DNA Viral Size Fraction Metagenomics for Human Stool Samples

Summary

We present a flexible protocol for generating high-quality viral size fraction metagenomes (viromes) from human stool samples. Originally developed for soil viromics, here this protocol was successfully applied to stool samples, enabling robust characterization of the human gut virosphere.

Abstract

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.

Introduction

Viromics, or the untargeted metagenomic sequencing of the viral size fraction of mixed communities, can provide insights into the diversity, evolution, and ecology of uncultured viruses. Since the early efforts to catalog gut viral communities over 20 years ago, which notably used methods developed for environmental samples like seawater¹, viromics approaches have continued to enable discoveries in viral ecology within the human microbiome². However, there is no single universal viromics protocol, and variations in techniques can impact sequencing results^3,4, ult....

Protocol

All subjects recruited for stool sample collection provided informed consent. Sample collection protocols were approved by the University of California, Davis Institutional Review Board, and the studies conform to the Declaration of Helsinki. This protocol is adapted from Emerson et al. (2022)¹⁷.

1. Preparation of materials

1. Prepare protein-enhanced phosphate-buffered saline (PPBS, 10x PBS, 1% K-citrate, 150 mM MgSO₄, 2% bovine serum albumin (BSA))¹⁸.
   ​NOTE: Other commonly used buffers in viromics protocols are saline magnesium (SM)

Results

An exploratory effort applying this protocol to a single frozen stool sample (processed in technical triplicate for viromes) alongside two soil samples demonstrated its efficacy in producing high-quality viromes from different sample types. Using quality-filtered sequencing data and de novo assembled contigs generated by this protocol, putative viral sequences were identified using an open-source virus identification tool²⁰. Reads mapped to the identified viral contigs were then .......

Discussion

This protocol presents a relatively straightforward and adaptable method for generating viromes. Proven in soils^14,15,16,21, it is successfully applied here to human stool samples. This protocol will best recover extracellular, intact DNA viruses and is not designed to intentionally recover intracellular viruses or prophage (though some may be recovered from contaminating cells), very large DNA.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA) DNase- and Protease-free Powder	Fisher BioReagents	BP9706100	For PPBS recipe (Step 1.1)
DNeasy PowerSoil Pro Kit	QIAGEN	47016	DNA extraction kit (Step 4.1)
KAPA DNA HyperPrep library kit	Roche	n/a	Libraries generated by this kit were sequenced using the Illumina NovaSeq 6000 platform (2 x 150 bp).
Magnesium sulfate (MgSO4), anhydrous, ≥99.5%	Sigma-Aldrich	M7506	For PPBS recipe (Step 1.1)
Phosphate-Buffered Saline (PBS), 10x, pH 7.4, RNase-free	Invitrogen	AM9624	For PPBS recipe (Step 1.1)
Polyethersulfone (PES) membrane syringe filters, sterile, 28 mm, 0.2 μm	Corning	431229	For syringe filtration (Step 2.9)
Polypropylene conical centrifuge tubes, sterile, 50 mL	Olympus Plastics	28-108
Potassium Citrate Monohydrate (Crystalline/Certified)	Fisher Chemical	P222	For PPBS recipe (Step 1.1)
Qubit  Assay Tubes	Invitrogen	Q32856	For DNA quantification (Step 5.1)
Qubit 4 Fluorometer	Invitrogen	Q33238	For DNA quantification (Step 5.1)
Qubit dsDNA Quantification Assay Kits	Invitrogen	Q32850	For DNA quantification (Step 5.1)
RQ1 RNase-Free DNase	Promega	PR-M6101	Includes the reaction buffer, DNase, and stop solution
Supor hydrophilic polyethersulfone (PES) membrane syringe filters, sterile, 32 mm, 5.00 µm	Cytiva (Formerly Pall Lab)	28150-956	For syringe filtration (Step 2.9)
Ultracentrifuge rotor: Type 50.2 Ti Fixed-Angle Rotor, 50.000 rpm, 302.000 x g, 12 x 39 m	Beckman Coulter	337901	We also use the Beckman Coulter Type 70 Ti Fixed-Angle Titanium Rotor (Cat. 337922). Centrifuge for 3 hours if using the 70 Ti.
Ultracentrifuge tubes: 26.3 mL Polycarbonate Bottle with Cap Assembly, 25 x 89mm	Beckman Coulter	355618	Compatible with Type 50.2 Ti (Cat. 337901) and Type 70 Ti (Cat. 337922) Beckman Coulter rotors
Ultracentrifuge: Beckman Instruments Optima LE-80K Floor Ultracentrifuge	Beckman Coulter	365668	We also use the Beckman Coulter L8-70M Refrigerated Ultracentrifuge (Cat. 344196)