Discovering and exploring the hidden diversity of human gut viruses using highly enriched virome samples

Abstract

Viruses are an abundant and crucial component of the human microbiome, but accurately discovering them via metagenomics is still challenging. Currently, the available viral reference genomes poorly represent the diversity in microbiome samples, and expanding such a set of viral references is difficult. As a result, many viruses are still undetectable through metagenomics even when considering the power of de novo metagenomic assembly and binning, as viruses lack universal markers. Here, we describe a novel approach to catalog new viral members of the human gut microbiome and show how the resulting resource improves metagenomic analyses. We retrieved >3,000 viral-like particles (VLP) enriched metagenomic samples (viromes), evaluated the efficiency of the enrichment in each sample to leverage the viromes of highest purity, and applied multiple analysis steps involving assembly and comparison with hundreds of thousands of metagenome-assembled genomes to discover new viral genomes. We reported over 162,000 viral sequences passing quality control from thousands of gut metagenomes and viromes. The great majority of the retrieved viral sequences (~94.4%) were of unknown origin, most had a CRISPR spacer matching host bacteria, and four of them could be detected in >50% of a set of 18,756 gut metagenomes we surveyed. We included the obtained collection of sequences in a new MetaPhlAn 4.1 release, which can quantify reads within a metagenome matching the known and newly uncovered viral diversity. Additionally, we released the viral database for further virome and metagenomic studies of the human microbiome.

EnVhog: an extended view of the viral protein space on Earth

“…Virome proteins were also compared to PHROG and EnVhog HMMs using HHBlits (e-value<1e-3). To evaluate the results obtained using more sensitive searches against EnVhog, the 1,159,978 complete proteins (prodigal; parameters -c -m -p meta) from human gut viral contigs (Zolfo et al 2024) were grouped into clusters (mmseqs; >70% of bidirectional coverage). For each of the 65,409 clusters, a multiple sequence alignment was computed (MMSeqs2 result2msa) and the corresponding HMM was compared to enVhog HMMs (HHblits; E-value<1e-3).…”

“…It should be noted that for the annotation of the latter dataset, its proteins were first clustered, with the resulting cluster HMMs being compared to the EnVhog HMMs. The same methodology was then applied to a set of human intestinal viral genomes (Zolfo et al 2024). Thanks to this more sensitive strategy, a homolog in EnVhog could be identified for over 97% of the proteins in these genomes.…”