Figure 3

Flowchart of the pre-S genotyping by TA cloning- and NGS-based analysis. The virus DNA is extracted from patient’s plasma. The pre-S gene is amplified from virus DNA following first and second rounds of nested PCR. For TA cloning-based analysis, the pre-S gene PCR products are visualized by using agarose gel electrophoresis. When only a single PCR band is seen in agarose gel, the products are directly subjected to DNA sequencing analysis. However, when two or more PCR bands are revealed, individual bands are first cloned into the TA vector for further DNA sequencing analysis. In contrast, for NGS-based analysis, the entire pre-S gene PCR products, including both visible and invisible bands, are directly subjected to NGS sequencing analysis without the complex procedures needed for TA cloning-based analysis.