Fig. 2: Primer design and tiled sequencing of HEV-3.
From: varVAMP: degenerate primer design for tiled full genome sequencing and qPCR
a Schematic overview of the data preparation steps preceding primer design. All full-length sequences of HEVs were downloaded from NCBI, sub-genotyped with fasta36 and clustered by similarity with vsearch. The clustering result was evaluated via phylogenetic tree construction. Clusters comprising multiple subgenotypes were aligned with MAFFT, and the MSA used as the input for varVAMP. Created in BioRender. Fuchs (2025) https://BioRender.com/csajk3b. b Phylogenetic tree of full-length HEV sequences constructed with IQ-TREE 2 (GTR + F + R10, 1000 bootstrap replicates). The vsearch clustering result for each sequence is displayed in colors and the HEV genotypes and subgenotypes are indicated at the respective branches (n number of sequences). c Agarose electrophoresis images of the individual PCR products for the cluster 2 (upper plot, representative plot out of 4 plots) and cluster 4 (lower plot, representative plot out of 5 plots) primer schemes tested with the supernatants of HEV-3 f or HEV-3 c stably infected PLC/PRF/5 cells, respectively. Triangles indicate bands at the expected molecular weight of the PCR products (kb kilobases). d Coverage plots of the Illumina sequencing results of the in c amplified PCR products for cluster 2 (upper plot) and cluster 4 (lower plot) mapped to their respective NCBI reference sequences MK089847 and MK089849. Below each coverage plot, the genomic start and stop positions of each amplicon are displayed as gray boxes with their respective amplicon number. Dotted lines indicate mean coverages. Coverage plots were created with BAMdash (individual coverage plots are given in Supplementary Fig. 2). e Genome recovery of HEV-3 persistently infected cell cultures and sub-genotyped HEV-3 positive blood samples subjected to their respective tiled amplicon workflow for cluster 2 (upper plot) or cluster 4 (lower plot). Genome recovery was calculated as the percentage of reference nucleotides covered at least 20 fold. All PCRs were performed in the singleplex setting.
