Methods for analyzing nucleic acids from single cells

Patent metadata

Field	Value
Publication number	US-10155981-B2
Application number	US-201715677957-A
Country	US
Kind code	B2
Filing date	Aug 15, 2017
Priority date	Aug 20, 2009
Publication date	Dec 18, 2018
Grant date	Dec 18, 2018

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 of analyzing nucleic acids from a plurality of single cells, the method comprising:
(a) providing a sample comprising a plurality of single cells, wherein each single cell of the plurality of single cells comprises a plurality of sample polynucleotides;
(b) generating a plurality of tagged polynucleotides from the plurality of sample polynucleotides, wherein each tagged polynucleotide comprises:
(i) a sequence from a sample polynucleotide of the plurality of sample polynucleotides; and
(ii) a multiplex identifier (MID) sequence comprising:
I. a first tag sequence associated with the single cell from which the sample polynucleotide is derived, wherein the first tag sequence is a different sequence for different single cells in the plurality of single cells; and
II. a second tag sequence distinguishing the sample polynucleotide from other sample polynucleotides derived from the same single cell;

(c) sequencing the plurality of tagged polynucleotides to obtain a plurality of identified polynucleotide sequences;
(d) using the first tag sequence to correlate the identified polynucleotide sequence with the single cell from which the identified polynucleotide sequence is derived; and
(e) using the second tag sequence to correlate the identified polynucleotide sequence with the sample polynucleotide from which the identified polynucleotide sequence is derived.

2. The method of claim 1 , wherein the method further comprises amplifying the tagged polynucleotides prior to the sequencing step (c).

3. The method of claim 1 , wherein the sample polynucleotides are selected from DNA and RNA.

4. The method of claim 3 , wherein the sample polynucleotides comprise mRNA.

5. The method of claim 1 , wherein the tagged polynucleotides are generated through at least one ligation reaction.

6. The method of claim 1 , wherein the tagged polynucleotides are generated through PCR or linear amplification of the plurality of sample polynucleotides using an adapter sequence comprising the MID sequence and an amplification primer.

Assignees

• 10X Genomics Inc

Inventors

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

Classifications

• C12Q1/6806

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

• C12N15/1065

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

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/6874 Primary

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title