METHODS AND SYSTEMS FOR SEQUENCING NUCLEIC ACIDS
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.