Microbial Diversity Research Based On High-throughput Sequencing Data Of 16S RRNA And Metagenome

Early microbiological research relies on artificial isolation and culture. But, most microorganisms cannot survive in laboratory conditions, which consequently hinders the understanding of the microorganism. The development of high-throughput DNA sequencing technology allows researchers to simultaneously sequencing multiple microbial genomes, and promotes the culture-independent metagenomics. Metagenomic studies are divided into two categories: one mainly focuses ontarget sequencing of species marker gene, such as 16 S rRNA gene; the other will sequencing whole genomes of all microorganisms. Since sequencing data of metagenomics is extremely large and complexity, there is no unified methodology or pipeline about the data processing and analysis. For this, we carried out the following three studies.Firstly, data processing pipeline for 16 S rRNA gene high-throughput sequencing was established, covering all the procedure from raw sequencing data to the final OTU abundance and taxonomy table. The OTU picking method was the combination of reference-based clustering and de novo clustering based-on heuristic clustering algorithm, which improved the efficiency of data usage.Embeddedsubsampling method in OTU picking strategy made it possible for being applied on large scale dataset, which was unmanageable for traditional methods.Secondly, based on direct short-read alignment, pipeline for microbial diversity studies using metagenome high-throughput sequencing data was built in this thesis. Compared to the traditional“short reads assembly, gene prediction and gene alignment” procedure, short reads were aligned with pre-defined clade-specific marker gene in this pipeline, speeding up the data analysis at the expense of losing acceptable classification accuracy and generalization ability.Thirdly, two kinds of established pipeline were applied in practical studies about micorbial community of Lake Taihu and human tongue dorsum.Based on 16 S rRNA gene pipeline, the variation of microbial community of Lake Taihuacross different sites and different time were comprehensively profiled at the condition of sequencing depth and sample numbers far more than similar studies, as well as the relationship between water bloom and microbial community. As a result, it’s concluded that temporal variation of microbial community was more significant that than of spatial variation with seasonal and annual periodic pattern. The relationship between microorganisms was heavily influenced by water bloom. Based on metagenome pipeline, microbial community oftongue dorsum samples from human withor without gastritis were studied at species-level. Significantly different species between gastritis samples and health samples were identified in this thesis. Meanwhile, correlations between microbial community and other variables, such as age, gender and BMI were explored by canonical correspondence analysis, partly verified the scientificity of tongue diagnosis in traditional Chinese medicine.