Description of a ‘plankton filtration bias’ in sequencing-based bacterial community analysis and of an Arduino microcontroller-based flowmeter device that can help to resolve it
Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer...
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| Vydané v: | PloS one Ročník 19; číslo 5; s. e0303937 |
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| Hlavní autori: | , , , , |
| Médium: | Journal Article |
| Jazyk: | English |
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United States
Public Library of Science
28.05.2024
Public Library of Science (PLoS) |
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| ISSN: | 1932-6203, 1932-6203 |
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| Abstract | Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias. |
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| AbstractList | Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias. Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias.Diversity studies of aquatic picoplankton (bacterioplankton) communities using size-class filtration, DNA extraction, PCR and sequencing of phylogenetic markers, require a robust methodological pipeline, since biases have been demonstrated essentially at all levels, including DNA extraction, primer choice and PCR. Even different filtration volumes of the same plankton sample and, thus, different biomass loading of the filters, can distort the sequencing results. In this study, we designed an Arduino microcontroller-based flowmeter that records the decrease of initial (maximal) flowrate as proxy for increasing biomass loading and clogging of filters during plankton filtration. The device was tested using freshwater plankton of Lake Constance, and total DNA was extracted and an 16S rDNA amplicon was sequenced. We confirmed that different filtration volumes used for the same water sample affect the sequencing results. Differences were visible in alpha and beta diversities and across all taxonomic ranks. Taxa most affected were typical freshwater Actinobacteria and Bacteroidetes, increasing up to 38% and decreasing up to 29% in relative abundance, respectively. In another experiment, a lake water sample was filtered undiluted and three-fold diluted, and each filtration was stopped once the flowrate had reduced to 50% of initial flowrate, hence, at the same degree of filter clogging. The three-fold diluted sample required three-fold filtration volumes, while equivalent amounts of total DNA were extracted and differences across all taxonomic ranks were not statistically significant compared to the undiluted controls. In conclusion, this work confirms a volume/biomass-dependent bacterioplankton filtration bias for sequencing-based community analyses and provides an improved procedure for controlling biomass loading during filtrations and recovery of equivalent amounts of DNA from samples independent of the plankton density. The application of the device can also avoid the distorting of sequencing results as caused by the plankton filtration bias. |
| Audience | Academic |
| Author | Kautz, Harald Fournier, Corentin Fiedler, Alexander Weidele, Maximilian Schleheck, David |
| AuthorAffiliation | 2 Scientific Engineering and Manufacturing Services, University of Konstanz, Konstanz, Germany 1 Department of Biology, Microbial Ecology and Limnic Microbiology, Limnological Institute, University of Konstanz, Konstanz, Germany University of Kalyani, INDIA |
| AuthorAffiliation_xml | – name: 2 Scientific Engineering and Manufacturing Services, University of Konstanz, Konstanz, Germany – name: 1 Department of Biology, Microbial Ecology and Limnic Microbiology, Limnological Institute, University of Konstanz, Konstanz, Germany – name: University of Kalyani, INDIA |
| Author_xml | – sequence: 1 givenname: Corentin surname: Fournier fullname: Fournier, Corentin – sequence: 2 givenname: Alexander surname: Fiedler fullname: Fiedler, Alexander – sequence: 3 givenname: Maximilian surname: Weidele fullname: Weidele, Maximilian – sequence: 4 givenname: Harald surname: Kautz fullname: Kautz, Harald – sequence: 5 givenname: David surname: Schleheck fullname: Schleheck, David |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/38805423$$D View this record in MEDLINE/PubMed |
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| CitedBy_id | crossref_primary_10_1080_02705060_2024_2413689 crossref_primary_10_1002_lno_70207 |
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| Copyright | Copyright: © 2024 Fournier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. COPYRIGHT 2024 Public Library of Science 2024 Fournier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. 2024 Fournier et al 2024 Fournier et al 2024 Fournier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Current address: Department of Biotechnology and Food Science, Norwegian University of Science and Technology, Gløshaugen, Norway Competing Interests: NO authors have competing interests. |
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| SubjectTerms | Bacteria - classification Bacteria - genetics Bacteria - isolation & purification Bacterioplankton Bias Biology and Life Sciences Biomass Computer and Information Sciences Deoxyribonucleic acid Dilution DNA DNA sequencing DNA, Bacterial - genetics Earth Sciences Ecology and Environmental Sciences Equivalence Ethylenediaminetetraacetic acid Filters Filtration Filtration - instrumentation Filtration - methods Flow rates Flowmeters Fresh water Freshwater lakes Gene sequencing Lake water Lakes - microbiology Membrane filters Microcontrollers Nucleotide sequence Phylogeny Picoplankton Plankton Plankton - genetics Polymerase chain reaction Pore size Rankings Relative abundance Research and analysis methods RNA, Ribosomal, 16S - genetics rRNA 16S Sequence Analysis, DNA - methods Statistical analysis Taxonomy Water Water analysis Water purification Water sampling |
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| Title | Description of a ‘plankton filtration bias’ in sequencing-based bacterial community analysis and of an Arduino microcontroller-based flowmeter device that can help to resolve it |
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