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PUBLICATIONS

A Sensitivity Analysis of Methodological Variables Associated with Microbiome Measurements

Published

Author(s)

Samuel Forry, Stephanie Servetas, Jennifer Dootz, Monique Hunter, Jason Kralj, James Filliben, Scott Jackson

Abstract

The particular experimental methods employed during metagenomic sequencing analyses of microbiome samples can significantly impact the resulting data but typically vary substantially between laboratories. In this study, a full factorial experimental design was used to compare the effects of a select set of methodological choices (sample, operator, lot, extraction kit, variable region, reference database) on the analysis of biologically diverse stool samples. For each parameter investigated, a main effect was calculated that allowed direct comparison both between methodological choices (bias effect) and between samples (real biological differences). Overall, methodological bias was found to be similar in magnitude to real biological differences, while also exhibiting significant taxa-to-taxa differences even between closely related genera. This investigation demonstrates a framework for quantitatively assessing the impact of methodological choices that could be routinely performed by individual laboratories to better understand their metagenomic sequencing measurements.
Citation
Scientific Reports
Pub Type
Journals

Download Paper

https://doi.org/10.1128/spectrum.00696-24
Local Download

Keywords

Microbiome, metagenomic sequencing, experimental design
Uncertainty quantification, Statistical analysis, Reference materials, Reference data, Microbial measurements, Human microbiome, Genomics, Experiment design, Documentary standards, Bioscience, Biometrology and Bioinformatics

Citation

Forry, S. , Servetas, S. , Dootz, J. , Hunter, M. , Kralj, J. , Filliben, J. and Jackson, S. (2025), A Sensitivity Analysis of Methodological Variables Associated with Microbiome Measurements, Scientific Reports, [online], https://doi.org/10.1128/spectrum.00696-24, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=956856 (Accessed May 21, 2026)
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Created January 14, 2025, Updated May 20, 2026
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