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A simple, cost-effective and automation-friendly direct PCR approach for bacterial community analysis

View ORCID ProfileFangchao Song, Jennifer V. Kuehl, Arjun Chandran, View ORCID ProfileAdam P. Arkin
doi: https://doi.org/10.1101/2021.03.01.433496
Fangchao Song
1Environmental Genomics & Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
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  • For correspondence: fsong@lbl.gov
Jennifer V. Kuehl
1Environmental Genomics & Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
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Arjun Chandran
2Department of Bioengineering, University of California at Berkeley, Berkeley, California, United States of America
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Adam P. Arkin
1Environmental Genomics & Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America
2Department of Bioengineering, University of California at Berkeley, Berkeley, California, United States of America
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ABSTRACT

Bacterial communities in water, soil, and humans play an essential role in environmental ecology and human health. PCR-based amplicon analysis, such as 16S ribosomal RNA sequencing, is a fundamental tool for quantifying and studying microbial composition, dynamics, and interactions. However, given the complexity of microbial communities, a substantial number of samples becomes necessary to analyses that parse the factors that determine microbial composition. A common bottleneck in performing these kinds of experiments is genomic DNA (gDNA) extraction, which is time-consuming, expensive, and often biased on the types of species. Direct PCR methods are a potentially simpler and more accurate alternative to gDNA extraction methods that do not require the intervening purification step. In this study, we evaluated three variations of direct PCR methods using diverse heterogeneous bacterial cultures, ZymoBIOMICS Microbial Community Standards, and groundwater. By comparing direct PCR methods with DNeasy blood and tissue kits and DNeasy Powersoil kits, we found a specific variant of the direct PCR method exhibits a comparable overall efficiency to the conventional DNeasy Powersoil protocol. We also found the method showed higher efficiency for extracting gDNA from the gram negative strains compared to DNeasy blood and tissue protocol. This direct PCR method is 1600 times cheaper ($0.34 for 96 samples), 10 times simpler (15 min hands-on time for 96 samples) than DNeasy Powersoil protocol. The direct PCR method can also be fully automated, and is compatible with small volume samples, thereby permitting scaling of samples and replicates needed to support high-throughput large-scale bacterial community analysis.

IMPORTANCE Understanding bacterial interaction and assembling in complex microbial communities using 16S ribosomal RNA sequencing normally requires a large experimental load. However, the current DNA extraction methods including cell disruption and genomic DNA purification are normally biased, costly, time and labor consuming, and not amenable to miniaturization by droplets or 1536 well plates due to the significant DNA loss during purification step for tiny volume and low cell density samples. Direct PCR method could potentially solve these problems. In this study, we demonstrate a direct PCR method which exhibits similar efficiency as the widely used method – DNeasy Powersoil protocol, while 1600 times cheaper and 10 times faster to execute. This simple, cost-effective, and automation friendly direct PCR based 16S ribosomal RNA sequencing method allows us to study the dynamics, microbial interaction and assembly of varying microbial communities in a high throughput fashion.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵† fsong{at}lbl.gov (FS); aparkin{at}lbl.gov (APA)

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-NC-ND 4.0 International license.
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Posted June 02, 2021.
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A simple, cost-effective and automation-friendly direct PCR approach for bacterial community analysis
Fangchao Song, Jennifer V. Kuehl, Arjun Chandran, Adam P. Arkin
bioRxiv 2021.03.01.433496; doi: https://doi.org/10.1101/2021.03.01.433496
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A simple, cost-effective and automation-friendly direct PCR approach for bacterial community analysis
Fangchao Song, Jennifer V. Kuehl, Arjun Chandran, Adam P. Arkin
bioRxiv 2021.03.01.433496; doi: https://doi.org/10.1101/2021.03.01.433496

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