METHODS AND COMPOSITIONS FOR NUCLEIC ACID ANALYSIS

• US 20120316074A1
• Filed: 04/25/2012
• Published: 12/13/2012
• Est. Priority Date: 04/25/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Saxonov, Serge
• Granted Patent: US 9,347,059 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/10 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 Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

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.

Claims

1. A method comprising:

a. subdividing a plurality of adaptors into a plurality of first partitions, wherein each of said first partitions has on average a first volume and wherein said adaptors comprise unique barcodes;

b. subdividing a sample comprising multiple polynucleotides into a plurality of second partitions, wherein each of said second partitions has on average a second volume, wherein said second volume is greater than said first volume;

c. merging at least one of said first partitions with at least one of said second partitions to form a merged partition; and

d. tagging one of said multiple polynucleotides, or fragment thereof, with at least one of said adaptors.

3. The method of claim 1 or 2, wherein said first partitions are droplets.

4. The method of claim 3, wherein said second partitions are droplets.

5. The method of claim 3, wherein said droplets are within an immiscible fluid.

6. The method of claim 1 or 2, wherein said polynucleotides are genomic DNA.

7. The method of claim 4, wherein said tagging comprises merging at least one first droplet comprising one of said adaptors with at least one second droplet comprising one of said polynucleotides.

8. The method of claim 1, wherein said first partitions are first droplets and said second partitions are second droplets;

and wherein, prior to said merging, said at least one second droplet comprises said at least one first droplet.

9. The method of claim 1, wherein said first partitions are first droplets and said second partitions are second droplets;

and wherein, prior to said merging, said at least one second droplet does not comprise said at least one first droplet.

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

14. The method of claim 7, further comprising modifying the temperature of said droplets.

15. The method of claim 7, wherein said merging comprises use of a controller such that each of said first droplets merges with each of said second droplets.

16. The method of claim 7, wherein said merging comprises randomly merging droplets comprising polynucleotides with droplets comprising adaptors.

17. The method of claim 1, further comprising pooling said adaptor-tagged polynucleotides, or fragments thereof.

18. The method of claim 1, further comprising analyzing said adaptor-tagged polynucleotides, or fragments thereof.

19. The method of claim 18, wherein said analyzing comprises sequencing said adaptor-tagged polynucleotides, or fragments thereof.

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

21. The method of claim 19, further comprising estimating the likelihood that any two sequence reads generated by said sequencing came from the same or different partitions.

22. The method of claim 6, wherein said sample is 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.

23. The method of claim 6, wherein said genomic DNA is high molecular-weight DNA.

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

25. The method of claim 24, wherein said polynucleotides fragments are generated by fragmenting said polynucleotides with an endonuclease.

26. The method of claim 1, wherein said polynucleotides are tagged by ligating said adaptors to said polynucleotides within a plurality of said merged partitions.

27. The method of claim 1, wherein said tagging is accomplished using transposons.

28. The method of claim 1, wherein said tagged polynucleotides are amplified.

29. The method of claim 28, wherein said amplification comprises a polymerase chain reaction.

30. The method of claim 1, wherein said polynucleotides are amplified before tagging.

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

32. The method of claim 1 or 2, wherein each of said second partitions comprises, on average, less than five of said multiple polynucleotides.

33. The method of claim 1, wherein said subdividing of said sample comprises emulsifying or mixing said sample with said second partitions.

35. The method of claim 30, wherein said amplification is multiple-displacement amplification.

2. A method comprising:

a. subdividing a plurality of adaptors into a plurality of first partitions, wherein each of said first partitions has on average a first volume and wherein said adaptors comprise unique barcodes;

b. subdividing a sample comprising multiple polynucleotides into a plurality of second partitions, wherein each of said second partitions has on average a second volume, wherein said second volume is less than said first volume;

c. merging at least one of said first partitions with at least one of said second partitions to form a merged partition; and

d. tagging one of said multiple polynucleotides, or fragment thereof, with at least one of said adaptors.

10. The method of claim 2, wherein said first partitions are first droplets and said second partitions are second droplets;

and wherein, prior to said merging, said at least one first droplet comprises said at least one second droplet.

11. The method of claim 2, wherein said first partitions are first droplets and said second partitions are second droplets;

and wherein, prior to said merging, said at least one first droplet does not comprise said at least one second droplet.

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

34. The method of claim 2, wherein said subdividing of said plurality of adaptors comprises emulsifying or mixing said plurality of adaptors with said second partitions.

36. A method comprising

a. partitioning organelles into a plurality of partitions, wherein each partition comprises on average less than five organelles per partition;

b. lysing said extracellular organelles in the plurality of partitions, wherein the lysing releases RNA from said organelles;

c. generating tagged cDNA from said released RNA in said plurality of partitions with adaptors comprising a barcode, wherein each partition in the plurality of partitions comprises adaptors with a unique barcode.

37. The method of claim 36, wherein said organelles are extracellular organelles.

38. The method of claim 36, wherein said organelles are exosomes.

39. The method of claim 36, wherein said generating tagged cDNA comprises reverse transcription of said released RNA with partition-specific barcoded primers.

40. The method of claim 36, further comprising sequencing said tagged cDNA.

41. The method of claim 36, further comprising determining if said tagged cDNA is from the same organelle.

42. A method comprising

a. partitioning microorganisms into a plurality of partitions,

b. obtaining polynucleotides from the microorganisms in the plurality of partitions;

and

c. tagging the polynucleotides in the plurality of partitions with adaptors comprising a barcode, wherein each partition in the plurality of partitions comprises adaptors with a unique barcode.

43. The method of claim 42, wherein each of said partitions comprises, on average, less than five microorganisms.

44. The method of claim 42, further comprising sequencing the tagged polynucleotides.

45. The method of claim 42, further comprising determining if the tagged polynucleotide fragments are from the same partition.