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Illumina sequencing is a representative tool for understanding the massive diversity of microbial eukaryotes in natural ecosystems. Here, we investigated the eukaryotic community in a pond (salinity of 2-4) on Dokdo (island) in the East Sea, Korea, using Illumina sequencing with primer sets for the V4 and V9 regions of 18S rDNA from 2016 to 2018 for the first time. Totally, 1,413 operational taxonomic units (OTUs) and 915 OTUs were detected using the V9 and V4 primer sets, respectively. Taxonomic analyses of these OTUs revealed that although the V4 primer set failed to describe the extant diversity for some major sub-division groups, the V9 primer set represented their diversity. Moreover, the rare taxa with <1% of total reads were exclusively detected using V9 primer set. Hence, the diversity of the eukaryotic community can vary depending on the choice of primers. The Illumina sequencing data of the V9 region of 18S rDNA may be advantageous for estimating the richness of the eukaryotic community including a rare biosphere, whereas the simultaneous application of two biomarkers may be suitable for understanding the molecular phylogenetic relationships. We strongly recommend both biomarkers be used to assess the diversity and phylogenetic relationship within the eukaryotic community in natural samples.The advent of next-generation sequencing (NGS) led to a substantial change in the previous knowledge about the microbial diversity in natural ecosystems 1,2 . NGS targeting the 18S rDNA is usually used to evaluate the diversity within all domains of life and provides large quantities of sequencing data for individual investigators. The 454 platform is hardly used anymore and a lot of other platforms are currently used far more widely. The Illumina platforms are representative tools for the investigation of the microbial community although this method can read a relatively short fragment of sequence (200 bp-500 bp) due to the technical limitations 3,4 . Illumina platform is a cost-effective tool per base (priced ~100 times lower than the 454 platform) and can now read longer sequences (200 bp-300 bp) than the initial platform 5,6 . Furthermore, the error rate of the Illumina platform is lower than that of the 454 platform 7 . However, considering the revolutionary application of the Illumina platform, our knowledge about the diversity and phylogenetic relationship of eukaryotes remains poor in field surveys such as those for brackish water 8 .Several studies on the diversity of eukaryotes noted that the V1-V2, V3, V4, and V9 regions of 18S rDNA have been used for better understanding the massive diversity of microbial community 9-11 . The V4 (expected amplicon size, 270 bp-387 bp) and V9 (expected amplicon size, 96 bp-134 bp) regions are considered to be the popular for metabarcoding 12,13 . Based on an in silico analysis, the V9 region of 18S rDNA offers the advantage to reveal the extant diversity of eukaryotes, whereas the V4 region of 18S rDNA is commonly used for studying the phylogenetic relationship of eukaryot...

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