DNA extraction, library preparation and sequencing
DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen), with the only modification from manufacturer instructions being an extended lysis time (overnight). DNA quality and quantity were assessed using 1.5% agarose gels and Qubit dsDNA HS assays. Next, ddRAD libraries were prepared following the protocol of Maas et al ., (2018), adapted from the original protocol of Peterson et al., (2012). We refer to the extensive protocol included in Maas et al. , (2018) for details, but describe here how we adapted it for S. diversicolor . In brief, genomic DNA (600ng) was double-digested using enzymes SphI-HF (rare-cutting) and MlucI (frequent-cutting) (See Supplementary Information S1 for example of a successful enzyme digestion). Size distribution of the fragments was assessed with the BioAnalyzer High Sensitivity Chip (Agilent). We used the spreadsheet provided in Petersonet al. , (2012) ”Locus count from Bioanalyzer % in region” to calculate the number of fragments to be expected assuming a genome size of 600Mb (common for sponges (Jeffery, Jardine and Ryan Gregory, 2013) and various size selections of RAD fragments. This number can subsequently be used to calculate the expected coverage when generating a known amount (Gb) of sequencing data. Custom-made sample-specific barcodes were ligated to the fragments to allow for the pooling of 21 samples per library, resulting in 8 libraries in total. The Sage Science Pippin Prep was used to size-select adapter-ligated fragments of length 500-575bp (indicating an insert size of 425-500bp). A trial was run for 8, 10 and 12 polymerase chain reaction cycles (PCR) reactions. In the end ten PCR cycles were chosen as a balance between DNA output and PCR duplication and were run on each library for enrichment and ligation of Illumina indices unique to each library pool. Quality and quantity of libraries throughout the process were checked using BioAnalyzer High Sensitivity chips (Agilent, Supplemental Information S2 for an example). Libraries were pooled at equimolar volumes and 150bp single-end sequenced on Illumina HiSeq 2500 at the Vincent J. Coates Genomic Sequencing Facility at UC Berkeley.