VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- GRADUATION THESIS TITLE: “COMPARATIVE ANALYSIS OF MICROBIAL COMMUNITIES ASSOCIATED WITH ACROPORA FORMOSA AND SEDIMENT IN PHU QUOC ISLAND.” Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ---------- ---------- GRADUATION THESIS TITLE: “COMPARATIVE ANALYSIS OF MICROBIAL COMMUNITIES ASSOCIATED WITH ACROPORA FORMOSA AND SEDIMENT IN PHU QUOC ISLAND.” Student name : NGUYEN PHAM DAN TRUONG Class : K63CNSHE Student’s code : 637372 Supervisor : NGUYEN HUY DUONG, MSc. NGUYEN VAN GIANG, Assoc. Department : MICROBIAL TECHNOLOGY Hanoi - 2022 COMMITMENT I hereby declare that the data and results stated in the thesis are honest and have never been published by anyone in other studies. In the references section, the graduations with references to papers and action information are mentioned.
I am completely responsible for the data of this thesis. Hanoi, January 9th, 2023 Sincerely Nguyen Pham Dan Truong i ACKNOWLEDGEMENTS Above all else, I would like to express my heartfelt gratitude to MSc. Nguyen Huy Duong, an officer of Bioinformatics Department at the Institute of Biotechnology of the Vietnam Academy of Science and Technology, who is my thesis advisor and has also provided me with invaluable guidance, input, and support throughout the thesis work. Following that, I'd want to offer my grateful to PhD.
Bui Van Ngoc with researchers at the Bioinformatics Department at the Institute of Biotechnology of the Vietnam Academy of Science and Technology, have assisted, provided technical support and imparted valuable knowledge as well as research experience. The knowledge and skills gained would be beneficial in the future it had been a warm and fruitful experience. It would not have been possible to complete this project without the enthusiastically guidelines, cordial supporting of Assoc. Nguyen Van Giang, Head of Department of Microbiology Technology at the Vietnam National University of Agriculture, who created opportunities for me to study and work directly at the Bioinformatics Department.
Without the enthusiastic facilitation of the Board of Directors at Vietnam National University of Agriculture and the teachers of Biotechnology falcuty, I would not have been able to acquire not only the professional foundation necessary to complete this report, but also the wealth of experience that has helped me take my first confident steps along my chosen career path. I would have to thank all the seniors in the lab, who also helped me during the preparation of this thesis. To wrap up, I'd want to express my gratitude to my family and all who have journeyed with me, encouraged me, and shared in my experiences. I sincerely thank you! ii Hanoi, Janury 9th, 2023 Sincerely Nguyen Pham Dan Truong iii Table of Contents COMMITMENT.
ii LIST OF TABLES. vi LIST OF FIGURES. vii LIST OF ABBREVIATIONS .1 State of problem .1 Coral and coral reef .2 Functional importance of coral reefs .3 The current situation of coral reefs .2 The coral microbiome .1 The coral holobiont: a multi-partite symbiotic organism .2 Microbiota of healthy corals .3 Microbial identification methods in coral reefs .1 Culture-based methods .2 Methods for unculturable microbes. Error! Bookmark not defined.4 Bioinformatics analysis of metagenomics .1 Methods in metagenomics .5 Current situation of domestic and international research.
MATERIALS AND METHODS .1 Total DNA extraction for 16S rRNA survey.2 Next-generation sequencing .1 Enhancing data reliability .1 Raw read quality profiles .2 Obtaining cleaned and chimera-free sequences. CONCLUSIONS AND RECOMMENDATIONS .49 v LIST OF TABLES Table 2. Overview of modern sequencing technologies. Sample name, sample abbreviation and sample collection location.
Abundance of archaeal phyla in the Acropora formosa mucus and the sediment (Abundance > 0. Abundance of archaeal genera in the Acropora formosa mucus and in the sediment (Abundance > 0. Statistical summary for alpha diversity indices for archaeal communities. Abundance of bacterial phyla in the Acropora formosa mucus and in the sediment (Abundance > 0.
Abundance of top 10 bacterial genera in the Acropora formosa mucus and in the sediment (Abundance > 0. Statistical summary for alpha diversity indices for bacterial communities. 37 vi LIST OF FIGURES Figure 2. Acropora formosa coral.
Forward read’s quality score of the archaeal data when sequencing. Reverse read’s quality score of the archaeal data when sequencing. Forward read’s quality score of the bacterial data when sequencing………. Reverse read’s quality score of the bacterial data when sequencing.
Quantity of reads that were kept after filtering step in the archaeal data…. Quantity of reads that were kept after filtering step in the bacterial data. Composition of microbiota at phylum level…… ……………………………26 Figure 4. Composition of microbiota at genus level.
Alpha diversity of archaeal biomes. Beta diversity of the archaeal community. Composition of microbiota at phylum level. Composition of microbiota at genus level.
Alpha diversity of bacterial community. Beta diversity of the bacterial community. Composition of microbiota at Class level. Composition of microbiota at Order level.
Composition of microbiota at Family level. Composition of microbiota at Class level. Composition of microbiota at Order level. Composition of microbiota at Family level.
58 vii LIST OF ABBREVIATIONS Abbreviation Full word AF Acropora formosa ANOSIM Analysis of similarities Automated Ribosomal Intergenic Spacer ARISA Analysis ASV Amplicon sequence variant DGGE Denaturing Gradient Gel Electrophoresis DNA Deoxyribonucleic Acid NGS Next-Generation Sequencing OTU Operational taxonomic unit PCoA Principal coordinates analysis PCR Polymerase Chain Reaction QC Quality score rRNA Ribosomal ribonucleic acid SE Sediment SST Sea surface temperatures Terminal Restriction Fragment Length T-RFLP Polymorphism viii ABSTRACT Coral reefs are among the most productive, complex and highly diverse ecosystems on the planet. Corals include a diverse range of microorganisms in their mucus, skeleton, and healthy tissue. These organisms, which include microalgae, bacteria, and archaea, assist corals in a variety of ways, including photosynthesis, nutrition intake, and infection resistance. Little is known about the similarities and differences in microbial diversity and composition across prokaryotic kingdoms and coral reef biotopes that are geographically near one another.
In this study, we compared archaea and bacteria communities in two distinct biotopes: the coral Acropora formosa and sediment in Phu Quoc Island using 16S rRNA metagenomics. Comparative analysis in composition at phylum level showed that Nanoarchaeota dominated the archaeal biomes in the coral mucus and the sediment, and there were significant differences in composition between them (p-value = 0. In bacterial data. Proteobacteria dominated in both the coral mucus and the sediment at the phylum level and there were significant differences in composition between them (p-value = 0.
Overall, the alpha diversity in the bacterial data is higher than in the archaeal data. In bacterial data, the composition diversity in the coral mucus and in the sediment is significant different through 4 indices Observed (p-value = 0. The archaeal data showed tthe composition diversity in the coral mucus and in the sediment with significant differences through 4 indices Observed (p-value = 0. The beta diversity in both archaeal data and bacterial data showed significant differences of the coral mucus community and the sediment community (p-value = 0.006 in archaeal data and p-value = 0.009 in bacterial data).
Biotope proved to be the main identifiable factor affecting composition. However, within the framework of this study, it stops at the level of genus classification of bacteria in the composition. Other molecular marker genes must be used in conjunction with functional genes for species or subspecies classification, as well as technologies such as long read sequencing or whole genome sequencing.1 State of problem Coral reefs are one of the most diverse ecosystems on the planet, contributing significant economic, social, and ecological value. In recent years, due to the change of the environment and the impact of humans such as overexploitation, vandalism, coral mining, tourism, etc., many coral reefs in the world have experienced the bleaching phenomenon, of which some coastal reefs in Vietnam are no exception.
As a result, research and coral protection are critical for marine biodiversity, which plays an important political, social, and economic role and is a top priority for the country. The coral microbiome is diverse and makes up the largest component of the organisms that live on corals and reefs; it is estimated that there are more than 100 million bacteria per square centimeter of a healthy coral. There are approximately a billion bacteria and 10 billion viruses per liter of seawater in coral reef areas. Corals have a lot of organisms in their mucus, skeleton, and healthy tissue.
These organisms, such as microalgae, bacteria, and archaea, help corals in many ways, such as photosynthesis, getting nutrients, and fighting off infections. Microbial biomes that live on corals have both beneficial and harmful effects on corals, and between them there is an extremely complex relationship. For example, in the coral Acropora tenuis, the bacterial genus Pseudoalteromonas can synthesize antibiotics that inhibit opportunistic microorganisms such as Vibrio coralliilyticus. Archaea also play key roles in processes such as the geochemical cycling of carbon, nitrogen, and sulfur.
This cycling activity, particularly the nitrogen cycle, is critical for oligotrophic coral reefs in order to breakdown organic matter and maintain high levels of primary production. Nitrogen is crucial for organisms and ecosystems because it is an essential component of proteins, nucleic acids, and cell wall elements, and it inhibits marine environment primary productivity. Therefore, the microbial identification will help us learn more about the variety and interactions of all the microbial biomes in the coral mucus layer. 1 Microorganisms are typically identified and evaluated by scientists by growing them in the appropriate environment and then determining their identity based on biochemical and physiological characteristics, combined with sample observation on electron microscopy, confocal microscopy, and fluorescence microscopy to assess the total number of microorganisms in the environment, as well as partially identify microorganisms based on known microbial morphology.
However, the number of cultured microorganisms on the disk exhibited an anomaly when it was less than the total number of microbial microorganisms under the microscope. Therefore, the culture method can only identify a small part of the microorganisms capable of growing well in the culture medium, so it is not possible to give an overview of the microbial biome in corals. Methods for separating genetic units such as: Denaturing Gradient Gel Electrophoresis (DGGE), Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) used before sequencing using the Sanger method. Although the DGGE, T-RFLP, and ARISA methods combined with Sanger sequencing produce good results, it is possible to completely evaluate environmental microorganisms.
But these methods are complex and time-consuming. In recent years, metagenomics has emerged as a new trend commonly used in microbial ecology, especially in research involving in-depth coverage of microbial biomes. This approach supports the analysis of microbial strains that cannot be cultured in the laboratory because it focuses on microbial genetic analysis through markers in highly conservative gene regions so that they can be easily classified. Moreover, the metagenomics method is increasingly developed with the next-generation sequencing method, Next-Generation Sequencing (NGS), and also integrates a number of bioinformatics tools and programming languages to save research time, visualize research data, and have high accuracy.
Therefore, 16S rRNA metagenomics analysis is an effective method for evaluating microbial diversity and composition in Acropora formosa mucus and sediment. Stemming from the above reasons, the implementation of the study “Comparative analysis of 2 microbial communities associated in Acropora formosa mucus and sediment in Phu Quoc Island.” is extremely important and urgent.2 Purpose Determination of diversity and identification of archaeal and bacterial communities residing in Acropora formosa mucus and sediment using metagenomics technology.3 Research contents Analyzing and processing data, data analysis, and visualization of data obtained by metagenomics approach and R programming language. Analysis of diversity and identification of microorganisms residing in coral mucus and sediment from the phylum to genus level.1 Coral and coral reef Corals are marine organisms of the phylum Cnidaria, class Anthozoa which comes from the Greek words άνθος (ánthos; "flower") and ζώα (zóa; "animals"), hence ανθόζωα (anthozoa) = "flower animals", a reference to the floral appearance of their perennial polyp stage.