Sequencing wheat BAC pools by 454 Titanium plus system

Although the clone-by-clone sequence strategy is more costly and labor intensive than Whole Genome Shotgun (WGS), it could provide more accurate and reliable assemble, which is essential to sequence the large and complex genomes, such as wheat and barley. Taking the advantage of the latest version of the 454 Titanium + system with longer mode read length (700 bp) and Multiplex Identifier (MID), more than 300 selected Aegilops tauschii BACs were sequenced. Among them, 256 non-overlapping BACs and 100 minimum tilling path (MTP) were selected based on the tausschii physical map. Five to sixteen pooled BACs were sequenced at the depth between 13x and 48x, with average read length ranging from 300 bp to 570 bp. A 3kb pair end library of pooled non-overlapping BAC and MTP BAC were made and sequenced 5 to 10 times coverage, respectively. The fragment sequences of each pool were assembled using GSAssembler and the paired sequences were added by Consed. The comparison of sequence assembly among the different strategy will be presented.