PCR products were purified with QIAquick gel extraction kit (Qiagen, Hilden, Germany) and direct nucleotide sequencing reaction was done using Big Dye Terminator kit version 3.1 (Applied Biosystems, Foster City, CA, USA) with external and internal primers described by Mallory et al.43 (link) and Kryazhimskiy and Plotkin20 (link). Sequencing reactions were performed on ABI3730 automated sequencer (Applied Biosystems).
Assembly of each sequence, multiple sequence alignment and phylogenetic analyses were done in MEGA11 software45 (link). Phylogenetic trees were reconstructed using Maximum Likelihood method with bootstrap test (1000 replicates) to assess the confidence of the output tree. Consensus sequences of each HBV isolate were submitted to a web-based tool for prediction of clinically relevant mutations (Geno2pheno [hbv] 2.0, Max-Planck-Institut für Informatik, Germany, available athttp://hbv.geno2pheno.org/index.php ).
Assembly of each sequence, multiple sequence alignment and phylogenetic analyses were done in MEGA11 software45 (link). Phylogenetic trees were reconstructed using Maximum Likelihood method with bootstrap test (1000 replicates) to assess the confidence of the output tree. Consensus sequences of each HBV isolate were submitted to a web-based tool for prediction of clinically relevant mutations (Geno2pheno [hbv] 2.0, Max-Planck-Institut für Informatik, Germany, available at
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