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Impact of DNA extraction, sample dilution, and reagent contamination on 16S rRNA gene sequencing of human feces


Culture-independent methods have granted the possibility to study microbial diversity in great detail, but technical issues pose a threat to the accuracy of new findings. Biases introduced during DNA extraction can result in erroneous representations of the microbial community, particularly in samples with low microbial biomass. We evaluated the DNA extraction method, initial sample biomass, and reagent contamination on the assessment of the human gut microbiota. Fecal samples of 200 mg were subjected to 1:10 serial dilutions; total DNA was obtained using two commercial kits and the microbiota assessed by 16S ribosomal RNA (rRNA) gene sequencing. In addition, we sequenced multiple technical controls. The two kits were efficient in extracting DNA from samples with as low as 2 mg of feces. However, in instances of lower biomass, only one kit performed well. The number of reads from negative controls was negligible. Both DNA extraction kits allowed inferring microbial consortia with similar membership but different abundances. Furthermore, we found differences in the taxonomic profile of the microbial community. Unexpectedly, the effect of sample dilution was moderate and did not introduce severe bias into the microbial inference. Indeed, the microbiota inferred from fecal samples was distinguishable from that of negative controls. In most cases, samples as low as 2 mg did not result in a dissimilar representation of the microbial community compared with the undiluted sample. Our results indicate that the gut microbiota inference is not much affected by contamination with laboratory reagents but largely impacted by the protocol to extract DNA.

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We thank participants who agreed to donate samples, the APOLO Scientific Computing Center at EAFIT University for hosting part of the bioinformatic resources employed in analyses, and the University of Michigan Medical School Host Microbiome Initiative for sequencing support.


This work was funded by Grupo Empresarial Nutresa. The funder has not had any role in designing or conducting the study; in the analysis or interpretation of the data; in the writing, review, or approval of the manuscript; and in the decision to submit the manuscript for publication.

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Correspondence to Juan S. Escobar.

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The authors declare that they have no conflict of interest.

Ethical approval

This study was conducted in accordance with the principles of the Declaration of Helsinki and had minimal risk according to the Colombian Ministry of Health (Resolution 008430 of 1993). This study was conducted with approval from the Bioethics Committee of SIU—University of Antioquia (approbation act 14-24-588 dated May 28, 2014).

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The participants were assured of anonymity and confidentiality. Written informed consent was obtained from them before beginning the study.

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Velásquez-Mejía, E.P., de la Cuesta-Zuluaga, J. & Escobar, J.S. Impact of DNA extraction, sample dilution, and reagent contamination on 16S rRNA gene sequencing of human feces. Appl Microbiol Biotechnol 102, 403–411 (2018).

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  • Contaminants
  • DNA reads
  • Sample biomass
  • Gut microbiota
  • Microbiome