Rapid Identification And Whole-genome Sequencing Of Viruses On Nanopore Sequencing Platform

Background and objectiveAcute viral infections and associated epidemics are the primary threats to public health.In epidemics,viruses,especially RNA viruses,could rapidly spread,frequently mutate,and have a potential risk of cross-species transmissions.The rapid viral identification and characterizing the viral genome sequences are important to the epidemic control.The conventional methods like qPCR and viral culturing methods are required our prior knowledge on the target viruses.The non-target and broad-spectrum detection methods are limited.More important,the conventional method could not provide viral sequences after the detection.In this study,we aim to ① Build up a rapid detection method for unknown RNA viruses;②Develop the high-throughput method for viral genome sequencing;③Evaluate the sensitivity and specificity of these methods and apply them in the viral epidemics.Methods1.For unknown viruses’ detection,random primer amplification together with ligation kit for library construction were used.The sensitivity and specificity of this method were evaluated through specific qPCR.The rapid PCR barcode method for library construction was further set up and evaluated the sensitivity and specificity by using different types of virus strains.2.Viral whole genome sequencing:whole genome sequencing of SARS-CoV-2 is performed by using multiplex PCR combined with Nanopore sequencing.The sensitivity and accuracy of the method were evaluated by comparing with Illumina sequencing,BGI sequencing and qPCR results.3.Using the metatranscriptomic sequencing set up in this study to rapidly identify the causative agent of an AGE outbreak in Guangdong,2020.Characterizing the viral genetics and analyzing the viruses transmission through the sequences generated by nanopore sequencing.4.Applying the multiplex PCR and Nanopore sequencing to characterize the virology of re-positive SARS-CoV-2 infection cases in Guangdong,2020 and to evaluate the transmission risks of the re-positive cases.Results1.By using the random primer PCR amplification and the Nanopore ligation kit,we can build a sequencing library within 8 hours,identify the potential pathogen in 12 hours.With this method,we obtained the whole genome of the norovirus in an AGE outbreak in 48 hours.The sequencing result imply the norovirus identified in this outbreak is a new recombinant variant.2.We further improved the metatranscriptomic sequencing by using random primer amplification and rapid PCR barcode kit which allowed us to finish the library construction in 5 hours.For detecting SARS-CoV-2,influenza A virus in oropharyngeal swabs,the sensitivity of this method was close to specific qPCR.For detecting enterovirus EVA71 and CVA6 in stool samples,the threshold of this method was around 4.0E+06 and 3.5E+07 copies/mL(cp/mL).3.The multiplex PCR together with Nanopore sequencing presented a relative high sensitivity and accuracy in SARS-CoV-2 whole genome sequencing.Over 80%sequencing coverage could be achieved for sample with viral loads as low as 5.70E+03 copies/mL.The viral consensus sequences generated by Nanopore sequencing were consistent with the sequence on the Illumina platform after being analyzed and corrected for errors.With this method,we sequenced samples of SARS-CoV-2 repositive cases in Guangdong 2020 and the results showed nonintact viral genome could be sequenced from all re-positive cases,indicating a relative low transmission risk.Conclusions1.For unknown viruses’ detection,we found the random primer amplification with different Nanopore sequencing methods could rapidly identify the viruses,while with different sensitivity for different types of samples.2.For the whole genome sequencing of SARS-CoV-2,the multiplex PCR with Nanopore sequencing could achieve a relative high sensitivity and accuracy.3.Based on the unknown viruses’ detection,a more comprehensive analysis of diarrheal outbreaks caused by recombinant noroviruses can be made,whereby the recombinant GII.12[P16]was first identified to cause outbreaks in China.4.Based on the whole genome sequencing of SARS-CoV-2,analysis of COVID19 repositive cases suggests that fragments of SARS-CoV-2 may remain in the cases without the complete viral genome.