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 identifying a protein as originating from a single cell, comprising:
(a) exposing said protein of said single cell to one or more antibodies such that an antibody of said one or more antibodies that is specific to said protein couples to said protein, wherein said antibody is conjugated to a nucleic acid antibody barcode molecule comprising an antibody barcode sequence;
(b) co-partitioning said single cell comprising said protein coupled to said antibody with a single bead in a partition, wherein said single bead comprises a plurality of nucleic acid cell barcode molecules each comprising a cell barcode sequence;
(c) performing one or more reactions to generate, from said nucleic acid antibody barcode molecule and a nucleic acid cell barcode molecule from said plurality of nucleic acid cell barcode molecules, a synthesized nucleic acid molecule comprising (i) said antibody barcode sequence or a complement thereof, and (ii) said cell barcode sequence or a complement thereof;
(d) using a sequencer to sequence at least a portion of said synthesized nucleic acid molecule or a derivative thereof, to identify said antibody barcode sequence and said cell barcode sequence, and
(e) using said antibody barcode sequence and said cell barcode sequence to identify said protein as originating from said single cell.
2. The method of claim 1 , wherein said plurality of nucleic acid cell barcode molecules comprises at least 100,000 nucleic acid cell barcode molecules.
3. The method of claim 1 , wherein (c) is performed in said partition.
4. The method of claim 3 , further comprising releasing or removing said synthesized nucleic acid molecule from said partition.
5. The method of claim 4 , further comprising subjecting said synthesized nucleic acid molecule to one or more additional reactions subsequent to releasing or removing said synthesized nucleic acid molecule from said partition.
6. The method of claim 5 , wherein said one or more additional reactions comprise polymerase chain reaction.
7. The method of claim 5 , wherein said one or more additional reactions comprise addition of one or more functional sequences to said synthesized nucleic acid molecule, wherein said one or more functional sequences are configured to permit attachment to a flow cell of said sequencer.
8. The method of claim 1 , wherein said single bead is a gel bead.
9. The method of claim 8 , further comprising, prior to (c), releasing said nucleic acid cell barcode molecule from said single bead.
10. The method of claim 9 , wherein said nucleic acid cell barcode molecule is released from said single bead upon exposure to a chemical stimulus in said partition.
11. The method of claim 1 , wherein said protein is a cell surface protein.
12. The method of claim 1 , wherein each of said plurality of nucleic acid cell barcode molecules comprises an identifier sequence separate from said cell barcode sequence, and wherein said identifier sequence is different for each nucleic acid cell barcode molecule of said plurality of nucleic acid cell barcode molecules.
13. The method of claim 1 , wherein said nucleic acid cell barcode molecule further comprises a sequence that is complementary to a portion of said nucleic acid antibody barcode molecule.
14. The method of claim 13 , wherein said sequence is a poly-thymine (poly-T) sequence.
15. The method of claim 14 , wherein (c) comprises hybridizing said poly-T sequence to said portion of said nucleic acid antibody barcode molecule, wherein said portion of said nucleic acid antibody barcode molecule comprises a poly-adenine (poly-A) sequence.
16. The method of claim 1 , wherein said one or more reactions comprise a nucleic acid amplification reaction.
17. The method of claim 1 , wherein said one or more reactions comprise a reverse transcription reaction.
18. The method of claim 1 , wherein said one or more reactions comprise a template switching reaction.
19. The method of claim 1 , wherein said synthesized nucleic acid molecule comprises a functional sequence that is configured to permit attachment to a flow cell of said sequencer.
20. The method of claim 1 , further comprising, subsequent to (c), adding one or more priming sequences to said synthesized nucleic acid molecule for primer extension of said synthesized nucleic acid molecule or a derivative thereof in said sequencer.
21. The method of claim 1 , wherein, in at least (a), said antibody is covalently attached to said nucleic acid antibody barcode molecule.
22. The method of claim 1 , wherein said partition is a droplet among a plurality of droplets.
23. The method of claim 1 , wherein said partition is a well among a plurality of wells.
24. The method of claim 1 , wherein, in (b), each of said plurality of nucleic acid cell barcode molecules comprises an identical cell barcode sequence, wherein said single bead is from a plurality of beads, and wherein said identical cell barcode sequence is different from cell barcode sequences of nucleic acid cell barcode molecules comprised in other beads of said plurality of beads.
25. The method of claim 10 , wherein said chemical stimulus is a reducing agent.
26. The method of claim 1 , wherein, in (b), said nucleic acid cell barcode molecule is covalently coupled to said single bead.
27. The method of claim 26 , wherein, in (b), said nucleic acid cell barcode molecule is coupled to said single bead via a disulfide bond.
28. The method of claim 1 , wherein said single bead comprises a disulfide bond.
29. The method of claim 1 , wherein said nucleic acid cell barcode molecule is not releasably coupled to said single bead.
30. The method of claim 1 , wherein said plurality of nucleic acid cell barcode molecules comprises at least 1,000,000 nucleic acid cell barcode molecules.
31. The method of claim 1 , wherein said nucleic acid cell barcode molecule is releasably coupled to said single bead.
32. The method of claim 24 , wherein said plurality of beads comprises at least 100,000 different cell barcode sequences.
33. The method of claim 32 , wherein said plurality of beads comprises at least 1,000,000 different cell barcode sequences.
34. The method of claim 10 , wherein said single bead is degradable upon exposure to said stimulus in said partition.
35. The method of claim 1 , wherein, in (b), said partition comprises an enzyme.
36. The method of claim 35 , wherein said enzyme is a restriction enzyme.
37. The method of claim 9 , wherein said chemical stimulus cleaves a linkage from said nucleic acid cell barcode molecule to said single bead.
38. The method of claim 37 , wherein said linkage is a disulfide bond.