This protocol describes a double digested restriction-site associated DNA (ddRADseq) procedure, that is a variation on the original RAD sequencing method (Davey & Blaxter 2011), which is used for de novo SNP discovery and genotyping. This protocol differs from the original ddRADseq protocol (Peterson et al 2012), in which the samples are pooled just after the ligation to adaptors (i.e. before size selection and PCR). The present ddRAD protocol as been slightly adapted from Alan Brelsford's protocol published in the supplementary material of this paper: Brelsford, A., Dufresnes, C. & Perrin, N. 2016. High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex. Heredity 116, 177–181 (2016). https://doi.org/10.1038/hdy.2015.83 In the present protocol, all samples are treated separately, in a microplate, until final PCR amplification performed before pooling. Despite being slightly more costly and time-consuming in the lab, it allows for fine adjustement of each sample representation in the final library pool, ensuring similar number of sequencing reads per sample in the final dataset. Briefly, genomic DNA from the samples are individually digested with 2 restriction enzymes (one rare-cutter and one more frequent cutter) then ligated to a barcoded adaptor (among 24 available) at one side, and a single adaptor at the other side, purified with magnetic beads, and PCR-amplified allowing the addition of a Illumina index (among 12 available) for multiplexing a maximum of 288 sample per library. Samples are then pooled in equimolar conditions after visualisation on an agarose gel. Purification and size selection is then performed before final quality control of the library and sequencing.
Fine structural changes in the endolymphatic sac induced by calcium loading in the tree frog, Hyla arborea japonica.