ddRADseq of Heterodontus francisci
Up to 1000ng genomic DNA from each individual was digested using enzymes BamHI and PstI. Custom adapters were appended to sticky ends (specific to each restriction enzyme). Rare-cutter adapters (BamHI) contained a 6-bp in-line barcode for demultiplexing. Size selection (PippinPrep) targeted fragments between 325 and 395 bp in length. Following size selection, Illumina sequencing adapters were appended via PCR (up to 12 cycles). Sequences matching bacterial and viral references in the MiniKraken2 (v1) database were removed from the sequence dataset(s). Sequences were trimmed to 95 bp (-t 95) and demultiplexed based on a unique 6-bp in-line barcode sequence (--inline_null). Reads were removed from the dataset if they contained adapter sequence, had uncalled bases (-c option), contained more than one mismatch in either the restriction enzyme site or the 6-bp barcode (single mismatches were corrected using the -r option), or had an average Phred33 quality score below 20 within a sliding window of 15% of the read length (parameters: -s 20, -w 0.15, -q).