ddRADSeq of Marmaronetta angustirostris: feather
DNA samples were processed following the double-digestion restriction-site associated DNA sequencing procedure (ddRAD-seq) adapted from Peterson et al., (2012) (PLOS One, 7: e37135) and described in Brown et al., (2016) (BMC Genomics, 17: 448) and Dicks et al., (2023) (Evolutionary Applications, 16: 111-125) with minor modifications. DNA was quantified using a Qubit Broad Range dsDNA Assay (Thermo Fisher Scientific) and normalized to 7 ng/l. We then digested the DNA with the restriction enzymes SbfI and SphI (New England Biolabs, Ipswich, MA, USA) and RAD-specific P1 and P2 paired-end adapters that included a unique pair of 5 or 7 bp barcodes was ligated to each sample. The samples were pooled and then gel electrophoresis extraction was used to select fragments between 400 and 700 bp. Following optimisation of PCR conditions, to identify the minimum number of PCR cycles required to produce sufficient product for sequencing, a bulk amplification of each library (400l) was conducted. The PCR comprised of 48 l library template, 12.8 L combined 10 M Illumina compatible P1 and P2 adapter specific primers, 200 l Q5 Hot Start HF 2x Master Mix (New England Biolabs) and 139.2 L nuclease-free water. Samples were sequenced across two libraries, and positive controls were used to confirm repeatability within and between libraries (Leigh et al., 2018, Molecular Ecology Resources, 18: 778-788). Each library was sequenced (150 bp, paired-end) on a full lane of an Illumina HiSeq.