High throughput sequencing of multiple transcripts

Patent metadata

Field	Value
Publication number	US-9708654-B2
Application number	US-201314407849-A
Country	US
Kind code	B2
Filing date	Jun 17, 2013
Priority date	Jun 15, 2012
Publication date	Jul 18, 2017
Grant date	Jul 18, 2017

Abstract

The present disclosure generally relates to sequencing two or more genes expressed in a single cell in a high-throughput manner. More particularly, the present disclosure relates to a method for high-throughput sequencing of pairs of transcripts co expressed in single cells (e.g., antibody VH and VL coding sequence) to determine pairs of polypeptide chains that comprise immune receptors.

First claim

What is claimed is:

1. A method comprising:
a) sequestering single cells and an mRNA capture agent into individual compartments;
b) lysing the cells and collecting mRNA transcripts with the mRNA capture agent;
c) isolating the mRNA from the compartments using the mRNA capture agent;
d) performing reverse transcription followed by PCR amplification on the captured mRNA; and
e) sequencing at least two distinct cDNA products amplified from a single cell,

wherein:
(i) the at least two distinct cDNA products comprise paired antibody VH and VL sequences, wherein the cells are B-cells; or
(ii) the at least two distinct cDNA products comprise paired T-cell receptor sequences, wherein the cells are T-cells.

2. The method of claim 1 , wherein the at least two distinct cDNA products comprise paired antibody VH and VL sequences, wherein the cells are B-cells.

3. The method of claim 1 , wherein the mRNA capture agent is a bead.

4. The method of claim 3 , wherein the beads are magnetic.

5. The method of claim 3 , wherein the bead comprises oligonucleotides which hybridize mRNA.

6. The method of claim 5 , wherein the oligonucleotides comprise at least one of poly(T) and primers specific to a transcript of interest.

7. The method of claim 2 , wherein the paired antibody VH and VL sequences are for an antibody that binds to an antigen of interest.

8. The method of claim 5 , wherein the beads are conjugated to an antigen of interest and the oligonucleotides are only conjugated to the beads in the presence of an antibody that binds to the antigen of interest.

9. The method of claim 1 , wherein the individual compartments are wells in a gel or microtiter plate.

10. The method of claim 1 , said individual compartments having a volume of less than 5 nL.

11. The method of claim 10 , wherein the compartments are sealed with a permeable membrane prior to step (c).

12. The method of claim 1 , wherein the individual compartments are microvesicles in an emulsion.

13. The method of claim 1 , wherein steps (a) and (b) are performed concurrently.

14. The method of claim 1 , wherein steps (a) and (b) comprise isolating single cells and an mRNA capture agent into individual microvesicles in an emulsion and in the presence of a cell lysis solution.

15. The method of claim 2 , comprising obtaining sequences from at least 10,000 individual cells or at least 5,000 individual paired antibody VH and VL sequences.

16. The method of claim 1 , wherein step (e) comprises:
linking VH and VL cDNAs by performing overlap extension reverse transcriptase polymerase chain reaction to link VH and VL cDNAs in single DNA molecules, wherein the at least two distinct cDNA products comprise paired antibody VH and VL sequences.

17. The method of claim 1 , wherein step (e) does not comprise the use of overlap extension reverse transcriptase polymerase chain reaction.

18. The method of claim 2 , wherein the VH and VL sequences are obtained by sequencing of distinct molecules.

19. The method of claim 1 , wherein the at least two distinct cDNA products comprise paired T-cell receptor sequences, wherein the cells are T-cells.

20. The method of claim 1 , wherein step (e) comprises linking cDNA by performing overlap extension reverse transcriptase polymerase chain reaction to link at least 2 transcripts into a single DNA molecule.

Assignees

• Univ Texas
• Univ Texas

Inventors

• Hunicke-Smith Scott
• Dekosky Brandon
• Ellington Andy
• Georgiou George

Classifications

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6806 Primary

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

• B01L3/502784

  specially adapted for droplet or plug flow, e.g. digital microfluidics · CPC title

• C12N15/1075

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title