High Throughput Sequencing For Clinical Microbe Screening

High-throughput sequencing,also known as next-generation sequencing(NGS)technology,has developed rapidly over the past decades.Nowadays,the sequencing throughput becomes larger,the sequencing speed becomes faster,the sequencing quality becomes higher,and the sequencing cost for a human genome has been reduced from $ 100 million to $ 1,000 or even to $ 100.High throughput sequencing has changed the traditional genomic analysis and brought a revolution for clinical microbiological testing.Based on NGS,the testing does not need to rely on culture but uses metagenomics and microbiology to study the interaction between the host and the microorganism.In recent years,a growing number of literature report on the use of NGS for infectious disease diagnosis and pathogen analysis,while some of them have also shown NGS as a single diagnostic tool for unknown infectious diseases.Using next generation sequencing,we only need to collect blood 1 ~ 2 m L,and within 2 days to complete pathogen identification,typing,drug resistance and virulence factor detection.Such a fast identification process allows a quick respond and control for major public health events.More and more researchers have applied NGS to disease-based pathophysiology,epidemiology,and clinical diagnostics.However,due to technical challenges such as pre-treatment of clinical samples,selection of sequencing strategies,analysis of data,and the lack of applicable regulation guidelines,we are currently not able to widely use NGS in practice.On May 13,2016,the Food and Drug Administration(FDA)published a draft guideline for the diagnosis of infectious disease.It is pointed that direct detection of NGS specimens can be used for microbiological identification,antimicrobial resistance and virulence marker detection.When applying NGS to clinic diagnosis,we need more supervision and management,and also need to pay more attention to specificity,sensitivity,minimum detection limit,repeatability and quality control.Such a milestone means that once the market supervision and standardization have established and sequencing costs has dropped down,NGS will work on clinical diagnosis efficiently.In this study,we use Ion Torrent PGM platform to detect three samples of unknown pathogen infection.Before doing NGS,we treated different types of specimens with different pretreatments,including sputasol,host cell remove,nucleic acids extraction and pathogen enrichment.Then we applied NGS to the treated specimens.At the third step,the sequencing data were assembled and then analyzed by the in-house species classification software.Finally,we generated the clinical feasibility reports.We used NGS to successfully diagnose three unknown pathogens,including Bacillus cereus,Hepatitis C virus and Aspergillus.These results were verified by real-time quantitative PCR and other methods.Our work provided timely and effective information for clinical treatments.The MDA method was performed using a Phi29 DNA polymerase kit.Phi29 DNA polymerase was used for rolling circle amplification of circular DNA,as well as amplifying liner DNA,named multiple displacement amplification(MDA).This method was one of the whole genome amplification developing rapidly in recent years,which was a non-specific amplification method to increase the amount of nucleic acids to meet the requirements of library reparation.In addition,multiple displacement amplification was adopted as an initial anellovirus enrichment tool,followed by high-throughput sequencing.After that,bioinformatic methods were used to analyze the sequencing reads as well as the status of virus infection in the blood samples.A total of 226 sequences from 33 individuals were determined as anelloviruses based on BLASTN or BLASTX analysis,of which 130 were complete genome sequences and the other 96 were ORF1 sequences.All of the samples were infected with two or more genera of TTV,TTMDV or TTMV.The common prevalence of anelloviruses in Chinese population was confirmed.Using BLASTN with default parameters for megablast,13.27% of the sequences could not be aligned to nucleotide database(NT)of the National Center for Biotechnology Information(NCBI).The results demonstrated the great genomic diversity in the anelloviruses carried by a Chinese population.TTV was separated into five genotypes: genotype 1 to 5.This classification was roughly supported by our phylogenetic analysis.Our data also suggested that genotype 2 is a subclade of genotype 3.In summary,this study used different sample pretreatment methods to treat clinical specimens from different sources and then sequenced them using Ion Torrent PGM sequencing platform.After that,we adopted in-house pathogen analysis software V1.0 to rapidly screen pathogens and finally provide information and guidance for clinical treatment.With the continuous development of sequencing technology,NGS will hopefully be applied to pathogen clinic diagnosis in the near future.