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

William Walters, Embriette R. Hyde, Donna Berg-Lyons, Gail Ackermann, Greg Humphrey, Alma Parada, Jack A. Gilbert, Janet K. Jansson, J. Gregory Caporaso, Jed A. Fuhrman, Amy Apprill, Rob Knight

Research output: Contribution to journalArticle

342 Scopus citations

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.

Original languageEnglish (US)
Article numbere00009-15
JournalmSystems
Volume1
Issue number1
DOIs
StatePublished - Jan 1 2016

Keywords

  • 16S
  • ITS
  • Marker genes
  • Microbial ecology
  • Primers

ASJC Scopus subject areas

  • Microbiology
  • Physiology
  • Biochemistry
  • Ecology, Evolution, Behavior and Systematics
  • Modeling and Simulation
  • Molecular Biology
  • Genetics
  • Computer Science Applications

Fingerprint Dive into the research topics of 'Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys'. Together they form a unique fingerprint.

  • Cite this

    Walters, W., Hyde, E. R., Berg-Lyons, D., Ackermann, G., Humphrey, G., Parada, A., Gilbert, J. A., Jansson, J. K., Gregory Caporaso, J., Fuhrman, J. A., Apprill, A., & Knight, R. (2016). Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys. mSystems, 1(1), [e00009-15]. https://doi.org/10.1128/mSystems.00009-15