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Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

Mahwash Jamy, Rachel Foster, Pierre Barbera, Lucas Czech, View ORCID ProfileAlexey Kozlov, Alexandros Stamatakis, David Bass, Fabien Burki
doi: https://doi.org/10.1101/627828
Mahwash Jamy
1Science for Life Laboratory, Program in Systematic Biology, Uppsala University, Uppsala, Sweden
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Rachel Foster
2Department of Life Sciences, Natural History Museum, London, UK
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Pierre Barbera
3Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
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Lucas Czech
3Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
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Alexey Kozlov
3Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
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  • ORCID record for Alexey Kozlov
Alexandros Stamatakis
3Computational Molecular Evolution Group, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany
4Institute of Theoretical Informatics, Karlsruhe Institute of Technology, Karlsruhe, Germany
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David Bass
2Department of Life Sciences, Natural History Museum, London, UK
5Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth, Dorset, UK
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  • For correspondence: fabien.burki@ebc.uu.se d.bass@nhm.ac.uk
Fabien Burki
1Science for Life Laboratory, Program in Systematic Biology, Uppsala University, Uppsala, Sweden
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  • For correspondence: fabien.burki@ebc.uu.se d.bass@nhm.ac.uk
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Abstract

High-throughput environmental DNA metabarcoding has revolutionized the analysis of microbial diversity, but this approach is generally restricted to amplicon sizes below 500 base pairs. These short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. However, new long-read sequencing technologies such as the Pacific Biosciences platform may provide sufficiently large sequence lengths to overcome the poor phylogenetic resolution of short amplicons. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain a ~4500 bp region of the eukaryotic rDNA operon spanning most of the small (18S) and large subunit (28S) ribosomal RNA genes. The CCS reads were first treated with a novel curation workflow that generated 650 high-quality OTUs containing the physically linked 18S and 28S regions of the long amplicons. In order to assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based and phylogenetic placement-based methods using shorter reads. The taxonomically-annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing to accurately derive the evolutionary origin of environmental diversity. A total of 1019 sequences were included, of which a majority (58%) corresponded to the new long environmental CCS reads. Comparisons to the 18S-only region of our amplicons revealed that the combined 18S-28S genes globally increased the phylogenetic resolution, recovering specific groupings otherwise missing. The long-reads also allowed to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Altogether, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

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Posted May 05, 2019.
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Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity
Mahwash Jamy, Rachel Foster, Pierre Barbera, Lucas Czech, Alexey Kozlov, Alexandros Stamatakis, David Bass, Fabien Burki
bioRxiv 627828; doi: https://doi.org/10.1101/627828
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Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity
Mahwash Jamy, Rachel Foster, Pierre Barbera, Lucas Czech, Alexey Kozlov, Alexandros Stamatakis, David Bass, Fabien Burki
bioRxiv 627828; doi: https://doi.org/10.1101/627828

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