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 for introducing unique barcode sequences into reaction containers or emulsion microdroplets, comprising:
 providing a pool of first sequences comprising the unique barcode sequences, wherein each of the unique barcode sequences is physically linked to a selection resistance gene;   providing a population of single cells;   transfecting the population of single cells with the pool of the first sequences;   selecting cells comprising one of the unique barcode sequences and the selection resistance gene; and   isolating each of the selected cells into the reaction containers or emulsion microdroplets.   
     
     
         2 . The method of  claim 1 , wherein the selection resistance gene comprises a gene that encodes resistance to gentamycin, neomycin, hygromycin, or puromycin. 
     
     
         3 . The method of  claim 1 , wherein each of the unique barcode sequences is further physically linked to a universal priming site. 
     
     
         4 . The method of  claim 1 , wherein each of the unique barcode sequences comprises at least six nucleotides. 
     
     
         5 . The method of  claim 1 , wherein the pool of the first sequences has greater than 1000-fold diversity in sequence. 
     
     
         6 . The method of  claim 5 , further comprising the step of sequencing the unique barcode sequences. 
     
     
         7 . The method of  claim 6 , further comprising the step of identifying at least one of the selected cells based on the sequencing. 
     
     
         8 . The method of  claim 1 , wherein each of the first sequences is further physically linked to a second polynucleic acid target sequence. 
     
     
         9 . The method of  claim 8 , wherein the second polynucleic acid target sequence is complementary to at least part of an RNA transcript. 
     
     
         10 . The method of  claim 9 , further comprising the step of synthesizing a fused polynucleic acid sequence comprising one of the unique barcode sequences and the second polynucleic acid target sequence. 
     
     
         11 . The method of  claim 10 , further comprising the step of sequencing the fused polynucleic acid sequence. 
     
     
         12 . The method of  claim 11 , further comprising the step of identifying at least one of the selected cells based on the sequencing.