Benchmarking of a time-saving and scalable protocol for the extraction of DNA from diverse viromes

Abstract

The virome, composed of viruses inhabiting diverse ecosystems, significantly influences microbial community dynamics and host health. The phenol-chloroform DNA extraction protocol for viromes, though effective, is time-intensive and requires the use of multiple toxic chemicals. This study introduces a streamlined, scalable protocol for DNA extraction using a commercially-available kit as an alternative, assessing its performance against the phenol-chloroform method across human fecal, mouse fecal, and soil samples. No significant differences in virome diversity or community composition were seen between methods. Most viral operational taxonomic units (vOTUs) were common to both methods, with only a small percentage unique to either approach. Alpha- and beta-diversity analyses showed no significant impact of the extraction method on virome composition, confirming the kit's efficacy and versatility on sample types beyond those officially supported by the manufacturer. While the kit approach offers benefits like reduced toxicity and increased throughput, it has limitations such as higher costs and potential issues reliably capturing low-abundance taxa. This protocol provides a viable option for large-scale virome studies, although the phenol-chloroform approach may still be preferable for specific sample types.

Keywords: Bacteriophage; DNA extraction; Human gut microbiota; Soil; Virome.

Figure 1. Overview of sample processing pipeline.

All samples underwent resuspension in sterile PBS, filtration to remove debris and bacterial or eukaryotic cells, chloroform treatment, and DNase treatment (with subsequent DNase inactivation). Samples were then split to be processed with either the phenol-chloroform extraction (PC) or QIAGEN QIAamp MinElute Virus Spin Kit (KIT) DNA extraction protocols. DNA libraries were prepared with the Illumina DNA Prep kit and sequenced on an Illumina MiSeq instrument with 150 bp paired-end reads. Approximate sample processing times are indicated for each step. The KIT protocol increases sample throughput by reducing protocol complexity and minimizing the number of incubation steps. Created in BioRender (Maurice, 2024).

Figure 2. PC and KIT extraction methods result in equivalent virome communities.

(A) The number of vOTUs detected only within the phenol-chloroform (PC), or kit (KIT) extraction method, or shared between both methods. (B) Observed richness (left) and Shannon diversity (right) of viromes according to environment and extraction method. No statistical differences were observed between extraction methods within any environment (Wilcoxon signed-rank test, p > 0.05). (C) Non-metric multidimensional scaling (NMDS) of Bray-Curtis distances of vOTUs between samples, colors representing sample environment and shapes representing extraction method (NMDS stress = 9.4 × 10⁻⁵). Tests for significant differences in Bray-Curtis dissimilarity were conducted using PERMANOVA (adonis2) with 999 permutations, and summary statistics are reported in the table.