Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

An Efficient Strategy For Full Length cDNA Sequence Finishing In Bos taurus

Johar Ali¹ , Brian Wynhoven¹ , Diana Palmquist¹ , Hye-Jung Elizabeth Chun¹ , Jerry Liu¹ , Robert Kirkpatrick¹ , Jeff Stott¹ , Sarah Barber¹ , George Yang¹ , Ryan Babakaiff¹ , Jaclyn Beland¹ , Steve Chand¹ , Luis Del Rio¹ , Lisa Dreolini¹ , Ruth Featherstone¹ , Susan Gibson¹ , Corey Matsuo¹ , Mike Mayo¹ , Jennifer Roger¹ , Miranda Tsai¹ , Dave Wong¹ , Stephen S. Moore² , Le Luo Guan² , Savio Lobo² , Yan Meng² , Masaaki Taniguchi² , Zhiquan Wang² , Kirsten Schreiber³ , Christa Prange³ , Nicole Shapiro³ , Carolyn Shenmen⁴ , Lukas Wagner⁴ , Lee Alexander⁵ , Mike MacNeil⁵ , Michael J. Brownstein⁶ , Asim Siddiqui¹ , Robert Holt¹ , Steven Jones¹ , Marco Marra¹

¹  Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, Vancouver, BC, Canada V5Z 1L3

²  Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5

³  Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA, USA 94550;4National Center for Biotechnology Information

⁴  National Center for Biotechnology Information; National Library of Medicine, Building 38A, Bethesda, MD, USA 20894

⁵  USDA-ARS, Fort Keogh LARRL, 243 , Fort Keogh Road, Miles City, MT, USA 59301

⁶  The J. Craig Venter Institute 9704 Medical Center Drive Rockville, MD 20850

Standard full length cDNA (fl-cDNA) sequence finishing approaches may require several rounds of primer walking and may take months before the sequence reaches the requisite accuracy level. To accelerate fl-cDNA sequence finishing we have experimented with designing sequencing primers using existing matching sequences. Primers were designed using sequences identified by a BLASTN search using the last 1/3 of the 5’ EST sequences versus the GenBank non-redundant database. The top 3 matching refseqs were downloaded and assembled by Phrap together with the appropriate 5’ EST sequence. To increase the success of the primer reactions any bovine mRNA refseq matching the 5’ EST was accepted as a template for primer design. Where there was no match to a bovine mRNA refseq, bovine genome sequences (exons only) or other closely related refseqs were utilized for primer design. Overall, the primer’s reactions success rate was 91% and we were able to finish 1979 clones in 3 rounds of reactions where it would have normally required 5 rounds of reactions to finish the same number of clones with the same quality standards. By applying this new strategy, fl-cDNA sequence finishing turn around time was reduced by well over a month from initial expectations and we were able to both meet and exceed our year one goal.