Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laborat...Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.展开更多
The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary...The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.展开更多
Amplicon sequencing of functional genes is a powerful technique to explore the diversity and abundance of microbes involved in biogeochemical processes. One such key process, denitrification, is of particular importan...Amplicon sequencing of functional genes is a powerful technique to explore the diversity and abundance of microbes involved in biogeochemical processes. One such key process, denitrification, is of particular importance because it can transform nitrate(NO3-) to N2 gas that is released to the atmosphere. In nitrogen limited alpine wetlands, assessing bacterial denitrification under the stress of wetland desertification is fundamental to understand nutrients, especially nitrogen cycling in alpine wetlands, and thus imperative for the maintenance of healthy alpine wetland ecosystems. We applied amplicon sequencing of the nirS gene to analyze the response of denitrifying bacterial community to alpine wetland desertification in Zoige, China. Raw reads were processed for quality, translated with frameshift correction, and a total of 95,316 nirS gene sequences were used for rarefaction analysis, and 1011 OTUs were detected and used in downstream analysis. Compared to the pristine swamp soil, edaphic parameters including water content, organic carbon, total nitrogen, total phosphorous, available nitrogen, available phosphorous and potential denitrification rate were significantly decreased in the moderately degraded meadow soil and in severely degraded sandy soil. Diversity of the soil nirS-type denitrifying bacteria communities increased along the Zoige wetland desertification, and Proteobacteria and Chloroflexi were the dominant denitrifying bacterial species. Genus Cupriavidus(formerly Wautersia), Azoarcus, Azospira, Thiothrix, and Rhizobiales were significantly(P<0.05) depleted along the wetland desertification succession. Soil available phosphorous was the key determinant of the composition of the nirS gene containing denitrifying bacterial communities. The proportion of depleted taxa increased along the desertification of the Zoige wetland, suggesting that wetland desertification created specific physicochemical conditions that decreased the microhabitats for bacterial denitrifiers and the denitrification related genetic diversity.展开更多
基金supported by the National Key Research and Development Program(grant number:2022YFC2305304).
文摘Objective Viral encephalitis is an infectious disease severely affecting human health.It is caused by a wide variety of viral pathogens,including herpes viruses,flaviviruses,enteroviruses,and other viruses.The laboratory diagnosis of viral encephalitis is a worldwide challenge.Recently,high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections.Thus,In this study,we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing.Methods We designed nine pairs of specific polymerase chain reaction(PCR)primers for the 12 viruses by reviewing the relevant literature.The detection ability of the primers was verified by software simulation and the detection of known positive samples.Amplicon sequencing was used to validate the samples,and consistency was compared with Sanger sequencing.Results The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×,and the sequence lengths were consistent with the sizes of the predicted amplicons.The sequences were verified using the National Center for Biotechnology Information BLAST,and all results were consistent with the results of Sanger sequencing.Conclusion Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis.It is also a useful tool for the high-volume screening of clinical samples.
基金supported by the National Key Research and Development Program of China(2022YFC2303401,2022YFC2304100,2016YFD0500301,2021YFC0863300)the Beijing Science and Technology Plan(Z211100002521017)the National Natural Science Foundation of China(82241080)。
文摘The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.
基金supported by the Natural Science Foundation of China (Grant No. 41201256)
文摘Amplicon sequencing of functional genes is a powerful technique to explore the diversity and abundance of microbes involved in biogeochemical processes. One such key process, denitrification, is of particular importance because it can transform nitrate(NO3-) to N2 gas that is released to the atmosphere. In nitrogen limited alpine wetlands, assessing bacterial denitrification under the stress of wetland desertification is fundamental to understand nutrients, especially nitrogen cycling in alpine wetlands, and thus imperative for the maintenance of healthy alpine wetland ecosystems. We applied amplicon sequencing of the nirS gene to analyze the response of denitrifying bacterial community to alpine wetland desertification in Zoige, China. Raw reads were processed for quality, translated with frameshift correction, and a total of 95,316 nirS gene sequences were used for rarefaction analysis, and 1011 OTUs were detected and used in downstream analysis. Compared to the pristine swamp soil, edaphic parameters including water content, organic carbon, total nitrogen, total phosphorous, available nitrogen, available phosphorous and potential denitrification rate were significantly decreased in the moderately degraded meadow soil and in severely degraded sandy soil. Diversity of the soil nirS-type denitrifying bacteria communities increased along the Zoige wetland desertification, and Proteobacteria and Chloroflexi were the dominant denitrifying bacterial species. Genus Cupriavidus(formerly Wautersia), Azoarcus, Azospira, Thiothrix, and Rhizobiales were significantly(P<0.05) depleted along the wetland desertification succession. Soil available phosphorous was the key determinant of the composition of the nirS gene containing denitrifying bacterial communities. The proportion of depleted taxa increased along the desertification of the Zoige wetland, suggesting that wetland desertification created specific physicochemical conditions that decreased the microhabitats for bacterial denitrifiers and the denitrification related genetic diversity.
