ArticlemSystems2026
Optimizing methods for virome analysis based on studies of a synthetic viral community.
Article in mSystems, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Studies of whole viral populations-the "virome"-are yielding exciting new insights into biological systems, but methods are still being optimized. Here, we describe generation and use of a synthetic viral community and its use to evaluate technical challenges arising in virome analysis. We spiked the mock community into different human sample types, then passed the samples through different virus enrichment protocols and analyzed by Illumina sequencing. Compared with direct metagenomic sequencing, VLP enrichment protocols greatly increased viral read yields from stool and saliva. Four methods for DNA amplification were compared, with three showing over-amplification of small circular ssDNA viruses, most notably GenomiPhi. Studies of viral particle stability in the presence of nuclease showed that most viral genomes were stable when protected in viral particles, but phage MS2 RNA was unexpectedly labile under some of the conditions tested. Comparison of Illumina 1,000-cycle sequencing versus 300-cycle sequencing showed that longer reads supported generation of longer viral genome assemblies. We tested bacteriophage T4 DNA modified with glucosyl-hydroxymethylcytosine (ghmC) and hydroxymethylcytosine (hmC) and found that both were readily detected, though the recovery of ghmC-modified DNA was reduced compared with T4 genomes with unmodified cytosine. These studies together with published data help provide guidance for virome researchers optimizing analytical protocols.IMPORTANCEA challenge in characterizing the human virome in health and disease is identifying optimal methods for enriching the viral content of samples. Due to the tremendous abundance and diversity of viruses, capturing as broad of a range of viruses as possible for analysis is difficult and potentially complicated by unrecognized biases. This report presents the use of a synthetic viral community for methods optimization in virome studies and illustrates the feasibility and challenges of current virus enrichment strategies for high-throughput virome analysis of different human sample types.
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