System and methods for massively parallel analysis of nucleic acids in single cells

Abstract

Methods and systems are provided for massively parallel genetic analysis of single cells in emulsion droplets or reaction containers. Genetic loci of interest are targeted in a single cell using a set of probes, and a fusion complex is formed by molecular linkage and amplification techniques. Methods are provided for high-throughput, massively parallel analysis of the fusion complex in a single cell in a population of at least 10,000 cells. Also provided are methods for tracing genetic information back to a cell using barcode sequences.

1 . A method of amplifying a T cell receptor sequence, comprising:
 (a) isolating each of a plurality of single cells into an individual compartment wherein each individual compartment is a microdroplet in an emulsion;   (b) obtaining transcripts from each of the single cells;   (c) reacting the transcripts from each of the single cells with a set of first probes and a set of second probes,
 i. wherein the set of first probes comprises (1) a first forward probe comprising a sequence complementary to a first subsequence of a first target sequence, and (2) a first reverse probe comprising a sequence complementary to a second subsequence of the first target sequence and a sequence complementary to a non-human, exogenous sequence; 
 ii. wherein the set of second probes comprises (1) a second forward probe comprising a sequence complementary to a first subsequence of a second target sequence, and (2) a second reverse probe comprising a sequence complementary to a second subsequence of the second target sequence and the non-human, exogenous sequence; and 
   (d) performing reverse transcription followed by PCR amplification, thereby generating a fused complex comprising the first target sequence and the second target sequence, wherein the first target sequence is a first gene transcript associated with a T cell.   
     
     
         2 . The method of  claim 1 , further comprising the step of sequencing the fused complex and the step of identifying a T cell based on the sequencing. 
     
     
         3 . The method of  claim 1 , wherein the second target sequence is a barcode sequence. 
     
     
         4 . The method of  claim 3 , wherein the barcode sequence is attached to a bead. 
     
     
         5 . The method of  claim 1 , wherein the first forward probe or the second forward probe is attached to a bead. 
     
     
         6 . The method of  claim 1 , wherein each individual compartment has an average volume of 1 nanoliter (nL). 
     
     
         7 . The method of  claim 1 , wherein the step (a) further comprises introducing an mRNA capture agent and a cell lysis solution into the individual compartments. 
     
     
         8 . The method of  claim 1 , comprising obtaining sequences from at least 10,000 individual cells or at least 5,000 fused complexes. 
     
     
         9 . The method of  claim 1 , wherein step (d) comprises:
 performing overlap extension reverse transcriptase polymerase chain reaction to link the first target sequence and the second target sequence.   
     
     
         10 . The method of  claim 1 , wherein the first target sequence and the second target sequence are different.