SEQUENTIAL SEQUENCING
The present invention provides improved methods, compositions and kits for short read next generation sequencing (NGS). The methods, compositions and kits of the present invention enable phasing of two or more nucleic acid sequences in a sample, i.e. determining whether the nucleic acid sequences (typically comprising regions of sequence variation) are located on the same chromosome and/or the same chromosomal fragment. Phasing information is obtained by performing multiple, successive sequencing reactions from the same immobilized nucleic acid template. The methods, compositions and kits provided herein are useful, for example, for haplotyping, SNP phasing, or for determining downstream exons in RNA-seq.
1 . A method for relating at least two nucleic acid sequences or regions of sequence variation to the same nucleic acid template, the method comprising:
a. creating a strand-oriented (i.e. directional) nucleic acid library;
b. sequencing the strand-oriented library with an oligonucleotide primer;
c. denaturing the first strands of the nucleic acid fragments in the library;
d. annealing a new oligonucleotide primer that is complementary to a conserved region or conserved regions within the nucleic acid fragments in the nucleic acid library;
e. sequencing the nucleic acid library with the new oligonucleotide primer; and
f. compiling data from first and second sequencing reads to map reads originating from the same nucleic acid fragments.
2 . The method of claim 1 , wherein the nucleic acid libraries are amplicons originating from conserved regions of sequence.
3 . The method of claim 2 , wherein the conserved regions are adjacent to variable regions.
4 . The method of claim 3 , wherein alignment of multiple variable regions enables differentiation and/or typing of related transcripts.
5 . The method of claim 3 , wherein alignment of multiple variable regions enables differentiation and/or typing of related micro-organisms.
6 . The method of claim 1 , wherein libraries are reduced complexity.
7 . The method of claim 6 , wherein reduced complexity is achieved by target capture.
8 . A method for distinguishing between two closely related nucleic acid sequences, the method comprising:
a. creating a strand-oriented nucleic acid library with closely related nucleic acid sequences as inserts;
b. sequencing the strand-oriented library with an oligonucleotide primer;
c. denaturing the first strands of the nucleic acid fragments in the library;
d. annealing a new oligonucleotide primer that is complementary to a conserved region or conserved regions within the nucleic acid fragments in the nucleic acid library;
e. sequencing the nucleic acid library with the new oligonucleotide primer; and
f. compiling data from first and second sequencing reads to map reads originating from the same nucleic acid fragments.