Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome
Date
2008-08-28
Authors
Quinn, Nicole L
Levenkova, Natasha
Chow, William
Bouffard, Pascal
Boroevich, Keith A
Knight, James R
Jarvie, Thomas P
Lubieniecki, Krzysztof P
Desany, Brian A
Koop, Ben F
Journal Title
Journal ISSN
Volume Title
Publisher
BioMed Central
Abstract
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.
Description
BioMed Central
Keywords
Citation
Quinn et al.: Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome. BMC Genomics 2008, 9:404.