In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. 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. Results: Samples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. GUT and Zymo Research) in comparison to samples frozen immediately upon collection.Methods: Human fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene 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. 2Department of Medical Microbiology and Infection Prevention, Virology and Immunology Research Group, University Medical Center Groningen, Groningen, Netherlandsīackground: Microbiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. 1Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands.Jolanda Kool 1, Liza Tymchenko 1, Sudarshan A.
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