Evaluation of different primers of the 18S rRNA gene to profile amoeba communities in environmental samples

Amoeboid protists, an assemblage of organisms belonging to different phylogenetic lineages, have drawn increasing attention due to their crucial ecological roles in various environments and their potential health risks. Currently, 18S rRNA gene sequencing is widely applied for the detection of amoebae. However, it is not clear which is the best primer pair for 18S rRNA gene amplification in amoebae. This study compared the four most commonly used primer pairs for revealing the diversity, composition, core species, and community assembly processes of amoebae in water and sediments. We found that the choice of primers artificially influences the detection of community composition of amoebae. We also found that short-read fragments may lead to mismatches in taxonomy and were not suitable for phylogenetic analyses. In contrast, full-length primers could detect the highest number of amoeba lineages and annotate 80% of reads belonging to amoebae to known species. However, full-length primers did not detect as many amoeba species as V4 primers. Moreover, we showed that beta diversity and community assembly determination were largely unaffected by primer choice, but different primers could influence our interpretations of the ecological process underlying stochasticity and determinism. This study indicates that full-length read sequencing and V4 region Illumina sequencing are suitable for profiling amoeba diversity in the environment.