Large fragment target enrichment and sequencing of the 4.4 Mb major histocompatibility complex by region-specific extraction and HiSeq 2500

Abstract

Due to the extensive polymorphism of the major histocompatibility complex (MHC), only limited progress has been made in high-resolution MHC sequencing, one of the initial requirements to elucidate the molecular immunopathology of several hundred MHC-associated diseases. We present an approach for sequencing the entire MHC with ≈ 1200 specific capture primers. Using target sites known to be highly conserved, Region-Specific Extraction (RSE, Generation Biotech) was used to target MHC segments of up to 40 kb length and enrich the contiguous 4.4 Mb MHC for sequencing. RSE uses a single primer extension-step that attaches a specific “handle” to genomic DNA target segments and firmly locks the selected DNA onto magnetic beads for capture. The captured segments extend far into both directions from any single capture point. This makes an RSE capture primer set essentially immune to changes in variable sections and avoids difficulties caused by genomic duplications, pseudo-genes, gene homologues and new sequence insertions. After RSE, unique oligonucleotide indexes were ligated to each DNA sample for parallel, paired-end 2 × 250 bp sequencing on a HiSeq2500 (Illumina). Read alignment (hg19 reference genome), local re-alignment, variant calling and annotation were performed by an in-house data analysis pipeline consisting of the CLC Genomics Workbench (Qiagen) and custom developed scripts. In our experiments, an up to 80-fold target enrichment was achieved, which provides an average coverage of about 200 across the 4.4 Mb target region. In samples with high coverage, > 90% of the target region can uniformly be sequenced with > 20× coverage. We expect that optimized target enrichment enables to sequence at least 16 samples per flow-cell with appropriate coverage for > 90% of the MHC region while maintaining the advantages of RSE in avoiding variation-dependent capture and allelic dropout. The combination of the RSE enrichment and massively parallel sequencing on HiSeq is a feasible approach for whole MHC sequencing. Further improvements in target enrichment efficiency and long-read/high-accuracy sequencing can turn it into a cost-effective and user-friendly tool in many clinical and research settings such as transplantation medicine, inflammatory or autoimmune disorders, pharmacogenomics, and many more. H. Rabenstein: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. B. Bangol: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. S. Linke: Employee; Company/Organization; Generation Biotech. R. Brumm: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. S. Poggel: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. S. Eck: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics. K. Ott: Employee; Company/Organization; Generation Biotech. J. Dapprich: Employee; Company/Organization; Generation Biotech. K. Hirv: Employee; Company/Organization; Center for Human Genetics and Laboratory Diagnostics.