A New BAC Ends Sequencing Approach to Improve Wheat 1B Chromosome Assembly

Bacterial artificial chromosome (BAC) libraries are still a valuable tool for de novo assembly of complex genomes, such as plants genomes. Shotgun sequencing of BACs, individually or by pools, produces first assemblies which usually need further improvement towards finished quality. We developed a new approach to obtain BAC ends libraries for Illumina sequencing (BES), overcoming the expensive and time consuming BAC ends Sanger sequencing. We applied this protocol to improve the initial assembly of wheat 1B long arm chromosome. To produce a reference sequence, a total of 6023 BAC clones representing the minimal tiling path of the 1B long arm physical map were sequenced by Illumina technology: paired end data from pools of about 10 BACs were combined to 5 kb mate pair reads to ensure the assembly of large sequence scaffolds. Contigs were generated by Newbler assembler using merged paired end data; then, they were scaffolded using SSPACE and mate pair sequences. Average scaffold size was 62 Kb and N50 about 231 Kb.  We tested the BES approach by preparing libraries from pools containing 384 BACs each. Illumina paired end reads were generated and used for scaffolding improvement with SSPACE. We observed that 62% of the previous assemblies benefited from this approach: scaffolds average size and N50 increased significantly (respectively 76 Kb and 354 kb on average). Therefore, the new method revealed useful for improving de novo assembly, especially in the case of challenging highly repeated genomes. Here we present the library protocol in details and discuss further assembly analyses.