Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent.

Area of Science:

• Virology
• Genomics
• Molecular Diagnostics

Background:

• Unbiased metagenomic sequencing offers potential for simultaneous detection of diverse viral nucleic acids in clinical samples.
• Viral genome sequences can predict phenotypes like drug susceptibility and neutralization serotypes.

Purpose of the Study:

• To compare laboratory method variables for optimizing viral metagenomics library preparation.
• To assess the impact of filtration, nuclease digestion, extraction methods, pre-amplification, and library kits on viral detection and genome coverage.

Main Methods:

• A biological reagent with 25 human viral pathogens was used to test different laboratory procedures.
• Variables included filtration, nuclease digestion, DNA/RNA extraction (silica spin columns, magnetic beads, Trizol), random RT-PCR pre-amplification, and library preparation kits (ScriptSeq, TruSeq, Nextera).

Main Results:

• Filtration and nuclease treatment slightly decreased viral reads and detection.
• Silica spin columns outperformed magnetic beads and Trizol for viral nucleic acid extraction.
• Pre-amplification increased viral reads but reduced virus detection and genome coverage.
• ScriptSeq yielded more viruses and greater genome coverage compared to TruSeq and Nextera.

Conclusions:

• Viral metagenomics successfully detected up to 22 viruses simultaneously in the tested reagent.
• Optimized methods, including ScriptSeq and silica spin columns, enhance viral detection capabilities.
• Further refinement is needed for complex biological samples like tissues and blood.