Benchmarking next-generation sequencing methods to investigate TTV species composition and estimate prevalence

SPEZIA, PIETRO G.; FILIPPINI FABIO; BAJ ANDREINA; NOVASSI FEDERICA; GENONI ANGELO; FERRANTE FRANCESCA D.; ECHAVARRIA MARCELA; REYES, NOELIA S.; PISTELLO MAURO; MAGGI FABRIZIO

Congreso; 7th National Congress of the Italian Society for Virology; 2023

BACKGROUNDIn recent years, there has been a growing interest in the use of Torquetenovirus (TTV), the major component of the human virome, for the prediction of post-transplant complications such as severe infections or graft rejection. The complete genetic characterization of TTV is poorly understood and constantly evolving due to its high degree of diversity. To overcome this challenge, the aim of the present study is to characterize the TTV species profiles in transplanted patients by comparing the gold standard method of metagenomic next-generation sequencing (mNGS) with a newly developed TTV amplicon-based NGS (TTV-NGS). The final objective is to assess the complexity and diversity of the TTV virome in a larger study population.MATERIALS AND METHODS: To compare two different NGS approaches, the gold standard mNGS and our in-house TTV-amplicon based approach (TTV-NGS), 20 plasma samples (10 liver transplant, 10 kidney transplant) were analyzed. A negative control was also included. Bioinformatic analysis of sequences was performed using an in-house bioinformatics pipeline. In addition, to determine the genetic diversity and prevalence of TTV, a larger population was examined. An additional 66 (52 kidney transplanted patients and 14 healthy subjects) from various geographical regions (Italy, Spain, Czech republic, Austria and Argentina) were added, reaching 86 patients. This analysis was conducted using our TTV-NGS method. The flowchart on the right shows a brief overview of the workflow.RESULTS: Point 1. Comparison of TTV-NGS and mNGS sequencing methods in 20 transplanted patients: the number of TTV genomes identified using the TTV-NGS sequencing method is significantly higher (209) than in mNGS sequencing (58) (1.A). Estimation of the degree of nucleotide identity of TTV-assembled genomes by both sequencing methods: high degree of identity between TTV-assembled isolates from the same patient by both methods was observed (mean 84.3%, 1.6% SEM), shown diagonally.In addiction, there is a moderate degree of nucleotide identity between the same TTV species from different samples (1.B). Point 2. The most common genus is TTV (2.A), in addition, many coinfections with TTMV have been observed. Only a few cases presented with exclusive TTMV infection. None showed exclusive TTMDV infection, which was only observed in association with TTV. The prevalence of TTV species was then evaluated, taking into consideration their geographical origin. Principal component analysis (PCA) did not reveal any significant variation in species composition across different geographical areas (2.B). Lastly, the estimated prevalence of different TTV species is showed (2.C). Heatmap displays the logarithm values of TTV species' mapped reads per million (log RPM) and the percentage of prevalence in the study population. There was no observed correlation between viral load and the number of TTV species (data not shown).CONCLUSIONS: The TTV-NGS method showed higher sensitivity than mNGS. It was able to improve the classification of TTV, especially in samples with low viral load. Furthermore, the prevalence of other anelloviruses was also assessed. TTMV (genus Betatorquevirus) was found in several patients, while TTMDV (genus Gammatorquevirus) was only present in a few cases. This study represents the first comparative analysis of different molecular approaches to characterize the TTV-virome. The novel in-house TTV-NGS method, based on amplicon sequencing, may represent a useful and simple tool for studying the dynamic viral composition of this so widespread but still poorly understood virus in its enormous genetic variability.