Seqstudio sequence analyzer

One DBS fin was taken to extract HBV DNA using the SMITEST EX‐R&D kit (Medical & Biological Laboratories Co., Ltd., Japan) and perform DNA quantification and genotype identification.
HBV DNA was quantified by real-time polymerase chain reaction (PCR) using the TaqMan Fast Advanced Master Mix reagent (Thermo Fisher Scientific, MA, USA) on Applied Biosystems StepOne (Thermo Fisher Scientific, MA, USA).
Using PrimeScript One-Step RT-PCR Kit Ver.2, HBV DNA was amplified by nested PCR targeting the overlapping surface-polymerase (SP) or the surface region of the viral genome in case the SP region amplification failed. PCR products were then sequenced by the Sanger sequencing method on SeqStudio Sequence Analyzer (Thermo Fisher Scientific, MA, USA) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, CA, USA). Then, HBV genotypes were identified by analyzing partial genome sequences with 103 reference strains retrieved from GenBank, using the neighbor-joining method with the Molecular Evolutionary Genetics Analysis software version 10 (Pennsylvania State University, PA, USA). The resulting phylogenetic trees are shown in Supplementary Figs. S1 and S2. Supplementary Table S1 shows the primers used for nested PCR reactions and genome sequencing^{13 (link),14 (link)}.

The extracted nucleic acid was amplified by means of nested RT-PCR as per the previously described method [6 (link)]. We used the in-house-designed primer set hCoV-Spike-D for the amplification as shown in Table 1. The amplified products underwent Sanger sequencing for partial genome sequences covering the targeted spike region with SeqStudio sequence Analyzer (Thermo Fisher Scientific, Applied Biosystems, Foster City, CA, USA) and BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Applied Biosystems, Foster City, CA, USA) and the corresponding primer set as shown in Table 2. First, 2 µL of amplified products was mixed with 4 µL of BigDyeTM Terminator v3.1 Ready Reaction Mix, 2 µL of Sequencing Buffer, 2 µL of 2 µM primer and 10 µL of RNase-free water and incubated at 96 °C for 1 min, followed by 25 cycles of denaturing, annealing, and elongation at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 4 min. Then, the products were purified by adding a mixture of 90 µL of SAM Solution and 20 µL of BigDye XterminatorTM bead solution. The mixture was thoroughly shaken for 30 min using the micro mixer (MX-4: SANKO JUNYAKU Co., Ltd., Chiyoda-ku Tokyo, Japan). Thereafter, sequencing was performed by SeqStudio sequence Analyzer following the manufacturer’s instructions.