Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations
ABSTRACT High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2020-04-01
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| Series: | mSphere |
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| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00052-20 |
| _version_ | 1818822266969915392 |
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| author | Kasia Piwosz Tanja Shabarova Jakob Pernthaler Thomas Posch Karel Šimek Petr Porcal Michaela M. Salcher |
| author_facet | Kasia Piwosz Tanja Shabarova Jakob Pernthaler Thomas Posch Karel Šimek Petr Porcal Michaela M. Salcher |
| author_sort | Kasia Piwosz |
| collection | DOAJ |
| description | ABSTRACT High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards. IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities. |
| first_indexed | 2024-12-18T23:21:22Z |
| format | Article |
| id | doaj.art-d5af1d57525746b3b8280c7880ab35dd |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-18T23:21:22Z |
| publishDate | 2020-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-d5af1d57525746b3b8280c7880ab35dd2022-12-21T20:47:57ZengAmerican Society for MicrobiologymSphere2379-50422020-04-015210.1128/mSphere.00052-20Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological InterpretationsKasia Piwosz0Tanja Shabarova1Jakob Pernthaler2Thomas Posch3Karel Šimek4Petr Porcal5Michaela M. Salcher6Centre Algatech, Institute of Microbiology, Czech Academy of Sciences, Třeboň, Czech RepublicInstitute of Hydrobiology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech RepublicLimnological Station, Institute of Plant and Microbial Biology, University of Zurich, Kilchberg, SwitzerlandLimnological Station, Institute of Plant and Microbial Biology, University of Zurich, Kilchberg, SwitzerlandInstitute of Hydrobiology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech RepublicInstitute of Hydrobiology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech RepublicInstitute of Hydrobiology, Biology Centre, Czech Academy of Sciences, České Budějovice, Czech RepublicABSTRACT High-throughput sequencing (HTS) of gene amplicons is a preferred method of assessing microbial community composition, because it rapidly provides information from a large number of samples at high taxonomic resolution and low costs. However, mock community studies show that HTS data poorly reflect the actual relative abundances of individual phylotypes, casting doubt on the reliability of subsequent statistical analysis and data interpretation. We investigated how accurately HTS data reflect the variability of bacterial and eukaryotic community composition and their relationship with environmental factors in natural samples. For this, we compared results of HTS from three independent aquatic time series (n = 883) with those from an established, quantitative microscopic method (catalyzed reporter deposition-fluorescence in situ hybridization [CARD-FISH]). Relative abundances obtained by CARD-FISH and HTS disagreed for most bacterial and eukaryotic phylotypes. Nevertheless, the two methods identified the same environmental drivers to shape bacterial and eukaryotic communities. Our results show that amplicon data do provide reliable information for their ecological interpretations. Yet, when studying specific phylogenetic groups, it is advisable to combine HTS with quantification using microscopy and/or the addition of internal standards. IMPORTANCE High-throughput sequencing (HTS) of amplified fragments of rRNA genes provides unprecedented insight into the diversity of prokaryotic and eukaryotic microorganisms. Unfortunately, HTS data are prone to quantitative biases, which may lead to an erroneous picture of microbial community composition and thwart efforts to advance its understanding. These concerns motivated us to investigate how accurately HTS data characterize the variability of microbial communities, the relative abundances of specific phylotypes, and their relationships with environmental factors in comparison to an established microscopy-based method. We compared results obtained by HTS and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) from three independent aquatic time series for both prokaryotic and eukaryotic microorganisms (almost 900 data points, the largest obtained with both methods so far). HTS and CARD-FISH data disagree with regard to relative abundances of bacterial and eukaryotic phylotypes but identify similar environmental drivers shaping bacterial and eukaryotic communities.https://journals.asm.org/doi/10.1128/mSphere.00052-20CARD-FISHamplicon sequencingbacterial communitiesbacterial community structurebacterial dynamicseukaryotic communities |
| spellingShingle | Kasia Piwosz Tanja Shabarova Jakob Pernthaler Thomas Posch Karel Šimek Petr Porcal Michaela M. Salcher Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations mSphere CARD-FISH amplicon sequencing bacterial communities bacterial community structure bacterial dynamics eukaryotic communities |
| title | Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations |
| title_full | Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations |
| title_fullStr | Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations |
| title_full_unstemmed | Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations |
| title_short | Bacterial and Eukaryotic Small-Subunit Amplicon Data Do Not Provide a Quantitative Picture of Microbial Communities, but They Are Reliable in the Context of Ecological Interpretations |
| title_sort | bacterial and eukaryotic small subunit amplicon data do not provide a quantitative picture of microbial communities but they are reliable in the context of ecological interpretations |
| topic | CARD-FISH amplicon sequencing bacterial communities bacterial community structure bacterial dynamics eukaryotic communities |
| url | https://journals.asm.org/doi/10.1128/mSphere.00052-20 |
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