the labeled amplicon 322. The universal primer 328' contains an adapter 336 and can hybridize with the universal PCR region 316 of the labeled amplicon 322. Thus, step 3 generates the adapter-labeled amplicon 338. In some embodiments, the nested PCR primer 332 and the second universal PCR primer 328' do not need to contain adapters 334 and 336. Instead, adapters 334 and 336 can ligate with the nested PCR product to generate the adapter-labeled amplicon 338.

[0129] As shown in step 4, the PCR product from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 334 and 336 can be used to perform one or more additional assays on the adapter-labeled amplicon 338. Adapters 334 and 336 can be hybridized with primers 340 and 342. One or more primers 340 and 342 may be PCR amplification primers. One or more primers 340 and 342 may be sequencing primers. One or more adapters 334 and 336 can be used for further amplification of the adapter-labeled amplicon 338. One or more adapters 334 and 336 can be used for sequencing of the adapter-labeled amplicon 338. Primer 342 may contain plate index 344, thereby allowing amplicons produced using the same set of barcodes or probabilistic barcodes 310 to be sequenced in a single sequencing reaction using next-generation sequencing (NGS).

[0130] Insight RNA Probing mRNA derived from formalin-fixed cells is cross-linked, making it difficult to release these molecules and capture them by barcoded particles (e.g., Rhapsody beads). To address this significant challenge, some embodiments provided herein offer RNA probes capable of binding to such mRNA, which can be generated by Insights reverse transcription to produce short cDNAs ligated to the probes. These probes can be readily captured by barcoded particles (e.g., Rhapsody beads), and the specificity of the probes can be improved and potential nonspecificity eliminated by using specific targeted panel primers. Targeted probe panels and primer panels, as well as Insights probe hybridization and RT kits, are provided herein. In some embodiments, methods and compositions are provided for performing RNA probing and amplification in a single-cell analysis system (e.g., Rhapsody) to extend the sample types to fixed cells (e.g., formalin-fixed cells). The methods and compositions disclosed herein enable single-cell transcriptome profiling of single cells from fixed samples by RNA probing assays and single-cell analysis systems (e.g., Rhapsody). In some embodiments, current single-cell analysis workflows (e.g., Rhapsody) cannot provide gene expression profiling solutions for cells from formalin-based fixatives because the mRNA is cross-linked and cannot be captured by conventional mRNA capture methods (e.g., poly(A / dT) capture). In some embodiments, while it is difficult to release mRNA from fixed cells without being bound by any particular theory, methods such as insight hybridization or FISH can be used to introduce small DNA probes into fixed cells, bind to target mRNA, and visualize the mRNA. However, currently, the number of probes that can be used to detect mRNA is limited due to the number of fluorescence detectors, and therefore, it is difficult to provide transcriptome solutions using these methods.In some embodiments of the compositions and methods provided herein, to obtain a further layer of specificity, mRNA sequence-specific probes can be used to bind to target mRNA and extend by Insights reverse transcription to obtain a sequence from the mRNA. Furthermore, in some embodiments disclosed herein, a large number of target panels can be designed without limiting the detection method. After probing / Insights RT, cells can be washed to remove unbound probes and loaded onto a single-cell analysis platform (e.g., Rhapsody). Probes bound to cDNA can be released from single cells and captured via capture sequences attached to the probes by oligonucleotide barcodes associated with barcoded particles (e.g., Rhapsody beads) via TSO capture oligos. The user can then use a targeted primer panel to amplify targeted genes from the beads along with cell labeling and UMI to analyze single-cell targeted mRNA profiling by sequencing. The disclosed compositions and methods can be extended to single-cell RNA-seq analysis of archived formalin-fixed cells. Furthermore, in some embodiments, methods and compositions are provided that enable spatial gene expression testing of FFPE-fixed tissue sections. Current single-cell analysis systems (e.g., Rhapsody) are limited to live cells, fresh isolated nuclei, or short-term stored cells. The disclosed compositions and methods enable the use of long-term stored formalin-fixed samples in single-cell analysis systems (e.g., Rhapsody).

[0131] Figures 4A–4D show schematic diagrams of non-limiting, exemplary workflows for gene expression analysis of fixed cells. The workflow may include a step of contacting a sample (e.g., a fixed sample containing fixed cells) with a plurality of probing oligonucleotides (step 400a). Each probing oligonucleotide may include a coupling sequence and a probe sequence configured to hybridize with a nucleic acid target in the sample. The 5' end of each probing oligonucleotide may be phosphorylated. The probing oligonucleotides may be able to enter the cells and / or nuclei of the sample (e.g., permeabilized cells and / or permeabilized nuclei of the sample). After the step of contacting the sample with the probing oligonucleotides, the workflow may include a step of removing one or more probing oligonucleotides from the plurality that have not come into contact with the sample. The step of removing one or more probing oligonucleotides that have not come into contact with the sample may include a step of removing one or more probing oligonucleotides that did not enter the cells of the sample. The workflow may include a step (step 400b) of extending multiple probing oligonucleotides hybridized to a copy of a nucleic acid target to produce multiple extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The workflow may include a step of contacting the sample with an extension reagent. The extension reagent may include a reverse transcription reagent (e.g., reverse transcriptase and dNTPs). Extension can be performed with incites and may include incites reverse transcription. In some embodiments, the cells of the sample remain intact during the extension step. The sample may include multiple cells, and the workflow may include a step of dissociating the sample to produce multiple single cells. The workflow may include a step of distributing the multiple single cells into multiple compartments. The workflow may include a step (step 400c) of contacting the multiple extended probing oligonucleotides with barcoded particles (e.g., Rhapsody beads).Barcoded particles (e.g., beads) may associate with multiple oligonucleotide barcodes containing one or more of the following: a cell label (CL), a molecular label (UMI), a first 5' universal sequence, and a 3' TSO (e.g., a capture sequence). The workflow may include the step of barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes to generate multiple barcoded probing oligonucleotides. The step of barcoding multiple extended probing oligonucleotides may include the steps of preparing a sprint oligonucleotide (e.g., a coupling oligonucleotide) containing a 5' coupling sequence complement and a 3' capture sequence complement; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5' coupling sequence complement of the coupling oligonucleotide; hybridizing the 3' capture sequence complement of the coupling oligonucleotide with the capture sequence of one of the oligonucleotide barcodes; and / or ligating the extended probing oligonucleotide to the hybridized oligonucleotide barcode. The workflow may include a step (step 400d) of amplifying a plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to a first universal sequence or its complement, and an amplification primer capable of hybridizing to a nucleic acid target or its complement, thereby generating a plurality of amplified barcoded probing oligonucleotides. The amplification primer may include a second universal sequence (e.g., an R2 primer sequence), and / or the first primer may include a third universal sequence. The workflow may include a step (step 400e) of obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides or their products.The step of obtaining sequencing data may include the step of binding the binding sites of sequencing primers and / or sequencing adapters to a plurality of barcoded probing oligonucleotides or their products. The workflow may include the step of determining the copy number of nucleic acid targets in the sample based on the number of molecular labels associated with the plurality of amplified barcoded probing oligonucleotides or their products. In some embodiments, the probing oligonucleotides include a predetermined spatial label, and the workflow includes the step of determining the spatial location and copy number of nucleic acid targets in the sample.

[0132] Some embodiments provide a method for labeling a nucleic acid target in a sample. In some embodiments, the method includes the step of contacting a sample containing a copy of a nucleic acid target with a plurality of probing oligonucleotides, each of which probing oligonucleotides includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target. The method may include the step of extending the plurality of probing oligonucleotides hybridized to the copy of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of barcoding the plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides, each of which oligonucleotide barcodes includes a molecular label, and each of the barcoded probing oligonucleotides includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular label sequence and a partial sequence of a nucleic acid target. The method may also include the step of determining the copy number of a nucleic acid target in a sample based on the number of molecular labels associated with the multiple barcoded probing oligonucleotides or their product.

[0133] Some embodiments provide a method for determining the copy number of a nucleic acid target in a sample. In some embodiments, the method includes contacting a sample containing a copy of the nucleic acid target with a plurality of probing oligonucleotides, each of which includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target. The method may include extending the plurality of probing oligonucleotides hybridized to a copy of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include barcoding the plurality of extended probing oligonucleotides or their products using a plurality of oligonucleotide barcodes to produce a plurality of barcoded probing oligonucleotides, each of which includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular label sequence and a partial sequence of a nucleic acid target. The method may also include the step of determining the copy number of a nucleic acid target in a sample based on the number of molecular labels associated with the multiple barcoded probing oligonucleotides or their product.

[0134] Some embodiments provide a method for determining the spatial location and copy number of a nucleic acid target in a sample. In some embodiments, the method includes the step of contacting each of two or more spatial locations in a sample containing copies of the nucleic acid target with a plurality of probing oligonucleotides, each of which includes a coupling sequence, a probe sequence configured to hybridize with the nucleic acid target, and a predetermined spatial label. In some embodiments, probing oligonucleotides that contact the same spatial location contain the same spatial label sequence, while probing oligonucleotides that contact separate spatial locations in the sample contain different spatial label sequences. The method may include the step of extending the plurality of probing oligonucleotides hybridized to copies of the nucleic acid target to produce a plurality of extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target. The method may include the step of generating multiple barcoded probing oligonucleotides by barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes, wherein each oligonucleotide barcode of the multiple oligonucleotide barcodes includes a molecular label, and each of the multiple barcoded probing oligonucleotides includes a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method may also include the step of obtaining sequencing data including multiple sequencing reads of the barcoded probing oligonucleotides or their products, wherein each of the multiple sequencing reads includes a spatial label sequence, a molecular label sequence, and a partial sequence of the nucleic acid target. The method may also include the step of determining the copy number of the nucleic acid target at each spatial location of the sample by counting the number of molecular labels having a distinct sequence associated with the nucleic acid target for each unique spatial label sequence associated with a distinct spatial location of the sample.

[0135] This method may include a step of contacting the sample with an elongation reagent. At least a portion of the contact step can be carried out in the presence of the elongation reagent. The entire contact step can be carried out in the presence of the elongation reagent. The contact step and the elongation step may be performed simultaneously. Elongation may be carried out with insights. The elongation may include insight reverse transcription. In some embodiments, the cells of the sample remain intact during the elongation step. The elongation reagent may include a reverse transcription reagent. The reverse transcription reagent may include reverse transcriptase and dNTPs. The reverse transcriptase may include viral reverse transcriptase. The viral reverse transcriptase may be mouse leukemia virus (MLV) reverse transcriptase or Moloney's mouse leukemia virus (MMLV) reverse transcriptase.

[0136] The step of barcoding multiple extended probing oligonucleotides or their products may include the steps of: preparing coupling oligonucleotides containing a 5' coupling sequence complement and a 3' capture sequence complement; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5' coupling sequence complement of the coupling oligonucleotide; hybridizing the 3' capture sequence complement of the coupling oligonucleotide with the capture sequence of an oligonucleotide barcode among the multiple oligonucleotide barcodes; and / or ligating the extended probing oligonucleotide to the hybridized oligonucleotide barcode. The method may include, prior to the step of ligating the extended probing oligonucleotide to the oligonucleotide barcode, a step of filling the gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode using a DNA polymerase lacking at least one of 5'-to-3' exonuclease activity and 3'-to-5' exonuclease activity. The step of ligating the extended probing oligonucleotide to the hybridized oligonucleotide barcode may be carried out using DNA ligase. The coupling oligonucleotide may be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a mixture thereof. The coupling oligonucleotide may contain non-natural nucleotides.The coupling oligonucleotides are at least approximately 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5 It may contain 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides, or a number or range of nucleotides between any two of these values. The coupling sequences are at least approximately 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 The probing oligonucleotides may contain 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides, or a number or range of nucleotides between any two of these values. The 5' end of each probing oligonucleotide may be phosphorylated. The probing oligonucleotides may be able to enter the cells and / or nuclei of the sample (e.g., permeabilized cells and / or permeabilized nuclei of the sample).This method may include, after the step of bringing the probing oligonucleotides into contact with the sample, a step of removing one or more probing oligonucleotides from among a plurality of probing oligonucleotides that have not come into contact with the sample, and the step of removing one or more probing oligonucleotides that have not come into contact with the sample may include a step of removing one or more probing oligonucleotides that have not entered the cells of the sample.

[0137] The contact step may include bringing the sample into contact with a device configured to position probing oligonucleotides (e.g., an inkjet device). The device may be a needle, a needle array, a tube, an aspiration device, an injection device, an electroporation device, a fluorescence-activated cell sorter device, an inkjet device, a microfluidic device, or any combination thereof. In some embodiments, the device brings separate spatial locations of the sample into contact at a specific speed. The spatial markers are at least approximately 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 The lengths of the nucleotides may be 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides, or a number or range of nucleotides between any two of these values. The two or more spatial locations may include at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 distinct spatial locations of the sample. In some embodiments, the spatial position of the sample corresponds to a region containing a number or range of cells between any two of these values: approximately 50 cells or less, approximately 45 cells or less, approximately 40 cells or less, approximately 35 cells or less, approximately 30 cells or less, approximately 25 cells or less, approximately 20 cells or less, approximately 15 cells or less, approximately 10 cells or less, approximately 9 cells or less, approximately 8 cells or less, approximately 7 cells or less, approximately 6 cells or less, approximately 5 cells or less, approximately 4 cells or less, approximately 3 cells or less, approximately 2 cells or less, approximately 1 cell or less.

[0138] Each oligonucleotide barcode in a plurality of oligonucleotide barcodes may contain a first universal sequence. In some embodiments, the step of obtaining sequencing data includes amplifying a plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or its complement, and an amplification primer capable of hybridizing to a nucleic acid target or its complement, thereby generating a plurality of amplified barcoded probing oligonucleotides. The step of obtaining sequencing data may include obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides or their products. The step of obtaining sequencing data may include binding sites of sequencing primers and / or sequencing adapters to the plurality of barcoded probing oligonucleotides or their products. The amplification primer may contain a second universal sequence, and / or the first primer may contain a third universal sequence. The first universal sequence, the second universal sequence, and / or the third universal sequence may be the same. The first universal sequence, the second universal sequence, and / or the third universal sequence may be different. The first universal sequence, the second universal sequence, and / or the third universal sequence may include binding sites of sequencing primers and / or sequencing adapters, their complementary sequences, and / or portions thereof. The sequencing adapter may include P5 sequences, P7 sequences, their complementary sequences, and / or portions thereof. The sequencing primers may include lead 1 sequencing primers, lead 2 sequencing primers, their complementary sequences, and / or portions thereof.

[0139] The sample may include a target panel of multiple nucleic acid targets, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or a number or range between any two of these values. Two or more nucleic acid targets in the target panel may be biomarkers. The biomarkers may be biomarkers for diseases or conditions. The diseases or conditions may be cancer, infectious diseases, viral infections, inflammatory diseases, neurodegenerative diseases, fungal diseases, bacterial infections, or any combination thereof. The contact step may include contacting the sample with a panel of probing oligonucleotides comprising two or more probing oligonucleotides, each comprising probe sequences configured to hybridize with a nucleic acid target among the plurality of nucleic acid targets. The step of determining the copy number of nucleic acid targets in the sample may include determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels having distinct sequences associated with each of the plurality of barcoded probing oligonucleotides or their products, each comprising the respective sequences of the plurality of nucleic acid targets. The method may include determining the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample by counting the number of molecular labels having distinct sequences associated with each of the plurality of nucleic acid targets for each unique spatial label sequence associated with a distinct spatial location of the sample.The amplification primers are panels of amplification primers configured to hybridize with multiple nucleic acid targets or their complements, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, The panel may include approximately 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or a number or range between any two of these values ​​of distinct amplification primers. As used herein, the term “panel” is given its ordinary meaning and also refers to a set of nucleic acids designed to hybridize with a set of target nucleic acid sequences or their products. For example, in some embodiments, the expression analysis of 200 genes can be performed using a panel of 200 different probing oligonucleotides designed to bind to 200 transcripts of the genes (e.g., a panel of probing oligonucleotides containing 200 different probing oligonucleotides). In some such embodiments, after these probing oligonucleotides are extended in insights, the extension product (extended probing oligonucleotide) can be barcoded as described herein and then amplified using a panel of 200 different amplification primers designed to bind to the sequences of 200 nucleic acid targets (or their complements). The nucleic acid targets may include nucleic acid molecules (e.g., ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, sample-indexed oligonucleotides, cell component-binding reagent-specific oligonucleotides, or any combination thereof).

[0140] Each molecular label of a plurality of oligonucleotide barcodes may contain at least six nucleotides. Each capture sequence of a plurality of oligonucleotide barcodes may contain at least four nucleotides. The plurality of oligonucleotide barcodes may be associated with a solid support, and one of the compartments may contain a single solid support. Each of the plurality of oligonucleotide barcodes may contain a cell label. Each cell label of the plurality of oligonucleotide barcodes may contain at least six nucleotides. Oligonucleotide barcodes associated with the same solid support may contain the same cell label. Oligonucleotide barcodes associated with different solid supports may contain different cell labels. The solid support may include synthetic particles, planar surfaces, or a combination thereof. The method may include the step of associating synthetic particles containing a plurality of oligonucleotide barcodes with cells in a compartment. The method may include the step of lysing cells after the step of associating synthetic particles with cells. The step of lysing cells may include heating cells, contacting cells with a surfactant, changing the pH of cells, or any combination thereof. Synthetic particles and single cells may be located within the same compartment, which may be a well or a droplet. At least one of a plurality of oligonucleotide barcodes may be immobilized or partially immobilized on the synthetic particle, and / or at least one of a plurality of oligonucleotide barcodes may be encapsulated or partially encapsulated within the synthetic particle. The synthetic particles may be disintegrable (e.g., disintegrable hydrogel particles). The synthetic particles may include beads.The beads may include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may include materials selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. Each oligonucleotide barcode in a plurality of oligonucleotide barcodes may include a linker functional group. The synthetic particles may include a solid support functional group. The support functional group and the linker functional group may be associated with each other, and the linker functional group and the support functional group can each be selected from the group consisting of C6, biotin, streptavidin, primary amine, aldehyde, ketone, and any combination thereof.

[0141] Sample analysis The sample may be physically divided or intact during the contact step. The sample may contain a single cell. The sample may contain multiple single cells. The sample may contain multiple cells, and the method may include a step of dissociating the sample to generate multiple single cells. The dissociation step may include chemical dissociation, enzymatic dissociation, and / or mechanical dissociation. One or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin may be used in the dissociation step. The method may include a step of distributing the multiple single cells into multiple compartments prior to the barcoding step, wherein one of the compartments contains a single cell from the multiple single cells, and in the compartment containing the single cells, an extended probing oligonucleotide may be brought into contact with the multiple oligonucleotide barcodes. In some embodiments, the method includes a step of lysing the single cells in the compartment containing the single cells by bringing the single cells into contact with a lysis buffer at 15-65°C. The lysis buffer may contain an active substance capable of dissociating protein-nucleic acid complexes.

[0142] The multiple cells may comprise one or more cell types. The one or more cell types can be selected from a group consisting of brain cells, cardiac cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, and circulating cells, or any combination thereof. The sample may comprise a biological sample, clinical sample, environmental sample, biological fluid, tissue, tissue section, or any combination thereof derived from the subject. The subject may be a human, mouse, dog, rat, or vertebrate. The method may include a step of determining the genotype, phenotype, or one or more gene mutations of the subject based on the spatial location of nucleic acid targets in the sample. The method may include a step of predicting the subject's susceptibility to one or more diseases, such as cancer or a genetic disorder. The method may include a step of determining the cell types of the multiple cells in the sample. In some embodiments, a drug can be selected based on the predicted responsiveness of the cell types of the multiple cells in the sample.

[0143] The method may include a step of imaging the sample, optionally an image of the sample before and / or after the contact step, and the imaging step may generate imaging data. The imaging step may include a step of staining the sample using staining, which may be fluorescence staining, negative staining, antibody staining, or any combination thereof. The staining step may include immunocytochemistry (ICC), immunohistochemical testing (IHC), immunofluorescence (IF), or any combination thereof. In some embodiments, the imaging step may include microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multiphoton microscopy, quantitative phase microscopy, surface-enhanced Raman spectroscopy, video recording, manual visual analysis, automated visual analysis, or any combination thereof. The method may include a step of correlating imaging data of one or more spatial locations of the sample with sequencing data. The method may include correlation analysis of spatial location imaging data with sequencing data. Correlation analysis may identify one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and / or cellular targets of candidate therapeutic agents. In some embodiments, the imaging step creates an image used to construct a map that physically represents the sample. In some embodiments, the map may be two-dimensional or three-dimensional. The method may include a step of mapping nucleic acid targets and / or cellular component targets onto the map of the sample. The method may include a step of mapping one or more single cells from a plurality of cells onto the map of the sample. Sequencing reads derived from the same single cell from a plurality of cells may contain the same cellular marker. These sequence reads may also contain sequences of spatial markers. Based on the association of cellular and spatial markers, the user can associate a single cell in the sample with its spatial location in the sample.

[0144] Cellular component targeting profiling The sample may contain multiple cell component targets, and the method may include the steps of: contacting the sample with multiple cell component binding reagents, each of which contains a cell component binding reagent-specific oligonucleotide containing a unique identifier sequence for the cell component binding reagent, and enabling the cell component binding reagent to specifically bind to at least one of the multiple cell component targets; barcoding the cell component binding reagent-specific oligonucleotides to generate multiple barcoded cell component binding reagent-specific oligonucleotides, each containing a sequence complementary to at least a portion of the unique identifier sequence and a molecular label sequence; and obtaining sequencing data comprising multiple sequencing reads of the multiple barcoded cell component binding reagent-specific oligonucleotides or their products, each of which contains at least a portion of the molecular label sequence and the unique identifier sequence. The step of obtaining sequencing data may include binding the binding site of a sequencing primer and / or sequencing adapter to the barcoded cell component binding reagent-specific oligonucleotides or their products.

[0145] This method may include a step of contacting a sample with multiple cell component binding reagents, followed by a step of removing one or more cell component binding reagents that have not come into contact with the sample. The step of removing one or more cell component binding reagents that have not come into contact with the sample may include a step of removing one or more cell component binding reagents that have not come into contact with at least one of the multiple cell component targets. The cell component targets may include intracellular proteins, carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, intracellular proteins, or any combination thereof. The cell component binding reagent-specific oligonucleotides may include a second molecular label, and at least 10 of the multiple cell component binding reagent-specific oligonucleotides may include different second molecular label sequences. The second molecular label sequences of at least two cell component binding reagent-specific oligonucleotides may be different, and the unique identifier sequences of at least two cell component binding reagent-specific oligonucleotides may be identical. The second molecular label sequences of at least two cell component binding reagent-specific oligonucleotides may be different, and the unique identifier sequences of at least two cell component binding reagent-specific oligonucleotides may be different. In some embodiments, the number of unique molecular label sequences associated with a unique identifier sequence for a cell component binding reagent capable of specifically binding to at least one cell component target in the sequencing data indicates the copy number of at least one cell component target in the sample. In some embodiments, the number of unique second molecular label sequences associated with a unique identifier sequence for a cell component binding reagent capable of specifically binding to at least one cell component target in the sequencing data indicates the copy number of at least one cell component target in the sample.

[0146] In some embodiments, cell component binding reagent-specific oligonucleotides are barcoded using the same plurality of oligonucleotide barcodes used to barcode the extended probing oligonucleotide. In some embodiments, the solid support comprises two or more plurality of oligonucleotide barcodes, each plurality comprising a distinct 3' target binding region or capture sequence. For example, in some embodiments described herein, the extended probing oligonucleotide is barcoded using a first plurality of oligonucleotide barcodes having a 3' capture sequence (e.g., TSO bait) configured to hybridize to a coupling oligonucleotide, and the cell component binding reagent-specific oligonucleotide is barcoded using a second plurality of oligonucleotide barcodes having a 3' poly(dT) sequence. In some such embodiments, the cell component binding reagent-specific oligonucleotide comprises a poly(dA) sequence.

[0147] In some embodiments, a method is provided for determining the spatial location and copy number of cellular component targets in a sample. Sequencing reads of multiple barcoded cellular component-binding reagent-specific oligonucleotides or their products may each include a cell-labeling sequence. As described above, the user can associate a single cell in the sample with its spatial location in the sample based on the association of the cell label with the spatial label. Thus, the user can determine the spatial location and copy number of cellular component targets in the sample based on the spatial label (and therefore the spatial location) associated with the cell label in the sequencing data.

[0148] Embodiments using cell component binding reagents (e.g., protein binding reagents) that associate with oligonucleotides (e.g., oligoconjugate antibodies (AbO) and oligoconjugate aptamers) for barcoding and / or for determining protein expression profiles in single cells and for sample tracking (e.g., tracking sample origin) are described in U.S. Patent Applications Publications 2018 / 0088112 and 2018 / 0346970; and International Publication WO / 2020 / 037065, the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in PCT Application Publication WO / 2021 / 163374, the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein may, in some embodiments, be used in conjunction with the systems, methods, compositions, and kits described in PCT applications WO / 2024 / 097719 and WO / 2024 / 097718, the contents of which are incorporated herein by reference in their entirety.

[0149] Fixative, release agent, and permeation agent The sample may include tissue, cell monolayers, fixed cells, tissue sections, or any combination thereof. The sample may include fresh tissue sections, frozen tissue sections, fixed tissue sections, formalin-fixed tissue sections, formalin-fixed paraffin-embedded (FFPE) tissue sections, acetone-fixed tissue sections, paraformaldehyde (PFA)-fixed tissue sections, and / or methanol-fixed tissue sections. The sample may include nuclear suspensions, such as fixed nuclear suspensions and / or permeabilized nuclear suspensions. In some embodiments, the sample has been in contact with one or more fixatives and / or permeabilizing agents. The sample may include cells, such as fresh cells, frozen cells, fixed cells, formalin-fixed cells, formalin-fixed paraffin-embedded (FFPE) cells, acetone-fixed cells, paraformaldehyde (PFA)-fixed cells, and / or methanol-fixed cells. The method may include a step of permeabilizing the sample and / or a step of fixing the sample. The step of fixing the sample may include the step of bringing the sample into contact with a fixative. The fixative may include a non-crosslinking fixative (e.g., methanol). The fixative may include a crosslinking agent. The crosslinking agent may include a cleavable crosslinking agent. Cleavable crosslinking agents include dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3,3'-dithiobispropionimidate (DTBP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), 6-(3(2-pyridyldithio This may include or be derived from succinimidyl hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene (SMPT), 3-(2-pyridyldithio)propionylhydrazide (PDPH), 2-((4,4'-adipentanamide)ethyl)-1,3'-dithiopropionate succinimidyl (SDAD, NHS-SS-diaziline), or any combination thereof.The cleavable crosslinking agent may include cleavable linkages selected from the group consisting of chemically cleavable linkages, photocleavable linkages, acid-unstable linkers, heat-sensitive linkages, enzymatically cleavable linkages, and combinations thereof. The cleavable crosslinking agent may be a thiol-cleavable crosslinking agent or may include a disulfide linker. The fixative may include paraformaldehyde (PFA), dithiobis(succinimidyl dithiopropionate (DSP), 3-(2-pyridyldithio)propionate succinimidyl (SPDP), CellCover, or combinations thereof. The step of fixing the sample and the step of permeabilizing the sample can be performed simultaneously.

[0150] The steps of immobilizing the sample and permeabilizing it can be carried out in the presence of a dual-function substance capable of both immobilizing and permeabilizing the sample. The dual-function substance may be methanol. The step of permeabilizing the sample may include contacting the sample with a permeabilizing agent. This method may include a step of contacting the sample with a plurality of probing oligonucleotides or a plurality of cell component binding reagents, followed by a step of removing the permeabilizing agent from the sample. The permeabilizing agent may be capable of (i) permeabilizing the cell membrane of a cell, or (ii) making the cell membrane permeable to probing oligonucleotides, cell component binding reagents, or both. The permeabilizing agent may include (i) a solvent, surfactant, or surfactant, (ii) BD Cytoperm, (iii) saponin or its derivatives, (iv) Triton X-100, (v) methanol or its derivatives, and / or (vi) digitonin or its derivatives. The substance capable of dissociating protein-nucleic acid complexes may include serine proteases with broad substrate specificity. A serine protease with broad substrate specificity may be proteinase K. The lysis buffer may contain a defixation agent. The defixation agent may contain a thiol, hydroxylamine, periodic acid, a base, or any combination thereof. The lysis buffer may contain DTT. The method may include a step of reversing the fixation of the sample and / or single cells. The step of reversing the fixation of the sample and / or single cells may include UV cutting, chemical treatment, heating, enzymatic treatment, or any combination thereof.

[0151] Blocking reagents, decoy oligonucleotides, and blocking oligonucleotides This method may include a step of contacting the sample with a blocking reagent, one or more decoy oligonucleotides, and / or one or more blocking oligonucleotides, prior to the steps of contacting the sample with multiple cell component binding reagents and / or multiple probing oligonucleotides. The methods and compositions provided herein can be used in conjunction with the methods and compositions described in PCT Patent Application No. PCT / US22 / 75661, filed on 30 August 2022, entitled “RNA Preservation and Recovery from Fixed Cells,” the contents of which are incorporated herein by reference in their entirety. The methods and compositions provided herein can be used in conjunction with blocking reagents, for example, those described in PCT Patent Application No. PCT / US22 / 75656, filed August 30, 2022, entitled “USE OF DECOY POLYNUCLEOTIDES IN SINGLE CELL MULTIOMICS,” the contents of which are incorporated herein by reference in their entirety. The step of contacting a sample with multiple cell component binding reagents can be carried out in the presence of the blocking reagent. The blocking reagent may comprise a plurality of oligonucleotides complementary to at least a portion of the cell component binding reagent-specific oligonucleotides. The blocking reagent may comprise an antibody or fragment thereof derived from a first species, and the blocking reagent may comprise serum derived from a first species. The sample may comprise one or more non-target nucleic acids, and the blocking reagent may comprise a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids. Each of the multiple decoy oligonucleotides may be capable of hybridizing to at least a portion of the non-target nucleic acid.In some embodiments, the decoy oligonucleotide contains a sequence complementary to at least a portion of the non-target nucleic acid; the decoy oligonucleotide contains a sequence identical or substantially similar to the sequence of the cell component binding reagent-specific oligonucleotide, the sequence may be 3 to 40 nucleotides in length; the decoy oligonucleotide has up to 50% sequence identity with respect to the cell component binding reagent-specific oligonucleotide; the decoy oligonucleotide does not contain UMI; the decoy oligonucleotide contains a random sequence, the random sequence may be approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length; The nucleotides do not contain any sequence having five or more, six or more, seven or more, or eight or more consecutive T or A nucleotides; the decoy oligonucleotides contain at least one G or C for every four, five, six or seven consecutive nucleotides; the decoy oligonucleotides contain one or more modified nucleotides; the decoy oligonucleotides contain a 5' modification, which may include a 5' amino acid modification factor C12 modification (5AmMC12); the decoy oligonucleotides contain a 3' modification, which may include a 3' dideoxy C modification (ddC); and / or the decoy oligonucleotides are 30 to 65 nucleotides in length.

[0152] The sample may contain one or more undesirable nucleic acid species, and the method may include the step of contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species, thereby reducing the reverse transcription of at least one of the one or more undesirable nucleic acid species. In some embodiments, the blocking oligonucleotide is contacted with the sample before contacting a plurality of probing oligonucleotides with the sample, the blocking oligonucleotide is contacted with the sample after contacting a plurality of probing oligonucleotides with the sample, and / or the blocking oligonucleotide is contacted with the sample when contacting a plurality of probing oligonucleotides with the sample. The method may include the step of preparing a blocking oligonucleotide that specifically binds to two or more undesirable nucleic acid species, optionally at least 10 or at least 100 undesirable nucleic acid species, in the sample.In some embodiments, the blocking oligonucleotide is locked nucleic acid (LNA), peptide nucleic acid (PNA), DNA, LNA / PNA chimera, LNA / DNA chimera, or PNA / DNA chimera. The blocking oligonucleotide specifically binds to within 100 nucleotides, 50 nucleotides, or 25 nucleotides from the 3' end of one or more undesirable nucleic acid species. The blocking oligonucleotide specifically binds to within 100 nucleotides from the 5' end of one or more undesirable nucleic acid species. The blocking oligonucleotide specifically binds to within 100 nucleotides from the central part of one or more undesirable nucleic acid species. The blocking oligonucleotides that specifically bind within the 3' poly(A) tail of one or more undesirable nucleic acid species may or may not contain non-native nucleotides, have a Tm of at least 50°C, at least 60°C, or at least 70°C, cannot function as a primer for reverse transcriptase or polymerase, and / or are 8 to 100 nucleotides long, 10 to 50 nucleotides long, 12 to 21 nucleotides long, 20 to 30 nucleotides long, or about 25 nucleotides long. The amount of one or more undesirable nucleic acid species may reach about 50%, about 60%, about 70%, or about 80% of the nucleic acid content of the sample. Undesirable nucleic acid species may be selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA, intron sequences, sequences of high abundance, and combinations thereof. One or more undesirable nucleic acid species may be mRNA molecules, and the blocking oligonucleotides may specifically bind within 10 nucleotides of the 3' poly(A) tail of one or more undesirable nucleic acid species.

[0153] kit In some embodiments, a composition (e.g., a kit) is provided. In some embodiments, the kit comprises: a plurality of probing oligonucleotides, each of which comprises a coupling sequence and a probe sequence configured to hybridize with a nucleic acid target, and the probing oligonucleotides may also comprise a predetermined spatial label; a coupling oligonucleotide comprising a 5' coupling sequence complement and a 3' capture sequence complement; a plurality of oligonucleotide barcodes, where the 3' end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, and the 5' end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; and capable of hybridizing to a first universal sequence, and further comprising the first A first primer which may contain a universal sequence 3; an amplification primer which may hybridize to a nucleic acid target or its complement and which may further contain a second universal sequence; a DNA ligase; an extension reagent, optionally a reverse transcription reagent, and optionally a reverse transcriptase and dNTPs; one or more fixatives; one or more permeabilizing agents; a crosslinking agent; a defixation agent; a lysis buffer; a plurality of cell component binding reagents, each of which contains a cell component binding reagent-specific oligonucleotide containing a unique identifier sequence for the cell component binding reagent, and which allows the cell component binding reagent to specifically bind to at least one of the plurality of cell component targets; a blocking reagent; one or more decoy oligonucleotides; and / or one or more blocking oligonucleotides.

[0154] Multiple probing oligonucleotides are a panel of probing oligonucleotides containing two or more multiple probing oligonucleotides, each multiple containing multiple nucleic acid targets, optionally at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, or 90. The panel may include probe sequences configured to hybridize with nucleic acid targets from a target panel of separate nucleic acid targets, each containing approximately 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or a number or range between any two of these values.

[0155] The amplification primers are a panel of amplification primers configured to hybridize with multiple nucleic acid targets or their complements, optionally containing at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, or 80 types. It may include a panel of separate amplification primers numbering approximately 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or a number or range between any two of these values.

[0156] term In at least some of the embodiments described above, one or more elements used in one embodiment may be interchangeably used in another embodiment, unless such substitution is technically impractical. Those skilled in the art will understand that various other omissions, additions, and modifications can 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 as defined by the appended claims. Those skilled in the art will recognize that, with respect to this and other processes and methods disclosed herein, the functions performed in those processes and methods may be achieved in different orders. Furthermore, the steps and operations outlined are given only as examples, and some of the steps and operations may be arbitrarily combined into fewer steps and operations, or expanded into additional steps and operations without diminishing the fundamental elements of the embodiments of the disclosure.

[0157] In connection with the use of substantially any plural and / or singular terms herein, a person skilled in the art can convert from plural to singular and / or singular to plural where appropriate in context and / or application. For clarity, various singular / plural substitutions may be explicitly described herein. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. Any reference to “or” herein is intended to include “and / or” unless otherwise specified.

[0158] In general, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims (e.g., the text of the appended claims), are intended to be "open" terms (for example, the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," and the term "includes" should be interpreted as "includes but is not limited to"). Furthermore, where a specific number of claims introduced is intended, such intent will be explicitly stated in the claim, and in the absence of such statement, such intent will be understood by those skilled in the art. For example, for the sake of understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the claims. However, the use of such phrases should not be interpreted as meaning that the introduction of a claim description by the indefinite article "a" or "an" means that any particular claim containing such introduced description is limited to embodiments containing only one such description (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce a claim description.Furthermore, even if a claim explicitly states that a specific number of claims have been introduced, a person skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (for example, an unmodified statement without other modifiers such as "two statements" means at least two statements, or two or more statements). Furthermore, when conventions similar to "at least one of A, B, and C, etc." are used, it is generally intended that such a construction is meant to be understood by a person skilled in the art (for example, "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B and C, etc.). Where conventions similar to "at least one of A, B, or C" are used, it is generally intended that such constructions are understood by those skilled in the art (for example, "a system having at least one of A, B, or C" would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B and C). Furthermore, it will be understood by those skilled in the art that substantially any disjunctive word and / or phrase representing two or more alternative terms should be understood, whether in the specification, claims, or drawings, as intended to include the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is understood to include the possibility of "A" or "B" or "A and B".

[0159] Furthermore, if any feature or aspect of the present disclosure is described by a Markush group, it will be recognized by those skilled in the art that the present disclosure may also be described by any individual member or subgroup of a member of the Markush group.

[0160] As will be understood by those skilled in the art, for all purposes, for example with respect to the provision of the specification, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. Any enumerated scope is sufficiently described and readily recognizable as being able to be divided into at least two, three, four, five, ten, etc., subscopes. As a non-limiting example, each scope considered herein can be readily broken down into a lower third, a middle third, an upper third, etc. Similarly, as will be understood by those skilled in the art, all expressions such as “maximum,” “at least,” “greater than,” and “less than” include the stated number and refer to a scope that can subsequently be broken down into subscopes as considered above. Finally, as will be understood by those skilled in the art, each scope includes its individual members. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0161] From the foregoing, it will be recognized that various embodiments of the present disclosure are described herein for illustrative purposes and that various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting and have the true scope and spirit set forth in the following claims.

Claims

1. A method for labeling nucleic acid targets in a sample, A step of contacting a sample containing a copy of a nucleic acid target with a plurality of probing oligonucleotides, wherein each probing oligonucleotide includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target, The steps include extending multiple probing oligonucleotides hybridized to a copy of a nucleic acid target to generate multiple extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target, and A step of generating multiple barcoded probing oligonucleotides by barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes, wherein each oligonucleotide barcode of the multiple oligonucleotide barcodes includes a molecular label, and each of the multiple barcoded probing oligonucleotides includes a sequence complementary to at least a portion of the molecular label, probe sequence, and nucleic acid target. A method that includes this.

2. A step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular labeling sequence and a nucleic acid target subsequence; The steps include determining the copy number of nucleic acid targets in a sample based on the number of molecular labels associated with multiple barcoded probing oligonucleotides or their products, and The method according to claim 1, further comprising:

3. A method for determining the copy number of nucleic acid targets in a sample, A step of contacting a sample containing a copy of a nucleic acid target with a plurality of probing oligonucleotides, wherein each probing oligonucleotide includes a coupling sequence and a probe sequence configured to hybridize with the nucleic acid target, The steps include extending multiple probing oligonucleotides hybridized to a copy of a nucleic acid target to generate multiple extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target, and A step of generating multiple barcoded probing oligonucleotides by barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes, wherein each oligonucleotide barcode of the multiple oligonucleotide barcodes includes a molecular label, and each of the multiple barcoded probing oligonucleotides includes a sequence complementary to at least a portion of the molecular label, probe sequence, and nucleic acid target; A step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a molecular labeling sequence and a nucleic acid target subsequence; The steps include determining the copy number of nucleic acid targets in a sample based on the number of molecular labels associated with multiple barcoded probing oligonucleotides or their products, and A method that includes this.

4. A method for determining the spatial position and copy number of nucleic acid targets in a sample, A step of contacting each of two or more spatial locations of a sample containing a copy of a nucleic acid target with a plurality of probing oligonucleotides, each of which probing oligonucleotides includes a coupling sequence, a probe sequence configured to hybridize with the nucleic acid target, and a predetermined spatial label. The steps include: a probing oligonucleotide that contacts the same spatial position contains the same spatial labeling sequence, and a probing oligonucleotide that contacts a different spatial position in the sample contains a different spatial labeling sequence; The steps include extending multiple probing oligonucleotides hybridized to a copy of a nucleic acid target to generate multiple extended probing oligonucleotides, each containing a sequence complementary to at least a portion of the nucleic acid target, and A step of generating multiple barcoded probing oligonucleotides by barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes, wherein each oligonucleotide barcode of the multiple oligonucleotide barcodes includes a molecular label, and each of the multiple barcoded probing oligonucleotides includes a sequence complementary to at least a portion of the molecular label, probe sequence, and nucleic acid target; A step of obtaining sequencing data comprising multiple sequencing reads of a barcoded probing oligonucleotide or its product, wherein each of the multiple sequencing reads comprises a spatially labeled sequence, a molecularly labeled sequence, and a nucleic acid target subsequence. The steps include: counting the number of molecular labels having distinct sequences associated with the nucleic acid target for each unique spatial label sequence associated with a distinct spatial position in the sample, and determining the copy number of the nucleic acid target at each spatial position in the sample; A method that includes this.

5. The step of barcoding multiple extended probing oligonucleotides or their products using multiple oligonucleotide barcodes is: The steps include preparing a coupling oligonucleotide containing a 5' coupling sequence complement and a 3' capture sequence complement, The steps include: hybridizing the coupling sequence of the extended probing oligonucleotide with the 5' coupling sequence complement of the coupling oligonucleotide; A step of hybridizing the 3' capture sequence complement of a coupling oligonucleotide with the capture sequence of an oligonucleotide barcode among multiple oligonucleotide barcodes, The steps include: Ligate the extended probing oligonucleotide to the hybridized oligonucleotide barcode; The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, comprising the step of filling the gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode using a DNA polymerase lacking at least one of 5'-to-3' exonuclease activity and 3'-to-5' exonuclease activity, prior to the step of ligating the extended probing oligonucleotide to an oligonucleotide barcode.

7. The method according to any one of claims 1 to 6, wherein the step of ligating the extended probing oligonucleotide to the hybridized oligonucleotide barcode is carried out using a DNA ligase.

8. The method according to any one of claims 1 to 7, wherein the coupling oligonucleotide is a single-stranded oligonucleotide, and the coupling oligonucleotide may contain at least six nucleotides.

9. The method according to any one of claims 1 to 8, wherein the coupling sequence comprises at least four nucleotides.

10. The method according to any one of claims 1 to 9, wherein the 5' end of each probing oligonucleotide is phosphorylated.

11. The method according to any one of claims 1 to 10, wherein the probing oligonucleotide can enter the cells and / or nuclei of the sample, and optionally the permeabilized cells and / or permeabilized nuclei of the sample.

12. The method according to any one of claims 1 to 11, further comprising the step of bringing a probing oligonucleotide into contact with a sample, followed by the step of removing one or more probing oligonucleotides from a plurality of probing oligonucleotides that have not come into contact with the sample, wherein the step of removing one or more probing oligonucleotides that have not come into contact with the sample may include the step of removing one or more probing oligonucleotides that have not entered the cells of the sample.

13. The method according to any one of claims 1 to 12, wherein the contact step includes bringing a sample into contact with a device configured to place a probing oligonucleotide, optionally an inkjet device.

14. The method according to any one of claims 1 to 13, wherein the device is a needle, a needle array, a tube, a suction device, an injection device, an electroporation device, a fluorescence-activated cell sorter device, an inkjet device, a microfluidic device, or any combination thereof.

15. The method according to any one of claims 1 to 14, wherein the device brings separate spatial positions of the sample into contact at a specific velocity.

16. The method according to any one of claims 1 to 15, wherein the spatial label is 6 to 60 nucleotides in length.

17. The method according to any one of claims 1 to 16, wherein the two or more spatial positions include at least three, four, five, six, seven, eight, nine, ten, twelve, fourteen, sixteen, eighteen, eighteen, eighteen, ten, twenty, threeteen, fourteen, eighteen, twenty, twenty, threeteen, fourteen, fifty, sixteen, seventeen, eighteen, eighteen, nineteen, or tenteen separate spatial positions of the sample.

18. The method according to any one of claims 1 to 17, wherein the spatial position of the sample corresponds to a region containing approximately 50 or fewer cells.

19. The method according to any one of claims 1 to 18, comprising the step of contacting a sample with an extension reagent.

20. The method according to any one of claims 1 to 19, wherein at least a portion of the contact step may be carried out in the presence of an extension reagent, or the entire contact step may be carried out in the presence of an extension reagent, and furthermore, the contact step and the extension step may be carried out simultaneously.

21. The method according to any one of claims 1 to 20, wherein the elongation is performed in an Insight, and the elongation may include an Insight reverse transcription, and further, the cells of the sample may remain intact during the elongation step.

22. The method according to any one of claims 1 to 21, wherein the extension reagent comprises a reverse transcription reagent, and the reverse transcription reagent may comprise a reverse transcriptase and dNTPs.

23. The method according to any one of claims 1 to 22, wherein the reverse transcriptase comprises a viral reverse transcriptase, and the viral reverse transcriptase may be mouse leukemia virus (MLV) reverse transcriptase or Moloney's mouse leukemia virus (MMLV) reverse transcriptase.

24. The method according to any one of claims 1 to 23, wherein the sample is physically divided or intact during the contact step.

25. The method according to any one of claims 1 to 24, wherein the sample comprises a single cell.

26. The method according to any one of claims 1 to 25, wherein the sample comprises a plurality of single cells.

27. The sample contains multiple cells, The method is The process may include a step of dissociating the sample to generate multiple single cells. The dissociation step may include chemical dissociation, enzymatic dissociation, and / or mechanical dissociation. The dissociation step may involve using one or more of the following: collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin. The method according to any one of claims 1 to 26.

28. Before the step of creating a barcode, A step of distributing multiple single cells into multiple compartments, wherein one of the compartments contains a single cell from multiple single cells, In a compartment containing a single cell, the steps include bringing an extended probing oligonucleotide into contact with multiple oligonucleotide barcodes. The method according to any one of claims 1 to 27, including the method described in any one of claims 1 to 27.

29. The method according to any one of claims 1 to 28, wherein in a compartment containing a single cell, the single cell is brought into contact with a lysis buffer at 15 to 65°C to lyse the single cell, and the lysis buffer may contain an active substance capable of dissociating protein-nucleic acid complexes.

30. The method according to any one of claims 1 to 29, wherein each oligonucleotide barcode of a plurality of oligonucleotide barcodes includes a first universal sequence.

31. The step of obtaining sequencing data is A step of amplifying a plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to a first universal sequence or its complement, and an amplification primer capable of hybridizing to a nucleic acid target or its complement, thereby generating a plurality of amplified barcoded probing oligonucleotides, The step of obtaining sequencing data includes obtaining sequencing data comprising multiple sequencing reads of amplified barcoded probing oligonucleotides or their products. The method according to any one of claims 1 to 30.

32. The method according to any one of claims 1 to 31, wherein the step of obtaining sequencing data includes the step of binding the binding sites of a sequencing primer and / or a sequencing adapter to a plurality of barcoded probing oligonucleotides or their products.

33. The method according to any one of claims 1 to 32, wherein the amplification primer comprises a second universal sequence, and / or the first primer comprises a third universal sequence.

34. (i) The first universal sequence, the second universal sequence, and / or the third universal sequence are the same, and / or (ii) The first universal sequence, the second universal sequence, and / or the third universal sequence are different, The method according to any one of claims 1 to 33.

35. The method according to any one of claims 1 to 34, wherein the first universal sequence, the second universal sequence, and / or the third universal sequence include binding sites of sequencing primers and / or sequencing adapters, complementary sequences thereof, and / or portions thereof.

36. The method according to any one of claims 1 to 35, wherein the sequencing adapter comprises a P5 sequence, a P7 sequence, complementary sequences thereof, and / or a portion thereof.

37. The method according to any one of claims 1 to 36, wherein the sequencing primer comprises a lead 1 sequencing primer, a lead 2 sequencing primer, complementary sequences thereof, and / or portions thereof.

38. The method according to any one of claims 1 to 37, wherein the sample comprises a target panel of multiple nucleic acid targets, optionally at least about two distinct nucleic acid targets.

39. The method according to any one of claims 1 to 38, wherein two or more nucleic acid targets of the target panel are biomarkers, the biomarkers may be biomarkers of a disease or condition, and further, the disease or condition may be cancer, an infectious disease, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof.

40. The method according to any one of claims 1 to 39, wherein the contact step includes contacting a sample with a panel of probing oligonucleotides comprising two or more probing oligonucleotides, each of which comprises a probe sequence configured to hybridize with a nucleic acid target among a plurality of nucleic acid targets.

41. The method according to any one of claims 1 to 40, wherein the step of determining the copy number of nucleic acid targets in a sample includes determining the copy number of each of a plurality of nucleic acid targets in a sample based on the number of molecular labels having distinct sequences associated with a plurality of barcoded probing oligonucleotides or products thereof, each containing the respective sequences of a plurality of nucleic acid targets.

42. The method according to any one of claims 1 to 41, comprising the step of determining the copy number of each of the multiple nucleic acid targets at each spatial position of the sample by counting the number of molecular labels having distinct sequences associated with each of the multiple nucleic acid targets for each unique spatial label sequence associated with a distinct spatial position of the sample.

43. The method according to any one of claims 1 to 42, comprising a panel of amplification primers configured to hybridize with a plurality of nucleic acid targets or their complements, optionally comprising a panel of at least two distinct amplification primers.

44. The method according to any one of claims 1 to 43, wherein the nucleic acid target comprises a nucleic acid molecule, and the nucleic acid molecule may comprise ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA including a poly(A) tail, sample-indexed oligonucleotide, cell component-binding reagent-specific oligonucleotide, or any combination thereof.

45. The method according to any one of claims 1 to 44, wherein the plurality of cells comprise one or more cell types, and the one or more cell types may be selected from the group consisting of brain cells, cardiac cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, and circulating cells, or any combination thereof.

46. The method according to any one of claims 1 to 45, wherein the sample comprises a biological sample, clinical sample, environmental sample, biological fluid, tissue, tissue section, or any combination thereof derived from the subject, and the subject may be a human, mouse, dog, rat, or vertebrate.

47. The method according to any one of claims 1 to 46, further comprising the step of determining the genotype, phenotype, or one or more gene mutations of a target based on the spatial position of nucleic acid targets in a sample.

48. The method according to any one of claims 1 to 47, further comprising the step of predicting the susceptibility of a subject to one or more diseases, optionally cancer or a genetic disease.

49. The method according to any one of claims 1 to 48, further comprising the step of determining the cell types of multiple cells in a sample.

50. The method according to any one of claims 1 to 49, comprising selecting a drug based on the predicted responsiveness of multiple cell types in a sample.

51. The method according to any one of claims 1 to 50, comprising the steps of imaging a sample, optionally, imaging the sample before and / or after the contact step, wherein imaging data may be generated by the imaging step.

52. The method according to any one of claims 1 to 51, wherein the step of imaging a sample includes the step of staining a sample using a stain, the stain being fluorescent staining, negative staining, antibody staining, or any combination thereof, and the staining step may include immunocytochemistry (ICC), immunohistochemical testing (IHC), immunofluorescence (IF), or any combination thereof.

53. The method according to any one of claims 1 to 52, wherein the imaging step includes microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multiphoton microscopy, quantitative phase microscopy, surface-enhanced Raman spectroscopy, video recording, manual visual analysis, automated visual analysis, or any combination thereof.

54. The method according to any one of claims 1 to 53, further comprising the step of associating imaging data with sequencing data of one or more spatial locations of a sample.

55. The method according to any one of claims 1 to 54, comprising correlation analysis of spatial position imaging data and sequencing data, wherein the correlation analysis may identify one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and / or cellular targets of candidate therapeutic agents.

56. The method according to any one of claims 1 to 55, wherein the imaging step produces an image used to construct a map for physically representing the sample, the map may be two-dimensional or three-dimensional.

57. The method according to any one of claims 1 to 56, comprising the step of mapping nucleic acid targets and / or cellular component targets onto a map of a sample.

58. The method according to any one of claims 1 to 57, comprising the step of mapping one or more single cells from a group of cells onto a map of a sample.

59. The method according to any one of claims 1 to 58, wherein the sample has been in contact with one or more types of fixatives and / or permeation agents.

60. The method according to any one of claims 1 to 59, wherein the sample comprises tissue, cell monolayer, fixed cells, tissue section, or any combination thereof, optionally including fresh tissue section, frozen tissue section, fixed tissue section, formalin-fixed tissue section, formalin-fixed paraffin-embedded (FFPE) tissue section, acetone-fixed tissue section, paraformaldehyde (PFA)-fixed tissue section, and / or methanol-fixed tissue section.

61. The method according to any one of claims 1 to 60, wherein the sample comprises a nuclear suspension, optionally, an immobilized nuclear suspension and / or a permeabilized nuclear suspension.

62. The method according to any one of claims 1 to 61, wherein the sample comprises cells, optionally fresh cells, frozen cells, fixed cells, formalin-fixed cells, formalin-fixed paraffin-embedded (FFPE) cells, acetone-fixed cells, paraformaldehyde (PFA)-fixed cells, and / or methanol-fixed cells.

63. The method according to any one of claims 1 to 62, comprising the steps of permeabilizing a sample and / or fixing a sample.

64. The method according to any one of claims 1 to 63, wherein the step of fixing a sample includes a step of bringing the sample into contact with a fixative, the fixative may include a non-crosslinking fixative, the non-crosslinking fixative may further include methanol, or the fixative may include a crosslinking agent.

65. The crosslinking agent comprises a cleavable crosslinking agent, where (a) the cleavable crosslinking agent is dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3,3'-dithiobispropionimidate (DTBP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamide)hexanoate (LC-SPDP), and 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio)toluene (b) The method according to claim 64, wherein the cleavable crosslinking agent may include or be derived from (SMPT), 3-(2-pyridyldithio)propionylhydrazide (PDPH), 2-((4,4'-adipentanamide)ethyl)-1,3'-dithiopropionate succinimidyl (SDAD, NHS-SS-diaziline), or any combination thereof, and / or (c) the cleavable crosslinking agent is a thiol-cleavable crosslinking agent or a disulfide linker.

66. The method according to any one of claims 1 to 65, wherein the fixative comprises paraformaldehyde (PFA), dithiobis(succinimidyl dithiopropionate (DSP), 3-(2-pyridyldithio)succinimidyl 3-(2-pyridyldithio)propionate (SPDP), CellCover, or a combination thereof.

67. The method according to any one of claims 1 to 66, wherein the step of fixing the sample and the step of permeabilizing the sample are performed simultaneously.

68. The method according to any one of claims 1 to 67, wherein the steps of fixing the sample and permeabilizing it are performed in the presence of a dual-function substance capable of fixing and permeabilizing the sample, and the dual-function substance may be methanol.

69. The method according to any one of claims 1 to 68, wherein the step of permeabilizing the sample includes the step of bringing the sample into contact with a permeabilizing agent.

70. The method according to any one of claims 1 to 69, comprising the step of contacting a sample with a plurality of probing oligonucleotides or a plurality of cell component binding reagents, followed by the step of removing a permeabilizing agent from the sample.

71. The permeabilizing agent can (i) permeabilize the cell membrane of a cell, and (ii) make the cell membrane permeable to probing oligonucleotides, cell component binding reagents, or both. The permeation agent comprises (i) a solvent, surfactant, or surfactant; (ii) BD Cytoperm; (iii) saponin or its derivative; (iv) Triton X-100, (v) methanol or its derivative, and / or (vi) digitonin or its derivative. The active substance capable of dissociating protein-nucleic acid complexes includes a serine protease having broad substrate specificity, and the serine protease having broad substrate specificity may be proteinase K, and / or The lysis buffer comprises (a) a defixation agent, the defixation agent may comprise a thiol, hydroxylamine, periodic acid, a base, or any combination thereof, and / or (b) DTT. The method according to any one of claims 1 to 70.

72. The method according to any one of claims 1 to 71, comprising the step of reversing the fixation of a sample and / or a single cell, wherein the step of reversing the fixation of a sample and / or a single cell may include UV cutting, chemical treatment, heating, enzymatic treatment, or any combination thereof.

73. The sample contains multiple cellular component targets, The method is A step of bringing multiple cell component binding reagents into contact with a sample, wherein each of the multiple cell component binding reagents contains a cell component binding reagent-specific oligonucleotide containing a unique identifier sequence for the cell component binding reagent, and the cell component binding reagent is capable of specifically binding to at least one of the multiple cell component targets, The steps include barcoding cell component-binding reagent-specific oligonucleotides to generate a plurality of barcoded cell component-binding reagent-specific oligonucleotides, each containing a sequence complementary to at least a portion of the unique identifier sequence and a molecular labeling sequence, and A step of obtaining sequencing data comprising multiple sequencing reads of multiple barcoded cell component-binding reagent-specific oligonucleotides or their products, wherein each of the multiple sequencing reads comprises at least a portion of a molecular label sequence and a unique identifier sequence. The method according to any one of claims 1 to 72, further comprising:

74. The method according to any one of claims 1 to 73, wherein the step of obtaining sequencing data includes the step of binding the binding site of a sequencing primer and / or a sequencing adapter to a barcoded cell component binding reagent-specific oligonucleotide or a product thereof.

75. The method according to any one of claims 1 to 74, comprising the step of bringing a plurality of cell component binding reagents into contact with a sample, followed by the step of removing one or more cell component binding reagents from the plurality that have not come into contact with the sample, wherein the step of removing one or more cell component binding reagents that have not come into contact with the sample may include the step of removing one or more cell component binding reagents that have not come into contact with at least one of the plurality of cell component targets.

76. The method according to any one of claims 1 to 75, wherein the cellular component target includes intracellular proteins, carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, intracellular proteins, or any combination thereof.

77. The method according to any one of claims 1 to 76, wherein the cell component binding reagent-specific oligonucleotide comprises a second molecular label, and at least 10 of the plurality of cell component binding reagent-specific oligonucleotides may comprise different second molecular label sequences.

78. The second molecular labeling sequences of at least two cell component binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two cell component binding reagent-specific oligonucleotides are identical, and / or The second molecular labeling sequences of at least two cell component binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two cell component binding reagent-specific oligonucleotides are different. The method according to any one of claims 1 to 77.

79. The number of unique molecular label sequences associated with a unique identifier sequence for a cell component binding reagent capable of specifically binding to at least one cell component target in the sequencing data indicates the copy number of at least one cell component target in the sample, and / or The number of unique second molecular label sequences associated with a unique identifier sequence for a cell component binding reagent capable of specifically binding to at least one cell component target in the sequencing data indicates the copy number of at least one cell component target in the sample. The method according to any one of claims 1 to 78.

80. The method according to any one of claims 1 to 79, comprising the step of contacting a sample with a blocking reagent, one or more decoy oligonucleotides, and / or one or more blocking oligonucleotides, prior to the step of contacting a sample with a plurality of cell component binding reagents and / or the step of contacting a plurality of probing oligonucleotides with the sample.

81. The method according to any one of claims 1 to 80, wherein the step of contacting a sample with multiple cell component binding reagents is performed in the presence of a blocking reagent.

82. The method according to any one of claims 1 to 81, wherein the blocking reagent comprises a plurality of oligonucleotides complementary to at least a portion of the cell component binding reagent-specific oligonucleotides.

83. The method according to any one of claims 1 to 82, wherein the blocking reagent comprises an antibody or fragment thereof derived from a first species, and the blocking reagent comprises serum derived from the first species.

84. The method according to any one of claims 1 to 83, wherein the sample comprises one or more non-target nucleic acids, and the blocking reagent comprises a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids.

85. The method according to any one of claims 1 to 84, wherein each of the multiple decoy oligonucleotides is capable of hybridizing to at least a portion of a non-target nucleic acid.

86. The decoy oligonucleotide contains a sequence complementary to at least a portion of the non-target nucleic acid. The decoy oligonucleotide contains a sequence identical or substantially similar to the sequence of the cell component binding reagent-specific oligonucleotide, and the sequence may be 3 to 40 nucleotides in length. The decoy oligonucleotide has up to 50% sequence identity with the cell component binding reagent-specific oligonucleotide. The decoy oligonucleotide does not contain UMI. The decoy oligonucleotide contains a random sequence, which may be approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. The decoy oligonucleotide does not contain any sequence having five or more, six or more, seven or more, or eight or more consecutive T or A atoms. The decoy oligonucleotide contains at least one G or C for every four, five, six, or seven consecutive nucleotides. The decoy oligonucleotide contains one or more modified nucleotides. The decoy oligonucleotide may include a 5' modification, and the 5' modification may include a 5' amino acid modification factor C12 modification (5AmMC12). The decoy oligonucleotide may include a 3' modification, and the 3' modification may include a 3' dideoxy C modification (ddC), and / or The decoy oligonucleotide is 30 to 65 nucleotides in length. The method according to any one of claims 1 to 85.

87. If the sample contains one or more undesirable nucleic acid species, The method is A step of contacting a sample with a blocking oligonucleotide, wherein the blocking oligonucleotide specifically binds to at least one of one or more undesirable nucleic acid species, This reduces the reverse transcription of at least one of one or more undesirable nucleic acid species by the blocking oligonucleotide. The method according to any one of claims 1 to 86.

88. Blocking oligonucleotides, Before contacting the sample with multiple probing oligonucleotides, bring them into contact with the sample; After contacting multiple probing oligonucleotides with the sample, and / or When bringing multiple probing oligonucleotides into contact with a sample, The method according to any one of claims 1 to 87.

89. The method according to any one of claims 1 to 88, comprising the step of preparing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species in a sample, optionally at least 10 or at least 100 undesirable nucleic acid species.

90. The blocking oligonucleotide is a locked nucleic acid (LNA), peptide nucleic acid (PNA), DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera. Blocking oligonucleotides specifically bind to within 100 nucleotides from the 3' end of one or more undesirable nucleic acid species. A blocking oligonucleotide specifically binds to within 100 nucleotides from the 5' end of one or more undesirable nucleic acid species, or a blocking oligonucleotide specifically binds to within 100 nucleotides from the central part of one or more undesirable nucleic acid species. Whether or not the blocking oligonucleotide contains non-natural nucleotides, The blocking oligonucleotide has a Tm of at least 50°C. Blocking oligonucleotides are unable to function as primers for reverse transcriptase or polymerase, and / or Blocking oligonucleotides are 8 to 100 nucleotides in length. The method according to any one of claims 1 to 89.

91. One or more undesirable nucleic acid species constitute approximately 50% to 80% of the nucleic acid content of the sample. Undesirable nucleic acid species are selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA, intron sequences, sequences with high abundance, and / or One or more undesirable nucleic acid species are mRNA molecules, and the blocking oligonucleotide specifically binds to within 10 nucleotides of the 3' poly(A) tail of one or more undesirable nucleic acid species. The method according to any one of claims 1 to 90.

92. Each molecular label of multiple oligonucleotide barcodes contains at least six nucleotides. Each capture sequence of multiple oligonucleotide barcodes contains at least four nucleotides. Multiple oligonucleotide barcodes are associated with a solid support, and one of the compartments contains a single solid support. Multiple oligonucleotide barcodes each contain a cell label, and each cell label of the multiple oligonucleotide barcodes may contain at least six nucleotides, and / or Among multiple oligonucleotide barcodes, those associated with the same solid support may contain the same cell label, while those associated with different solid supports may contain different cell labels. The method according to any one of claims 1 to 91.

93. The method according to any one of claims 1 to 92, wherein the solid support comprises synthetic particles, a planar surface, or a combination thereof.

94. The method according to any one of claims 1 to 93, comprising the step of associating synthetic particles containing a plurality of oligonucleotide barcodes with cells in a compartment.

95. The process includes a step of associating synthetic particles with cells, followed by a step of lysing the cells, the cell lysing step may include a step of heating the cells, a step of contacting the cells with a surfactant, a step of changing the pH of the cells, or any combination thereof. Synthetic particles and single cells are in the same compartment, and the compartment may be a well or a droplet. At least one of multiple oligonucleotide barcodes is immobilized or partially immobilized on the synthetic particle, or at least one of multiple oligonucleotide barcodes is encapsulated or partially encapsulated within the synthetic particle. The synthetic particles are decayable, and are arbitrarily decayable hydrogel particles. The synthetic particles may include beads, and the beads may include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, antibiotin microbeads, antifluorescent dye microbeads, or any combination thereof, and / or The synthetic particles include materials selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The method according to any one of claims 1 to 94.

96. Each of the multiple oligonucleotide barcodes contains a linker functional group. The synthetic particles contain functional groups for a solid support. The support functional group and the linker functional group are associated with each other, and the linker functional group and the support functional group may be selected from the group consisting of C6, biotin, streptavidin, primary amine, aldehyde, ketone, and any combination thereof. The method according to any one of claims 1 to 95.

97. A plurality of probing oligonucleotides, each of which comprises a coupling sequence and a probe sequence configured to hybridize with a nucleic acid target, and the probing oligonucleotides may also contain a predetermined spatial label. Coupling oligonucleotides containing a 5' coupling sequence complement and a 3' capture sequence complement, Multiple oligonucleotide barcodes, wherein the 3' end of each oligonucleotide barcode is associated with a solid support, and the 5' end of each oligonucleotide barcode contains a capture sequence, A first primer that can hybridize to a first universal sequence and may further contain a third universal sequence, Amplification primers capable of hybridizing to nucleic acid targets or their complements, and which may further contain a second universal sequence, DNA ligase, Extension reagent, optionally reverse transcription reagent, and optionally reverse transcriptase and dNTPs. One or more types of fixatives, One or more types of permeation agents, Crosslinking agent, Fixation release agent, lysis buffer, Multiple cell component binding reagents, each of which contains a cell component binding reagent-specific oligonucleotide containing a unique identifier sequence for the cell component binding reagent, and the cell component binding reagents are capable of specifically binding to at least one of the multiple cell component targets. Blocking reagent, One or more decoy oligonucleotides, and / or One or more blocking oligonucleotides A kit that includes this.

98. The kit according to claim 97, wherein the plurality of probing oligonucleotides comprises a panel of probing oligonucleotides comprising two or more plurality of probing oligonucleotides, each plurality comprising a probe sequence configured to hybridize with a plurality of nucleic acid targets, optionally a nucleic acid target from a target panel of at least about two distinct nucleic acid targets.

99. A kit according to any one of claims 97 to 98, comprising a panel of amplification primers configured to hybridize with a plurality of nucleic acid targets or their complements, optionally comprising a panel of at least about two distinct amplification primers.

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