A combined approach of rolling-circle amplification-single site restriction endonuclease digestion followed by next generation sequencing to characterize the whole genome and intra-host variants of human Torque teno virus

Abstract

Torque Teno Virus (TTV) was initially associated with post-transfusion hepatitis, but growing evidence of its ubiquity in humans is compatible to no apparent clinical significance. TTV is a small non-enveloped virus with a circular single-negative-stranded DNA genome, belonging to the Anelloviridae family. Currently, TTVs are divided in seven phylogenetic groups and are further classified into 21 species. Studies about diversity of TTV in different conditions are receiving increasing interest and in this sense, sequencing of whole genomes for better genetic characterization becomes even more important. Since its discovery in 1997, few TTV complete genomes have been reported worldwide. This is probably due, among other reasons, to the great genetic heterogeneity among TTV strains that prevents its amplification and sequencing by conventional PCR and cloning methods. In addition, although metagenomics approach is useful in these cases, it remains a challenging tool for viromic analysis. With the aim of contributing to the expansion of the TTV whole genomes dataset and to study intra-host variants, we employed a methodology that combined a rolling-circle amplification approach followed by EcoRI digestion, generating a DNA fragment of ∼4Kb consistent with TTV genome length which was sequenced by Illumina next generation sequencing. A genogroup 3 full-length consensus TTV genome was obtained and co-infection with other species (at least those with a single EcoRI cleavage site) was not identified. Additionally, bioinformatics analysis allowed to identify the spectrum of TTV intra-host variants which provides evidence of a complex evolution dynamics of these DNA circular viruses, similarly to what occurs with RNA viruses.