Methods and compositions for nucleic acid analysis

• US 9,885,034 B2
• Filed: 09/22/2014
• Issued: 02/06/2018
• Est. Priority Date: 04/25/2011
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Saxonov, Serge
• Primary Examiner(s): Martinell, James
• Application Number:
  US14/493,268
• Publication Number:
  US 20150011432A1
• Time in Patent Office: 1,233 Days
• Field of Search: None
• 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 for sample preparation, comprising:

a. combining;

i. a first droplet comprising an oligonucleotide barcode and

ii. a target analyte

into a partition, wherein said first droplet is burstable, fusible, or mergeable upon application of a treatment; and

b. applying said treatment to said first droplet to release said oligonucleotide barcode to said target analyte.

2. The method of claim 1, wherein said partition is a well.

3. The method of claim 1, wherein said partition is a second droplet containing the first droplet.

4. The method of claim 1, wherein said treatment is selected from the group consisting of a chemical stimulus, a thermal stimulus, an electrical stimulus, a photo stimulus, a pressure stimulus, or any combination thereof.

5. The method of claim 1, further comprising attaching said oligonucleotide barcode to said target analyte.

6. The method of claim 5, wherein the target analyte is a nucleic acid.

7. The method of claim 6, wherein the nucleic acid is cell-free.

8. The method of claim 6, wherein the attaching comprises ligation of the oligonucleotide barcode to the nucleic acid.

9. The method of claim 6, wherein the attaching comprises nucleic acid amplification.

10. The method of claim 9, wherein the amplification comprises polymerase chain reaction (PCR).

11. The method of claim 9, wherein the amplification comprises amplification of a polynucleotide on a bead.

12. The method of claim 1, wherein said target analyte is a nucleic acid.

13. The method of claim 12, wherein said nucleic acid is selected from the group consisting of single stranded nucleic acid, double stranded nucleic acid, nucleic acid containing portions of both double stranded and single stranded sequence, DNA, genomic DNA, mitochondrial DNA, cDNA, RNA, mRNA, rRNA, siRNA, miRNA, cRNA, tRNA, rRNA, viral RNA, and a DNA/RNA hybrid.

14. The method of claim 12, wherein said nucleic acid is genomic DNA.

15. The method of claim 12, wherein the nucleic acid is cell-free.

16. The method of claim 1, wherein the first droplet comprises an adaptor oligonucleotide that comprises the barcode oligonucleotide.

17. The method of claim 16, wherein the barcode oligonucleotide is 4 to 6 base pairs long.

18. The method of claim 16, wherein the barcode oligonucleotide is 4-10 base pairs long.

19. The method of claim 16, wherein the adaptor oligonucleotide is DNA.

20. The method of claim 16, wherein the adaptor oligonucleotide is RNA.

21. The method of claim 1, wherein the partition comprises a polymerase.

22. The method of claim 21, wherein the polymerase is a DNA polymerase.

23. The method of claim 1, wherein barcodes of unique sequence are linked to different copies of the target analyte.

24. The method of claim 1, wherein target analytes from different samples comprise sample-specific barcodes.