OR05: COMPLETE RESEQUENCING OF EXTENDED GENOMIC REGIONS USING FOSMID TARGET CAPTURE AND SINGLE MOLECULE REAL-TIME (SMRT®) LONG READ SEQUENCING TECHNOLOGY

Abstract

Aim The knowledge of complete haplotype structure and underlying genetic variation is often key in understanding gene function, phenotype, and medically relevant traits such as susceptibility to disease and individual response to drugs. However, genome wide SNP arrays and shotgun sequencing methods are limited in their ability to identify phased genetic variations at high resolution over extended localized regions. Methods To address this, we improved methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generation that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As specific applications, we targeted subregions of the human major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, two of the most polymorphic genomic regions in humans. Results For the MHC region, an 800 kb segment of the MHC class II region including the highly polymorphic DR, DQ, and DP genes was targeted. Complete phased sequences were obtained encompassing the DR4 common extended haplotype – a haplotype associated with autoimmune disorders - from four class II heterozygous individuals. For the KIR locus, which spans some 200 kb including tandem repeats of highly homologous polymorphic loci, we targeted several KIR haplotype heterozygous individuals. Conclusions Complete phased sequences for all haplotypes were obtained from all individuals with a high level of data accuracy. The results demonstrate the approach is amenable to cost-effective scale-up to generate scores to hundreds of phased chromosomal sequences of extended lengths that encompass targeted genomic regions associated with disease.