Methods for nucleic acid analysis
The present disclosure provides methods and processes for increasing the efficiency and accuracy of nucleic acid sequencing using techniques such as polymerase chain reaction (PCR). The methods described herein can be used to achieve clonal amplification even with a greater than Poisson distribution of beads and/or nucleic acid templates into an emulsion. A PCR method may comprise generating a partition (e.g., a droplet) comprising at least two beads and/or at least two nucleic acid molecules and generating clonal amplification products corresponding to the nucleic acid molecule, at least a subset of which may be attached to a bead.
1. A method for nucleic acid processing, comprising:
(a) providing a plurality of partitions, wherein a partition of said plurality of partitions comprises (i) at least two beads of a plurality of beads, comprising a first bead and a second bead, wherein said first bead comprises a first primer and said second bead comprises a second primer, wherein said first primer and said second primer have different sequences, (ii) at least two nucleic acid molecules of a plurality of nucleic acid molecules, comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein said first nucleic acid molecule and said second nucleic acid molecule have different nucleic acid sequences, wherein said first nucleic acid molecule comprises a first adaptor and said second nucleic acid molecule comprises a second adaptor, wherein said first adaptor and said second adaptor have different sequences, wherein said first primer has sequence complementarity with said first adaptor and said second primer has sequence complementarity with said second adaptor, and (iii) one or more reagents;
(b) in said partition, with said first primer hybridized to said first adaptor and said second primer hybridized to said second adaptor, (i) using said first nucleic acid molecule and said one or more reagents to generate one or more first amplification products of said first nucleic acid molecule, wherein at least a subset of said one or more first amplification products is attached to said first bead, and (ii) using said second nucleic acid molecule and said one or more reagents to generate one or more second amplification products of said second nucleic acid molecule, wherein at least a subset of said one or more second amplification products is attached to said second bead;
(c) recovering said first bead and said second bead from said partition; and
(d) assaying (i) a first amplification product of said at least said subset of said one or more first amplification products attached to said first bead, to identify a sequence of said first nucleic acid molecule and (ii) a second amplification product of said at least said subset of said one or more second amplification products attached to said second bead, to identify a sequence of said second nucleic acid molecule.
2. The method of claim 1 , wherein (a) comprises bringing (i) a first solution comprising said plurality of nucleic acid molecules and (ii) a second solution comprising said plurality of beads in contact with a fluid that is immiscible with said first solution and said second solution, to generate said plurality of partitions.
3. The method of claim 1 , wherein said first bead has attached thereto a plurality of primers comprising said first primer for performing one or more amplification reactions using said first nucleic acid molecule, and (b) comprises using primer molecules of said plurality of primers comprising said first primer to conduct said one or more amplification reactions to generate said at least said subset of said one or more first amplification products attached to said first bead.
4. The method of claim 1 , wherein each of at least 80% of said plurality of partitions comprises two or more beads of said plurality of beads.
5. The method of claim 1 , wherein each of at least 80% of said plurality of partitions comprises three or more beads of said plurality of beads.
6. The method of claim 1 , wherein said one or more reagents comprise nucleic acid molecules comprising priming sequences.
7. The method of claim 6 , wherein said nucleic acid molecules comprising said priming sequences further comprise unique molecular identifier sequences.
8. The method of claim 6 , wherein said nucleic acid molecules comprising said priming sequences further comprise barcode sequences.
9. The method of claim 1 , wherein said one or more reagents comprise one or more polymerizing enzymes.
10. The method of claim 1 , wherein said plurality of partitions is a plurality of droplets.
11. The method of claim 1 , wherein (d) comprises sequencing said first amplification product of said at least said subset of said one or more first amplification products attached to said first bead and said second amplification product of said at least said subset of said one or more second amplification products attached to said second bead.
12. The method of claim 1 , wherein, in (a), wherein said first bead comprises a plurality of first primers comprising said first primer attached to said first bead, and wherein said second bead comprises a plurality of second primers comprising said second primer attached to said second bead.
13. The method of claim 12 , wherein (b) comprises:
(1) generating a first extension product of said first nucleic acid molecule which comprises a first sequence complementary to said first primer of said plurality of first primers, binding said first extension product to said first primer on said first bead, and extending said first primer to generate said first amplification product of said at least said subset of said one or more first amplification products attached to said first bead, and
(2) generating a second extension product of said second nucleic acid molecule which comprises a second sequence complementary to said second primer of said plurality of second primers, binding said second extension product to said second primer on said second bead, and extending said second primer to generate said second amplification product of said at least said subset of said one or more second amplification products attached to said second bead.
14. The method of claim 1 , further comprising generating said plurality of partitions, wherein each of at least 50% of said plurality of partitions comprises at least one bead and at least one nucleic acid template.
15. The method of claim 1 , further comprising generating said plurality of partitions, wherein each of at least 80% of said plurality of partitions comprise at least one bead and at least one nucleic acid template.
16. The method of claim 1 , wherein, subsequent to (c), said first bead is monoclonal and said second bead is monoclonal.
17. The method of claim 1 , wherein said first primer does not have sequence complementarity with said second adaptor and said second primer does not have sequence complementarity with said first adaptor.
18. The method of claim 1 , wherein said first primer does not hybridize with said second adaptor and said second primer does not hybridize with said first adaptor.
19. The method of claim 1 , wherein said plurality of nucleic acid molecules comprises isolated nucleic acid molecules.