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 generating a fused nucleic acid complex comprising least two nucleic acid sequences from a single cell or subpopulation of cells contained within a population of at least 10,000 cells, comprising:
 isolating the single cell or subpopulation of cells;   providing a set of nucleic acid probes to said isolated single cell or subpopulation of cells comprising a first probe comprising a sequence that is complementary to a first subsequence of a first target nucleic acid, a second probe comprising a sequence that is complementary to a second subsequence of the first target nucleic acid and a sequence that is complementary to an exogenous sequence, a third probe comprising a sequence that is complementary to the portion of the second probe comprising the sequence that is complementary to the exogenous sequence and a sequence that is complementary to a first subsequence of a second target nucleic acid, and a fourth probe comprising a sequence that is complementary to a second subsequence of the second target nucleic acid sequence, wherein the first target nucleic acid or the second target nucleic acid comprises an endogenous sequence;   amplifying the first and second target nucleic acid sequences independently, wherein the first target nucleic acid sequence is amplified using the first probe and the second probe, and wherein the second target nucleic acid sequence is amplified using the third probe and the fourth probe;   hybridizing the exogenous sequence to its complement; and   amplifying the first target nucleic acid sequence, the second target nucleic acid sequence, and the exogenous sequence using the first and fourth probes, thereby generating a fused nucleic acid complex.   
     
     
         2 . The method of  claim 1 , further comprising performing a bulk sequencing reaction to generate sequence information for at least 100,000 fused complexes from at least 10,000 cells within the population of cells, wherein the sequence information is sufficient to co-localize the first target nucleic acid sequence and the second target nucleic acid sequence to the single cell or subpopulation of cells from the population of at least 10,000 cells. 
     
     
         3 . The method of  claim 1 , wherein the single cell or subpopulation of cells is isolated from at least one mammalian subject. 
     
     
         4 . The method of  claim 1 , wherein the single cell or subpopulation of cells comprises T cells, B cells, or plasma blast cells. 
     
     
         5 . The method of  claim 1 , wherein the first target nucleic acid sequence comprises a heavy chain immunoglobulin sequence and the second target nucleic acid sequence comprises a light chain immunoglobulin sequence. 
     
     
         6 . The method of  claim 1 , wherein the target nucleic acids comprise alpha and beta T cell receptor subunit sequences. 
     
     
         7 . The method of  claim 1 , further comprising recombinantly expressing in a host cell one or more polypeptide chains encoded by the first and second target sequences of the fused nucleic acid complex. 
     
     
         8 . The method of  claim 7 , further comprising isolating the expressed one or more polypeptide chains. 
     
     
         9 . The method of  claim 7 , wherein the expressed one or more polypeptide chains are expressed in a mammalian cell, a yeast cell, or a bacterial cell. 
     
     
         10 . The method of  claim 7 , wherein the one or more polypeptide chains are expressed on the surface of the host cell or secreted by the host cell. 
     
     
         11 . The method of  claim 7 , wherein at least 100,000 of the target sequences are expressed in a plurality of the host cells. 
     
     
         12 . A polyclonal antibody composition comprising the expressed one or more polypeptide chains produced according to the method of  claim 7 . 
     
     
         13 . A single chain variable fragments (scFv), an antibody fragment, or a full-length antibody comprising the expressed one or more polypeptide chains produced according to the method of  claim 7 . 
     
     
         14 . A method of expressing one or more polypeptide chains, comprising:
 culturing an engineered microorganism comprising an expression construct comprising the first target nucleic acid of the fused nucleic acid construct of  claim 1  under conditions sufficient to express a polypeptide chain encoded by the first target nucleic acid.   
     
     
         15 . A method of expressing one or more polypeptides, comprising:
 culturing an engineered microorganism comprising an expression construct comprising the second target nucleic acid of the fused nucleic acid construct of  claim 1  under conditions sufficient to express a polypeptide encoded by the second target nucleic acid.   
     
     
         16 . The method of  claim 15 , wherein the engineered microorganism further comprises an expression construct comprising the first target nucleic acid sequence and wherein the engineered microorganism is cultured under conditions sufficient to express a polypeptide encoded by the first target nucleic acid sequence. 
     
     
         17 . The method of  claim 16 , wherein a single expression construct comprises the first target nucleic acid sequence and the second target nucleic acid. 
     
     
         18 . The method of  claim 15 , further comprising isolating the expressed polypeptide encoded by the expression construct. 
     
     
         19 . A method of generating a recombinantly-expressed polypeptide chain, comprising:
 providing a set of nucleic acid probes, comprising a first probe comprising a sequence that is complementary to a first target nucleic acid sequence and a second probe comprising a sequence that is complementary to a second target nucleic acid sequence;   introducing the set of nucleic acid probes into the single cell or subpopulation of cells contained within a population of at least 10,000 cells;   adding a third probe to the single cell or subpopulation of cells, wherein the third probe comprises a sequence that is complementary to a subsequence from the first target nucleic acid sequence and a sequence that is complementary to a subsequence from the second target nucleic acid sequence, thereby generating a fused nucleic acid complex comprising the first target nucleic acid and the second target nucleic acid;   amplifying the fused nucleic acid complex; and   recombinantly expressing in a host cell one or more polypeptide chains encoded by the first and second target sequences of the fused nucleic acid complex.   
     
     
         20 . A method of generating a recombinantly-expressed polypeptide chain, comprising:
 culturing an engineered microorganism comprising an expression construct comprising a sequence encoding one or more polypeptide chains encoded by the first or second target sequences of the fused nucleic acid complex of  claim 19  under conditions sufficient to recombinantly express the polypeptide chain; and   isolating the recombinantly expressed polypeptide chain.