Sample and library preparation approaches for the analysis of the virome of irrigation water.

BACKGROUND

The virome (i.e. community of mainly RNA and DNA eukaryotic viruses and bacteriophages) of waters is yet to be extensively explored. In particular, the virome of waters used for irrigation could therefore potentially carry viral pathogens that can contaminate fresh produce. One problem in obtaining viral sequences from irrigation waters is the relatively low amount of virus particles, as well as the presence of human, bacterial and protozoan cells. The present aimed study was to compare different processing, amplification, and sequencing approaches for virome characterization in irrigation waters.

RESULTS

Our analyses considered percentages of viral reads, values for diversity indices and number of families found in sequencing results. The results obtained suggest that enrichment protocols using two (bezonase and microccocal nuclease) or four enzymes at once (bezonase, microccocal nuclease, DNAse and RNase), regardless of an Amicon filtration step, are more appropriate than separated enzymatic treatments for virome characterization in irrigation water. The NetoVIR protocol combined with the ScriptSeq v2 RNA-Seq Library (P0-L20 protocol) showed the highest percentages of RNA viruses and identified the higher number of families.

CONCLUSION

Although virome characterization applied in irrigation waters is an important tool for protecting public health by informing on circulating human and zoonotic infections, optimized and standardized procedures should be followed to reduce the variability of results related to either the sample itself and the downstream bioinformatics analyses. Our results show that virome characterization can be an important tool in the discovery of pathogenic viruses in the environment and can be used to inform and optimize reference-based detection methods provided that appropriate and rigorous controls are included. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.