A refined protocol for high throughput multiplexed sequencing of animal-associated eukaryotic diversity

The goal of this study was to develop and test a refined protocal for using "universal non-metazoan" PCR primers to amplify the V4 hypervariable region of the 18S gene. The 574*f-UNonMet_DB (Bass & Del Campo, 2020) and 574*f-1132r (Hugerth et al., 2014) primer sets were tested on a range of sample types to determine the proportion of metazoan sequences that were amplified in a real-world scenario. In addition, the effect of temperature and multiple stage amplification was also tested.