Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome

Nicole L Quinn 2 Natasha Levenkova 1 William Chow 2 Pascal Bouffard 1 Keith A Boroevich 2 James R Knight 1 Thomas P Jarvie 1 Krzysztof P Lubieniecki 2 Brian A Desany 1 Ben F Koop 0 Timothy T Harkins 3 William S Davidson 2 0 Department of Biology, University of Victoria , Victoria , Canada 1 454 Life Sciences , Branford , USA 2 Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby , Canada 3 Roche Applied Science , Indianapolis , USA Background: With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated 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 and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library. Results: An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30 coverage allowed 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 assembly comprising 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 was comparable 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 data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, 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 Sanger technology. - Background The salmonids (salmon, trout and charr) are of considerable environmental, economic and social importance. They contribute to ecosystem health by providing food sources for predators such as bears, eagles, sea lions and whales. As an increasingly popular food choice for humans, salmonid species contribute to local and global economies through fisheries, aquaculture and sport fishing. In addition, they have distinct social importance as they are a traditional food source for indigenous peoples, and play a significant role in their culture and spirituality. Salmonids are also of great scientific interest. The common ancestor of salmonids underwent a whole genome duplication event between 20 and 120 million years ago [1,2]. Thus, the extant salmonid species are considered pseudo-tetraploids whose genomes are in the process of reverting to a stable diploid state. More is known about the biology of salmonids than any other fish group, and in the past 20 years, more than 20,000 reports have been published on their ecology, physiology and genetics. Salmonids, with their genome duplication and wealth of biological data, are excellent model organisms for studying evolutionary processes, fates of duplicated genes and the genetic and physiological processes associated with complex behavioral phenotypes [3]. It is surprising therefore, that no salmonid genome has been sequenced to date. The Atlantic salmon (Salmo salar) is an ideal representative salmonid for genome sequencing given the popularity of this species for aquaculture as well as the extensive genomic resources that are available. The current genomic resources include: a BAC library [4], restriction enzyme fingerprint physical map comprising 223,781 BACs in ~4,300 contigs [5], 207,869 BAC-end sequences that cover ~3.5% of the genome sequence, a linkage map with ~1,600 markers, ~600 of which are integrated with the physical map [6], and > 432,000 ESTs [7,8]. The haploid C-value for Atlantic salmon is estimated to be 3.27 pg [9], or a genome size of approximately 3 109 bp, which is very comparable to the sizes of mammalian genomes. The Atlantic salmon genome is highly repetitive, and at least 14 different DNA transposon families whose members are ~1.5 kb have been described [10]. Although five fish genomes have been sequenced (medaka, Oryzias latipes; tiger pufferfish, Takifugu rubripes; green spotted pufferfish, Tetraodon nigriviridis; zebrafish, Danio rerio and stickleback, Gasterosteus aculeatus), they represent euteleostei lineages, and often very derived species that have been separated from salmonids for at least 200 million years [11]. The complexity of the Atlantic salmon genome combined with the lack of a closely related guide sequence means that sequencing and assembly will be extremely challenging. Conventional Sanger sequencing of paired end templates (24 kb plasmids, 40 kb fosmids, or ~150 kb BACs) using fluorescent di-deoxy chain terminators and capillary electrophoresis revolutionized the field of genomics (reviewed in [12]). Although this approach remains the gold standard for sequence and assembly quality, limitations with respect to cost, labor-intensiveness and speed, which are largely due to the necessity of generating and arraying cloned shotgun libraries and isolating template DNA for sequencing, have fueled the demand for new approaches to DNA sequencing. In recent years, several novel high-throughput sequencing platforms have entered the market including the SOLiD system by Applied Biosystems [13], the Solexa technology [14], now owned by Illumina, the recently released true Single Molecule Sequencing (tSMS) platform by Helicos [15] and the 454 platform [16], now owned by Roche. Most of these are targeted to the goal of re-sequencing an entire human genome for < $1,000 [17]. This next generation of genome sequencing stands to have major scientific, economic and cultural implications with respect to applications such as personalized medicine, metagenomics and large-scale polymorphism studies on organisms of commercial value whose genomes have already been sequenced. However, the ability of these technologies to sequence the genomes of complex or (...truncated)