Heterozygote targeting and whole transcriptome amplification

Abstract

The invention relates to heterozygote targeting and whole transcriptome amplification. The disclosure herein includes systems, methods, compositions, and kits for labeling nucleic acid targets. In some embodiments, the methods include the use of target-specific primers and target-non-specific primers during library preparation (e.g., heterozygote library preparation). In some embodiments, the methods increase the abundance of selected low-abundance targets during the preparation of cDNA libraries and / or sequencing libraries.

Description

[0001] This application is a divisional application of an application filed on February 12, 2020, with application number 202080014409.2, and invention name “Hybrid Targeting and Whole Transcriptome Amplification”.

[0002] Related Applications

[0003] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Serial No. 62 / 805,956, filed on February 14, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes. background

[0005] field

[0006] The present disclosure relates generally to the field of molecular biology, such as determining gene expression using molecular barcoding.

[0007] Description of the Prior Art

[0008] Current technology allows for the measurement of gene expression of single cells in a massively parallel manner (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to multiple (A) mRNA molecules from individual cells when each cell is co-localized with barcoded reagent beads in a compartment. However, nucleic acids in cells are typically in a wide concentration range that ranges over several orders of magnitude. This greatly increases the amount of sequencing or screening necessary for a comprehensive assessment of the sample. There is a need for systems and methods for increasing the abundance of low-abundance nucleic acids during library preparation.

[0009] Overview

[0010] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide, each oligonucleotide comprising a first universal sequence and a barcode sequence; extending more than one oligonucleotide hybridized to more than one nucleic acid target to produce more than one first-strand barcoded polynucleotide; and using more than one first-strand barcoded polynucleotide as a template to synthesize more than one second-strand barcoded polynucleotide to produce more than one double-stranded barcoded polynucleotide. The method may include adding the sequence of an adapter (e.g., connecting the adapter) to more than one double-stranded barcoded polynucleotide, wherein the adapter comprises a second universal sequence. The method may include amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and a second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of a nucleic acid target, the first universal sequence, the second universal sequence, a complementary sequence thereof, and / or a portion thereof, and amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets, the first universal sequence, the second universal sequence, a complementary sequence thereof, and / or a portion thereof.

[0011] In some embodiments, the method includes random priming and extension using random primers and a first more than one barcoded amplicon and / or a second more than one barcoded amplicon or its product as a template. In some embodiments, random priming and extension include adding the sequence of a sequencing adapter to the first more than one barcoded amplicon and the second more than one barcoded amplicon or its product. In some embodiments, the method includes extending and / or amplifying using a target-specific primer and a first more than one barcoded amplicon and / or a second more than one barcoded amplicon or its product as a template. In some embodiments, extension and / or amplification includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon and / or the second more than one barcoded amplicon. In some embodiments, the sequencing adapter comprises a binding site for a sequencing primer (e.g., Illumina read 1 and read 2 sequences) or a sequencing adapter (e.g., Illumina P5 and P7 sequences), its complementary sequence, or a portion thereof.

[0012] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide, each oligonucleotide comprising a first universal sequence and a barcode sequence; extending more than one oligonucleotide hybridized to more than one nucleic acid target to produce more than one first-strand barcoded polynucleotide; and using more than one first-strand barcoded polynucleotide as a template to synthesize more than one second-strand barcoded polynucleotide to produce more than one double-stranded barcoded polynucleotide. The method may include adding the sequence of an adapter (e.g., by connecting the adapter or PCR) to more than one double-stranded barcoded polynucleotide, wherein the adapter comprises a second universal sequence. The method may include amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of a nucleic acid target, a first universal sequence, a second universal sequence, a complement thereof, and / or a portion thereof. The method may include extension and / or amplification using a target-specific primer and a first one or more barcoded amplicons or products thereof as a template.

[0013] In some embodiments, the method includes random priming and extension using random primers and a first more than one barcoded amplicon or its product as a template. In some embodiments, the method includes amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets, thereby generating a second more than one barcoded amplicon comprising sequences of two or more of more than one nucleic acid targets and / or a portion thereof. In some embodiments, performing random priming and extension includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon and the second more than one barcoded amplicon or its product. In some embodiments, performing extension and / or amplification includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon. In some embodiments, the sequencing adapter comprises a sequencing primer binding site or a sequencing primer. In some embodiments, the sequencing primer is a read 1 sequencing primer or a read 2 sequencing primer.

[0014] In some embodiments, amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets includes amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets and primers capable of amplifying a first universal sequence. In some embodiments, amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets includes amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets and primers capable of amplifying a second universal sequence. In some embodiments, extending more than one oligonucleotide to generate more than one first-strand barcoded polynucleotide includes reverse transcribing one or more nucleic acid targets in more than one nucleic acid targets. In some embodiments, more than one first-strand barcoded polynucleotide comprises a barcoded single-stranded cDNA molecule. In some embodiments, extending more than one oligonucleotide to generate more than one first strand barcoded polynucleotide comprises nucleic acid extension of one or more of the more than one oligonucleotides that hybridize to one or more nucleic acid targets of more than one nucleic acid targets. In some embodiments, an adaptor is attached to the 5' end, the 3' end, or both of the more than one double-stranded barcoded polynucleotide. In some embodiments, the method comprises obtaining sequence information of the first more than one barcoded amplicon and / or the second more than one barcoded amplicon or a product thereof.

[0015] In some embodiments, the first more than one barcoded amplicon includes a whole transcriptome amplification (WTA) product. In some embodiments, the first more than one barcoded amplicon corresponds to at least 10% of the mRNA of a single cell. In some embodiments, the first more than one barcoded amplicon corresponds to at least 50% of the mRNA of a single cell. In some embodiments, the first more than one barcoded amplicon corresponds to at least 90% of the mRNA of a single cell. In some embodiments, the second more than one barcoded amplicon corresponds to at most 5% of the mRNA of a single cell. In some embodiments, the second more than one barcoded amplicon corresponds to at most 10% of the mRNA of a single cell. In some embodiments, the second more than one barcoded amplicon corresponds to at most 20% of the mRNA of a single cell.

[0016] In some embodiments, the adapter is a single-stranded polynucleotide. In some embodiments, the adapter is a double-stranded polynucleotide. In some embodiments, the length of the adapter or the chain of the adapter is 2-30 nucleotides. In some embodiments, the first universal sequence and the second universal sequence are the same. In some embodiments, the first universal sequence and the second universal sequence are different. In some embodiments, each of more than one amplicon comprises at least a portion of the following: the first universal sequence, the second universal sequence, or both.

[0017] In some embodiments, adding the sequence of the adapter to more than one double-stranded barcoded polynucleotide includes connecting the adapter to more than one double-stranded barcoded polynucleotide. Adding the sequence of the adapter to more than one double-stranded barcoded polynucleotide can include adding the sequence of the adapter to more than one double-stranded barcoded polynucleotide by nucleic acid extension or amplification. Amplification includes polymerase chain reaction. Amplifying more than one double-stranded barcoded polynucleotide can include amplifying more than one double-stranded barcoded polynucleotide by linear amplification. Linear amplification can include 1-100 cycles of amplification. Amplifying more than one double-stranded barcoded polynucleotide can include amplifying more than one double-stranded barcoded polynucleotide by polymerase chain reaction (PCR). PCR can include 1-100 cycles of PCR.

[0018] In some embodiments, extending and / or amplifying includes extending and / or amplifying for 1-100 cycles. Amplification may include polymerase chain reaction (PCR) amplification. In some embodiments, randomly priming and extending and / or amplifying includes randomly priming and extending and / or amplifying for 1-100 cycles. Amplification may include polymerase chain reaction (PCR) amplification.

[0019] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide; and extending the more than one oligonucleotide in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotide and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. The method may include amplifying more than one first-strand barcoding polynucleotide using primers capable of hybridizing to a first universal sequence and TSO or a portion thereof to synthesize a first more than one second-strand barcoding polynucleotide and produce a first more than one double-stranded barcoding polynucleotide, each second-strand barcoding polynucleotide comprising a sequence of a nucleic acid target, a first universal sequence, TSO or a portion thereof; and amplifying more than one first-strand barcoding polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize a second more than one second-strand barcoding polynucleotide to produce a second more than one double-stranded barcoding polynucleotide, each second-strand barcoding polynucleotide comprising a sequence of a nucleic acid target or a portion thereof. The method may include amplifying the first more than one double-stranded barcoding polynucleotide and / or the second more than one double-stranded barcoding polynucleotide using primers capable of hybridizing to a sequence or subsequence of the first universal sequence and TSO, thereby producing more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets and / or a portion thereof. The method can include amplifying a first one or more double-stranded barcoded polynucleotide and / or a second one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the one or more nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the one or more nucleic acid targets and / or a portion thereof.

[0020] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide; and extending the more than one oligonucleotide in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to produce more than one first-strand barcoded polynucleotides comprising TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. The method may include amplifying more than one first-strand barcoded polynucleotides using primers capable of hybridizing with the first universal sequence and TSO or a portion thereof to synthesize a first more than one second-strand barcoded polynucleotide and produce a first more than one double-stranded barcoded polynucleotide, each second-strand barcoded polynucleotide comprising a sequence of a nucleic acid target, a first universal sequence, TSO or a portion thereof. The method may include amplifying a first one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and a sequence or subsequence of a TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof, and amplifying the first one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0021] In some embodiments, the method includes amplifying more than one first-strand barcoding polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets to synthesize a second more than one second-strand barcoding polynucleotide to produce a second more than one double-stranded barcoding polynucleotide, each second-strand barcoding polynucleotide comprising a sequence of a nucleic acid target or a portion thereof. In some embodiments, each of the more than one oligonucleotides comprises a first universal sequence and a barcode sequence. In some embodiments, the template switching oligonucleotide comprises a second universal sequence. In some embodiments, the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. In some embodiments, each of the more than one oligonucleotides comprises a second universal sequence. In some embodiments, the method includes amplifying a first more than one second-strand barcoding polynucleotide and / or a second more than one second-strand barcoding polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets. In some embodiments, a first more than one second strand barcoded polynucleotide and / or a second more than one second strand barcoded polynucleotide is amplified using primers capable of hybridizing to two or more of the more than one nucleic acid targets and a primer capable of hybridizing to the first universal sequence. In some embodiments, a first more than one second strand barcoded polynucleotide and / or a second more than one second strand barcoded polynucleotide is amplified using primers capable of hybridizing to two or more of the more than one nucleic acid targets and a primer capable of hybridizing to a sequence or subsequence of TSO.

[0022] In some embodiments, amplifying more than one first strand barcoded polynucleotide includes amplifying more than one first strand barcoded polynucleotide for 1-100 cycles. Amplifying more than one first strand barcoded polynucleotide may include linear amplification of more than one first strand barcoded polynucleotide. Amplifying more than one first strand barcoded polynucleotide may include polymerase chain reaction (PCR) amplification of more than one first strand barcoded polynucleotide. Amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide may include amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide for 1-100 cycles. Amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide may include amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide by linear amplification. Amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides may include amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides by polymerase chain reaction (PCR) amplification.

[0023] In some embodiments, the method includes dividing the first more than one second strand barcoded polynucleotide and the second more than one second strand barcoded polynucleotide into two pools, wherein amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide is performed using (1) primers that can hybridize to the first universal sequence and a sequence or subsequence of TSO in one reaction using one of the two pools, and using (2) primers that can hybridize to two or more of the more than one nucleic acid targets in another reaction using the other of the two pools. In some embodiments, the method includes obtaining sequence information of the first more than one barcoded amplicons and / or the second more than one barcoded amplicons or their products.

[0024] In some embodiments, the method includes extending and / or amplifying using a target-specific primer and a first more than one barcoded amplicon and / or a second more than one barcoded amplicon as a template. In some embodiments, extending more than one oligonucleotide to produce more than one first-strand barcoded polynucleotides includes reverse transcribing one or more nucleic acid targets in more than one nucleic acid target to produce a barcoded single-stranded cDNA molecule. In some embodiments, extending more than one oligonucleotide to produce more than one first-strand barcoded polynucleotides includes nucleic acid extension of oligonucleotides in more than one oligonucleotides hybridized with one or more nucleic acid targets in more than one nucleic acid target to produce a barcoded single-stranded nucleic acid molecule. In some embodiments, the reverse transcriptase includes a viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase. In some embodiments, the viral reverse transcriptase is a Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0025] In some embodiments, two or more of the more than one nucleic acid targets include mRNAs of underexpressed genes.

[0026] The total number of occurrences of two or more of the more than one nucleic acid targets may include mRNA as at most 1% of the mRNA of a single cell. The total number of occurrences of two or more of the more than one nucleic acid targets may include mRNA as at most 5% of the mRNA of a single cell. The total number of occurrences of two or more of the more than one nucleic acid targets may include mRNA as at most 10% of the mRNA of a single cell.

[0027] In some embodiments, the barcode sequence comprises a molecular marker, a cell marker, and wherein the oligonucleotide comprises a target-specific region or any combination thereof. In some embodiments, the target-specific region comprises an oligo dT sequence, a random sequence, a target-specific sequence, or a combination thereof. In some embodiments, more than one nucleic acid target comprises DNA, mRNA, or a combination thereof. In some embodiments, more than one nucleic acid target is mRNA. In some embodiments, each of more than one oligonucleotide comprising a first universal sequence and a barcode sequence is fixed on a solid support. In some embodiments, the solid support is a bead. In some embodiments, at least two of the more than one oligonucleotides fixed on a single solid support comprise different molecular markers. In some embodiments, the more than one nucleic acid attached to the solid support comprises the same cell marker. In some embodiments, more than one nucleic acid target is a nucleic acid from a single sample. In some embodiments, the sample is a single cell. In some embodiments, the sample comprises more than one cell. In some embodiments, the single cell is the following or more than one cell includes the following: brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, circulating cells, or a combination thereof. In some embodiments, hybridization of more than one nucleic acid target with more than one oligonucleotide occurs in a partition. In some embodiments, the partition is a microwell or a droplet. In some embodiments, the method includes lysing a single cell to release more than one nucleic acid target. In some embodiments, each of the barcoded amplicons comprises a barcode sequence. In some embodiments, the barcode sequence comprises a molecular marker, a cell marker, or a combination thereof. In some embodiments, the method includes sequencing the barcoded amplicons to generate more than one sequencing read comprising a molecular marker, a cell marker, or any combination thereof. In some embodiments, the method includes analyzing more than one sequencing read using a cell marker. In some embodiments, the method includes analyzing more than one sequencing read using a molecular marker.

[0028] The disclosure of this article also provides the following items:

[0029] 1. A method for labeling a nucleic acid target, the method comprising:

[0030] (a) hybridizing more than one nucleic acid target with more than one oligonucleotide, each of the oligonucleotides comprising a first universal sequence and a barcode sequence;

[0031] (b) extending the more than one oligonucleotides hybridized to the more than one nucleic acid targets to generate more than one first-strand barcoded polynucleotides;

[0032] (c) synthesizing more than one second-strand barcoded polynucleotide using the more than one first-strand barcoded polynucleotide as a template to generate more than one double-stranded barcoded polynucleotide;

[0033] (d) adding the sequence of an adaptor to the more than one double-stranded barcoded polynucleotide, wherein the adaptor comprises a second universal sequence; and

[0034] (e) amplifying the more than one double-stranded barcoded polynucleotides:

[0035] (e1) amplifying the more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of the nucleic acid target, the first universal sequence, the second universal sequence, a complementary sequence thereof, and / or a portion thereof, and

[0036] (e2) amplifying the more than one double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets, the first universal sequence, the second universal sequence, a complementary sequence thereof, and / or a portion thereof.

[0037] 2. The method according to item 1, comprising (f1) extending and / or amplifying using a target-specific primer and the first one or more barcoded amplicons and / or the second one or more barcoded amplicons or their products as templates.

[0038] 3. The method according to item 2, wherein (f1) extension and / or amplification includes adding the sequence of the binding site of the sequencing primer and / or sequencing adapter, its complementary sequence and / or part thereof to the first one or more barcoded amplicons and / or the second one or more barcoded amplicons.

[0039] 4. The method according to any one of items 1-3, comprising (f2) using random primers and the first one or more barcoded amplicons and / or the second one or more barcoded amplicons or their products as templates for random priming and extension and / or amplification.

[0040] 5. A method according to item 4, wherein (f2) performing random priming and extension and / or amplification includes adding sequences of binding sites for sequencing primers and / or sequencing adapters, their complementary sequences and / or parts thereof to the first one or more barcoded amplicons and the second one or more barcoded amplicons or their products.

[0041] 6. A method according to any one of items 3-5, wherein the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof or a portion thereof.

[0042] 7. The method according to any one of items 3-6, wherein the sequencing primer comprises a read 1 sequencing primer, a read 2 sequencing primer, a complementary sequence thereof or a portion thereof.

[0043] 8. A method for labeling a nucleic acid target, the method comprising:

[0044] (a) hybridizing more than one nucleic acid target with more than one oligonucleotide, each of the oligonucleotides comprising a first universal sequence and a barcode sequence;

[0045] (b) extending the more than one oligonucleotides hybridized to the more than one nucleic acid targets to generate more than one first-strand barcoded polynucleotides;

[0046] (c) synthesizing more than one second-strand barcoded polynucleotide using the more than one first-strand barcoded polynucleotide as a template to generate more than one double-stranded barcoded polynucleotide;

[0047] (d) adding the sequence of an adaptor to the more than one double-stranded barcoded polynucleotides, wherein the adaptor comprises a second universal sequence;

[0048] (e1) amplifying the more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of the nucleic acid target, the first universal sequence, the second universal sequence, a complementary sequence thereof, and / or a portion thereof;

[0049] (f1) extending and / or amplifying using a target-specific primer and the first one or more barcoded amplicons or products thereof as templates.

[0050] 9. The method according to item 8, comprising (f2) performing random priming and extension and / or amplification using random primers and the first one or more barcoded amplicons or their products as templates.

[0051] 10. A method according to item 9, wherein (f2) performing random priming and extension and / or amplification includes adding the sequence of a sequencing adapter to the first one or more barcoded amplicons or their products.

[0052] 11. A method according to any one of items 8-10, wherein (f1) extending and / or amplifying includes adding sequences of binding sites for sequencing primers and / or sequencing adapters, their complementary sequences and / or parts thereof to the first one or more barcoded amplicons.

[0053] 12. The method according to item 10 or 11, wherein the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof or a portion thereof.

[0054] 13. The method according to any one of items 10-12, wherein the sequencing primer comprises a read 1 sequencing primer, a read 2 sequencing primer, a complementary sequence thereof or a portion thereof.

[0055] 14. A method according to any one of items 8-13, comprising (e2) amplifying the more than one double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby producing a second more than one barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0056] 15. A method according to any one of items 1-7 and 14, wherein (e2) amplifying the more than one double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets comprises amplifying the more than one double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets and primers capable of amplifying the first universal sequence.

[0057] 16. A method according to any one of items 1-7 and 14-15, wherein (e2) amplifying the more than one double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets comprises amplifying the more than one double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets and primers capable of amplifying the second universal sequence.

[0058] 17. The method according to any one of items 1-16, wherein (b) extending the more than one oligonucleotides to generate the more than one first-strand barcoded polynucleotides comprises reverse transcribing one or more of the more than one nucleic acid targets.

[0059] 18. The method according to item 17, wherein the more than one first-strand barcoded polynucleotides comprise barcoded single-stranded cDNA molecules.

[0060] 19. A method according to any one of items 1-18, wherein (b) extending the more than one oligonucleotides to produce the more than one first-strand barcoded polynucleotides includes performing nucleic acid extension on one or more of the more than one oligonucleotides hybridized to one or more of the more than one nucleic acid targets.

[0061] 20. The method according to any one of items 1-19, wherein the sequence of the adaptor is added to the 5' end, the 3' end, or both of the more than one double-stranded barcoded polynucleotides.

[0062] 21. The method according to any one of items 1-20, comprising (g) obtaining sequence information of the first one or more barcoded amplicons and / or the second one or more barcoded amplicons or products thereof.

[0063] 22. The method of any one of items 1-21, wherein the first one or more barcoded amplicons comprise whole transcriptome amplification (WTA) products.

[0064] 23. The method according to any one of items 1-22, wherein the first one or more barcoded amplicons correspond to at least 10%, at least 50% or at least 90% of the mRNA of a single cell.

[0065] 24. The method according to any one of items 1-23, wherein the second one or more barcoded amplicons correspond to at most 5%, at most 10% or at most 20% of the mRNA of a single cell.

[0066] 25. A method according to any one of items 1-24, wherein the adaptor is a single-stranded polynucleotide.

[0067] 26. A method according to any one of items 1-24, wherein the adaptor is a double-stranded polynucleotide.

[0068] 27. The method according to any one of items 1-26, wherein the length of the adaptor or the chain of the adaptor is 2-30 nucleotides.

[0069] 28. A method according to any one of items 1-27, wherein the first universal sequence and the second universal sequence are identical.

[0070] 29. A method according to any one of items 1-27, wherein the first universal sequence and the second universal sequence are different.

[0071] 30. The method of any one of items 1-29, wherein each of the more than one amplicons comprises at least a portion of: the first universal sequence, the second universal sequence, or both.

[0072] 31. The method according to any one of items 1-30, wherein (d) adding the sequence of the adaptor to the more than one double-stranded barcoded polynucleotide comprises ligating the adaptor to the more than one double-stranded barcoded polynucleotide.

[0073] 32. A method according to any one of items 1-30, wherein (d) adding the sequence of the adapter to the more than one double-stranded barcoded polynucleotide comprises adding the sequence of the adapter to the more than one double-stranded barcoded polynucleotide by nucleic acid extension or amplification, optionally wherein the amplification comprises a polymerase chain reaction.

[0074] 33. The method according to any one of items 1-32, wherein amplifying the more than one double-stranded barcoded polynucleotide comprises amplifying the more than one double-stranded barcoded polynucleotide by linear amplification, optionally wherein the linear amplification comprises 1-100 cycles of amplification.

[0075] 34. The method according to any one of items 1-33, wherein amplifying the more than one double-stranded barcoded polynucleotide comprises amplifying the more than one double-stranded barcoded polynucleotide by polymerase chain reaction (PCR), optionally wherein the PCR comprises 1-100 cycles of PCR.

[0076] 35. The method according to any one of items 1-34, wherein (f1) performing the extension and / or amplification comprises performing 1-100 cycles of extension and / or amplification, optionally wherein the amplification comprises polymerase chain reaction (PCR) amplification.

[0077] 36. A method according to any one of items 1-35, wherein (f2) performing random priming and extension and / or amplification comprises performing 1-100 cycles of random priming and extension and / or amplification, optionally wherein the amplification comprises polymerase chain reaction (PCR) amplification.

[0078] 37. A method for labeling a nucleic acid target, the method comprising:

[0079] (a) hybridizing more than one nucleic acid target with more than one oligonucleotide;

[0080] (b) extending the more than one oligonucleotides in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence;

[0081] (c) amplifying the more than one first-strand barcoded polynucleotides,

[0082] (c1) amplifying the more than one first strand barcoding polynucleotides using primers capable of hybridizing to the first universal sequence and the TSO or a portion thereof to synthesize first more than one second strand barcoding polynucleotides and produce first more than one double-stranded barcoding polynucleotides, each of the second strand barcoding polynucleotides comprising a sequence of the nucleic acid target, the first universal sequence, the TSO or a portion thereof; and

[0083] (c2) amplifying the more than one first-strand barcoding polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize a second more than one second-strand barcoding polynucleotide to produce a second more than one double-stranded barcoding polynucleotide, each of the second-strand barcoding polynucleotides comprising a sequence of the nucleic acid target or a portion thereof; and

[0084] (d1) amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides using primers that are capable of hybridizing to the first universal sequence and a sequence or a subsequence of the TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the one or more nucleic acid targets and / or a portion thereof.

[0085] 38. The method according to item 37 further includes (d2) amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby producing a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0086] 39. A method for labeling a nucleic acid target, the method comprising:

[0087] (a) hybridizing more than one nucleic acid target with more than one oligonucleotide;

[0088] (b) extending the more than one oligonucleotides in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence;

[0089] (c1) amplifying the more than one first strand barcoding polynucleotides using primers capable of hybridizing to the first universal sequence and the TSO or a portion thereof to synthesize first more than one second strand barcoding polynucleotides and produce first more than one double-stranded barcoding polynucleotides, each of the second strand barcoding polynucleotides comprising a sequence of the nucleic acid target, the first universal sequence, the TSO or a portion thereof; and

[0090] (d) amplifying the first one or more double-stranded barcoded polynucleotides:

[0091] (d1) amplifying the first one or more double-stranded barcoded polynucleotides using primers capable of hybridizing to the first universal sequence and a sequence or subsequence of the TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof, and

[0092] (d2) amplifying the first one or more double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0093] 40. The method according to item 39 further includes (c2) amplifying the more than one first-chain barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize more than one second-chain barcoded polynucleotides to produce more than one second-chain barcoded polynucleotides, each of which comprises the sequence of the nucleic acid target or a portion thereof.

[0094] 41. A method according to any one of items 37-40, wherein each of the more than one oligonucleotide comprises a first universal sequence and a barcode sequence.

[0095] 42. A method according to item 41, wherein the template switching oligonucleotide comprises a second universal sequence.

[0096] 43. A method according to any one of items 37-40, wherein the template switching oligonucleotide comprises a first universal sequence and a barcode sequence.

[0097] 44. A method according to any one of items 37-40, wherein each of the more than one oligonucleotides comprises a second universal sequence.

[0098] 45. A method according to any one of items 37-44, wherein (c2) amplifying the more than one first-chain barcoded polynucleotides includes amplifying the first more than one second-chain barcoded polynucleotides and / or the second more than one second-chain barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets and primers capable of hybridizing to the first universal sequence.

[0099] 46. ​​A method according to any one of items 37-44, wherein (c2) amplifying the more than one first-chain barcoded polynucleotides comprises amplifying the first more than one second-chain barcoded polynucleotides and / or the second more than one second-chain barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets and primers capable of hybridizing to a sequence or subsequence of the TSO.

[0100] 47. The method according to any one of items 37-46, wherein amplifying the more than one first-strand barcoded polynucleotides comprises amplifying the more than one first-strand barcoded polynucleotides for 1-100 cycles.

[0101] 48. The method according to any one of items 37-47, wherein amplifying the more than one first-strand barcoded polynucleotides comprises linearly amplifying the more than one first-strand barcoded polynucleotides.

[0102] 49. The method according to any one of items 37-47, wherein amplifying the more than one first-strand barcoded polynucleotides comprises performing polymerase chain reaction (PCR) amplification on the more than one first-strand barcoded polynucleotides.

[0103] 50. The method of any one of items 37-49, wherein amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides comprises amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides for 1-100 cycles.

[0104] 51. The method according to any one of items 37-50, wherein amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides comprises amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides by linear amplification.

[0105] 52. The method of any one of items 37-50, wherein amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides comprises amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides by polymerase chain reaction (PCR) amplification.

[0106] 53. The method according to any one of items 37-52, comprising dividing the first more than one second-strand barcoded polynucleotides and / or the second more than one second-strand barcoded polynucleotides into two pools, wherein amplifying the first more than one double-stranded barcoded polynucleotides and / or the second more than one double-stranded barcoded polynucleotides is performed using (d1) primers capable of hybridizing to the first universal sequence and the sequence or subsequence of the TSO in one reaction using one of the two pools, and using (d2) primers capable of hybridizing to two or more of the more than one nucleic acid targets in another reaction using the other of the two pools.

[0107] 54. The method according to any one of items 37-53, comprising (e) obtaining sequence information of the first one or more barcoded amplicons and / or the second one or more barcoded amplicons or their products.

[0108] 55. A method according to any one of items 37-54, wherein (b) extending the more than one oligonucleotides to produce the more than one first-strand barcoded polynucleotides includes reverse transcribing one or more of the more than one nucleic acid targets to produce a barcoded single-stranded cDNA molecule.

[0109] 56. A method according to any one of items 37-55, wherein (b) extending the more than one oligonucleotides to produce the more than one first-strand barcoded polynucleotides includes performing nucleic acid extension on oligonucleotides among the more than one oligonucleotides that hybridize to one or more nucleic acid targets of the more than one nucleic acid targets to produce barcoded single-stranded nucleic acid molecules.

[0110] 57. A method according to any one of items 37-56, wherein the reverse transcriptase comprises a viral reverse transcriptase, optionally wherein the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0111] 58. A method according to any one of items 1-57, wherein two or more of the more than one nucleic acid targets include mRNAs of underexpressed genes.

[0112] 59. The method of any one of items 1-58, wherein the total number of occurrences of two or more of the more than one nucleic acid targets comprises an mRNA that is at most 1%, at most 5%, or at most 10% of the mRNA of a single cell.

[0113] 60. A method according to any one of items 1-59, wherein the barcode sequence comprises a molecular marker, a cellular marker, and wherein the oligonucleotide comprises a target-specific region or any combination thereof, and optionally the target-specific region comprises an oligo dT sequence, a random sequence, a target-specific sequence or a combination thereof.

[0114] 61. The method according to any one of items 1-60, wherein the more than one nucleic acid target comprises DNA, mRNA or a combination thereof.

[0115] 62. A method according to any one of items 1-60, wherein the more than one nucleic acid target is mRNA.

[0116] 63. The method according to any one of items 1-62, wherein each of more than one oligonucleotide comprising the first universal sequence and the barcode sequence is immobilized on a solid support, optionally wherein the solid support is a bead.

[0117] 64. The method according to item 63, wherein at least two of the more than one oligonucleotides immobilized on a single solid support comprise different molecular labels.

[0118] 65. A method according to any one of items 63-64, wherein the more than one oligonucleotides immobilized on the solid support comprise the same cellular marker.

[0119] 66. A method according to any one of items 1-65, wherein the more than one nucleic acid targets are nucleic acids from a single sample.

[0120] 67. A method according to item 66, wherein the sample is a single cell.

[0121] 68. A method according to item 66, wherein the sample comprises more than one cell.

[0122] 69. A method according to item 67 or 68, wherein the single cell is the following or the more than one cell includes the following: a brain cell, a heart cell, a cancer cell, a circulating tumor cell, an organ cell, an epithelial cell, a metastatic cell, a benign cell, a primary cell, a circulating cell or a combination thereof.

[0123] 70. The method according to any one of items 1-68, wherein hybridizing more than one nucleic acid target with more than one oligonucleotide occurs in partitions.

[0124] 71. A method according to item 70, wherein the partitions are microwells or droplets.

[0125] 72. A method according to item 70 or 71, comprising lysing a single cell to release the more than one nucleic acid target.

[0126] 73. A method according to any one of items 1-72, wherein each of the barcoded amplicons comprises a barcode sequence, and optionally the barcode sequence comprises a molecular marker, a cellular marker, or a combination thereof.

[0127] 74. The method according to item 73 further includes sequencing the barcoded amplicons to generate more than one sequencing read comprising molecular markers, cellular markers or any combination thereof.

[0128] 75. The method according to item 74 further comprises analyzing the more than one sequencing reads using the cell marker.

[0129] 76. The method according to item 74 or 75 further comprises analyzing the more than one sequencing reads using the molecular markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 Non-limiting exemplary bar codes are illustrated.

[0132] Figure 2 A non-limiting exemplary workflow for barcoding and digital counting is shown.

[0133] Figure 3 is a schematic representation showing a non-limiting exemplary method for generating an indexed library of targets barcoded at the 3 '-end from more than one target.

[0134] Figure 4 is a schematic representation of a non-limiting exemplary workflow for performing whole transcriptome analysis (WTA).

[0135] Figure 5 is a schematic representation of a non-limiting exemplary workflow for determining the expression profile of a set of target genes using a set of target-specific multiplex primers.

[0136] Figure 6 is a schematic representation of a non-limiting exemplary workflow for hybrid cDNA library preparation followed by non-hybrid sequencing library preparation.

[0137] Figure 7 is a schematic representation of a non-limiting exemplary workflow for performing a non-hybrid cDNA library preparation followed by a hybrid sequencing library preparation.

[0138] Figure 8 is a schematic representation of a non-limiting exemplary workflow of an alternative single-cell RNA sequencing (scRNA-seq) embodiment including methods of hybrid cDNA library preparation and / or hybrid sequencing library preparation as disclosed herein.

[0139] Fig. 9 is a schematic representation of a non-limiting exemplary workflow for hybrid cDNA library preparation followed by hybrid sequencing library preparation.

[0140] Fig.10 is a schematic representation of a non-limiting exemplary workflow for performing non-hybrid cDNA library preparation followed by separation of amplicons into pools for separate non-hybrid sequencing library preparation and hybrid sequencing library preparation. Detailed Description

[0142] In the following detailed description, reference is made to the accompanying drawings which form a part of this document. In the accompanying drawings, similar symbols generally identify similar parts unless the context indicates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the accompanying drawings can be arranged, replaced, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein and form a part of the present disclosure.

[0143] All patents, published patent applications, other publications, and sequences from GenBank, and other databases mentioned herein are incorporated by reference in their entirety with respect to the relevant art.

[0144] Quantification of small amounts of nucleic acids, such as messenger ribonucleotide (mRNA) molecules, is clinically important for determining genes expressed in cells, for example, at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules, such as mRNA molecules, is also very challenging, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is the digital polymerase chain reaction (PCR). Ideally, PCR produces identical copies of molecules in each cycle. However, PCR may have the disadvantage that each molecule is replicated with a random probability, and this probability varies depending on the PCR cycle and the gene sequence, which leads to amplification bias and inaccurate gene expression measurements. Random barcodes with unique molecular markers, also known as molecular indices (MIs), can be used to count the number of molecules and correct for amplification bias. Methods such as Precise TM Assay (Cellular Research, Inc. (Palo Alto, CA)) and Rhapsody TM Random barcoding of the assay (Becton, Dickinson and Company (Franklin Lakes, NJ)) can correct for bias introduced by PCR and library preparation steps by labeling mRNA during reverse transcription (RT) using molecular markers (ML).

[0145] Precise TM The assay can utilize a non-depleting pool of random barcodes with a large number (e.g., 6561 to 65536) of unique molecular marker sequences on poly (T) oligonucleotides to hybridize with all poly (A)-mRNAs in the sample during the RT step. The random barcode can include a universal PCR priming site. During RT, the target gene molecule reacts randomly with the random barcode. Each target molecule can hybridize with the random barcode to generate randomly barcoded complementary ribonucleotide (cDNA) molecules. After labeling, the randomly barcoded cDNA molecules from the microwells of the microplate can be pooled into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to generate the number of reads, the number of random barcodes with unique molecular marker sequences, and the number of mRNA molecules.

[0146] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide, each oligonucleotide comprising a first universal sequence and a barcode sequence; extending more than one oligonucleotide hybridized to more than one nucleic acid target to produce more than one first-strand barcoded polynucleotide; and using more than one first-strand barcoded polynucleotide as a template to synthesize more than one second-strand barcoded polynucleotide to produce more than one double-stranded barcoded polynucleotide. The method may include adding the sequence of an adapter (e.g., connecting the adapter) to more than one double-stranded barcoded polynucleotide, wherein the adapter comprises a second universal sequence. The method may include amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and a second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of a nucleic acid target, the first universal sequence, the second universal sequence, a complementary sequence thereof, and / or a portion thereof, and amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0147] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide, each oligonucleotide comprising a first universal sequence and a barcode sequence; extending more than one oligonucleotide hybridized to more than one nucleic acid target to produce more than one first-strand barcoded polynucleotide; and using more than one first-strand barcoded polynucleotide as a template to synthesize more than one second-strand barcoded polynucleotide to produce more than one double-stranded barcoded polynucleotide. The method may include adding the sequence of an adapter (e.g., connecting the adapter) to more than one double-stranded barcoded polynucleotide, wherein the adapter comprises a second universal sequence. The method may include amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of a nucleic acid target, a first universal sequence, a second universal sequence, a complement thereof, and / or a portion thereof. The method may include extension and / or amplification using a target-specific primer and a first one or more barcoded amplicons or products thereof as a template.

[0148] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide; and extending the more than one oligonucleotide in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotide and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. The method may include amplifying more than one first strand barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and TSO or a portion thereof to synthesize a first more than one second strand barcoded polynucleotide and produce a first more than one double strand barcoded polynucleotide, each second strand barcoded polynucleotide comprising a sequence of a nucleic acid target, a first universal sequence, TSO or a portion thereof; and amplifying more than one first strand barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize a second more than one second strand barcoded polynucleotide to produce a second more than one double strand barcoded polynucleotide, each second strand barcoded polynucleotide comprising a sequence of a nucleic acid target or a portion thereof. The method may include amplifying the first more than one double strand barcoded polynucleotide and / or the second more than one double strand barcoded polynucleotide using primers capable of hybridizing to a sequence or subsequence of the first universal sequence and TSO, thereby producing a first more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0149] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide; and extending the more than one oligonucleotide in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to produce more than one first-strand barcoded polynucleotides comprising TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. The method may include amplifying more than one first-strand barcoded polynucleotides using primers capable of hybridizing with the first universal sequence and TSO or a portion thereof to synthesize a first more than one second-strand barcoded polynucleotide and produce a first more than one double-stranded barcoded polynucleotide, each second-strand barcoded polynucleotide comprising a sequence of a nucleic acid target, a first universal sequence, TSO or a portion thereof. The method may include amplifying a first one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and a sequence or subsequence of a TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof, and amplifying the first one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0150] definition

[0151] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of the present disclosure, the following terms are defined below.

[0152] As used herein, the term "adapter" may mean a sequence that promotes the amplification or sequencing of an associated nucleic acid. The associated nucleic acid may include a target nucleic acid. The associated nucleic acid may include one or more of the following: a spatial marker, a target marker, a sample marker, an indexing label, or a barcode sequence (e.g., a molecular marker). The adaptor may be linear. The adaptor may be a pre-adenylated adaptor. The adaptor may be double-stranded or single-stranded. One or more adaptors may be located at the 5' or 3' end of the nucleic acid. When the adaptor includes a known sequence at the 5' and 3' ends, the known sequence may be the same or different sequences. The adaptor located at the 5' and / or 3' ends of the polynucleotide may hybridize with one or more oligonucleotides fixed on the surface. In some embodiments, the adaptor may include a universal sequence. The universal sequence may be a region of a nucleotide sequence common to two or more nucleic acid molecules. Two or more nucleic acid molecules may also have regions of different sequences. Therefore, for example, the 5' adaptor may include the same and / or universal nucleic acid sequence, and the 3' adaptor may include the same and / or universal sequence. Universal sequences that can be present in different members of more than one nucleic acid molecule can allow the use of a single universal primer complementary to the universal sequence to replicate or amplify multiple different sequences. Similarly, at least one, two (e.g., a pair) or more universal sequences that can be present in different members of a set of nucleic acid molecules can allow the use of at least one, two (e.g., a pair) or more single universal primers complementary to the universal sequence to replicate or amplify multiple different sequences. Therefore, universal primers include sequences that can hybridize with such universal sequences. Molecules with target nucleic acid sequences can be modified to attach universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of different target nucleic acid sequences. One or more universal primers attached to target nucleic acid can provide sites for the hybridization of universal primers. One or more universal primers attached to target nucleic acid can be identical or different from each other.

[0153] As used herein, the term "associated" or "associated with" may mean that two or more species can be identified as being co-localized at a certain point in time. Association may mean that two or more species are or have been in similar containers. Association may be an informatics association. For example, digital information about two or more species may be stored and may be used to determine that one or more of the species are co-localized at a certain point in time. Association may also be a physical association. In some embodiments, two or more associated species are "connected," "attached," or "fixed" to each other or to a common solid or semi-solid surface. Association may refer to a covalent or non-covalent means for attaching a marker to a solid or semi-solid support, such as a bead. Association may be a covalent bond between a target and a marker. Association may include hybridization between two molecules, such as a target molecule and a marker.

[0154] As used herein, the term "complementary" can refer to the ability of accurate pairing between two nucleotides. For example, if the nucleotides at a given position of a nucleic acid can be hydrogen bonded with the nucleotides of another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules can be "partial", wherein only some of the nucleotides in the nucleotides are combined, or when there is complete complementarity between the single-stranded molecules, this complementarity can be complete. If the first nucleotide sequence is complementary to the second nucleotide sequence, the first nucleotide sequence can be called the "complement" of the second sequence. If the first nucleotide sequence is complementary to the sequence opposite to the second sequence (that is, the nucleotide order is opposite), the first nucleotide sequence can be called the "reverse complement" of the second sequence. As used herein, a "complementary" sequence can refer to the "complement" or "reverse complement" of a sequence. It can be understood from the present disclosure that if a molecule can hybridize with another molecule, it can be complementary or partially complementary to the molecule it hybridizes with.

[0155] 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 markers that have been associated with the target in the sample. This method, which may be stochastic in nature, converts the problem of counting molecules from one of locating and identifying identical molecules to a series of yes / no digital questions about detecting a set of predefined markers.

[0156] As used herein, the term "label" or labels can refer to a nucleic acid encoding associated with a target in a sample. The label can be, for example, a nucleic acid label. The label can be a fully or partially amplifiable label. The label can be a fully or partially sequenceable label. The label can be a part of a natural nucleic acid that can be identified as being different. The label can be a known sequence. The label can include a junction region of a nucleic acid sequence, such as a junction region of a natural sequence and a non-natural sequence. As used herein, the term "label" can be used interchangeably with the term "index", "label" or "label-label". Labels can convey information. For example, in various embodiments, labels can be used to determine the identity of a sample, the source of a sample, the identity of a cell, and / or a target.

[0157] As used herein, the term "non-depleting reservoir" may refer to a pool of barcodes (e.g., random barcodes) composed of many different markers. A non-depleting reservoir may include a large number of different barcodes such that when the non-depleting reservoir is associated with a target pool, each target may be associated with a unique barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random selection and depends on the number of copies of the same target molecule in the set compared to the diverse markers. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and then analysis of the number of detected barcodes allows calculation of the number of target molecules present in the original set or sample. When the ratio of the number of copies of the target molecules present to the number of unique barcodes is low, the labeled target molecules are highly unique (i.e., the probability that more than one target molecule is labeled by a given marker is very low).

[0158] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. Nucleic acid may include nucleotides. Nucleic acid may be exogenous or endogenous to a cell. Nucleic acid may be present in a cell-free environment. Nucleic acid may be a gene or a fragment thereof. Nucleic acid may be DNA. Nucleic acid may be RNA. Nucleic acid may include one or more analogs (e.g., altered backbones, sugars, or nucleobases). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xenonucleic acid, morpholinos, locked nucleic acids, diol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" are used interchangeably.

[0159] Nucleic acid can comprise one or more modifications (e.g., base modification, backbone modification) to provide new or enhanced features (e.g., improved stability) to nucleic acid. Nucleic acid can comprise nucleic acid affinity tags. Nucleoside can be a base-sugar combination. The base portion of a nucleoside can be a heterocyclic base. The two most common categories of such heterocyclic bases are purine and pyrimidine. Nucleotide can be a nucleoside further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides including furanose, the phosphate group can be linked to the 2', 3' or 5' hydroxyl portion of the sugar. In forming nucleic acid, the phosphate group can covalently link adjacent nucleosides to each other to form a linear polymer compound. Then the respective ends of this linear polymer compound can be further joined to form a cyclic compound; however, linear compounds are generally suitable. In addition, linear compounds can have internal nucleotide base complementarity, and therefore can be folded in a manner that produces a completely or partially double-stranded compound. In nucleic acid, the phosphate group can generally be referred to as the internucleoside backbone forming the nucleic acid. The connection or backbone can be a 3' to 5' phosphodiester connection.

[0160] Nucleic acid can comprise modified backbone and / or modified internucleoside connection.Modified backbone can comprise those backbones that retain phosphorus atom in backbone and those backbones that do not have phosphorus atom in backbone.Wherein contain suitable modified nucleic acid backbone of phosphorus atom and can comprise, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphates, such as 3'-alkylene phosphate, 5'-alkylene phosphate, chiral phosphate, phosphite, phosphoramide comprising 3'-aminophosphoramide and aminoalkylphosphoramide, phosphorodiamidate, thiophosphoramide, alkylthiophosphate, alkylthiophosphotriester, selenophosphate and boric acid phosphate (boranophosphates) with normal 3'-5' connection, analogs of 2'-5' connection, and those with reversed polarity, wherein one or more internucleotide connection is 3' to 3', 5' to 5' or 2' to 2' connection.

[0161] Nucleic acids can comprise polynucleotide backbones formed by: short-chain alkyl or cycloalkyl nucleoside interlinkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside interlinkages, or one or more short-chain heteroatomic or heterocyclic nucleoside interlinkages. These can include those having: morpholino linkages (partially formed from the sugar moiety of nucleosides); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; backbones containing olefins; sulfamic acid salt backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and those having mixed N, O, S and CH 2 Other substances that make up the components.

[0162] Nucleic acid can include nucleic acid mimics.Term " mimics " can be intended to include polynucleotides in which only furanose ring or furanose ring and internucleotide connection are replaced by non-furanose groups, and only the replacement of furanose ring can also be called sugar substitute.Heterocyclic base part or modified heterocyclic base part can be kept to hybridize with appropriate target nucleic acid.A kind of such nucleic acid can be peptide nucleic acid (PNA).In PNA, the sugar backbone of polynucleotide can be replaced by backbone containing amide, particularly aminoethylglycine backbone.Nucleotide can be retained and directly or indirectly bonded to the nitrogen-nitrogen atom of the amide part of backbone.The backbone in PNA compound can include two or more aminoethylglycine units connected, and this provides the backbone containing amide for PNA.Heterocyclic base part can be directly or indirectly bonded to the nitrogen-nitrogen atom of the amide part of backbone.

[0163] The nucleic acid can include a morpholino backbone structure. For example, the nucleic acid can include a 6-membered morpholino ring replacing a ribose ring. In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside connections can replace phosphodiester connections.

[0164] Nucleic acid can include morpholino units (e.g., morpholino nucleic acids) having a connection to a heterocyclic base attached to a morpholino ring. A linking group can connect morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds can have less undesirable interactions with cell proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds in the morpholino category can be connected using different linking groups. Another class of polynucleotide mimics can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose rings commonly present in nucleic acid molecules can be replaced by cyclohexenyl rings. Phosphoramidite monomers protected by CeNA DMT can be prepared using phosphoramidite chemistry and used for oligomeric compound synthesis. Incorporating CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylic acid can form a complex with a nucleic acid complement having a stability similar to that of a natural complex. Additional modifications can include locked nucleic acids (LNA), in which a 2'-hydroxyl group is connected to the 4' carbon atom of a sugar ring to form a 2'-C, 4'-C-oxymethylene connection to form a bicyclic sugar moiety. The linker can be a methylene (-CH 2 ), a group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNA and LNA analogs can show very high duplex thermal stability (Tm = +3°C to +10°C) with complementary nucleic acids, stability to 3'-exonuclease degradation, and good solubility properties.

[0165] Nucleic acids may also include modifications or substitutions of nucleobases (often referred to simply as "bases"). As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)), and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases may include other synthetic as well as natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine, and other pyrimidine bases. Alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido (5,4-b) (1,4) benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido (5,4-b) (1,4) benzothiazin-2 (3H) -one), G-clamps such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido (5,4-(b) (1,4) benzoxazin-2 (3H) -one), phenoxazine cytidine (1H-pyrimido (5,4-b) (1,4) benzothiazin-2 (3H) -one), Thiazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0166] As used herein, the term "sample" may refer to a composition comprising a target. Suitable samples for analysis by the methods, devices and systems disclosed herein include cells, tissues, organs or organisms.

[0167] As used herein, the term "sampling device" or "device" may refer to a device that can take a portion of a sample and / or place the portion on a substrate. The sampling device may refer to, for example, a fluorescence activated cell sorter (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a cascade, and / or an ultramicrotome.

[0168] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which more than one barcode (e.g., a random barcode) may be attached. A solid support may include any type of solid, porous or hollow sphere, ball, socket, cylinder or other similar shape, composed of plastic, ceramic, metal or polymeric material (e.g., hydrogel), on which nucleic acids may be fixed (e.g., covalently or non-covalently). A solid support may include discrete particles that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as a cube, a cuboid, a cone, a cylinder, a cone, an ellipse or a disc, etc. The shape of the bead may be non-spherical. More than one solid support spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead".

[0169] As used herein, the term "random barcode" may refer to a polynucleotide sequence comprising a tag of the present disclosure. A random barcode may be a polynucleotide sequence that can be used for random barcoding. A random barcode may be used to quantify a target in a sample. A random barcode may be used to control errors that may occur after a tag is associated with a target. For example, a random barcode may be used to assess amplification or sequencing errors. A random barcode associated with a target may be referred to as a random barcode-target or a random barcode-tag-target.

[0170] As used herein, the term "gene-specific random barcode" may refer to a polynucleotide sequence comprising a marker and a gene-specific target binding region. A random barcode may be a polynucleotide sequence that can be used for random barcoding. A random barcode may be used to quantify a target in a sample. A random barcode may be used to control errors that may occur after a marker is associated with a target. For example, a random barcode may be used to assess amplification or sequencing errors. A random barcode associated with a target may be referred to as a random barcode-target or a random barcode-tag-target.

[0171] As used herein, the term "random barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Random barcoding can utilize a recursive Poisson strategy to associate and quantify labels associated with a target. As used herein, the term "random barcoding" can be used interchangeably with "random labeling."

[0172] As used herein, the term "target" may refer to a composition that can be associated with a barcode (e.g., a random barcode). Exemplary suitable targets for analysis by the methods, devices, and systems disclosed herein include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, etc. The target may be single-stranded or double-stranded. In some embodiments, the target may be a protein, peptide, or polypeptide. In some embodiments, the target is a lipid. As used herein, "target" may be used interchangeably with "species."

[0173] As used herein, the term "reverse transcriptase" may refer to a group of enzymes with reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Typically, such enzymes include but are not limited to retroviral reverse transcriptases, retrotransposon reverse transcriptases, reverse transcriptases of plasmids, reverse transcriptases of retroviruses, bacterial reverse transcriptases, reverse transcriptases of group II introns, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, reverse transcriptases of plasmids, reverse transcriptases of retroviruses, and group II intron reverse transcriptases. Examples of type 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 can include many types of non-retroviral reverse transcriptases (i.e., retroviral, type II intron, and diversity-generating reverse transcriptase elements, etc.).

[0174] The terms "universal adapter primer," "universal primer adapter," or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can be used to hybridize with a barcode (e.g., a random barcode) to generate a gene-specific barcode. The universal adapter sequence, for example, can be a known sequence that is common to all barcodes used in the methods of the present disclosure. For example, when multiple targets are labeled using the methods disclosed herein, each target-specific sequence can be connected to the same universal adapter sequence. In some embodiments, more than one universal adapter sequence can be used in the methods disclosed herein. For example, when multiple targets are labeled using the methods disclosed herein, at least two target-specific sequences are connected to different universal adapter sequences. The universal adapter primer and its complement can be included in two oligonucleotides, one of which contains a target-specific sequence and the other contains a barcode. For example, the universal adapter sequence can be a portion of an oligonucleotide containing a target-specific sequence to generate a nucleotide sequence complementary to a target nucleic acid. A second oligonucleotide containing a barcode and a complement of the universal adapter sequence can hybridize with the nucleotide sequence and generate a target-specific barcode (e.g., a target-specific random barcode). In some embodiments, the universal adapter primer has a different sequence than the universal PCR primer used in the methods of the disclosure.

[0175] Barcode

[0176] Barcoding, such as random barcoding, has been described in, for example, Fu et al., Proc Natl Acad Sci USA, 2011 May 31, 108(22):9026-31; U.S. Patent Application Publication No. US2011 / 0160078; Fan et al., Science, 2015 February 6, 347(6222):1258367; U.S. Patent Application Publication No. US2015 / 0299784 and PCT Application Publication No. WO2015 / 031691; the contents of each of these, including any supporting or supplementary information or materials, are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein can be random barcodes, which can be polynucleotide sequences that can be used to randomly label (e.g., barcodes, tags) a target. A barcode may be referred to as a random barcode if the ratio of the number of different barcode sequences in the random barcode to the number of occurrences of any target to be labeled is, or is about, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target may be an mRNA species that includes mRNA molecules having identical or nearly identical sequences. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences in a stochastic barcode to the number of occurrences of any target 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 sequences of a stochastic barcode can be referred to as molecular markers.

[0177] A barcode, such as a random barcode, can include one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. Figure 1An exemplary barcode 104 with spatial tags is shown. The barcode 104 can include a 5' amine that can link the barcode to the solid support 105. The barcode can include universal tags, dimensional tags, spatial tags, cell tags, and / or molecular tags. The order of the different tags (including but not limited to universal tags, dimensional tags, spatial tags, cell tags, and molecular tags) in the barcode can be changed. For example, Figure 1 As shown in , the universal label can be the label on the most 5' side (5'-most label), and the molecular label can be the label on the most 3' side (3'-most label). Spatial labels, dimensional labels and cell labels can be in any order. In some embodiments, universal labels, spatial labels, dimensional labels, cell labels and molecular labels are in any order. The barcode can include a target binding region. The target binding region can interact with the target (e.g., target nucleic acid, RNA, mRNA, DNA) in the sample. For example, the target binding region can include an oligo (dT) sequence that can interact with the poly (A) tail of mRNA. In some cases, the label of the barcode (e.g., universal label, dimensional label, spatial label, cell label and barcode sequence) can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides.

[0178] Mark, for example, cell marker, can include a set of unique defined length of nucleic acid subsequences, for example, each has seven nucleotides (equivalent to the number used in some Hamming error correction codes), which can be designed to provide error correction capabilities. The error correction subsequence group comprising seven nucleotide sequences can be designed so that any paired combination of the sequences in the group exhibits a defined "genetic distance" (or number of mismatched bases), for example, a set of error correction subsequences can be designed to exhibit a genetic distance of three nucleotides. In this case, the examination of the error correction sequence in the sequence data set of the target nucleic acid molecule through labeling (described more fully below) can allow detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequence for generating error correction codes can vary, for example, their length can be as follows, or about as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or the nucleotides of the number or range between any two values ​​in these values. In some embodiments, nucleic acid subsequences of other lengths can be used to generate error correction codes.

[0179] The barcode may include a target binding region. The target binding region may interact with a target in a sample. The target may be, or include, ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each containing a poly (A) tail, or any combination thereof. In some embodiments, more than one target may include deoxyribonucleic acid (DNA).

[0180] In some embodiments, the target binding region may include an oligo (dT) sequence that can interact with the poly (A) tail of the mRNA. One or more of the markers of the barcode (e.g., universal markers, dimensional markers, spatial markers, cell markers, and barcode sequences (e.g., molecular markers)) may be separated from another or two of the remaining markers of the barcode by a spacer sequence. The spacer sequence may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides. In some embodiments, the markers of the barcode are not separated by a spacer sequence.

[0181] General Tags

[0182] The barcode may include one or more universal markers. In some embodiments, for all barcodes in the barcode group (attached to a given solid support), one or more universal markers may be the same. In some embodiments, for all barcodes attached to more than one bead, one or more universal markers may be the same. In some embodiments, the universal marker may include a nucleic acid sequence that can hybridize with a sequencing primer. A sequencing primer may be used to sequence the barcode comprising the universal marker. A sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer related to a high-throughput sequencing platform. In some embodiments, the universal marker may include a nucleic acid sequence that can hybridize with a PCR primer. In some embodiments, the universal marker may include a nucleic acid sequence that can hybridize with a sequencing primer and a PCR primer. The nucleic acid sequence of the universal marker that can hybridize with a sequencing or PCR primer may be referred to as a primer binding site. The universal marker may include a sequence that can be used to initiate transcription of the barcode. The universal marker may include a sequence that can be used to extend the region within the barcode or barcode. The length of the universal tag can be, or about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. For example, the universal tag can include at least about 10 nucleotides. The length of the universal tag can be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal tag sequence to enable the barcode to be cleaved from the support.

[0183] Dimension tagging

[0184] The barcode may include one or more dimensional markers. In some embodiments, the dimensional marker may include a nucleic acid sequence that provides information about the dimension in which the marker (e.g., random marker) occurs. For example, the dimensional marker may provide information about the time when the target is barcoded. The dimensional marker may be associated with the time of barcoding (e.g., random barcoding) in the sample. The dimensional marker may be activated at the time of the marker. Different dimensional markers may be activated at different times. The dimensional marker provides information about the order in which the target, the target group, and / or the sample are barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. In the G1 phase of the cell cycle, the cell may be pulsed again with a barcode (e.g., a random barcode). In the S phase of the cell cycle, the cell may be pulsed again with a barcode, and so on. The barcode at each pulse (e.g., each phase of the cell cycle) may include different dimensional markers. In this way, the dimensional marker provides information about which targets are marked in which phase of the cell cycle. Dimensional markers may interrogate many different biological times. Exemplary biological events may include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., cell, cell colony) may be labeled before and / or after treatment with a drug and / or therapy. Changes in the copy number of different targets may indicate the response of the sample to the drug and / or therapy.

[0185] Dimensional markers can be activatable. Activatable dimensional markers can be activated at a specific time point. Activatable markers can be, for example, constitutively activated (for example, not closed). The activatable dimensional markers can be, for example, reversibly activated (for example, the activatable dimensional markers can be turned on and off). Dimensional markers can be, for example, reversibly activated at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times. Dimensional markers can be reversibly activated, for example, at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times. In some embodiments, the dimensional markers can be activated by fluorescence, light, chemical events (for example, cleavage, connection of another molecule, modified addition (for example, pegylation, sumoylation (sumoylate), acetylation, methylation, deacetylation, demethylation), photochemical events (for example, light locking (photocaging)) and the introduction of non-natural nucleotides.

[0186] In some embodiments, the dimensional mark can be the same for all bar codes (e.g., random bar codes) attached to a given solid support (e.g., beads), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100% of the bar codes on the same solid support can contain the same dimensional mark. In some embodiments, at least 60% of the bar codes on the same solid support can contain the same dimensional mark. In some embodiments, at least 95% of the bar codes on the same solid support can contain the same dimensional mark.

[0187] More than one solid support (e.g., beads) may represent up to 10 6 The length of the dimension tag can be as follows, or about as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The length of the dimension tag can be at least as follows, or at most as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. The dimension tag can include about 5 to about 200 nucleotides. The dimension tag can include about 10 to about 150 nucleotides. The dimension tag can include a length of about 20 to about 125 nucleotides.

[0188] Space Marking

[0189] The barcode may include one or more spatial markers. In some embodiments, the spatial marker may include a nucleic acid sequence that provides information about the spatial orientation of a target molecule associated with the barcode. The spatial marker may be associated with a coordinate in a sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed with reference to a substrate. The spatial marker may be referenced to a two-dimensional or three-dimensional grid. The coordinate may be fixed with reference to a landmark. Landmarks may be identifiable in space. Landmarks may be structures that may be imaged. Landmarks may be biological structures, such as anatomical landmarks. Landmarks may be cell landmarks, such as organelles. Landmarks may be non-natural landmarks, such as structures with identifiable markers (such as color coding, barcodes, magnetism, fluorescence, radioactivity, or unique size or shape). Spatial markers may be associated with physical partitions (e.g., holes, containers, or droplets). In some embodiments, more than one spatial marker may be used together to encode one or more positions in space.

[0190] The spatial label can be the same for all barcodes attached to a given solid support (e.g., beads), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support can be the following, or about the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support can be at least the following, or at most the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. In some embodiments, at least 60% of the barcodes on the same solid support can contain the same spatial label. In some embodiments, at least 95% of the barcodes on the same solid support can contain the same spatial label.

[0191] More than one solid support (e.g., beads) may represent up to 10 6 The length of the spatial marker can be as follows, or about as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or the nucleotides of the number or range between any two values ​​in these values. The length of the spatial marker can be at least as follows, or at most as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. The spatial marker can comprise about 5 to about 200 nucleotides. The spatial marker can comprise about 10 to about 150 nucleotides. The spatial marker can comprise a length of about 20 to about 125 nucleotides.

[0192] Cell labeling

[0193] The barcode (e.g., random barcode) may include one or more cell markers. In some embodiments, the cell marker may include a nucleic acid sequence that provides information for determining which target nucleic acid is from which cell. In some embodiments, the cell marker is the same for all barcodes attached to a given solid support (e.g., beads), but is different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support may be as follows, or about as follows: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support may be as follows, or about as follows: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. For example, at least 60% of the barcodes on the same solid support may contain the same cell marker. As another example, at least 95% of the barcodes on the same solid support can comprise the same cellular marker.

[0194] More than one solid support (e.g., beads) may represent up to 10 6 The length of the cell marker can be as follows, or about as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The length of the cell marker can be at least as follows, or at most as follows: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. For example, the cell marker can include about 5 to about 200 nucleotides. As another example, the cell marker can include about 10 to about 150 nucleotides. As another example, the cell marker can include a length of about 20 to about 125 nucleotides.

[0195] Barcode sequence

[0196] The barcode can include one or more barcode sequences. In some embodiments, the barcode sequence can include a nucleic acid sequence that provides identification information of a specific type of target nucleic acid species that hybridizes to the barcode. The barcode sequence can include a nucleic acid sequence that provides a count of a specific occurrence (e.g., provides a rough approximation) of a target nucleic acid species that hybridizes to the barcode (e.g., target binding region).

[0197] In some embodiments, a set of different barcode sequences are attached to a given solid support (e.g., a bead). In some embodiments, there may be the following, or about the following unique molecular marker sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 , or a number or range between any two of these values. For example, more than one barcode may include about 6561 barcode sequences with different sequences. As another example, more than one barcode may include about 65536 barcode sequences with different sequences. In some embodiments, there may be at least the following, or at most the following unique barcode sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Species or 10 9 The unique molecular marker sequence can be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular marker sequence is partially or completely surrounded by particles (e.g., hydrogel beads).

[0198] In different embodiments, the length of the barcode can be different. For example, the length of the barcode can be, or about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. As another example, the length of the barcode can be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0199] Molecular markers

[0200] The barcode (e.g., a random barcode) can include one or more molecular markers. The molecular marker can include a barcode sequence. In some embodiments, the molecular marker can include a nucleic acid sequence that provides identification information for a specific type of target nucleic acid species that hybridizes to the barcode. The molecular marker can include a nucleic acid sequence that provides a count of a specific occurrence of a target nucleic acid species that hybridizes to the barcode (e.g., a target binding region).

[0201] In some embodiments, a set of different molecular markers are attached to a given solid support (e.g., a bead). In some embodiments, there may be the following, or about the following unique molecular marker sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 , or a number or range between any two of these values. For example, more than one barcode may include about 6561 molecular markers with different sequences. As another example, more than one barcode may include about 65536 molecular markers with different sequences. In some embodiments, there may be at least the following, or at most the following unique molecular marker sequences: 10 2 10 3 10 4 10 5 10 6 10 7 10 8 Species or 10 9 A barcode having a unique molecular marker sequence can be attached to a given solid support (e.g., a bead).

[0202] For barcoding using more than one stochastic barcode (e.g., stochastic barcoding), the ratio of the number of different molecular marker sequences to the number of occurrences of any target can be, or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target can be an mRNA species that includes mRNA molecules having identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular marker sequences to the number of occurrences of any target 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.

[0203] The length of a molecular marker can be, or is about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The length of a molecular marker can be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0204] Target binding region

[0205] The barcode may include one or more target binding regions, such as capture probes. In some embodiments, the target binding region may be hybridized 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, such as a cell nucleic acid to be analyzed), for example, with a specific gene sequence. In some embodiments, the target binding region may include a nucleic acid sequence that can be attached (e.g., hybridized) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region may include a nucleic acid sequence that can specifically hybridize with a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode may then be connected to any nucleic acid molecule comprising a sequence complementary to a restriction site overhang.

[0206] 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 a variety of target nucleic acids without relying on a specific sequence of the target nucleic acid. For example, the target binding region may include a random polymer sequence, a poly (dA) sequence, a poly (dT) sequence, a poly (dG) sequence, a poly (dC) sequence, or a combination thereof. For example, the target binding region may be an oligo (dT) sequence hybridized with a poly (A) tail on an mRNA molecule. A random polymer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or a higher polymer sequence of any length. In some embodiments, the target binding region of all barcodes attached to a given bead is the same. In some embodiments, the target binding region of more than one barcode attached to a given bead may include two or more different target binding sequences. The length of the target binding region can be, or is about, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The length of the target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. For example, the mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to produce a cDNA molecule with a poly (dC) tail. The barcode can include a target binding region with a poly (dG) tail. Upon base pairing between the poly (dG) tail of the barcode and the poly (dC) tail of the cDNA molecule, the reverse transcriptase converts the template strand from the cellular RNA molecule to the barcode and continues to replicate to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains the sequence of a barcode (such as a molecular tag) on ​​the 3' end of the cDNA molecule.

[0207] In some embodiments, the target binding region can include an oligo (dT) that can hybridize to an mRNA comprising a polyadenylated end. The target binding region can be gene specific. For example, the target binding region can be configured to hybridize to a specific region of the target. The length of the target binding region can be, or is about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, or a number or range of nucleotides between any two of these values. The length of the target binding region can be at least, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The length of the target binding region can be about 5-30 nucleotides. When the barcode comprises a gene-specific target binding region, the barcode may be referred to herein as a gene-specific barcode.

[0208] Directional characteristics

[0209] Random barcodes (e.g., random barcodes) may include one or more directional properties that can be used to orient (e.g., align) the barcodes. The barcodes 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 facilitate positioning the barcodes in a known manner. In this way, the directional properties may be used to develop a known map of the barcodes in the sample. Exemplary directional properties may include electrophoretic migration (e.g., based on the size of the barcode), isoelectric point, spin, conductance, and / or self-assembly. For example, a barcode having a directional property of self-assembly may self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) upon activation.

[0210] Affinity characteristics

[0211] Barcodes (e.g., random barcodes) may include one or more affinity properties. For example, spatial labels may include affinity properties. Affinity properties may include chemical and / or biological moieties that may promote binding of a barcode to another entity (e.g., a cell receptor). For example, affinity properties may include antibodies, for example, antibodies that are specific to a particular portion (e.g., a receptor) on a sample. In some embodiments, antibodies may guide barcodes to specific cell types or molecules. Targets at and / or near specific cell types or molecules may be labeled (e.g., randomly labeled). In some embodiments, because antibodies may guide barcodes to specific locations, affinity properties may provide spatial information in addition to the nucleotide sequence of the spatial label. The antibody may be a therapeutic antibody, such as a monoclonal antibody or a polyclonal antibody. The antibody may be humanized or chimeric. The antibody may be a naked antibody or a fusion antibody.

[0212] Antibodies can be full-length (ie, naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) immunoglobulin molecules (eg, IgG antibodies) or immunologically active (ie, specifically binding) portions of immunoglobulin molecules (eg, antibody fragments).

[0213] The antibody fragment can be, for example, a part of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include a separated fragment consisting of the variable region of the antibody, such as a "Fv" fragment consisting of the variable region of the heavy chain and the light chain and a recombinant single-chain polypeptide molecule ("scFv protein") in which the variable region of the light chain and the heavy chain is connected by a peptide linker. Exemplary antibodies can include, but are not limited to, cancer cell antibodies, viral antibodies, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0214] Universal adapter primer

[0215] The barcode can comprise one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific random barcode, can comprise a universal adapter primer. A universal adapter primer can refer to a nucleotide sequence that is universal across all barcodes. A universal adapter primer can be used to construct a gene-specific barcode. The length of the universal adapter primer can be, or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, or a number or range of nucleotides between any two of these values. The length of the universal adapter primer can be at least, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The length of the universal adapter primer can be 5-30 nucleotides.

[0216] Connectors

[0217] When the barcode comprises more than one marker type (e.g., more than one cell marker or more than one barcode sequence, such as a molecular marker), the markers may be interspersed with adapter tag sequences. The length of the adapter tag sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. The length of the adapter tag sequence may be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides. In some cases, the length of the adapter tag sequence is 12 nucleotides. The adapter tag sequence can be used to facilitate the synthesis of the barcode. The adapter tag may include an error correction (e.g., Hamming) code.

[0218] Solid support

[0219] In some embodiments, the barcodes disclosed herein, such as random barcodes, can be associated with a solid support. For example, the solid support can be a synthetic particle. In some embodiments, some or all barcode sequences (such as molecular markers of random barcodes (e.g., first barcode sequences)) of more than one barcode on a solid support have a difference of at least one nucleotide. The cell markers of the barcodes on the same solid support can be the same. The cell markers of the barcodes on different solid supports can have a difference of at least one nucleotide. For example, the first cell marker of the first more than one barcode on the first solid support can have the same sequence, and the second cell marker of the second more than one barcode on the second solid support can have the same sequence. The first cell marker of the first more than one barcode on the first solid support and the second cell marker of the second more than one barcode on the second solid support can have a difference of at least one nucleotide. The cell marker can be, for example, about 5-20 nucleotides long. The barcode sequence can be, for example, about 5-20 nucleotides long. The synthetic particle can be, for example, a bead.

[0220] The beads can be, for example, silica beads, controlled pore glass beads, magnetic beads, Dynabeads, cross-linked dextran / sepharose beads, beaded cellulose, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose, nylon, silicone, or any combination thereof.

[0221] In some embodiments, the beads can be polymeric beads functionalized with barcodes or random barcodes, such as deformable beads or gel beads (such as gel beads from 10X Genomics (San Francisco, CA)). In some embodiments, the gel beads can include polymer-based gels. For example, the gel beads can be produced by encapsulating one or more polymer precursors into droplets. After exposing the polymer precursors to a promoter (e.g., tetramethylethylenediamine (TEMED)), the gel beads can be produced.

[0222] In some embodiments, the particles can be destructible (e.g., soluble, degradable). For example, the polymer beads can dissolve, melt, or degrade, for example, under desired conditions. The desired conditions can include environmental conditions. The desired conditions can cause the polymer beads to dissolve, melt, or degrade in a controlled manner. The gel beads can dissolve, melt, or degrade due to chemical stimulants, physical stimulants, biological stimulants, thermal stimulants, magnetic stimulants, electrical stimulants, light stimulants, or any combination thereof.

[0223] Analytes and / or reagents (such as oligonucleotide barcodes) can, for example, be coupled / fixed to the inner surface of gel beads (e.g., accessible interior by diffusion of oligonucleotide barcodes and / or materials for generating oligonucleotide barcodes), and / or the outer surface of gel beads or any other microcapsule described herein. Coupling / fixation can be by any form of chemical bonding (e.g., covalent bonds, ionic bonds) or physical phenomena (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the coupling / fixation of reagents to gel beads or any other microcapsule described herein can be reversible, such as, for example, by unstable parts (e.g., by chemical crosslinkers, including chemical crosslinkers described herein). After applying a stimulus, the unstable part can be cleaved and release the immobilized reagent. In some embodiments, the unstable part is a disulfide bond. For example, in the case where the oligonucleotide barcode is fixed to the gel beads by a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the beads. The labile moiety can be included as part of a gel bead or microcapsule, as part of a chemical linker that connects a reagent or analyte to a gel bead or microcapsule, and / or as part of a reagent or analyte. In some embodiments, at least one of the more than one barcodes can be immobilized on a particle, partially immobilized on a particle, encapsulated in a particle, partially encapsulated in a particle, or any combination thereof.

[0224] In some embodiments, the gel beads may include a wide variety of different polymers, including but not limited to polymers, thermosensitive polymers, photosensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. The polymers may include but are not limited to materials such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methyl methacrylate) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(o-phthalaldehyde) (PPA), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

[0225] Many chemical stimuli can be used to trigger the destruction, dissolution or degradation of beads. Examples of these chemical changes can include, but are not limited to, pH-mediated changes in the bead wall, disintegration of the bead wall by chemical cleavage of crosslinks, triggered depolymerization of the bead wall, and bead wall switching reactions. Bulk changes can also be used to trigger the destruction of beads.

[0226] The volumetric or physical changes of the microcapsules by various stimuli also provide many advantages in designing the capsules to release agents. Volumetric or physical changes occur on a macroscopic scale, where bead rupture is the result of mechanical-physical forces caused by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in the porosity of the bead wall.

[0227] Biostimulants can also be used to trigger the destruction, dissolution or degradation of the beads. Generally, biotriggers are similar to chemical triggers, but many examples use biomolecules or molecules common in living systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, etc. For example, the beads may include polymers with peptide crosslinks that are sensitive to cleavage by specific proteases. More specifically, one example may include microcapsules containing GFLGK peptide crosslinks. Upon addition of a biotrigger (such as the protease cathepsin B), the peptide crosslinks of the shell wall are cleaved and the contents of the beads are released. In other cases, the protease can be heat activated. In another example, the beads include a shell wall containing cellulose. The addition of chitosan hydrolase serves as a biotrigger for the cleavage of cellulose bonds, the depolymerization of the shell wall, and the release of its internal contents.

[0228] The beads can also be induced to release their contents upon application of a thermal stimulus. Changes in temperature can result in various changes in the beads. Changes in heat can result in melting of the beads, causing the bead wall to disintegrate. In other cases, heat can increase the internal pressure of the internal components of the beads, causing the beads to rupture or explode. In still other cases, heat can cause the beads to transform into a shrunken, dehydrated state. Heat can also act on the thermosensitive polymers within the bead wall, causing the beads to break down.

[0229] Including magnetic nanoparticles in the bead wall of the microcapsule can allow for triggered rupture of the beads and for guiding the beads in an array. The device of the present disclosure may include magnetic beads for any purpose. In one example, Fe 3 O 4 Incorporation of nanoparticles into polyelectrolyte-containing beads triggers the rupture of the beads in the presence of an oscillating magnetic field stimulus.

[0230] The beads may also break, dissolve, or degrade due to electrical stimulation. Similar to the magnetic particles described in the previous section, electrosensitive beads can allow for triggered rupture of the beads as well as other functions such as alignment in an electric field, conductivity, or redox reactions. In one example, beads containing electrosensitive materials align in an electric field so that the release of an internal agent can be controlled. In other examples, the electric field can induce a redox reaction within the bead wall itself, which can increase porosity.

[0231] The beads can also be disrupted using a light stimulus. Many light triggers are possible and can include systems using various molecules such as nanoparticles and chromophores that can absorb photons of a specific wavelength range. For example, metal oxide coatings can be used as capsule triggers. 2 UV irradiation of polyelectrolyte capsules can result in the disintegration of the bead wall. In yet another example, photo-switchable materials, such as azobenzene groups, can be incorporated into the bead wall. Upon application of UV or visible light, chemicals such as these undergo reversible cis-to-trans isomerization upon absorption of photons. In this regard, incorporation of photonswitches results in the bead wall being able to disintegrate or become more porous upon application of a light trigger.

[0232] For example, in Figure 2 In the non-limiting example of barcoding (e.g., random barcoding) illustrated in FIG, after introducing cells (such as single cells) into more than one microwell of a microwell array at block 208, beads can be introduced into more than one microwell of the microwell array at block 212. Each microwell can contain one bead. The beads can contain more than one barcode. The barcode can contain a 5' amine region attached to the bead. The barcode can contain a universal tag, a barcode sequence (e.g., a molecular tag), a target binding region, or any combination thereof.

[0233] The barcodes disclosed herein can be associated (e.g., attached) with a solid support (e.g., a bead). The barcodes associated with the solid support can each include a barcode sequence selected from the group including: at least 100 or 1000 barcode sequences with a unique sequence. In some embodiments, different barcodes associated with the solid support can include barcodes with different sequences. In some embodiments, a certain percentage of the barcodes associated with the solid support include the same cell marker. For example, the percentage can be the following, or about the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. As another example, the percentage can be at least the following, or at most the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. In some embodiments, the barcodes associated with the solid support can have the same cell marker. The barcodes associated with different solid supports may have different cell markers selected from the group consisting of at least 100 or 1000 cell markers having unique sequences.

[0234] The barcode disclosed herein can be associated (e.g., attached) with a solid support (e.g., a bead). In some embodiments, more than one target in a sample can be barcoded with a solid support including more than one synthetic particle associated with more than one barcode. In some embodiments, a solid support may include more than one synthetic particle associated with more than one barcode. The spatial marking of more than one barcode on different solid supports may have a difference of at least one nucleotide. The solid support may, for example, include more than one barcode in two or three dimensions. The synthetic particles may be beads. The beads may be silica beads, controlled pore glass beads, magnetic beads, Dynabeads, cross-linked dextran / agarose gel beads, beaded cellulose, polystyrene beads, or any combination thereof. The solid support may include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support may be free floating. In some embodiments, the solid support may be embedded in a semi-solid or solid array. The barcode may not be associated with the solid support. The barcode may be a single nucleotide. A barcode may be associated with the substrate.

[0235] As used herein, the terms "tethered," "attached," and "fixed" can be used interchangeably and can refer to covalent or non-covalent means for attaching a barcode to a solid support. Any of a variety of different solid supports can be used as a solid support for attaching pre-synthesized barcodes or for in situ solid phase synthesis of barcodes.

[0236] In some embodiments, the solid support is a bead. The bead may include one or more types of solid, porous or hollow spheres, balls, sockets, cylinders or other similar shapes, on which nucleic acids can be fixed (e.g., covalently or non-covalently). The bead may be, for example, made of plastic, ceramic, metal, polymeric material, or any combination thereof. The bead may be, or include, spherical (e.g., microspheres) or discrete particles having a non-spherical or irregular shape, such as a cube, a cuboid, a cone, a cylinder, a cone, an ellipse or a disc, etc. In some embodiments, the shape of the bead may be non-spherical.

[0237] The beads may include a variety of materials, including but not limited to paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe 3 O 4 ; 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, cross-linked agarose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof.

[0238] In some embodiments, the beads (e.g., beads to which labels are attached) are hydrogel beads. In some embodiments, the beads include a hydrogel.

[0239] Some embodiments disclosed herein include one or more particles (e.g., beads). Each of the particles may include more than one oligonucleotide (e.g., barcode). Each of more than one oligonucleotide may include a barcode sequence (e.g., a molecular marker sequence), a cell marker, and a target binding region (e.g., an oligo (dT) sequence, a gene-specific sequence, a random polymer, or a combination thereof). The cell marker sequence of each of more than one oligonucleotide may be the same. The cell marker sequences of the oligonucleotides on different particles may be different, so that the oligonucleotides on different particles may be identified. In different embodiments, the number of different cell marker sequences may be different. In some embodiments, the number of cell marker sequences can be the following, or about the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 10 ... 6 , 10 7 , 10 8 , 10 9, a number or range between any two of these values, or more. In some embodiments, the number of cell marker sequences can be at least the following, or at most the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 or 10 9 In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the more than one particles comprise an oligonucleotide having the same cellular sequence. In some embodiments, the more than one particles comprising oligonucleotides having the same cellular sequence may be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none of the more than one particles have the same cell marker sequence.

[0240] More than one oligonucleotide on each particle can comprise different barcode sequences (e.g., molecular markers). In some embodiments, the number of barcode sequences can be, or about, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 , 10 9, or a number or range between any two of these values. In some embodiments, the number of barcode sequences can be at least the following, or at most the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 or 10 9 For example, at least 100 of the more than one oligonucleotides comprise different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, numbers or ranges between any two of these values, or more of the more than one oligonucleotides comprise different barcode sequences. Some embodiments provide more than one particle comprising a barcode. In some embodiments, the ratio of the occurrence (or copies or number) of the target to be labeled and the different barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or more. In some embodiments, each of the more than one oligonucleotides further comprises a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or a microparticle.

[0241] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 microns to 50 microns. In some embodiments, the diameter of the beads can be below, or about below: 0.1 microns, 0.5 microns, 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 6 microns, 7 microns, 8 microns, 9 microns, 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, or a number or range between any two of these values.

[0242] The diameter of bead can be relevant to the diameter of the hole of substrate.In some embodiments, the diameter of bead can be longer or shorter than the diameter of hole below or approximately below: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any two values ​​in these values ​​or scope.The diameter of bead can be relevant to the diameter of cell (for example, the unicellular being retained by the hole of matrix).In some embodiments, the diameter of bead can be longer or shorter than the diameter of hole at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.The diameter of bead can be relevant to the diameter of cell (for example, the unicellular being retained by the hole of matrix). In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the cell by less than or about less than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or range between any two of these values. In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the cell by at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300%.

[0243] The beads can be attached to and / or embedded in a substrate. The beads can be attached to and / or embedded in a gel, a hydrogel, a polymer, and / or a matrix. The spatial position of the beads in a substrate (e.g., a gel, a matrix, a scaffold, or a polymer) can be identified using a spatial tag present on a barcode on the beads that can be used as a positional address.

[0244] Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, beads, antibody conjugated beads (e.g., anti-immunoglobulin beads), protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin beads, anti-fluorescent dye beads, and BcMag TM Carboxyl-terminated magnetic beads.

[0245] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to cause them to fluoresce in one fluorescent optical channel or more than one optical channel. The beads can be associated with iron oxide or chromium oxide to cause them to be paramagnetic or ferromagnetic. The beads can be identifiable. For example, a camera can be used to image the beads. The beads can have a detectable code associated with the beads. For example, the beads can contain a barcode. For example, due to swelling in an organic or inorganic solution, the beads can change size. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible.

[0246] The solid support (e.g., bead) can be visualized. The solid support can include a visualization label (e.g., a fluorescent dye). The solid support (e.g., bead) can be etched with an identifier (e.g., a number). The identifier can be visualized by imaging the bead.

[0247] The solid support may comprise soluble, semi-soluble or insoluble material. When the solid support comprises a linker, a scaffold, a building block or other reactive moiety attached thereto, the solid support may be referred to as "functionalized", whereas when the solid support lacks such reactive moieties attached thereto, the solid support may be referred to as "non-functionalized". The solid support may be used freely in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.

[0248] Solid supports can include films, paper, plastics, coating surfaces, flat surfaces, glass, slides, chips, or any combination thereof. Solid supports can take the form of resins, gels, microspheres or other geometric shapes. Solid supports can include silicon dioxide chips, particulates, nanoparticles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, films, microplates, slides, plastic materials, including porous plates or films (e.g., formed by polyethylene, polypropylene, polyamide, polyvinylidene fluoride), and / or wafers (wafers), combs, pins (pins) or needles (needles) (e.g., suitable for combined synthesis or analysis of needle arrays), or flat surfaces such as the pits (pit) of wafers (e.g., silicon wafers) or the beads in the array of nanoliter holes, with wafers with or without the pits at the bottom of the filter.

[0249] The solid support may comprise a polymer matrix (eg, gel, hydrogel). The polymer matrix may be capable of permeating intracellular spaces (eg, around organelles). The polymer matrix may be capable of being pumped throughout the circulatory system.

[0250] Substrates and microwell arrays

[0251] As used herein, substrate may refer to a type of solid support. Substrate may refer to a solid support that may contain a barcode or a random barcode of the present disclosure. For example, the substrate may include more than one micropore. For example, the substrate may be a hole array including two or more micropores. In some embodiments, the micropore may include a small reaction chamber of a defined volume. In some embodiments, the micropore may retain one or more cells. In some embodiments, the micropore can only retain one cell. In some embodiments, the micropore may retain one or more solid supports. In some embodiments, the micropore can only retain one solid support. In some embodiments, the micropore retains a single cell and a single solid support (e.g., a bead). The micropore may contain a barcode reagent of the present disclosure.

[0252] Barcoding method

[0253] The present disclosure provides a method for estimating the number of different targets at different locations in a body sample (e.g., a tissue, an organ, a tumor, a cell). The method may include placing a barcode (e.g., a random barcode) near the sample, lysing the sample, associating different targets with the barcode, amplifying the target and / or digitally counting the target. The method may also include analyzing and / or visualizing the information obtained from the spatial markers on the barcode. In some embodiments, the method includes visualizing more than one target in the sample. Mapping more than one target to a map of the sample may include generating a two-dimensional map or a three-dimensional map of the sample. Two-dimensional maps and three-dimensional maps may be generated before or after barcoding more than one target in the sample (e.g., random barcoding). Visualizing more than one target in the sample may include mapping more than one target to a map of the sample. Mapping more than one target to a map of the sample may include generating a two-dimensional map or a three-dimensional map of the sample. Two-dimensional maps and three-dimensional maps may be generated before or after barcoding more than one target in the sample. In some embodiments, two-dimensional maps and three-dimensional maps may be generated before or after lysing the sample. Lysing the sample before or after generating the two-dimensional map or the three-dimensional map may include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0254] In some embodiments, barcoding more than one target comprises hybridizing more than one barcode to more than one target to generate barcoded targets (e.g., randomly barcoded targets). Barcoding more than one target can comprise generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets can be performed with a solid support comprising more than one barcode (e.g., random barcodes).

[0255] Bring the sample and barcode into contact

[0256] The present disclosure provides methods for contacting a sample (e.g., a cell) with a substrate of the present disclosure. Samples including, for example, cells, organs, or tissue slices can be contacted with a barcode (e.g., a random barcode). For example, the cells can be contacted by a gravity flow, in which the cells settle and produce a monolayer. The sample can be a tissue slice. The slice can be placed on a substrate. The sample can be one-dimensional (e.g., forming a flat surface). The sample (e.g., a cell) can be spread on a substrate, for example, by growing / culturing the cells on a substrate.

[0257] When the barcode is in proximity to the target, the target can hybridize to the barcode. The barcodes can be contacted at a non-exhaustive ratio such that each different target can be associated with a different barcode of the present disclosure. To ensure effective association between the target and the barcode, the target can be cross-linked to the barcode.

[0258] Cell lysis

[0259] After the distribution of cells and barcodes, cells can be lysed to release target molecules. Cell lysis can be accomplished by any of a variety of means, such as by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis or optical lysis. Cells can be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, lithium dodecyl sulfate, Triton X-100, Tween-20 or NP-40), an organic solvent (e.g., methanol or acetone) or a digestive enzyme (e.g., proteinase K, pepsin or trypsin), or any combination thereof. In order to increase the association of the target and the barcode, the diffusion rate of the target molecule can be changed by, for example, lowering the temperature of the lysate and / or increasing the viscosity of the lysate.

[0260] In some embodiments, the sample can be lysed using filter paper. The filter paper can be soaked with the lysis buffer on the filter paper. The filter paper can be applied to the sample with pressure, which can promote the lysis of the sample and the hybridization of the target of the sample with the substrate.

[0261] In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin and trypsin. The lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris HCl. The lysis buffer can include at least about 0.01M, 0.05M, 0.1M, 0.5M or 1M or more Tris HCl. The lysis buffer can include up to about 0.01M, 0.05M, 0.1M, 0.5M or 1M or more Tris HCl. The lysis buffer can include about 0.1M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can include a salt (e.g., LiCl). The salt concentration in the lysis buffer can be at least about 0.1, 0.5, or 1 M or more. The salt concentration in the lysis buffer can be up to about 0.1, 0.5, or 1 M or more. In some embodiments, the salt concentration in the lysis buffer is about 0.5 M. The lysis buffer can contain a detergent (e.g., SDS, lithium dodecyl sulfate, triton X, Tween, NP-40). The concentration of the detergent in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or more. The concentration of the detergent in the lysis buffer can be up to about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6% or 7% or more. In some embodiments, the concentration of the detergent in the lysis buffer is about 1% lithium dodecyl sulfate. The time used in the lysis method can depend on the amount of detergent used. In some embodiments, the more detergent is used, the less time is required for lysis. The lysis buffer can contain a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer can be at least about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM or 30mM or higher. The concentration of the chelating agent in the lysis buffer can be at most about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM or 30mM or higher. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10mM. The lysis buffer can contain a reducing agent (e.g., β-mercaptoethanol, DTT). The concentration of the reducing agent in the lysis buffer can be at least about 1mM, 5mM, 10mM, 15mM or 20mM or higher.The concentration of the reducing agent in the lysis buffer can be up to about 1 mM, 5 mM, 10 mM, 15 mM or 20 mM or higher. In some embodiments, the concentration of the reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer can include about 0.1 M TrisHCl, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA and about 5 mM DTT.

[0262] The lysis can be carried out at a temperature of about 4°C, 10°C, 15°C, 20°C, 25°C or 30°C. The lysis can be carried out for about 1 minute, 5 minutes, 10 minutes, 15 minutes or 20 minutes or longer. The lysis cells can include at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000 or 700,000 or more target nucleic acid molecules. The lysis cells can include at most about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000 or 700,000 or more target nucleic acid molecules.

[0263] Attaching barcodes to target nucleic acid molecules

[0264] After cell lysis and nucleic acid molecules are released from the cells, the nucleic acid molecules can be randomly associated with the barcodes of the co-located solid support. The association can include hybridizing the target recognition region of the barcode with the complementary portion of the target nucleic acid molecule (e.g., the oligo (dT) of the barcode can interact with the poly (A) tail of the target). The assay conditions for hybridization (e.g., buffer pH, ionic strength, temperature, etc.) can be selected to promote the formation of specific stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with more than one probe on the substrate (e.g., hybridized with the probe on the substrate). When the probe includes an oligo (dT), the mRNA molecule can hybridize with the probe and be reverse transcribed. The oligo (dT) portion of the oligonucleotide can act as a primer for the first chain synthesis of the cDNA molecule. For example, in Figure 2 In the non-limiting example of barcoding illustrated in FIG, at block 216, the mRNA molecule can be hybridized to the barcode on the bead. For example, a single-stranded nucleotide fragment can be hybridized to the target binding region of the barcode.

[0265] Attachment can also include connecting the target recognition region of the barcode to a portion of the target nucleic acid molecule. For example, the target binding region can include a nucleic acid sequence that may be able to specifically hybridize with a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure can also include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to produce a restriction site overhang. The barcode can then be connected to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to connect two fragments.

[0266] For example, in Figure 2 In the non-limiting example of barcoding illustrated in FIG, labeled targets (e.g., target-barcode molecules) from more than one cell (or more than one sample) can then be pooled, e.g., into a tube, at block 220. The labeled targets can be pooled by, for example, retrieving the barcodes and / or beads to which the target-barcode molecules are attached.

[0267] Retrieval of a solid support-based collection of attached target-barcode molecules can be implemented by using magnetic beads and an externally applied magnetic field. After the target-barcode molecules have been pooled, all further processing can be performed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., there is no need to first pool labeled target nucleic acid molecules from more than one cell.

[0268] Reverse transcription or nucleic acid extension

[0269] The present disclosure provides methods for using reverse transcription (e.g., Figure 2 The method of producing a target-barcode conjugate by a barcode or nucleic acid extension. The target-barcode conjugate may include a barcode and a complementary sequence of all or a portion of a target nucleic acid (i.e., a barcoded cDNA molecule, such as a random barcoded cDNA molecule). Reverse transcription of the associated RNA molecule may occur by adding a reverse transcription primer together with a reverse transcriptase. The reverse transcription primer may be an oligo (dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The length of the oligo (dT) primer may be 12-18 nucleotides, or may be about 12-18 nucleotides, and binds to the endogenous poly (A) tail at the 3' end of the mammalian mRNA. The random hexanucleotide primer may bind to the mRNA at multiple complementary sites. The target-specific oligonucleotide primer typically selectively triggers the mRNA of interest.

[0270] In some embodiments, the reverse transcription of mRNA molecules into labeled RNA molecules can occur by adding reverse transcription primers. In some embodiments, the reverse transcription primer is an oligo (dT) primer, a random hexanucleotide primer or a target-specific oligonucleotide primer. Typically, the length of the oligo (dT) primer is 12-18 nucleotides, and is combined with the endogenous poly (A) tail at the 3' end of mammalian mRNA. Random hexanucleotide primers can be combined with mRNA at multiple complementary sites. Target-specific oligonucleotide primers are usually selectively triggered by mRNA of interest.

[0271] In some embodiments, the target is a cDNA molecule. For example, a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, can be used to reverse transcribe the mRNA molecule to produce a cDNA molecule with a poly (dC) tail. The barcode can include a target binding region with a poly (dG) tail. During base pairing between the poly (dG) tail of the barcode and the poly (dC) tail of the cDNA molecule, the reverse transcriptase converts the template strand from the cell RNA molecule to the barcode and continues to copy to the 5' end of the barcode. By doing so, the resulting cDNA molecule includes a sequence of a barcode (such as a molecular marker) at the 3' end of the cDNA molecule.

[0272] Reverse transcription can occur repeatedly to produce multiple labeled cDNA molecules. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The method can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0273] Amplification

[0274] One or more nucleic acid amplification reactions can be performed (e.g., Figure 2The amplification reaction can be performed in a multiplexed manner, wherein more than one target nucleic acid sequence is amplified simultaneously. The amplification reaction can be used to add sequencing adapters to the nucleic acid molecules. The amplification reaction can include at least a portion of the amplified sample label (if present). The amplification reaction can include at least a portion of the amplified cell label and / or barcode sequence (e.g., molecular marker). The amplification reaction can include at least a portion of the amplified sample label, cell label, spatial label, barcode sequence (e.g., molecular marker), target nucleic acid, or a combination thereof. The amplification reaction can include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100% of more than one nucleic acid, or a number or range between any two of these values. The method can also include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of a target-barcode molecule comprising a sample marker, a cell marker, a spatial marker, and / or a barcode sequence (e.g., a molecular marker).

[0275] In some embodiments, polymerase chain reaction (PCR) can be used for amplification. As used herein, PCR can refer to a reaction for amplifying a specific DNA sequence in vitro by primer extension while a complementary strand of DNA. As used herein, PCR can include a derivative form of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR and assembly PCR.

[0276] The amplification of labeled nucleic acid can include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), amplification based on nucleic acid sequences (NASBA), chain displacement amplification (SDA), real-time SDA, rolling circle amplification, or ring-to-ring amplification. Other non-PCR-based amplification methods include multiple cycles of DNA synthesis and transcription of DNA synthesis driven by DNA-dependent RNA polymerase or RNA-guided amplification to amplify DNA or RNA targets, ligase chain reaction (LCR) and Qβ replicase (Qβ) methods, the use of palindromic probes, chain displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, primers hybridized to nucleic acid sequences and amplification methods of cleavage of the resulting duplex prior to extension reactions and amplification, chain displacement amplification, rolling circle amplification, and branch extension amplification (RAM) using nucleic acid polymerases lacking 5' exonuclease activity. In some embodiments, amplification does not produce circularized transcripts.

[0277] In some embodiments, the methods disclosed herein also include 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 randomly labeled amplicon). The labeled amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The single-stranded molecule can include DNA, RNA, or a combination thereof. Amplification can include the use of one or more non-natural nucleotides.

[0278] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable or triggerable nucleotides. Examples of non-natural nucleotides can include but are not limited to peptide nucleic acids (PNA), morpholino nucleic acids and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of amplified reactions. Adding non-natural nucleotides can be used to identify the product of a specific cycle or time point in an amplified reaction.

[0279] Carrying out one or more amplification reactions can include using one or more primers.One or more primers can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides.One or more primers can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides.One or more primers can include less than 12-15 nucleotides.One or more primers can anneal to at least a portion of more than one labeled target (e.g., a randomly labeled target).One or more primers can anneal to 3' ends and / or 5' ends of more than one labeled target.One or more primers can anneal to the internal region of more than one labeled target. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910 , 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides. One or more primers can include a group of fixed primers. One or more primers can include at least one or more custom primers. One or more primers can include at least one or more control primers. One or more primers can include at least one or more gene specific primers.

[0280] One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers may anneal to a first sample marker, a second sample marker, a spatial marker, a cell marker, a barcode sequence (e.g., a molecular marker), a target, or any combination thereof. One or more primers may include universal primers and custom primers. Custom primers may be designed to amplify one or more targets. The target may include a subset of total nucleic acids in one or more samples. The target may include a subset of total 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.

[0281] Any amplification scheme can be used in the method of the present disclosure. For example, in one scheme, the first round of PCR can use gene-specific primers and primers for universal Illumina sequencing primer 1 sequence to amplify the molecules attached to the beads. The second round of PCR can use nested gene-specific primers flanking Illumina sequencing primer 2 sequences, and primers for universal Illumina sequencing primer 1 sequence to amplify the first PCR product. The third round of PCR adds P5 and P7 and sample indexes to convert PCR products into Illumina sequencing libraries. Sequencing using 150bp x 2 sequencing can reveal cell markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and sample indexes on index 1 reads.

[0282] In some embodiments, nucleic acid can be removed from substrate using chemical cleavage. For example, chemical groups or modified bases present in nucleic acid can be used to promote its removal from solid support. For example, enzymes can be used to remove nucleic acid from substrate. For example, nucleic acid can be removed from substrate by restriction endonuclease digestion. For example, nucleic acid containing dUTP or ddUTP can be removed from substrate using uracil-d-glycosylase (UDG). For example, nucleic acid can be removed from substrate using enzymes (such as, base excision repair enzymes, such as apurinic / apyrimidinic (AP) endonucleases) that perform nucleotide excision. In some embodiments, nucleic acid can be removed from substrate using photocleavable groups and light. In some embodiments, nucleic acid can be removed from substrate using cleavable joints. For example, cleavable joints can include at least one of the following: biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig protein A, light labile joints, acid or base labile joint groups, or aptamers.

[0283] When the probe is gene specific, the molecule can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, after the nucleic acid has been synthesized (e.g., reverse transcribed), it can be amplified. Amplification can be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acid.

[0284] In some embodiments, amplification can be performed on a substrate, for example, with bridge amplification. Homopolymer tails can be added to cDNA to produce compatible ends for bridge amplification using oligo (dT) probes on substrates. In bridge amplification, a primer complementary to the 3' end of a template nucleic acid can be the first primer of each pair of primers covalently attached to a solid particle. When a sample containing a template nucleic acid contacts a particle and performs a single thermal cycle, the template molecule can be annealed to the first primer, and the first primer is extended in the forward direction by adding nucleotides to form a duplex molecule, which is composed of a template molecule and a newly formed DNA chain complementary to the template. In the heating step of the next cycle, the duplex molecule can be denatured, the template molecule is released from the particle, and the complementary DNA chain is attached to the particle by the first primer. In the annealing stage of the subsequent annealing and extension steps, the complementary chain can be hybridized with the second primer, which is complementary to the segment (segment) of the complementary chain at the position removed from the first primer. This hybridization can cause complementary strands to form a bridge between the first primer and the second primer, and the bridge is fixed to the first primer by a covalent bond and fixed to the second primer by hybridization. In the extension phase, by adding nucleotides in the same reaction mixture, the second primer can extend in the reverse direction, thereby converting the bridge into a double-stranded bridge. Then start the next cycle, and the double-stranded bridge can be denatured to produce two single-stranded nucleic acid molecules, one end of each single-stranded nucleic acid molecule is attached to the particle surface through the first primer and the second primer respectively, and the other end of each single-stranded nucleic acid molecule is unattached. In the annealing and extension steps of this second cycle, each chain can hybridize with other complementary primers not previously used on the same particle to form a new single-stranded bridge. Extend the two previously unused primers that are now hybridized to convert two new bridges into double-stranded bridges.

[0285] The amplification reaction can include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of more than one nucleic acid.

[0286] Amplification of labeled nucleic acid may include PCR-based methods or non-PCR-based methods. Amplification of labeled nucleic acid may include exponential amplification of labeled nucleic acid. Amplification of labeled nucleic acid may include linear amplification of labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for amplifying a specific DNA sequence in vitro by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, repression PCR, semi-repression PCR, and assembly PCR.

[0287] In some embodiments, the amplification of the labeled nucleic acid includes a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), chain displacement amplification (SDA), real-time SDA, rolling circle amplification, or ring-to-ring amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-guided DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ), the use of palindromic probes, chain displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, primers hybridized to nucleic acid sequences and the resulting duplexes cleaved before extension reactions and amplification, chain displacement amplification using nucleic acid polymerases lacking 5' exonuclease activity, rolling circle amplification, and / or branch extension amplification (RAM).

[0288] In some embodiments, the method disclosed herein also includes performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample label or a molecular identifier tag. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid of the present invention can include a synthetic or altered nucleic acid.

[0289] In some embodiments, the method includes repeatedly amplifying the labeled nucleic acid to produce more than one amplicon. The method disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the method includes performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0290] Amplification can also include adding one or more control nucleic acids to one or more samples including more than one nucleic acid. Amplification can also include adding one or more control nucleic acids to more than one nucleic acid. The control nucleic acid can include a control label.

[0291] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable and / or triggerable nucleotides. Examples of non-natural nucleotides include but are not limited to peptide nucleic acids (PNA), morpholino nucleic acids and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of an amplified reaction. Adding non-natural nucleotides can be used to identify the product of a specific cycle or time point in an amplified reaction.

[0292] Carrying out one or more amplification reactions can include using one or more primers. One or more primers can include one or more oligonucleotides. One or more oligonucleotides can include at least about 7-9 nucleotides. One or more oligonucleotides can include less than 12-15 nucleotides. One or more primers can anneal to at least a portion of more than one labeled nucleic acid. One or more primers can anneal to the 3' end and / or 5' end of more than one labeled nucleic acid. One or more primers can anneal to the internal region of more than one labeled nucleic acid. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910 , 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides. One or more primers may include a set of fixed primers. One or more primers may include at least one or more custom primers. One or more primers may include at least one or more control primers. One or more primers may include at least one or more housekeeping gene primers. One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers can anneal to the first sample tag, the second sample tag, a molecular identifier label, a nucleic acid or their product. One or more primers can include universal primers and custom primers. Custom primers can be designed to amplify one or more target nucleic acids. Target nucleic acids can include a subset of total nucleic acids in one or more samples. In some embodiments, primers are probes attached to an array of the present disclosure.

[0293] In some embodiments, barcoding more than one target in a sample (e.g., randomly barcoding) further comprises generating an index library of barcoded targets (e.g., randomly barcoded targets) or an index library of barcoded fragments of the targets. The barcode sequences of different barcodes (e.g., molecular markers of different random barcodes) can be different from each other. Generating an index library of barcoded targets comprises generating more than one index polynucleotide from more than one target in a sample. For example, for an index library of barcoded targets comprising a first index target and a second index target, the marker region of the first index polynucleotide can have the following difference from the marker region of the second index polynucleotide, have about the following difference, have at least the following difference, or have at most the following difference: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range of nucleotides between any two of these values. In some embodiments, generating an index library of barcoded targets includes contacting more than one target (e.g., mRNA molecules) with more than one oligonucleotide including a poly (T) region and a tag region; and performing first-strand synthesis using a reverse transcriptase to generate single-stranded labeled cDNA molecules each comprising a cDNA region and a tag region, wherein the more than one target includes at least two mRNA molecules of different sequences, and the more than one oligonucleotide includes at least two oligonucleotides of different sequences. Generating an index library of barcoded targets can also include amplifying single-stranded labeled cDNA molecules to generate double-stranded labeled cDNA molecules; and performing nested PCR on double-stranded labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method can include generating adapter-tagged amplicons.

[0294] Barcoding (e.g., random barcoding) can include the use of nucleic acid barcodes or tags to label a single nucleic acid (e.g., DNA or RNA) molecule. In some embodiments, it involves adding a DNA barcode or tag to a cDNA molecule, since the cDNA molecule is generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters can be added for sequencing using, for example, next generation sequencing (NGS). For example, in Figure 2 At block 232 of the method, the sequencing results can be used to determine the sequence of cellular markers, molecular markers, and nucleotide fragments of one or more copies of the target.

[0295] Figure 3is a schematic illustration of a non-limiting exemplary method for generating an index library of barcoded targets (e.g., random barcoded targets), such as an index library of barcoded mRNAs or fragments thereof. As shown in step 1, the reverse transcription process can encode each mRNA molecule with a unique molecular marker sequence, a cell marker sequence, and a universal PCR site. In particular, by hybridizing (e.g., randomly hybridizing) a set of barcodes (e.g., random barcodes) 310 with a poly (A) tail region 308 of an RNA molecule 302, an RNA molecule 302 can be reverse transcribed to produce a labeled cDNA molecule 304 (including a cDNA region 306). Each of the barcodes 310 can include a target binding region, such as a poly (dT) region 312, a marker region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0296] In some embodiments, the cell marker sequence may include 3 to 20 nucleotides. In some embodiments, the molecular marker sequence may include 3 to 20 nucleotides. In some embodiments, each of more than one random barcode also includes one or more of a universal marker and a cell marker, wherein the universal marker of more than one random barcode on the solid support is the same, and the cell marker of more than one random barcode on the solid support is the same. In some embodiments, the universal marker may include 3 to 20 nucleotides. In some embodiments, the cell marker includes 3 to 20 nucleotides.

[0297] 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 length of the barcode sequence or molecular label 318 may be below, may be about below, may be at least below, or may be at most below: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values. The length of the cell label 320 can be below, can be about below, can be at least below, or can be at most below: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values. The length of the universal label can be below, can be about below, can be at least below, or can be at most below: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values. The universal label of more than one random barcode on the solid support can be the same, and the cell labels of more than one random barcode on the solid support are the same. The length of a dimensional marker can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values.

[0298] In some embodiments, the labeling area 314 can include the following, including about the following, including at least the following, or including at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any two of these values ​​of different labels, such as barcode sequences or molecular labels 318 and cell labels 320. The length of each marker can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of nucleotides between any two of these values. A set of barcodes or random barcodes 310 can contain, can be about, can be at least, or can be at most 10, 20, 40, 50, 70, 80, 90, 100 nucleotides. 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 20 A barcode or random barcode 310 having a value of 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, 59, 60, 61, 62,

[0299] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled into 1 tube and PCR amplified using the 1st PCR primer pool and the 1st universal PCR primer. Pooling is possible due to the unique labeling region 314. In particular, the labeled cDNA molecules 304 can be amplified to produce nested PCR labeled amplicons 322. Amplification can include multiplex PCR amplification. Amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, in a single reaction volume, multiplex PCR amplification can utilize the following, utilize about the following, utilize at least the following, or utilize at most the following: 10, 20, 40, 50, 70, 80, 90, 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 10 20 Amplification can include using a first PCR primer pool 324 including custom primers 326A-C targeting specific genes and universal primers 328. Custom primers 326 can hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. Universal primers 328 can hybridize to universal PCR region 316 of the labeled cDNA molecule 304.

[0300] like Figure 3As shown in step 3 of , the product from the PCR amplification in step 2 can be amplified with a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the amplicon 322 of the nested PCR marker can be further amplified by nested PCR. Nested PCR can include multiplex PCR performed in a single reaction volume using a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328'. The nested PCR primer pool 330 can contain the following, contain about the following, contain at least the following, or contain at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any two of these values ​​different nested PCR primers 332. Nested PCR primer 332 may contain adapter 334 and hybridize to a region within cDNA portion 306″ of labeled amplicon 322. Universal primer 328′ may contain adapter 336 and hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 produces adapter-tagged amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328′ may not contain adapters 334 and 336. Instead, adapters 334 and 336 may be ligated to the product of nested PCR to produce adapter-tagged amplicon 338.

[0301] 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 adapter-tagged amplicon 338. Adapters 334 and 336 can hybridize with primers 340 and 342. One or more primers 340 and 342 can be PCR amplification primers. One or more primers 340 and 342 can be sequencing primers. One or more adapters 334 and 336 can be used for further amplification of adapter-tagged amplicon 338. One or more adapters 334 and 336 can be used to sequence adapter-tagged amplicon 338. Primer 342 can contain a plate index 344 so that the amplicon generated using the same set of barcodes or random barcodes 310 can be sequenced using next generation sequencing (NGS) in one round of sequencing reactions.

[0302] Hybrid library preparation

[0303] In recent years, next generation sequencing (NGS) has provided a high-throughput method for evaluating gene expression profiles. During the preparation of libraries for NGS, samples with heterogeneous cDNA species are amplified by PCR to obtain sufficient sample quantities and attach NGS-compatible adapters. The sequencing process captures the number of reads for each gene from the PCR-amplified library sample to explain the gene expression level. Gene expression levels in biological samples may vary greatly. For example, it has been described that gene expression levels follow three major categories: 1) "high expressers", consisting of 5-10 genes, accounting for ~20% of cell mRNA; 2) "intermediate expressers", consisting of 50-200 genes, accounting for 40%-60% of cell mRNA; and 3) "low expressers", consisting of 10,000-20,000 genes, accounting for the rest of the cell mRNA fraction. A challenge for molecular biology and molecular genetics is to be able to efficiently and effectively capture this highly dynamic gene expression profile in order to distinguish different cell types and phenotypes in the sample. Because different genes are expressed at a wide range of levels, PCR amplification can cause a skew in native gene expression. For example, a gene with 1 molecule of cDNA will require 40 cycles of PCR to achieve the same representation as a gene with 1000 molecules of cDNA in 30 cycles. In heterogeneous cDNA samples, PCR is often performed with an excess of cycles to fully amplify low expressers; in these cases, the native gene expression profile is often skewed by the predominant PCR products of high expressers. A method to correct for this bias in PCR products is molecular indexing; however, high expressers such as ribosomal protein mRNAs, mitochondrial mRNAs, or housekeeping genes often dominate the sequencing run and contribute little to the experimental interpretation, making sequencing of molecular index counts expensive.

[0304] Previous efforts to increase the relative abundance of low-abundance species in nucleic acid samples include library normalization methods. A library normalization method or strategy includes hybridizing a library with another set of nucleic acids such as genomic DNA from a source organism in which the sequence is consistently represented and retaining the hybridized fraction. Another library normalization method or strategy is based on the concentration dependence of solution hybridization (for example, when a group of dsDNA molecules are denatured, they will hybridize again at a rate proportional to the square of their original concentration). Exemplary library normalization methods are described in U.S. Patent Application Publications US2017 / 0073730 and 2017 / 0342484; the contents of each of these patent application publications are incorporated herein by reference in their entirety. In some embodiments, the methods, compositions, and kits disclosed herein can complement or supplement these library normalization strategies. In some embodiments, the methods, compositions, and kits disclosed herein can avoid using physical and enzymatic separation of ssDNA and dsDNA fractions during library normalization. In some embodiments, the methods, compositions, and kits disclosed herein can avoid the need to hybridize a library to another set of nucleic acids. The disclosure herein includes systems and methods for increasing the relative abundance of low-abundance species in a sample by hybrid library preparation (e.g., hybrid cDNA library preparation and / or hybrid sequencing library preparation).

[0305] Since the single cell molecular index full transcriptome amplification (WTA) analysis does not use target-specific primers during library amplification, the assay provides the advantage of being able to discover biology (discovery biology). The performance of the scRNA-seq using a set of target-specific multiple primers has higher sensitivity (especially for low-abundance targets) relative to the WTA analysis, but this method cannot target the entire transcriptome, and therefore cannot be used for discovery biology. The method disclosed herein combines the advantages of both WTA and target-specific group scRNA-seq methods, and combines the use of target-specific primers and target non-specific primers during the preparation of cDNA libraries and / or sequencing libraries. The method disclosed herein can cause the sensitivity of low-abundance transcripts to increase, detect new transcripts, detect new cell types and / or reduce sequencing costs.

[0306] In some embodiments, methods disclosed herein, compositions and kits increase the abundance of the low-abundance target selected during cDNA library preparation and / or sequencing library preparation.In some embodiments, the abundance of the low-abundance target selected during cDNA library preparation and / or sequencing library preparation includes heterozygote library preparation (for example, heterozygote cDNA library preparation, heterozygote sequencing library preparation).In some embodiments, heterozygote cDNA library preparation includes: (a) using non-target specific primers (for example, random primers, WTA primers, primers annealed to universal sequences) to carry out full transcriptome cDNA amplification, and (b) target-specific cDNA amplification is carried out with target-specific primers.In some embodiments, heterozygote sequencing library preparation includes: (a) using non-target specific primers (for example, random primers, WTA primers, primers annealed to universal sequences) extension and / or amplification;With (b) using target-specific primers extension and / or amplification. In some embodiments, the method includes using non-target specific primers (e.g., WTA primers) and a group of target-specific primers during the preparation of cDNA library. In some embodiments, the method includes using non-target specific primers and a group of target-specific primers during the preparation of sequencing library. In some embodiments, the method includes using non-target specific primers and a group of target-specific primers during the preparation of cDNA library and sequencing library.

[0307] Some embodiments disclosed herein provide a method for increasing the relative abundance of one or more low-abundance species during cDNA library preparation and / or heterozygote sequencing library preparation.As used herein, "target" can be used interchangeably with "species".In some embodiments, the method for increasing the relative abundance of one or more low-abundance species includes a method for preparing a heterozygote cDNA library as disclosed herein and / or a heterozygote sequencing library preparation.In some embodiments, increasing the relative abundance of one or more low-abundance species includes preparing a cDNA library with WTA (e.g., random) primers and target-specific primers.In some embodiments, increasing the relative abundance of one or more low-abundance species includes preparing a sequencing library with non-target-specific primers and target-specific primers.

[0308] As used herein, "species" refers to polynucleotides that are identical to each other, are complements or reverse complements of each other, or can hybridize to each other, or are transcripts from the same genetic locus, or encode the same protein or fragments thereof, etc. (e.g., single-stranded polynucleotides, such as cDNA molecules, sample index oligonucleotides for sample tracking, and protein-specific oligonucleotides for determining protein expression profiles). As used herein, "target" can be used interchangeably with "species". In some embodiments, members of a species (e.g., copies or occurrences) are at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% homologous to each other or to its complement. In some embodiments, members of a species are transcripts from the same genetic locus, and transcripts can have the same or different lengths. In some embodiments, species are cDNA or mRNA.

[0309] As used herein, "high abundance species" refers to species that are present in large quantities, for example, the species may be below, about below, at least below, or at least about below: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50% or more. In some embodiments, a sample may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000 or more high abundance species. In some embodiments, the sum of all high abundance species accounts for at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or more of the nucleic acid molecules or species in the sample. In some embodiments, high abundance species may include polynucleotides encoding one or more ribosomal proteins. In some embodiments, high abundance species may include polynucleotides encoding one or more mitochondrial proteins. In some embodiments, high abundance species may include polynucleotides encoding one or more housekeeping proteins. In some embodiments, high abundance species may include sequences of sample indexing oligonucleotides (also referred to herein as sample tag sequences or sample indexing indices). In some embodiments, high abundance species may include oligonucleotide sequences for sample tracking. In some embodiments, high abundance species may include sample indexing sequences for identifying the sample source of one or more cells of a sample. In some embodiments, high abundance species may include unique identifier sequences for cell component binding reagents (e.g., antibodies). In some embodiments, high abundance species may include oligonucleotide sequences conjugated (or pre-conjugated) with cell component binding reagents. In some embodiments, high abundance species may include oligonucleotide sequences conjugated or pre-conjugated with antibodies that may be referred to herein as antibody oligonucleotides (abbreviated as "AbOligo" or "AbO").

[0310] As used herein, "medium abundance species" refers to species that exist in an amount lower than at least one species and higher than at least one other species. In some embodiments, the medium abundance species may be below, at least below, about below, or at least about below: 10%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01% of nucleic acid molecules or species in a sample, or a range between any two of these values. In some embodiments, a sample may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000 or more medium abundance species. In some embodiments, the total amount of all medium abundance species accounts for about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30% of nucleic acid molecules in a sample, or a range between any two of the above values. In some embodiments, the medium abundance species may include polynucleotides encoding one or more housekeeping proteins. In some embodiments, the medium abundance species may include an oligonucleotide sequence for sample tracking. In some embodiments, the medium abundance species may include a sample indexing sequence for identifying the sample source of one or more cells of a sample. In some embodiments, the medium abundance species may include a unique identifier sequence for a cell component binding reagent (e.g., an antibody). In some embodiments, the medium abundance species may include an oligonucleotide sequence conjugated (or pre-conjugated) to a cell component binding reagent. In some embodiments, the medium abundance species may include an oligonucleotide sequence conjugated to or pre-conjugated with an antibody that may be referred to herein as an antibody oligonucleotide (abbreviated as "AbOligo" or "AbO").

[0311] As used herein, "low abundance species" refers to species present in low amounts, for example, the species may be below, about below, less than, or less than about below: 1%, 0.1%, 0.01%, 0.001%, 0.0001% or less of nucleic acid molecules or species in a sample. In some embodiments, a sample may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000 or more low abundance species. In some embodiments, the total amount of all low abundance species accounts for less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1% or less of nucleic acid molecules in a sample. In some embodiments, low abundance species may include polynucleotides encoding one or more transcription factors. In some embodiments, low abundance species may include polynucleotides encoding one or more T cell receptors. In some embodiments, low abundance species may include polynucleotides encoding one or more antibodies.

[0312] In some embodiments, two or more of the more than one nucleic acid targets include low abundance species. In some embodiments, two or more of the more than one nucleic acid targets include low abundance species and / or medium abundance species. In some embodiments, two or more of the more than one nucleic acid targets include mRNAs of low expressed genes. In some embodiments, the percentage of nucleic acid targets representing low abundance species can be, or be about: 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 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%, 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%, 8 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%, 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%, or a number or range between any two of these values.In some embodiments, the percentage of nucleic acid targets that represent low abundance species can be at least the following, or at most the following: 0.000000001%, 0.00000001%, 0.0000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 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%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 7%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 9%, 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% or 100%.

[0313] In some embodiments, the hybrid library includes the product of hybrid cDNA library preparation and / or sequencing cDNA library preparation. In some embodiments, the hybrid library includes the amplicon derived from the hybrid cDNA library preparation and / or sequencing cDNA library preparation. In some embodiments, the hybrid library includes the product of one or more downstream methods of the present disclosure, and the one or more downstream methods use the product of hybrid cDNA library preparation and / or sequencing cDNA library preparation as a template. In some embodiments, compared with the sequencing of non-hybrid libraries, the sequencing of the hybrid library produced by the method of the present disclosure leads to an increase in the number of reads of one or more low-abundance species (e.g., nucleic acid targets, low-expressed mRNA). In some embodiments, the increase in the number of reads of one or more low abundance species in a hybrid library compared to a non-hybrid library is at least 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100% or more, and overlapping ranges therein). In some embodiments, sequencing of a hybrid library generated by the methods of the present disclosure results in an increase in the number of reads of one or more low abundance species (e.g., nucleic acid targets, lowly expressed mRNAs) relative to the total number of reads compared to sequencing of a non-hybrid library. In some embodiments, the increase in the number of reads of one or more low abundance species relative to the total number of reads in the hybrid library compared to a non-hybrid library is at least 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100% or more, and overlapping ranges therein). In some embodiments, sequencing of a hybrid library generated by the methods of the present disclosure results in an increase in the number of molecular indices (MIs) of one or more low abundance species (e.g., nucleic acid targets, lowly expressed mRNAs) compared to sequencing of a non-hybrid library. In some embodiments, the increase in the number of MIs of one or more low abundance species relative to the total number of reads in a hybrid library compared to a non-hybrid library is at least 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100% or more, and ranges of overlap therein).

[0314] In some embodiments, the methods disclosed herein can increase the relative abundance of one or more low-abundance species after hybrid cDNA library preparation and / or hybrid sequencing library preparation (compared to non-hybrid cDNA library preparation and / or non-hybrid sequencing library preparation). For example, the methods disclosed herein can increase the relative abundance of at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000 or more low-abundance species after hybrid cDNA library preparation and / or hybrid sequencing library preparation (compared to non-hybrid cDNA library preparation and / or non-hybrid sequencing library preparation). In some embodiments, the methods disclosed herein can increase the relative abundance of each of one or more low-abundance species by at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 250%, 500%, 1000% or more, and overlapping ranges therein) after a hybrid cDNA library preparation and / or a hybrid sequencing library preparation (compared to a non-hybrid cDNA library preparation and / or a non-hybrid sequencing library preparation). In some embodiments, the methods disclosed herein can increase the relative abundance of at least one of the one or more low-abundance species by at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 250%, 500%, 1000% or more, and overlapping ranges therein) after a hybrid cDNA library preparation and / or a hybrid sequencing library preparation (compared to a non-hybrid cDNA library preparation and / or a non-hybrid sequencing library preparation). In some embodiments, the methods disclosed herein can increase the relative abundance of total low-abundance species after a hybrid cDNA library preparation and / or a hybrid sequencing library preparation (compared to a non-hybrid cDNA library preparation and / or a non-hybrid sequencing library preparation) by at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 250%, 500%, 1000% or more, and overlapping ranges therein).

[0315] In some embodiments, the methods disclosed herein can selectively increase the relative abundance of one or more low-abundance species after hybrid cDNA library preparation and / or hybrid sequencing library preparation (compared to non-hybrid cDNA library preparation and / or non-hybrid sequencing library preparation), so that after hybrid cDNA library preparation and / or hybrid sequencing library preparation, the abundance of low-abundance species is increased relative to one or more high-abundance species. In some embodiments, the methods and compositions disclosed herein can increase, increase by about, increase by at least, or increase by at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%) of the relative abundance of each of the one or more low-abundance species after hybrid cDNA library preparation and / or hybrid sequencing library preparation (compared to non-hybrid cDNA library preparation and / or non-hybrid sequencing library preparation). , 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 250%, 500%, 1000% or more, and overlapping ranges therein), such that the abundance of the low abundance species is increased relative to one or more high abundance species after hybrid cDNA library preparation and / or hybrid sequencing library preparation.

[0316] In some embodiments, the sequencing of the hybrid library produced by the methods of the present disclosure produces similar gene expression profiles and / or protein expression profiles (e.g., gene expression group results) of one or more targets compared to the sequencing of the non-hybrid library. In some embodiments, the difference in gene expression profiles and / or protein expression profiles of one or more targets between the hybrid library and the non-hybrid library can be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or a number or range between any two of these values. In some embodiments, the R between the expression profiles of the hybrid library and the non-hybrid library is ... 2 The correlation can be 0.6, 0.7, 0.8, 0.9, 0.990, 0.999, 1.0 and overlapping ranges therein. Methods for testing correlation between expression profiles of library results are well understood in the art (e.g., overlay of tSNE plots).

[0317] It will be understood that by increasing the sequencing reads of medium or low abundance species in the hybrid library, and / or reducing the sequencing reads of high abundance species in the non-hybrid library, the hybrid library can improve sequencing efficiency. In the hybrid library produced by the method of the present disclosure, less abundant (e.g., rarer) nucleic acid targets can be more easily identified than in the non-hybrid library. The sequencing reads of less abundant targets in the hybrid library can constitute a larger portion of the total reads compared to the non-hybrid library. The sequencing reads of one or more less abundant targets in the hybrid library can include at least 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or 500% or more reads compared to the reads of the same target in the non-hybrid library. The number of sequencing reads for one or more less abundant targets in the normalized library may be at least 1, 2, 3, 4, 5, or 6 or more times greater than the number of sequencing reads for the same target in the non-hybrid library.

[0318] The methods described herein for generating hybrid libraries can increase the sequencing reads of medium-abundance nucleic acid targets and / or low-abundance nucleic acid targets in more than one nucleic acid (e.g., nucleic acid library). For example, the sequencing reads of more than one medium-abundance nucleic acid target can be at least 30%, at least 20%, at least 10%, at least 5% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target can be at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2%, at least 1% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target are at least 5% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target are at least 10% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target are at least 20% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target are at least 30% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target are at least 40% of the total sequencing reads of the hybrid library. In some embodiments, the sequencing reads of more than one low-abundance nucleic acid target are at least 50% of the total sequencing reads of the hybrid library.

[0319] In some embodiments, the preparation of the hybrid cDNA library includes: (a) performing full transcriptome cDNA amplification with non-target specific primers (e.g., random primers, WTA primers, primers annealed to universal sequences) to produce a first more than one barcoded amplicon (e.g., a first more than one double-stranded barcoded polynucleotide); and (b) performing target-specific cDNA amplification with target-specific primers to produce a second more than one barcoded amplicon (e.g., a second more than one double-stranded barcoded polynucleotide). In some embodiments, the preparation of the hybrid sequencing library includes: (a) extension and / or amplification using non-target specific primers (e.g., random primers, primers annealed to universal sequences) to produce a first more than one barcoded amplicon; and (b) extension and / or amplification using target-specific primers to produce a second more than one barcoded amplicon.

[0320] In some embodiments, the ratio of non-target specific primers to target-specific primers ranges from 1:100 to 100:1. In some embodiments, the ratio of non-target specific primers to target-specific primers is at most 10:1. In some embodiments, the ratio of non-target specific primers to target-specific primers is at most 100:1. In some embodiments, the ratio of non-target specific primers to target-specific primers is at most 1:1000. In some embodiments, the ratio of non-target specific primers to target-specific primers is at least 1:10. In some embodiments, the ratio of non-target specific primers to target-specific primers is at least 1:100. In some embodiments, the ratio of non-target specific primers to target-specific primers is at least 1:1000.

[0321] In some embodiments, the ratio of non-target specific primers to target specific primers can be, or about: 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 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, 1:2 19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55 , 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1 :92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or a number or range between any two of the recited values.In some embodiments, the ratio of non-target specific primers to target specific primers can be at least the following, or at most the following: 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 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, 1:2 :18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53 , 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1: 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000 or 1:10000.

[0322] In some embodiments, the ratio of non-target specific primers to target specific primers can be, or about: 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 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 :1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1 , 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1 1, 2:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of the recited values.In some embodiments, the ratio of non-target specific primers to target specific primers can be at least the following, or at most the following: 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 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 8:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、52:1、53:1 , 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89 :1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1 or 10000:1.

[0323] In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons ranges from 1:100 to 100:1. In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons is at most 10:1. In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons is at most 100:1. In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons is at most 1:1000. In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons is at least 1:10. In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons is at least 1:100. In some embodiments, the ratio of the number of the first more than one barcoded amplicons to the number of the second more than one amplicons is at least 1:1000.

[0324] In some embodiments, the ratio of the number of the first one or more barcoded amplicons to the number of the second one or more amplicons can be, or about the following: 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 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, 1:2 :17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54 54、1:55、1:56、1:57、1:58、1:59、1:60、1:61、1:62、1:63、1:64、1:65、1:66、1:67、1:68、1:69、1:70、1:71、1:72、1:73、1:74、1:75、1:76、1:77、1:78、1:79、1:80、1:81、1:82、1:83、1:84、1:85、1:86、1:87、1:88、1:89、1:90、1: 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or a number or range between any two of the recited values.In some embodiments, the ratio of the number of the first one or more barcoded amplicons to the number of the second one or more amplicons can be at least the following, or at most the following: 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1 :52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88 8, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000 or 1:10000.

[0325] In some embodiments, the ratio of the number of the first one or more barcoded amplicons to the number of the second one or more amplicons can be, or about the following: 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 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 7:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、52:1、53:1、54 :1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91 :1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, or a number or range between any two of the recited values.In some embodiments, the ratio of the number of the first one or more barcoded amplicons to the number of the second one or more amplicons can be at least the following, or at most the following: 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 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、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、5 2:1、53:1、54:1、55:1、56:1、57:1、58:1、59:1、60:1、61:1、62:1、63:1、64:1、65:1、66:1、67:1、68:1、69:1、70:1、71:1、72:1、73:1、74:1、75:1、76:1、77:1、78:1、79:1、80:1、81:1、82:1、83:1、84:1、85:1、86:1、87:1、88: 1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1 or 10000:1.

[0326] In some embodiments, one or more nucleic acid targets include a group of target genes. In some embodiments, one or more target-specific primers include a group of target-specific multiple primers. One or more primers for multiplex PCR can include a first gene-specific primer and a nested gene-specific primer designed to anneal downstream of the first gene-specific primer. In different embodiments, the number of different target-specific primers in a group of target-specific multiple primers can be different. In some embodiments, the number of different target-specific primers in a group of target-specific multiple primers can be as follows, or about as follows: 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, 1100, 1250, 1500, 2000, 3000 or a number or range between any two of these values. In some embodiments, the number of different target-specific primers in a set of target-specific multiplex primers can be at least the following, or at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1250, 1500, 2000 or 3000.

[0327] In some embodiments, the method includes performing non-hybrid cDNA library preparation, followed by hybrid sequencing library preparation. In some embodiments, the method includes performing hybrid cDNA library preparation, followed by non-hybrid sequencing library preparation. In some embodiments, the method includes performing hybrid cDNA library preparation, followed by hybrid sequencing library preparation. In embodiments in which both hybrid cDNA library preparation and hybrid sequencing library preparation are performed, target-specific cDNA amplification primers and target-specific sequencing library primers can be directed to the same group of nucleic acid targets (e.g., annealing to the same group of transcripts). In some such embodiments, target-specific cDNA amplification primers and target-specific sequencing library primers can anneal to the same region of the same nucleic acid target, or can anneal to different regions of the same nucleic acid target. In embodiments in which both hybrid cDNA library preparation and hybrid sequencing library preparation are performed, target-specific cDNA amplification primers and target-specific sequencing library primers can be directed to a group of different nucleic acid targets (e.g., annealing to one or more different transcripts).

[0328] During the preparation of the hybrid cDNA library and the preparation of the hybrid sequencing library, the nucleic acid target (e.g., low-abundance transcript) of the target-specific primer can be the same. During the preparation of the hybrid cDNA library and the preparation of the hybrid sequencing library, one or more of the nucleic acid targets (e.g., low-abundance transcripts) of the target-specific primer can be different. In some embodiments, one or more nucleic acid targets are selectively extended and / or amplified by target-specific primers during the preparation of the hybrid cDNA library, and are selectively extended and / or amplified by target-specific primers during the preparation of the hybrid sequencing library. In some embodiments, one or more nucleic acid targets are selectively extended and / or amplified by target-specific primers during the preparation of the hybrid cDNA library, and are not selectively extended and / or amplified by target-specific primers during the preparation of the hybrid sequencing library. In some embodiments, one or more nucleic acid targets are not selectively extended and / or amplified by target-specific primers during the preparation of the hybrid cDNA library, and are selectively extended and / or amplified by target-specific primers during the preparation of the hybrid sequencing library. In some embodiments, one or more nucleic acid targets (e.g., low abundance transcripts) are selectively extended and / or amplified by multiple target-specific primers (e.g., target-specific primers amplify alternative transcripts of the same gene) during hybrid cDNA library preparation and / or hybrid sequencing library preparation.

[0329] In different embodiments, the amplification cycle number during the preparation of hybrid cDNA library and / or hybrid sequencing library can be different.In some embodiments, method disclosed herein can use less PCR circulation during the preparation of cDNA library and / or sequencing library, to realize the sufficient amplification of low abundance species (for example, low expresser).In some embodiments, the amplification cycle number during the preparation of hybrid cDNA library can be less than the amplification cycle number during the preparation of non-hybrid cDNA library, or less about: 1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100, or the number or scope between any two values ​​in these values. In some embodiments, the number of amplification cycles during hybrid sequencing library preparation can be less than, or about less than, the number of amplification cycles during non-hybrid sequencing library preparation by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range between any two of these values.

[0330] In some embodiments, the sample includes an unnormalized nucleic acid library, a partially normalized nucleic acid library, or a nucleic acid library normalized by other methods, such as a cDNA library, a genomic DNA library, etc. In some embodiments, the sample may include a pooled unnormalized nucleic acid library, such as a pooled unnormalized nucleic acid library constructed from more than one unnormalized nucleic acid library each representing a single cell. In some embodiments, the unnormalized nucleic acid library is a cDNA library. In some embodiments, the unnormalized nucleic acid library is a genomic library. In some embodiments, the methods, compositions, and kits disclosed herein can complete or supplement library normalization strategies known in the art. Exemplary library normalization methods are described in U.S. Patent Application Publications US 2017 / 0073730 and 2017 / 0342484; the contents of each of these patent application publications are incorporated herein by reference in their entirety. The methods disclosed herein are combined with a variety of 3'-based transcript counting methods (e.g., Rhapsody TM Assay (Becton, Dickinson and Company (Franklin Lakes, NJ)), Chromium TM Compatible with Single Cell 3' Solution (10X Genomics (San Francisco, CA)).

[0331] Whole transcriptome analysis (WTA)

[0332] Figure 4Schematic illustration of a non-limiting exemplary workflow for full transcriptome analysis using adapter ligation and random priming. Barcode 408 (e.g., random barcode) can be attached (e.g., conjugated, covalently attached, non-covalently attached) to solid support 407 (e.g., beads). Barcode 408 can include a target binding region (e.g., poly (dT) tail 403t), which can be combined with RNA transcripts (e.g., polyadenylated RNA transcripts 401) or other nucleic acid targets by poly (dA) tail 402r for labeling or barcoding (e.g., unique labeling). Reverse transcription reaction 400a can be performed on a solid support. Excess barcodes attached to the solid support can be removed (e.g., by washing, by a magnet). Reverse transcription reaction 400a can produce cDNA 409 of the first strand label attached to solid support 407 (beads illustrated here). The first strand labeled cDNA 409 may include cDNA 402c1 (the reverse complement of RNA sequence 402r) and may include a poly (dT) sequence 403t. The first strand labeled cDNA 409 may also include a number of tags, such as a unique molecular index (UMI) 404, a cell label (CL) 405, and a universal PCR handle (Univ) 406a (which may include or may be, for example, a binding site for a sequencing library amplification primer, such as a read 1 sequencing primer). The universal PCR handle may include a first universal primer, its complementary sequence, its reverse complementary sequence, its partial sequence, or a combination thereof.

[0333] The first strand labeled cDNA 409 can undergo second strand synthesis 400b to produce a double strand labeled cDNA molecule 410. The synthesis of the second strand can be carried out by contacting the labeled cDNA molecule-mRNA hybrid with a nicking enzyme (such as RNase H), which can form a nick in the mRNA 401 hybridized with the labeled cDNA molecule 409, thereby producing a nicked mRNA. The nicked mRNA can be used as a primer and extended using a polymerase (e.g., DNA Pol I) to incorporate the sequence of the first strand. The polymerase can include 5'-3' exonuclease activity. The polymerase can degrade the downstream mRNA nick used as a second strand synthesis primer. A ligase can be used to connect the extended sequences together to produce a second strand (e.g., a double strand labeled cDNA molecule 410 containing an antisense cDNA). The double strand labeled cDNA molecule 410 can be end-blunted and A-tailed at the free end to prepare for the adapter connection 400c. The double-stranded labeled cDNA molecule 410 can be contacted with an adapter 421. The adapter 421 can be single-stranded, partially double-stranded, or completely double-stranded. The adapter 421 can include a 5' overhang, which can include a first or second universal primer sequence. The adapter 421 can include a free 5' phosphate (P), which can be connected to the 3' hydroxyl of the double-stranded labeled cDNA molecule 410. The adapter 421 can be connected to the two chains of the double-stranded labeled cDNA molecule 410, thereby generating an adapter-connected double-stranded labeled cDNA molecule 420.

[0334] In some embodiments, the adapter comprises a sequence of a first universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof. In some embodiments, the first universal primer comprises an amplification primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof. In some embodiments, the first universal primer comprises a sequencing primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof. In some embodiments, the sequencing primer comprises an Illumina sequencing primer. In some embodiments, the sequencing primer comprises a portion of an Illumina sequencing primer. In some embodiments, the sequencing primer comprises a P7 sequencing primer. In some embodiments, the sequencing primer comprises a portion of a P7 sequencing primer. In some embodiments, the sequencing primer comprises a sequencing library amplification primer. In some embodiments, the sequencing primer comprises a read 1 sequencing primer. In some embodiments, the sequencing primer comprises a read 2 sequencing primer. In some embodiments, the adapter comprises a sequence of a second universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof. In some embodiments, the first universal sequence and the second universal sequence are identical. In some embodiments, the first universal sequence and the second universal sequence are different.

[0335] The term "adapter" can refer to a single-stranded, partially double-stranded or double-stranded oligonucleotide of at least 2, 5, 10, 15, 20 or 25 bases that can be connected to the end of a nucleic acid. The adaptor sequence can include, for example, a priming site, a complement of the priming site, and a recognition site for an endonuclease, a common sequence, and a promoter. The adaptor can be completely or substantially double-stranded. A double-stranded adaptor can include two oligonucleotides that are at least partially complementary. The adaptor can be phosphorylated or unphosphorylated on one or both chains. The adaptor can have a double-stranded portion and a single-stranded overhang end that is completely or partially complementary to the overhang (e.g., produced by a restriction enzyme or a polymerase). In some embodiments, the adaptor is a sequencing adaptor. In some embodiments, the adaptor is a single-stranded polynucleotide. In some embodiments, the adaptor is a double-stranded polynucleotide. In some embodiments, the length of the chain of the adaptor or adaptor is 2-30 nucleotides.In some embodiments, an adaptor (or a chain of adaptors) can comprise at least 2 nucleotides, e.g., at least 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, 59, 60, 61, 62, 63, 64, 65 , 44, 45, 46, 47, 48, 49, or 50 nucleotides, including ranges between any two of the recited values, for example, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-2 0, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14 , 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12 or 10-11 nucleotides.The adapter (or a strand of the adapter) can comprise a nucleic acid sequence of at least 2 nucleotides of the sequence of the first universal primer, the amplification primer, the sequencing primer, the complementary sequence thereof, the partial sequence thereof, or a combination thereof, for example, at least 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, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, including ranges between any two of the listed values, for example, 2-50, 2-45, 2-40, 2-35, 2-30, 2-25, 2-20, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 6-50 of the sequence of the first universal primer, an amplification primer, a sequencing primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof. , 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25 , 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides.

[0336] The adapter-ligated double-stranded labeled cDNA molecules 420 can be denatured 400d to produce adapter-ligated labeled cDNA molecules 430, which can be used as a template for full-length cDNA amplification using primers 431 and 432 (which anneal to the first and / or second universal primer sequences and adapters in the universal PCR handle), thereby producing amplified adapter-ligated labeled cDNA molecules 440. The product 440 can be contacted with a random primer 442 (e.g., a degenerate primer having a length of less than, about less than, at least less than, or at most less than: 4, 5, 6, 7, 8, 9, 10 or more nucleotides) and undergo random priming 440f. The random primer 442 can include an overhang (e.g., a binding site for a sequencing library amplification primer, such as a read 2 sequencing primer) that includes a universal PCR handle (Univ) 406b. Random primers 442 can bind to different positions along the coding sequence of all transcripts and extend to produce extension products 450 (e.g., linear amplification products). Extension products 450 include cDNA 402c2 with different lengths depending on the binding site of random primers 442. Extension products 450 can be amplified with sequencing library amplification primers 451 and 452. Library amplification 400g can add sequencing adapters 461 and 462 (e.g., P5 and P7 sequences) and sample indexes 463 (e.g., i5, i7) through the overhangs in the library forward primer 451 and the library reverse primer 452. Library amplicon 460 can be sequenced and subjected to the downstream methods of the present disclosure. Paired end sequencing to generate 150 bp x 2 sequencing reads can reveal cell markers, unique molecular indexes, poly(A) tails and / or genes (or partial sequences of genes) on read 1, genes (or partial sequences of genes) and / or poly(A) tails on read 2, and sample indexes on reads of index 1. Methods, compositions, systems, devices and kits for whole transcriptome amplification using random barcodes have been previously disclosed, for example, in U.S. Patent Publication No. 2016 / 0312276, the contents of which are expressly incorporated herein by reference in their entirety.

[0337] In some embodiments, each of the more than one amplicon comprises at least a portion of: a first universal sequence, a second universal sequence, or both. In some embodiments, the first universal sequence and the second universal sequence are identical. In some embodiments, the first universal sequence and the second universal sequence are different.

[0338] Expression profiling using target-specific multiplex primer sets

[0339] Figure 5506a (which may include or may be, for example, a binding site for a sequencing library amplification primer, such as a binding site for a read 1 sequencing primer). The universal PCR handle may include a first universal primer, a complementary sequence thereof, a partial sequence thereof, or a combination thereof. Reverse transcription reaction 500a can be performed on a solid support. Excess barcodes attached to the solid support can be removed (e.g., by washing, by a magnet). The reverse transcription reaction can produce a first strand labeled cDNA 510. The first strand labeled cDNA 510 can include cDNA 501c1 (the reverse complementary sequence of the mRNA sequence 501). The first strand labeled cDNA 510 can undergo a first round of multiplex amplification ("multiplex PCR 1500b") using a set of target-specific PCR 1 reverse primers 512 and a universal oligonucleotide forward primer 511 comprising a universal primer sequence (or its complement). Multiplex PCR 1 500b can include 1-30 cycles (e.g., 15 cycles). Multiplex PCR 1 amplicon 520 can include cDNA 501c2 (whose length depends on the binding site of PCR 1 reverse primer 512 within cDNA 501c1). Multiplex PCR 1 amplicon 520 can undergo a second multiplex amplification ("Multiplex PCR 2 500c") using a set of nested target-specific PCR 2 reverse primers 521 and a universal oligonucleotide forward primer 511 comprising a universal primer sequence (or its complement). Nested target-specific PCR 2 reverse primers 521 can include an overhang, which can include or can be, for example, a universal PCR handle (Univ) 506b. Multiplex PCR 2 500c can include 1-30 cycles (e.g., 8 cycles). Multiplex PCR 2 amplicon 530 can include cDNA 501c3 (whose length depends on the binding site of PCR 2 reverse primer 521 within cDNA 501c2). Multiplex PCR 2 amplicon 530 can undergo a third round of amplification (library amplification 500c) using sequencing library amplification primers 531 and 532.The library amplification 500d can add sequencing adapters 541 and 542 (e.g., P5 and P7 sequences) and sample indexes 543 (e.g., i5, i7) through the overhangs in the library forward primer 531 and the library reverse primer 532. The library amplicon 540 can be sequenced and subjected to the downstream methods of the present disclosure. Sequencing using 150bp×2 sequencing can reveal cell markers, unique molecular indexes, poly(A) tails and / or genes (or partial sequences of genes) on read 1, genes (or partial sequences of genes) and / or poly(A) tails on read 2, and sample indexes on index 1 reads.

[0340] Hybrid cDNA library preparation and / or hybrid sequencing library preparation

[0341] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide, each oligonucleotide comprising a first universal sequence and a barcode sequence; extending more than one oligonucleotide hybridized to more than one nucleic acid target to produce more than one first-strand barcoded polynucleotide; and synthesizing more than one second-strand barcoded polynucleotide using more than one first-strand barcoded polynucleotide as a template to produce more than one double-stranded barcoded polynucleotide. The method may include connecting an adapter to more than one double-stranded barcoded polynucleotide, wherein the adapter comprises a second universal sequence. The method may include amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and a second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of a nucleic acid target, the first universal sequence, the second universal sequence, a complement thereof, and / or a portion thereof, and amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0342] In some embodiments, the method includes using random primers and the first more than one barcoded amplicon and / or the second more than one barcoded amplicon or its product as a template for random priming and extension. In some embodiments, performing random priming and extension includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon and the second more than one barcoded amplicon or its product. In some embodiments, the method includes using a target-specific primer and the first more than one barcoded amplicon and / or the second more than one barcoded amplicon or its product as a template for extension and / or amplification. In some embodiments, performing extension and / or amplification includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon and / or the second more than one barcoded amplicon. In some embodiments, the sequencing adapter comprises a sequencing primer binding site or a sequencing primer. In some embodiments, the sequencing primer is a read 1 sequencing primer or a read 2 sequencing primer.

[0343] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide, each oligonucleotide comprising a first universal sequence and a barcode sequence; extending more than one oligonucleotide hybridized to more than one nucleic acid target to produce more than one first-strand barcoded polynucleotide; and using more than one first-strand barcoded polynucleotide as a template to synthesize more than one second-strand barcoded polynucleotide to produce more than one double-stranded barcoded polynucleotide. The method may include connecting an adapter to more than one double-stranded barcoded polynucleotide, wherein the adapter comprises a second universal sequence. The method may include amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first more than one barcoded amplicon comprising a sequence of a nucleic acid target, a first universal sequence, a second universal sequence, a complement thereof, and / or a portion thereof. The method may include extending and / or amplifying using a target-specific primer and the first more than one barcoded amplicon or a product thereof as a template.

[0344] In some embodiments, the method includes random priming and extension using random primers and a first more than one barcoded amplicon or its product as a template. In some embodiments, the method includes amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets, thereby generating a second more than one barcoded amplicon comprising sequences of two or more of more than one nucleic acid targets and / or a portion thereof. In some embodiments, performing random priming and extension includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon and the second more than one barcoded amplicon or its product. In some embodiments, performing extension and / or amplification includes adding the sequence of a sequencing adapter to the first more than one barcoded amplicon. In some embodiments, the sequencing adapter comprises a sequencing primer binding site or a sequencing primer. In some embodiments, the sequencing primer is a read 1 sequencing primer or a read 2 sequencing primer.

[0345] In some embodiments, amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of more than one nucleic acid targets comprises amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of more than one nucleic acid targets and primers capable of amplifying a first universal sequence. In some embodiments, amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of more than one nucleic acid targets comprises amplifying more than one double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of more than one nucleic acid targets and primers capable of amplifying a second universal sequence. In some embodiments, extending more than one oligonucleotide to generate more than one first-strand barcoded polynucleotide comprises reverse transcribing one or more of the more than one nucleic acid targets.

[0346] In some embodiments, more than one first-strand barcoded polynucleotide comprises a barcoded single-stranded cDNA molecule. In some embodiments, more than one first-strand barcoded polynucleotide comprises a barcoded sequence of an antibody oligonucleotide molecule. In some embodiments, extending more than one oligonucleotide to produce more than one first-strand barcoded polynucleotide includes nucleic acid extension of one or more oligonucleotides in more than one oligonucleotide hybridized with one or more nucleic acid targets of more than one nucleic acid target. In some embodiments, an adapter is connected to the 5' end of more than one double-stranded barcoded polynucleotide. In some embodiments, an adapter is connected to the 3' end of more than one double-stranded barcoded polynucleotide. In some embodiments, an adapter is connected to both the 5' end and the 3' end of more than one double-stranded barcoded polynucleotide. In some embodiments, the method includes obtaining sequence information of the first more than one barcoded amplicon and / or the second more than one barcoded amplicon or its product. In some embodiments, each of the more than one amplicon comprises at least a portion of: a binding site for a sequencing library amplification primer (eg, read 1 sequencing primer, read 2 sequencing primer).

[0347] In some embodiments, two or more of more than one nucleic acid target include mRNA of low expression gene. Two or more of more than one nucleic acid target can include low abundance species. Two or more of more than one nucleic acid target can include medium abundance species. Two or more of more than one nucleic acid target can include low abundance species and medium abundance species. In some embodiments, the first more than one barcoded amplicon includes a full transcriptome amplification (WTA) product.

[0348] The first one or more barcoded amplicons may correspond to at least 10% of the mRNA of the single cell. The first one or more barcoded amplicons may correspond to at least 50% of the mRNA of the single cell. The first one or more barcoded amplicons may correspond to at least 90% of the mRNA of the single cell. In some embodiments, the first one or more barcoded amplicons can correspond to at least 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%, 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%, %, 52%, 53%, 54%, 55%, 56%, 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%, or a value or range between any two of these values.

[0349] The second more than one barcoded amplicon may correspond to at most 5% of the mRNA of a single cell. The second more than one barcoded amplicon may correspond to at most 10% of the mRNA of a single cell. The second more than one barcoded amplicon may correspond to at most 20% of the mRNA of a single cell. In some embodiments, the second more than one barcoded amplicon may correspond to at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% of the mRNA of a single cell. , 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%, or a value or range between any two of these values.

[0350] Adding the sequence of the adapter to more than one double-stranded barcoded polynucleotide includes connecting the adapter to more than one double-stranded barcoded polynucleotide. Adding the sequence of the adapter to more than one double-stranded barcoded polynucleotide can include adding the sequence of the adapter to more than one double-stranded barcoded polynucleotide by nucleic acid extension or amplification such as 1-100 cycles of extension or amplification. Amplification includes polymerase chain reaction. Amplifying more than one double-stranded barcoded polynucleotide can include amplifying more than one double-stranded barcoded polynucleotide by linear amplification. For example, linear amplification can include 1-100 cycles of amplification. Amplifying more than one double-stranded barcoded polynucleotide can include amplifying more than one double-stranded barcoded polynucleotide by polymerase chain reaction (PCR). PCR can include 1-100 cycles of PCR. In some embodiments, extending and / or amplifying includes performing 1-100 cycles of extension and / or amplification. Amplification can include polymerase chain reaction (PCR) amplification. In some embodiments, performing random priming and extension and / or amplification comprises performing 1-100 cycles of random priming and extension and / or amplification.Amplification may comprise polymerase chain reaction (PCR) amplification.

[0351] In different embodiments, the number of cycles of extension (e.g., nucleic acid extension) and / or amplification (e.g., linear amplification, PCR amplification) can be different. In some embodiments, amplification can include the following, or include about the following cycles of amplification: 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, 60, 70, 80, 90, 100, or a number or range between any two of these values. In some embodiments, linear amplification can include at least the following, or at most the following cycles of amplification: 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, 60, 70, 80, 90, or 100.

[0352] Heterozygous cDNA library preparation followed by non-heterozygous sequencing library preparation

[0353] Figure 6600a. A schematic illustration of a non-limiting exemplary workflow for performing hybrid cDNA library preparation followed by non-hybrid sequencing library preparation. A barcode 608 (e.g., a random barcode) can include a target binding region (e.g., a poly(dT) tail 606t) that can bind to an RNA transcript (e.g., a polyadenylated RNA transcript 602) or other nucleic acid target via a poly(dA) tail 606a for labeling or barcoding (e.g., a unique tag). The barcode 608 can be attached (e.g., conjugated, covalently attached, non-covalently attached) to a solid support (e.g., a bead). The reverse transcription reaction 600a can produce a first strand labeled cDNA 616. The first strand labeled cDNA 616 can include cDNA 604c1 (the reverse complement of RNA sequence 604r) and can include a poly(dT) sequence 606t. The first strand labeled cDNA 616 may also include a number of tags, such as a unique molecular index (UMI) 614, a cell label (CL) 612, and a universal PCR handle (Univ) 610a (which may include or may be, for example, a binding site for a sequencing library amplification primer, such as a read 1 sequencing primer). The universal PCR handle may include the first universal primer, its complementary sequence, its reverse complementary sequence, its partial sequence, or a combination thereof.

[0354] The first strand labeled cDNA 616 can undergo second strand synthesis 600b to produce a double strand labeled cDNA molecule 618. The synthesis of the second strand can be carried out by contacting the labeled cDNA molecule-mRNA hybrid with a nicking enzyme (such as RNase H), which can form a nick in the mRNA 602 hybridized with the labeled cDNA molecule 616, thereby producing a nicked mRNA. The nicked mRNA can be used as a primer and extended using a polymerase (e.g., DNA Pol I) to incorporate the sequence of the first strand. The polymerase can include 5'-3' exonuclease activity. The polymerase can degrade the downstream mRNA nick used as a second strand synthesis primer. A ligase can be used to connect the extended sequences together to produce a second strand (e.g., a double strand labeled cDNA molecule 618 containing an antisense cDNA). The double strand labeled cDNA molecule 618 can be end-blunted and A-tailed at the free end to prepare for the adapter connection 600c. The double-stranded labeled cDNA molecule 618 can be contacted with an adapter 622. The adapter 622 can be single-stranded, partially double-stranded, or completely double-stranded. The adapter 622 can include a 5' overhang, which can include a first or second universal primer sequence. The adapter 622 can include a free 5' phosphate (P), which can be connected to the 3' hydroxyl of the double-stranded labeled cDNA molecule 618. The adapter 622 can be connected to the two chains of the double-stranded labeled cDNA molecule 618, thereby generating an adapter-connected double-stranded labeled cDNA molecule 620.

[0355] The cDNA molecule 620 of the double-stranded mark that adapter connects can be used as the template of cDNA amplification 600d.Amplification 600d can include 1-30 circulation (for example, 18-20 circulation).CDNA amplification 600d can include full-length cDNA amplification and target-specific (for example, gene-specific) cDNA amplification.The cDNA molecule 620 of the double-stranded mark that adapter connects can be used as the template for full-length cDNA amplification (the primer 624 and 626n anneal to the first and / or second universal primer sequence and adapter (and therefore non-target specific)) for using primer 624 and 626n, so as to produce the cDNA molecule 630w of the mark that the adapter of amplification connects.The cDNA molecule 630w of the mark that the adapter of amplification connects can include WTA cDNA sequence 604c2w. The adapter-ligated double-stranded labeled cDNA molecule 620 can be used as a template for target-specific (e.g., gene-specific) primers 626s (e.g., target-specific multiplex primer sets) and primers 624 to produce amplified adapter-ligated labeled cDNA molecules 630s. Primers 626s can be specific to low-expression genes. Amplified adapter-ligated labeled cDNA molecules 630s can include gene-specific cDNA sequences 604c2s.

[0356] The cDNA amplicons 630w and 630s can be denatured 600e to produce adapter-ligated labeled cDNA molecules 634. The adapter-ligated labeled cDNA molecules 634 can be contacted with random primers 636 (e.g., degenerate primers having a length of less than, about less than, at least less than, or at most less than: 4, 5, 6, 7, 8, 9, 10 or more nucleotides) and undergo random primer extension 600f. The random primers 636 can include an overhang including a universal PCR handle (Univ) 610b (e.g., a binding site for a sequencing library amplification primer, such as a read 2 sequencing primer). The random primers 636 can bind to different positions along the coding sequence of all transcripts and extend to produce extension products 638 (e.g., linear amplification products). The extension products 638 include cDNAs 604c3 having different lengths depending on the binding site of the random primers 600f. Extension product 638 can be amplified with sequencing library amplification primers 640 and 642. Size selection, followed by library amplification 600g can add sequencing adapters 646 and 648 (e.g., P5 and P7 sequences) and sample index 650 (e.g., i5, i7) through the overhangs in the library forward primer 640 and the library reverse primer 642. Library amplicon 644 can be sequenced and subjected to the downstream methods of the present disclosure. Paired end sequencing to generate 150bp x 2 sequencing reads can reveal cell markers, unique molecular indexes, poly (A) tails and / or genes (or partial sequences of genes) on read 1, genes (or partial sequences of genes) and / or poly (A) tails on read 2, and sample indexes on index 1 reads.

[0357] In some embodiments, each of the more than one amplicon comprises at least a portion of: a first universal sequence, a second universal sequence, or both. In some embodiments, the first universal sequence and the second universal sequence are identical. In some embodiments, the first universal sequence and the second universal sequence are different.

[0358] Non-hybrid cDNA library preparation followed by heterozygous sequencing library preparation

[0359] Figure 7Schematic illustration of a non-limiting exemplary workflow for performing non-hybrid cDNA library preparation followed by hybrid sequencing library preparation. Barcode 708 (e.g., random barcode) can include a target binding region (e.g., poly (dT) tail 706t), which can bind to RNA transcripts (e.g., polyadenylated RNA transcripts 702) or other nucleic acid targets via poly (dA) tail 706a for labeling or barcoding (e.g., unique labeling). Barcode 708 can be attached (e.g., conjugated, covalently attached, non-covalently attached) to a solid support (e.g., beads). Reverse transcription reaction 700a can produce first strand labeled cDNA 716. First strand labeled cDNA 716 can include cDNA 704c1 (reverse complement of RNA sequence 704r) and can include poly (dT) sequence 706t. The first strand labeled cDNA 716 may also include a number of tags, such as a unique molecular index (UMI) 714, a cell label (CL) 712, and a universal PCR handle (Univ) 710a (which may include or may be, for example, a binding site for a sequencing library amplification primer, such as a read 1 sequencing primer). The universal PCR handle may include the first universal primer, its complementary sequence, its reverse complementary sequence, its partial sequence, or a combination thereof.

[0360] The first strand labeled cDNA 716 can undergo second strand synthesis 700b to produce a double-stranded labeled cDNA molecule 718. The synthesis of the second strand can be carried out by contacting the labeled cDNA molecule-mRNA hybrid with a nicking enzyme (such as RNase H), which can form a nick in the mRNA 702 hybridized with the labeled cDNA molecule 716, thereby producing a nicked mRNA. The nicked mRNA can be used as a primer and extended using a polymerase (e.g., DNA Pol I) to incorporate the sequence of the first strand. The polymerase can include 5'-3' exonuclease activity. The polymerase can degrade the downstream mRNA nick used as a second strand synthesis primer. A ligase can be used to connect the extended sequences together to produce a second strand (e.g., a double-stranded labeled cDNA molecule 718 containing an antisense cDNA). The double-stranded labeled cDNA molecule 718 can be end-blunted and A-tailed at the free end to prepare for the adapter connection 700c. The double-stranded labeled cDNA molecule 718 can be contacted with an adapter 722. The adapter 722 can be single-stranded, partially double-stranded, or completely double-stranded. The adapter 722 can include a 5' overhang, which can include a first or second universal primer sequence. The adapter 722 can include a free 5' phosphate (P), which can be connected to the 3' hydroxyl of the double-stranded labeled cDNA molecule 718. The adapter 722 can be connected to the two chains of the double-stranded labeled cDNA molecule 718, thereby generating an adapter-connected double-stranded labeled cDNA molecule 720.

[0361] The cDNA molecule 720 of the double-stranded mark of adapter connection can be used as the template for full-length cDNA amplification 700d (for example, WTA).Amplification 700d can include 1-30 cycles (for example, 18-20 cycles).The cDNA molecule 720 of the double-stranded mark of adapter connection can be used as the template for full-length cDNA amplification (the primers 724 and 726 are annealed to the first and / or second universal primer sequence and adapter in the universal PCR handle) using primers 724 and 726, so as to produce the cDNA molecule 730 (" WTA product ") of the mark of the adapter connection of amplification.The WTA product 730 of the mark of the adapter connection of amplification can include WTA cDNA sequence 704c2.

[0362] WTA product 730 can be denatured 700e, and can be contacted with random primer 736r (e.g., length is less than, is about less than, is at least less than, or is at most less than degenerate primer: 4, 5, 6, 7, 8, 9, 10 or more nucleotides) and target-specific (e.g., gene-specific) primer 736s, and undergo random and gene-specific primer extension 700f (e.g., hybrid library sequencing preparation). Random primer 736r can include an overhang (e.g., sequencing library amplification primer, such as a binding site for read 2 sequencing primer) comprising a universal PCR handle (Univ) 710b. Random primer 736r can bind to different positions along the coding sequence of all transcripts and extend to produce extension products (e.g., linear amplification products). The extension product 738 of random and gene-specific primer extension 700f comprises cDNA 704c3, and this cDNA 704c3 has different lengths according to the binding site of primer 736r and 736s.Extension product 738 can be amplified with sequencing library amplification primer 740 and 742.Size selection, library amplification 700g can add sequencing adapter 746 and 748 (for example, P5 and P7 sequences) and sample index 750 (for example, i5, i7) by the overhang in library forward primer 740 and library reverse primer 742 subsequently.Library amplicon 744 can be sequenced and subjected to the downstream method of the present disclosure.Paired end sequencing in order to produce 150bp x 2 sequencing reads can reveal cell markers, unique molecular index, poly (A) tail and / or gene (or partial sequence of gene) on read 1, gene (or partial sequence of gene) and / or poly (A) tail on read 2, and sample index on index 1 read. In some embodiments, each of the more than one amplicon comprises at least a portion of: a first universal sequence, a second universal sequence, or both. In some embodiments, the first universal sequence and the second universal sequence are identical. In some embodiments, the first universal sequence and the second universal sequence are different.

[0363] Combination of heterozygous / non-hybrid cDNA library preparation and heterozygous / non-hybrid sequencing library preparation

[0364] Figure 8 is a schematic representation of a non-limiting exemplary workflow of an alternative single cell RNA sequencing (scRNA-seq) embodiment including methods of hybrid cDNA library preparation and / or hybrid sequencing library preparation as disclosed herein. In some embodiments, whole transcriptome analysis (e.g. Figure 4The workflow of the method (depicted in FIG) includes: (1) cell capture and barcoding of transcripts using oligonucleotides (the oligonucleotides contain a single-stranded sequence of deoxythymidine (dT) and a unique molecular index (UMI)) 800a; (2) PCR adapter ligation 800b; (3) whole transcriptome amplification using ligated PCR adapters and 3' adapters 800c; and (4) random priming and PCR to attach Illumina sequencing adapters 800e. In some embodiments, a set of target-specific multiplex primers (such as Figure 5The workflow for determining the expression profile of a set of target genes (depicted in ) includes: (1) cellular capture and barcoding of transcripts using oligonucleotides (the oligonucleotides contain a single-stranded sequence of deoxythymidine (dT) and a unique molecular index (UMI)) 800a; (2) PCR adapter ligation 800b; (3) cDNA library preparation with multiplex target-specific primer sets 800d; and (4) random priming and PCR to attach Illumina sequencing adapters 800e. In some embodiments, a workflow for performing the methods disclosed herein comprising hybrid cDNA library preparation followed by non-hybrid sequencing library preparation includes: (1) cellular capture and barcoding of transcripts using oligonucleotides comprising a single-stranded sequence of deoxythymidine (dT) and a unique molecular index (UMI) 800a; (2) PCR adapter ligation 800b; (3) whole transcriptome amplification using ligated PCR adapters and 3' adapters 800c and cDNA library preparation with multiplex target-specific primer sets 800d; and (4) random priming and PCR to attach Illumina sequencing adapters 800e. In some embodiments, a workflow for performing the methods disclosed herein comprising non-hybrid cDNA library preparation followed by hybrid sequencing library preparation includes: (1) cellular capture and barcoding of transcripts using oligonucleotides comprising a single-stranded sequence of deoxythymidine (dT) and a unique molecular index (UMI) 800a; (2) PCR adapter ligation 800b; (3) whole transcriptome amplification using ligated PCR adapters and 3' adapters 800c; and (4) amplification with gene-specific primers 800f and random priming and PCR to attach Illumina sequencing adapters 800e. In some embodiments, a workflow for performing the methods disclosed herein comprising hybrid cDNA library preparation followed by hybrid sequencing library preparation includes: (1) cell capture and barcoding of transcripts using oligonucleotides comprising a single-stranded sequence of deoxythymidine (dT) and a unique molecular index (UMI) 800a; (2) PCR adapter ligation 800b; (3) whole transcriptome amplification 800c using ligated PCR adapters and 3' adapters and cDNA library preparation 800d with multiplex target-specific primer sets; and (4) amplification 800f with gene-specific primers and random priming and PCR to attach Illumina sequencing adapters 800e.

[0365] Alternative method for heterozygous cDNA library preparation followed by non-heterzygous sequencing library preparation

[0366] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide; and extending the more than one oligonucleotide in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotide and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. The method may include amplifying more than one first strand barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and TSO or a portion thereof to synthesize a first more than one second strand barcoded polynucleotide and produce a first more than one double strand barcoded polynucleotide, each second strand barcoded polynucleotide comprising a sequence of a nucleic acid target, a first universal sequence, TSO or a portion thereof; and amplifying more than one first strand barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize a second more than one second strand barcoded polynucleotide to produce a second more than one double strand barcoded polynucleotide, each second strand barcoded polynucleotide comprising a sequence of a nucleic acid target or a portion thereof. The method may include amplifying the first more than one double strand barcoded polynucleotide and / or the second more than one double strand barcoded polynucleotide using primers capable of hybridizing to a sequence or subsequence of the first universal sequence and TSO, thereby producing more than one barcoded amplicon comprising a sequence of two or more of the more than one nucleic acid targets and / or a portion thereof. The method can include amplifying a first one or more double-stranded barcoded polynucleotide and / or a second one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the one or more nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the one or more nucleic acid targets and / or a portion thereof.

[0367] The disclosure herein includes methods for labeling nucleic acid targets. In some embodiments, the method includes: hybridizing more than one nucleic acid target with more than one oligonucleotide; and extending the more than one oligonucleotide in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to produce more than one first-strand barcoded polynucleotides comprising TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. The method may include amplifying more than one first-strand barcoded polynucleotides using primers capable of hybridizing with the first universal sequence and TSO or a portion thereof to synthesize a first more than one second-strand barcoded polynucleotide and produce a first more than one double-stranded barcoded polynucleotide, each second-strand barcoded polynucleotide comprising a sequence of a nucleic acid target, a first universal sequence, TSO or a portion thereof. The method may include amplifying a first one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to a first universal sequence and a sequence or subsequence of a TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof, and amplifying the first one or more double-stranded barcoded polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

[0368] In some embodiments, the method includes amplifying more than one first-strand barcoding polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets to synthesize a second more than one second-strand barcoding polynucleotide to produce a second more than one double-stranded barcoding polynucleotide, each second-strand barcoding polynucleotide comprising a sequence of a nucleic acid target or a portion thereof. In some embodiments, each of the more than one oligonucleotides comprises a first universal sequence and a barcode sequence. In some embodiments, the template switching oligonucleotide comprises a second universal sequence. In some embodiments, the template switching oligonucleotide comprises a first universal sequence and a barcode sequence. In some embodiments, each of the more than one oligonucleotides comprises a second universal sequence. In some embodiments, the method includes amplifying a first more than one second-strand barcoding polynucleotide and / or a second more than one second-strand barcoding polynucleotide using primers capable of hybridizing with two or more of more than one nucleic acid targets. In some embodiments, a first more than one second strand barcoded polynucleotide and / or a second more than one second strand barcoded polynucleotide is amplified using primers capable of hybridizing to two or more of the more than one nucleic acid targets and a primer capable of hybridizing to the first universal sequence. In some embodiments, a first more than one second strand barcoded polynucleotide and / or a second more than one second strand barcoded polynucleotide is amplified using primers capable of hybridizing to two or more of the more than one nucleic acid targets and a primer capable of hybridizing to a sequence or subsequence of TSO.

[0369] In some embodiments, amplifying more than one first strand barcoded polynucleotide includes amplifying more than one first strand barcoded polynucleotide for 1-100 cycles. Amplifying more than one first strand barcoded polynucleotide may include linear amplification of more than one first strand barcoded polynucleotide. Amplifying more than one first strand barcoded polynucleotide may include polymerase chain reaction (PCR) amplification of more than one first strand barcoded polynucleotide. Amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide may include amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide for 1-100 cycles. Amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide may include amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide by linear amplification. Amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides may include amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides by polymerase chain reaction (PCR) amplification.

[0370] In different embodiments, the number of cycles of extension (e.g., nucleic acid extension) and / or amplification (e.g., linear amplification, PCR amplification) can be different. In some embodiments, amplification can include the following, or include about the following cycles of amplification: 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, 60, 70, 80, 90, 100, or a number or range between any two of these values. In some embodiments, linear amplification can include at least the following, or at most the following cycles of amplification: 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, 60, 70, 80, 90, or 100.

[0371] In some embodiments, the method comprises extending and / or amplifying using a target-specific primer and a first more than one barcoded amplicon and / or a second more than one barcoded amplicon as a template. In some embodiments, extending more than one oligonucleotide to produce more than one first-strand barcoded polynucleotide comprises reverse transcribing one or more nucleic acid targets of more than one nucleic acid targets to produce barcoded single-stranded cDNA molecules. In some embodiments, extending more than one oligonucleotide to produce more than one first-strand barcoded polynucleotide comprises nucleic acid extension of oligonucleotides in more than one oligonucleotide hybridized to one or more nucleic acid targets of more than one nucleic acid targets to produce barcoded single-stranded nucleic acid molecules.

[0372] Fig. 9Schematic representation of a non-limiting exemplary workflow for performing hybrid cDNA library preparation followed by hybrid sequencing library preparation. A barcode 908 (e.g., a random barcode) can include a target binding region (e.g., poly(dT)VN 907) that can bind to an RNA transcript (e.g., polyadenylated RNA transcript 902) or other nucleic acid target via a poly(dA) tail 906a for labeling or barcoding (e.g., a unique tag). In some embodiments, the 3' end of the poly(dT)VN sequence 907 includes at least one "VN" doublet (where each "V" is any one of A, C, or G, and where "N" is any one of A, G, C, or T). In some embodiments, the barcode is associated with a solid support (e.g., a bead). In some embodiments, the bead can be a polymeric bead functionalized with a barcode or a random barcode, such as a deformable bead or a gel bead (such as a gel bead from 10X Genomics (San Francisco, CA)). In some embodiments, the gel beads may include a polymer-based gel. For example, the gel beads may be produced by encapsulating one or more polymer precursors into droplets. After the polymer precursors are exposed to a promoter (e.g., tetramethylethylenediamine (TEMED)), the gel beads may be produced.

[0373] A reverse transcription reaction 900a can be performed. During reverse transcription 900a, upon reaching the 5' end of barcode 908, the terminal transferase activity of an enzyme (e.g., a reverse transcriptase such as Moloney murine leukemia virus (MMLV)) adds new additional nucleotides (e.g., deoxycytidine, CCC 916) to the 3' end of the newly synthesized cDNA sequence strand 904c1 (the reverse complement of RNA sequence 904r). These CCC bases 916 can act as anchoring sites for a template switching oligonucleotide 918 that includes a sequence complementary to the tail sequence (e.g., rGrGrG 920). The template switching oligonucleotide 918 can include at least a portion of the first universal sequence, the second universal sequence, or both. Upon base pairing between rGrGrG 920 and the additional deoxycytidine stretch 916, the enzyme "switches" the template strand from the barcode 908 to the template switching oligonucleotide 918, and replication continues to the 5' end of the template switching oligonucleotide 918. Thus, the resulting first strand labeled cDNA 923 comprises the reverse complement sequence of the template switching oligonucleotide 918. The first strand labeled cDNA 923 may comprise cDNA 904c1 (the reverse complement sequence of RNA sequence 904r). The first strand labeled cDNA 923 may also comprise a number of tags, such as a unique molecular index (UMI) 914, a partitioning label (PL) 912, and a read 1 sequence 910a.

[0374] The term "template switching" may refer to the ability of a reverse transcriptase to switch from an initial nucleic acid sequence template to the 3' end of a new nucleic acid sequence template, which has little or no complementarity with the 3' end of a nucleic acid synthesized by the initial template. An example of template switching is the ability of a reverse transcriptase to switch from an initial nucleic acid sequence template / primer substrate to the 3' end of a new nucleic acid sequence template, which has little or no complementarity with the 3' end of a nucleic acid primer chain. Template switching allows, for example, the preparation of a DNA copy using a reverse transcriptase that switches from an initial nucleic acid sequence template to the 3' end of a new nucleic acid sequence template, which has little or no complementarity with the 3' end of a DNA synthesized by the initial template, thereby allowing the synthesis of a continuous product DNA that directly connects an adapter sequence to a target oligonucleotide sequence without being connected. Template switching may include the connection of an adapter, homopolymer tailing (e.g., polyadenylation), a random primer, or an oligonucleotide to which a polymerase may be associated. In any of the above-mentioned embodiments, template switching may be used to introduce a universal adapter sequence or its complement.

[0375] The cDNA 923 of the first strand labeling of the reverse complementary sequence comprising template switching oligonucleotide 918 can undergo reaction to produce a template for amplification 900b (e.g., denaturation), producing labeled cDNA molecule 923. The cDNA molecule 923 of labeling can be used as a template for cDNA amplification 900c. Amplification 900c can include 1-30 cycles (e.g., 18-20 cycles). CDNA amplification 900c can include full-length cDNA amplification and target-specific (e.g., gene-specific) cDNA amplification. The cDNA molecule 923 of labeling can be used as a template for full-length cDNA amplification using primers 924 and 926n, which anneal to the sequence (and therefore non-target specific) in read segment 1 910a and template switching oligonucleotide 918, thereby producing amplified labeled cDNA molecule 930w. The cDNA molecule 930w of the labeling of amplification can include cDNA sequence 904c2w. The labeled cDNA molecule 923 can be used as a template for target-specific (e.g., gene-specific) primers 926g (e.g., target-specific multiplex primer sets) and primers 924 to produce amplified labeled cDNA molecules 930s. Primers 926g can be specific to low-expression genes. Amplified labeled cDNA molecules 930s can include gene-specific cDNA sequences 904c2s.

[0376] Amplified labeled cDNA molecules 930s and amplified labeled cDNA molecules 930w can undergo process 900d, including enzymatic fragmentation, end repair, A-tailing, adapter ligation (including read 2 910b), and sample index PCR (adding library index 938 and P5 936 and P7 940 through sample index primer overhangs), thereby generating library amplicon 934. Library amplicon 934 can be sequenced and subjected to downstream methods of the present disclosure. Paired end sequencing to generate 150bp x 2 sequencing reads can reveal cell markers, unique molecular indexes, poly (A) tails and / or genes (or partial sequences of genes) on read 1, genes (or partial sequences of genes) and / or poly (A) tails on read 2, and sample indexes on index 1 reads.

[0377] In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase. In some embodiments, the viral reverse transcriptase is a Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0378] The cDNA library amplification product can be fragmented to produce more than one nucleic acid fragment. For example, the cDNA library amplification product can be fragmented for adapter connection. Fragmentation can include using mechanical (Covaris focused electroacoustic, nebulizer, sonication, vortex) or enzymatic (for example, fragmentase) fragmentation. Alternatively, the labeled amplicon can be fragmented by labeling (Nextera). In some embodiments, fragmentation can be partial fragmentation so that the fragments of the target nucleic acid molecule can have different lengths. Fragmentation can be performed by, for example, sonication, restriction enzyme digestion or any other suitable method. In some embodiments, two or more of more than one nucleic acid fragment have the same 5' end but different 3' ends. In some embodiments, each of more than one nucleic acid fragment has a length between 50nt and 10,000nt. In some embodiments, more than one nucleic acid fragment includes at least 2 nucleic acid fragments. In some embodiments, more than one nucleic acid fragment includes at least 10 nucleic acid fragments. In some embodiments, more than one nucleic acid fragment includes at least 100 nucleic acid fragments. In some embodiments, more than one nucleic acid fragment includes at least 1,000 nucleic acid fragments. In some embodiments, more than one nucleic acid fragment includes at least 10,000 nucleic acid fragments. In some embodiments, fragmentation includes restriction digestion of the first more than one amplicon. In some embodiments, at least 50% of more than one nucleic acid fragment include different lengths. In some embodiments, at least 80% of more than one nucleic acid fragment include different lengths. In some embodiments, at least 90% of more than one nucleic acid fragment include different lengths. In some embodiments, the fragments can be subjected to purification (e.g., washing) to remove fragments that do not contain molecular barcodes.

[0379] The method of connecting the adapter to the nucleic acid fragment is well known. The adapter can be double-stranded, single-stranded or partially single-stranded. In some aspects, the adapter is formed by two oligonucleotides with a region of complementarity (e.g., complete complementarity of about 10 to 30 or about 15 to 40 bases); so that when the two oligonucleotides are hybridized together, they form a double-stranded region. Optionally, any one or both oligonucleotides can have a region that is not complementary to another oligonucleotide, and form a single-stranded overhang at one end or both ends of the adapter. The single-stranded overhang can be about 1 to about 8 bases, or about 2 to about 4 bases. The overhang can be complementary to the overhang produced by cleavage with a restriction enzyme to promote "sticky ends" connection. The adapter can include other features, such as primer binding sites and restriction sites.

[0380] Non-heterozygous cDNA library preparation followed by split pooling of heterozygous and non-heterozygous sequencing library preparations

[0381] In some embodiments, the method includes dividing the first more than one second strand barcoded polynucleotide and the second more than one second strand barcoded polynucleotide into two pools, wherein amplification of the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide is performed (1) in one reaction using one of the two pools using primers that can hybridize to the first universal sequence and a sequence or subsequence of the TSO, and (2) in another reaction using the other of the two pools using primers that can hybridize to two or more of the more than one nucleic acid targets. In some embodiments, the method includes obtaining sequence information of the first more than one barcoded amplicons and / or the second more than one barcoded amplicons or products thereof. In some embodiments, the amplicons of the first pool and the second pool are re-pooled before obtaining the sequence information.

[0382] In some embodiments, the method includes dividing the first more than one second strand barcoded polynucleotide and the second more than one second strand barcoded polynucleotide into three pools, wherein the third pool includes another set of target-specific primers (different from the primers in the first two pools) for amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide. In some embodiments, the method includes dividing the first more than one second strand barcoded polynucleotide and the second more than one second strand barcoded polynucleotide into four or more pools, wherein each pool includes a different set of primers for amplifying the first more than one double-stranded barcoded polynucleotide and / or the second more than one double-stranded barcoded polynucleotide.

[0383] In various embodiments, the number of cycles of amplification (e.g., linear amplification, PCR amplification) of the first more than one second strand barcoded polynucleotides and / or the second more than one second strand barcoded polynucleotides of the two (or more) pools can be different. In some embodiments, the amplification can include the following, or include about the following cycles of amplification: 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, 60, 70, 80, 90, 100, or a number or range between any two of these values. In some embodiments, amplification can include at least the following, or at most the following cycles of amplification: 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, 60, 70, 80, 90, or 100.

[0384] Fig.101006a) for binding to an RNA transcript (e.g., polyadenylated RNA transcript 1002) or other nucleic acid target for labeling or barcoding (e.g., unique labeling). In some embodiments, the 3' end of the poly (dT) VN sequence 1007 comprises at least one "VN" doublet (where each "V" is any one of A, C, or G, and where "N" is any one of A, G, C, or T). In some embodiments, the barcode is associated with a solid support (e.g., a bead). In some embodiments, the beads can be polymeric beads functionalized with barcodes or random barcodes, such as deformable beads or gel beads (such as gel beads from 10XGenomics (San Francisco, CA)). In some embodiments, the gel beads can include polymer-based gels. For example, the gel beads can be produced by encapsulating one or more polymer precursors into droplets. After exposing the polymer precursors to a promoter (e.g., tetramethylethylenediamine (TEMED)), the gel beads can be produced.

[0385] A reverse transcription reaction 1000a can be performed. During reverse transcription 1000a, upon reaching the 5' end of the barcode 1008, the terminal transferase activity of an enzyme (e.g., a reverse transcriptase such as Moloney murine leukemia virus (MMLV)) adds new additional nucleotides (e.g., deoxycytidine, CCC 1016) to the 3' end of the newly synthesized cDNA sequence strand 1004c1 (the reverse complement of the RNA sequence 1004r). These CCC bases 1016 can act as anchoring sites for a template switching oligonucleotide 1018 that includes a sequence complementary to the tail sequence (e.g., rGrGrG 1020). The template switching oligonucleotide 1018 can include at least a portion of the first universal sequence, the second universal sequence, or both. Upon base pairing between rGrGrG1020 and the additional deoxycytidine stretch 1016, the enzyme "switches" the template strand from the barcode 1008 to the template switching oligonucleotide 1018, and continues replication to the 5' end of the template switching oligonucleotide 1018. Thus, the resulting first strand labeled cDNA 1023 comprises the reverse complement sequence of the template switching oligonucleotide 1018. The first strand labeled cDNA 1023 may comprise cDNA 1004c1 (the reverse complement sequence of RNA sequence 1004r). The first strand labeled cDNA 1023 may also comprise a number of tags, such as a unique molecular index (UMI) 1014, a partitioning label (PL) 1012, and a read 1 sequence 1010a.

[0386] The first strand labeled cDNA 1023 comprising the reverse complement sequence of the template switching oligonucleotide 1018 can undergo a reaction to produce a template for amplification 1000b (e.g., denaturation), producing a labeled cDNA molecule 1023. The labeled cDNA molecule 1023 can be used as a template for cDNA amplification 1000c. Amplification 1000c can include 1-30 cycles (e.g., 18-20 cycles). The labeled cDNA molecule 1023 can be used as a template for full-length cDNA amplification using primers 1024 and 1026, which anneal to the sequence in read segment 1 1010a and the template switching oligonucleotide 1018 (and are therefore non-target specific), thereby producing an amplified labeled cDNA molecule 1030. The amplified labeled cDNA molecule 1030 can include a cDNA sequence 1004c2.

[0387] More than one amplified labeled cDNA molecules 1030 can be divided into two pools. In one pool, the amplified labeled cDNA molecules 1030 can be subjected to PCR 1000d2 (which adds read 2 1010b, library index 1038, and P5 1036 and P7 1040 through primer overhangs) with target-specific (e.g., gene-specific) primers to produce library amplicon 1034s. In another pool, the amplified labeled cDNA molecules 1030 undergo process 1000d1, including enzymatic fragmentation, end repair, A-tailing, adapter ligation (including read 2 1010b) and sample index PCR (adding library index 1038 and P5 1036 and P7 1040 through sample index primer overhangs), thereby producing library amplicon 1034w. Library amplicons 1034w and 1034s can be sequenced and subjected to downstream methods of the present disclosure.

[0388] the term

[0389] In at least some previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0390] Those skilled in the art will appreciate that for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in different orders. In addition, the steps and operations outlined are provided only as examples, and some steps and operations may be optional, combined into fewer steps and operations, or expanded to additional steps and operations without departing from the essence of the disclosed embodiments.

[0391] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art may convert from the plural to the singular and / or from the singular to the plural, where appropriate for the context and / or application. For the sake of clarity, various singular / plural arrangements may be expressly set forth herein. As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural reminders. Unless otherwise indicated, any reference to "or" in this article is intended to encompass "and / or".

[0392] Those skilled in the art will understand that, in general, the terms used herein, and particularly in the appended claims (e.g., the bodies of the appended claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). Those skilled in the art will further understand that if a specific number of an introduced claim statement is intended, such an expectation will be expressly stated in the claim, and in the absence of such a statement, no such expectation is present. For example, as an aid to understanding, the following appended claims may contain the use of the prepositions "at least one" and "one or more" to introduce claim statements. However, the use of such phrases should not be construed as implying that the introduction of a claim recitation by the indefinite article "a" or "an" will limit any specific claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the preposition "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim recitations. Furthermore, even if a specific number of introduced claim recitations is explicitly stated, one skilled in the art will recognize that such a statement should be interpreted as meaning at least the stated number (e.g., merely stating "two statements" without other modifiers means at least two statements, or two or more statements). Furthermore, in those cases where a convention similar to “at least one of A, B, and C, etc.” is used, such syntactic structure is generally intended in a sense that a person skilled in the art would understand the convention to mean (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).In those cases where a convention similar to "at least one of A, B, or C, etc." is used, such syntactic construction is generally intended in a sense that one skilled in the art would understand the convention to mean (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One skilled in the art would further understand that, in practice, any transitional words and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0393] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0394] As will be understood by those skilled in the art, for any and all purposes, such as in providing a written description, all ranges disclosed herein also include any and all possible sub-ranges and sub-range combinations thereof. Any enumerated range can be easily considered to fully describe and enable the same range to be divided into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily divided into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages ​​such as "up to", "at least", "greater than", "less than", etc. include the cited numbers and refer to the ranges that can be subsequently divided into sub-ranges as discussed above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, etc.

[0395] From the foregoing, it will be understood that for illustrative purposes, various embodiments of the present disclosure have been described herein, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Therefore, the various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. A method for labeling a nucleic acid target, the method comprising: include: (a) hybridizing more than one nucleic acid target with more than one oligonucleotide; (b) extending the more than one oligonucleotides in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence; (c) amplifying the more than one first-strand barcoded polynucleotides, (c1) amplifying the more than one first strand barcoding polynucleotides using primers capable of hybridizing to the first universal sequence and the TSO or a portion thereof to synthesize first more than one second strand barcoding polynucleotides and produce first more than one double-stranded barcoding polynucleotides, each of the second strand barcoding polynucleotides comprising a sequence of the nucleic acid target, the first universal sequence, the TSO or a portion thereof; and (c2) amplifying the more than one first-strand barcoding polynucleotide using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize a second more than one second-strand barcoding polynucleotide to produce a second more than one double-stranded barcoding polynucleotide, each of the second-strand barcoding polynucleotides comprising a sequence of the nucleic acid target or a portion thereof; and (d1) amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides using primers that are capable of hybridizing to the first universal sequence and a sequence or a subsequence of the TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the one or more nucleic acid targets and / or a portion thereof.

2. The method according to claim 1, further comprising (d2) amplifying the first one or more double-stranded barcoded polynucleotides and / or the second one or more double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

3. A method for labeling a nucleic acid target, the method comprising: include: (a) hybridizing more than one nucleic acid target with more than one oligonucleotide; (b) extending the more than one oligonucleotides in the presence of a reverse transcriptase and a template switching oligonucleotide (TSO) to generate more than one first-strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the more than one oligonucleotides and / or the template switching oligonucleotide comprises a first universal sequence and a barcode sequence; (c1) amplifying the more than one first strand barcoding polynucleotides using primers capable of hybridizing to the first universal sequence and the TSO or a portion thereof to synthesize first more than one second strand barcoding polynucleotides and produce first more than one double-stranded barcoding polynucleotides, each of the second strand barcoding polynucleotides comprising a sequence of the nucleic acid target, the first universal sequence, the TSO or a portion thereof; and (d) amplifying the first one or more double-stranded barcoded polynucleotides: (d1) amplifying the first one or more double-stranded barcoded polynucleotides using primers capable of hybridizing to the first universal sequence and a sequence or subsequence of the TSO, thereby generating a first one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof, and (d2) amplifying the first one or more double-stranded barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets, thereby generating a second one or more barcoded amplicons comprising sequences of two or more of the more than one nucleic acid targets and / or a portion thereof.

4. The method according to claim 3, further comprising (c2) amplifying the more than one first-strand barcoding polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets to synthesize second more than one second-strand barcoding polynucleotides to produce second more than one double-stranded barcoding polynucleotides, each of which comprises a sequence of the nucleic acid target or a portion thereof.

5. The method of any one of claims 1-4, wherein each of the more than one oligonucleotides comprises a first universal sequence and a barcode sequence. The method of claim 5 , wherein the template switching oligonucleotide comprises a second universal sequence.

7. The method of any one of claims 1-6, wherein the template switching oligonucleotide comprises a first universal sequence and a barcode sequence.

8. The method of any one of claims 1-7, wherein each of the more than one oligonucleotides comprises a second universal sequence.

9. The method according to any one of claims 1 to 8, wherein (c2) amplifying the more than one first-strand barcoded polynucleotides comprises amplifying the first more than one second-strand barcoded polynucleotides and / or the second more than one second-strand barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets and a primer capable of hybridizing to the first universal sequence.

10. The method of any one of claims 1 to 9, wherein (c2) amplifying the more than one first strand barcoded polynucleotides comprises amplifying the first more than one second strand barcoded polynucleotides and / or the second more than one second strand barcoded polynucleotides using primers capable of hybridizing to two or more of the more than one nucleic acid targets and primers capable of hybridizing to a sequence or a subsequence of the TSO.

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