Selection of dna adaptor orientation by hybrid capture

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 selective capture of defined molecules. The resulting multi-adaptor constructs are then used in massively-parallel nucleic acid sequencing techniques.

1 . A method for 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 one strand of the first adaptor comprises a capture sequence;   (c) ligating first and second arms of a second adaptor to the linearized first library constructs to form second library constructs, wherein at least one strand of one of the second adaptor arms comprises a functional group;   (d) capturing functionalized double-stranded second library constructs and discarding un-functionalized second library constructs;   (e) eluting single-stranded nucleic acids without a functional group from the captured double-stranded functionalized second library constructs; and   (f) capturing the capture sequence in the one strand of the first adaptor, thereby selecting for library constructs having a desired orientation of the second adaptor with respect to the first adaptor.   
     
     
         2 . The method of  claim 1 , further comprising repeating processes (b) through (f) until a desired number of adaptors have been inserted into the nucleic acid library constructs. 
     
     
         3 . The method of  claim 1 , 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 functional group is biotin and the functionalized double-stranded second library constructs are captured by a strepavidin column. 
     
     
         6 . The method of  claim 1 , wherein the first and second adaptors further comprise Type IIs endonuclease recognition sites. 
     
     
         7 . A method for selecting for orientation of two adaptors with respect to one another in library constructs comprising:
 (a) obtaining a target nucleic acid;   (b) ligating a first adaptor to the target nucleic acid to produce first library constructs, wherein one strand of the first adaptor comprises a capture sequence;   (c) ligating first and second arms of a second adaptor to the linearized first library constructs to form second library constructs;   (d) amplifying the second library constructs with a functionalized primer complementary to an end of one strand of the second adaptor;   (d) capturing functionalized amplified double-stranded second library constructs and discarding unfunctionalized second library constructs;   (e) eluting single-stranded nucleic acids without a functional group from the captured double-stranded functionalized second library constructs; and   (f) capturing the capture sequence in the one strand of the first adaptor, thereby selecting for library constructs having a desired orientation of the second adaptor with respect to the first adaptor.   
     
     
         8 . The method of  claim 7 , wherein the first library constructs are cut with a restriction endonuclease after being circularized. 
     
     
         9 . The method of  claim 7 , wherein each adaptor is ligated to the target nucleic acid as two adaptor arms. 
     
     
         10 . The method of  claim 7 , wherein the functional group is biotin and the functionalized double-stranded second library constructs are captured by a strepavidin column. 
     
     
         11 . The method of  claim 7 , wherein each adaptor further comprises one or more endonuclease recognition sites. 
     
     
         12 . The method of  claim 11 , wherein the endonuclease recognition sites are 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 at least one of the plurality of adaptors has a desired orientation with respect to at least one of the 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 13 , 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 of the 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 18 , 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 18 , 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 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 inserting interspersed adaptors in target nucleic acid, wherein said kit comprises:
 b) a first double-stranded adaptor;   c) a functionalized second double stranded adaptor;   d) reagents for capturing functionalized second double-stranded adaptor; and   (e) reagents for capturing one strand of the first double-stranded adaptor.   
     
     
         25 . The kit of  claim 24  further comprising:
 a) a ligase;   b) a first Type IIs restriction endonuclease;   c) a second Type IIs restriction endonuclease; or   d) a functionalized third adaptor.