Ion Channels Target-Sequencing:Imantodes_cenchoa_2671
We collected or received loans of tissue samples from 40 snakes from different locations across Colombia. We obtained mouth swabs, muscle tissue, or liver tissue, subsequently stored in 70% ethanol or RNAlater (Life Technologies, Carlsbad, CA) at 80 C.  We used target enrichment and Illumina sequencing to obtain a complete sequence of 74 genes from 14 gene families involved in neurological functions, and therefore putatively involved in toxin resistance, and two nuclear genes commonly used in phylogenetic reconstruction (RAG2 and c-mos). We extracted the DNA using the Qiagen DNeasy kit and quantified it using a Qubit dsDNA HD Assay Kit (Thermo Fisher Scientific). We reconcentrated some samples using a SpeedVac until they reached a minimum DNA concentration threshold of 40 ng/ul. We fragmented DNA to obtain 400-bp fragments by running three cycles of 30 seconds using a Brandon 2800 sonicator, and prepared 40 genomic DNA libraries using the Accel-NGS 1S Plus Swift bioscience library preparation kit for the Illumina platform, following the recommendations of the manufacturer, except for the purification step when we used the AMPure XP beads (Beckman Coulter) for an average size selection of 400 bp. We indexed each sample using dual identifiers (Dual-indexing barcodes) from the Swift bioscience 1S Plus Dual Indexing kit. For a final quality check, we used Agilent High Sensitivity DNA kit and the Qubit dsDNA HD Assay kit. On average we obtained libraries with 400 bp fragment sizes at 4 nM. Library preparations were performed at the Universidad de los Andes. We enriched the target genes using a custom set of MYbaits in-solution (MYcroarray) baits, designed by the company using gene annotations extracted from the Python bivittatus (Genbank accession number: AEQU02000001.1), and Ophiophagus hanna genomes (PRJNA201683), or gene sequences from Thamnophis sirtalis, Erythrolamprus reginae and Protobothrops mucrosquamatus generated in previous studies. We chose the most relaxed design of the probes (Relaxed-4) to account for the wide phylogenetic breadth of our samples. We pooled 2 to 3 libraries for each capture, depending on the DNA concentration, to perform 16 enrichment reactions using the provider's protocol, and we purified the enriched libraries using Dynabeads MyOne Streptavidin C1 (Life Technologies). Finally, we sequenced all libraries using one lane of the Illumina NextSeq500 platform (150-bp paired-end reads) at ACGT, Inc, Wheeling, IL.