Bigdye v3.1 chemistry sequencing kit

Total nucleic acid was extracted from all samples using an automated MagNA Pure LC robot and a MagNA Pure LC Total Nucleic Acid Isolation kit (Roche), and eluted with 50 μl elution buffer according to the manufacturer’s instructions. cDNA was generated using a high-temperature capability SuperScript VILO cDNA Synthesis kit (Invitrogen) on an ABI PRISM 9700 PCR system. The PCR conditions following reverse transcriptase were: 25°C for 10 min, 42°C for 90 min and 85°C for 5 min.
Amplification of the NS5B gene region was done as described previously (Forbi et al., 2012 (link)). Nested NS5B amplicons derived from the PCR amplification were purified (PCR purification kit; Qiagen) and sequenced with their respective nested primers using a BigDye v3.1 chemistry sequencing kit (Applied Biosystems) with an automated sequencer (ABI 3130xl; Applied Biosystems) as described previously (Forbi et al., 2012 (link)).

Amplification of the NS5b-gene region was done as we previously described [Forbi et al., 2012 (link)] using the LightCycler^® 480 software (Version 1.5.0.SP3, Roche, Indianapolis, IN). Samples that were NS5b-positive were subjected to HVR1 amplification. The E1/E2 junction region (306 nt), which contains the HVR1 region, was amplified using the nested PCR protocol described in Ramachandran et al. [2008] (link). Nested NS5b and HVR1 amplicons derived from the PCR amplification were purified (Millipore multiscreen PCR filter plate, Billerica, MA) and sequenced using their respective nested primers, and the BigDye v3.1 chemistry sequencing kit (Applied Biosystems) by an automated sequencer (ABI 3130xl, Applied Biosystems, Foster City, CA) as previously described [Forbi et al., 2012 (link)].