Abstract
The smallest size fractions of plankton, nano- and pico-plankton, have been highlighted due to they accomplish key functions in marine ecosystems. However, the knowledge about some of them is scarce because they are difficult or impossible to be detected and identified with non-DNA-based methodologies. Here we have evaluated five DNA extraction protocols (MT1–MT5) and seven bioinformatic pipelines (P1–P7) to find the best protocol for detecting most of the eukaryotic species of nano- and pico-plankton present in an environmental sample using Ion Torrent technology. The protocol MT3 was the most reproducible methodology, showing less variation among samples, good DNA quality and sufficient quantity to amplify and sequence the eukaryote species, offering the best results after sequencing. For bioinformatic analyses, P1 and P7 resulted in the highest percentage of detection for the difficult-to-detect species in mock communities. However, only P1 avoided the confusion with other closed species during the taxonomic assignment. The final protocols, MT3-P1 (free) and MT3-P7 (private), showed good and consistent results when they were applied to an environmental sample, being a valuable tool to study the eukaryotes present in environmental samples of nano- and pico-plankton, even for the genera that are difficult to be detected by other techniques.
Highlights
The study of biodiversity is essential to understand the dynamics and functions of ecosystems, as well as their sustainability and resilience (Fonseca et al, 2010; Brose and Hillebrand, 2016)
Different protocols were tested for two critical steps of metabarcoding analyses of marine environmental samples, DNA extraction and bioinformatic analyses, focusing on nano- and pico-plankton eukaryotes as target species
MT2 and MT3 were the best among the DNA extraction methods analyzed because of the yield and the reproducibility of the results, but from the samples extracted with MT3 was possible to identify more eukaryotic taxa during the taxonomic assignment
Summary
The study of biodiversity is essential to understand the dynamics and functions of ecosystems, as well as their sustainability and resilience (Fonseca et al, 2010; Brose and Hillebrand, 2016). The study of plankton entails difficulties due to the small size of most of these organisms and the fact that they can be present in very low abundances or as part of small populations (Jerde et al, 2011; Zhan et al, 2013). Morphological and anatomical characteristics have been used to identify the organisms present in the samples (Zhan et al, 2013). These techniques are time consuming and require expert taxonomists for each target group (Abad et al, 2016), and high-resolution images
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