Comparison of PCR versus PCR-Free DNA library preparation for characterising the human faecal virome

Hsieh, Shen-Yuan, Tariq, Mohammad A., Telatin, Andrea, Ansorge, Rebecca, Adriaenssens, Evelien M., Savva, George M., Booth, Catherine, Wileman, Tom, Hoyles, Lesley and Carding, Simon R. (2021) Comparison of PCR versus PCR-Free DNA library preparation for characterising the human faecal virome. Viruses, 13 (10). ISSN 1999-4915

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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 lowerabundance 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.

Item Type: Article
Additional Information: Funding Information: This work was supported in part by the UK Biotechnology and Biological Sciences Research Council (BBSRC) and BBSRC Institute Strategic Program grant BB/R012490/1 to the Gut Microbes and Health Research Programme and its constituent projects BBS/E/F/000PR10353 and BBS/E/F/000PR10356 410 (S.R.C.).We would like to acknowledge the assistance of Moreno Zolfo and Nicola Segata (University of Trento, Italy) with ViromeQC analysis and providing mapping data. We would also like to thank the donors who participated in this study. Funding Information: Funding: This work was supported in part by the UK Biotechnology and Biological Sciences Research Council (BBSRC) and BBSRC Institute Strategic Program grant BB/R012490/1 to the Gut Microbes and Health Research Programme and its constituent projects BBS/E/F/000PR10353 and BBS/E/F/000PR10356 410 (S.R.C.). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
Uncontrolled Keywords: bacteriophage,pcr bias,virome,infectious diseases,virology ,/dk/atira/pure/subjectarea/asjc/2700/2725
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Faculty of Science > School of Biological Sciences
Faculty of Medicine and Health Sciences > School of Health Sciences
Related URLs:
Depositing User: LivePure Connector
Date Deposited: 02 Nov 2021 01:58
Last Modified: 20 Nov 2021 01:39
URI: https://ueaeprints.uea.ac.uk/id/eprint/81940
DOI: 10.3390/v13102093

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