Open AccessMultiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes
Author(s) -
Beier Sebastian,
Himmelbach Axel,
Schmutzer Thomas,
Felder Marius,
Taudien Stefan,
Mayer Klaus F. X.,
Platzer Matthias,
Stein Nils,
Scholz Uwe,
Mascher Martin
Publication year - 2016
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12511
Subject(s) - biology , shotgun sequencing , hybrid genome assembly , illumina dye sequencing , sequence assembly , genome , computational biology , dna sequencing , bacterial artificial chromosome , shotgun , reference genome , genetics , k mer , whole genome sequencing , gene , transcriptome , gene expression
Summary 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 ( BAC s) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BAC s ( Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BAC s 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.