Compositions and methods for sample processing
This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.
1. A system comprising:
a plurality of gel beads comprising a plurality of barcode sequences, wherein a gel bead of said plurality of gel beads comprises (i) a first nucleic acid molecule comprising a first barcode sequence and a first capture ligand configured to capture a ribonucleic acid (RNA) molecule; and (ii) a second nucleic acid molecule comprising a second barcode sequence and a second capture ligand configured to capture a deoxyribonucleic acid (DNA) molecule, wherein said first barcode sequence comprises a first barcode sequence segment and said second barcode sequence comprises a second barcode sequence segment, wherein said first barcode sequence segment and said second barcode sequence segment are identical; and
a microfluidic device comprising a plurality of fluidically connected channel junctions arranged in series, wherein channel junctions of said plurality of fluidically connected channel junctions comprise a plurality of inlet channels, wherein said plurality of fluidically connected channel junctions comprises a channel junction comprising a first inlet channel and a second inlet channel, wherein said channel junction is fluidically connected to a droplet generation channel junction, wherein said first inlet channel is configured to provide said plurality of gel beads and said second inlet channel is configured to provide said plurality of cells, and wherein, during use, said plurality of fluidically connected channel junctions co-partitions said plurality of gel beads and a plurality of cells into a plurality of droplets, wherein a droplet of said plurality of droplets comprises: (i) said gel bead, and (ii) a cell of said plurality of cells.
2. The system of claim 1 , wherein said first barcode sequence comprises an additional barcode sequence segment, wherein said first barcode sequence segment and said additional barcode sequence segment are separated by one or more nucleotides.
3. The system of claim 1 , wherein said first capture ligand or said second capture ligand is an N-mer sequence.
4. The system of claim 3 , wherein said N-mer sequence is a random N-mer sequence.
5. The system of claim 3 , wherein said N-mer sequence is a target-specific N-mer sequence.
6. The system of claim 5 , wherein said target-specific N-mer sequence is a poly-T sequence.
7. The system of claim 1 , wherein said first capture ligand is a poly-T sequence and wherein said second capture ligand is a random N-mer sequence.
8. The system of claim 1 , wherein said first nucleic acid molecule or said second nucleic acid molecule is covalently attached to said gel bead.
9. The system of claim 8 , wherein said first nucleic acid molecule or said second nucleic acid molecule is covalently attached to said gel bead via a moiety comprising a labile bond.
10. The system of claim 9 , wherein said labile bond is selected from the group consisting of a thermally cleavable bond, a disulfide bond, and a UV-sensitive bond.
11. The system of claim 9 , wherein said labile bond comprises an ester linkage, a vicinal diol linkage, a Diels-Alder linkage, a sulfone linkage, a silyl ester linkage, a glycosidic linkage, a peptide linkage, or a phosphodiester linkage.
12. The system of claim 1 , wherein said first nucleic acid molecule or said second nucleic acid molecule is releasable from said gel bead through cleavage of a linkage between said first nucleic acid molecule or said second nucleic acid molecule and said gel bead.
13. The system of claim 12 , wherein said first nucleic acid molecule or said second nucleic acid molecule is releasable upon application of a stimulus.
14. The system of claim 13 , wherein said stimulus is a chemical stimulus.
15. The system of claim 14 , wherein said chemical stimulus is a reducing agent.
16. The system of claim 1 , wherein said plurality of gel beads is a plurality of degradable gel beads.
17. The system of claim 16 , wherein said plurality of degradable gel beads comprises chemically cleavable cross-linking.
18. The system of claim 17 , wherein said chemically cleavable cross-linking comprises disulfide cross-linking.
19. The system of claim 1 , wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises a sequencing primer sequence.
20. The system of claim 1 , wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises a sequence capable of attachment to a flow cell of a sequencer.
21. The system of claim 1 , wherein said first nucleic acid molecule or said second nucleic acid molecule further comprises one or more phosphorothioate nucleotide analogues.
22. The system of claim 21 , wherein said one or more phosphorothioate nucleotide analogues include 5 to 10 internucleotide linkages towards either a 3′ or a 5′ terminus of said first nucleic acid molecule or said second nucleic acid molecule.
23. The system of claim 21 , wherein said one or more phosphorothioate nucleotide analogues include a 3′ or 5′ terminal internucleotide linkage.
24. The system of claim 1 , wherein an additional gel bead of said plurality of gel beads comprises:
a third nucleic acid molecule comprising a third capture ligand configured to capture an RNA molecule; and
a fourth nucleic acid molecule comprising a fourth capture ligand configured to capture a DNA molecule.
25. The system of claim 24 , wherein said third nucleic acid molecule comprises a third barcode sequence comprising a third barcode sequence segment and wherein said fourth nucleic acid molecule comprises a fourth barcode sequence comprising a fourth barcode sequence segment, wherein said third barcode sequence segment and said fourth barcode sequence segment are identical.
26. The system of claim 25 , wherein said third barcode sequence and said fourth barcode sequence are different than said first barcode sequence and said second barcode sequence.
27. The system of claim 1 , wherein said droplet of said plurality of droplets further comprises one or more reagents configured to facilitate a reaction on (i) said RNA molecule or a derivative thereof, or (ii) said DNA molecule or a derivative thereof.
28. The system of claim 1 , wherein said first nucleic acid molecule further comprises a third barcode sequence and said second nucleic acid molecule further comprises a fourth barcode sequence, wherein said third barcode sequence and said fourth barcode sequence have different sequences.
29. The system of claim 28 , wherein said third barcode sequence and said fourth barcode sequence are random N-mer sequences.