DNA reference reagents isolate biases in microbiome profiling: a global multi-lab study
mSystems, vol. 10, pp. e0046625
Abstract
When profiling the human gut microbiome, technical biases introduced by analytical approaches impede translational research, reducing data reliability and study comparability. Here, through a global study involving 23 labs, we analyzed a wide range of sequencing and bioinformatic approaches for the taxonomic profiling of two well-defined DNA reference reagents (RRs) comprised of 20 common gut bacteria. Through both shotgun and 16S rRNA gene amplicon sequencing, we aimed to isolate sources of bias and understand their impact on microbiome profiling accuracy. Importantly, minimum quality criteria (MQC) were established and are used to evaluate profiling performance. We found that the variability of shotgun sequencing data sets was greater than that of 16S rRNA gene amplicon sequencing and isolated sources of bias in wet and dry lab steps, such as sequencing depth, primer and database choices, rarefaction, and 16S copy number adjustment. This study presents well-defined RRs and MQC to combat technical bias, paving the way for reliable and comparable microbiome research.IMPORTANCEThis benchmark paper highlights the true level of variability in microbiome data across the world and across sectors, underscoring the critical need for the use of WHO International DNA Gut Reference Reagents (RRs) to elevate the quality of data in microbiome research. This global study is the first of its kind, revealing the reality of the bias in the field, comprehensively testing methodologies used by leading laboratories across the world, but also providing avenues for workflow optimization, to accelerate innovation and translational research and move the field forward.
Authors 89
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Medicines and Healthcare products Regulatory Agency
Affiliation as printed
Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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Medicines and Healthcare products Regulatory Agency
Affiliation as printed
Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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Medicines and Healthcare products Regulatory Agency
Affiliation as printed
Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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Medicines and Healthcare products Regulatory Agency
Affiliation as printed
Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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UNSW Sydney · St George Hospital
Affiliation as printed
UNSW Microbiome Research Centre, St George Hospital
UNSW Microbiome Research Centre, St George Hospital, Kogarah, New South Wales, Australia
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Affiliation as printed
NSW Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales
NSW Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia
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Affiliation as printed
Diagnostic & Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz
Diagnostic & Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria
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Medical University of Graz · BioTechMed-Graz
Affiliation as printed
BioTechMed-Graz
Diagnostic & Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz
BioTechMed-Graz, Graz, Austria
Diagnostic & Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria
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Affiliation as printed
Core Facility Molecular Biology, Center for medical research, Medical University of Graz
Core Facility Molecular Biology, Center for medical research, Medical University of Graz, Graz, Austria
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Affiliation as printed
Core Facility Molecular Biology, Center for medical research, Medical University of Graz
Core Facility Molecular Biology, Center for medical research, Medical University of Graz, Graz, Austria
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University of Vienna · Medical University of Vienna
Affiliation as printed
Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, UBB University of Vienna
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna
Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, UBB University of Vienna, Vienna, Austria
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna, Vienna, Austria
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University of Vienna · Medical University of Vienna
Affiliation as printed
Department of Laboratory Medicine, Medical University of Vienna
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna
Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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University of Vienna · Medical University of Vienna
Affiliation as printed
Department of Laboratory Medicine, Medical University of Vienna
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna
Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria
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University of Vienna · Medical University of Vienna
Affiliation as printed
Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, UBB University of Vienna
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna
Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, UBB University of Vienna, Vienna, Austria
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna, Vienna, Austria
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University of Vienna · Medical University of Vienna
Affiliation as printed
Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, UBB University of Vienna
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna
Department of Microbiology and Ecosystem Science, Centre for Microbiology and Environmental Systems Science, UBB University of Vienna, Vienna, Austria
Joint Microbiome Facility of the Medical University of Vienna, University of Vienna, Vienna, Austria
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Affiliation as printed
Department of Molecular and Cellular Biology, University of Guelph
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Affiliation as printed
Department of Molecular and Cellular Biology, University of Guelph
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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Clinical Microbiomics (Denmark)
Affiliation as printed
Clinical Microbiomics A/S
Clinical Microbiomics A/S, Copenhagen, Denmark
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Clinical Microbiomics (Denmark)
Affiliation as printed
Clinical Microbiomics A/S
Clinical Microbiomics A/S, Copenhagen, Denmark
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Clinical Microbiomics (Denmark)
Affiliation as printed
Clinical Microbiomics A/S
Clinical Microbiomics A/S, Copenhagen, Denmark
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Clinical Microbiomics (Denmark)
Affiliation as printed
Clinical Microbiomics A/S
Clinical Microbiomics A/S, Copenhagen, Denmark
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Universität Hamburg · German Rheumatism Research Centre · University Medical Center Hamburg-Eppendorf
Affiliation as printed
Bioinformatics and Computational Biology, University Heart & Vascular Centre Hamburg, University Medical Centre Hamburg-Eppendorf
German Rheumatology Research Center Berlin, A Leibniz Institute
Bioinformatics and Computational Biology, University Heart & Vascular Centre Hamburg, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
German Rheumatology Research Center Berlin, A Leibniz Institute, Berlin, Germany
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German Rheumatism Research Centre
Affiliation as printed
German Rheumatology Research Center Berlin, A Leibniz Institute
German Rheumatology Research Center Berlin, A Leibniz Institute, Berlin, Germany
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German Rheumatism Research Centre · Technische Universität Berlin
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Chair of Cytometry, Institute of Biotechnology, Technische Universität Berlin
German Rheumatology Research Center Berlin, A Leibniz Institute
German Rheumatology Research Center Berlin, A Leibniz Institute, Berlin, Germany
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Affiliation as printed
Probiotics Microbiome Convergence Center, Soonchunhyang University
Probiotics Microbiome Convergence Center, Soonchunhyang University, Asan-si, South Korea
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Affiliation as printed
Probiotics Microbiome Convergence Center, Soonchunhyang University
Probiotics Microbiome Convergence Center, Soonchunhyang University, Asan-si, South Korea
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Affiliation as printed
Probiotics Microbiome Convergence Center, Soonchunhyang University
Probiotics Microbiome Convergence Center, Soonchunhyang University, Asan-si, South Korea
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Affiliation as printed
Probiotics Microbiome Convergence Center, Soonchunhyang University
Probiotics Microbiome Convergence Center, Soonchunhyang University, Asan-si, South Korea
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Affiliation as printed
BiowaveW
Department of Obstetrics and Gynecology, Ewha College of Medicine
Probiotics Microbiome Convergence Center, Soonchunhyang University
Department of Obstetrics and Gynecology, Ewha College of Medicine, Seoul, South Korea
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National Institute of Genomic Medicine · Universidad Nacional Autónoma de México
Affiliation as printed
Instituto de Biotecnologia UNAM
Unidad de Vinculación Cientifica UNAM-INMEGEN, Instituto Nacional de Medicina Genomica
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National Institute of Genomic Medicine · Universidad Nacional Autónoma de México
Affiliation as printed
Instituto de Biotecnologia UNAM
Unidad de Vinculación Cientifica UNAM-INMEGEN, Instituto Nacional de Medicina Genomica
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Erasmus MC · Erasmus University Rotterdam
Affiliation as printed
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, the Netherlands
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Erasmus MC · Erasmus University Rotterdam
Affiliation as printed
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, the Netherlands
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Erasmus MC · Erasmus University Rotterdam
Affiliation as printed
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, the Netherlands
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Erasmus MC · Erasmus University Rotterdam
Affiliation as printed
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center
Laboratory of Population Genomics, Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, the Netherlands
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Anoecim Robecca Geelen Aachen Center for Microbiome Analyses and Therapeutics Department of Medical Microbiology
Leiden University Medical Center
Affiliation as printed
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands
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Quinten R. Ducarmon Aachen Center for Microbiome Analyses and Therapeutics Department of Medical Microbiology
Leiden University Medical Center
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Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands
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Wiep Klaas Smits Aachen Center for Microbiome Analyses and Therapeutics Department of Medical Microbiology
Leiden University Medical Center
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Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands
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Ed J. Kuijper Aachen Center for Microbiome Analyses and Therapeutics Department of Medical Microbiology
Leiden University Medical Center
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Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands
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Romy Daniëlle Zwittink Aachen Center for Microbiome Analyses and Therapeutics Department of Medical Microbiology
Leiden University Medical Center
Affiliation as printed
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center
Center for Microbiome Analyses and Therapeutics, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands
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Affiliation as printed
Department of Medical Microbiology, Infectious Diseases and Infection Prevention, School for Nutrition and Translational Research in Metabolism, Maastricht University
Department of Medical Microbiology, Infectious Diseases and Infection Prevention, School for Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands
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Affiliation as printed
Department of Medical Microbiology, Infectious Diseases and Infection Prevention, School for Nutrition and Translational Research in Metabolism, Maastricht University
Department of Medical Microbiology, Infectious Diseases and Infection Prevention, School for Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands
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Affiliation as printed
Department of Medical Microbiology, Infectious Diseases and Infection Prevention, School for Nutrition and Translational Research in Metabolism, Maastricht University
Department of Medical Microbiology, Infectious Diseases and Infection Prevention, School for Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, the Netherlands
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National Institute for Public Health and the Environment
Affiliation as printed
Center for infectious disease control, Dutch National Institute for Public Health and the Environment
Center for infectious disease control, Dutch National Institute for Public Health and the Environment, Bilthoven, the Netherlands
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National Institute for Public Health and the Environment
Affiliation as printed
Center for infectious disease control, Dutch National Institute for Public Health and the Environment
Center for infectious disease control, Dutch National Institute for Public Health and the Environment, Bilthoven, the Netherlands
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National Institute for Public Health and the Environment
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Center for infectious disease control, Dutch National Institute for Public Health and the Environment
Center for infectious disease control, Dutch National Institute for Public Health and the Environment, Bilthoven, the Netherlands
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Affiliation as printed
Department of Medical Microbiology, Faculty of Medicine, Kırklareli University
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Department of Genetics, Faculty of Veterinary Medicine, Harran University
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Affiliation as printed
Genomics Birmingham, Institute of Cancer & Genomic Sciences, University of Birmingham
Genomics Birmingham, Institute of Cancer & Genomic Sciences, University of Birmingham, Birmingham, United Kingdom
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Affiliation as printed
Genomics Birmingham, Institute of Cancer & Genomic Sciences, University of Birmingham
Genomics Birmingham, Institute of Cancer & Genomic Sciences, University of Birmingham, Birmingham, United Kingdom
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Quadram Institute · Earlham Institute
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Earlham Institute and Quadram Institute Bioscience
Earlham Institute and Quadram Institute Bioscience, Norwich, United Kingdom
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Quadram Institute · Earlham Institute
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Earlham Institute and Quadram Institute Bioscience
Earlham Institute and Quadram Institute Bioscience, Norwich, United Kingdom
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Medicines and Healthcare products Regulatory Agency
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Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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Medicines and Healthcare products Regulatory Agency
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Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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Medicines and Healthcare products Regulatory Agency
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Medicines and Healthcare Products Regulatory Agency
Medicines and Healthcare Products Regulatory Agency, London, United Kingdom
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Imperial College London
Imperial College London, London, United Kingdom
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Imperial College London
Imperial College London, London, United Kingdom
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Imperial College London
Imperial College London, London, United Kingdom
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AlphaBiomics Ltd
AlphaBiomics Ltd., London, United Kingdom
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AlphaBiomics Ltd
AlphaBiomics Ltd., London, United Kingdom
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AlphaBiomics Ltd
AlphaBiomics Ltd., London, United Kingdom
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Source BioScience (United Kingdom)
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Source BioScience UK LTD
Source BioScience UK LTD, Nottingham, United Kingdom
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Source BioScience (United Kingdom)
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Source BioScience UK LTD
Source BioScience UK LTD, Nottingham, United Kingdom
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Quadram Institute Bioscience-Gut Microbes and Health Research Programme
Quadram Institute Bioscience-Gut Microbes and Health Research Programme, Norwich, United Kingdom
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Quadram Institute Bioscience-Gut Microbes and Health Research Programme
Quadram Institute Bioscience-Gut Microbes and Health Research Programme, Norwich, United Kingdom
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Quadram Institute Bioscience-Gut Microbes and Health Research Programme
Quadram Institute Bioscience-Gut Microbes and Health Research Programme, Norwich, United Kingdom
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Affiliation as printed
Quadram Institute Bioscience-Gut Microbes and Health Research Programme
Quadram Institute Bioscience-Gut Microbes and Health Research Programme, Norwich, United Kingdom
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Affiliation as printed
Quadram Institute Bioscience-Gut Microbes and Health Research Programme
Quadram Institute Bioscience-Gut Microbes and Health Research Programme, Norwich, United Kingdom
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National Institute of Standards and Technology
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National Institute of Standards and Technology
National Institute of Standards and Technology, Gaithersburg, Maryland, USA
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National Institute of Standards and Technology
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National Institute of Standards and Technology
National Institute of Standards and Technology, Gaithersburg, Maryland, USA
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National Institute of Standards and Technology
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National Institute of Standards and Technology
National Institute of Standards and Technology, Gaithersburg, Maryland, USA
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National Institute of Standards and Technology
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National Institute of Standards and Technology
National Institute of Standards and Technology, Gaithersburg, Maryland, USA
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National Institute of Standards and Technology
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National Institute of Standards and Technology
National Institute of Standards and Technology, Gaithersburg, Maryland, USA
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National Institute of Standards and Technology
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National Institute of Standards and Technology
National Institute of Standards and Technology, Gaithersburg, Maryland, USA
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Affiliation as printed
Department of Physiology and Biophysics, Weill Cornell Medicine
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA
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Affiliation as printed
Department of Physiology and Biophysics, Weill Cornell Medicine
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA
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Affiliation as printed
Department of Physiology and Biophysics, Weill Cornell Medicine
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA
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Affiliation as printed
Department of Physiology and Biophysics, Weill Cornell Medicine
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA
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Affiliation as printed
Department of Physiology and Biophysics, Weill Cornell Medicine
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, New York, USA
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Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Affiliation as printed
Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Affiliation as printed
Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Affiliation as printed
Diversigen, Inc
Diversigen, Inc., New Brighton, Minnesota, USA
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Affiliation as printed
Illumina, Inc
Illumina, Inc., San Diego, California, USA
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Illumina, Inc
Illumina, Inc., San Diego, California, USA
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Affiliation as printed
Illumina, Inc
Illumina, Inc., San Diego, California, USA
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