2025-10-24_Duan_Optimizing-Methods-for-Virome

Studies of whole viral populations--the "virome"--are yielding exciting newinsights into biological systems, but methods are still being optimized. Here wedescribe generation and use of a synthetic viral community to assess severaltechnical challenges important in virome analysis. Our mock community wascomprised of phages lambda, T4, M13, MS2, and phi6, together with adenoassociated virus (AAV), murine hepatitis virus (MHV), and vaccinia virus (VV).We spiked the mock community into different human sample types, includingstool, saliva, oropharyngeal (OP) wash, and bronchoalveolar lavage (BAL), thenpassed the samples through different virus enrichment protocols and analyzed byIllumina sequencing. Compared to direct metagenomic sequencing, VLPenrichment protocols greatly increased viral read yields from virus-rich samplessuch as from stool and saliva. Three VLP enrichment work flows were compared,and each was found to have strengths and weaknesses. Four methods for DNAamplification were compared, with three showing over-amplification of smallcircular ssDNA viruses, most notably GenomiPhi. Studies of viral particle stabilityin the presence of nuclease showed that most viral genomes were stable whenprotected in viral particles, but phage MS2 RNA was unexpectedly labile undersome of the conditions tested. Comparison of Illumina 1000-cycle sequencingversus 300-cycle sequencing showed that longer reads supported generation oflonger viral genome assemblies. Bacteriophage DNA can be modified by at least12 different chemistries, raising the question of whether these modificationsmight block recovery in virome analytical protocols. We tested bacteriophage T4 DNA modified with glucosyl-hydroxymethylcytosine (ghmC) andhydroxymethylcytosine (hmC), and found that both were readily detected, thoughthe recovery of ghmC-modified DNA was reduced. These studies together withpublished data help provide guidance for virome researchers optimizinganalytical protocols.