Development of a cost-effective metabarcoding strategy for analysis of the marine phytoplankton community

Abstract

We developed a cost-effective metabarcoding strategy to analyze phytoplankton community structure using the Illumina MiSeq system. The amplicons (404–411 bp) obtained by end-pairing of two reads were sufficiently long to distinguish algal species and provided barcode data equivalent to those generated with the Roche 454 system, but at less than 1/20th of the cost. The original universal primer sequences targeting the 23S rDNA region and the PCR strategy were both modified, and this resulted in higher numbers of eukaryotic algal sequences by excluding non-photosynthetic proteobacterial sequences supporting effectiveness of this strategy. The novel strategy was used to analyze the phytoplankton community structure of six water samples from the East/Japan Sea: surface and 50 m depths at coastal and open-sea sites, with collections in May and July 2014. In total, 345 operational taxonomic units (OTUs) were identified, which covered most of the prokaryotic and eukaryotic algal phyla, including Dinophyta, Rhodophyta, Ochrophyta, Chlorophyta, Streptophyta, Cryptophyta, Haptophyta, and Cyanophyta. This highlights the importance of plastid 23S primers, which perform better than the currently used 16S primers for phytoplankton community surveys. The findings also revealed that more efforts should be made to update 23S rDNA sequences as well as those of 16S in the databases. Analysis of algal proportions in the six samples showed that community structure differed depending on location, depth and season. Across the six samples evaluated, the numbers of OTUs in each phylum were similar but their relative proportions varied. This novel strategy would allow laboratories to analyze large numbers of samples at reasonable expense, whereas this has not been possible to date due to cost and time. In addition, we expect that this strategy will generate a large amount of novel data that could potentially change established methods and tools that are currently used in the realms of oceanography and marine ecology.

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

“…Thus, currently, some universal metabarcoding assays result in low taxonomic resolution. OTU analyses, which make up the bulk of eDNA metabarcoding research to date, have been proposed to alleviate some of the problems associated with universal primer sets (Yoon et al, ). However, OTU metrics are known to inflate biodiversity estimates (Diaz et al, ).…”

A metabarcoding comparison of windward and leeward airborne algal diversity across the Ko‘olau mountain range on the island of O'ahu, Hawai‘i1

“…Yoon et al. () recently provided modifications to the UPA primers and cycling conditions that resulted in a substantial decrease in the proportion of heterotrophic bacterial reads obtained in an Illumina metabarcoding study of marine phytoplankton, which holds promise for increasing the efficiency of future NGS applications of this marker. Sauvage et al.…”

“…In our results here, we recovered a large fraction of heterotrophic bacterial OTUs (33.3% of total). Yoon et al (2016) recently provided modifications to the UPA primers and cycling conditions that resulted in a substantial decrease in the proportion of heterotrophic bacterial reads obtained in an Illumina metabarcoding study of marine phytoplankton, which holds promise for increasing the efficiency of future NGS applications of this marker. Sauvage et al (2016) recently demonstrated that the tufA plastid marker can be amplified and sequenced with the Illumina platform for both cyanobacteria and eukaryotic algae in marine systems, although this marker is also unable to amplify cladophoralean taxa and may pose additional problems in amplifying trentepohlialean taxa.…”

“…, Yoon et al. ). This 23S ribosomal DNA marker amplifies and sequences from most algal groups tested (with the exception of cladophoralean green algae, Saunders and Kucera , and possibly dinoflagellates, Sherwood and Presting ) and has the advantage of simultaneously yielding data for both eukaryotic algae and cyanobacteria (Sherwood and Presting ).…”

“…Metabarcoding of algal communities is an emerging field, and researchers are currently evaluating suitable sets of universal primers, building reference sequence databases, and developing bioinformatic pipelines for data processing of NGS reads (e.g., Sauvage et al 2016). The universal plastid amplicon (UPA) marker has been used extensively in DNA barcoding applications of red algae (e.g., Clarkston andSaunders 2010, Sherwood et al 2010a), as one of several markers amplified in broad algal taxonomic surveys (Sherwood et al 2010b(Sherwood et al , 2014b, and has been successfully employed in NGS applications (Steven et al 2012, Letendu et al 2014, Yoon et al 2016. This 23S ribosomal DNA marker amplifies and sequences from most algal groups tested (with the exception of cladophoralean green algae, Saunders and Kucera 2010, and possibly dinoflagellates, Sherwood and Presting 2007) and has the advantage of simultaneously yielding data for both eukaryotic algae and cyanobacteria (Sherwood and Presting 2007).…”