Abstract
Environmental DNA (eDNA) metabarcoding is an emerging tool to estimate diversity by combining DNA from the environmental samples and the high-throughput sequencing. Despite its wide use in estimating eukaryotic diversity, many factors may bias the results. Maker choice and reference databases are among the key issues in metabarcoding analyses. In the present study, we compared the performance of a novel 28S rRNA gene marker designed in this study and two commonly used 18S rRNA gene markers (V1-2 and V9) in estimating the eukaryotic diversity in the deep-sea sediments. The metabarcoding analyses based on the sediment surveys of the Okinawa Trough found that more eukaryotic taxa were discovered by 18S V9 than 28S and 18S V1-2, and that 18S V9 also performed better in metazoan recovery than the other two markers. Although a broad range of taxa were detected by the three metabarcoding markers, only a small proportion of taxa were shared between them even at the phylum level. The non-metric multidimensional scaling (NMDS) analysis also supported that communities detected by the three markers were distinct from each other. In addition, different communities were resolved by different reference databases (NCBI nt vs. SILVA) for the two 18S markers. Combining the three markers, annelids were found to be the most abundant (44.9%) and diverse [179 operational taxonomic units (OTUs)] metazoan group in the sediments of the Okinawa Trough. Therefore, multiple independent markers are recommended to be used in metabarcoding analyses during marine diversity surveys, especially for the poorly understood deep-sea sediments.
Highlights
About 8.7 million eukaryotic species are predicted to inhabit Earth with ∼2.2 million in the ocean, most of them are not described (Mora et al, 2011)
Given the importance of marker choice in environmental DNA (eDNA) matabarcoding studies, it is critical to evaluate the performance of different markers used in the diversity surveys based on the eDNA, because different markers could perform differently in assessing eukaryotic diversity (e.g., Tanabe et al, 2016; Zhang et al, 2020)
We provided a novel 28S rRNA gene marker targeting the metazoans, and presented the evaluation of this and two commonly used 18S rRNA gene markers (18S V1-2 and 18S V9) in assessing the benthic diversity of the deep-sea benthic communities by metabarcoding analyses
Summary
About 8.7 million eukaryotic species are predicted to inhabit Earth with ∼2.2 million in the ocean, most of them are not described (Mora et al, 2011). Fast, efficient, and reliable methods are required for estimating ecosystem biodiversity. The environmental DNA (eDNA) coupled with high-throughput sequencing is an emerging tool to assess eukaryotic biodiversity (Bik et al, 2012a). The eDNA derived from the environmental samples (e.g., sediments, soil, water, etc.) is an efficient, eDNA Metabarcoding for Deep-Sea Biodiversity non-invasive, and easy-to-standardize sampling approach (Thomsen and Willerslev, 2015). The eDNA metabarcoding method has been widely used in biodiversity surveys, there are still pitfalls and limitations for this emerging method (Ruppert et al, 2019)
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