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Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study

Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared diffe...

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Autores principales: Yap, Min, O’Sullivan, Orla, O’Toole, Paul W., Cotter, Paul D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9713316/
https://www.ncbi.nlm.nih.gov/pubmed/36466696
http://dx.doi.org/10.3389/fmicb.2022.1036643
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author Yap, Min
O’Sullivan, Orla
O’Toole, Paul W.
Cotter, Paul D.
author_facet Yap, Min
O’Sullivan, Orla
O’Toole, Paul W.
Cotter, Paul D.
author_sort Yap, Min
collection PubMed
description Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches.
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spelling pubmed-97133162022-12-02 Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study Yap, Min O’Sullivan, Orla O’Toole, Paul W. Cotter, Paul D. Front Microbiol Microbiology Although high-throughput DNA sequencing-based methods have been of great value for determining the composition of microbial communities in various environments, there is the potential for inaccuracies arising from the sequencing of DNA from dead microorganisms. In this pilot study, we compared different sequencing-based methods to assess their relative accuracy with respect to distinguishing between viable and non-viable cells, using a live and heat-inactivated model community spiked into bovine milk. The methods used were shotgun metagenomics with and without propidium monoazide (PMA) treatment, RNA-based 16S rRNA sequencing and metatranscriptomics. The results showed that methods were generally accurate, though significant differences were found depending on the library types and sequencing technologies. Different molecular targets were the basis for variations in the results generated using different library types, while differences in the derived composition data from Oxford Nanopore Technologies-and Illumina-based sequencing likely reflect a combination of different sequencing depths, error rates and bioinformatics pipelines. Although PMA was successfully applied in this study, further optimisation is required before it can be applied in a more universal context for complex microbiomes. Overall, these methods show promise and represent another important step towards the ultimate establishment of approaches that can be applied to accurately identify live microorganisms in milk and other food niches. Frontiers Media S.A. 2022-11-17 /pmc/articles/PMC9713316/ /pubmed/36466696 http://dx.doi.org/10.3389/fmicb.2022.1036643 Text en Copyright © 2022 Yap, O’Sullivan, O’Toole and Cotter. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Yap, Min
O’Sullivan, Orla
O’Toole, Paul W.
Cotter, Paul D.
Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study
title Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study
title_full Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study
title_fullStr Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study
title_full_unstemmed Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study
title_short Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study
title_sort development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: a pilot study
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9713316/
https://www.ncbi.nlm.nih.gov/pubmed/36466696
http://dx.doi.org/10.3389/fmicb.2022.1036643
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