Next Generation Sequencing for the Analysis of Parvovirus B19 Genomic Diversity

Parvovirus B19 (B19V) is a ssDNAhuman virus, responsible for an ample range of clinical 11manifestations. Sequencing of B19V DNA from clinical samples is frequently reported in the litera-12ture, to assign genotype (genotypes 1-3) and for finer molecular epidemiological tracing. The in-13creasing availability of Next Generation Sequencing (NGS) with its depth of coverage potentially 14yields information on intrinsic sequence heterogeneity, however integration of this information in 15analysis of sequence variation is not routinely obtained. The present work investigated genomic 16sequence heterogeneity within and between B19V isolates, by application of NGS techniques, and 17by the development of a novel dedicated bioinformatic tool and analysis pipeline, yielding infor-18mation on two newly defined parameters. The first, α-diversity, is a measure of the amount and 19distribution of position-specific, normalised Shannon Entropy as a measure of intra-sample se-20quence heterogeneity. The second, σ-diversity, is a measure of the amount of inter-sample sequence 21heterogeneity, also incorporating information on α-diversity. Based on these indexes, further cluster 22analysis can be performed. A set of 24 high-titre viremic samples was investigated. Of these, 23 23samples were genotype 1 and onesample was genotype 2. Genotype 1 isolates showed low α-di-24versity values, only a few samples showing distinct position-specific polymorphisms; a few genet-25ically related clusters emerged when analysing inter-sample distances, correlated to the year of iso-26lation; the single genotype 2 isolate showed the highest α-diversity, even if not presenting polymor-27phisms, and was an evident outlier when analysing inter-sample distance. In conclusion, NGS anal-28ysis and the bioinformatic tool and pipeline developed and used in the present work can be consid-29ered an effective tool for investigating sequence diversity, an observable that can be incorporated 30into the quasispecies theory framework to yield a better insight into viral evolution dynamics.