Sanger Sequencing of Hepatitis E Virus

PCR products with bands at the expected sizes were enzymatically cleaned up using the ExoSAP-IT™ Express PCR Product Clean Up kit (Thermo Fisher Scientific^®, Berlin, Germany). Sanger sequencing was performed directly on both strands at the sequencing facility of the Robert Koch Institute, Berlin, Germany, using 0.5 µl Big Dye version 3.1 (Life Technologies, Applied Biosystems, Darmstadt, Germany). The resulting aHEV sequences were compared with reference aHEV strains available at GenBank. Multiple sequence alignments with reference strains were performed using the MAFFT method with default options in Geneious Prime software version 2020.0.5 (Biomatters, New Zealand). Phylogenetic trees were constructed by maximum likelihood method with 1000 standard non-parametric bootstrap replicates using IQ-TREE 1.6.12 [42 (link)]. The best fitting model was determined using the substitution model test included in IQ-TREE [43 (link)]. Trees were graphically adjusted using iTOL [44 (link)].