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. A method for cellular analysis, the method comprising:
(a) providing a cell comprising a protein and an mRNA;
(b) contacting an antibody with the protein of the cell, wherein the antibody is associated with a first nucleic acid molecule comprising a barcode sequence that corresponds to the antibody;
(c) placing the cell in a partition comprising a second nucleic acid molecule, the second nucleic acid molecule comprising a barcode sequence that corresponds to the partition;
(d) generating a third nucleic acid molecule from the first nucleic acid molecule and the second nucleic acid molecule, wherein the third nucleic acid molecule comprises the barcode sequence that corresponds to the antibody and the barcode sequence that corresponds to the partition; and
(e) generating a fourth nucleic acid molecule from the mRNA and the second nucleic acid molecule, the fourth nucleic acid molecule comprising a sequence from the mRNA or reverse complement thereof and the barcode sequence that corresponds to the partition.
2. The method according to claim 1 , further comprising:
(f) capturing the third nucleic acid molecule or the fourth nucleic acid molecule with an oligonucleotide molecule comprising a capture sequence.
3. The method according to claim 2 , further comprising capturing the third nucleic acid molecule and the fourth nucleic acid molecule with oligonucleotide molecules that comprise the same capture sequence.
4. The method according to claim 2 , wherein the third nucleic acid molecule or the fourth nucleic acid molecule is capable of hybridizing to the capture sequence.
5. The method according to claim 2 , wherein the capture sequence comprises a poly-thymine (poly-T) sequence.
6. The method according to claim 1 , further comprising ligating a fifth nucleic acid molecule to the third nucleic acid molecule.
7. The method according to claim 6 , wherein the fifth nucleic acid molecule comprises a biotin tag.
8. The method according to claim 6 , wherein the fifth nucleic acid molecule comprises a flow cell attachment sequence, a sequencing primer binding site, and a sample index sequence.
9. The method according to claim 6 , wherein the fifth nucleic acid molecule comprises a flow cell attachment sequence and a sequencing primer binding site.
10. The method according to claim 6 , wherein the fifth nucleic acid molecule comprises a unique molecular sequence segment.
11. The method according to claim 1 , further comprising ligating a fifth nucleic acid molecule to the fourth nucleic acid molecule.
12. The method according to claim 11 , wherein the fifth nucleic acid molecule comprises a biotin tag.
13. The method according to claim 11 , wherein the fifth nucleic acid molecule comprises a flow cell attachment sequence, a sequencing primer binding site, and a sample index sequence.
14. The method according to claim 11 , wherein the fifth nucleic acid molecule comprises a flow cell attachment sequence and a sequencing primer binding site.
15. The method according to claim 11 , wherein the fifth nucleic acid molecule comprises a unique molecular sequence segment.
16. The method according to claim 1 , wherein the mRNA comprises a synthetic nucleic acid sequence.
17. The method according to claim 1 , wherein (d) and (e) are performed simultaneously.
18. The method according to claim 1 , wherein (d) and (e) are performed in the partition.
19. The method according to claim 18 , wherein the partition comprises a ligase and/or a reverse transcriptase.
20. The method according to claim 1 , wherein the partition is a well.
21. The method according to claim 20 , wherein the well is a microwell.
22. The method according to claim 1 , wherein the third nucleic acid molecule and/or the fourth nucleic acid molecule comprise a flow cell attachment sequence, a sequencing primer binding site, and a sample index sequence.
23. The method according to claim 1 , wherein the third nucleic acid molecule and/or the fourth nucleic acid molecule comprise a flow cell attachment sequence and a sequencing primer binding site.
24. A method for cellular analysis, the method comprising:
(a) providing a cell comprising a protein and an mRNA;
(b) contacting an antibody with the protein of the cell, wherein the antibody is associated with a first nucleic acid molecule comprising a barcode sequence that corresponds to the antibody;
(c) placing the cell in a partition comprising a second nucleic acid molecule, the second nucleic acid molecule comprising a barcode sequence that corresponds to the partition;
(d) generating a third nucleic acid molecule from the first nucleic acid molecule and the second nucleic acid molecule, wherein the third nucleic acid molecule comprises the barcode sequence that corresponds to the antibody and the barcode sequence that corresponds to the partition;
(e) generating a fourth nucleic acid molecule from the mRNA and the second nucleic acid molecule, the fourth nucleic acid molecule comprising a sequence from the mRNA or reverse complement thereof and the barcode sequence that corresponds to the partition; and
(f) capturing the third nucleic acid molecule and the fourth nucleic acid molecule with oligonucleotide molecules comprising a common capture sequence.
25. A method for cellular analysis, the method comprising:
(a) providing a cell comprising a cell surface feature and a polynucleotide molecule;
(b) contacting a cell surface feature binding group with the cell surface feature of the cell, wherein the cell surface feature binding group is associated with a first nucleic acid molecule comprising a barcode sequence that corresponds to the cell surface feature binding group;
(c) placing the cell in a partition comprising a second nucleic acid molecule, the second nucleic acid molecule comprising a barcode sequence that corresponds to the partition;
(d) generating a third nucleic acid molecule from the first nucleic acid molecule and the second nucleic acid molecule, wherein the third nucleic acid molecule comprises the barcode sequence that corresponds to the cell surface feature binding group and the barcode sequence that corresponds to the partition; and
(e) generating a fourth nucleic acid molecule from the polynucleotide molecule and the second nucleic acid molecule, the fourth nucleic acid molecule comprising a sequence from the polynucleotide molecule or reverse complement thereof and the barcode sequence that corresponds to the partition.
26. The method according to claim 25 , wherein the polynucleotide molecule comprises a synthetic nucleic acid sequence.
27. The method according to claim 25 , wherein the polynucleotide molecule is a messenger ribonucleic acid (RNA).
28. The method according to claim 25 , wherein the cell surface feature is selected from the group consisting of a protein, an antigen, and a receptor.
29. The method according to claim 25 , wherein the cell surface feature binding group is an antibody or an antibody fragment.
30. The method according to claim 25 , further comprising:
(f) capturing the third nucleic acid molecule or the fourth nucleic acid molecule with an oligonucleotide molecule comprising a capture sequence.
31. The method according to claim 25 , wherein the cell surface feature is a protein, and wherein the protein is coupled to a surface of the cell.
32. The method according to claim 25 , wherein the second nucleic acid molecule is attached to a support.
33. The method according to claim 32 , wherein the second nucleic acid molecule is releasably attached to the support.
34. The method according to claim 32 , wherein the second nucleic acid molecule is reversibly coupled to the support through a chemical crosslink.
35. The method according to claim 32 , wherein the second nucleic acid molecule is reversibly coupled to the support through a disulfide bond.
36. The method according to claim 33 , wherein the partition further comprises an agent capable of releasing the second nucleic acid molecule from the support.
37. The method according to claim 36 , wherein the agent comprises a chemical reducing agent.
38. The method according to claim 32 , wherein the support is a bead.
39. The method according to claim 38 , wherein the bead is a solid bead.
40. The method according to claim 38 , wherein the bead is a magnetic bead.
41. The method according to claim 38 , wherein the bead comprises a polymer.
42. The method according to claim 38 , wherein the bead comprises a hydrogel.
43. The method according to claim 38 , wherein the bead is degradable upon exposure to a stimulus in the partition.
44. The method according to claim 25 , wherein the second nucleic acid molecule comprises a unique molecular identifier (UMI) sequence.
45. The method according to claim 25 , wherein the third nucleic acid molecule or derivative thereof, comprises a UMI sequence.
46. The method according to claim 25 , further comprising attaching a UMI sequence to the third nucleic acid molecule or derivative thereof.
47. The method according to claim 25 , wherein the fourth nucleic acid molecule or derivative thereof, comprises a UMI sequence.
48. The method according to claim 25 , further comprising attaching a UMI sequence to the fourth nucleic acid molecule or derivative thereof.
49. The method according to claim 25 , wherein the second nucleic acid molecule comprises a sequence complementary to a sequence of the first nucleic acid molecule.
50. The method according to claim 25 , wherein the second nucleic acid molecule comprises a sequence complementary to a sequence of the polynucleotide molecule.
51. The method according to claim 25 , further comprising sequencing the third nucleic acid molecule or derivative thereof, and the fourth nucleic acid molecule or derivative thereof.
52. The method according to claim 38 , wherein the bead is a semi-solid bead.