Abstract
BackgroundTranscriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism. However, because many genes have tissue-specific expression patterns, developing a complete transcriptome usually requires a 'discovery pool' of individuals to be sacrificed in order to harvest mRNA from as many different types of tissue as possible. This hinders transcriptome development in large, charismatic and endangered species, many of which stand the most to gain from such approaches. To circumvent this problem in a model pinniped species, we 454 sequenced cDNA from testis, heart, spleen, intestine, kidney and lung tissues obtained from nine adult male Antarctic fur seals (Arctocephalus gazella) that died of natural causes at Bird Island, South Georgia.ResultsAfter applying stringent quality control criteria based on length and annotation, we obtained 12,397 contigs which, in combination with 454 data previously obtained from skin, gave a total of 23,096 unique contigs. Homology was found to 77.0% of dog (Canis lupus familiaris) transcripts, suggesting that the combined assembly represents a substantial proportion of this species' transcriptome. Moreover, only 0.5% of transcripts revealed sequence similarity to bacteria, implying minimal contamination, and the percentage of transcripts involved in cell death was low at 2.6%. Transcripts with immune-related annotations were almost five-fold enriched relative to skin and represented 13.2% of all spleen-specific contigs. By reference to the dog, we also identified transcripts revealing homology to five class I, ten class II and three class III genes of the Major Histocompatibility Complex and derived the putative genomic distribution of 17,121 contigs, 2,119 in silico mined microsatellites and 9,382 single nucleotide polymorphisms.ConclusionsOur findings suggest that transcriptome development based on samples collected post mortem may greatly facilitate genomic studies, not only of marine mammals but also more generally of species that are of conservation concern.
Highlights
Transcriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism
The resulting transcriptomes can in turn be mined for Single Nucleotide Polymorphisms (SNPs) which have already proven powerful for gene mapping [6] and are likely to become increasingly important in conservation genetics since they allow the characterisation of population structure and genetic diversity with unprecedented resolution [7]
To circumvent this problem in a natural population of Antarctic fur seals, we 454 sequenced cDNA from various tissues obtained at necropsy from nine adult males that died of natural causes
Summary
Transcriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism. Because many genes have tissue-specific expression patterns, developing a complete transcriptome usually requires a 'discovery pool' of individuals to be sacrificed in order to harvest mRNA from as many different types of tissue as possible This hinders transcriptome development in large, charismatic and endangered species, many of which stand the most to gain from such approaches. Parallel sequencing approaches such as Roche 454 and Illumina HiSeq are transforming the study of non-model organisms by dramatically increasing sequencing depth and coverage in return for a greatly reduced investment in time, labour and resources [1] This has facilitated the development of transcriptomes, which provide access to the coding sequences of literally thousands of genes and can be mined for genetic markers for use in genome scans, quantitative trait loci mapping and various other applications [2,3]. One potential solution, supported by recent expression studies of human cadavers [12] and slaughtered domestic pigs [13], is to sequence tissues obtained shortly after animals have died of natural causes
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