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Processing Method Affects Oral and Gut Microbiome Composition

OBJECTIVES: Conducting research on the microbiome has become prevalent, particularly in the area of precision nutrition as nutritional strategies targeting the microbiome are increasingly explored. However, differences in sample processing methods for generating microbial genetic sequencing data cou...

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Autores principales: Superdock, Dorothy, Poole, Angela
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9194270/
http://dx.doi.org/10.1093/cdn/nzac063.021
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author Superdock, Dorothy
Poole, Angela
author_facet Superdock, Dorothy
Poole, Angela
author_sort Superdock, Dorothy
collection PubMed
description OBJECTIVES: Conducting research on the microbiome has become prevalent, particularly in the area of precision nutrition as nutritional strategies targeting the microbiome are increasingly explored. However, differences in sample processing methods for generating microbial genetic sequencing data could influence observed microbial composition. The objective of this study was to process human oral and gut samples using several common methods to examine the influence of each on microbiota composition. METHODS: We collected fecal, saliva, and dental swab samples from 10 individuals. Fecal samples were 1) fresh, 2) lyophilized (freeze-dried), 3) frozen then ground in liquid nitrogen, or 4) stored at room temperature (RT) for 72 hours, frozen, then ground in liquid nitrogen. Saliva and dental swabs were 1) fresh (saliva), 2) subjected to the Human Microbiome Project (HMP) initial processing protocol (saliva), or 3) frozen whole upon collection and split into different fractions after thawing: supernatant, pellet, or mixed supernatant and pellet (both saliva and dental). We performed 16S rRNA gene sequencing, used QIIME2 to identify amplicon sequence variants (ASVs) and calculate diversity metrics, and used MaAsLin2 to evaluate the effect of treatment types on relative abundances of microbes. RESULTS: For fecal samples, we found significant differences in the relative abundances of 51 ASVs between fresh and lyophilized, four ASVs between fresh and liquid nitrogen post-72 hour at RT, and two ASVs between fresh and liquid nitrogen treated samples. There were differences among both saliva and dental swab fractions. For saliva, the relative abundances of 15 ASVs differed significantly between both mixed and pellet fractions and mixed and supernatant fractions. For dental swabs, the relative abundances of 19 ASVs differed significantly between mixed and pellet fractions and 10 between mixed and supernatant fractions. For all reported significant results, q < 0.05. CONCLUSIONS: Relative abundances are significantly affected by processing methods for both gut and oral microbiome samples. Therefore it is crucial to exercise caution when comparing microbial composition results across studies that use different processing methods. FUNDING SOURCES: Research reported in this abstract was supported by a NIH NIDDK T32 Award.
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spelling pubmed-91942702022-06-14 Processing Method Affects Oral and Gut Microbiome Composition Superdock, Dorothy Poole, Angela Curr Dev Nutr Methods OBJECTIVES: Conducting research on the microbiome has become prevalent, particularly in the area of precision nutrition as nutritional strategies targeting the microbiome are increasingly explored. However, differences in sample processing methods for generating microbial genetic sequencing data could influence observed microbial composition. The objective of this study was to process human oral and gut samples using several common methods to examine the influence of each on microbiota composition. METHODS: We collected fecal, saliva, and dental swab samples from 10 individuals. Fecal samples were 1) fresh, 2) lyophilized (freeze-dried), 3) frozen then ground in liquid nitrogen, or 4) stored at room temperature (RT) for 72 hours, frozen, then ground in liquid nitrogen. Saliva and dental swabs were 1) fresh (saliva), 2) subjected to the Human Microbiome Project (HMP) initial processing protocol (saliva), or 3) frozen whole upon collection and split into different fractions after thawing: supernatant, pellet, or mixed supernatant and pellet (both saliva and dental). We performed 16S rRNA gene sequencing, used QIIME2 to identify amplicon sequence variants (ASVs) and calculate diversity metrics, and used MaAsLin2 to evaluate the effect of treatment types on relative abundances of microbes. RESULTS: For fecal samples, we found significant differences in the relative abundances of 51 ASVs between fresh and lyophilized, four ASVs between fresh and liquid nitrogen post-72 hour at RT, and two ASVs between fresh and liquid nitrogen treated samples. There were differences among both saliva and dental swab fractions. For saliva, the relative abundances of 15 ASVs differed significantly between both mixed and pellet fractions and mixed and supernatant fractions. For dental swabs, the relative abundances of 19 ASVs differed significantly between mixed and pellet fractions and 10 between mixed and supernatant fractions. For all reported significant results, q < 0.05. CONCLUSIONS: Relative abundances are significantly affected by processing methods for both gut and oral microbiome samples. Therefore it is crucial to exercise caution when comparing microbial composition results across studies that use different processing methods. FUNDING SOURCES: Research reported in this abstract was supported by a NIH NIDDK T32 Award. Oxford University Press 2022-06-14 /pmc/articles/PMC9194270/ http://dx.doi.org/10.1093/cdn/nzac063.021 Text en © The Author 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods
Superdock, Dorothy
Poole, Angela
Processing Method Affects Oral and Gut Microbiome Composition
title Processing Method Affects Oral and Gut Microbiome Composition
title_full Processing Method Affects Oral and Gut Microbiome Composition
title_fullStr Processing Method Affects Oral and Gut Microbiome Composition
title_full_unstemmed Processing Method Affects Oral and Gut Microbiome Composition
title_short Processing Method Affects Oral and Gut Microbiome Composition
title_sort processing method affects oral and gut microbiome composition
topic Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9194270/
http://dx.doi.org/10.1093/cdn/nzac063.021
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