High efficiency construction of dna libraries

Abstract

The invention provides a method for efficient DNA library construction and targeted genetic analyses of the libraries.

1 - 60 . (canceled) 
     
     
         61 . A kit comprising:
 (i) a ligation strand oligonucleotide comprising an anchor sequence;   (ii) a non-ligation partner strand oligonucleotide, and   (iii) a repair oligonucleotide, wherein the repair oligonucleotide is longer than the ligation strand oligonucleotide.   
     
     
         62 . The kit of  claim 61 , wherein the ligation strand oligonucleotide and non-ligation partner strand oligonucleotide are configured to form a double-stranded DNA (dsDNA) pre-adaptor, wherein the non-ligation partner strand oligonucleotide is at least partially hybridized to the ligation strand oligonucleotide. 
     
     
         63 . The kit of  claim 61 , wherein the non-ligation partner strand oligonucleotide comprises a modification at the 3′ terminus that prevents ligation to the 5′ end of an end-repaired DNA fragment and/or adaptor dimer formation. 
     
     
         64 . The kit of  claim 61 , wherein the ligation strand oligonucleotide further comprises one or more of
 (i) one or more unique read codes;   (ii) a PCR primer binding site;   (iii) one or more sample codes for sample multiplexing; and/or   (iv) one or more primer binding sites for DNA sequencing.   
     
     
         65 . The kit of  claim 61 , wherein the repair oligonucleotide comprises an anchor sequence. 
     
     
         66 . The kit of  claim 65 , wherein the repair oligonucleotide further comprises one or more of:
 (i) one or more unique read codes;   (ii) a PCR primer binding site;   (iii) one or more sample codes for sample multiplexing; and/or   (iv) one or more primer binding sites for DNA sequencing.   
     
     
         67 . The kit of  claim 65 , wherein the anchor sequence of the ligation strand oligonucleotide is at least partially complementary to the anchor sequence of the repair oligonucleotide. 
     
     
         68 . The kit of  claim 61 , further comprising a first ligase configured to ligate the ligation strand oligonucleotide to the 3′ end of an end-repaired DNA fragment at a first temperature. 
     
     
         69 . The kit of  claim 68 , further comprising a second ligase configured to ligate the repair oligonucleotide to the 5′ end of the end-repaired DNA fragment at a second temperature. 
     
     
         70 . The kit of  claim 69 , wherein the second temperature is higher than the first temperature and results in the displacement of the partner strand oligonucleotide from the ligation strand oligonucleotide. 
     
     
         71 . The kit of  claim 69 , wherein the first ligase is different from the second ligase. 
     
     
         72 . The kit of  claim 69 , wherein the second ligase is a thermostable ligase. 
     
     
         73 . The kit of  claim 69 , wherein the first temperature is 22° C. or lower and the second temperature is 37° C. or higher. 
     
     
         74 . The kit of  claim 61 , further comprising a phosphatase, wherein the phosphatase is configured to remove a terminal phosphate residue of a DNA fragment. 
     
     
         75 . The kit of  claim 61 , further comprising one or more DNA end-repair enzymes. 
     
     
         76 . The kit of  claim 61 , further comprising a kinase, wherein the kinase is configured to add a phosphate group to the 5′ terminal nucleotide of each strand of an end-repaired DNA fragment. 
     
     
         77 . The kit of  claim 61 , further comprising one or more reagents for repairing damage of one or more DNA fragments. 
     
     
         78 . The kit of  claim 77 , wherein the damage is a deaminated cytosine (Uracil), an abasic site, methylation of guanine to O6MeG, one or more DNA nicks, one or more DNA gaps, or a thymine dimer. 
     
     
         79 . The kit of  claim 69 , wherein the ligation strand oligonucleotide is configured to hybridize to the repair oligonucleotide, and wherein the ligation strand oligonucleotide is configured to be extended at its 3′ end using the repair oligonucleotide as a template. 
     
     
         80 . The kit of  claim 79 , wherein the extended ligation strand oligonucleotide is ligated to the 3′ end of an end-repaired DNA fragment, and wherein the repair oligonucleotide is ligated to the 5′ end of the end-repaired DNA fragment. 
     
     
         81 . The kit of  claim 61 , further comprising a capture probe module comprising a capture probe sequence and a tail sequence. 
     
     
         82 . A method for adaptor ligation to one or more DNA fragments comprising:
 (a) removing the terminal phosphate residues of the one or more DNA fragments;   (b) treating the dephosphorylated DNA fragments with one or more end-repair enzymes to generate end-repaired DNA fragments;   (c) ligating one or more double-stranded DNA (dsDNA) pre-adaptors to the 3′ end of each strand of the end-repaired DNA fragments to form pre-adaptor/end-repaired DNA complexes,   wherein each dsDNA pre-adaptor comprises (i) a ligation strand oligonucleotide that is ligated to the 3′ end of each strand of the end-repaired DNA fragments and comprises an anchor sequence; and (ii) a non-ligation partner strand oligonucleotide;   (d) displacing the non-ligation partner strand oligonucleotide from the pre-adaptor/end-repaired DNA complexes with a repair oligonucleotide to form adaptor/end-repaired DNA complexes, wherein each adaptor comprises the ligation strand oligonucleotide and the repair oligonucleotide; and   (e) treating the adaptor/end-repaired DNA complexes with one or more enzymes to form one or more contiguous dsDNA fragments each comprising an adaptor molecule ligated to each end of the DNA fragment.   wherein step (c) is performed at a first temperature and step (d) is performed at a second temperature, wherein the second temperature is higher than the first temperature and results in the displacement of the partner strand oligonucleotide from the ligation strand oligonucleotide.   
     
     
         83 . A method of diagnosing a genetic disease in a subject comprising:
 (a) obtaining DNA fragments from a biological sample of a subject;   (b) removing the terminal phosphate residues of the DNA fragments to generate dephosphorylated DNA fragments;   (c) treating the dephosphorylated DNA fragments with one or more end-repair enzymes to generate end-repaired DNA fragments;   (d) ligating one or more double-stranded DNA (dsDNA) pre-adaptors to the 3′ end of each strand of the end-repaired DNA fragments to form pre-adaptor/end-repaired DNA complexes, wherein each dsDNA pre-adaptor comprises (i) a ligation strand oligonucleotide that is ligated to the 3′ end of each strand of the end-repaired DNA fragments; and (ii) a non-ligation partner strand oligonucleotide;   (e) displacing the non-ligation partner strand oligonucleotide from the pre-adaptor/end-repaired DNA complexes with a repair oligonucleotide to form adaptor/end-repaired DNA complexes, wherein each adaptor comprises the ligation strand oligonucleotide and the repair oligonucleotide;   (f) treating the adaptor/end-repaired DNA complexes with one or more enzymes to form one or more contiguous, tagged dsDNA fragments, wherein the one or more tagged dsDNA fragments comprise a tagged dsDNA library;   (g) amplifying the tagged dsDNA library to generate a library of DNA clones;   (h) performing a quantitative genetic analysis of one or more target genetic loci in the library of DNA clones, wherein the one or more target genetic loci are associated with the genetic disease, and wherein the detection of one or more genetic lesions in the one or more target genetic loci is diagnostic of the genetic disease.   
     
     
         84 . A method for constructing a DNA library comprising:
 (a) removing the terminal phosphate residues of one or more DNA fragments to generate dephosphorylated DNA fragments;   (b) treating the dephosphorylated DNA fragments with one or more end-repair enzymes to generate end-repaired DNA fragments;   (c) ligating one or more double-stranded DNA (dsDNA) pre-adaptors to the 3′ end of each strand of the end-repaired DNA fragments to form pre-adaptor/end-repaired DNA complexes, wherein each dsDNA pre-adaptor comprises (i) a ligation strand oligonucleotide that is ligated to the 3′ end of each strand of the end-repaired DNA fragments; and (ii) a non-ligation partner strand oligonucleotide;   (d) displacing the non-ligation partner strand oligonucleotide from the pre-adaptor/end-repaired DNA complexes with a repair oligonucleotide to form adaptor/end-repaired DNA complexes, wherein each adaptor comprises the ligation strand oligonucleotide and the repair oligonucleotide; and   (e) forming a DNA library comprising one or more tagged dsDNA fragments by treating the adaptor/end-repaired DNA complexes with one or more enzymes to form one or more contiguous dsDNA fragments each comprising an adaptor molecule ligated to each end of the DNA fragment; and   (f) amplifying the DNA library to generate a library of DNA clones.