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Plant Biotechnol J. 2016 Jul;14(7):1511-22. doi: 10.1111/pbi.12511. Epub 2016 Jan 23.

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

Author information

1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Stadt Seeland, Germany.
2
Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Jena, Germany.
3
Plant Genome and System Biology (PGSB), Helmholtz Center Munich, German Research Center for Environmental Health (GmbH), Neuherberg, Germany.

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.

KEYWORDS:

Hordeum vulgare; bacterial artificial chromosome; barley; hierarchical shotgun sequencing; mate-pair sequencing; paired-end sequencing; physical map; sequence assembly

PMID:
26801048
PMCID:
PMC5066668
DOI:
10.1111/pbi.12511
[Indexed for MEDLINE]
Free PMC Article

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