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Article: Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

TitleComparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water
Authors
Issue Date2017
Citation
Scientific Reports, 2017, v. 7, n. 1 How to Cite?
Abstract© 2017 The Author(s). The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA-and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA-and PMA-based methods in detecting live bacterial cells in water.
Persistent Identifierhttp://hdl.handle.net/10722/254463

 

DC FieldValueLanguage
dc.contributor.authorLi, Ru-
dc.contributor.authorTun, Hein Min-
dc.contributor.authorJahan, Musarrat-
dc.contributor.authorZhang, Zhengxiao-
dc.contributor.authorKumar, Ayush-
dc.contributor.authorFernando, Dilantha-
dc.contributor.authorFarenhorst, Annemieke-
dc.contributor.authorKhafipour, Ehsan-
dc.date.accessioned2018-06-19T15:40:37Z-
dc.date.available2018-06-19T15:40:37Z-
dc.date.issued2017-
dc.identifier.citationScientific Reports, 2017, v. 7, n. 1-
dc.identifier.urihttp://hdl.handle.net/10722/254463-
dc.description.abstract© 2017 The Author(s). The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA-and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA-and PMA-based methods in detecting live bacterial cells in water.-
dc.languageeng-
dc.relation.ispartofScientific Reports-
dc.titleComparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water-
dc.typeArticle-
dc.description.natureLink_to_subscribed_fulltext-
dc.identifier.doi10.1038/s41598-017-02516-3-
dc.identifier.scopuseid_2-s2.0-85025144758-
dc.identifier.volume7-
dc.identifier.issue1-
dc.identifier.spagenull-
dc.identifier.epagenull-
dc.identifier.eissn2045-2322-

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