دورية أكاديمية
Reducing bias in microbiome research:Comparing methods from sample collection to sequencing
| العنوان: | Reducing bias in microbiome research:Comparing methods from sample collection to sequencing |
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| المؤلفون: | Kool, Jolanda, Tymchenko, Liza, Shetty, Sudarshan A., Fuentes, Susana |
| المصدر: | Kool , J , Tymchenko , L , Shetty , S A & Fuentes , S 2023 , ' Reducing bias in microbiome research : Comparing methods from sample collection to sequencing ' , Frontiers in Microbiology , vol. 14 , 1094800 . https://doi.org/10.3389/fmicb.2023.1094800Test |
| سنة النشر: | 2023 |
| المجموعة: | University of Groningen research database |
| مصطلحات موضوعية: | 16S rRNA gene sequencing, gut, human studies, microbiome, microbiota, reproducible analysis |
| الوصف: | Background: Microbiota 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. Methods: Human 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. Results: Samples 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. Conclusion: Our study reaffirms the importance of the mechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison ... |
| نوع الوثيقة: | article in journal/newspaper |
| وصف الملف: | application/pdf |
| اللغة: | English |
| العلاقة: | https://research.rug.nl/en/publications/0a4f9770-0fea-48f7-ba44-233868f77053Test |
| DOI: | 10.3389/fmicb.2023.1094800 |
| الإتاحة: | https://doi.org/10.3389/fmicb.2023.1094800Test https://hdl.handle.net/11370/0a4f9770-0fea-48f7-ba44-233868f77053Test https://research.rug.nl/en/publications/0a4f9770-0fea-48f7-ba44-233868f77053Test https://pure.rug.nl/ws/files/695138828/Reducing_bias_in_microbiome_research_Comparing_methods_from_sample_collection_to_sequencing.pdfTest |
| حقوق: | info:eu-repo/semantics/openAccess |
| رقم الانضمام: | edsbas.1D9693EA |
| قاعدة البيانات: | BASE |
| DOI: | 10.3389/fmicb.2023.1094800 |
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