Sequencing of PCR products and nucleotide sequence analysis of HBV and HDV
SEPARATION
Before sequencing, PCR products of HBV/HDV were purified from primer and nucleotide residues using ExoSAP-IT™ PCR Product Cleanup Reagent (Applied Biosystems™, USA) according to the manufacturer’s instructions. The purified PCR products were sequenced in two directions, using a forward and reverse FHBS1-RHBS1 or FHBS2-RHBS2 primers for HBV and HD-1,2 or HD-3,4 primers for HDV (developed by ThermoFisher ScientificTM, USA), in accordance with the manufacturer’s instructions for BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™, USA).Electrophoresis and separation of labeled fragments were performed on a 3130 Genetic Analyzer (Applied Biosystems™, USA). Before electrophoresis, the PCR mixtures were cleaned of terminator residues using BigDye XTerminator (Applied Biosystems™, USA), in accordance with the manufacturer’s instructions. The analysis of the primary data was carried out using the Sequencing Analysis Software program (Applied Biosystems™, USA). The alignment and assembly of HBV and HDV nucleotide sequences was performed using the DNA Baser Sequence Assembly Software program.