Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

Abstract

Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5' end, allowing for a range of different 3' primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies. IMPORTANCE We continue to uncover a wealth of information connecting microbes in important ways to human and environmental ecology. As our scientific knowledge and technical abilities improve, the tools used for microbiome surveys can be modified to improve the accuracy of our techniques, ensuring that we can continue to identify groundbreaking connections between microbes and the ecosystems they populate, from ice caps to the human body. It is important to confirm that modifications to these tools do not cause new, detrimental biases that would inhibit the field rather than continue to move it forward. We therefore demonstrated that two recently modified primer pairs that target taxonomically discriminatory regions of bacterial and fungal genomic DNA do not introduce new biases when used on a variety of sample types, from soil to human skin. This confirms the utility of these primers for maintaining currently recommended microbiome research techniques as the state of the art.

Keywords: 16S; ITS; marker genes; microbial ecology; primers.

Figures

FIG 1

Comparison of the original 515f/806r primer pair and the new, modified 515f/806rB primer pair. (A) Procrustes plot of original and modified 515f/806r constructs, with unweighted UniFrac metric, M2 = 0.111. (B) Original and modified 515f/806r constructs, weighted UniFrac metric M2 = 0.196. (C) Pie charts illustrating the mean relative abundance of phyla present (all studies combined) in samples amplified with the old 515f/806r construct or with the modified 515f/806rB construct. (D) Taxa scatterplots for the original and modified 515f/806r primers. Phyla plots are shown for American Gut fecal, American Gut skin, agricultural soils, EMP Rice Rhizome, Body Farm 1, Body Farm 2, mouse decomposition, and Sloan built environment samples. Outlier samples have been removed from the results shown.

FIG 2

Comparison of the modified 515f/806rB V4 primer pair and the 515f (modified)/926r primer pair. (A) Procrustes plot of modified 515f/806r and 515f/926r constructs, Bray-Curtis dissimilarity M2 = 0.058. (B) Pie charts illustrating the mean relative abundance of phyla present (all studies combined) in samples amplified with the modified 515f/806rB construct or with the 515f/926r construct. (C) Taxa scatterplots for the modified 515f/806rB construct and the 515f/926r construct. Phyla plots are shown for American Gut fecal, American Gut skin, agricultural soils, EMP Rice Rhizome, Body Farm 1, Body Farm 2, mouse decomposition, and Sloan built environment samples. Outlier samples have not been removed from the data shown.

FIG 3

Comparison of the original ITS primer pair and the new, modified ITS primer pair. (A) Procrustes plot of original and modified ITS1 constructs, Bray-Curtis dissimilarity M2 = 0.363. (B) Pie charts illustrating the mean relative abundance of phyla present (all studies combined) in samples amplified with the old ITS construct and the modified ITS construct. (C) Taxa scatterplots for the original and modified ITS primers. Phyla plots are shown for American Gut fecal, American Gut skin, agricultural soils, EMP Rice Rhizome, Body Farm 1, Body Farm 2, mouse decomposition, and Sloan built environment samples. Outlier samples have been removed from the data shown.