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 sample preparation, the method comprising:
(a) contacting a plurality of cells with a plurality of beads comprising a plurality of oligonucleotides comprising barcode sequences to form a plurality of constructs, each construct of the plurality of constructs comprising a cell of the plurality of cells and a bead of the plurality of beads, wherein the cell comprises polynucleotides,
(b) encapsulating the plurality of constructs together in a matrix, and
(c) generating a plurality of nucleic acid molecules comprising the barcode sequences of oligonucleotides of the plurality of oligonucleotides, each nucleic acid molecule of the plurality of nucleic acid molecules comprising a barcode sequence of the barcode sequences or complement thereof and at least a portion of a polynucleotide of the polynucleotides, or complement thereof, of the cell.
2. The method according to claim 1 , wherein (c) comprises hybridizing the polynucleotides to the oligonucleotides.
3. The method according to claim 2 , wherein: (i) the polynucleotides comprise a poly-A sequence; (ii) the oligonucleotides comprise a poly-T sequence; and (iii) the hybridizing occurs between the poly-A sequence of the polynucleotides and the poly-T sequence of the oligonucleotides.
4. The method according to claim 1 , further comprising determining a sequence of the plurality of nucleic acid molecules.
5. The method according to claim 1 , wherein the plurality of constructs comprise one or more chemical linkers.
6. The method according to claim 5 , wherein the one or more chemical linkers are selected from the group consisting of disulfide linkers, biotin linkers, streptavidin linkers, and avidin linkers.
7. The method according to claim 1 , wherein the plurality of constructs comprise one or more feature binding groups.
8. The method according to claim 7 , wherein the one or more feature binding groups are selected from the group consisting of antibodies, antibody fragments, and cell surface receptor binding molecules.
9. The method according to claim 7 , wherein the one or more feature binding groups are biotinylated.
10. The method according to claim 7 , wherein the one or more feature binding groups are linked to one or more beads of the plurality of beads.
11. The method according to claim 10 , wherein the one or more feature binding groups are linked to one or more beads of the plurality of beads via chemical conjugation.
12. The method according to claim 10 , wherein the one or more feature binding groups are linked to the one or more beads of the plurality of beads via biotin, streptavidin, or avidin.
13. The method according to claim 8 , wherein the one or more feature binding groups are antibodies.
14. The method according to claim 13 , wherein the antibodies are biotinylated.
15. The method according to claim 13 , wherein the antibodies bind a cell surface feature present on the cell.
16. The method according to claim 1 , wherein encapsulating the plurality of constructs comprises exposing the plurality of constructs to a polymer precursor material capable of being polymerized into a matrix upon application of a stimulus to the polymer precursor material.
17. The method according to claim 16 , wherein the stimulus is selected from the group consisting of a thermal stimulus, a photo stimulus, and a chemical stimulus.
18. The method according to claim 16 , wherein the stimulus initiates crosslinking of the polymer precursor material.
19. The method according to claim 1 , wherein the matrix is a gel matrix.
20. The method according to claim 19 , wherein the gel matrix is a hydrogel.
21. The method according to claim 1 , wherein each of the plurality of nucleic acid molecules comprises a unique barcode sequence.
22. The method according to claim 1 , wherein each of the plurality of beads comprises an oligonucleotide comprising a distinct barcode sequence.
23. The method according to claim 1 , further comprising, prior to (c), lysing the cell.
24. The method according to claim 1 , wherein the plurality of cells comprises at least 10,000 cells.
25. The method according to claim 1 , wherein (c) comprises ligating the polynucleotides to the oligonucleotides of the plurality of oligonucleotides.
26. A method for sample preparation, the method comprising:
(a) contacting a plurality of eukaryotic organelles with a plurality of beads comprising a plurality of oligonucleotides comprising barcode sequences to form a plurality of constructs, each construct of the plurality of constructs comprising a eukaryotic organelle of the plurality of eukaryotic organelles and a bead of the plurality of beads, wherein the eukaryotic organelle comprises polynucleotides,
(b) encapsulating the plurality of constructs together in a matrix,
(c) generating a plurality of nucleic acid molecules comprising the barcode sequences of the oligonucleotides, each nucleic acid molecule of the plurality of nucleic acid molecules comprising a barcode sequence of the barcode sequences or complement thereof and at least a portion of a polynucleotide of the polynucleotides, or complement thereof, of the eukaryotic organelle.
27. The method according to claim 26 , wherein (c) comprises hybridizing the polynucleotides to the oligonucleotides.
28. The method according to claim 27 , wherein: (i) the polynucleotides comprise a poly-A sequence; (ii) the oligonucleotides comprise a poly-T sequence; and (iii) the hybridizing occurs between the poly-A sequence of the polynucleotides and the poly-T sequence of the oligonucleotides.
29. The method according to claim 26 , further comprising determining a sequence of the plurality of nucleic acid molecules.
30. The method according to claim 26 , wherein the plurality of constructs comprise one or more chemical linkers.
31. The method according to claim 30 , wherein the one or more chemical linkers are selected from the group consisting of disulfide linkers, biotin linkers, streptavidin linkers, and avidin linkers.
32. The method according to claim 26 , wherein the plurality of constructs comprises one or more feature binding groups.
33. The method according to claim 32 , wherein the one or more feature binding groups are selected from the group consisting of antibodies, antibody fragments, and surface receptor binding molecules.
34. The method according to claim 32 , wherein the one or more feature binding groups are biotinylated.
35. The method according to claim 32 , wherein the one or more feature binding groups are linked to one or more beads of the plurality of beads.
36. The method according to claim 35 , wherein the one or more feature binding groups are linked to one or more beads of the plurality of beads via chemical conjugation.
37. The method according to claim 35 , wherein the one or more feature binding groups are linked to the one or more beads of the plurality of beads via biotin, streptavidin, or avidin.
38. The method according to claim 33 , wherein the one or more feature binding groups are antibodies.
39. The method according to claim 38 , wherein the antibodies are biotinylated.
40. The method according to claim 38 , wherein the antibodies bind a surface feature present on the eukaryotic organelle.
41. The method according to claim 26 , wherein encapsulating the plurality of constructs comprises exposing the plurality of constructs to a polymer precursor material capable of being polymerized into a matrix upon application of a stimulus to the polymer precursor material.
42. The method according to claim 41 , wherein the stimulus is selected from the group consisting of a thermal stimulus, a photo stimulus, and a chemical stimulus.
43. The method according to claim 42 , wherein the stimulus initiates crosslinking of the polymer precursor material.
44. The method according to claim 26 , wherein the matrix is a gel matrix.
45. The method according to claim 44 , wherein the gel matrix is a hydrogel.
46. The method according to claim 26 , wherein each of the plurality of nucleic acid molecules comprises a unique barcode sequence.
47. The method according to claim 26 , wherein each of the plurality of beads comprises an oligonucleotide comprising a distinct barcode sequence.
48. The method according to claim 26 , further comprising, prior to (c), lysing the eukaryotic organelle.
49. The method according to claim 26 , wherein the plurality of eukaryotic organelles comprises at least 10,000 eukaryotic organelles.
50. The method according to claim 26 , wherein (c) comprises ligating the polynucleotide:
to the oligonucleotides of the plurality of oligonucleotides.
51. The method according to claim 1 , wherein the polynucleotides are ribonucleic acid (RNA).
52. The method according to claim 51 , wherein the RNA is messenger RNA (mRNA).
53. The method according to claim 26 , wherein the polynucleotides are ribonucleic acid (RNA).
54. The method according to claim 53 , wherein the RNA is messenger RNA (mRNA).