METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.
1 - 27 . (canceled)
28 . A method for cellular analysis, the method comprising:
(a) providing a cell comprising an analyte and a nucleic acid sequence;
(b) coupling an antibody to the analyte, wherein the antibody is associated with a first nucleic acid molecule comprising a first barcode sequence;
(c) using the first nucleic acid molecule and a second nucleic acid molecule to generate a third nucleic acid molecule, wherein the second nucleic acid molecule comprises a second barcode sequence, and wherein the third nucleic acid molecule comprises:
(i) the first barcode sequence or complement thereof, and
(ii) the second barcode sequence or complement thereof; and
(d) using the nucleic acid sequence and a fourth nucleic acid molecule to generate a fifth nucleic acid molecule, wherein the fourth nucleic acid molecule comprises the second barcode sequence, and wherein the fifth nucleic acid molecule comprises:
(i) a sequence from the nucleic acid sequence or complement thereof, and
(ii) the second barcode sequence or complement thereof.
29 . The method according to claim 28 , wherein, prior to (c), the cell is placed in a partition comprising the second nucleic acid molecule.
30 . The method according to claim 28 , further comprising:
(e) capturing the third nucleic acid molecule or the fifth nucleic acid molecule with an oligonucleotide molecule comprising a capture sequence.
31 . The method according to claim 30 , capturing the third nucleic acid molecule and the fifth nucleic acid molecule with oligonucleotide molecules that comprise identical capture sequences.
32 . The method according to claim 31 , wherein the identical capture sequences comprise poly-thymine (poly-T) sequences.
33 . The method according to claim 28 , wherein the second nucleic acid molecule or the fourth nucleic acid molecule comprises a unique molecular sequence segment.
34 . The method according to claim 30 , further comprising sequencing the third nucleic acid molecule or derivative thereof and sequencing the fifth nucleic acid molecule or derivative thereof.
35 . The method according to claim 30 , further comprising:
(e) identifying the first barcode sequence and the second barcode sequence; and
(f) using the first barcode sequence and the second barcode sequence identified in (e) to identify the analyte of the cell and identify the analyte as derived from the cell, respectively.
36 . The method according to claim 28 , wherein the analyte is a protein.
37 . The method according to claim 36 , wherein the protein is located on an extracellular surface of the cell.
38 . The method according to claim 28 , wherein the nucleic acid sequence is a ribonucleic acid (RNA) sequence.
39 . The method according to claim 38 , wherein the RNA sequence is a messenger RNA (mRNA) sequence.
40 . The method according to claim 39 , wherein the mRNA sequence comprises a synthetic nucleic acid sequence.
41 . The method according to claim 28 , wherein (c) and (d) are performed simultaneously.
42 . The method according to claim 28 , wherein (c) and (d) are performed in a partition among a plurality of partitions.
43 . The method according to claim 42 , wherein the partition comprises a ligase or a reverse transcriptase.
44 . The method according to claim 42 , further comprising co-partitioning the cell with a bead, wherein the bead comprises the second nucleic acid molecule or the fourth nucleic acid molecule.
45 . The method according to claim 44 , wherein the bead is a gel bead.
46 . The method according to claim 42 , wherein the partition is a droplet.
47 . The method according to claim 42 , wherein the partition is a well.
48 . The method according to claim 47 , wherein the well is a microwell.
49 . The method according to claim 44 , wherein bead comprises at least 1,000 nucleic acid molecules comprising the second barcode sequence.
50 . The method according to claim 28 , further comprising attaching a sixth nucleic acid molecule to the third nucleic acid molecule or derivative thereof.
51 . The method according to claim 50 , wherein the sixth nucleic acid molecule comprises a biotin tag.
52 . The method according to claim 50 , wherein the sixth nucleic acid molecule comprises a flow cell attachment sequence.
53 . The method according to claim 50 , wherein the sixth nucleic acid molecule comprises a unique molecular sequence segment.
54 . The method according to claim 28 , further comprising ligating a sixth nucleic acid molecule to the fourth nucleic acid molecule.
55 . The method according to claim 54 , wherein the sixth nucleic acid molecule comprises a biotin tag.
56 . The method according to claim 54 , wherein the sixth nucleic acid molecule comprises a flow cell attachment sequence.
57 . The method according to claim 54 , wherein the sixth nucleic acid molecule comprises a unique molecular sequence segment.