Single Cell Nucleic Acid Detection and Analysis

Abstract

Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.

1 - 24 . (canceled) 
     
     
         25 . A method, comprising:
 (a) providing a sample comprising a set of double-stranded cDNA molecules, each double-stranded cDNA molecule including first and second complementary strands;   (b) tagging said double-stranded cDNA molecules with a set of duplex tags;   (c) sequencing at least some of said tagged strands to produce a set of sequence reads;   (d) sorting sequence reads into paired sequence reads and unpaired sequence reads, wherein (i) each paired read corresponds to sequence reads generated from a first tagged strand and a second differently tagged complementary strand derived from an original parent cDNA molecule in said set, and (ii) each unpaired read represents a first tagged strand having no second differently tagged complementary strand derived from an original parent cDNA molecule represented among said sequence reads in said set of sequence reads; and   (e) quantifying at least: (i) said paired sequence reads, and (ii) said unpaired sequence reads.   
     
     
         26 . The method of  claim 25 , further comprising detecting copy number in said sample, wherein said copy number is based at least in part on said quantification of said paired sequence reads and said unpaired sequence reads that map to each of one or more genetic loci. 
     
     
         27 . The method of  claim 26 , wherein detecting copy number in said sample further comprises one or more of (i) comparing said quantification of said paired and said unpaired sequence reads that map to each of one or more genetic loci to a reference standard copy number, and (ii) comparing said quantification of said paired and said unpaired sequence reads that map to each of one or more genetic loci to a quantification of paired and unpaired sequence reads that map to one or more different genetic loci. 
     
     
         28 . The method of  claim 25 , wherein the tagging step further comprises attaching duplex tags to ends of each of said double-stranded cDNA molecules in said sample, and wherein said set of duplex tags comprise tag sequences that are sufficiently unique so that individually tagged double-stranded cDNA molecules can be substantially differentiated from other tagged double-stranded polynucleotide molecules. 
     
     
         29 . The method of  claim 25 , wherein the duplex tags are present in Y shaped adapters. 
     
     
         30 . The method of  claim 26 , wherein the duplex tags are present in Y shaped adapters. 
     
     
         31 . The method of  claim 27 , wherein the duplex tags are present in Y shaped adapters. 
     
     
         32 . The method of  claim 28 , wherein the duplex tags are present in Y shaped adapters.