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

Abstract

Small subunit ribosomal RNA (SSU rRNA) amplicon sequencing comprehensively profiles microbiomes, but results will only be accurate if PCR primers perfectly match environmental sequences. To evaluate whether primers commonly used in microbial oceanography match naturally-occurring organisms, we compared primers with > 300 million rRNA sequences retrieved from globally-distributed metagenomes. The best-performing 16S primers were 515Y/926R and 515Y/806RB which perfectly matched most sequences (~0.95). Considering Cyanobacteria/Chloroplast 16S, 515Y/926R had the highest coverage (0.99), making it ideal for quantifying phytoplankton. For 18S sequences, 515Y/926R performed best (0.88), followed by V4R/V4RB (18S-specific; 0.82). Using Atlantic and Pacific BioGEOTRACES field samples, we demonstrate high correspondence between 515Y/926R amplicons (generated as part of this study) and metagenomic 16S rRNA (median R2=0.98, n=272), indicating amplicons can produce equally accurate community composition data versus shotgun metagenomics. Since our pipeline identifies missed taxa, we suggest modifications to improve coverage of biogeochemically-important oceanic microorganisms - a strategy applicable to any environment with metagenomic data.Significance StatementQuantification of taxonomically-informative marker genes using PCR amplification and high-throughput sequencing is a low-cost technique for monitoring distributions and changes of microbial communities across space and time. In order to maximize this procedure’s effectiveness, it is essential that environmental organisms match PCR primer sequences exactly. In this study, we developed a software pipeline to evaluate how well commonly-used primers match primer-binding regions from globally-distributed short-read oceanic metagenomes. Our results demonstrate common primer sets vary widely in performance, and that including additional degenerate bases is a simple strategy to maximize environmental coverage. Written in the reproducible snakemake workflow language and publicly accessible, our pipeline provides a general-purpose tool to guide rational design of PCR primers for any environment with metagenomic data.

Comparison of Two 16S rRNA Primers (V3–V4 and V4–V5) for Studies of Arctic Microbial Communities

“…Therefore, the observed inconsistency in the representation of some taxonomic groups (i.e., SAR11 clade) may also result from these limitations. In order to further investigate the quantitative performance of the primer sets, further investigation, using techniques such as mock communities ( Yeh et al, 2019 ) or metagenomics ( McNichol et al, 2020 ), is required.…”

“…Therefore, the unique conditions and the currently available technologies constrain year-round microbial observations to PCR-based approaches (i.e., 16S rRNA gene amplicon sequencing), which can be realized with low concentrations of DNA ( Thomas et al, 2012 ). Metagenomics approaches suggest that the functional capacity of marine microbial communities is strongly linked to their taxonomic composition ( Galand et al, 2018 ; McNichol et al, 2020 ). Thus, when supported by curated taxonomic databases (e.g., SILVA 16S rRNA gene reference; Quast et al, 2013 ), 16S rRNA gene amplicon sequencing provides an affordable high-throughput tool for addressing traditional community ecology questions, especially under the constrained sampling conditions of the Arctic marine environment.…”

“…For the investigation of marine microbiomes, an alternative primer set 515F-Y/926R (targeting the V4–V5 hypervariable regions of the 16S rRNA gene), which is also able to capture the diversity of the archaeal communities, has been developed by Parada et al (2016) . Currently, both V3–V4 and V4–V5 primer sets are widely used in studies of marine microbial communities and were extensively tested using mock and natural communities of temperate waters (e.g., Wear et al, 2018 ; Willis et al, 2019 ; McNichol et al, 2020 ). However, no study has systematically tested the performance of these primer sets on microbial communities of the Arctic Ocean.…”

Long-term patterns of an interconnected core marine microbiota

“…OTUs assigned to Metazoa, Streptophyta, nucleomorph, chloroplast, and mitochondria were removed before further analysis. Archaea were removed from downstream analyses as the used primers are not optimal for recovering this domain [79].…”

Disentangling temporal associations in marine microbial networks

“…The PR2 classification was used when the taxonomic assignment from SILVA and PR2 disagreed. We removed ASVs that identified as Metazoa, Streptophyta, plastids, mitochondria, and Archaea since the 341F-primer was not optimal for recovering this domain (McNichol et al ., 2020).…”