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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). This protocol is an update of the protocol from Claire Daguin Thiebaut et al. (dx.doi.org/10.17504/protocols.io.bv4tn8wn) adapted for micro-algae. The following protocol is intended for the construction of individual ddRADseq libraries from genomic DNA of various macro-algae samples (Kelp). In the present protocol, we added a genomic DNA purification step to eliminate the inhibitors of PCR and Ligation present in macro-algae (polysaccharides). Moreover, all samples are treated separately until final PCR amplification (Rad Taq enrichment step) 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 reads between samples. Finally, we have defined new P1 adapters (barcodes) with variable sequences and variable sizes (6bp to 13bp) to increase the efficiency of the Illumina sequencing. Briefly, purified genomic DNA from the samples are individually digested with 2 restriction enzymes PstI/HhaI or PstI/MesI (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 24 available) for multiplexing a maximum of 576 samples 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. This protocol has proven its effectiveness in several genetic studies of marco-algae populations.
Mauger et al. (Tue,) studied this question.