title: Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.
abstract: 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.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Beier
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Himmelbach
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmutzer
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Felder
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Taudien
authors.affiliation: Plant Genome and System Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Neuherberg, Germany.
authors.forename: Klaus F X
authors.initials: KF
authors.lastname: Mayer
authors.affiliation: Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Platzer
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Stein
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Scholz
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Mascher
mesh_terms: D001709:Biotechnology / Q000379:methods; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018745:Genome, Plant; D001467:Hordeum / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: Hordeum vulgare; bacterial artificial chromosome; barley; hierarchical shotgun sequencing; mate-pair sequencing; paired-end sequencing; physical map; sequence assembly