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The Use of Degenerate Primers in qPCR Analysis of Functional Genes Can Cause Dramatic Quantification Bias as Revealed by Investigation of nifH Primer Performance

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Abstract

The measurement of functional gene abundance in diverse microbial communities often employs quantitative PCR (qPCR) with highly degenerate oligonucleotide primers. While degenerate PCR primers have been demonstrated to cause template-specific bias in PCR applications, the effect of such bias on qPCR has been less well explored. We used a set of diverse, full-length nifH gene standards to test the performance of several universal nifH primer sets in qPCR. We found significant template-specific bias in all but the PolF/PolR primer set. Template-specific bias caused more than 1000-fold mis-estimation of nifH gene copy number for three of the primer sets and one primer set resulted in more than 10,000-fold mis-estimation. Furthermore, such template-specific bias will cause qPCR estimates to vary in response to beta-diversity, thereby causing mis-estimation of changes in gene copy number. A reduction in bias was achieved by increasing the primer concentration. We conclude that degenerate primers should be evaluated across a range of templates, annealing temperatures, and primer concentrations to evaluate the potential for template-specific bias prior to their use in qPCR.

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Acknowledgements

This work was supported in part by the Cornell University NSF-IGERT program in Biogeochemistry and Environmental Biocomplexity, the NSF Microbial Observatories Program, grant number [MCB-0447586], and the Soil Processes Program of the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number [2005-35107-15266].

We would like to thank Dr. Sean Berthrong and Dr. Peter Bergholz for guidance with performing qPCR and Dr. Olena Vatamaniuk, Dr. Stephen Zinder, and Dr. Joel Kostka for use of their qPCR machines. We thank the staff of the USDA ARS Culture Collection for their service providing strains used in this work. We also thank those individuals who provided strains or DNA samples including Dr. Eric Triplett and Kelsey Gano, Dr. Jay Keasling, Dr. Aindrila Mukhopadhyay, and Eric Luning, Dr. Derek Lovley and Joy Ward, and Dr. David Benson and Ying Huang.

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  1. School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA

    John Christian Gaby & Daniel H. Buckley

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  1. John Christian Gaby
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Correspondence to Daniel H. Buckley.

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Gaby, J.C., Buckley, D.H. The Use of Degenerate Primers in qPCR Analysis of Functional Genes Can Cause Dramatic Quantification Bias as Revealed by Investigation of nifH Primer Performance. Microb Ecol 74, 701–708 (2017). https://doi.org/10.1007/s00248-017-0968-0

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