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

References

Dec 1, 1990·Molecular and Cellular Probes·K H Rand, H Houck
May 2, 2000·Journal of Clinical Microbiology·C E CorlessA J Fox
Mar 28, 2002·Applied and Environmental Microbiology·Leonid A KulakovJohn F O'Hanlon
Aug 6, 2002·Journal of Industrial Microbiology & Biotechnology·M B McAlisterK L Ogden
Jun 6, 2006·Journal of Molecular Evolution·Jason EvansJames Foster
Jul 6, 2006·Applied and Environmental Microbiology·T Z DeSantisG L Andersen
Jun 30, 2009·Applied and Environmental Microbiology·Larry M FeinsteinChristopher B Blackwood
Nov 7, 2009·Science·Elizabeth K CostelloRob Knight
Jan 19, 2010·Proceedings of the National Academy of Sciences of the United States of America·William J BrazeltonJohn A Baross
Jun 7, 2011·The Open Microbiology Journal·Birgitte SmithKaren Angeliki Krogfelt
Jun 28, 2011·Bioinformatics·Robert C EdgarRob Knight
Sep 14, 2011·Annual Review of Pathology·Kathryn J Pflughoeft, James Versalovic
Jun 16, 2012·Nature·UNKNOWN Human Microbiome Project Consortium
Jul 5, 2012·Applied Microbiology and Biotechnology·Feng Guo, Tong Zhang
Aug 17, 2012·Nutrition Reviews·Willem M de Vos, Elisabeth A J de Vos
Nov 30, 2012·Nucleic Acids Research·Christian QuastFrank Oliver Glöckner
Oct 30, 2015·Science·A P AlivisatosUNKNOWN Unified Microbiome Initiative Consortium
Aug 24, 2016·Frontiers in Nutrition·Jacobo de la Cuesta-Zuluaga, Juan S Escobar

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Citations

Jan 16, 2019·Current Protocols in Mouse Biology·Máximo E LangeG Douglas Inglis
Dec 18, 2019·Clinical Chemistry·Robert Schlaberg
Jun 7, 2019·Foodborne Pathogens and Disease·Heather A CarletonPeter Gerner-Smidt
May 7, 2019·Frontiers in Microbiology·Kristýna FiedorováTomáš Freiberger
Apr 19, 2019·FEMS Microbiology Ecology·Bastian V H HornungEd J Kuijper
Mar 14, 2021·Forensic Science International. Genetics·Ana NeckovicAnnalisa Durdle
Apr 15, 2021·Reproductive Biomedicine Online·Anna OberleMichael Feichtinger
May 15, 2021·Science & Justice : Journal of the Forensic Science Society·Ana NeckovicAnnalisa Durdle
Jul 3, 2021·Foods·Cristina SánchezMaría Paz Lorenzo

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Datasets Mentioned

BETA
PRJNA385915

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

MetaHIT
UCHIME
R
MoBio
Mothur
PERMANOVAs
Clearcut
PERMANOVA
GUniFrac
Silva

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