Investigation of human gut microbiota using different DNA isolation methods

The human microbiota is most dense in the gut. The change in the homeostatic structure of the human gut microbiota in a way that may adversely affect human health is called intestinal dysbiosis. In recent years, next-generation sequencing methods, especially 16S rRNA amplicon sequencing, have been widely used in studies because they provide sensitive, low-cost and high-throughput data. In this thesis study, bacterial genomic DNA isolations were performed from stool samples taken from volunteers(n=3) with three different methods(Phenol-Chloroform, QIAamp Powerfecal DNA Kit, ZymoBIOMICS Miniprep Kit) and 16S rRNA gene V3-V4 regions of their products were amplified by PCR and analyzed in Illumina Miseq device. As a result of 16S amplicon sequencing, an average of 83,633 read successes was achieved. However, the average number of taxonomically classified reads was determined as 40,603, and the most meaningful data obtained at this reading depth was performed at the phylum level. The most comprehensive data is provided with samples obtained from commercial kits with filters, Qiagen(7 phyla) and Zymo(6 phyla). Although the amount of DNA obtained with Phenol-Chloroform(5 phyla) was high, it was seen that it was more unsuccessful in terms of bacterial representation compared to the filter kits. In the comparisons between samples, it was observed that the detection of the Bacteroidetes phylum was more effective in filtered(Qiagen) isolations. According to the results of this study, the success of bacterial genomic DNA isolation method in performing DNA isolation of all bacteria directly affects the microbial composition regardless of biological parameters.