An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities

Abstract Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to...

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Autores principales:	Denise M. O’Sullivan, Ronan M. Doyle, Sasithon Temisak, Nicholas Redshaw, Alexandra S. Whale, Grace Logan, Jiabin Huang, Nicole Fischer, Gregory C. A. Amos, Mark D. Preston, Julian R. Marchesi, Josef Wagner, Julian Parkhill, Yair Motro, Hubert Denise, Robert D. Finn, Kathryn A. Harris, Gemma L. Kay, Justin O’Grady, Emma Ransom-Jones, Huihai Wu, Emma Laing, David J. Studholme, Ernest Diez Benavente, Jody Phelan, Taane G. Clark, Jacob Moran-Gilad, Jim F. Huggett
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8468c62cda324455a0cd7a1fb1c847ee
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spelling	oai:doaj.org-article:8468c62cda324455a0cd7a1fb1c847ee2021-12-02T16:51:26ZAn inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities10.1038/s41598-021-89881-22045-2322https://doaj.org/article/8468c62cda324455a0cd7a1fb1c847ee2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-89881-2https://doaj.org/toc/2045-2322Abstract Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.Denise M. O’SullivanRonan M. DoyleSasithon TemisakNicholas RedshawAlexandra S. WhaleGrace LoganJiabin HuangNicole FischerGregory C. A. AmosMark D. PrestonJulian R. MarchesiJosef WagnerJulian ParkhillYair MotroHubert DeniseRobert D. FinnKathryn A. HarrisGemma L. KayJustin O’GradyEmma Ransom-JonesHuihai WuEmma LaingDavid J. StudholmeErnest Diez BenaventeJody PhelanTaane G. ClarkJacob Moran-GiladJim F. HuggettNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Denise M. O’Sullivan Ronan M. Doyle Sasithon Temisak Nicholas Redshaw Alexandra S. Whale Grace Logan Jiabin Huang Nicole Fischer Gregory C. A. Amos Mark D. Preston Julian R. Marchesi Josef Wagner Julian Parkhill Yair Motro Hubert Denise Robert D. Finn Kathryn A. Harris Gemma L. Kay Justin O’Grady Emma Ransom-Jones Huihai Wu Emma Laing David J. Studholme Ernest Diez Benavente Jody Phelan Taane G. Clark Jacob Moran-Gilad Jim F. Huggett An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
description	Abstract Despite the advent of whole genome metagenomics, targeted approaches (such as 16S rRNA gene amplicon sequencing) continue to be valuable for determining the microbial composition of samples. Amplicon microbiome sequencing can be performed on clinical samples from a normally sterile site to determine the aetiology of an infection (usually single pathogen identification) or samples from more complex niches such as human mucosa or environmental samples where multiple microorganisms need to be identified. The methodologies are frequently applied to determine both presence of micro-organisms and their quantity or relative abundance. There are a number of technical steps required to perform microbial community profiling, many of which may have appreciable precision and bias that impacts final results. In order for these methods to be applied with the greatest accuracy, comparative studies across different laboratories are warranted. In this study we explored the impact of the bioinformatic approaches taken in different laboratories on microbiome assessment using 16S rRNA gene amplicon sequencing results. Data were generated from two mock microbial community samples which were amplified using primer sets spanning five different variable regions of 16S rRNA genes. The PCR-sequencing analysis included three technical repeats of the process to determine the repeatability of their methods. Thirteen laboratories participated in the study, and each analysed the same FASTQ files using their choice of pipeline. This study captured the methods used and the resulting sequence annotation and relative abundance output from bioinformatic analyses. Results were compared to digital PCR assessment of the absolute abundance of each target representing each organism in the mock microbial community samples and also to analyses of shotgun metagenome sequence data. This ring trial demonstrates that the choice of bioinformatic analysis pipeline alone can result in different estimations of the composition of the microbiome when using 16S rRNA gene amplicon sequencing data. The study observed differences in terms of both presence and abundance of organisms and provides a resource for ensuring reproducible pipeline development and application. The observed differences were especially prevalent when using custom databases and applying high stringency operational taxonomic unit (OTU) cut-off limits. In order to apply sequencing approaches with greater accuracy, the impact of different analytical steps needs to be clearly delineated and solutions devised to harmonise microbiome analysis results.
format	article
author	Denise M. O’Sullivan Ronan M. Doyle Sasithon Temisak Nicholas Redshaw Alexandra S. Whale Grace Logan Jiabin Huang Nicole Fischer Gregory C. A. Amos Mark D. Preston Julian R. Marchesi Josef Wagner Julian Parkhill Yair Motro Hubert Denise Robert D. Finn Kathryn A. Harris Gemma L. Kay Justin O’Grady Emma Ransom-Jones Huihai Wu Emma Laing David J. Studholme Ernest Diez Benavente Jody Phelan Taane G. Clark Jacob Moran-Gilad Jim F. Huggett
author_facet	Denise M. O’Sullivan Ronan M. Doyle Sasithon Temisak Nicholas Redshaw Alexandra S. Whale Grace Logan Jiabin Huang Nicole Fischer Gregory C. A. Amos Mark D. Preston Julian R. Marchesi Josef Wagner Julian Parkhill Yair Motro Hubert Denise Robert D. Finn Kathryn A. Harris Gemma L. Kay Justin O’Grady Emma Ransom-Jones Huihai Wu Emma Laing David J. Studholme Ernest Diez Benavente Jody Phelan Taane G. Clark Jacob Moran-Gilad Jim F. Huggett
author_sort	Denise M. O’Sullivan
title	An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
title_short	An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
title_full	An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
title_fullStr	An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
title_full_unstemmed	An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
title_sort	inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/8468c62cda324455a0cd7a1fb1c847ee
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An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communities

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