Comparison Of 4 Viromic Methods And Which Application In Marmot Intestinal Samples

As an important part of microorganisms,viruses can not only cause various types of infections of the host,but also play a vital role in the homeostasis and physiological health of humans and animals.In recent years,many infectious diseases have emerged due to frequent appearance of new viruses with animal origin.,which highlights that the discovery of new viruses and the continuously monitor and control of the virus carried by natural hosts are extremely important.Viral metagenomics is the application of metagenomics in the field of virology,which is a powerful tool for virus tracking and tracing,the appropriate viromic technology and program can effectively improve the efficiency and accuracy of viromic research.At present,there are many virus metagenomics technologies in the world,but their performance has not been systematically assessed.In this study,we constructed artificial virus samples containing different titers and different nucleic acid types to compar ed the sensitivity,specificity and the ability to cover the whole genome of Meta-transcriptome(MTT),Metagenomic(MTG),Sequence-independent single primer application(SIA)and Multiple displacement amplification(MDA),also explored the relationship between viral load,sequencing depth and the number and coverage of reads.Then we applied these four methods to analyze the intestinal samples of Himalayan marmots in Kashgar,Xinjiang,further verifying the respective advantages of the four methods,providing technical solutions and alternative plan for future viromic research,according to the results of the viromic,the detection and genetic evolution analysis of key viruses are also carried out.With a known viral load,it was found that the MTT has the highest sensitivity,the MTT and MDA are superior to the SIA and MTG in terms of sensitivity and coverage,the MTT has high specificity and coverage for RNA viruses;the SIA has the ability to capture both RNA and DNA viruses,but the detection rate of reads is less than MTT and MDA.MTG and MDA are specific for DNA viruses and the MDA has a strong coverage and detection rate for circular DNA viruses.The viral load is positively correlated with the number of reads and the coverage of the whole genome,and the number of reads and the coverage can be effectively improved with the sequencing depth increasing.Afterwards,through the application of these four methods,the virome of intestinal samples of the marmots revealed 30 families of viruses,among which 12 are vertebrate virus,6 are plant virus,and insect virus and phages are 1 and 12 respectively.Further comparison found that the results obtained from four methods applied in real sample are consistent with simulated samples.Here,7 viruses were selected for detection,and it was found that the bocaparvovirus had the highest positive rate of 93.5%,followed by the circovirus with 71%.The phylogenetically analysis of astrovirus,hepatitis A virus,adenovirus,and bocaparvovirus revealed that astrovirus and adenovirus were significantly divergent from currently known viruses and are prototypes of new species;the hepatitis A virus and bocaparvoviruses showed little difference from the reference strain.Taken together,for the purpose of exploring the whole virome,SIA alone or the combination of MTT and MDA can be used,though the latter will increase the cost of research,but has good sensitivity.Viral load is positively correlated with the number of reads and coverage,indicating that the number of reads can reflect the abundance of virus community components,and increasing the sequencing depth can effectively improve the detection ability and coverage.We conducted analyses of intestinal samples of Himalayan marmots,verified the application scenarios of the four viromic methods and the detection of viruses and phylogenetically analyzed revealed the diversity and prevalence of viruses carried by Himalayan marmots in Kashgar.