Effect of different DNA polymerases on 16S rDNA amplification for gut microbiota classification

Abstract

Background: Gut microbiota plays vital role in enhancing the intestinal immune system and influencing nutrient
metabolism. To study these microbial communities, 16S rDNA sequencing is commonly employed. However, the
variability in reagents used for 16S rDNA amplification, especially in terms of DNA polymerases, can affect the
quality and accuracy of the data.

Objective: This study aims to examine the effect of two commercially high-fidelity DNA polymerases on the gut
microbiota profile based on full-length 16S rDNA amplification.

Methods: Fecal samples from lung cancer patients at King Chulalongkorn Memorial Hospital were collected for
DNA extraction with the ZymoBIOMICSTM DNA Miniprep Kit. The extracted DNA was amplified using the Ultra
HiFidelity PCR Kit and KOD OneTM PCR Master Mix, then sequenced with Oxford Nanopore Technologies. The
filtered sequences were clustered, polished, and classified using NanoCLUST, and visualized by
MicrbiomeAnalyst.

Results: Gel electrophoresis showed that DNA polymerases from different PCR kits yielded identical PCR products.
The KOD OneTM PCR Master Mix (Toyobo) produced a more intense band, indicating possible overamplification,
which may have reduced microbial richness and certain bacterial taxa abundance. No significant differences were
noted in Shannon diversity, beta diversity, or the top 20 bacterial species’ relative abundance between the PCR
kits.

Conclusion: These results suggest that the choice of high-fidelity DNA polymerases can influence gut microbiota
diversity and classification. This highlights the importance of selecting appropriate PCR kits and optimizing
amplification conditions in future microbiome research.