Specimen were collected in the central Philippines during several collection expeditions in 2011–2015. Specimen identification was initially performed by morphological examination and later verified by sequence analysis of the cytochrome oxidase c subunit 1 (COI) gene. Venom glands were dissected and stored in RNAlater at −80 °C until further processing. Total RNA was isolated from venom glands using TRIzol®Reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) or the RNeasy kit (Qiagen, Germantown, MD, USA) following the manufacturers’ instructions. RNA integrity, quantity, and purity were determined on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were prepared and sequenced on an Illumina HiSeq 2000 instrument (Sanger/Illumina 1.9 reads, 101 bp or 125 bp paired-end, Illumina, San Diego, CA, USA). Publicly available Illumina datasets were used for the venom gland transcriptomes of C. marmoreus (specimen 2), C. virgo (specimen 2), C. coronatus, and C. ebraeus [10 (link)].
Adapter clipping and quality trimming of raw reads were performed using fqtrim software (Version 0.9.4,http://ccb.jhu.edu/software/fqtrim/ ) and PRINSEQ (Version 0.20.4 [38 (link)]). After processing, sequences shorter than 70 bps and those containing more than 5% ambiguous bases (Ns) were discarded. De novo transcriptome assembly was performed using Trinity Version 2.0.5 [39 (link)] with a kmer size for building De Bruijn Graphs of 31, a minimum kmer coverage of 10, and a minimum glue of 10. Assembled transcripts were annotated using Blastx ((NCBI-Blast-2.2.28+, [40 (link)]) against conotoxin sequences extracted from the ConoServer [5 (link)] and UniProt databases [34 (link)].
Adapter clipping and quality trimming of raw reads were performed using fqtrim software (Version 0.9.4,
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