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) generating a plurality of droplets with a plurality of beads and a plurality of nucleic acid molecules, wherein a droplet of said plurality of droplets comprises (i) a bead of said plurality of beads, wherein said bead comprises a first primer comprising a first primer sequence, wherein said droplet does not comprise any other beads, (ii) at least two nucleic acid molecules of said 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 and each of said first nucleic acid molecule and said second nucleic acid molecule comprises a first adaptor sequence, wherein said first primer sequence does not have sequence complementarity with said first adaptor sequence, and (iii) one or more reagents comprising a second primer within said droplet, said second primer not attached to any beads and comprising a first portion and a second portion, wherein said first portion is configured to hybridize to said first adaptor sequence, and wherein said second portion comprises a second primer sequence that corresponds to said first primer sequence, wherein a ratio of a concentration of said second primer within said droplet to a concentration of said first primer in said droplet is on an order of 10 - 1 or less;
(b) in said droplet, with said second primer hybridized to said first adaptor sequence of said first nucleic acid molecule, (i) using said one or more reagents to generate one or more extension products of said first nucleic acid molecule comprising said second primer sequence or reverse complement thereof, (ii) attaching an extension product of said one or more extension products to said bead by annealing said second primer sequence or reverse complement thereof to said first primer sequence and extending said first primer, and (iii) generating amplification products of said first nucleic acid molecule attached to said bead, thereby monoclonally amplifying said first nucleic acid molecule on said bead;
(c) recovering said bead from said droplet; and
(d) assaying an amplification product of said amplification products attached to said bead, to identify a sequence of said first 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 droplets.
3. The method of claim 1 , wherein said bead has attached thereto a plurality of first primers comprising said first primer for performing one or more amplification reactions to generate said amplification products of said first nucleic acid molecule attached to said bead.
4. The method of claim 1 , wherein each of at least 50 % of said plurality of droplets comprises two or more beads of said plurality of beads.
5. The method of claim 1 , wherein each of at least 50 % of said plurality of droplets comprises one or more nucleic acid molecules of said plurality of nucleic acid molecules.
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 (d) comprises sequencing said amplification product while said amplification product is attached to said bead.
11. The method of claim 1 , further comprising generating said plurality of droplets, wherein each of at least 50% of said plurality of droplets comprises at least one bead and at least one nucleic acid template.
12. The method of claim 1 , further comprising generating said plurality of droplets, wherein each of at least 80% of said plurality of droplets comprise at least one bead and at least one nucleic acid template.
13. The method of claim 1 , wherein said plurality of nucleic acid molecules comprises isolated nucleic acid molecules.
14. The method of claim 10 , wherein said bead is placed on a planar substrate for said sequencing.
15. The method of claim 1 , wherein at most 50% of said plurality of droplets are unoccupied by said plurality of beads or said plurality of nucleic acid molecules.
16. The method of claim 1 , wherein said one or more reagents comprises first solution primers not attached to said bead, wherein each of said first solution primers comprises said first primer sequence.
17. The method of claim 1 , wherein said one or more reagents comprises third solution primers not attached to said bead, wherein said at least two nucleic acid molecules each comprises a second adaptor sequence different from said first adaptor sequence, and wherein each of said third solution primers comprises a third primer sequence that is identical or a reverse complement to said second adaptor sequence.
18. The method of claim 1 , wherein said ratio of said concentration of said second primer within said droplet to said concentration of said first primer in said droplet is on an order of 10 −2 or less.
19. The method of claim 1 , wherein said ratio of said concentration of said second primer within said droplet to said concentration of said first primer in said droplet is on an order of 10 −3 or less.
20. The method of claim 1 , wherein said bead of said plurality of beads comprises a plurality of first primers comprising said first primer, and wherein a ratio of a concentration of said second primer within said droplet to a concentration of said plurality of first primers in said droplet is on an order of 10 -1 or less.
21. The method of claim 1 , wherein said droplet has a diameter of about 1 nanometer (nm) to about 1 millimeter (mm).
22. The method of claim 1 , wherein said first adapter sequence of said first nucleic acid molecule and said second nucleic acid molecule comprises a same sequence.