Nucleic acid encoding reactions

Patent metadata

Field	Value
Publication number	US-10501786-B2
Application number	US-201514723872-A
Country	US
Kind code	B2
Filing date	May 28, 2015
Priority date	May 20, 2011
Publication date	Dec 10, 2019
Grant date	Dec 10, 2019

Abstract

Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.

First claim

What is claimed is:

1. A method of analyzing nucleic acids in or associated with single particles, the method comprising:
a) capturing particles in separate reaction volumes, such that a plurality of reaction volumes each comprise a single particle, wherein the separate reaction volumes are droplets in an emulsion;
b) producing barcoded reaction products from nucleic acids in or associated with the single particle in each of the separate reaction volumes, wherein producing the reaction products comprises incorporating barcoded adaptor nucleotide sequences into the reaction products using a plurality of different single-stranded barcoded adaptor molecules for each separate reaction volume, wherein each adaptor molecule comprises: (i) a nucleotide sequence comprising a primer binding site, (ii) a degenerate tail sequence, and (iii) a barcode nucleotide sequence, wherein each plurality of different single-stranded barcoded adaptor molecules comprises the same barcode;
c) pooling the barcoded reaction products;
d) amplifying the barcoded reaction products using primers that bind the primer binding sites, to produce templates for bidirectional DNA sequencing; and
e) sequencing the barcoded reaction products.

2. The method of claim 1 , wherein the particles are cells.

3. The method of claim 2 , further comprising lysing the cells to release cell contents, after step a) and prior to step b).

4. The method of claim 2 , wherein the distribution of cells in the separate reaction volumes approximates a Poisson distribution.

5. The method of claim 4 , wherein the cells are diluted to produce a highest fraction of reaction volumes having only one cell.

6. The method of claim 2 , wherein step a) further comprises binding the cells to a solid support.

7. The method of claim 6 , wherein the solid support has a binding partner distributed across its surface.

8. The method of claim 7 , wherein the binding partner is an antibody.

9. The method of claim 6 , wherein the solid support is a bead.

10. The method of claim 1 , wherein the particles are disrupted to release interior components, after step a) and prior to step b).

11. The method of claim 1 , wherein the particles are beads.

12. The method of claim 11 , wherein the beads are primer-coated.

13. The method of claim 1 , wherein step b) of producing reaction products comprises reverse transcription.

14. The method of claim 1 , wherein step b) of producing reaction products comprises amplification.

15. The method of claim 13 , wherein step b) of producing reaction products comprises amplification.

16. The method of claim 1 , further comprising f) associating sequencing results obtained in e) with single particles.

17. The method of claim 1 , wherein the barcoded adaptor molecule is a non-specific degenerate adaptor.

Assignees

• Fluidigm Corp

Inventors

• Anderson Megan
• Chen Peilin
• Fowler Brian
• Jones Robert C
• Kaper Fiona
• Lebofsky Ronald
• May Andrew

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · 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

• C12Q1/6855 Primary

  Ligating adaptors · CPC title