| dc.description.abstract | Due to its fundamental impact on overall health, the gut microbiome has emerged as one of the most popular research subjects over the last decade. It is believed that there are associations between gut microbial perturbation and several disorders such as obesity, diabetes, and cancer. Surprisingly, gut microbiota might also play a role in non-digestive disorders such as depression and autism. Therefore, it is crucial to analyze the metagenomic (microbial composition) and metatranscriptomic (the functional gene expression of the microbiota) of a healthy person in order to comprehend how this complex ecology preserves human health.
In this master thesis, we conducted an optimization method in nucleic acid extraction, to produce high-quality DNA and RNA for metagenomic and metatranscriptomic studies. A longitudinal study was performed to determine the natural variation in the genomic and transcriptomic profile of the gut microbiome over the course of a month in four-time points (days 0, 7, 14, and 28). Lastly, a freezing and thawing (FT) cycle study was performed to investigate the sensitivity of genomic and transcriptomic profiles of the fecal microbiome to repeated freezing and thawing. Due to difficulties with rRNA abundance even after rRNA depletion, metatranscriptomic sequencing could not be completed in this study.
This master thesis has established the nucleic acid extraction method to study metagenomics and metatranscriptomics. We obtained high-quality DNA for metagenomic 16S amplicon studies by optimizing the extraction procedures. The RNA yield and purity were likewise excellent, however, there were some issues with RNA integrity. The longitudinal study showed that overall participants' phyla distributions were dominated by Firmicutes, followed by Bacteroidetes and Actinobacteria with each participant's gut microbiota containing between 300 and 650 species. Diversity analysis reveals that the majority of participants had stable diversity over 28 days, with each participant having a different microbial composition that can vary greatly from one healthy person to another. Venn diagram revealed that after one month, the majority of participants seem to have maintained around 60 and 80% of their genera. Based on our findings that the more prevalent the taxa, the more their abundance varied over a period of 28 days, Clostridia, Bacteroidia, Actinobacteria, and Erysipelotrichia were the four classes assumed to be the active members of the gut microbiota. The FT cycles study revealed that throughout FT cycles the species diversity was reduced even though there was no significant difference. Composition analysis showed that each participant had a unique microbial composition and abundance but shows a pattern throughout FT cycles, where some genera decline while others were rising. Seven different genera from the classes of Clostridia and Coriobacteria had considerably increased in abundance throughout FT cycles, while nine genera from the classes of Bacteroidia, Clostridia, and Deltaproteobacteria experienced a sharp decline among all participants.
Taken together, longitudinal studies show that most individuals can maintain the stability of their gut microbiota composition, though some taxa's abundance varies over a one-month period. Based on the findings from the FT cycle study, we suggested only once freezing and thawing fecal samples to get the closest representation of the original microbial composition of the gut. | |