Evaluating the Performance of De Novo Assembly Methods for Venom-Gland Transcriptomics.

Venom-gland transcriptomics is a key tool in the study of the evolution, ecology, function, and pharmacology of animal venoms. In particular, gene-expression variation and coding sequences gained through transcriptomics provide key information for explaining functional venom variation over both ecological and evolutionary timescales. The accuracy and usefulness of inferences made through transcriptomics, however, is limited by the accuracy of the transcriptome assembly, which is a bioinformatic problem with several possible solutions. Several methods have been employed to assemble venom-gland transcriptomes, with the Trinity assembler being the most commonly applied among them. Although previous evidence of variation in performance among assembly software exists, particularly regarding recovery of difficult-to-assemble multigene families such as snake venom metalloproteinases, much work to date still employs a single assembly method. We evaluated the performance of several commonly used de novo assembly methods for the recovery of both nontoxin transcripts and complete, high-quality venom-gene transcripts across eleven snake and four scorpion transcriptomes. We varied k-mer sizes used by some assemblers to evaluate the impact of k-mer length on transcript recovery. We showed that the recovery of nontoxin transcripts and toxin transcripts is best accomplished through different assembly software, with SDT at smaller k-mer lengths and Trinity being best for nontoxin recovery and a combination of SeqMan NGen and a seed-and-extend approach implemented in Extender as the best means of recovering a complete set of toxin transcripts. In particular, Extender was the only means tested capable of assembling multiple isoforms of the diverse snake venom metalloproteinase family, while traditional approaches such as Trinity recovered at most one metalloproteinase transcript. Our work demonstrated that traditional metrics of assembly performance are not predictive of performance in the recovery of complete and high quality toxin genes. Instead, effective venom-gland transcriptomic studies should combine and quality-filter the results of several assemblers with varying algorithmic strategies.