Sequential Sequencing

摘要

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.

主权项

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.