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 composition, comprising: a plurality of polymer beads, wherein a polymer bead of said plurality of polymer beads comprises a plurality of nucleic acid barcode molecules covalently attached thereto, wherein a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises: (i) a cell barcode sequence, (ii) an identification barcode sequence configured to quantify a polynucleotide, and (iii) a capture sequence configured to hybridize to said polynucleotide,
wherein said plurality of polymer beads comprises 10,000 polymer beads,
wherein said plurality of polymer beads comprises a plurality of cell barcode sequences,
wherein said plurality of cell barcode sequences comprises 10,000 different cell barcode sequences,
wherein said plurality of nucleic acid barcode molecules comprises an identical cell barcode sequence,
wherein said plurality of nucleic acid barcode molecules comprises a plurality of identification barcode sequences,
wherein said plurality of identification barcode sequences comprises random sequences, and
wherein said plurality of identification barcode sequences comprises 10,000 different identification barcode sequences.
2. The composition of claim 1 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules further comprises an adaptor sequence.
3. The composition of claim 2 , wherein said adaptor sequence comprises one or more sequences selected from the group consisting of a primer binding sequence and a sequence configured to attach to a flow cell of a sequencer.
4. The composition of claim 2 , wherein said primer binding sequence is a sequencing primer binding sequence.
5. The composition of claim 1 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is releasable from said polymer bead upon application of a stimulus.
6. The composition of claim 5 , wherein said stimulus comprises a thermal stimulus, a chemical stimulus, an electrical stimulus, a magnetic stimulus, a photo-stimulus, a biological stimulus, or a combination thereof.
7. The composition of claim 1 , wherein said polymer bead is a gel bead.
8. The composition of claim 7 , wherein said gel bead is degradable upon application of a stimulus.
9. The composition of claim 8 , wherein said stimulus comprises a thermal stimulus, a chemical stimulus, an electrical stimulus, a magnetic stimulus, a photo stimulus, biological stimulus, or a combination thereof.
10. The composition of claim 8 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is releasable from said gel bead upon application of said stimulus.
11. The composition of claim 1 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules comprises a uracil-containing nucleotide.
12. The composition of claim 1 , wherein said plurality of polymer beads comprises 100,000 polymer beads and wherein said plurality of cell barcode sequences comprises 100,000 different cell barcode sequences.
13. The composition of claim 1 , wherein said plurality of nucleic acid barcode molecules comprises 10,000 nucleic acid barcode molecules.
14. The composition of claim 1 , wherein said plurality of nucleic acid barcode molecules comprises 100,000 nucleic acid barcode molecules.
15. The composition of claim 1 , wherein said plurality of nucleic acid barcode molecules comprises 1,000,000 nucleic acid barcode molecules.
16. The composition of claim 1 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is single-stranded.
17. The composition of claim 1 , wherein said polymer bead comprises an acrylic polymer.
18. The composition of claim 1 , wherein said polymer bead comprises polyacrylamide.
19. The composition of claim 1 , wherein said polymer bead has a diameter of at least 10 micrometers.
20. The composition of claim 1 , wherein said polymer bead has a diameter of at most 50 micrometers.
21. The composition of claim 1 , wherein said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules is covalently attached to said polymer bead via a chemical linker.
22. The composition of claim 21 , wherein said chemical linker comprises a labile bond configured to release said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules.
23. The composition of claim 22 , wherein said labile bond is a disulfide bond.