Abi 3730xi dna analyser

All primers used in this study (Supplementary Table S6) were designed by the Primer Premier 5.0 software (http://www.premierbiosoft.com/; last accessed Jan 2019) and synthesized by the BGI Tech (Shenzhen, China). PCR was performed in a total volume of 15 μl, containing 7.5 μl of GC buffer I, 50 ng of DNA, 1 μl of forward and reverse primers (10 mM), 0.24 μl of dNTPs (25 mM), and 0.15 μl of LA Taq polymerase (Takara, Dalian). PCR was carried out in a Veriti 96-Well Thermal Cycler (Applied Biosystems, USA) with the following procedure: denaturing at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, annealing at 55–65 °C for 45 s, and 72 °C for extension (1 kb min^–1), and finally extension at 72 °C for 10 min. PCR products were purified by an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Hangzhou), cloned into the pEASY-T1 Cloning Vector, and then transformed into Trans1-T1 competent cells by the heat shock method (TransGen Biotech, Beijing). Conjugative plasmids were extracted with an AxyPrep Plasmid Miniprep Kit (Axygen Biosciences) and sequenced by an ABI 3730XI DNA Analyser (Applied Biosystems).

All viral RNA extractions were performed using the QIAamp Viral RNA Mini Kit (Qiagen) using on-column DNase digestion (Qiagen). Reverse transcription was performed with the Uni12 primer using the Verso cDNA kit (Thermo Scientific). PCR reactions were performed using Pfu Ultra II fusion HS polymerase (Stratagene #600674-51) according to the manufacturer's protocol. PCR products were purified for sequencing by Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare). Sequencing reactions were performed using BigDye^® Terminator v1.2 Cycle Sequencing Kit (Applied Biosystems) (according to the manufacturer's instructions) and samples were run on an ABI 3730xI DNA Analyser.

Primers were designed by Premier 5.0 (http://www.premierbiosoft.com/) and synthesized by BGI Tech (Shenzhen, China). PCR was conducted in reaction volumes of 15 mL containing 50–100 ng DNA, 7.5 mL buffer, 2.4 mL of 2.5 mm deoxynucleotide triphosphates, 0.5 mL of 10 mm forward and reverse primers, and 0.15 mL of LA Taq polymerase (TaKaRa Biotechnology). PCR was carried out in a Veriti 96‐Well Thermal Cycler (Applied Biosystems) with the following steps: denaturing at 95 °C for 5 min, followed by 32 cycles of 95 °C for 30 s, annealing at 55–63°C for 1 min, 72 °C for extension (1 kb/min) and a final extension of 72 °C for 10 min. PCR products were purified by purification kit DP‐1502 (TIANGEN), then cloned by the pGEM‐T Easy Cloning Vector (TIANGEN), and transformed into Escherichia coli cells by the heat shock method. Plasmids were extracted with a DP‐1002 Kit (TIANGEN). DNA was sequenced by an ABI 3730XI DNA Analyser (Applied Biosystems). Sequence alignments and SNP identification were made using DNASTAR (http://www.dnastar.com/). The digestion sites of restriction endonucleases were detected by Primer Premier 5.0. Two restriction endonucleases used in the study were produced by New England Biolabs (Beijing).