Metabarcoding revolutionized our understanding of diversity and ecology of microorganisms in different habitats. However, it is also associated with several inherent biases, one of which is associated with intragenomic diversity of a molecular barcode. Here, we compare intragenomic variability of the V9 region of the 18S rRNA gene in 19 eukaryotic phyla abundant in marine plankton. The level of intragenomic variability is comparable across all the phyla, and in most genomes and transcriptomes one V9 sequence and one OTU is predominant. However, most of the variability observed at the barcode level is probably caused by sequencing errors and is mitigated by using a denoising tool, DADA2. The SWARM algorithm commonly used in metabarcoding studies is not optimal for collapsing genuine and erroneous sequences into a single OTU, leading to an overestimation of diversity in metabarcoding data. For an unknown reason, SWARM inflates diversity of eupelagonemids more than that of other eukaryotes.

Department of Biology and Ecology Faculty of Science University of Ostrava Ostrava Czech Republic

Department of Molecular Biology Faculty of Science University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

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