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

Authors :
Shen-Yuan Hsieh
Mohammad A. Tariq
Andrea Telatin
Rebecca Ansorge
Evelien M. Adriaenssens
George M. Savva
Catherine Booth
Tom Wileman
Lesley Hoyles
Simon R. Carding
Source :
Viruses, Vol 13, Iss 10, p 2093 (2021)
Publication Year :
2021
Publisher :
MDPI AG, 2021.

Abstract

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

Details

Language :
English
ISSN :
19994915
Volume :
13
Issue :
10
Database :
Directory of Open Access Journals
Journal :
Viruses
Publication Type :
Academic Journal
Accession number :
edsdoj.58e25ecd5fd247dba2d66cfcabb3f044
Document Type :
article
Full Text :
https://doi.org/10.3390/v13102093