Selection of dna adaptor orientation by amplification

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 by employing amplification procedures. 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 library constructs comprising:
 (a) obtaining target nucleic acid;   (b) ligating a first adaptor to the target nucleic acid to produce first library constructs wherein the first adaptor comprises a first desired strand and a first undesired strand;   (c) ligating a second adaptor to the first library constructs to produce second library constructs, wherein the second adaptor comprises a second desired strand and a second undesired strand;   (d) circularizing the second library constructs; and   (e) 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 when the primers bind to different strands of the circularized second library constructs, the second library constructs are amplified exponentially and wherein when the primers bind to the same strands of the circularized second library constructs, the second library constructs are amplified non-exponentially.   
     
     
         2 . The method of  claim 1 , wherein the first library constructs are circularized between the two ligating steps. 
     
     
         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 or more adaptors with respect to one another in library constructs comprising:
 (a) obtaining target nucleic acid;   (b) ligating a first adaptor to the target nucleic acid to produce first library constructs wherein the first adaptor comprises a first desired strand and a first undesired strand;   (c) ligating a second adaptor to the first library constructs to produce second library constructs, wherein the second adaptor comprises a second desired strand and a second undesired strand;   (d) circularizing the second library constructs;   (e) 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 when the primers bind to different strands of the circularized second library constructs, the second library constructs are amplified exponentially and wherein when the primers bind to the same strands of the circularized second library constructs, the second library constructs are amplified non-exponentially; and   (f) repeating processes (b) through (e) until a desired number of adaptors have been inserted into the nucleic acid library constructs.   
     
     
         8 . The method of  claim 7 , wherein the first library constructs are circularized between the two ligating steps. 
     
     
         9 . The method of  claim 8 , wherein the first library constructs are cut with a restriction endonuclease after being circularized. 
     
     
         10 . The method of  claim 7 , wherein the first adaptor is ligated to the target nucleic acid as two adaptor arms. 
     
     
         11 . The method of  claim 7 , wherein the second adaptor is ligated to the first library constructs as two adaptor arms. 
     
     
         12 . The method of  claim 7 , wherein the first and second adaptors further comprise Type IIs endonuclease recognition sites. 
     
     
         13 . An amplicon made by amplification of a circular library construct comprising target nucleic acid interspersed with a plurality of adaptors, wherein one of the plurality of adaptors has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         14 . The amplicon of  claim 13 , wherein each of the plurality of adaptors has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         15 . The amplicon of  claim 14 , wherein one or more of the adaptors comprises a restriction endonuclease recognition site. 
     
     
         16 . The amplicon of  claim 15 , wherein the restriction endonuclease recognition site is a Type IIs restriction endonuclease recognition site. 
     
     
         17 . The amplicon of  claim 13 , 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. 
     
     
         18 . 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. 
     
     
         19 . The multiplicity of amplicons of  claim 18 , wherein each of the plurality of adaptors has a desired orientation with respect to at least one other of the plurality of adaptors. 
     
     
         20 . The multiplicity of amplicons of  claim 19 , 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. 
     
     
         21 . The multiplicity of amplicons of  claim 19 , wherein one or more of the adaptors comprises a restriction endonuclease recognition site. 
     
     
         22 . The multiplicity of amplicons of  claim 21 , wherein the restriction endonuclease recognition site is a Type IIs restriction endonuclease recognition site. 
     
     
         23 . The multiplicity of amplicons of  claim 18 , wherein the 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. 
     
     
         24 . A kit for selectively amplifying 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;   (b) a second double-stranded adaptor, wherein said second adaptor comprises a restriction site for a second Type IIs restriction endonuclease; and   (c) primers complementary to one strand of each of said first and second adaptors.