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

被引:1496
作者
Walters, William [1 ]
Hyde, Embriette R. [2 ]
Berg-Lyons, Donna [3 ]
Ackermann, Gail [2 ]
Humphrey, Greg [2 ]
Parada, Alma [4 ]
Gilbert, Jack A. [5 ,6 ,7 ,8 ,9 ,10 ]
Jansson, Janet K. [11 ]
Caporaso, J. Gregory [12 ]
Fuhrman, Jed A. [4 ]
Apprill, Amy [13 ]
Knight, Rob [2 ,14 ,15 ]
机构
[1] Cornell Univ, Dept Mol Biol & Genet, Ithaca, NY USA
[2] Univ Calif San Diego, Dept Pediat, La Jolla, CA 92093 USA
[3] Univ Colorado, BioFrontiers Inst, Boulder, CO 80309 USA
[4] Univ Southern Calif, Dept Biol Sci, Los Angeles, CA 90089 USA
[5] Argonne Natl Lab, Biosci Div BIO, 9700 S Cass Ave, Argonne, IL 60439 USA
[6] Univ Chicago, Dept Ecol & Evolut, 940 E 57th St, Chicago, IL 60637 USA
[7] Univ Chicago, Dept Surg, 5841 S Maryland Ave, Chicago, IL 60637 USA
[8] Univ Chicago, Inst Genom & Syst Biol, Chicago, IL 60637 USA
[9] Marine Biol Lab, Woods Hole, MA 02543 USA
[10] Field Museum Nat Hist, Chicago, IL 60605 USA
[11] Pacific Northwest Natl Lab, Earth & Biol Sci Directorate, Richland, WA 99352 USA
[12] No Arizona Univ, Dept Biol Sci, Box 5640, Flagstaff, AZ 86011 USA
[13] Woods Hole Oceanog Inst, Dept Marine Chem & Geochem, Woods Hole, MA 02543 USA
[14] Univ Calif San Diego, Dept Comp Sci, La Jolla, CA 92093 USA
[15] Univ Calif San Diego, Dept Engn, La Jolla, CA 92093 USA
基金
美国国家科学基金会;
关键词
microbial ecology; marker genes; primers; 16S; ITS; DESIGN;
D O I
10.1128/mSystems.00009-15
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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.
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页数:10
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