Title Page
국문 초록
Abstract
Contents
Ⅰ. Introduction 14
Ⅱ. Material and Method 18
1. Sampling of Human Fecal Sample 18
2. 16S rRNA gene amplicon sequencing for community analysis of human gut bacteria 18
2.1. DNA Extraction from Fecal Samples 18
2.2. Amplification of 16S rRNA gene sequence from DNA samples 18
2.3. Analyze 16S rRNA gene sequences from fecal samples. 19
3. Set Up and Maintain an Anaerobic Culture Environment 20
4. Isolation and Cultivation of Human Gut Bacteria 21
4.1. Obtaining clinical sample feces 21
4.2. Designing an intestinal environment-mimicking medium for culturing gut bacteria 21
4.3. Human gut bacteria culture based on High-throughput cultivation(HTC) method 29
4.4. Attempts to obtain a single culture using anaerobic high-throughput cultivation based on the dilution-extinction method. 30
4.1. Cultivation of human gut bacteria based on plate-based cultivation. 34
5. Phylogenetic Analysis of Cultured Bacteria 34
5.1. DNA extraction and 16S rRNA gene amplification (based on HTC method) 34
5.2. DNA extraction and 16S rRNA gene amplification (based on Conventional plating method) 35
5.3. Phylogenetic analysis based on the 16S rRNA gene 36
6. Preparation of stocks for long-term storage of isolated microorganisms 36
7. Analysis of dsDNA Virus in the Human Gut 36
7.1. Viral DNA extraction from fecal samples 36
7.2. DNA sequencing and trimming 37
7.3. Cluster analysis of viruses in the human gut using Phanta 38
7.4. Cluster analysis of viruses in the human gut using Metaphage 39
Ⅲ. Result 41
1. 16S rRNA gene amplicon sequencing for community analysis of human gut bacteria 41
1.1. DNA Extraction from Fecal Samples 41
1.2. Analyze 16S rRNA gene sequence from fecal samples. 46
2. Cultivation of Anaerobic Bacteria in the Human Gut 49
2.1. Designing an intestinal environment-mimicking medium for cultivation of intestinal bacteria 49
2.2. High-Throughput Cultivation of Human Gut Bacteria 51
2.3. Cultivation of human gut bacteria based on plate-based cultivation. 58
2.4. Phylogenetic analysis based on the 16S rRNA gene 60
2.5. Comparison of species in HTC-based and plate-based cultured bacteria 60
3. Analysis of Human Gut dsDNA Viruses 62
3.1. Extraction and sequencing of viral DNA from fecal samples 62
3.2. Analysis of human gut viruses using Phanta 65
3.3. Analysis of human gut viruses using Metaphage 67
Ⅳ. Discussion 75
Ⅴ. References 77
Table 1. The sequence of primers (515FY, 805RB) were used to amplify 16S rRNA genes. 19
Table 2. Composition of YCGMs (Food Biotechnology Lab, Yonsei University) and Mucin medium. 23
Table 3. Composition of ASW(modified with sodium sulfate for electron acceptor). 24
Table 4. Composition of LNHM(Low Nutrient Heterotrophic Medium) added after sterilization and filtration. 25
Table 5. Composition of MHM (Microbiome HTC medium) basic type, designed to simulate intestinal environment, except for carbon source. 26
Table 6. Composition of vitamin mixture and trace metal. 27
Table 7. MHM composition for Microbiome HTC based on dilution-to-extinction. 28
Table 8. MHM composition for Microbiome HTC based on dilution-to-extinction. 28
Table 9. MHM composition for Microbiome HTC based on dilution-to-extinction. 28
Table 10. Information of samples used in Microbiome HTC. 31
Table 11. The number of inoculating cells by Microbiome HTC based on dilution-to-extinction according to condition. 32
Table 12. DNA QC result of the extracted DNA samples. 41
Table 13. Raw Data Statistics 43
Table 14. Summary of anaerobic HTC experiments using human fecal samples. 54
Table 15. Taxonomic distribution of the MH-2, MH-4, MH-5 and MH-6 HTC isolates. 56
Table 16. Taxonomic distribution of the isolates based on plates. 59
Table 17. QC results of extracted viral DNA of Normal people. 62
Table 18. QC results of extracted viral DNA of UC patients. 64
Figure 1. High throughput culture's workflow. 30
Figure 2. Phanta's workflow 39
Figure 3. Metaphage's workflow 40
Figure 4. Plot of quality for sequencing read. 47
Figure 5. Barplot of 16S rRNA gene amplicon in the Order level. 47
Figure 6. Barplot of 16S rRNA gene amplicon in the Family level. 48
Figure 7. Barplot of 16S rRNA gene amplicon in the Genus level. 48
Figure 8. PCoA Plot of 16S rRNA gene amplicon data. 49
Figure 9. Taxonomic distribution of the MH-2, MH-4, MH-5 and MH-6 HTC isolates. 58
Figure 10. Taxonomic distribution of the isolates based on conventional plating method. 60
Figure 11. Comparison of captured species between culture-based and 16S rRNA gene amplicon based. 61
Figure 12. Sankey plots obtained via Pavian. 66
Figure 13. Classification with Phanta. 66
Figure 14. Metaphage assembly statistics. 67
Figure 15. Assembly statistics by Metpahge. 67
Figure 16. vOTUs Miner Comparison. 68
Figure 17. Quality check of vOTUs by CheckV. 69
Figure 18. Heatmap of abundance for vOTUs. 70
Figure 19. Alpha diversity with Chao1. 71
Figure 20. Alpha diversity with Shannon. 71
Figure 21. Beta diversity with Bray Curtis. 72
Figure 22. Taxonomic assignment of vOTUs. 73
Figure 23. Graphanalyzer result for vOTU_2030. 74