For samples with mixed genotype infections, PCR cover HBV S gene (nt136-1110) was performed using the primers and thermocycling conditions descirbed by Sugauchi et al [10] . For samples needing further recombination analysis, PCR was performed using the primers and thermocycling conditions described by Günther to obtain full-length HBV genome [11] . Alternatively, a nested PCR was used to produce two overlapping fragments in subjects with low HBV DNA levels as described by Sugauchi et al [12] . The spanning of fragment A cover nucleotides 2813 to 1824, and fragment B included nucleotides 1821 to 237. LA-Taq (TAKARA, Japan) and high-fidelity polymerase COD-FX (TOYOBO, Japan) were used to produce amplimers for cloning and direct sequencing respectively. Finally, Fragment C (HBV nt56-nt1824) was obtained from a PCR amplification of Y2 HBV-DNA to which an aliquot of genotype B HBV-DNA had been added. The purpose of this experiment with in-tube control of genotype B was to determine if the recombinant clones were being generated during the PCR amplification. PCR products were gel-purified and cloned into the PMD19-T vector (TAKARA, Japan) according to the manufacturer's instructions, and used to transform JM109 competent cells (TAKARA, Japan). A minimum of 15 clones were sequenced from subjects with a mixed-strain infection and three clones were sequenced from family members with a single-strain infection. All sequencing of clones and PCR products was performed by Invitrogen Ltd. (Shanghai, China).