Combining Next Generation Sequencing Strategies For Improved Assembly Of Wheat Bacterial Artificial Chromosomes

As part of the Canadian-funded “Canadian Triticum Advancement through Genomics (CTAG)” project, we are working to generate a high quality reference sequence of chromosome 6D.  To test our assembly strategy we are evaluating several hybrid co-assembly strategies.  For this study, we selected 25 wheat BACs, with an average insert size of 150 KB for sequencing with both Roche 454 (Titanium and Flex+) and Illumina (paired end and mate-pair libraries) platforms.    As expected, co-assembly of Roche 454 Titanium and Illumina paired-end data improved the assembly of all BACs tested.  However, the number of Illumina paired-end reads required to generate assemblies with the fewest scaffolds and largest scaffold size was BAC dependent, suggesting that an iterative sampling of paired-end data may be required to generate the best assemblies.  In some cases, addition of Illumina-generated sequence from large insert (3-5 kb and/or 8-10 kb) mate pair libraries was effective at collapsing Roche 454 Flex Plus generated contigs into a single scaffold.  Co-assembly of sequence data of overlapping BACs appeared to be more effective in generating large, single super scaffolds than independent assembly of each BAC.  We are currently evaluating other strategies and platforms which could also be useful for assembly of BACs from a minimum tilling path generated from physical mapping.   These preliminary experiments will be useful in selecting a final sequencing/assembly strategy for generation of a reference sequence of chromosome 6D of wheat.