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Beier S.,Leibniz Institute of Plant Genetics and Crop Plant Research | Himmelbach A.,Leibniz Institute of Plant Genetics and Crop Plant Research | Schmutzer T.,Leibniz Institute of Plant Genetics and Crop Plant Research | Felder M.,Leibniz Institute on Aging Fritz Lipmann Institute FLI Jena Germany | And 6 more authors.
Plant Biotechnology Journal | Year: 2016

Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path. © 2015 The Authors.

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