Document Type
Article
Publication Date
6-8-2024
Abstract
Recent microbiome research has incorporated a higher number of samples through more participants in a study, longitudinal studies, and metanalysis between studies. Physical limitations in a sequencing machine can result in samples spread across sequencing runs. Here we present the results of sequencing nearly 1000 16S rRNA gene sequences in fecal (stabilized and swab) and oral (swab) samples from multiple human microbiome studies and positive controls that were conducted with identical standard operating procedures. Sequencing was performed in the same center across 18 different runs. The simplified mock community showed limitations in accuracy, while precision (e.g., technical variation) was robust for the mock community and actual human positive control samples. Technical variation was the lowest for stabilized fecal samples, followed by fecal swab samples, and then oral swab samples. The order of technical variation stability was inverse of DNA concentrations (e.g., highest in stabilized fecal samples), highlighting the importance of DNA concentration in reproducibility and urging caution when analyzing low biomass samples. Coefficients of variation at the genus level also followed the same trend for lower variation with higher DNA concentrations. Technical variation across both sample types and the two human sampling locations was significantly less than the observed biological variation. Overall, this research providing comparisons between technical and biological variation, highlights the importance of using positive controls, and provides semi-quantified data to better understand variation introduced by sequencing runs.
Source Publication
Applied Microbiology and Biotechnology
Recommended Citation
Hoisington, A.J., Stamper, C.E., Ellis, J.C. et al. Quantifying variation across 16S rRNA gene sequencing runs in human microbiome studies. Appl Microbiol Biotechnol 108, 367 (2024). https://doi-org.afit.idm.oclc.org/10.1007/s00253-024-13198-z
Comments
This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2024.
This article is published by SpringerNature, licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Funding note: U.S. Department of Veterans Affairs (U.S. Department of Veterans Affairs) Mindsource Brain Injury Network (140755)
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