Methods and systems for sequencing nucleic acids

Abstract

The present invention provides methods and systems for sequencing long nucleic acid fragments.

1 . A method for sequencing a target nucleic acid, comprising:
 (a) sequencing one or more bases of a target nucleic acid by extending a first sequencing primer hybridized to said target nucleic acid to generate a first primer extension product, thereby obtaining a first sequence read;   (b) releasing said first primer extension product from said target nucleic acid;   (c) hybridizing a second sequencing primer to said target nucleic acid;   (d) generating a second primer extension product by extending said second sequencing primer through limited extension; and   (e) sequencing one or more bases of said target nucleic acid by further extending said second primer extension product to generate a third primer extension product, thereby obtaining a second sequence read.   
     
     
         2 . The method of  claim 1 , wherein said first sequencing primer and said second sequencing primer are the same. 
     
     
         3 . The method of  claim 1 , wherein said first sequencing primer and said second sequencing primer are different. 
     
     
         4 . The method of  claim 1 , wherein said limited extension is carried out by pulse extension. 
     
     
         5 . The method of  claim 4 , wherein said pulse extension is carried out by allowing the extending reaction to last 30 to 60 seconds. 
     
     
         6 . The method of  claim 1 , wherein said limited extension is carried out by using a nucleic acid polymerase and one or more sets of nucleotides, wherein each set comprises not more than three different nucleotides. 
     
     
         7 . The method of  claim 6 , wherein said extending is with more than one set of nucleotides. 
     
     
         8 . The method of  claim 7 , wherein said set of nucleotides comprises one, two or three different nucleotides. 
     
     
         9 . The method of  claim 1 , further comprises repeating steps (b) to (e) by releasing the primer extension product generated in step (e), thereby obtaining one or more additional sequence reads. 
     
     
         10 . The method of  claims 1 , wherein the sequence of said target nucleic acid is determined by assembling said first and second sequence reads. 
     
     
         11 . The method  claim 1 , wherein said sequencing is by extending the sequencing primer using a labeled reversible terminator. 
     
     
         12 . The method of  claim 6 , wherein prior to a subsequent addition of a set of nucleotides a washing step is performed. 
     
     
         13 . The method of  claim 6 , wherein prior to a subsequent addition of a set of nucleotides a nucleotide degradation step is performed. 
     
     
         14 . The method of  claim 1 , wherein said target nucleic acid is attached to a substrate. 
     
     
         15 . The method of  claim 14 , wherein said substrate is a flat surface or bead. 
     
     
         16 . The method of  claim 14 , wherein said substrate is a flow cell. 
     
     
         17 . The method of  claim 14 , wherein said substrate comprises glass. 
     
     
         18 . The method of  claim 14 , wherein said target nucleic acid is attached to said substrate via a capture probe. 
     
     
         19 . The method of  claim 1 , further comprising analyzing said sequencing results to provide a diagnosis, prognosis, or theranosis for a subject. 
     
     
         20 . The method of  claim 1 , comprising sequencing a plurality of target nucleic acids. 
     
     
         21 . The method of  claim 9 , wherein the sequence of said target nucleic acid is determined by assembling said first, second, and at least one of the one or more additional sequence reads.