Method of making a paired tag library for nucleic acid sequencing

Abstract

The present disclosure relates to methods and compositions for making paired tags and paired tag libraries.

1 . A method of forming a paired tag comprising a first tag sequence and a second tag sequence that together comprise at least part of a polynucleotide of interest, said method comprising:
 ligating a first end and a second end of a double stranded polynucleotide of interest to an adaptor thereby forming a circular nucleic acid molecule, wherein the circular nucleic acid molecule comprises a first nick between the first end of the double stranded polynucleotide of interest and the adaptor, and a second nick between the second end of the double stranded polynucleotide of interest and the adaptor, wherein the first nick and the second nick are on different strands of the circular nucleic acid molecule than the second nick; and   performing a nick translation reaction wherein at least one nick is translated into the polynucleotide of interest.   
     
     
         2 . The method of  claim 1 , further comprising:
 allowing the nick translation reaction to proceed for a specific time; and   terminating the nick translation reaction.   
     
     
         3 . The method of  claim 1 , wherein the first end and the second end of the double stranded polynucleotide of interest lack 5′ phosphate residues. 
     
     
         4 . The method of  claim 1 , further comprising terminating the nick translation reaction before the first nick and the second nick are translated past each other. 
     
     
         5 . The method of  claim 1 , wherein the first nick and the second nick are translated more than 10 bases, further comprising the step of cleaving the circular nucleic acid molecule at the first nick and at the second nick. 
     
     
         6 . The method of  claim 5 , wherein the first nick and the second nick are translated less than 500 bases. 
     
     
         7 . The method of  claim 6 , wherein the first nick and the second nick are translated less than 200 bases. 
     
     
         8 . The method of  claim 7 , wherein the first nick and the second nick are translated less than 100 bases. 
     
     
         9 . The method of  claim 5 , wherein the first nick and the second nick are translated between about 20 bases to about 50 bases. 
     
     
         10 . The method of  claim 1 , wherein at least one nick is translated less than 500 bases. 
     
     
         11 . The method of  claim 10 , wherein at least one nick is translated more than 27 bases and less than 500 bases. 
     
     
         12 . The method of  claim 11 , wherein at least one nick is translated less than 200 bases. 
     
     
         13 . The method of  claim 1 , wherein at least one nick is translated from 28 bases to about 50 bases. 
     
     
         14 . The method of  claim 1 , wherein the circular nucleic acid molecule comprises a first gap between the first end of the double stranded polynucleotide of interest and the adaptor. 
     
     
         15 - 80 . (canceled) 
     
     
         81 . A nicked linking polynucleotide comprising:
 a first adaptor comprising a first adaptor strand that is hybridized to a second adaptor strand, wherein the first adaptor strand lacks a phosphate group on its 5′ end;   a second adaptor comprising a third adaptor strand that is hybridized to a fourth adaptor strand, wherein the third adaptor strand lacks a phosphate group on its 5′ end; and   a linking polynucleotide comprising a first linking strand hybridized to a second linking strand, wherein the linking polynucleotide comprises a first end and a second end, wherein the first adaptor is attached to the first end of the linking polynucleotide such that a nick is present where the first adaptor strand lacks a phosphate group on its 5′ end, wherein the second linking strand is attached to the second adaptor strand, wherein the second adaptor is attached to the second end of the linking polynucleotide such that a nick is present where the third adaptor strand of the second adaptor lacks a phosphate group on its 5′ end, wherein the second strand of the linking polynucleotide is attached to the fourth adaptor strand.   
     
     
         82 . The nicked linking polynucleotide of  claim 81 , further comprising a phosphate group on a 5′ end of the second adaptor strand. 
     
     
         83 . The nicked linking polynucleotide of  claim 82 , further comprising a phosphate group on a 5′ end of the fourth adaptor strand. 
     
     
         84 . The nicked linking polynucleotide of  claim 83 , wherein there is no phosphate group on the further comprising a phosphate group on a 3′ end of the second adaptor strand. 
     
     
         85 - 91 . (canceled) 
     
     
         92 . A plurality of circular nucleic acid molecules, the individual circular molecules in the plurality comprising:
 (a) an a first and a second adaptor sequence common to the plurality of circular nucleic acid molecules; and   (b) a target sequence region that differs between different molecules of the plurality of circular molecules of the plurality of circular nucleic acid molecules, wherein each target sequence region comprises a fragment of genomic DNA, wherein each of the circular nucleic acid molecules comprise a first and a second nick in the target sequence, one on each strand of the circular nucleic acid molecule, wherein the first and second nick are each located at different positions within the target sequence.   
     
     
         93 . The molecules of  claim 92 , wherein the first and the second adaptor sequences comprise a double-stranded nucleic acid sequence.