Abi3130 genetic analyzer automated sequencer

Total DNA was extracted from muscle fragments following the protocol of the Canadian Center for DNA-Barcoding (CCDB), available at http://www.ccdb.ca. A segment of the 5' region of the mitochondrial COI gene was amplified using different combinations of primers, including L5698-Asn [65 (link)], FishF1, FishF2, FishR1 and FishR2 [15 (link)], C_FishF1t1–C_FishR1t1 cocktail [66 ], and H7271-COI [67 ]. Polymerase chain reactions (PCR) were run in a 12.5 μl volume containing: 1 μl DNA (concentration 50 ng/μl), 0.25 μl each of the forward and reverse primers (concentration 10 mM), 1.25 μl of reaction buffer, 0.2 μl of 200 mM dNTPs mix, 0.37 μl of MgCl₂ and 0.0625 μl (5 units/μl) of Platinum Taq DNA polymerase (Invitrogen).
The samples were amplified in a Veriti^® 96-well thermocycler (Applied Biosystems), with initial denaturation of 5 minutes at 96°C followed by 35 cycles at 96°C for 45 seconds, 54°C for 45 seconds, 72°C for 1 minute, and final extension at 72°C for 1 minute. The amplified PCR products were cleaned up with ExoSAP-IT (USB Corporation) and sequenced in both directions using the BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies) in an ABI3130 Genetic Analyzer automated sequencer (Applied Biosystems).

The PCR products were purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany), and the nucleotide sequencing was carried out with primers HEV-F2 and HEV-R1 using an ABI 3130 Genetic Analyzer Automated Sequencer (Applied Biosystems, Foster City, CA) and a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) according to the manufacturer’s instructions. Sequence analysis was performed using the Genetyx ver.11.0.4 software program (Genetyx Corp., Tokyo).

The RNA was extracted from the purified virus particles by using a MagNA Pure LC system with a MagNA Pure LC Total Nucleic Acid isolation kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s recommendations. The nucleotide sequences of the entire genomes were determined by NGS as described previously^{24 (link)}. The 3′-terminus nucleotide sequence was further confirmed by RT-PCR. The reverse transcription was performed by using Superscript^™ II RNase H⁻ reverse transcriptase (Invitrogen, Carlsbad, CA) and oligonucleotide (dT) primer TX30SXN (5′-GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′)^{25 (link)}. The 3′-terminal sequence was amplified by PCR using the primers, camF7879 (5′-ATGGACACGTAGCGCTGCTA-3′) and TX30SXN. The nucleotide sequencing was carried out with the primer camF7879 using an ABI 3130 Genetic Analyzer automated sequencer (Applied Biosystems, Foster City, CA).