BAC-by-BAC Sequencing with ABI Solid and Roche 454 In Complex Genomes.

The complete BAC-by-BAC sequencing of many large plant genomes, such as those of wheat, barley and other economically important plants, is currently prohibitively expensive if traditional Sanger technology is employed. New sequencing technologies, such as Roche 454 (454 Life Sciences), Solexa (Illumina Inc), and SOLiD (Applied BioSystems), allow sequencing with much higher throughputs and at greatly reduced costs. We developed a strategy for minimum tiling path (MTP) BAC sequencing of plant genomes of any size with a combination of two next-generation sequencing platforms (Roche 454 and SOLiD) and two-dimensional (2-D) BAC DNA pooling. A set of non-overlapping BACs are chosen from MTP BAC contigs. Then one DNA pool with all selected BACs for Roche 454 sequencing with paired ends, and 2-D BAC pools for SOLiD sequencing are produced and sequenced. An algorithm and its implementation, the Pool2BAC software package, were developed for assignment of Roche 454 reads of pooled BAC DNAs into individual BAC clones based on the information of SOLiD reads of 2-D BAC pools mapping to Roche 454 reads of pooled BACs. Subsequently assigned Roche 454 reads of individual BACs can be assembled separately. The simulation and experimental results of 4x4 and 8x7 BAC pools showed that more than 90% of the gene-space and repeated reads of pooled BACs were correctly assigned to individual BACs, and 95% of assembled contigs of a BAC were correctly mapped to reference BAC sequences, which cover 93% of gene spaces. Preliminary results suggest that the 2-D BAC pooling technique can be scaled up for sequencing of large pools containing hundreds of BAC clones, thus opening the door to sequencing of plant genomes of any size. Higher dimensions (24x24) of BAC DNA pooling for genome sequencing are being evaluated.