Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via multiplex metabarcoding
- Published
- Accepted
- Subject Areas
- Biodiversity, Biotechnology, Ecology, Marine Biology, Molecular Biology
- Keywords
- Multiplex Metabarcoding, High-Throughput Sequencing, South Pacific Ocean, symbiosis, Marine Ecology, Tridacna, Biodiversity
- Copyright
- © 2018 Pochon et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2018. Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via multiplex metabarcoding. PeerJ Preprints 6:e27313v1 https://doi.org/10.7287/peerj.preprints.27313v1
Abstract
High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam (Tridacna maxima) samples obtained from nine French Polynesian locations and developed a multiplex metabarcoding method that pools and simultaneously sequences multiple Symbiodiniaceae genes for in-depth biodiversity assessments. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three multiplexed genes investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. Multiplex metabarcoding offers significant analytical cost savings while providing exceptional phylogenetic information and sequence coverage.
Author Comment
Dinoflagellates in the family Symbiodiniaceae are critical components of coral reefs ecosystem in their role as obligate symbiotic partners of benthic marine invertebrate taxa. Recent advances in sequencing technologies now enable in-depth characterization of free-living and symbiotic Symbiodiniaceae, although most studies focus on using a single genetic marker. In this proofof- concept study, we used a small set of symbiotic giant clam (Tridacna maxima) samples obtained from nine French Polynesian locations and developed a multiplex metabarcoding method that pools and simultaneously sequences multiple Symbiodiniaceae genes for in-depth biodiversity assessments. Our results showed that the technique effectively recovered similar proportions of sequence reads and Symbiodiniaceae diversity among the three multiplexed genes investigated per sample enabling a more comprehensive assessment of the diversity present. We believe that this study will generate great interest among Coral Reefs readers and make a substantial contribution for future research in the field.
Supplemental Information
Tridacna maxima samples used in this pilot study
Identification numbers, collection localities and date collected for the twelve samples of Tridacna maxima investigated in this study.
Primer sets used in this study
List of primers used for generating PCR amplicons. Illumina adaptors are shown in bold.
Sequence counts and blast annotations for the 21 amplicon samples analyzed in multiplex and individually (controls) over three distinct genes (23S, ITS2, LSU)
Sequence counts and blast annotations for the 21 amplicon samples analyzed in multiplex and individually (controls) over three distinct genes (23S, ITS2, LSU). Sheet 1 includes the merged counts and dereplicated data; Sheet 2 includes exact 23S sequence matches against the Takabayashi et al. (2012) database and NCBI; Sheet 3 includes retained 23S genotypes following the 0.05% abundance threshold; Sheet 4 includes exact ITS2 sequence matches against the GeoSymbio database and NCBI; Sheet 5 includes retained ITS2 genotypes following the 0.05% abundance threshold; Sheet 6 includes exact LSU sequence matches against the Pochon et al. (2012) database and NCBI; and Sheet 7 includes retained LSU genotypes following the 0.05% abundance threshold.
Percentage comparison of each Symbiodiniaceae sub-generic type recovered using the three markers in ‘Multiplex’ versus single ‘Control’ markers
Percentage comparison of each Symbiodiniaceae sub-generic type recovered using the three markers in ‘Multiplex’ versus single ‘Control’ markers (see Table 1). The proportion of each sub-generic type between ‘Multiplex’ and ‘Control’ is almost identical for the 23S marker, but shows some minor differences for the ITS2 and LSU markers. For example, four ITS2 types were detected in the ‘Multiplex’ but not in the ‘Control’ samples, and there were five instances where LSU types were detected in the ‘Control’ but not in the ‘Multiplex’ samples. These minor differences are likely attributable to PCR or sequencing biases.
Unrooted circled trees of Symbiodiniaceae genotypes recovered in this study
Unrooted circled trees of Symbiodiniaceae genotypes inferred using the Neighbor-Joining method, with (A) 11 23 Ssequences, (B) 46 ITS2 sequences, and (C) 51 LSU sequences.
Distribution of Symbiodiniaceae genera (i.e. clades) in Tridacna maxima obtained from each of the three datasets
Distribution of Symbiodiniaceae genera (i.e. clades) in Tridacna maxima obtained from each of the three datasets (left to right: 23S, ITS2, and LSU) per sample identification (S141-152).
Extended Methods, Results, and Discussion
This file provides additional details on Methods and Results, as well as additional Discussion (not included in the intentionally short main manuscript).