P0679 Sequencing Brachypodium Insertional Mutants Using TDNA-Seq

Mandy Hsia , USDA, Albany, CA
Ronan O'Malley , The Salk Institute, La Jolla, CA
Alana Clark , USDA-ARS Western Regional Research Center, Albany, CA
Amy Anderton , USDA-ARS Western Regional Research Center, Albany, CA
Jennifer Bragg , USDA-ARS-WRRC, Albany, CA
Rita Nieu , USDA-ARS Western Regional Research Center, Albany, CA
Sandra Kelly , Agriculture and Agri-Food Canada, Winnipeg, MB, Canada
Mark Jordan , Agriculture and Agri-Food Canada, Winnipeg , MB, Canada
Vera Thole , John Innes Centre, Norwich, United Kingdom
Gao Caixia , Chinese Academy of Sciences, Beijing, China
Philippe Vain , John Innes Centre, Norfolk, United Kingdom
Joseph Ecker , The Salk Institute, La Jolla, CA
John Vogel , USDA, ARS, Western Regional Research Center, Albany, CA
Brachypodium distachyon (Brachypodium) has recently emerged as powerful experimental model for the grasses. A growing collection of genomic resources has been developed including a complete genome sequence, resequenced genomes, microarrays, large germplasm collections and numerous bioinformatic tools. We are adding to these resources by continuing to produce T-DNA lines and flanking sequence tags (FSTs). Our goal is to create 35,000 more lines over the next 3 years. To improve FST sequencing efficiency, we adopted a strategy based on four dimensional pooling and Illumina sequencing. This approach, called TDNA-Seq, is being used to resequence existing Arabidopsis T-DNA collections and offers significant advantages over Sanger sequencing of individual T-DNA lines. Using TDNA-Seq, 10,000 lines can be sequenced simultaneously from only 40 DNA pools. Furthermore, since there is no interference between sequencing templates, the number of FSTs obtained approaches the number of T-DNA inserts. Thus, we expect to get approximately 1.5 FSTs per line which is a significantly more than the 0.75 FSTs per line we obtain with Sanger sequencing. We are currently sequencing 8,000 T-DNA lines, 7,731 lines from the WRRC and 269 lines from Canada. The next batch of lines from the International Brachypodium Tagging Consortium (IBTC) to be sequenced will include 5,000 lines from the JIC, ~4,000 lines from the WRRC, ~500 lines from the CAS and several hundred more lines from AAFC. In addition, we have established collaborations to sequence T-DNA lines produced by two other members of the IBTC, but they have not yet begun to produce lines.