Data from: Wangensteen, O.S., Palacín, C., Guardiola, M. & Turon, X. DNA metabarcoding of littoral hard-bottom communities: high diversity and database gaps revealed by two molecular markers
Biodiversity assessment of marine hard-bottom communities is hindered by the high diversity and size-ranges of the organisms present. We developed a DNA metabarcoding protocol for biodiversity characterization of structurally complex natural marine hard-bottom communities. We used two molecular markers: the “Leray fragment” of mitochondrial cytochrome c oxidase (COI), for which a novel primer set was developed, and the V7 region of ribosomal RNA 18S. Eight different marine littoral communities from two National Parks in Spain (one in the Atlantic Ocean and another in the Mediterranean Sea) were studied. Samples were sieved into three size fractions from where DNA was extracted separately. Bayesian clustering was used for delimiting molecular operational taxonomic units (MOTUs) and custom reference databases were constructed for taxonomic assignment. Despite applying conservative stringent filters, we found high values for MOTU richness (2,510 and 9,679 MOTUs with 18S and COI, respectively), suggesting that these communities host a large amount of yet undescribed eukaryotic biodiversity. Significant gaps are still found in sequence reference databases, which currently prevent the complete taxonomic assignment of the detected sequences. 85% of 18S MOTUs of and 64% of COI MOTUs could be identified to phylum or lower taxonomical level. Nevertheless, those unassigned were mostly rare MOTUs with low numbers of reads, and assigned MOTUs comprised over 90% of the total sequence reads. The identification rate might be significantly improved in the future, as reference databases are further completed. Our results show that marine metabarcoding, currently applied mostly to plankton or sediments, can be adapted to structurally complex hard bottom samples, and emerges as a robust, fast, objective and affordable method for comprehensively characterizing the diversity of marine benthic communities dominated by macroscopic seaweeds and colonial or modular sessile metazoans, allowing for standardized biomonitoring of these ecologically important communities. The 18S marker lacks species-level resolution and thus cannot be recommended to assess the detailed taxonomic composition of these communities. Our new universal primers for COI can potentially be used for biodiversity assessment with high taxonomic resolution in a wide array of marine, terrestrial or freshwater eukaryotic communities.
Steps to reproduce
Files metabarpark_18S.demultiplexed.fastq and metabarpark_COI.demultiplexed.fastq contain the paired-end aligned and demultiplexed metabarcoding reads for the two markers analysed in the work: 18S (V7-region) and COI (Leray region), respectively. The metadata for the included samples are in files Samples_metabarpark_18S.csv and Samples_metabarpark_COI.csv. These fastq files were obtained using the OBITools pipeline from Miseq raw reads. First, the illuminapairedend command was used and only reads with an alignment quality score > 40 were selected. Then the ngsfilter command was used, to demultiplex the samples and remove primer sequences. The "sample" attribute with the sample information for every read was created by ngsfilter during the demultiplexing step. Note: File metabarpark_COI.demultiplexed.fastq has been splitted into five parts to facilitate file transfer, due to its large size. After downloading the five parts, they should be gunzipped individually and then concatenated.