A garter snake transcriptome: pyrosequencing, de novo assembly, and sex-specific differences

The reptiles, characterized by both diversity and unique evolutionary adaptations, provide a comprehensive system for comparative studies of metabolism, physiology, and development. However, molecular resources for ectothermic reptiles are severely limited, hampering our ability to study the genetic basis for many evolutionarily important traits such as metabolic plasticity, extreme longevity, limblessness, venom, and freeze tolerance. Here we use massively parallel sequencing (454 GS-FLX Titanium) to generate a transcriptome of the western terrestrial garter snake (Thamnophis elegans) with two goals in mind. First, we develop a molecular resource for an ectothermic reptile; and second, we use these sex-specific transcriptomes to identify differences in the presence of expressed transcripts and potential genes of evolutionary interest.

Using sex-specific pools of RNA (one pool for females, one pool for males) representing 7 tissue types and 35 diverse individuals, we produced 1.24 million sequence reads, which averaged 366 bp in length after cleaning. Assembly of the cleaned reads from both sexes with NEWBLER and MIRA resulted in 96,379 contigs containing 87% of the cleaned reads. Over 34% of these contigs and 13% of the singletons were annotated based on homology to previously identified proteins. From these homology assignments, additional clustering, and ORF predictions, we estimate that this transcriptome contains ~13,000 unique genes that were previously identified in other species and over 66,000 transcripts from unidentified protein-coding genes. Furthermore, we use a graph-clustering method to identify contigs linked by NEWBLER-split reads that represent divergent alleles, gene duplications, and alternatively spliced transcripts. Beyond gene identification, we identified 95,295 SNPs and 31,651 INDELs. From these sex-specific transcriptomes, we identified 190 genes that were only present in the mRNA sequenced from one of the sexes (84 female-specific, 106 male-specific), and many highly variable genes of evolutionary interest.

This is the first large-scale, multi-organ transcriptome for an ectothermic reptile. This resource provides the most comprehensive set of EST sequences available for an individual ectothermic reptile species, increasing the number of snake ESTs 50-fold. We have identified genes that appear to be under evolutionary selection and those that are sex-specific. This resource will assist studies on gene expression and comparative genomics, and will facilitate the study of evolutionarily important traits at the molecular level.