DNA extraction with inhibitor removal outperforms more simple methods in DNA metabarcoding of homogenized unsorted bulk samples

Characterization of freshwater benthic biodiversity using DNA metabarcoding may allow more cost-effective environmental assessments than the current morphological based assessment methods. Especially DNA metabarcoding methods where sorting or pre-sorting of samples are avoided all together are interesting, since time between sampling and taxonomic identification is reduced. Due to the presence of PCR inhibitors in crude samples, DNA extraction protocols become crucial for maximum DNA recovery and sample replicability. We sampled freshwater invertebrates from six river and lake sites and extracted DNA from homogenized bulk samples, using three different, easily available methods: the HotSHOT approach, the Qiagen DNeasy Blood & Tissue Kit and the Qiagen DNeasy PowerPlant Pro Kit. The performance of the selected extraction methods were evaluated by measuring DNA yield and metazoan diversity to see if the PowerPlant kit with an included PCR-inhibitor removal step performed better than other methods. In our experiment, the PowerPlant kit extractions resulted in the highest DNA yield and a significant and strong correlation of sample weight and DNA yield, while the DNA yields of the Blood & Tissue kit and HotSHOT method did not correlate with the sample weights. The repeatability of the community composition measurements were also better in the samples extracted with the PowerPlant Kit than in the samples extracted with the HotSHOT method or the Blood & Tissue Kit, and subsampling using Blood & Tissue kit and HotSHOT extraction failed to describe the same community in the lake samples. For extraction of DNA from crude bulk samples of freshwater macroinvertebrates with high specimen abundance, we recommend using a DNA extraction protocol that removes potential PCR-inhibitors. But low-abundance samples are less time-consuming to sort and will likely provide more stable results if specimens are sorted prior to DNA extraction.