Computational protocol: Molecular epidemiological study of hepatitis B virus in blood donors from five Chinese blood centers

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Protocol publication

[…] The PCR products were analyzed by electrophoresis in 1.5 % agarose gels and purified using a commercially available kit (NucleoSpin Extract II kit, Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer’s instructions. Purified products were used as templates in cycle sequencing reactions. Nucleotide sequences were determined from both strands using primers BS1 and BS3, and the resulting sequences were read directly with a genetic analyzer (ABI 3730, Applied Biosystems). For the single sequence with no heterogeneous sites, the HBV genotypes were determined by phylogenetic analysis with a panel of reference sequences from genotypes A to H retrieved from GenBank. The GenBank accession numbers for the reference strains were AB076678, AF536524, AJ012207, and AB194951 for genotype A; AB073851, AB106885, AB010290, AB205119, AB073834, AY293309, AF121245, AB033554, AB031267, AB219426, and AB219427 for genotype B; AB074047, AF223961, AB031262, AB014374, AB014360, AB026814, X75656, AB105172, GQ377635, Y18855, AB241110, AB241109, and AF241410 for genotype C; AY161157, AF151735, AB078033, AB090269, AJ132335, AY902776, AB048701, AB033559, DQ315779, and DQ315780 for genotype D; AB091255 and AB194948 for genotype E; AB036909 and AY090458 for genotype F; AB064311 and 064313 for genotype G; and AY090457 for genotype H. Nucleotide sequences were multiply aligned using the CLUSTAL_X (version 1.83) program []. The alignments were then used to construct phylogenetic trees for each subalignment using the neighbor-joining method implemented by the MEGA program []. The statistical validity of the neighbor-joining trees was assessed by bootstrap re-sampling with 1000 replicates. If the amplified sequence had heterogeneous sites identified by sequencing both strands, multiplex PCR was used for genotyping as reported previously []. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Hepatitis B virus, Homo sapiens
Diseases Hepatitis B