Assembly Improvement Tricks: Application To The 1Gb Wheat Chromosome 3B (3BSEQ project)

The 3BSEQ project aims at obtaining an annotated sequence of chromosome 3B, the largest bread wheat chromosome (1Gb) , and at exploiting this knowledge to develop tilling arrays of the 3B gene space for further functional and structural characterizations .The project takes advantage of the potential offered by the next generation sequencing to develop an original strategy and deliver a high quality draft sequence of the chromosome.

We sequenced 8452 BAC clones comprising the minimal tiling path established during the construction of the physical map. As sequencing each BAC individually would have been too costly and labor-intensive, we used a pooling strategy to combine BACs from the same physical contigs in one or more pools of all, DNA was extracted from each BAC individually using a standard method and then, pools corresponding to 10 equimolar amounts of BAC DNA were mixed. Each pool was used to create a mate pair library of 454-Roche Titanium fragments through the re-circularization of 8kb fragments. In addition, we produced pair end Illumina reads of sorted chromosome 3B at 82x coverage. The first 454 sequence assembly resulted in 16136 scaffolds (293806 contigs) with a N50 of 275 kb and an average of 12.5 scaffolds per physical contigs. To reduce the number of contigs and improve the scaffolding (resulting on 159587 contigs and 5109 scaffolds with a N50 of 463 kb, respectively), we developed a strategy to improve the continuity based on Newbler work files. The method uses the Illumina reads to fill gaps and correct scaffold consensus errors. Details of this strategy will be presented.