Optimizing methods for virome analysis based on studies of a synthetic viral community

Duan, J.; Marques, A. D.; Hogenauer, M.; Hwang, Y.; Zhang, Y.; Timperman, A.; Higgins, S.; Wilson, N. G.; Fitts, E. A.; Lim, H. K.; Bittinger, K.; Moustafa, A. M.; Collman, R. G.; Bushman, F. D.

doi:10.1101/2025.10.23.683462

bioRxiv · 10.1101/2025.10.23.683462

Optimizing methods for virome analysis based on studies of a synthetic viral community

Duan, J.·Marques, A. D.·Hogenauer, M.·Hwang, Y.·Zhang, Y.·Timperman, A.·Higgins, S.·Wilson, N. G.·Fitts, E. A.·Lim, H. K.·Bittinger, K.·Moustafa, A. M.·Collman, R. G.·Bushman, F. D.

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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 to assess several technical challenges important in virome analysis. Our mock community was comprised of phages lambda, T4, M13, MS2, and phi6, together with adeno-associated virus (AAV), murine hepatitis virus (MHV), and vaccinia virus (VV). We spiked the mock community into different human sample types, including stool, saliva, oropharyngeal (OP) wash, and bronchoalveolar lavage (BAL), then passed the samples through different virus enrichment protocols and analyzed by Illumina sequencing. Compared to direct metagenomic sequencing, VLP enrichment protocols greatly increased viral read yields from virus-rich samples such as from stool and saliva. Three VLP enrichment work flows were compared, and each was found to have strengths and weaknesses. 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 1000-cycle sequencing versus 300-cycle sequencing showed that longer reads supported generation of longer viral genome assemblies. Bacteriophage DNA can be modified by at least 12 different chemistries, raising the question of whether these modifications might block recovery in virome analytical protocols. 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. These studies together with published data help provide guidance for virome researchers optimizing analytical protocols.

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Duan, J., Marques, A. D., Hogenauer, M., Hwang, Y., Zhang, Y., Timperman, A., Higgins, S., Wilson, N. G., Fitts, E. A., Lim, H. K., Bittinger, K., Moustafa, A. M., Collman, R. G., Bushman, F. D.. 2025-10-23. Optimizing methods for virome analysis based on studies of a synthetic viral community. https://doi.org/10.1101/2025.10.23.683462

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