Comprehensive single-PCR 16S and 18S rRNA community analysis validated with mock communities and denoising algorithms

Abstract

Universal SSU rRNA primers allow comprehensive quantitative profiling of natural communities by simultaneously amplifying templates from Bacteria, Archaea, and Eukaryota in a single PCR reaction. Despite the potential to show all rRNA gene relative gene abundances, they are rarely used due to concerns about length bias against 18S amplicons and bioinformatic challenges converting mixed 16S/18S sequences into amplicon sequence variants. We thus developed 16S and 18S rRNA mock communities and a bioinformatic pipeline to validate this three-domain approach. To test for length biases, we mixed eukaryotic and prokaryotic mocks before PCR, and found consistent two-fold underestimation of longer 18S sequences due to sequencing but not PCR bias. Using these mocks, we show universal V4-V5 primers (515Y/926R) outperformed eukaryote-specific V4 primers in observed vs. expected abundance correlations and sequences with single mismatches to the primer were strongly underestimated (3-8 fold). A year of monthly time-series data from a protist-enriched 1.2-80 m size fraction yielded an average of 9% 18S, 17% chloroplast 16S, and 74% prokaryote 16S rRNA gene amplicons. These data demonstrate the potential for universal primers to generate quantitative and comprehensive microbiome profiles, although gene copy and genome size variability should be considered - as for any quantitative genetic analysis.