Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons
A peer-reviewed article of this Preprint also exists.
Author and article information
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 tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rational for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach decribed here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage.
Cite this as
2019. Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons. PeerJ Preprints 7:e27313v2 https://doi.org/10.7287/peerj.preprints.27313v2note This preprint is not peer-reviewed. You may wish to reference the subsequent peer-reviewed version of this article.
Author comment
The previous pre-print version was improved and reformatted for peer-review submission in PeerJ.
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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).
Additional Information
Competing Interests
Xavier Pochon is serving as an Academic Editor for PeerJ.
Author Contributions
Xavier Pochon conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Patricia Wecker contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Michael Stat authored or reviewed drafts of the paper, approved the final draft.
Veronique Berteaux-Lecellier conceived and designed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Gael Lecellier conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
Raw sequence data were submitted to the BioProject Archive under accession PRJNA471926 (SRR7181922-SRR7181942)
Data Deposition
The following information was supplied regarding data availability:
Raw sequence data were submitted to the BioProject Archive under accession PRJNA471926 (SRR7181922-SRR7181942)
Funding
This work was supported by Cawthron Institute Internal Investment Fund (IIF) #BST16931 and by the French National Research Center (CNRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.