OR01: AUTOMATED ASSEMBLY OF COMPLEX IMMUNOGENETIC HAPLOTYPES USING LONG-READ SINGLE MOLECULE, REAL-TIME SEQUENCING OF FOSMIDS

Abstract

Aim To implement an automated workflow for the assembly of complex immunogenetic regions using pooled fosmids and SMRT® Sequencing. Methods Here we present a simple automated workflow for the assembly of complete haplotypes from pools of tiled fosmids using a single library preparation and sequencing run with a quick turnaround time using an automated pipeline. The long reads generated in SMRT® Sequencing together with the hierarchical genome assembly process (HGAP) algorithm make it possible to fully assemble data with high accuracy even when the underlying structure is highly repetitive. Long reads are first preassembled into highly accurate error corrected reads before an overlap layout consensus (OLC) assembly to generate complete fosmid sequences. Fosmid sequences are trimmed of any vector before being overlapped to from a continuous haplotype. Results We show complete assembly of ∼150–200 kb haplotypes of the KIR locus, characterized by a large number of tandem repeats, from a single sequencing run. The assembly has a high base accuracy when compared to known references. Discussion The ability to haplotype and sequence complex immunogenetic regions will facilitate the integration of long-range LD in Conserved Extended Haplotypes in the MHC region and LRC region. Local fine-scale LD data will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, means that sequence assembly of complete haplotypes is difficult and often requires expert knowledge. This simple and robust assembly approach can be scaled-up allowing a complex genomic region to be sequenced in a population genetics setup. Such sequencing would be of high value in disease association research. R.J. Hall: Employee; Company/Organization; Pacific Biosciences. K. Eng: Employee; Company/Organization; Pacific Biosciences. L. Hon: Employee; Company/Organization; Pacific Biosciences. D.E. Geraghty: Employee; Company/Organization; Scisco Genetics Inc. S. Ranade: Employee; Company/Organization; Pacific Biosciences.