Sequential sequencing

Abstract

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.

Description

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61 / 801,600, filed Mar. 15, 2013, which application is incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] Short read next generation sequencing (NGS) analysis has some limitations in both research and diagnostics. One key drawback is the problem of phasing. That is, when interrogating multiple loci of sequence variation, it is often impossible to determine which loci are co-located on the same chromosome or on the same chromosomal fragment. One example of a phasing problem occurs in diploid organisms in which two parental chromosomes, one from the mother and one from the father, are inherited, resulting in two copies of each gene (except for the genes carried on the sex chromosomes). Within each copy of the two copies of a gene in a diploid cell are regions of sequence variation, or loci, that fall within distinct sequence types known as alleles. Thus, allelic variation across ...

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