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 comprising:
 (a) preparing a sequencing library by (i) ligating cypher polynucleotides to double-stranded target DNA molecules to form double-stranded cypher-target DNA complexes, and (ii) amplifying each strand of the double-stranded cypher-target DNA complexes to produce a set of copies of an original first strand and a set of distinct yet related copies of an original second strand for each of the double-stranded cypher-target DNA complexes;   (b) capturing cypher-target DNA complex amplification products by hybridization to substrate bound oligonucleotides to provide enriched cypher-target DNA products;   (c) sequencing the enriched cypher-target DNA products to produce a plurality of first-strand sequencing reads and a plurality of second-strand sequencing reads; and   (d) for at least some of the enriched cypher-target DNA products, comparing the first-strand sequencing reads and second-strand sequencing reads to produce an error-corrected sequence of a corresponding double-stranded target DNA molecule;   wherein the error-corrected sequence has only nucleotide bases at which the first-strand sequencing reads and second-strand sequencing reads are in agreement, such that a mutation in the error-corrected sequence is identified as a true mutation.   
     
     
         40 . The method of  claim 39 , wherein the double-stranded target DNA molecules comprise a heterogenous mixture of DNA. 
     
     
         41 . The method of  claim 39 , wherein each of the double-stranded cypher-target DNA complexes comprises a cypher polynucleotide at both ends. 
     
     
         42 . The method of  claim 39 , wherein each of the cypher polynucleotides comprises an identifier tag that alone or in combination with an end of a double-stranded target DNA molecule uniquely identifies the double-stranded target DNA molecule to which it is ligated. 
     
     
         43 . The method of  claim 39 , wherein the cypher polynucleotides comprise a random or partially random sequence. 
     
     
         44 . The method of  claim 39 , wherein each of the double-stranded cypher-target DNA complexes comprises (i) a first strand comprising a first random or partially random nucleic acid sequence, and (ii) a second strand comprising a second random or partially random nucleic acid sequence. 
     
     
         45 . The method of  claim 39 , wherein (i) the cypher polynucleotides each comprise an identifier sequence, (ii) at least two cypher polynucleotides comprise the same identifier sequence and are ligated to different double-stranded target DNA molecules, (iii) end sequences of the double-stranded target DNA molecules together with associated identifier sequences form cyphers at both ends of the double-stranded target DNA molecules; and (iv) individual double-stranded cypher-target DNA complexes comprise different pairs of cyphers. 
     
     
         46 . The method of  claim 39 , wherein each of the double-stranded cypher-target DNA complexes comprises a unique identifier sequence on each strand, wherein the identifier sequences link each strand with its original complementary strand. 
     
     
         47 . The method of  claim 39 , wherein the cypher polynucleotides comprise a capture sequence for hybridization to the substrate bound oligonucleotides. 
     
     
         48 . The method of  claim 39 , wherein the ligating cypher polynucleotides to double-stranded target DNA molecules comprises ligation of complementary overhangs. 
     
     
         49 . The method of  claim 48 , wherein the double-stranded target DNA molecules are produced by specific cleavage with a restriction endonuclease. 
     
     
         50 . The method of  claim 39 , wherein the double-stranded target DNA molecules subjected to the ligating comprise double-stranded DNA fragments that range in size from 100 to 1,000 nucleotides. 
     
     
         51 . The method of  claim 50 , wherein the double-stranded DNA fragments range in size from 150 to 500 nucleotides. 
     
     
         52 . The method of  claim 39 , wherein the preparing a sequencing library further comprises purifying the double-stranded cypher-target DNA complexes prior to the amplifying. 
     
     
         53 . A method of generating error-corrected sequences of nucleic acid molecules, the method comprising:
 (a) providing a sequencing library comprising a plurality of adapter-tagged nucleic acid molecules, wherein the sequencing library is generated from a plurality of double-stranded polynucleotides;   (b) purifying a plurality of the adapter-tagged nucleic acid molecules to provide enriched adapter-target nucleic acid molecules, wherein the enriched adapter-target nucleic acid molecules comprise nucleic acid molecules that map to specific genomic regions;   (c) sequencing the enriched adapter-target nucleic acid molecules to obtain a plurality of sequencing reads;   (d) grouping sequencing reads of the plurality of sequencing reads based on adapter bar code sequences, wherein a group of sequencing reads comprises sequencing reads for a first strand and complementary second strand of one of the adapter-tagged nucleic acid molecules; and   (e) within each group of sequencing reads:
 (i) comparing the sequencing reads for the first-strand with the sequencing reads for the complementary second-strand of the same adapter-tagged nucleic acid molecule; and 
 (ii) generating error-corrected sequences of the adapter-tagged nucleic acid molecule, wherein the error-corrected sequences have only nucleotide bases at which the sequencing reads for the first-strand and complementary second-strand are in agreement. 
   
     
     
         54 . The method of  claim 53 , wherein the enriched adapter-target nucleic acid molecules comprise adapter-tagged nucleic acid molecules generated from double-stranded polynucleotide fragments that range in size from 100 to 1,000 nucleotides. 
     
     
         55 . The method of  claim 54 , wherein the double-stranded polynucleotide fragments range in size from 150 to 500 nucleotides. 
     
     
         56 . The method of  claim 53 , wherein the purifying comprises hybridizing adapter-tagged nucleic acid molecules comprising a hybridization sequence to substrate bound oligonucleotides comprising a sequence complementary to the hybridization sequence. 
     
     
         57 . The method of  claim 53 , wherein (i) the purifying comprises amplification with amplification primers, and (ii) the sequencing is targeted to specific genomic regions. 
     
     
         58 . The method of  claim 53 , wherein (i) the purifying comprises hybridizing adapter-tagged nucleic acid molecules to substrate bound oligonucleotides, and (ii) the adapter-tagged nucleic acid molecules comprise an adapter-derived capture sequence for hybridization to the substrate bound oligonucleotides.