Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities
Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its o...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1151907/full |
| _version_ | 1797845531922268160 |
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| author | Iva Stojan Iva Stojan Željka Trumbić Ivana Lepen Pleić Danijela Šantić |
| author_facet | Iva Stojan Iva Stojan Željka Trumbić Ivana Lepen Pleić Danijela Šantić |
| author_sort | Iva Stojan |
| collection | DOAJ |
| description | Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study. |
| first_indexed | 2024-04-09T17:41:34Z |
| format | Article |
| id | doaj.art-4bed64bd7bdb4177857dc43f2d6263fd |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-09T17:41:34Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-4bed64bd7bdb4177857dc43f2d6263fd2023-04-17T05:23:25ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-04-011410.3389/fmicb.2023.11519071151907Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communitiesIva Stojan0Iva Stojan1Željka Trumbić2Ivana Lepen Pleić3Danijela Šantić4Laboratory of Microbiology, Institute of Oceanography and Fisheries, Split, CroatiaDoctoral Study of Biophysics, Faculty of Science, University of Split, Split, CroatiaUniversity Department of Marine Studies, University of Split, Split, CroatiaLaboratory for Aquaculture, Institute of Oceanography and Fisheries, Split, CroatiaLaboratory of Microbiology, Institute of Oceanography and Fisheries, Split, CroatiaRecent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1151907/fullDNA extractionmock communitymarine bacteria16S rRNAdirect PCRDNA metabarcoding |
| spellingShingle | Iva Stojan Iva Stojan Željka Trumbić Ivana Lepen Pleić Danijela Šantić Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities Frontiers in Microbiology DNA extraction mock community marine bacteria 16S rRNA direct PCR DNA metabarcoding |
| title | Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities |
| title_full | Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities |
| title_fullStr | Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities |
| title_full_unstemmed | Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities |
| title_short | Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities |
| title_sort | evaluation of dna extraction methods and direct pcr in metabarcoding of mock and marine bacterial communities |
| topic | DNA extraction mock community marine bacteria 16S rRNA direct PCR DNA metabarcoding |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1151907/full |
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