Viral DNA Extraction and Sequencing from Raja clavata Spleen

DNA was extracted from the spleen tissue from each of the ten Raja clavata. Briefly, approximately of 12 mg of tissue was homogenized with 300 µl of SM buffer (0.1 M NaCl, 50 mM Tris/HCl-pH 7.4, 10 mM MgSO₄) and disrupted using a bioruptor. The homogenized sample was centrifuged at 10.000 rpm for 2 min and 200 µl of the supernatant was used to isolate viral DNA using the High Pure Viral Nucleic Acid Kit (Roche Diagnostic, USA), according to manufacturer’s specifications. The extracted viral nucleic acid was then enriched for circular DNA molecules using the rolling circle amplification (RCA) reaction with the TempliPhi™ kit (GE Healthcare, USA). The products from RCA were quantified using Qubit™ dsDNA HS Assay kit (Thermo Fisher Scientific, USA), pooled equimolarly, and sent to Macrogen Inc. (Korea) for library preparation (Nextera DNA XT) and sequencing on an Illumina Novaseq 6000. Following Illumina sequencing, the resulting pair-end-reads were trimmed using Trimmomatic [31 (link)], host genome sequences were removed using the RefSeq genome of the Rajiform Amblyraja radiata available at NCBI (RefSeq accession number GCF_010909765.2) as a reference with Bowtie2 [32 (link)]. The remaining reads were de novo assembled using Megahit v1.2.9 [33 (link)].