Exome Capture Method for Complex Genomes

Miscanthus is a perennial grass and a candidate bioenergy crop. The Miscanthus genome creates a challenge for variant discovery and genotyping because of its abundant repeats, the presence of a genome scale duplication event with little subsequent divergence, and high heterozygosity due to its self-incompatible breeding system. This complexity necessitates the discovery of sequence variants that distinguish paralogs from alleles, which are most frequent in the low- copy non-coding fraction of the Miscanthus genespace. To enrich for such sequences from Miscanthus genomes, we developed an exome capture array using a solution-based hybridization method (Nimblegen SeqCap EZ Choice). The probe set was designed to capture exons and flanking intronic sequences predicted from alignment of transcriptome, fosmid, and genomic reads from Miscanthus to the largely syntenic Sorghum genome. To facilitate haplotype discovery, we selected for large insert sizes (approximately 400-500bp) that were subsequently sequenced at high depth as 100x75 and 150x150 paired- end reads on the Illumina HiSeq 2000, as well as on the 454 Roche GS FLX+ platform. We report here the sequencing and assembly of exome capture libraries prepared from a doubled haploid Miscanthus sinensis plant where all allelic variation has been fixed. Our exome capture method proved to be successful in acquiring high sequence depth for haplotypes that define paralogous sequences between the two Miscanthus subgenomes.