| In recent years,the emerging and re-emerging infectious diseases have brought serious challenges to public health and livestock and poultry breeding in China.Nanopore sequencing is a new generation of highthroughput sequencing technology developed by Oxford Nanopore Technologies.Due to its characteristics of portability,long reading and real-time data processing,it can provide pathogen information for rapid diagnosis and treatment of epidemic diseases,thus revolutionizing the application mode of traditional high-throughput sequencing technology in related fields.In order to evaluate the sequencing performance of nanopore sequencing technology in unknown pathogens in DNA or RNA viruses,this study used the nanopore technology to quickly confirm an outbreak of farmed wild boar in Arxan region of Inner Mongolia.In result,213 ASFV sequences which covered 28.1% of the whole genome sequence were obtainted within 150 minutes since we received the sample.Then,1826 African Swine Fever Virus(ASFV)sequences were sequenced by nanopore ligation,covering 99% ASFV genome,with an average 5.02 x sequencing depth.The early detection of African swine fever was realized through this study,which is of great significance in prevebping the epidemic of virus in wild boar population.In order to further expand the application of nanopore sequencing technology in the rapid diagnosis of viral diseases,this study used the nanopore RAD004 strategy to sequence and explore a positive disease material of mixed infection of Porcine circovirus(PCV)and Porcine Bocavirus(PBo V),then successfully detected 17 single-strand linear PBo V sequences,with a sequencing depth of 2.4x,cover 87% of the genome with an accuracy of 92%.However,this strategy is not suitable for nanopores sequencing of single-stranded circular DNA viruses.Furthermore,in order to explore the application of nanopore sequencing in RNA viruses.this study performed direct c DNA sequencing of nanopore after adding Poly A to the end of single negative strand rabies virus.After sequencing for 12 h,1.03 G data was obtained,with the coverage of 8.7%.According to the LSK109 process,3.44 g data were obtained after 12 h sequencing,among which 577 were RABV sequences.Further,by designing specific primers,the nanopore amplicon sequencing of the rabies virus L gene was performed to obtain 1558 RABV sequences with a sequencing depth up to 58 x and the accuracy of 99.2%.Subsequebply,all the data sequenced by ligation method were used for splicing to obtain 80.5% of the whole genome.In addition,PCS109 kit was used to sequence the segment Alongshan virus(ALSV)from low initial samples.The 599 M data contained 18 ALSV sequence,which covered 4 segments,The N50 was only 208 bp after PCR amplified,but the 1531 bp of NS3-like gene coding region was amplified using this short sequence as a reference,which assisted the research progress of the whole genome of ALSV.The above results show that the nanopore sequencing platform can not only be used for the rapid identification of emerging and re-emerging pathogens,but also can validate and optimize the whole genome sequencing process of DNA/RNA viruses by nanopore sequencing and the related bioinformation analysis protocols.It promotes the application of nanopore sequencing in the field of virus disease prevention and control,and provides a convenient,real-time and efficient gene sequencing solution for the research of molecular virology,which is of great significance in the prevention and control of zoonoses. |
