blue crab totivirus
Total RNA was extracted from blue crab, and the viral dsRNA was revealed by electrophoresis gel. Then, the isolated dsRNA was agarose gel purified. Purified dsRNA of the two viruses (CsTLV1 and CsTLV2) was used for cDNA synthesis with barcoded octamers (5-GGCGGAGCTCTGCAGATATC-NNNNNNNN-3). The resulting cDNA was amplified by PCR using the barcode primers (5-GGCGGAGCTCTGCAGATATC-3). PCR products of 250-500 bp were obtained by agarose gel purification. The DNA library was constructed and sequenced in a 2  250 paired-end configuration on the Illumina MiSeq platform with a MiSeq Reagent kit v3. Terminal sequences were determined using a SMARTer RACE 5'/3' Kit (Takara Bio, San Jose, CA, USA) with purified dsRNA as the initial template. Sequence verification and filling of gaps between contigs were achieved with Sanger sequencing.