Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

Area of Science:

• Microbiology
• Virology
• Bioinformatics

Background:

• The human gut harbors a complex viral community, the virome, primarily composed of bacteriophages.
• The virome is poorly characterized due to high genetic diversity and limited tools, often termed "viral dark matter".
• Study design, including sequencing and library preparation, significantly impacts virome characterization.

Purpose of the Study:

• To compare PCR and PCR-free library preparation methods for human fecal virome characterization.
• To assess the impact of these methods on the recovery of viral genomes and diversity metrics.

Main Methods:

• Viral DNA extraction from fecal samples of three healthy donors.
• Sequencing using the Illumina platform with both PCR and PCR-free library preparation.
• Comparative analysis of sequence data, focusing on viral operational taxonomic units (vOTUs) and diversity indices.

Main Results:

• Most variation in the virome reflected individual-specific differences.
• PCR-free methods showed improved recovery of low-abundance viral genomes compared to PCR methods.
• PCR amplification reduced alpha-diversity indices (Chao1 and Simpson), indicating a loss of rare vOTUs.

Conclusions:

• Methodological choices, specifically PCR vs. PCR-free library preparation, significantly influence gut virome studies.
• PCR-free methods are recommended for accurately characterizing low-abundance viruses and rare members of the gut virome.
• Biases introduced by PCR are likely negligible for abundant viruses but critical for comprehensive virome analysis.