期刊论文详细信息
Frontiers in Microbiology
Reducing bias in microbiome research: Comparing methods from sample collection to sequencing
Microbiology
Jolanda Kool1  Liza Tymchenko1  Susana Fuentes1  Sudarshan A. Shetty2 
[1] Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands;Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands;Department of Medical Microbiology and Infection Prevention, Virology and Immunology Research Group, University Medical Center Groningen, Groningen, Netherlands;
关键词: human studies;    microbiota;    reproducible analysis;    microbiome;    gut;    16S rRNA gene sequencing;   
DOI  :  10.3389/fmicb.2023.1094800
 received in 2022-11-11, accepted in 2023-02-22,  发布年份 2023
来源: Frontiers
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【 摘 要 】

BackgroundMicrobiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. To investigate and identify potential biases introduced by technical variations, we compared several approaches throughout the entire workflow of a microbiome study, from sample collection to sequencing, using commercially available mock communities (from bacterial strains as well as from DNA) and multiple human fecal samples, including a large set of positive controls created as a random mix of several participant samples.MethodsHuman fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene·GUT and Zymo Research) in comparison to samples frozen immediately upon collection. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls.ResultsSamples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). These stabilized samples stored at RT were different from immediately frozen samples, where the relative abundance of Bacteroidota was higher and Actinobacteriota and Firmicutes were lower. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. The DNA extraction had a significant impact on the microbial composition, also observed with the use of different Illumina barcodes during library preparation and sequencing, while no batch effect was observed in replicate runs.ConclusionOur study reaffirms the importance of the mechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison of storage conditions revealed that the bias was limited in RT samples preserved in stabilization systems, and these may be a suitable compromise when logistics are challenging due to the size or location of a study. Moreover, to reduce the effect of contaminants in fecal microbiota profiling studies, we suggest the use of ~125 pg input DNA and 25 PCR cycles as optimal parameters during library preparation.

【 授权许可】

Unknown   
Copyright © 2023 Kool, Tymchenko, Shetty and Fuentes.

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