Bigdye3.1 terminator kit

The BAC-end sequences for selected BAC-clones were obtained with the universal M13 Reverse (5′-CAGGAAACAGCTATGAC -3′) and T7 forward (5′-TAATACGACTCACTATAGGG-3′) primers using the BigDye3.1 Terminator kit (Applied Biosystems, USA). Each 20 μl reaction contained ~200 μg of BAC-DNA, 1.5 μl of BigDye 3.1, 0.25 pM of specific forward, or reverse primer, 4 μl of5X buffer and deionized water. After preliminary denaturation at 95 °C for 5 min, 80 cycles were run at 95 °C for 30s, 55 °C for 15 s, and 60 °C for 4 min. The reaction products were precipitated using ethanol, and separated in a 3730XL DNA Analyzer (Perkin Elmer Cetus, USA).
BAC clones marked by 5S rDNA were included in the pool of 134 BAC-clones belonging to different locations of chromosome 5BS, and were collectively sequenced as one sample on an IonTorrent platform (Thermo Fisher Scientific). The sequencing and assembly were described in Nesterov et al. [23 (link)].

The snPCR product band of size 464 bp was excised, gel-purified, and sequenced using a Big Dye 3.1 Terminator Kit (Applied Biosystems, USA) and a SeqStudio Genetic Analyser (Thermo Fisher Scientific, USA). Sequences were edited using the BioEdit software (version 7.2), and one sequence was submitted to GenBank with an accession number of MK636574.
Four different genotyping PCR assays were performed to type CyHV-3 to the right strain. PCR assays targeting the thymidine kinase (TK) gene and ORF 136 were performed using the published primers and protocols [3 (link), 14 (link)]. PCR amplification targeting markers I and II was performed as previously outlined by Bigarre et al. [6 (link)]. PCR products were sequenced and analysed as mentioned above, and molecular phylogenetic analysis was performed by the maximum likelihood method based on the Tamura 3-parameter model in MEGA6 software.

Total DNA was extracted from the tissues using the Wizard Genomic DNA Purification kit (Promega) following the manufacturer’s protocol. The concentration and purity of the DNA were evaluated in a Nanodrop 2000 (Thermo Scientific), and whenever necessary, the samples were electrophoresed in a 1% agarose gel stained with GelRed to evaluate the quality of the DNA under an ultraviolet transilluminator.
The COI barcoding region was amplified by Polymerase Chain Reaction (PCR), using the primers FishF1 and FishR1 [29 (link)]. The PCRs were run in a final volume of 15 μl, containing 2.4 μl of dNTPs (1.25 mM), 1.5 μl of 10 X buffer solution, 0.5 μl of MgCl₂ (50 mM), 0.3 μl of each primer (10 pmol/μl), 1–3 μl of genomic DNA (100 ng/μl), 0.12 μl of Taq DNA polymerase (5 U/μl), and water to complete the final reaction volume. The cycling conditions were based on the protocol described by Silva et al. [10 (link)].
The PCR products were purified using the polyethylene glycol-8000 M protocol [30 (link)], and the sequencing reaction was based on the dideoxyterminal method [31 (link)] using the Big Dye 3.1 terminator kit (Applied Biosystems) following the manufacturer’s instructions. The samples were sequenced in an ABI 3500XL Genetic Analyzer (Applied Biosystems, Inc.).