Establishment of a high throughput sequencing technology for detection of SFTS virus genome / 中华实验和临床病毒学杂志

Objective@#The objective of this study was to establish a next generation sequencing (NGS) method for severe fever with thrombocytopenia syndrome virus(SFTS).@*Methods@#SFTS virus RNA was extracted from the patient serum inoculated and isolated by Vero cells. Two methods of random primer sequencing and oligo(dT) beads selection sequencing were used for library construction. The libraries were built based on the best amplification and purification conditions. Whole genome sequencing was performed on NGS platform.@*Results@#There were significant differences in data of 3 virus between the two methods.The sample was sequenced by random primer sequencing showed low coverage and depth. However, three samples were sequenced by oligo(dT) beads selection showed coverage was over 99% and depth was over 900.The alignment rate of database from NCBI was more than 90%. The initial detection quality of this method was 300ng RNA.@*Conclusions@#In this study, we used the method of oligo(dT) beads selection to build libraries, and established a SFTS virus genome detection based on next generation sequencing.

Identification of unclassified influenza A virus using high-throughput sequencing technology / 中华实验和临床病毒学杂志

Objective@#To identify the avian influenza virus subtype from the avian and environmental samples using the Ion Torrent new-generation semiconductor sequencing technology and to establish a high-throughput sequencing method to identify unclassified influenza A virus.@*Methods@#Virus RNA was extracted from the nine avian swab and environmental samples and real-time RT-PCR was carried out to detect universal fluA, H5N1, H7N9 and H9N2. The whole genome of influenza A virus was amplified by PathAmpFluA kit. Sequencing library was prepared using Next Fast DNA Fragmentation & Library Prep Set for Ion Torrent kit and high-throughput sequencing was done by Ion Torrent Personal Genome Machine(PGM). Data from the PGM was processed and quality evaluated using Ion TorrentSuite v3.0 software. Sequence assembly and influenza database blast were carried out by FluAtyping v4.0 and PathogenAnalyzer bioinformatics software to identify the influenza A virus subtype of these nine samples.@*Results@#The results of real-time RT-PCR for universal fluA of these nine samples were positive but the results for H5N1, H7N9 and H9N2 were negative. Seven subtypes of influenza A virus were identified by high-throughput sequencing and bioinformatics analysis: six samples were H2N3, H5N6, H5N8, H7N1, H7N7, H11N3 subtype respectively and three samples were H6N6 subtype.@*Conclusions@#Avian influenza virus has many subtypes in the environment of Zhejiang province. Ion Torrent semiconductor sequencing technology is suitable for fast identification of unclassified influenza virus for avian influenza environment monitoring.