Benchmarking protocols for the metagenomic analysis of stream biofilm viromes.

Meriem Bekliz,Tom J Battin,Hannes Peter,Jade Brandani,Massimo Bourquin

doi:10.7717/peerj.8187

Abstract

Viruses drive microbial diversity, function and evolution and influence important biogeochemical cycles in aquatic ecosystems. Despite their relevance, we currently lack an understanding of their potential impacts on stream biofilm structure and function. This is surprising given the critical role of biofilms for stream ecosystem processes. Currently, the study of viruses in stream biofilms is hindered by the lack of an optimized protocol for their extraction, concentration and purification. Here, we evaluate a range of methods to separate viral particles from stream biofilms, and to concentrate and purify them prior to DNA extraction and metagenome sequencing. Based on epifluorescence microscopy counts of viral-like particles (VLP) and DNA yields, we optimize a protocol including treatment with tetrasodium pyrophosphate and ultra-sonication to disintegrate biofilms, tangential-flow filtration to extract and concentrate VLP, followed by ultracentrifugation in a sucrose density gradient to isolate VLP from the biofilm slurry. Viromes derived from biofilms sampled from three different streams were dominated by Siphoviridae, Myoviridae and Podoviridae and provide first insights into the viral diversity of stream biofilms. Our protocol optimization provides an important step towards a better understanding of the ecological role of viruses in stream biofilms.

Highlights

Viruses are the smallest and most abundant biological entities on Earth, typically outnumbering their prokaryotic and eukaryotic hosts by an order of magnitude (Rohwer, 2003; Sime-Ngando, 2014)
Protocols for the extraction of viral-like particles (VLP) In order to establish an optimized stream biofilm sample-to-sequence pipeline, we explored a variety of protocols for the concentration, extraction and purification of VLP (Fig. 1)
Biofilm properties Bacterial abundance ranged between 4.1 × 1011 cells m−2 at the lowest stream (SNG) and 2.3 × 109 cells m−2 at the uppermost stream (VDN; Table 1)

Summary

Introduction

Viruses are the smallest and most abundant biological entities on Earth, typically outnumbering their prokaryotic and eukaryotic hosts by an order of magnitude (Rohwer, 2003; Sime-Ngando, 2014). Many of these strides have only recently been possible with the advent of molecular tools such as metagenomic sequencing (Rosario & Breitbart, 2011; Brum & Sullivan, 2015; Roux et al, 2016, 2017). One may differentiate between untargeted approaches, which have produced a wealth of viral sequences (Paez-Espino et al, 2016) and virome sequencing, in which the viral fraction is purified prior to sequencing, ensuring that the sequencing effort is targeted towards viral nucleic acids (Rosario & Breitbart, 2011)

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