Application Research Of Metagenomic Second-generation Sequencing Technology In Virus Pathogen Detection

With increased urbanization and globalization,viruses previously confined to small,remote geographic areas now spread around the world.Classical viral diseases are invading novel populations.For example,we have seen the transmission of Middle East Respiratory Syndrome coronavirus from camel to human in Arabian countries,and its spread to remote areas in east Asia;the transmission of Yellow fever virus(YFV)from Africa to Asia;and the transmission of Zika virus from South America to the whole world.Even in areas with poor transportation,viruses have caused new threats to public health,such as the outbreak of Ebola in 2014-16.The first critical step of viral disease control and prevention is identification of viral pathogens.Traditionally,serological tests and PCR-based molecular methods are used as first-line identification methods on the outbreak of epidemic diseases.In particular,in most cases,real-time quantitative PCR(qPCR)is considered the gold standard for virus identification with high sensitivity and accuracy.However,the limitation of real-time qPCR is that the design of specific primers and probes relies on prior knowledge of the pathogen's DNA sequence.Thus,sequence-independent methods of virus identification need to be developed.The development of next-generation sequencing(NGS),with unprecedented sequencing throughput,has promised to address this problem.With the use of random oligonucleotides instead of predefined primers,unbiased sequencing is able to identify any virus sequence,known or unknown,local or imported,including never reported pathogens.However,unbiased sequencing is very sensitive to background interference from the host and environment.Clinical samples with low viral load need enrichment of viral nucleic acids.This research aimed to establish a metagenomic NGS platform based on Ion Torrent PGM/Proton technology and apply this platform to epidemic emergencies,identification of unknown pathogens and screening of pathogen spectra.Additionally,we intended to develop a virus-targeted amplification method to improve the detection sensitivity of traditional NGS.The main content of this research was:1.In March 2016,a Chinese worker living in Angola became ill and returned to China for medical treatment.Initial detection results indicated suspected YFV.This was the first imported case of Yellow fever in China.Metagenomic NGS was applied to a clinical serum sample from the patient.Nucleic acid extraction,library construction,sequencing and bioinformatic analysis were completed within 30 h.A total of 10,823 sequencing reads were generated.After bioinformatic analysis,the consensus sequence assembled from hit reads covered 99.92%of the genome of the strain Angola71.The average sequencing depth was 157.96 × coverage.The Ct value from real-time qPCR was 21.56.2.Human torque tenovirus(TTV)was detected by metagenomic sequencing in 10 serum samples from patients with fever of unexplained origin.After assembling sequencing reads and calculating the consensus sequence coverage,all reads were assigned to the genera Alphatorquetenovirus,Betatorquetenovirus and Gammatorquetenovirus.The genomes and their evolution were characterized.The role of TTV as a signature of immune level was also discussed.The phenomenon that TTV was the only viral pathogen detected may be due to the pathogenicity of TTV,or to a low level immune response ofthe host to other infections.A low level of immunity in the host caused by non-infection factors is another possible reason for this phenomenon.3.Surveillance of febrile jaundice in the Sierra Leone-China Friendship P3 Biosafety Laboratory was carried out.We profiled the pathogen spectra in archived YFV-negative serum from 100 patients in Sierra Leone who presented with unexplained febrile jaundice,using metagenomic NGS.The most frequently identified sequencing reads belonged to pathogens including cytomegalovirus(86%),Epstein-Barr virus(53%),hepatitis C virus(33%),rhinovirus(27%),hepatitis A virus(20%),coxsackievirus(10%),Ebola virus(8%),hepatitis E virus(8%),lyssavirus(4%),leptospirosis(4%),chikungunya virus(2%),Crimean-Congo hemorrhagic fever virus(1%),and hepatitis B virus(1%).This distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.4.A viral sequence-independent targeted amplification(VSITA)approach using a set of non-ribosomal and virus-enriched octamers(V8)was developed and compared with traditionally-used random hexamers(N6).A minimum 30 hexamers matching to viral reference sequences(sense and antisense)were selected from a dataset of 4096(46)random hexamers(N6).Two random nucleotides were added to the 5'-end of the selected hexamers,and 480(30×42)octamers were obtained(V8).Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in reverse transcription followed by qPCR.Ten serum samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were also used in comparison of V8 and N6 enrichment performance followed by NGS analysis.In general,the VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the 45 predefined clinical samples.Moreover,VSITA combined with NGS enabled detection of more viruses and achieved more viral read hits and higher viral genome coverage in the 20 clinical samples with diarrhea or fever of unknown origin.Conclusions:1.An Ion Torrent PGM/Proton-based integrated metagenomic sequencing pipeline was established.This pipeline included pretreatment of clinical samples,nucleic acid extraction,library construction,sequencing,and virus identification pipeline(VIP)bioinformatic analysis.This pipeline provided excellent technical support in an emergency epidemic situation,identification of undefined pathogens,and screening of pathogen spectra.2.This pipeline was tested in the emergency response to the first imported case of yellow fever in China.A whole genome sequence of YFV was obtained and its phylogenetic relationships determined within 30 h.3.The identification of undefined pathogens in clinical samples of fever of unknown origin was performed using this metagenomic sequencing pipeline.A group of sequences belonging to human Anelloviridae were identified.Bioinformatic analysis indicated that all the sequences belonged to the genera Alphatorquetenovirus,Betatorquetenovirus and Gammatorquetenovirus.4.During work in the Sierra Leone-China Friendship P3 Biosafety Laboratory,the metagenomic sequencing pipeline based on Ion Torrent Proton was applied to clinical samples from febrile jaundice patients which were negative for YFV by real-time qPCR.The pathogen spectrum was screened and analyzed.The data contributed to local public health work and clinical diagnosis.5.A novel virus sequence-independent targeted amplification method was developed.Compared with traditional NGS,evaluation results from clinical samples analyzed by VSITA showed higher sensitivity,viral read percentage and viral genome coverage.