Bias of library preparation for virome characterization in untreated and treated wastewaters

Gloria Sánchez,Alba Pérez-Cataluña,Enric Cuevas-Ferrando,Walter Randazzo

doi:10.1016/j.scitotenv.2020.144589

Abstract

The use of metagenomics for virome characterization and its implementation for wastewater analyses, including wastewater-based epidemiology, has increased in the last years. However, the lack of standardized methods can led to highly different results. The aim of this work was to analyze virome profiles in upstream and downstream wastewater samples collected from four wastewater treatment plants (WWTPs) using two different library preparation kits. Viral particles were enriched from wastewater concentrates using a filtration and nuclease digestion procedure prior to total nucleic acid (NA) extraction. Sequencing was performed using the ScriptSeq v2 RNA-Seq (LS) and the NEBNext Ultra II RNA (NB) library preparation kits. Cleaned reads and contigs were annotated using a curated in-house database composed by reads assigned to viruses at NCBI. Significant differences in viral families and in the ratio of detection were shown between the two library kits used. The use of LS library showed Virgaviridae, Microviridae and Siphoviridae as the most abundant families; while Ackermannviridae and Helleviridae were highly represented within the NB library. Additionally, the two sequencing libraries produced outcomes that differed in the detection of viral indicators. These results highlighted the importance of library selection for studying viruses in untreated and treated wastewater. Our results underline the need for further studies to elucidate the influence of sequencing procedures in virome profiles in wastewater matrices in order to improve the knowledge of the virome in the water environment.

Highlights

The reuse of water, including for irrigation, cooling, and other non-potable applications is an emerging topic due to climate change and water scarcity
Treatment and regeneration of household sewage water in urban regions are usually performed by wastewater treatment plants (WWTPs); they are not always able to completely eliminate the microbiological risks present in treated wastewaters (Chalmers et al, 2010; Randazzo et al, 2019; Sano et al, 2016)
Fecal bacteria have traditionally been used as indicators for the presence of pathogenic microorganisms even though they fail to detect the presence of human pathogenic enteric viruses (Eslamian, 2016; Gerba et al, 2013; Kitajima et al, 2014)

Summary

Introduction

The reuse of water, including for irrigation, cooling, and other non-potable applications is an emerging topic due to climate change and water scarcity. Several viruses (i.e. crAssphage, Pepper mild mottle virus, adenovirus, polyomavirus, ...) have been proposed as indicators because of their similarity to pathogenic viruses in terms of environmental stability and resistance to wastewater sanitation treatments (Farkas et al, 2020). The presence of human enteric viruses in treated wastewaters has been well documented (Gerba et al., 2018; Randazzo et al, 2019; Sano et al, 2016), posing public health risk-related concerns because of their stability into the environment. The viruses most commonly detected in untreated and treated wastewaters include human norovirus, adenovirus (AdV), enterovirus (EV), sapovirus (SaV), astrovirus (HAstV), rotavirus A (RV), and hepatitis A and E viruses (HAV and HEV)

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