Oct 29, 2017

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

Applied Microbiology and Biotechnology
Eliana P Velásquez-MejíaJuan S Escobar

Abstract

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....Continue Reading

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Mentioned in this Paper

DNA Contamination
Impacted Tooth
Chemical Extraction
Nucleic Acid Sequencing
Laboratory
Ribosomal RNA
Microbial Consortia
Evaluation
Metagenomics
Extraction

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