Abstract
A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.
Highlights
Generation sequencing (NGS) has revolutionised the discovery of novel viruses in humans and animals in various ecosystems[9,10,11]
Our study individually evaluated the performance of each step of the procedure and systematically studied their effect on a unique mock-virome an artificial bacterial community, as well as on 16S rRNA to investigate bias introduced during each step and to obtain the most favourable virus-to-bacteria ratio
In this study we investigated bias that can be introduced during various sample treatment procedures commonly used in viral metagenomics studies on artificial, well-characterised communities of viruses and bacteria
Summary
Generation sequencing (NGS) has revolutionised the discovery of novel viruses in humans and animals in various ecosystems[9,10,11]. Key-steps for studying viromes are the enrichment for virus-like-particles (VLPs) from a sample and the performance of random amplification, if the starting material needs to be increased before NGS library preparation[16] These steps should preferentially avoid losses of any types of viruses and minimise bias introduced during sample preparation steps. Li and colleagues pooled samples (allantoic fluid, cell culture and faecal material) containing twenty five human viruses to compare different purification and amplification methods[19] Their protocol was not able to recover all viruses and showed that recovery of viral reads was highly diverse when different sample preparation methods were used. The modular approach of our study allows researchers to customise sample preparation depending on their needs or particular virus(es) of interest
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