Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines

ABSTRACT When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the perf...

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Autores principales:	Q. R. Ducarmon, B. V. H. Hornung, A. R. Geelen, E. J. Kuijper, R. D. Zwittink
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	microbiota DNA extraction positive controls negative controls bioinformatics 16S rRNA gene amplicon sequencing Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c2fbe2ccd157439fb50811695e35b20b
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spelling	oai:doaj.org-article:c2fbe2ccd157439fb50811695e35b20b2021-12-02T18:15:47ZToward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines10.1128/mSystems.00547-192379-5077https://doaj.org/article/c2fbe2ccd157439fb50811695e35b20b2020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00547-19https://doaj.org/toc/2379-5077ABSTRACT When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples. IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.Q. R. DucarmonB. V. H. HornungA. R. GeelenE. J. KuijperR. D. ZwittinkAmerican Society for MicrobiologyarticlemicrobiotaDNA extractionpositive controlsnegative controlsbioinformatics16S rRNA gene amplicon sequencingMicrobiologyQR1-502ENmSystems, Vol 5, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	microbiota DNA extraction positive controls negative controls bioinformatics 16S rRNA gene amplicon sequencing Microbiology QR1-502
spellingShingle	microbiota DNA extraction positive controls negative controls bioinformatics 16S rRNA gene amplicon sequencing Microbiology QR1-502 Q. R. Ducarmon B. V. H. Hornung A. R. Geelen E. J. Kuijper R. D. Zwittink Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines
description	ABSTRACT When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples. IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.
format	article
author	Q. R. Ducarmon B. V. H. Hornung A. R. Geelen E. J. Kuijper R. D. Zwittink
author_facet	Q. R. Ducarmon B. V. H. Hornung A. R. Geelen E. J. Kuijper R. D. Zwittink
author_sort	Q. R. Ducarmon
title	Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines
title_short	Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines
title_full	Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines
title_fullStr	Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines
title_full_unstemmed	Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines
title_sort	toward standards in clinical microbiota studies: comparison of three dna extraction methods and two bioinformatic pipelines
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/c2fbe2ccd157439fb50811695e35b20b
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Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines

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