Methods and compositions for nucleic acid analysis

Patent metadata

Field	Value
Publication number	US-9347059-B2
Application number	US-201213456121-A
Country	US
Kind code	B2
Filing date	Apr 25, 2012
Priority date	Apr 25, 2011
Publication date	May 24, 2016
Grant date	May 24, 2016

Abstract

Provided herein are methods, compositions, and kits for assays, many of which involve amplification reactions such as digital PCR or droplet digital PCR. The assays may be used for such applications as sequencing, copy number variation analysis, and others. In some cases, the assays involve subdividing a sample into multiple partitions (e.g., droplets) and merging the partitions with other partitions that comprise adaptors with barcodes.

First claim

What is claimed is:

1. A method comprising:
a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets in a fluid that is immiscible to the first droplets;
b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is greater than the first volume, wherein the second partitions are second droplets in a immiscible fluid that is immiscible to the second droplets;
c. applying a treatment to fuse the at least one second partition with at least one first droplet to form a fused partition; and
d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof.

2. A method comprising:
a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets in a fluid that is immiscible to the first droplets;
b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is less than the first volume, wherein the second partitions are second droplets in a fluid that is immiscible to the second droplets;
c. applying a treatment to fuse the at least one first partition with the at least one second droplet to form a fused partition; and
d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof.

3. The method of claim 1 or 2 , wherein the sample polynucleotides are genomic DNA.

4. The method of claim 1 , wherein at least one second droplet does not comprise the at least one first droplet.

5. The method of claim 2 , wherein at least one first droplet does not comprise the at least one second droplet.

6. The method of claim 1 , wherein the second volume is at least two times the volume of the first volume.

7. The method of claim 2 , wherein the first volume is at least two times the volume of the second volume.

8. The method of claim 1 or 2 , wherein the treatment comprises modifying the temperature of the droplets.

9. The method of claim 1 or 2 , wherein the fusing comprises use of a controller such that each of the first droplets fuses with each of the second droplets.

10. The method of claim 1 or 2 , wherein the fusing comprises randomly fusing droplets comprising polynucleotides with droplets comprising adaptors.

11. The method of claim 1 , further comprising pooling the tagged polynucleotides, or fragments thereof.

12. The method of claim 1 , further comprising analyzing the adaptor tagged polynucleotides, or fragments thereof.

13. The method of claim 12 , wherein the analyzing comprises sequencing the tagged polynucleotides, or fragments thereof.

14. The method of claim 1 , further comprising determining whether the tagged polynucleotides, or fragments thereof, were located in the same partition.

15. The method of claim 13 , further comprising estimating a likelihood that any two sequence reads generated by the sequencing came from the same or different partitions.

16. The method of claim 13 , wherein the polynucleotides are partitioned so that it is unlikely that a given partition comprises two or more polynucleotides, or fragments thereof, from the same locus but from different chromosomes.

17. The method of claim 3 , wherein the genomic DNA is greater than 10,000 bases or base pairs.

18. The method of claim 1 or 2 , further comprising fragmenting the sample polynucleotides within the second partitions to form polynucleotide fragments.

19. The method of claim 18 , wherein the polynucleotide fragments are generated by fragmenting the polynucleotides with an endonuclease.

20. The method of claim 1 , wherein the sample polynucleotides are tagged by ligating the adaptors to the sample polynucleotides within a plurality of the fused partitions.

21. The method of claim 1 , wherein the tagging is accomplished using transposons.

22. The method of claim 1 , wherein the tagged polynucleotides are amplified.

23. The method of claim 22 , wherein the amplification comprises a polymerase chain reaction.

24. The method of claim 1 , wherein the polynucleotides are amplified before tagging.

25. The method of claim 1 or 2 , wherein each of the first partitions comprises, on average, less than five adaptors.

26. The method of claim 1 or 2 , wherein each of the second partitions comprises, on average, less than five of the sample polynucleotides.

27. The method of claim 1 , wherein the generating the plurality of second partitions comprises emulsifying or mixing the sample with the second partitions.

28. The method of claim 2 , wherein the generating the plurality of first partitions comprises emulsifying or mixing the plurality of adaptors with the second partitions.

29. The method of claim 24 , wherein the amplification is multiple-displacement amplification.

30. The method of claim 1 , wherein the second volume is on average greater than 4-fold more than the first volume.

31. The method of claim 1 , wherein at least one second droplet comprises at least one first droplet.

32. The method of claim 2 , wherein first volume is on average greater than 4-fold more than the second volume.

33. The method of claim 2 , wherein at least one second droplet comprises at least one first droplet.

34. A method comprising:
a. generating a plurality of first partitions comprising adaptors, wherein each of the first partitions has on average a first volume and wherein the adaptors comprise unique barcodes, wherein the first partitions are first droplets;
b. generating a plurality of second partitions comprising sample polynucleotides, wherein each of the second partitions has on average a second volume, wherein the second volume is greater than the first volume or the second volume is less than the first volume, wherein the second partitions are second wells;
c. applying a treatment to fuse the at least one second partition with at least one first droplet to form a fused partition; and
d. tagging one of the sample polynucleotides, or fragment thereof, with at least one of the adaptors in the fused partition to form tagged polynucleotides or fragments thereof.

35. The method of claim 34 , wherein the second volume is on average greater than 4-fold more than the first volume.

36. The method of claim 34 , wherein the tagged polynucleotides are amplified.

Assignees

• Saxonov Serge
• Bio Rad Laboratories

Inventors

• Saxonov Serge

Classifications

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12N15/1075

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title