Adams, S. H. | bioRxiv Science

bioRxiv2025

DNA extraction and virome processing methods strongly influence recovered human gut viral community characteristics

Accurately characterising the human gut virome is critical to understanding virus-microbiome-host interactions. However, widely used methods introduce biases that complicate data interpretation and limit cross-study comparability. For instance, multiple-displacement amplification (MDA) preferentially amplifies single-stranded DNA viruses, while total metagenomes are dominated by non-viral sequences, reducing viral signal. These traditional methods have not been systematically compared to viral size-fraction metagenomes (viromes) prepared without MDA. To address this, we applied four common methods for characterising human gut viral community composition (total metagenomes, viromes with/ without DNase treatment (to remove free DNA), and MDA viromes) to a human stool sample, with technical triplicates for each approach. MDA biased viral community composition to a shocking degree: Microviridae formed [~]90% of MDA viromes compared to just 2% of non-MDA viromes. Removing ssDNA viruses from data analyses substantially reduced, but did not eliminate, MDA bias. Metagenomes were enriched for putative temperate phages and predicted Bacillota-phages, whereas predicted Bacteroidetes-phages dominated all viromes, suggesting that metagenomes and viromes select for different populations within the total viral community. DNase treatment had little-to-no effect on virome richness or community composition. This proof-of-principle experiment demonstrates that preparatory methods for viral community analysis can lead to substantially different conclusions from the same faecal sample, and we provide a comprehensive omic data analysis framework for comparing laboratory methodologies for viral ecology. With sufficient DNA yields now easily achievable from human gut viromes without the use of MDA, our results suggest that this biased amplification method should be avoided in human gut virome studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/690293v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@36252org.highwire.dtl.DTLVardef@2c06f0org.highwire.dtl.DTLVardef@7b7cceorg.highwire.dtl.DTLVardef@13ec804_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

microbiology↗

bioRxiv2018

Metaproteomics Reveals Potential Mechanisms by which Dietary Resistant Starch Supplementation Attenuates Chronic Kidney Disease Progression in Rats

BackgroundResistant starch is a prebiotic metabolized by the gut bacteria. It has been shown to attenuate chronic kidney disease (CKD) progression in rats. Previous studies employed taxonomic analysis using 16S rRNA sequencing and untargeted metabolomics profiling. Here we expand these studies by metaproteomics, gaining new insight into the host-microbiome interaction.\n\nMethodsDifferences between cecum contents in CKD rats fed a diet containing resistant starch with those fed a diet containing digestible starch were examined by comparative metaproteomics analysis. Taxonomic information was obtained using unique protein sequences. Our methodology results in quantitative data covering both host and bacterial proteins.\n\nResults5,834 proteins were quantified, with 947 proteins originating from the host organism. Taxonomic information derived from metaproteomics data surpassed previous 16S RNA analysis, and reached species resolutions for moderately abundant taxonomic groups. In particular, the Ruminococcaceae family becomes well resolved - with butyrate producers and amylolytic species such as R. bromii clearly visible and significantly higher while fibrolytic species such as R. flavefaciens are significantly lower with resistant starch feeding. The observed changes in protein patterns are consistent with fiber-associated improvement in CKD phenotype. Several known host CKD-associated proteins and biomarkers of impaired kidney function were significantly reduced with resistant starch supplementation. Data are available via ProteomeXchange with identifier PXD008845.\n\nConclusions- Metaproteomics analysis of cecum contents of CKD rats with and without resistant starch supplementation reveals changes within gut microbiota at unprecedented resolution, providing both functional and taxonomic information. Proteins and organisms differentially abundant with RS supplementation point toward a shift from mucin degraders to butyrate producers.

systems biology↗