ddRAD sequencing of Helix hybrid zone: foot tissue
Double-digest restriction-site-associated DNA library conctructed following a slightly modified qaddRAD protocol of Franchini et al. (2017). 200-400 ng genomic DNA from each sample digested with FastDigest PstI and MspI and ligated to adaptors containing 6 bp inner barcodes and four random bases for the recognition of PCR duplicates. Products purified and double size-selected with AmpureXP beads (adding successively 0.45 and 0.35 the sample volume). Twelve digested/ligated samples, each with a different inner barcode combination, pooled by equal DNA weight (based on Qubit concentration measurement). The pools amplified by 12 or 15 PCR cycles with Phusion Polymerase and primers containing 8 bp barcodes and Illumina adaptors. PCR performed in at least eight separate tubes. After a purification step with 0.8 AmpureXP beads, the DNA concentration and fragment length distribution of each pool measured with TapeStation (HS D1000 assay). The 16 pools then pooled equimolarly with respect to the molarity of fragments of the targeted size range and the final pool concentrated using a vacuum centrifuge concentrator. Fragments in the range 400-500 bp selected using a Blue Pippin electrophoresis system (Sage Science, Beverly, USA). Paired-end sequencing (2  150 bp) performed on one Illumina HiSeq X lane after adding 20% of the PhiX Control library. Stacks 2.60 used to filter and demultiplex the raw sequencing data. PCR duplicates removed based on four random bases within the ligated adaptor sequence using the clone_filter module. Retained reads then demultiplexed according to the inner barcodes using the process_radtags module (-P -c -q --score-limit 15 -r -D --len-limit 120 --barcode-dist-2 1 --barcode-dist-1 1).