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 processing or analyzing one or more components from a cell, comprising:
(a) providing (i) a porous matrix comprising said one or more components of said cell, wherein said one or more components comprise a nucleic acid molecule, and (ii) a plurality of nucleic acid barcode molecules for barcoding said nucleic acid molecule; and
(b) partitioning said porous matrix and said plurality of nucleic acid barcode molecules into a partition, wherein upon partitioning, said partition comprises said porous matrix and said plurality of nucleic acid barcode molecules.
2. The method of claim 1 , further comprising performing one or more reactions on said nucleic acid molecule.
3. The method of claim 2 , wherein said one or more reactions comprise a reaction selected from the group consisting of nucleic acid modification, nucleic acid amplification, nucleic acid extension, nucleic acid insertion, nucleic acid cleavage, reverse transcription, and a combination thereof.
4. The method of claim 2 , wherein said one or more reactions are performed in said partition.
5. The method of claim 2 , wherein said one or more reactions are performed subsequent to (a).
6. The method of claim 1 , further comprising using a nucleic acid barcode molecule from said plurality of nucleic acid barcode molecules and said nucleic acid molecule to generate a barcoded nucleic acid molecule.
7. The method of claim 6 , further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to sequencing.
8. The method of claim 7 , further comprising subjecting said barcoded nucleic acid molecule or derivative thereof to one or more reactions prior to said sequencing.
9. The method of claim 8 , further comprising recovering said barcoded nucleic acid molecule or derivative thereof from said partition and subsequently performing said one or more reactions.
10. The method of claim 8 , wherein said one or more reactions comprise addition of a functional sequence to said barcoded nucleic acid molecule or derivative thereof, which functional sequence permits attachment of said barcoded nucleic acid molecule or derivative thereof to a flow cell of a sequencer during said sequencing.
11. The method of claim 1 , wherein said porous matrix comprises said cell, and wherein said porous matrix comprising said cell is subjected to conditions sufficient to lyse said cell to generate said one or more components.
12. The method of claim 11 , wherein said porous matrix is subjected to said conditions sufficient to lyse said cell in said partition.
13. The method of claim 11 , wherein said conditions sufficient to lyse said cell comprise exposing said porous matrix to a lysis agent.
14. The method of claim 1 , wherein said partition is a droplet.
15. The method of claim 1 , wherein said partition is a well.
16. The method of claim 1 , wherein said partition further comprises additional reagents.
17. The method of claim 1 , wherein said nucleic acid molecule is a messenger ribonucleic acid molecule.
18. The method of claim 1 , further comprising recovering said nucleic acid molecule or a derivative thereof from said partition.
19. The method of claim 18 , further comprising performing one or more reactions on said nucleic acid molecule or derivative thereof subsequent to said recovering said nucleic acid molecule or derivative thereof from said partition.
20. The method of claim 1 , wherein (a) comprises providing a barcode bead, wherein said barcode bead comprises said plurality of nucleic acid barcode molecules, and (b) comprises partitioning said porous matrix and said barcode bead into said partition.
21. The method of claim 20 , wherein one or more nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules are coupled to a surface of said barcode bead or enclosed within said barcode bead.
22. The method of claim 20 , wherein said barcode bead is a gel bead.
23. The method of claim 20 , wherein said barcode bead comprises a disulfide bond.
24. The method of claim 20 , wherein said barcode bead is degradable upon application of a stimulus.
25. The method of claim 24 , further comprising releasing said plurality of nucleic acid barcode molecules upon application of said stimulus.
26. The method of claim 24 , wherein said stimulus is a chemical or biological stimulus, and wherein said partition comprises said stimulus.
27. The method of claim 24 , wherein said stimulus is a reducing agent.
28. The method of claim 1 , wherein said porous matrix is degradable upon application of a stimulus.
29. The method of claim 28 , wherein said stimulus is a chemical or biological stimulus, and wherein said partition comprises said stimulus.
30. The method of claim 1 , wherein a given nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises an identifier sequence that uniquely identifies said given nucleic acid barcode molecule among other nucleic acid barcode molecules of said plurality of nucleic acid barcode molecules.
31. The method of claim 30 , wherein said identifier sequence is a random sequence.
32. The method of claim 6 , wherein said nucleic acid molecule is a deoxynucleic acid (DNA) and wherein said barcoded nucleic acid comprises a sequence of said DNA molecule, or a complement thereof.
33. The method of claim 6 , wherein said nucleic acid molecule is a ribonucleic acid (RNA) molecule and wherein said barcoded nucleic acid comprises a sequence of said RNA molecule, or a complement thereof.
34. The method of claim 2 , wherein said performing one or more reactions on said nucleic acid molecule generates cDNA.
35. The method of claim 3 , wherein said nucleic acid amplification reaction comprises polymerase chain reaction (PCR).
36. The method of claim 16 , wherein said additional reagents comprise one or more members selected from the group consisting of a polymerase, a ligase, an endonuclease, and a reverse transcriptase.
37. The method of claim 27 , wherein said reducing agent comprises DTT.
38. The method of claim 29 , wherein said stimulus comprises a reducing agent.
39. The method of claim 1 , wherein said plurality of nucleic acid barcode molecules comprise a common barcode sequence.
40. The method of claim 20 , wherein said plurality of nucleic acid barcode molecules comprise a common barcode sequence.
41. The method of claim 1 , wherein said plurality of nucleic barcode molecules comprise a poly-thymine (poly-T) sequence.
42. The method of claim 1 , wherein said partition comprises a template switching oligonucleotide.
43. The method of claim 11 , wherein said conditions sufficient to lyse said cell comprise one or more members selected from the group consisting of electroporation, thermal cellular disruptions, acoustic cellular disruption, or mechanical cellular disruption.
44. The method of claim 13 , wherein said lysis agent comprises one or more members selected from the group consisting of a lysis enzyme, an ionic surfactant, and a non-ionic surfactant.
45. The method of claim 44 , wherein said lysis enzyme is selected from one or more members of the group consisting of lysozyme, achromopeptidase, lysostaphin, labiase, kitalase, and lyticase.
46. The method of claim 44 , wherein said ionic surfactant comprises sarkosyl or sodium dodecyl sulfate.
47. The method of claim 44 , wherein said non-ionic surfactant comprises TritonX-100 or Tween 20.
48. The method of claim 1 , further comprising prior to (a), encapsulating said one of more components of said cell in said porous matrix.
49. The method of claim 48 , wherein said encapsulating comprises (i) combining a fluid comprising said one or more components of said cell with a polymeric precursor material, and (ii) applying a stimulus to generate said porous matrix from said polymeric precursor material.
50. The method of claim 49 , wherein said stimulus is a thermal stimulus, a photo-stimulus, or a chemical stimulus.
51. The method of claim 49 , wherein said stimulus is a polymerization initiator.