Sample Processing Method And Application Of Bacterial Metagenome And Viral Metagenome Sequencing

With the development of high-throughput sequencing technology,the sequencing throughput is increasing,such as the Hi Seq X Ten sequencer,which can generate 1.8T data volume per single run.The cost of sequencing drops dramatically,such as the single run of the PGM sequencer,which the price is less than 10,000 yuan.The sequencing rate is also getting faster and faster,such as the time for a single run of the PGM sequencer is within 24 hours.The sequencing quality is getting higher and higher,such as the PGM sequencer can produce a read length of about 400 bp.The degree of sequencing automation is also getting higher and higher,such as the Miseq sequencing platform in this article,the average manual operation is less than 5h and so on.Based on the high-throughput sequencing platform,this study performed metagenomic sequencing of different unknown pathogen-infected samples,and adopted different sample processing methods according to the source,type and detection purpose of the samples.It is specifically divided into the following two parts: 1.Establishment of screening methods for pathogenic bacteria combined with targeted sequencing and shotgun sequencing,2.Sample processing methods and applications for virus metagenomic sequencingEstablishing screening methods for pathogenic bacteria combined with targeted sequencing and shotgun sequencingHigh-throughput sequencing techniques also have certain flaws when screening for bacterial fungal protozoa in samples.Because the concentration of bacterial and fungi in the patient are very low,and some samples are taken by patients who have been treated with antibiotics,recovered patients,or patients who are locally infected with bacterial fungi but have not entered the bloodstream,their bacterial and fungi are detected more difficult.In this study,bacteria and fungi in the samples were detected by establishing a pathogen screening method that combines targeted sequencing and shotgun sequencing.First,the analytical method designed by our laboratory was verified by downloading known bacterial sequences.In the four groups of specimens analyzed by computer simulation,the reads detection ratio of the corresponding bacterial species was the highest among the total bacteria reads,which proved that the analysis method was feasible.Second,experimental verification was performed using four known bacteria and three fungal specimens.Among the seven samples,the proportion of reads detected in the corresponding species and genera was the highest among the total reads of the specimen,which proved that the test method was feasible.Both simulation data and experiment data with known bacteria showed that our analysis algorithm and experiment method can effectively detect target pathogens using short reads generated from 16S/ITS full-length sequence amplification products.Finally,this method was applied to clinical samples,Rhodococcus and Pseudomonas were detected in clinical cerebrospinal fluid samples,and Staphylococcus aureus was detected in blood and saliva.Sample processing method and application of virus metagenomic sequencingIn recent years,there have been many large-scale infectious diseases caused by viruses worldwide,such as the SARS outbreak in China in 2003,the outbreak of avian influenza in 2004 swept the United States and most of the Asian countries,the outbreak of Ebola virus in West Africa in 2014,etc.In these situations,it becomes very urgent to quickly detect the virus in the sample.Routine virus detection,such as the isolation and culture of viruses,serological tests,morphological examinations and so on.But these conventional methods have certain deficiencies,such as long time required to find unknown viruses.This study was based on high-throughput sequencing and the detection of vector insect samples and clinical samples.In the six samples,four mixed samples were found to contain two or more suspicious viruses,which revealing the diversity of viruses carried by mosquitoes.In a clinical bronchoalveolar lavage fluid sample,a circovirus was discovered by MDA amplification.The similarity to the currently known closest PoSCV5-like circovirus was 89.9%,which is a diagnosis of disease.Provide a reference.In summary,this paper uses different sample processing methods for different types of samples and different detection purposes.Then uses high-throughput sequencing platforms for sequencing,and explores new data analysis methods to screen the type of pathogens in infected samples.The method is important for the prevention and clinical treatment of new infectious diseases.