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Comparing DNA Extraction and 16s Amplification Methods for Plant-Associated Bacterial Communities

Cecelia Giangacomo, Mohsen Mohseni, Lynsey Kovar, View ORCID ProfileJason G. Wallace
doi: https://doi.org/10.1101/2020.07.23.217901
Cecelia Giangacomo
1Department of Biology, University of Georgia, Athens, Georgia
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Mohsen Mohseni
2Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia
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Lynsey Kovar
3Institute of Bioinformatics, University of Georgia, Athens, Georgia
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Jason G. Wallace
2Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia
4Department of Crop & Soil Sciences, University of Georgia, Athens, Georgia
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  • ORCID record for Jason G. Wallace
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Abstract

Plant-associated microbes play important roles in global ecology and agriculture. The most common method to profile these microbial communities is amplicon sequencing of the bacterial 16s rRNA gene. Both the DNA extraction and PCR amplification steps of this process are subject to bias, especially since the latter requires some way to exclude DNA from plant organelles, which would otherwise dominate the sample. We compared several common DNA extraction kits and 16s rRNA amplification protocols to determine the relative biases of each and to make recommendations for plant microbial researchers. For DNA extraction, we found that, as expected, kits optimized for soil were the best for soil, though each still included a distinct “fingerprint” of its own biases. Plant samples were less clear, with different species having different “best” options. For 16s amplification, we find that using peptide nucleic acid (PNA) clamps provides the least taxonomic distortion, while chloroplast-discriminating primers are easy and inexpensive but present significant bias in the results. We do not recommend blocking oligos, as they involved a more complex protocol and showed significant taxonomic bias in the results. Further methods development will hopefully result in protocols that are even more reliable and less biased.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://www.github.com/wallacelab/paper-giangacomo-16s-methods

  • https://www.ncbi.nlm.nih.gov/bioproject/PRJNA646931

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted July 24, 2020.
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Comparing DNA Extraction and 16s Amplification Methods for Plant-Associated Bacterial Communities
Cecelia Giangacomo, Mohsen Mohseni, Lynsey Kovar, Jason G. Wallace
bioRxiv 2020.07.23.217901; doi: https://doi.org/10.1101/2020.07.23.217901
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Comparing DNA Extraction and 16s Amplification Methods for Plant-Associated Bacterial Communities
Cecelia Giangacomo, Mohsen Mohseni, Lynsey Kovar, Jason G. Wallace
bioRxiv 2020.07.23.217901; doi: https://doi.org/10.1101/2020.07.23.217901

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