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. 2010 Oct 13:11:564.
doi: 10.1186/1471-2164-11-564.

Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches

Mohamed Salem  1 , Caird E Rexroad 3rd, Jiannan Wang, Gary H Thorgaard, Jianbo Yao
Affiliations

Characterization of the rainbow trout transcriptome using Sanger and 454-pyrosequencing approaches

Mohamed Salem et al. BMC Genomics. .

Abstract

Background: Rainbow trout are important fish for aquaculture and recreational fisheries and serves as a model species for research investigations associated with carcinogenesis, comparative immunology, toxicology and evolutionary biology. However, to date there is no genome reference sequence to facilitate the development of molecular technologies that utilize high-throughput characterizations of gene expression and genetic variation. Alternatively, transcriptome sequencing is a rapid and efficient means for gene discovery and genetic marker development. Although a large number (258,973) of EST sequences are publicly available, the nature of rainbow trout duplicated genome hinders assembly and complicates annotation.

Results: High-throughput deep sequencing of the Swanson rainbow trout doubled-haploid transcriptome using 454-pyrosequencing technology yielded ~1.3 million reads with an average length of 344 bp, a total of 447 million bases. De novo assembly of the sequences yielded 151,847 Tentative Consensus (TC) sequences (average length of 662 bp) and 224,391 singletons. A combination assembly of both the 454-pyrosequencing ESTs and the pre-existing sequences resulted in 161,818 TCs (average length of 758 bp) and 261,071 singletons. Gene Ontology analysis of the combination assembly showed high similarities to transcriptomes of other fish species with known genome sequences.

Conclusion: The 454 library significantly increased the suite of ESTs available for rainbow trout, allowing improved assembly and annotation of the transcriptome. Furthermore, the 454 sequencing enables functional genome research in rainbow trout, providing a wealth of sequence data to serve as a reference transcriptome for future studies including identification of paralogous sequences and/or allelic variation, digital gene expression and proteomic research.

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Figures

Figure 1
Figure 1
Average length distribution of assemblies and pyrosequencing reads of the rainbow trout ESTs. Average lengths of the combination assembly contigs are more than those of the 454-pyrosequencing assembly over the 100-700 bp length range, indicating that addition of the Sanger-based data improved assembly of the short 454-pyrosequenceing reads up to 700 bp. However, over the 700-2000 bp length range, average contig lengths of the combination assembly are less than those of the 454-pyrosequencing assembly indicating that contigs longer than 700 bp of the combination assembly were mainly derived from the 454-pyroseqeuncing data, possibly because most of the 454-pyrosequencing reads were 400-600 bp. Number of the 454-pyrosequencing reads is divided by 10 for scaling.
Figure 2
Figure 2
Gene Ontology (GO) assignment (2nd level GO terms) of the rainbow trout Sanger-based and 454-pyrosequncing combination assembly. Biological processes constitute majority of GO assignment of the transcripts (17,694 counts, 39%), followed by cellular components (16,452 counts, 36%) and molecular function (11,160 counts, 25%).
Figure 3
Figure 3
BLASTx top-hit species distribution of gene annotations showing high homology to fish species with known genome sequences. Only 5.7% of the BLAST hits matched rainbow trout protein sequences due to the limited number of the rainbow trout proteins (6,915) that are currently available in the NCBI database (compared to 141,396 proteins in Zebrafish).
Figure 4
Figure 4
Gene Ontology (2nd level GO terms) comparison of the rainbow trout combination assembly showing high similarity to the transcriptome of Zebrafish (with known genome sequence).
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