The Application Of Metagenomics Technology In Pathogen Detection

Environmental microbe is the most widely distributed biological type which contains the most species, the largest number on the earth. It’s estimated that there are existing for more than 1030 organisms on the earth. The traditional research method of microbiology is mainly based on microbial pure culture and isolation. So far, the vast majority of microbe(more than 99%) can not be obtained depending on the method mentioned above, which greatly limits the study of microbiology. With the development of high-throughput sequencing technology, metagenomics develops rapidly and plays a more and more important role in pathogen detection.Metagenomic sequencing does not screen pure cultures of different microbial communities, but determinates the nucleic acid of all microbe directly, it can avoid the deviation of the experimental pollution. 16 S r DNA sequencing and whole genome sequencing are the two main kinds of metagenomic studies. 16 S r DNA sequencing research object is the 16 S r DNA of environmental samples, whole genome sequencing only determinates the extracted microbial genome directly.This research is based on metagenomic sequencing, exploring the pathogenic organisms which may exist in a sample with unknown etiology, providing clues to reveal the disease occurrence and development.We extracted bacterial nucleic acid of 10 foot rot disease samples, amplified high variable region of 16 S r DNA V1~V2. After sequencing, we analyzed the flora composition, found that Staphylococcus aureus and Streptococcus associated with wounds fester were detected in all samples.16S r DNA sequencing has many aspects needing improvement, such as bacterial nucleic acid extraction, hyper variable region selection, universal primer selection, sequencing platform selection and data analysis strategy choice. We experimented seven kinds of methods for bacterial nucleic acid extraction, after sequencing, we analyzed the content of various bacteria and relative distribution of simulation samples, looking for a broad-spectrum, highly efficient bacterial DNA extracting method.In the screening of a unknown etiology fever patient samples, we did virus metagenomic sequencing, found a virus genome designated IME-16 which belongs to Microviridae and showed relatively large differences with previous reported Microviridae genomes. we have done genome annotation, evolution analysis and protein structure analysis on it, suggesting that IME-16 may be a remote clad of alphaviruses.In the virus metagenomic sequencing of a serum samples from patients with hepatitis B, we optimized the bioinformatics analysis method, speed up data analysis. We also found some Anelloviridae virus genomes which are related to hepatitis in some previous reported paper, there are 10 strains of Torque Teno Midi Virus(TTMDV) genomes and 3 strains of Torque Teno Virus(TTV) genomes, they are all showing large differences with previously reported Anelloviridae virus genomes, we did genome annotation and evolution analysis of these genomes, confirmed that they are belong to the novel Anelloviridae virus.In summary, we adopted metagenomic sequencing, screening a number of unknown etiology foot rot disease samples, a serum of unknown etiology fever sample and a hepatitis patient serum sample, we discovered some pathogenic bacteria associated with related disease and some complete genomes of new type virus, at the same time, we improved the bacterial nucleic acid extraction method and data analysis method. In short, with the improvement of metagenomic sequencing method and analysis technology, metagenomics will make a significant contribution to pathogen detection.