Methods for analyzing nucleic acids

Patent metadata

Field	Value
Publication number	US-10907207-B2
Application number	US-202016984034-A
Country	US
Kind code	B2
Filing date	Aug 3, 2020
Priority date	Aug 20, 2009
Publication date	Feb 2, 2021
Grant date	Feb 2, 2021

Abstract

Aspects of the present invention include analyzing nucleic acids from single cells using methods that include using tagged polynucleotides containing multiplex identifier sequences.

First claim

What is claimed:

1. A method for biological analysis, the method comprising:
(a) extracting a plurality of polynucleotides from a site within a tissue sample;
(b) generating a plurality of tagged polynucleotides from the plurality of polynucleotides and a plurality of oligonucleotide tags, wherein a tagged polynucleotide of the plurality of tagged polynucleotides comprises: (i) a first tag sequence corresponding to the site; and (ii) a second tag sequence distinguishing the tagged polynucleotide from other tagged polynucleotides of the plurality of tagged polynucleotides;
(c) sequencing the plurality of tagged polynucleotides to determine sequences corresponding to the plurality of polynucleotides, including the first tag sequence and the second tag sequence; and
(d) using the sequences determined in step (c) to count polynucleotides for multiple different polynucleotides extracted from the tissue sample.

2. The method of claim 1 , wherein the method further comprises extracting a second plurality of polynucleotides from a second site within the tissue sample.

3. The method of claim 2 , wherein the method further comprises generating a second plurality of tagged polynucleotides from the second plurality of polynucleotides and a second plurality of oligonucleotide tags, wherein a tagged polynucleotide of the second plurality of tagged polynucleotides comprises: (i) a third tag sequence corresponding to the second site; and (ii) a fourth tag sequence distinguishing the tagged polynucleotide from other tagged polynucleotides of the second plurality of tagged polynucleotides.

4. The method of claim 3 , wherein the method further comprises sequencing the second plurality of tagged polynucleotides to determine sequences corresponding to the second plurality of polynucleotides, including the third tag sequence and the fourth tag sequence.

5. The method of claim 4 , wherein the method further comprises using the determined sequences corresponding to the second plurality of polynucleotides to count polynucleotides for multiple different polynucleotides derived from the tissue sample.

6. The method of claim 3 , wherein the method further comprises pooling the plurality of tagged polynucleotides and the second plurality of tagged polynucleotides.

7. The method of claim 3 , wherein the generating step is performed in a well of a plurality of wells.

8. The method of claim 3 , wherein an oligonucleotide tag of the second plurality of oligonucleotide tags further comprises a hybridization sequence that is configured to hybridize to the second plurality of polynucleotides.

9. The method of claim 3 , wherein substantially every polynucleotide of the second plurality of polynucleotides is associated with the same third tag sequence.

10. The method of claim 3 , wherein an oligonucleotide tag of the second plurality of oligonucleotide tags further comprises a sequencing adaptor.

11. The method of claim 1 , wherein the generating step is performed in a well of a plurality of wells.

12. The method of claim 1 , wherein an oligonucleotide tag of the plurality of oligonucleotide tags further comprises a hybridization sequence that is configured to hybridize to the plurality of polynucleotides.

13. The method of claim 1 , wherein substantially every polynucleotide of the plurality of polynucleotides is associated with the same first tag sequence.

14. The method of claim 1 , wherein an oligonucleotide tag of the plurality of oligonucleotide tags further comprises a sequencing adaptor.

15. The method of claim 1 , wherein sequencing step comprises hybridizing the plurality of tagged polynucleotides or derivatives thereof to a solid support via the sequencing adaptor or derivative thereof.

Assignees

• 10X Genomics Inc

Inventors

• Brenner Sydney
• Mikawa Gi
• Osborne Robert
• Slatter Andrew

Classifications

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12N15/1065

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title