DNA Viral Size Fraction Metagenomics for Human Stool Samples.

Area of Science:

• Microbiology
• Virology
• Molecular Biology

Background:

• The human virosphere, comprising viruses in the microbiome, is crucial for understanding health.
• Accurate characterization of viral communities requires robust DNA extraction methods.
• Existing methods may not be universally applicable across diverse laboratory settings.

Purpose of the Study:

• To develop and present a versatile laboratory protocol for enriching and extracting DNA from extracellular DNA viruses in human stool.
• To provide a flexible framework adaptable to various equipment and resource availabilities.
• To facilitate accurate measurements of viral community composition in the human virosphere.

Main Methods:

• The protocol involves sample lysis with a buffer (e.g., protein-enhanced phosphate buffered saline), centrifugation, and filtration (e.g., 0.2 µm) to isolate viral particles.
• Viral particle concentration (e.g., ultracentrifugation) and nuclease treatment to remove free nucleic acids precede virion lysis.
• DNA extraction follows lysis, preparing samples for sequencing.

Main Results:

• A primary protocol for human stool viromic DNA extraction is demonstrated.
• Multiple options and deviations are provided for different steps, enhancing protocol flexibility.
• The protocol is designed for broad applicability across laboratories with standard molecular biology equipment.

Conclusions:

• The presented protocol offers a adaptable approach for high-quality viromic DNA extraction from human stool.
• Flexibility in methods allows for customization based on available resources.
• This work supports more accurate measurements of the human virosphere, advancing microbiome research.