Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotechnology / methods
  • Chromosomes, Artificial, Bacterial / genetics*
  • Genome, Plant*
  • Hordeum / genetics*
  • Sequence Analysis, DNA / methods*

Associated data

  • GENBANK/AF252830
  • GENBANK/AF285443
  • GENBANK/AY268139