Metabarcode diversity from single eukaryotic cells. Radiolaria Single Cells

Metabarcoding is a powerful tool for exploring microbial diversity in the environment, but accurate interpretation is impeded by diverse technical and biological biases that remain poorly understood. We investigated the diversity of the V4 and V9 regions of the 18S rRNA gene from single radiolarian cells using 454 pyrosequencing. For six different cells, an average of 309 (V4) and 634 (V9) amplicons were assigned to Radiolaria. At the 97% identity level, most amplicons belonged to a single OTU, but up to 5 OTUs were found. Duplicate PCR and sequencing from the same DNA extract had only 9 to 16 % of unique amplicons in common. The non-common amplicons were fewer in number but formed more OTUs than common amplicons, and they contained unexpected mutations in highly-conserved regions. This demonstrates that many amplicons are PCR and pyrosequencing-produced artifacts leading to diversity overestimates. Replication therefore appears essential for accurate interpretation of large and complex metabarcoding datasets.