Sequencing and Assembling a Pseudomolecule for the Wheat Chromosome 6B

The International Wheat Genome Sequencing Consortium (IWGSC) makes effort to obtain a high quality reference genome sequence of the bread wheat (cv. Chinese Spring), which is anchored to the genetic and physical maps.  As a part of this international effort, we focus on sequencing of both (short and long) arms of chromosome 6B utilizing a minimum tiling path (MTP) BAC-by-BAC sequencing strategy.  
A total of 7,573 MTP BAC clones were selected based on the physical mapping and then sequenced individually using 96-tag sequencing system with GS FLX (Roche).  We found that up to 87% of these clones were successfully sequenced.  For BAC clones with the successes on draft sequencing, we checked their sequence overlapping with neighboring BAC clones on the physical map, and it was confirmed that more than 95% of these BAC clones were truly overlapped.  Two kinds of mate-pair libraries (3~4 kb, 8~11 kb) using pooled BAC clones was constructed and sequenced with MiSeq (24 tags/run, Illumina) to make a map-based and high-quality pseudomolecule of the wheat chromosome 6B.  The assemblies of draft sequences from the BAC clones confirmed their overlapping were manually ordered using the output of SSPACE, which resulted in the first version of wheat chromosome 6B pseudomolecule (648.30 Mb).  The number of scaffolds (> 10kb) was 2,935 and the size of the largest scaffold was about 3.1 Mb.  
This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan. (Genomics-based Technology for Agricultural Improvement, NGB1003)