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Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection |
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| Authors: | Kleopatra Leontidou Ion L Abad-Recio Verena Rubel Sabine Filker Martin Däumer Alexander Thielen Anders Lanzén Thorsten Stoeck |
| Institution: | 1. Ecology Group, Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau, Kaiserslautern, Germany
Contribution: Data curation (lead), Formal analysis (lead), Investigation (equal), Methodology (equal), Software (lead), Visualization (lead), Writing - original draft (equal);2. Marine Ecosystems Functioning, AZTI, Marine Research, Basque Research and Technology Alliance, Pasia, Gipuzkoa, Spain Contribution: Data curation (equal), Formal analysis (equal), Investigation (equal), Software (equal), Writing - review & editing (equal);3. Ecology Group, Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau, Kaiserslautern, Germany Contribution: Resources (equal), Writing - review & editing;4. Molecular Ecology Group, Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau, Kaiserslautern, Germany Contribution: Funding acquisition (equal), Methodology (equal), Resources (equal), Writing - review & editing (equal);5. SeqIT, Laboratory for Molecular Diagnostics and Services, Kaiserslautern, Germany Contribution: Investigation (equal), Methodology, Writing - review & editing (equal);6. SeqIT, Laboratory for Molecular Diagnostics and Services, Kaiserslautern, Germany Contribution: Investigation (equal), Methodology (equal), Writing - review & editing (equal);7. Marine Ecosystems Functioning, AZTI, Marine Research, Basque Research and Technology Alliance, Pasia, Gipuzkoa, Spain IKERBASQUE, Basque Foundation for Science, Bilbao, Bizkaia, Spain Contribution: Conceptualization (equal), Investigation (equal), Methodology (equal), Supervision (equal), Validation (equal), Writing - review & editing (equal);8. Ecology Group, Rheinland-Pfälzische Technische Universität Kaiserslautern-Landau, Kaiserslautern, Germany |
| Abstract: | Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3–V4 region to investigate whether the RC-PCR reveals more of the ‘unseen’ diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3–V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject. |
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