Abstract
Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole exome of barley. The Exome Library is provided as a liquid array containing biotinylated probes (Roche/NimbleGen). Subsequently, genomic shotgun DNA fragments hybridized to the Exome Library are affinity-purified using streptavidin coated magnetic beads. The captured library is PCR-amplified and sequenced using high-throughput short read sequencing-by-synthesis.
Keywords: Sequence capture, Targeted resequencing, Exome, Sequencing-by-synthesis, Barley
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Supplementary Materials
Table 1. List of oligonucleotides
Acknowledgments
The generation of DNA Illumina multiplex (IM) libraries was essentially as described (Meyer and Kircher, 2010). The operations leading to the captured library were based on the manufacturer’s instructions (Roche/NimbleGen, NimbleGen Arrays User’s Guide, Plant Sequence Capture Illumina Optimized, 2010) and as described previously (Mascher et al., 2013). The financial support provided by the grants GABI-BARLEX (FKZ 0314000 to N.S.) and TRITEX (FKZ 0315954 to N.S.) by the German Ministry of Education and Research (BMBF) is gratefully acknowledged.
References
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