|Genetic barcoding of marine zooplankton using the MinIon sequencer|
Willemse, S. (2018). Genetic barcoding of marine zooplankton using the MinIon sequencer. MSc Thesis. Faculty of Bioscience Engineering, Gent University: Gent. 64 pp.
Biomonitoring is a way to determine the biodiversity and ecological status of an ecosystem. This is important in a world where species are lost at an alarming rate. Animal species can be identified during biomonitoring with various methods. One of these is metabarcoding which uses genetic markers isolated from a DNA extract from the biological community under study. These can then be sequenced with one of the many available platforms. Recently, the British company Oxford Nanopore Technologies has developed a revolutionary technology (MinION) has made sequencing much more accessible. In this research, it was investigated whether it is possible to use this MinION sequencer for metabarcoding of marine zooplankton communities. Each step of the protocol was optimised, after which it was applied to samples taken during a biomonitoring campaign on the North Sea. Different DNA extraction techniques are tested, after which PCR amplification and purification are optimized by selection of an optimal primer set, PCR master mix, inhibition prevention measures and purification protocols. The amplicons were then sequenced and subdivided into species using a reference database. Finally, a comparison was made with a ZooScan analysis in which species are determined based on their morphology. The results largely conform to ZooScan determinations and show no discrepancies from what was found in the literature. Although further research in data processing is required, it can be concluded that metabarcoding with MinION sequencing is a valuable innovation in the field of biomonitoring.