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

Plant Biotechnol. J. 14, 1511-1522 (2016)
Publishers Version DOI PMC
Open Access Gold
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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.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Hordeum Vulgare ; Bacterial Artificial Chromosome ; Barley ; Hierarchical Shotgun Sequencing ; Mate-pair Sequencing ; Paired-end Sequencing ; Physical Map ; Sequence Assembly; Map-based Cloning; Physical Map; Transposable Elements; Provides Insights; Hordeum-vulgare; Barley Genome; Evolution; Wheat; Dna; Gene
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