Synthetic virome mock community, human samples, Philadelphia

Studies of whole viral populations--the "virome"--are yielding exciting new</p><p>insights into biological systems, but methods are still being optimized. Here we</p><p>describe generation and use of a synthetic viral community to assess several</p><p>technical challenges important in virome analysis. Our mock community was</p><p>comprised of phages lambda, T4, M13, MS2, and phi6, together with adenoassociated virus (AAV), murine hepatitis virus (MHV), and vaccinia virus (VV).</p><p>We spiked the mock community into different human sample types, including</p><p>stool, saliva, oropharyngeal (OP) wash, and bronchoalveolar lavage (BAL), then</p><p>passed the samples through different virus enrichment protocols and analyzed by</p><p>Illumina sequencing. Compared to direct metagenomic sequencing, VLP</p><p>enrichment protocols greatly increased viral read yields from virus-rich samples</p><p>such as from stool and saliva. Three VLP enrichment work flows were compared,</p><p>and each was found to have strengths and weaknesses. Four methods for DNA</p><p>amplification were compared, with three showing over-amplification of small</p><p>circular ssDNA viruses, most notably GenomiPhi. Studies of viral particle stability</p><p>in the presence of nuclease showed that most viral genomes were stable when</p><p>protected in viral particles, but phage MS2 RNA was unexpectedly labile under</p><p>some of the conditions tested. Comparison of Illumina 1000-cycle sequencing</p><p>versus 300-cycle sequencing showed that longer reads supported generation of</p><p>longer viral genome assemblies. Bacteriophage DNA can be modified by at least</p><p>12 different chemistries, raising the question of whether these modifications</p><p>might block recovery in virome analytical protocols. We tested bacteriophage T4 DNA modified with glucosyl-hydroxymethylcytosine (ghmC) and</p><p>hydroxymethylcytosine (hmC), and found that both were readily detected, though</p><p>the recovery of ghmC-modified DNA was reduced. These studies together with</p><p>published data help provide guidance for virome researchers optimizing</p><p>analytical protocols.

Classified as human gut / stool / oral cavity / lower respiratory shotgun DNA metagenome of age unknown by opus_adjudicated with confidence 0.55. Assay shotgun_dna.

• host study.description · “spiked the mock community into different human sample types”
• body site study.description · “stool, saliva, oropharyngeal (OP) wash”
• body site study.description · “bronchoalveolar lavage (BAL)”

323 runs over 323 samples · mean 0.69 Gbp per run (median 0.33 Gbp, total 222.73 Gbp; 323 runs with a base count) · layout PAIRED 323 · platform ILLUMINA 323 · models NextSeq 2000 (323) · 12% of runs with a Sandpiper profile · first public 2025-10-27 – 2026-02-07

Duan J first · Marques AD · Hogenauer M · Hwang Y · Zhang Y · Timperman A · Higgins S · Wilson NG · Fitts EA · Lim HK · Bittinger K · Moustafa AM · Collman RG · Bushman FD last

3 of the 4 core fields are below 50% coverage: age_category, health_condition, subject_id. Share metadata

PRJNA1348896.csv.gz (7.8 KB) · parquet (80.9 KB) — all 323 samples, every field with route and confidence · PRJNA1348896_determinations.csv.gz (10.4 KB) — 1,938 evidence rows