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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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-11-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1036643/full |
| _version_ | 1811224479339118592 |
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| author | Min Yap Min Yap Orla O’Sullivan Orla O’Sullivan Paul W. O’Toole Paul W. O’Toole Paul D. Cotter Paul D. Cotter |
| author_facet | Min Yap Min Yap Orla O’Sullivan Orla O’Sullivan Paul W. O’Toole Paul W. O’Toole Paul D. Cotter Paul D. Cotter |
| author_sort | Min Yap |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-12T08:50:09Z |
| format | Article |
| id | doaj.art-1235fde436814df6a94590b1dc5a7607 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-12T08:50:09Z |
| publishDate | 2022-11-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-1235fde436814df6a94590b1dc5a76072022-12-22T03:39:36ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-11-011310.3389/fmicb.2022.10366431036643Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot studyMin Yap0Min Yap1Orla O’Sullivan2Orla O’Sullivan3Paul W. O’Toole4Paul W. O’Toole5Paul D. Cotter6Paul D. Cotter7Teagasc Food Research Centre, Moorepark, Fermoy, IrelandSchool of Microbiology, University College Cork, Cork, IrelandTeagasc Food Research Centre, Moorepark, Fermoy, IrelandAPC Microbiome Ireland, Cork, IrelandSchool of Microbiology, University College Cork, Cork, IrelandAPC Microbiome Ireland, Cork, IrelandTeagasc Food Research Centre, Moorepark, Fermoy, IrelandAPC Microbiome Ireland, Cork, IrelandAlthough 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.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1036643/fullnext-generation sequencingmicrobial viabilityIlluminaOxford nanoporePMAmilk microbiome |
| spellingShingle | Min Yap Min Yap Orla O’Sullivan Orla O’Sullivan Paul W. O’Toole Paul W. O’Toole Paul D. Cotter Paul D. Cotter Development of sequencing-based methodologies to distinguish viable from non-viable cells in a bovine milk matrix: A pilot study Frontiers in Microbiology next-generation sequencing microbial viability Illumina Oxford nanopore PMA milk microbiome |
| 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 | next-generation sequencing microbial viability Illumina Oxford nanopore PMA milk microbiome |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.1036643/full |
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