Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Hierarchical shotgun sequencing is crucial for assembling complex plant genomes.
• High-throughput sequencing necessitates parallel processing of numerous large-insert clones.
• Bacterial artificial chromosomes (BACs) are key for constructing reference genomes.

Purpose of the Study:

• To develop a multiplexed pipeline for shotgun sequencing and assembling individual BACs.
• To enable parallel processing of a large number of BACs using Illumina sequencing.
• To improve the contiguity, completeness, and gene space representation of genome assemblies.

Main Methods:

• Developed a multiplexed pipeline for shotgun sequencing of bacterial artificial chromosomes (BACs).
• Utilized the Illumina sequencing platform for high-throughput data generation.
• Employed a parallelized computational pipeline for sequence assembly of individual BACs.

Main Results:

• Successfully sequenced 668 barley BACs in a single Illumina HiSeq 2000 lane.
• Achieved average BAC assemblies with eight scaffolds, representing >98% of genomic inserts.
• Demonstrated superior contiguity, completeness, and gene space representation compared to whole-genome shotgun assemblies.

Conclusions:

• The developed multiplexed pipeline enables rapid, high-quality BAC assembly.
• This approach is suitable for assembling map-based reference sequences of complex plant and animal genomes.
• The methods facilitate efficient sequencing of numerous clones for genome projects.