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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Nature Publishing Group
2021-05-01
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Series: | Scientific Reports |
Online Access: | https://doi.org/10.1038/s41598-021-89881-2 |
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Article |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
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 |
spellingShingle |
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 Scientific Reports |
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 Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2021-05-01 |
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. |
url |
https://doi.org/10.1038/s41598-021-89881-2 |
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doaj-8468c62cda324455a0cd7a1fb1c847ee2021-05-23T11:31:31ZengNature Publishing GroupScientific Reports2045-23222021-05-0111111410.1038/s41598-021-89881-2An inter-laboratory study to investigate the impact of the bioinformatics component on microbiome analysis using mock communitiesDenise M. O’Sullivan0Ronan M. Doyle1Sasithon Temisak2Nicholas Redshaw3Alexandra S. Whale4Grace Logan5Jiabin Huang6Nicole Fischer7Gregory C. A. Amos8Mark D. Preston9Julian R. Marchesi10Josef Wagner11Julian Parkhill12Yair Motro13Hubert Denise14Robert D. Finn15Kathryn A. Harris16Gemma L. Kay17Justin O’Grady18Emma Ransom-Jones19Huihai Wu20Emma Laing21David J. Studholme22Ernest Diez Benavente23Jody Phelan24Taane G. Clark25Jacob Moran-Gilad26Jim F. Huggett27Molecular Biology, National Measurement Laboratory, LGCDepartment of Microbiology, Virology and Infection Control, Great Ormond Street Hospital for Children NHS TrustMolecular Biology, National Measurement Laboratory, LGCMolecular Biology, National Measurement Laboratory, LGCMolecular Biology, National Measurement Laboratory, LGCDepartment of Infection, Immunity and Inflammation, UCL Great Ormond Street Institute of Child Health and Reubens Centre of Paediatric Virology and MetagenomicsInstitute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, UKEInstitute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, UKEDepartment of Bacteriology, TDI, National Institute for Biological Standards and ControlDepartment of Bacteriology, TDI, National Institute for Biological Standards and ControlSchool of Biosciences, Cardiff UniversityPathogens and Microbes, Wellcome Sanger Institute, Wellcome Genome CampusPathogens and Microbes, Wellcome Sanger Institute, Wellcome Genome CampusDepartment of Health System Management, School of Public Health, Faculty of Health Sciences, Ben-Gurion University of the NegevEuropean Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome CampusEuropean Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome CampusDepartment of Microbiology, Virology and Infection Control, Great Ormond Street Hospital for Children NHS TrustMedical Microbiology Research Laboratory, Bob Champion Research and Educational Building, University of East AngliaMedical Microbiology Research Laboratory, Bob Champion Research and Educational Building, University of East AngliaDepartment of Biological and Geographical Sciences, School of Applied Sciences, University of HuddersfieldSchool of Biosciences, University of SurreySchool of Biosciences, University of SurreyBiosciences, University of ExeterFaculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical MedicineFaculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical MedicineFaculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical MedicineDepartment of Health System Management, School of Public Health, Faculty of Health Sciences, Ben-Gurion University of the NegevMolecular Biology, National Measurement Laboratory, LGCAbstract 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.https://doi.org/10.1038/s41598-021-89881-2 |