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

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...

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Autores principales: Hsieh, Shen-Yuan, Tariq, Mohammad A., Telatin, Andrea, Ansorge, Rebecca, Adriaenssens, Evelien M., Savva, George M., Booth, Catherine, Wileman, Tom, Hoyles, Lesley, Carding, Simon R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8537689/
https://www.ncbi.nlm.nih.gov/pubmed/34696523
http://dx.doi.org/10.3390/v13102093
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author Hsieh, Shen-Yuan
Tariq, Mohammad A.
Telatin, Andrea
Ansorge, Rebecca
Adriaenssens, Evelien M.
Savva, George M.
Booth, Catherine
Wileman, Tom
Hoyles, Lesley
Carding, Simon R.
author_facet Hsieh, Shen-Yuan
Tariq, Mohammad A.
Telatin, Andrea
Ansorge, Rebecca
Adriaenssens, Evelien M.
Savva, George M.
Booth, Catherine
Wileman, Tom
Hoyles, Lesley
Carding, Simon R.
author_sort Hsieh, Shen-Yuan
collection PubMed
description 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.
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spelling pubmed-85376892021-10-24 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 Carding, Simon R. Viruses Article 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. MDPI 2021-10-18 /pmc/articles/PMC8537689/ /pubmed/34696523 http://dx.doi.org/10.3390/v13102093 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hsieh, Shen-Yuan
Tariq, Mohammad A.
Telatin, Andrea
Ansorge, Rebecca
Adriaenssens, Evelien M.
Savva, George M.
Booth, Catherine
Wileman, Tom
Hoyles, Lesley
Carding, Simon R.
Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
title Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
title_full Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
title_fullStr Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
title_full_unstemmed Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
title_short Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome
title_sort comparison of pcr versus pcr-free dna library preparation for characterising the human faecal virome
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8537689/
https://www.ncbi.nlm.nih.gov/pubmed/34696523
http://dx.doi.org/10.3390/v13102093
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