Assessing the Feasibility of GS FLX Pyrosequencing for Sequencing the Atlantic salmon Genome

Background: With a whole genome duplication event and wealth of biological data, salmonids are excellent modelorganisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processesassociated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced.Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture andthe genomic resources available. However, the size and complexity of the genome combined with the lack of a sequencedreference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sangersequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility ofusing the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eightpooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GSFLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of ashotgun library.Results: An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverageallowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage)were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assemblycomprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5×coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly wascomparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly.Conclusion: These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our datademonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complexgenomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently,for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sangertechnology.