Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Abstract

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

A chromosome conformation capture ordered sequence of the barley genome

“…Analysis of sequence and haplotype diversity. Ninety-six two-row spring (n = 48) and winter (n = 48) homozygous inbred elite barley lines (Supplementary Table 5.1) were subjected to exome capture using the barley Roche NimbleGen exome capture liquid array 94 and sequenced on the Illumina HiSeq 2500 platform. An average of 2 × 21,876,780 paired-end Illumina reads per sample was generated.…”

A homolog of Blade-On-Petiole 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence

“…Sample preparation was done with the Illumina TruSeq DNA Sample Preparation Kit based on the manufacturer's instructions. Different samples or pools were individually indexed and captured together by a single liquid array capture assay (Mascher et al, 2013a;Himmelbach et al, 2014). Sequences of recombinant pools were mapped against the wholegenome shotgun assembly of cv Bowman with BWA 0.5.9 (Li and Durbin, 2009) using the parameter -q 15 for quality trimming.…”

“…2A). DNAs of individuals belonging to the same class were then pooled for target-enrichment shotgun resequencing (Mascher et al, 2013a). More than 30 million properly paired sequence reads were obtained per sample (Supplemental Table S1), and singlenucleotide polymorphism (SNP) frequencies were determined between the pooled, sequence-enriched DNA samples and wild-type cv Bowman and visualized in the context of the physical/genetic map of barley (Fig.…”

“…Marker scores and phenotype scores for each recombinant are represented by a color code for simplification (yellow = wild type, green = heterozygote, and red = mutant). Genotypes with identical marker haplotype/ phenotype combinations were pooled for target enrichment resequencing (Mascher et al, 2013a). B, Obtained sequence reads of the individual pools were mapped to the reference cv Bowman (Mayer et al, 2012) for SNP discovery and determination of SNP frequencies.…”

Transcriptome profiling reveals mosaic genomic origins of modern cultivated barley

“…To validate the above results, we also conducted a case study on five additional cultivars (Cultivar-additional, cv. Borwina, Kindred, Vogelsanger Gold, Steptoe and Harrington) containing exome capture data (25). After aligning the reads in each sample against the WGS contigs of cv.…”