DNA Viral Size Fraction Metagenomics for Human Stool Samples

Understanding the healthy human virosphere (the viral component of the microbiome) requires accurate measurements of viral community composition across a diverse range of viral types. Building on prior experience with soil viral community ecology methods, here we demonstrate a series of laboratory approaches for enriching and extracting DNA from extracellular DNA viruses in human stool samples. A working primary protocol is presented, along with options for deviations at different steps. The general approach involves adding a liquid buffer (default: protein-enhanced phosphate buffered saline, PPBS) to facilitate removal of free viral particles from the stool matrix, centrifugation to separate the liquid fraction containing viral particles, filtration (default: 0.2 µm pore size) to remove most cells, concentration of viral particles (default: ultracentrifugation), removal of free nucleic acids with nucleases prior to virion lysis, and then DNA extraction for sequencing. Alternative techniques, including different buffers, filter sizes, and concentration methods, are also noted. Overall, multiple options for generating high-quality viromic DNA for sequencing are offered. Rather than tailoring the approach to specific equipment and resources, the protocol's flexibility should make it broadly applicable across labs with varying standard molecular biology equipment.