Soil protist richness and composition affected by primer pair and annealing temperature

Protists are a diverse and understudied group of microbial eukaryotic organisms especially in terrestrial environments. Advances in molecular methods are increasing our understanding of the distribution and functions of these creatures; however, there is a vast array of choices researchers make including barcoding genes, primer pairs, PCR settings, and bioinformatic choices that can impact the outcome of protist community surveys. Here, we tested four commonly used primer pairs targeting the V4 and V9 regions of the 18S rRNA gene using different PCR annealing temperatures and processed the sequences with different bioinformatic parameters in ten diverse soils to evaluate how primer pair, amplification parameters, and bioinformatic choices influence the composition and richness of target and non-target taxa using Illumina sequencing. Our results showed that annealing temperature influenced sequencing depth and target species richness for most primer pairs, and that merging forward and reverse sequencing reads for the V4 primer pairs dramatically reduced the number of sequences and species richness of protists. The composition of protist taxa was similar between datasets of primers targeting the same 18S rRNA gene region (e.g., V4 or V9); however, datasets from primers targeting the V4 18S rRNA region resolved 87% of the total number of protist taxa in all datasets compared to those prepared with primers targeting the V9 18S rRNA region (21.5%). There was limited overlap of protist taxa between datasets targeting the two different gene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affect the results and conclusions about protist diversity and community composition.