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

By William Walters, Embriette R. Hyde, Donna berg-Lyons, Gail L Ackermann, Gregory Humphrey, A. Parada, Jack A. Gilbert, J. Jansson, J. Gregory Caporaso, Jed A. Fuhrman and 4 more. mSystems, 2015

1,905 citations recorded by the source corpus.

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. 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.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/eae34f0c44ee2c66f7381765931f0a9798fef0e0

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Citations in the corpus

Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data (2017, methodology): “Sample DNA was extracted with the PowerSoil®-HTP 96 well Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) and then PCR amplified in 2–4 replicate 75-μL reactions using Golay barcoded primers targeting the V4 region of the bacterial 16S rRNA gene [51].”
Bifidobacteria-mediated immune system imprinting early in life (2020, methodology): “Briefly, the V4 region of the 16S rRNA genewas amplified and sequenced using primers 515f and 806r as previously described with recent modifications (Caporaso et al., 2011; Walters et al., 2015).”
Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease (2016, positive): “Briefly, DNA was extracted using a MoBio Power soil kit (Carlsbad, CA), and the V4 region of the 16S rRNA gene was amplified using barcoded primers (Walters et al., 2016).”
Deterministic selection dominates microbial community assembly in termite mounds (2020, methodology): “The bacterial 16S rRNA gene and fungal ITS region were ampli ed with the primers 515FmodF/806RmodR [45] and ITS1F/ITS2R [46], respectively, on the CFX96 TouchTM PCR Detection System (Bio-Rad, Herculers, USA).”
The Fungal Mycobiome Promotes Pancreatic Oncogenesis via MBL Activation (2019, positive): “PCR was initially performed using the primer set ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA −3’) and ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’) 14 ; each with overhang adapter sequences (IDT, Coralville, IA) using 2x Kapa HiFi Hotstart ReadyMix DNA polymerase (KapaBiosystems, Wilmington, MA).”
Microbiome Helper: a Custom and Streamlined Workflow for Microbiome Research (2017, methodology): “The bacterial HMP Mock Community “B” sample (BEI Resources) was processed using 16S V4-V5 (515F 926R [28]) and V6-V8 (B969F BA1406R [27]) primer sets through our on-bench and bioinformatics workflow.”