VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ******* GRADUATION THESIS “16S METAGENOMICS ANALYSIS OF ENDOMETRIAL MICROBIOTA IN VIETNAMESE WOMAN” HA NOI, 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ******* GRADUATION THESIS “16S METAGENOMICS ANALYSIS OF ENDOMETRIAL MICROBIOTA IN VIETNAMESE WOMAN” Student’s name : Le Doan Quoc ID : 610760 Class : K61CNSHE Supervisor : Dr. Pham Dinh Minh Prof. Phan Huu Ton HA NOI, 2021 STATEMENT OF ORIGINAL AUTHORSHIP The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other education institution. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.
Signature: Date: 2 ACKNOWLEDGEMENTS This thesis, like any other, would not have been possible without the involvement and support of many people. They have all earned my deepest gratitude, even if it has been poorly expressed at times. First and foremost, my sincere thank goes to my principal supervisors, Dr. Pham Dinh Minh and Prof.
Phan Huu Ton, for their ongoing support and suggestions through my graduation thesis. Without them, this project would not have been possible. I would like to thank to all Staff at Gentis Company who have had to put up with me going through all kinds of emotional swings also deserve my thanks. They were all very friendly and helpful to me, I was lucky to study in a great working environment.
Last, but not least, I thank my family and friends for all their help, encouragement and for supporting me through all these years. 3 TABLE OF CONTENT STATEMENT OF ORIGINAL AUTHORSHIP. 3 TABLE OF CONTENT. i LIST OF ABBREVIATIONS.
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PART ONE: INTRODUCTION. Aims and requirements. 12 PART TWO: LITERATURE REVIEW. The importance of 16S metagenomics.
Choosing the Appropriate Target Region. Error! Bookmark not defined. Taxonomic Classification: The Growing of 16S rRNA Databanks. Error! Bookmark not defined.
Bioinformatics pipelines for micrbial 16S rRNA amplicon sequencingError! Bookmark not defined. Human Endometrial Microbiota. Error! Bookmark not defined.Relevant researches over the world. Error! Bookmark not defined.
Error! Bookmark not defined. Error! Bookmark not defined. Error! Bookmark not defined. PART THREE: MATERIALS AND METHODS.
Location and duration of the study. Error! Bookmark not defined. Error! Bookmark not defined. DNA Purification from Body Fluids.
Error! Bookmark not defined. Amplication V3-V4 region on 16S micrbial genesError! Bookmark not defined. Error! Bookmark not defined. PCR Clean-up.
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Library quantification, Normalization, and PoolingError! Bookmark not defined. Error! Bookmark not defined. Error! Bookmark not defined. Amplicon bioinformatics: from raw reads to tablesError! Bookmark not defined.
Filter and trim. Error! Bookmark not defined. Learn the Error Rates. Error! Bookmark not defined.
Merge paired reads. Error! Bookmark not defined. Construct sequence table. Error! Bookmark not defined.
Error! Bookmark not defined. Error! Bookmark not defined. RESULTS AND DISCUSSION. Error! Bookmark not defined.Quality score of sequences.
Error! Bookmark not defined. Filter and Trim. Error! Bookmark not defined. Finding true sequence variants.
Error! Bookmark not defined. Merging Paired Reads. Error! Bookmark not defined. Contructing ASV table and removing chimerasError! Bookmark not defined.
Error! Bookmark not defined. Visualizing alpha-diversity. Error! Bookmark not defined. Abundance bar plot.
Error! Bookmark not defined. Error! Bookmark not defined. Error! Bookmark not defined. 6 LIST OF ABBREVIATIONS µL microlitre Bp Base pair CP Common region CR Common region DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphotphate dsDNA Double DNA mL milliliter PCR Polymerase Chain Reaction RCA Rolling circle amplification RE Restriction enzyme REP Replacation protein rpm revolutions per minute NGS Next Generation Sequencing ssDNA Singe strand DNA TAE Tris – acetate – EDTA Taq Thermus aquatic 7 ABSTRACT Diagnosis of bacteria with NGS methods has facilitated the research of low biomass microbiomes in tissues and organs previously considered sterile, for instance, the endometrium.
Therefore, an abnormal endometrial microbiota has been proved to link with implantation failure, pregnancy loss, and other gynecological and obstetrical conditions. Future investigation of the endometrial microbiota could enhance a further insight of bacterial communities’ role in both physiology and pathophysiology, with the hope that can aid the ability to increase of pregnancy rate and create a healthy pregnancy. 16S rRNA gene sequencing, or simply 16S sequencing, utilizes PCR to target and amplify portions of the hypervariable regions (V1-V9) of the bacterial 16S rRNA gene1. In this project, sequences generated from Illumina MiSeq was created with V3-V4 region.
After sequencing, raw data is analyzed with a bioinformatics pipeline which includes trimming, error correction, and comparison to a 16S reference database. In this study, the method is applied for a small sample of three Vietnamese Women with the hope that could lead to a little contribution to the development of research relevant to endometrial microbiota as well as study using 16S metagenomics pipeline. 8 PART ONE: INTRODUCTION 1. Introduction The main part of microbial cells that can be seen in microscope and shown to be living with various staining procedures cannot be invoked to create colonies on Petri plates.
Actually, there was only 0.1 to 1 percentage of the living bacteria when culture soils with standard conditions. Furthermore, when culture microbes in aquatic environments the number of microbes could be thousand times lower. Moderate and high throughput nutrients were put to help the existing problems. But the result of cultivation of microbe in isolation continues to be low.
The problem could be harder because for most of the time these bacteria need community to grow. Under laboratory cultivation conditions, this always favor the identification of organisms that can live best with these special nutrients. This mean in turn not appropriate for those microbes which are dominant or most influential in the native environment of bacteria. These limitations have given the rise of culture-independent methods for identifying and enumerating microbes in the community.
Over the decades these gradually have played a bigger role in the isolation of bacteria. The technique has been used widely is rRNA phylotyping, which is predominant among microbes. This 9 powerful technique is based on databases of rRNA gene sequences. These databases nowadays are enriched overtime from researchers.
By identify the rRNA genes of organisms, these were compared to the branch to position. This could infer the nearest branch to infer that organism could have similar biology and ecology with the closest relavtives. The polymerase chain reaction makes rRNA genes to be detected and copied directly from environmental samples, then cloned and sequenced. If the environmental sample contains many types of organisms, there will be many different rRNA sequences, the diversity of which will be a measure of the complexity of the community.
With the development in metagenomics, not-yet-cultivated microbes in biotechnology has been explored in terms of DNA-base investigation. Research fields like ecology, community biology, and microbiome research has significantly changed their views when metagenome term was coined 20 years ago. Furthermore, it has led to several important identification of biomolecules that have a huge potential application to bio-based industrial processes. Laboratories over the world has been investigate the novel functional genes which called function-driven metagenomics.
In another way, this creates more helpful applications relevant to the diversity of biocatalysts and valuable biomolecules. Metagenomics infer to both a research technique and research field. Metagenomics, the field can be defined as the genomic analysis of microbial DNA from microbial environment. Metagenomics tools allows the diversity analysis of independent-culturable or previously unknown microbes.
This is important as only around 1-2% of bacteria can be cultured in the laboratory. The ability to identify 10 microbes without a priori knowledge of what a sample contains is opening new doors in disciplines like microbial ecology, virology, microbiology, environmental sciences and biomedical research. Sequencing based examination of the metagenome has become a powerful tool for generating novel hypotheses. The 16S rRNA gene is a taxonomic genomic marker that is common to almost all bacteria and archaea.
The marker allows one to examine genetic diversity in microbial communities, specifically what microbes are present in a sample. While some estimates of relative abundance within similar samples can be made, drawing conclusions across different sample types is not recommended due to amplification artifacts introduced during PCR. 16S rRNA sequencing is accomplished by designing primers to the entire 16S locus or targeting multiple hypervariable domains within the gene. The nine variable regions of the 16s rRNA gene are flanked by conserved stretches in the majority of bacteria.
Conserved regions can be used as targets for PCR primers, designed upstream and downstream of the variable domains. These hypervariable regions provide the species-species signature necessary for identification. After these domains have been amplified, sequencing related primers are either ligated or added by a second PCR step. Aims and requirements 1.1 Aims The major aim of the project is to create 16S metagenomics bioinformatics for endometrial microbiota analysis in which the result includes the compositions of microbiota in biopsies of endometrium.
Most of the steps in wet lab were done by high skilled staff. The bioinformatics pipeline in this project could lead to more findings for similar experiment. To extract DNA from endometrial biopsies for PCR and further analysis. This step requires proper kit to retain a necessary amount of DNA.
Using primers to amplify the target gene, 16S, to make copies of V3-V4 region of 16S gene. Cleaning the PCR products from previous step to eliminate or reduce contamination. Sequencing DNA sample by NGS technology, ILLUMINA sequencing technology was applied in this project. In fact, MISEQ device was used.
Analyzing the sequences from sequencer to investigate composition of microbiota in the sample by using modern pipeline bioinformatics DADA2. 12 PART TWO: LITERATURE REVIEW 2. The importance of 16S metagenomics The ribosome plays a key role in the work of fundamental cellular processes since it a part of interpreting the genetic code delivered by mRNA and construct the chain of amino acids with the resembling of transfer RNA, which eventually folds into fully functional proteins, this is extremely complex process called translation. Every living thing all has a similar machinery at a basic that mean from an evolutionary perspective, the whole process is extremely conserved.
In fact, ribosome is built up from only dozens of distinct proteins plus a few distinct RNA molecules, which are called ribosomal RNA (rRNA). Prokaryotic ribosomes account for 65% rRNA and 35% ribosomal proteins. The whole structure of the ribosome is composed of two multimeric subunits, which are the large subunit (50S), which contains two rRNA molecules (5S and 23S), and the small subunit (30S), which contains a single rRNA molecule (16S). There are scientific reasons that make 16S rRNA an almost perfect candidate for the study of prokaryotic phylogenesis, a so- called molecular clock.
In fact, three following features make 16S rRNA so nicely fitting. First, 16S rRNA is the basic present part in all microbes. And the second, the reason is with low probability of lateral gene transfer. Finally, the extremely conserved scaffolding by ribosomal proteins makes its sequence extremely conserved in certain regions, while other regions not directly involved in the stabilization and exposed to solvent are relatively free from evolutionary constraints, so they were extremely variable.
In total, we can count nine variable regions (V1 to 13 V9) of different size in the approximately 1500 bp constituting a full 16S rRNA molecule13. (A) Two opposite sides of the 3D structure of the prokaryotic ribosome with ribosomal proteins, 23S rRNA, 16S rRNA and 5S rRNA in white, green, red and blue, respectively. (B) Two opposite sides of the 3D structure of the prokaryotic ribosome small subunit (SSU) with ribosomal proteins and 16S rRNA in white and red, respectively.