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 nucleic acid processing, comprising:
(a) providing a partition comprising a nucleic acid molecule and a bead, wherein said bead comprises a barcode oligonucleotide molecule that is coupled to said bead through a photo-labile linkage, and wherein said barcode oligonucleotide molecule comprises a barcode sequence;
(b) applying a photo-stimulus to release said barcode oligonucleotide molecule from said bead, which photo-stimulus cleaves said photo-labile linkage; and
(c) using (i) said nucleic acid molecule and (ii) said barcode oligonucleotide molecule released from said bead to generate a barcoded nucleic acid molecule, wherein said barcoded nucleic acid molecule comprises said barcode sequence or complement thereof.
2. The method of claim 1 , wherein said barcode oligonucleotide molecule comprises a primer sequence.
3. The method of claim 2 , wherein said barcode oligonucleotide molecule hybridizes to said nucleic acid molecule via said primer sequence.
4. The method of claim 1 , wherein said nucleic acid molecule is derived from a sample nucleic acid molecule.
5. The method of claim 4 , further comprising, performing a nucleic acid extension reaction using said sample nucleic acid molecule to generate said nucleic acid molecule.
6. The method of claim 1 , wherein (a) further comprises providing one or more primers in said partition.
7. The method of claim 6 , wherein (c) comprises using said one or more primers to generate said barcoded nucleic acid molecule in said partition.
8. The method of claim 1 , wherein (c) comprises performing a nucleic acid extension reaction on said nucleic acid molecule or derivative thereof to generate said barcoded nucleic acid molecule.
9. The method of claim 1 , further comprising, providing a lysate from a single cell.
10. The method of claim 9 , wherein said single cell is a cancer cell, a cell derived from a solid tumor, a cell derived from a hematological malignancy, or a circulating tumor cell.
11. The method of claim 9 , wherein said lysate comprises a sample nucleic acid molecule.
12. The method of claim 11 , wherein said nucleic acid molecule is generated from said sample nucleic acid molecule.
13. The method of claim 1 , wherein said nucleic acid molecule comprises genomic deoxyribonucleic acid (gDNA) or is generated from gDNA.
14. The method of claim 1 , wherein, in (a), said partition is a partition among a plurality of partitions.
15. The method of claim 1 , wherein said partition is a microwell.
16. The method of claim 1 , wherein said partition is a droplet.
17. The method of claim 1 , wherein said partition comprises a lysate from a single cell.
18. The method of claim 17 , wherein said single cell is a cancer cell, a cell derived from a solid tumor, a cell derived from a hematological malignancy, or a circulating tumor cell.
19. The method of claim 17 , wherein said lysate comprises a sample nucleic acid molecule.
20. The method of claim 19 , wherein said nucleic acid molecule is generated from said sample nucleic acid molecule.
21. The method of claim 1 , wherein said partition comprises a set of primer pairs for amplifying gDNA.
22. The method of claim 14 , wherein said barcode sequence identifies said partition from another partition of said plurality of partitions.
23. The method of claim 1 , further comprising removing said barcoded nucleic acid molecule or a derivative thereof from said partition.
24. The method of claim 23 , wherein said partition is a droplet in an emulsion, and wherein said removing comprises breaking said emulsion.
25. The method of claim 1 , further comprising amplifying said barcoded nucleic acid molecule or a derivative thereof to provide a plurality of amplified barcoded nucleic acid molecules.
26. The method of claim 1 , further comprising attaching a functional sequence to said barcoded nucleic acid molecule or a derivative thereof.
27. The method of claim 26 , wherein said functional sequence is a sequencing primer or a sample index sequence.
28. The method of claim 1 , wherein said barcoded nucleic acid molecule or a derivative thereof is sequenced.
29. The method of claim 1 , wherein said bead comprises a plurality of barcode oligonucleotide molecules, which plurality of barcode oligonucleotide molecules comprises said barcode oligonucleotide molecule.
30. The method of claim 29 , wherein an additional barcode oligonucleotide molecule of said plurality of barcode oligonucleotide molecules comprises a primer sequence.
31. The method of claim 1 , wherein said partition further comprises nucleic acid extension reagents.
32. The method of claim 1 , wherein (b) occurs prior to (c).
33. The method of claim 1 , wherein said partition comprises a nuclease inactivating agent.
34. The method of claim 33 , wherein said nuclease inactivating agent is proteinase K.
35. The method of claim 2 , wherein said bead comprises an additional barcode oligonucleotide molecule comprising an additional primer sequence.
36. The method of claim 35 , wherein said primer sequence or said additional primer sequence is a targeted primer sequence.
37. The method of claim 23 , wherein said barcoded nucleic acid molecule or derivative thereof is sequenced.
38. The method of claim 25 , wherein said amplified barcoded nucleic acid molecules or derivatives thereof are sequenced.
39. The method of claim 14 , wherein said barcode sequence is unique to said partition of said plurality of partitions.
40. The method of claim 14 , further comprising pooling said barcoded nucleic acid molecule or derivative thereof from said partition with other barcoded nucleic acid molecules or derivatives thereof from other partitions of said plurality of partitions.
41. The method of claim 1 , wherein said nucleic acid molecule comprises ribonucleic acid (RNA).
42. The method of claim 1 , wherein said nucleic acid molecule is derived from RNA.
43. The method of claim 1 , wherein said barcode oligonucleotide molecule comprises a poly-T sequence.
44. The method of claim 43 , wherein said barcode oligonucleotide molecule hybridizes to said nucleic acid molecule via said poly-T sequence.