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.