High throughput sequencing of multiple transcripts

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.

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.   
     
     
         2 . The method of  claim 1 , further defined as a method for obtaining a plurality of 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 , further defined as a method for obtaining paired antibody VH and VL sequences for an antibody that binds to an antigen of interest. 
     
     
         8 . The method of  claim 3 , wherein the beads are conjugated to the 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 - 13 . (canceled) 
     
     
         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 - 16 . (canceled) 
     
     
         17 . 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. 
     
     
         18 . The method of  claim 1 , wherein step (e) does not comprise the use of overlap extension reverse transcriptase polymerase chain reaction. 
     
     
         19 . The method of  claim 2 , 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 molecules. 
     
     
         20 . The method of  claim 2 , wherein step (e) does not comprise the use of overlap extension reverse transcriptase polymerase chain reaction and wherein the VH and VL cDNAs are separate molecules. 
     
     
         21 . The method of  claim 2 , wherein the VH and VL sequences are obtained by sequencing of distinct molecules. 
     
     
         22 . The method of  claim 2 , further comprising identifying the paired antibody VH and VL sequences comprises performing a probability analysis of the sequences. 
     
     
         23 - 27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein sequestering the single cells comprises introducing the cells to a device comprising a plurality of microwells so that the majority of cells are captured as single cells. 
     
     
         29 . The method of  claim 1 , wherein step (e) comprises sequencing of two or more transcripts covalently linked to the same bead. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 1 , further comprising determining natively paired transcripts using probability analysis. 
     
     
         32 - 33 . (canceled) 
     
     
         34 . A system comprising:
 a) an aqueous fluid phase exit disposed within an annular flowing oil phase; and   b) an aqueous fluid phase, wherein the aqueous phase fluid comprises a suspension of cells and is dispersed within the flowing oil phase, resulting in emulsified droplets with low size dispersity comprising an aqueous suspension of cells.   
     
     
         35 - 43 . (canceled) 
     
     
         44 . A composition comprising an emulsion having a plurality of individual microvesicles, said microvesicles comprising a bead with immobilized oligonucleotides for priming of reverse transcription and individual B-cells, which have been disrupted to release mRNA transcripts from individual B-cells. 
     
     
         45 - 64 . (canceled)