Compositions and methods for accurately identifying mutations

Abstract

The present disclosure provides compositions and methods for accurately detecting mutations by uniquely tagging double stranded nucleic acid molecules with dual cyphers such that sequence data obtained from a sense strand can be linked to sequence data obtained from an anti-sense strand when sequenced, for example, by massively parallel sequencing methods.

1 .- 38 . (canceled) 
     
     
         39 . A method of sequencing DNA, the method comprising:
 (a) preparing a sequence library from a sample comprising a plurality of double-stranded DNA molecules from a biological source, wherein preparing the sequence library comprises ligating adapters to the plurality of double-stranded DNA molecules to generate adapter-DNA molecules having a first strand and a second strand;   (b) for each of a plurality of the adapter-DNA molecules:
 (i) sequencing the first and second strands to produce first-strand sequence reads and distinct yet related second-strand sequence reads; and 
 (ii) comparing the first-strand sequence reads and second-strand sequence reads to identify one or more sequence correspondences between the first- and second-strand sequence reads; and 
   (c) comparing the one or more sequence correspondences to a reference sequence to identify a sequence variation as a true mutation, wherein a true mutation is a sequence variation relative to the reference sequence that is consistent between the first-strand sequence reads and second-strand sequence reads for at least one of the adapter-DNA molecules.   
     
     
         40 . The method of  claim 39 , further comprising identifying non-complementary bases between the first-strand sequence reads and the second-strand sequence reads as resulting from experimental or biological error in one strand. 
     
     
         41 . The method of  claim 39 , wherein prior to comparing the first-strand sequence reads and second-strand sequence reads, the method comprises grouping the first-strand sequence reads and second-strand sequence reads based on at least an adapter bar code sequence. 
     
     
         42 . The method of  claim 39 , wherein the adapters comprise bar code sequences selected from a plurality of distinct bar code sequences. 
     
     
         43 . The method of  claim 42 , wherein each bar code sequence is 6 to 8 nucleotides in length. 
     
     
         44 . The method of  claim 42 , wherein each bar code sequence is a double-stranded portion of the respective adapter. 
     
     
         45 . The method of  claim 42 , wherein (i) at least two adapters comprise the same bar code sequence and are ligated to different double-stranded DNA molecules, (ii) end sequences of the double-stranded DNA molecules together with associated bar code sequences form cyphers at both ends of the adapter-DNA molecules; and (iii) individual adapter-target complexes comprise different pairs of cyphers 
     
     
         46 . The method of  claim 39 , wherein the plurality of double-stranded DNA molecules comprises DNA originating from a circulating tumor cell or a cancer cell. 
     
     
         47 . The method of  claim 39 , wherein the sample comprises a tissue obtained from the biological source. 
     
     
         48 . The method of  claim 39 , wherein prior to sequencing, the method further comprises purifying a plurality of adapter-DNA molecules, wherein the purified adapter-DNA molecules comprise nucleic acid molecules that map to specific genomic regions. 
     
     
         49 . The method of  claim 39 , wherein for each of a plurality of the adapter-DNA molecules prior to sequencing, the method further comprises amplifying each original strand of the adapter-DNA molecules to produce a set of copies of the first strand and a set of copies of the second strand. 
     
     
         50 . The method of  claim 39 , further comprising generating an error-corrected sequence, wherein the error-corrected sequence has only nucleotide bases at which the first-strand sequence reads and second-strand sequence reads are in agreement. 
     
     
         51 . A method of sequencing nucleic acid molecules from a biological source, the method comprising:
 (a) providing a sample from the biological source, wherein the sample comprises a plurality of double-stranded nucleic acid molecules,   (b) attaching adapters to individual double-stranded nucleic acid molecules to generate adapter-nucleic acid molecules; and   (c) for each of a plurality of the adapter-nucleic acid molecules:
 (i) generating a set of copies of an original first strand of the adapter-nucleic acid molecule and a set of distinct yet related copies of an original second strand of the adapter-nucleic acid molecule; 
 (ii) sequencing one or more copies of the original first and second strands to produce first- and second-strand sequence reads; 
 (iii) comparing the first- and second-strand sequence reads to identify corresponding base calls that are in agreement; and 
 (iv) generating an error-corrected sequence, wherein the error-corrected sequence has only base calls at which the first- and second-strand sequence reads are in agreement. 
   
     
     
         52 . The method of  claim 51 , further comprising:
 mapping the first- and second-strand sequence reads to a reference sequence to identify sequences corresponding to the reference sequence; and   analyzing sequences corresponding to the reference sequence for each of a plurality of the adapter-nucleic acid molecules to detect the presence of one or more genomic mutations in the sample.   
     
     
         53 . The method of  claim 52 , wherein the one or more genomic mutations comprise a cancer biomarker. 
     
     
         54 . The method of  claim 52 , wherein the biological source is a human, wherein the sample comprises a heterogeneous mixture, and wherein detecting the presence of one or more genomic mutations in the sample characterizes a heterogeneity of the sample. 
     
     
         55 . The method of  claim 54 , wherein the heterogeneous mixture is derived from a tumor. 
     
     
         56 . The method of  claim 54 , wherein the heterogeneous mixture comprises circulating tumor cells or blood. 
     
     
         57 . The method of  claim 51 , wherein the error-corrected sequence comprises a reconstructed sequence of the original first strand of the adapter-nucleic acid molecule and a reconstructed sequence of the original second strand of the adapter-nucleic acid molecule. 
     
     
         58 . The method of  claim 51 , wherein prior to sequencing, the method further comprises purifying a plurality of the adapter-nucleic acid molecules, wherein the purified adapter-nucleic acid molecules comprise nucleic acid molecules that map to specific genomic regions. 
     
     
         59 . The method of  claim 51 , wherein prior to sequencing, the adapter-nucleic acid molecules or copies thereof are selectively enriched by hybridization to substrate bound oligonucleotides. 
     
     
         60 . The method of  claim 51 , wherein prior to comparing the first- and second-strand sequence reads, the method further comprises grouping the first- and second-strand sequence reads based on at least an adapter bar code sequence. 
     
     
         61 . A method of generating sequence reads for a double-stranded target nucleic acid molecule, the method comprising:
 (a) amplifying each original strand of the double-stranded target nucleic acid molecule to produce a set of copies of an original first strand of the double-stranded target nucleic acid molecule and a set of distinct yet related copies of an original second strand of the double-stranded target nucleic acid molecule;   (b) sequencing the amplified target nucleic acid molecules generated from each original strand to produce first- and second-strand sequence reads;   (c) comparing the first- and second-strand sequence reads; and   (d) identifying corresponding base calls in the first- and second-strand sequence reads, wherein corresponding base calls for which first- and second-strand sequence reads are in agreement are identified as true.   
     
     
         62 . The method of  claim 61 , further comprising identifying non-complementary bases between the first- and second-strand sequence reads as experimental errors or sites of DNA damage. 
     
     
         63 . The method of  claim 62 , further comprising generating an error-corrected sequence for the double-stranded target nucleic acid molecule, wherein the error-corrected sequence comprises nucleotide bases at which the first- and second-strand sequence reads are in agreement. 
     
     
         64 . The method of  claim 61 , wherein the double-stranded target nucleic acid molecule comprises a deaminated cytosine. 
     
     
         65 . The method of  claim 64 , wherein the method further comprises enzymatically treating the double-stranded target nucleic acid molecule to repair damaged ends thereof. 
     
     
         66 . The method of  claim 63 , further comprising comparing the error-corrected sequence to a reference sequence to detect the presence of a genetic mutation in the double-stranded target nucleic acid molecule. 
     
     
         67 . The method of  claim 61 , wherein prior to amplifying each original strand of the double-stranded target nucleic acid molecule, the method comprises ligating adapters to each end of the double-stranded target nucleic acid molecule, and wherein the adapters comprise an overhang. 
     
     
         68 . The method of  claim 67 , wherein the adapters comprise bar code sequences, and wherein each bar code sequence is 6 to 8 nucleotides in length.