Measurements of soil protist richness and community composition are influencedby primer pair, annealing temperature, and bioinformatics choices

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 options 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 differentPCR annealing temperatures and processed the sequences with differentbioinformatic parameters in 10 diverse soils to evaluate how primer pair, amplificationparameters, and bioinformatic choices influencethe composition and richness of protist and non-protist taxa using Illumina sequencing. Our results showed that annealing temperature influencedsequencing depth and protist taxon 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 taxon richness of protists. The data sets of primers that targeted the same 18S rRNA gene region (e.g., V4 or V9) had similar protist community compositions; however, data sets from primers targeting the V4 18S rRNA gene region detected a greater number of protist taxa compared to those prepared with primers targeting the V9 18S rRNA region. There was limited overlap of protist taxa between data sets targeting the two differentgene regions (80/549 taxa). Together, we show that laboratory and bioinformatic choices can substantially affectthe results and conclusions about protist diversity and community composition using metabarcoding.