Plant & Animal Genomes XVI Conference

January 12-16, 2008
Town & Country Convention Center
San Diego, CA

A Comparison Of 454 Vs Traditional Sequencing Approaches

Gemma M. Payne¹ , John C. McEwan¹ , Rudiger Brauning¹ , Brendon Hill² , Nessa H. O'Sullivan¹ , V. Hutton Oddy³ , Frank W. Nicholas⁴ , International Sheep Genomics Consortium⁵

¹  AgResearch, Invermay Agricultural Centre PB 50034, Mosgiel, NZ

²  454 Life Sciences,16 Commercial Street, Branford, CT, USA

³  University of New England, Armidale, NSW, Australia

⁴  University of Sydney, Sydney, NSW, Australia

⁵  http://www.sheephapmap.org

The International Sheep Genomics Consortium’s aims to skim sequence the ovine genome to identify SNPs for creation of a 60K SNP chip. This project necessitates fast and cost-effective sequencing. Roche 454 sequencing technology is a new technology based on pyrosequencing. It is claimed to be simple, cost-effective and quick. A pilot study was set up to research the usefulness and test the advantages and disadvantages of this technology compared to traditional Sanger sequencing. Four BACs from the Chori-243 ovine BAC library were selected for sequencing with the 454 GS20 by 454 Life Sciences. These BACs had previously been sequenced using Sanger technology as part of the ENCODE project. Resultant 454 raw read coverage ranged between 29 to 45X of the reference assembly. The reads were assembled de novo into contigs using Newbler, Phrap and CAP3 software programs. The Newbler assembler performed best in assembling raw 454 reads with 95.3-99.6% of the coverage of the Sanger sequenced BACs. Comparison of the Sanger and Newbler assembled sequence identified 0.08% indels and 0.03-0.06% different base calls between the two assemblies. Our conclusion was that Roche 454 sequencing is a cost-effective alternative to Sanger sequencing for some sequencing applications.