Modahl, C., et al. Exploring toxin evolution: venom protein transcript sequencing and transcriptome-guided high-throughput proteomics. 97-127
Studying animal toxin evolution requires sequences of these proteins and peptides, and transcript sequences allow for the construction of cladograms and evaluation of selection pressures from nonsynonymous and synonymous nucleotide mutation ratios. In addition, these translated sequences can be useful as custom databases for peptide identifications within venoms and for better proteomic quantification. Obtaining these transcripts is achieved by sequencing cDNA originating from venom gland tissue or venom. This chapter provides the methodology for (1) targeted sequencing of transcripts from a single venom protein family (RNA isolation and 3′RACE [rapid amplification of cDNA ends]), (2) generation of a venom gland transcriptome with next-generation sequencing (NGS) technology (de novo transcriptome assembly, toxin transcript identification, quantification, and positive selection analysis), and (3) combined high-throughput proteomics to identify secreted venom components. Transcriptomics has become fundamental for studying toxin evolution, but it creates many challenges for scientists who are unfamiliar with working with RNA, managing large NGS datasets and executing the required programs, particularly considering that there is an overabundance of available software in this field and not all perform optimally for venom gland transcriptome assembly. This chapter provides one pipeline for the integration of both low- and high-throughput transcriptomics with proteomics to characterize venoms.