Selection of dna adaptor orientation by nicking

Abstract

Aspects described and claimed herein provide methods to insert multiple DNA adaptors into a population of circular target DNAs at defined positions and orientations with respect to one another using nicking reactions. The resulting multi-adaptor constructs are then used in massively-parallel nucleic acid sequencing techniques.

1 . A method for enriching or selecting for orientation of two adaptors with respect to one another in nucleic acid library constructs comprising:
 obtaining target nucleic acids;   ligating a first adaptor to the target nucleic acids to produce first library constructs, wherein one strand of the first adaptor comprises a first nickable site;   ligating a second adaptor to the first library constructs to produce second library constructs, wherein one strand of the second adaptor comprises a second nickable site;   circularizing the second library constructs;   nicking the second library constructs to form a mixture of library constructs with nicks on both strands and library constructs with nicks on one strand and no nicks on the other strand; and   subjecting the library constructs to circle dependant amplification, wherein the strands without nicks will be amplified exponentially and the strands with nicks will be amplified non-exponentially, thereby enriching for library constructs having a desired orientation of the second adaptor with respect to the first adaptor.   
     
     
         2 . The method of  claim 1 , wherein the first library constructs circularized between the ligating processes. 
     
     
         3 . The method of  claim 2 , wherein the first library constructs are cut with a restriction endonuclease after being circularized. 
     
     
         4 . The method of  claim 1 , wherein the first adaptor is ligated to the target nucleic acid as two adaptor arms. 
     
     
         5 . The method of  claim 1 , wherein the second adaptor is ligated to the first library constructs as two adaptor arms. 
     
     
         6 . The method of  claim 1 , wherein the first and second adaptors further comprise Type IIs endonuclease recognition sites. 
     
     
         7 . A method for enriching or selecting for orientation of two adaptors with respect to one another in nucleic acid library constructs comprising:
 (a) obtaining target nucleic acids;   (b) ligating a first adaptor to the target nucleic acids to produce first library constructs, wherein one strand of the first adaptor comprises a first nickase site;   (c) ligating a second adaptor to the first library constructs to produce second library constructs, wherein one strand of the second adaptor comprises a second nickase site;   (d) circularizing the second library constructs;   (e) nicking the second library constructs with a nickase to form a mixture of library constructs with nicks on both strands and library constructs with nicks on one strand and no nicks on the other strand; and   (f) subjecting the library constructs to circle dependant amplification, wherein the strands with no nicks will be amplified exponentially and the strands with nicks will be amplified non-exponentially, thereby enriching for orientation of the second adaptor with respect to the first adaptor in the nucleic acid library constructs.   
     
     
         8 . The method of  claim 7 , wherein processes (b) through (f) until a desired number of adaptors have been inserted into the nucleic acid library constructs. 
     
     
         9 . The method of  claim 7 , wherein the first adaptor is ligated to the target nucleic acid as two adaptor arms. 
     
     
         10 . The method of  claim 7 , wherein the second adaptor is ligated to the first library constructs as two adaptor arms. 
     
     
         11 . An amplicon made by amplification of a circular library construct comprising target nucleic acid interspersed with a plurality of adaptors, wherein at least one of the plurality of adaptors has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         12 . The amplicon of  claim 11 , wherein each of the plurality of adaptors has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         13 . The amplicon of  claim 11 , wherein one or more of the adaptors comprises a restriction endonuclease recognition site. 
     
     
         14 . The amplicon of  claim 13 , wherein the restriction endonuclease recognition site is a Type IIs restriction endonuclease recognition site. 
     
     
         15 . The amplicon of  claim 11 , wherein each adaptor of the plurality of adaptors further comprise a different anchor primer binding site at a 5′ and 3′ end of each of the plurality of adaptors. 
     
     
         16 . A multiplicity of amplicons of circular library constructs, wherein each amplicon comprises target nucleic acid interspersed with a plurality of adaptors, wherein at least one of the plurality of adaptors has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         17 . The multiplicity of amplicons of  claim 16 , wherein each of the plurality of adaptors in each amplicon has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         18 . The multiplicity of amplicons of  claim 16 , wherein the target nucleic acid is genomic DNA, cDNA or RNA, and wherein the multiplicity of amplicons comprises substantially all of genomic DNA, cDNA or RNA of interest. 
     
     
         19 . The multiplicity of amplicons of  claim 16 , wherein one or more of the adaptors comprises a restriction endonuclease recognition site. 
     
     
         20 . The multiplicity of amplicons of  claim 19 , wherein the restriction endonuclease recognition site is a Type IIs restriction endonuclease recognition site. 
     
     
         21 . The multiplicity of amplicons of  claim 16 , wherein each adaptor of the plurality of adaptors further comprise a different anchor primer binding site at a 5′ and 3′ end of each of the plurality of adaptors. 
     
     
         22 . A kit for selecting for desired orientations of multiple adaptors in library constructs, wherein said kit comprises:
 a) a first double-stranded adaptor, wherein said first adaptor comprises a recognition site for a first Type IIs restriction endonuclease and a nickable site;   b) a second double-stranded adaptor, wherein said second adaptor comprises a restriction site for a second Type IIs restriction endonuclease and a nickable site; and   c) primers complementary to both strands of each of said first and second adaptors.   
     
     
         23 . The kit for selecting for desired orientation of multiple adaptors in library constructs of  claim 22 , further comprising: Type IIs restriction endonucleases, polymerases or dNTPs. 
     
     
         24 . A method for enriching or selecting for orientation of two adaptors with respect to one another in library constructs comprising:
 obtaining target nucleic acids;   ligating a first adaptor to the target nucleic acids to produce first library constructs, wherein the undesired strand of the first adaptor comprises a first nickable site;   ligating a second adaptor to the first library constructs to produce second library constructs, wherein the undesired strand of the second adaptor comprises a second nickable site;   circularizing the second library constructs;   nicking the second library constructs to form a mixture of library constructs with nicks on both strands and library constructs with nicks on one strand and no nicks on the other strand; and   amplifying the circularized second library constructs with primers that bind to the desired strand of the first adaptor and the desired strand of the second adaptor, wherein the desired strands without nicks will be amplified exponentially and the strands with nicks will be amplified non-exponentially, thereby enriching for library constructs having a desired orientation of the second adaptor with respect to the first adaptor.