Study On Multiple In-solution Hybridization Capture For The Whole Genome Of Three Diarrhea-causing Viruses

Objective: Viral diarrhea has been a serious problem globally,especially as Rotavirus A(RVA),Norovirus(NoV),and Human Astrovirus(HAstV)are more common.In particular,without gastrointestinal symptoms,HAstV can also cause severe central nervous system infections.Whole genome sequencing plays a crucial role in the tracing of virus origin and monitoring of virus transmission patterns during disease outbreaks.Under the challenge of high host/background genome in clinical samples,this study designed probes for the whole genome of three common diarrhea-causing viruses,combining in-solution hybridization capture with next-generation sequencing(NGS)technology,and directly captured the whole genome of the virus from clinical samples,providing technical support for the identification,traceability analysis and mutation regularity of infectious disease pathogens.Methods: In this research,48 fecal specimens were taken from 48 diarrhea sufferers in 4sentinel hospitals in China,and the three most frequent diarrheal virus infection samples were examined using RT-qPCR.Meanwhile we screened 1615 reference genomes according to RVA,NoV GⅠ/GⅡ,and HAstV,designed in-solution hybridization capture probes.And then combined with Next Generation Sequencing.Finally,Complete genome capture of four genotypes of three diarrhea-causing viruses was achieved.Results: 1.Probe design: a total of 56547 probes with a length of 100 bp were designed and synthesized,with a total coverage area of 8142.32 Kb.The coverage of all available RVA,NoV GⅠ/GⅡ,and HAstV sequences in the NCBI database was up to 99.8693%,and the capture depth of each genome region was uniform and effective.2.Genome coverage:the average circulation threshold(CT)of 50 viral samples was 21.83(7.35-36.91).An average of 3584721(1126 ～ 9621440)Reads were generated per sample.Excluding two e outlier,the mean coverage of the whole genome was 99.55%(range 94.56% to 100%).Conclusion: 1.In this study,RVA had the highest detection rate among the three viruses detected by RT-qPCR(accounting for 50%),indicating that the population in the corresponding areas of the 4 sentinel hospitals monitored in China was mostly infected with RVA.2.In this study,we designed and synthesized a total of 56547 probes for 4genotypes of 3 diarrhea-causing viruses,and validated 48 samples of 50 viruses(24 RVA,7NoV GⅠ,10 NoV GⅡ,and 5 HAstV)with CT values ranging from 7.35 to 36.91.The whole genome of the virus was successfully captured from clinical samples with CT value > 35,and the average coverage rate of the whole genome was 98.90%,which confirmed the availability of multiple In-Solution probes capturing methods of diarrhea-causing virus in the clinic and provided technical support for the identification,tracing,and analysis of infectious disease pathogens as well as the study of variation rules.