Evaluating and Improving SSU rRNA PCR Primer Coverage for Bacteria, Archaea, and Eukaryotes Using Metagenomes from Global Ocean Surveys

Small subunit ribosomal RNA (SSU rRNA) amplicon sequencing can quantitatively and comprehensively profile natural microbiomes, representing a critically important tool for studying diverse global ecosystems. However, results will only be accurate if PCR primers perfectly match the rRNA of all organisms present. To evaluate how well marine microorganisms across all 3 domains are detected by this method, we compared commonly-used primers with > 300 million rRNA gene sequences retrieved from globally-distributed marine metagenomes. The best-performing primers when comparing to 16S rRNA of Bacteria and Archaea were 515Y/926R and 515Y/806RB, which perfectly matched over 96% of all sequences. Considering Cyanobacteria and Chloroplast 16S rRNA, 515Y/926R had the highest coverage (99%), making this set ideal for quantifying marine primary producers. For eukaryotic 18S rRNA sequences, 515Y/926R also performed best (88%), followed by V4R/V4RB (18S rRNA-specific; 82%) - demonstrating that the 515Y/926R combination performs best overall for all 3 domains. Using Atlantic and Pacific Ocean samples, we demonstrate high correspondence between 515Y/926R amplicon abundances (generated for this study) and metagenomic 16S rRNA (median R2=0.98, n=272), indicating amplicons can produce equally accurate community composition data versus shotgun metagenomics. Our analysis also revealed that expected performance of all primer sets could be improved with minor modifications, pointing toward a nearly-completely universal primer set that could accurately quantify biogeochemically-important taxa in ecosystems ranging from the deep-sea to the surface. In addition, our reproducible bioinformatic workflow can guide microbiome researchers studying different ecosystems or human health to similarly improve existing primers and generate more accurate quantitative amplicon data.