Abstract
BackgroundViruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias.ResultsWe assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 μm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 μm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies.ConclusionsBenchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes.
Highlights
Viruses are key players regulating microbial ecosystems
Low-speed centrifugation, filtration and random amplification methods alter composition of mock viral communities To evaluate the potential effect of purification and random amplification methods on metagenomic studies of viruses, we prepared two mock viral communities (Table 1), each composed of seven DNA viruses with different morphologic and genetic features
We avoided the use of bacteria as indicator of contamination because the protocol included two consecutive low-speed centrifugation steps and 0.45 μm filtration that reduced the number of colonies in pure cultures of Escherichia coli, Staphylococcus aureus and Roseobacter litoralis by at least 7–8 logarithmic units (Additional file 4: Table S4)
Summary
Viruses are key players regulating microbial ecosystems. Exploration of viral assemblages is possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. A variety of physical virus-like particle (VLP) enrichment protocols has been employed to increase their relative ratio, enabling deep exploration of viral assemblages [17, 18, 20,21,22,23,24,25] Most of these protocols combine low-speed centrifugation, 0.22–0.45 μm filtration, or ultracentrifugation in density gradients to remove cellular contamination and concentrate VLPs, with nuclease treatment for elimination of unprotected genetic material. Some studies propose that both strategies efficiently reduce bacterial contamination in host-associated samples [35,36,37], while 0.22 μm filtration diminished viral DNA yields recovered from human faeces by half in comparison to the use of 0.45 μm filters [38]. This may be explained, at least in part, by the filter retention of giant eukaryotic viruses such as those recently found in human samples [39, 40], or large bacteriophages [41]
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