Jamy, Mahwash, Foster, Rachel, Barbera, Pierre, Czech, Lucas, Kozlov, Alexey, Stamatakis, Alexandros, Bending, G. D., Hilton, Sally, Bass, David and Burki, Fabien (2020) Long-read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity. Molecular Ecology Resources, 20 (2). pp. 429-443. doi:10.1111/1755-0998.13117

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Abstract

High‐throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long‐read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500‐bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high‐quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. 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 methods. 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 us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, 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.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history Q Science > QP Physiology Q Science > QR Microbiology
Divisions:	Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	Phylogeny, Phylogeny -- Molecular aspects, Protista , Recombinant DNA, Microbial ecology
Journal or Publication Title:	Molecular Ecology Resources
Publisher:	Wiley-Blackwell Publishing Ltd.
ISSN:	1755-098X
Official Date:	March 2020
Dates:	Date Event March 2020 Published 9 November 2019 Available 31 October 2019 Accepted
Date of first compliant deposit:	29 November 2019
Volume:	20
Number:	2
Page Range:	pp. 429-443
DOI:	10.1111/1755-0998.13117
Status:	Peer Reviewed
Publication Status:	Published
Publisher Statement:	This is the peer reviewed version of the following article: Jamy, M, Foster, R, Barbera, P, et al. Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity. Mol Ecol Resour. 2019; 00: 1– 15. https://doi.org/10.1111/1755-0998.13117, which has been published in final form at https://doi.org/10.1111/1755-0998.13117. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
Access rights to Published version:	Restricted or Subscription Access
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID UNSPECIFIED Science for Life Laboratory http://dx.doi.org/10.13039/501100009252 NE/H009426/1 [EPSRC] Engineering and Physical Sciences Research Council http://dx.doi.org/10.13039/501100000266 FC1214 Department for Environment, Food and Rural Affairs http://dx.doi.org/10.13039/501100000277 UNSPECIFIED Klaus Tschira Stiftung http://dx.doi.org/10.13039/501100007316
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