• anellovirus
• bio-informatics
• next-generation sequencing
• real-time pcr
• torque teno virus

Torque teno virus (TTV) represents the main component of human virome, as commonly reported in several recent studies. Due to the high degree of diversity, the genetic characterization of TTV over time is poorly understood and constantly evolving. To overcome this challenge, this study aims to investigate the individual TTV profiles in 20 transplanted patients and 14 healthy subjects by using three different molecular approaches: metagenomic next-generation sequencing (mNGS), NGS-based on TTV-amplicons (TTV-NGS) and semi-quantitative TTV species-specific PCRs (ssPCR). Bioinformatic analysis of the sequences was performed by setting up an in-house bioinformatics pipeline. Our pipeline was also validated using datasets simulating NGS sequencing. A high degree of genetic complexity was identified in TTV species in healthy and transplanted subjects. Both NGS methods resulted in a good performance in terms of accuracy and efficiency, however , the TTV-NGS method showed higher sensitivity than mNGS. The ssPCRs revealed a good degree of concordance with TTV-NGS method, although some TTV species were sub-optimally amplified. A slight but statistically significant correlation between the TTV mapped reads per million (RPM) and the threshold cycle value (CT) observed in the ssPCR was found. Finally, the prevalence of other anelloviruses was estimated. TTMV (Betatorquevirus) was present in the 8 subjects, and TTMDV (Gammatorquevirus) was found in only one case.
This study represents the first comparative study between different molecular approaches, next generation sequencing (NGS) and semi-quantitative real-time PCR species-specific (ssPCR) to characterize the TTV-virome present in solid organ transplant recipients and healthy subjects. The development of in-house methods based on real-time PCR and TTV-NGS are important and cost-effective tools for investigating the dynamic viral composition of this virus, so widespread.