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| 1 | VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens显示文摘Objective Unbiased next generation sequencing(NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification(VSITA) approach using a set of non-ribosomal and virus-enriched octamers(V8) was developed and compared with traditionally used random hexamers(N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR(qP CR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences(sense and antisense) were selected from a dataset of random 4,096(4~6) hexamers(N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480(30 × 4~2) octamers(V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples. | ZHANG Yi ZHANG Chen LI Bo LI Yang HE Xiao Zhou LI Acher WU Wei DUAN Su Xia QIU Fang Zhou WANG Ji SHEN Xin Xin YANG Meng Jie LI De Xin MA Xue Jun | 2018 | Biomedical and Environmental Sciences2018，31，4： | 3 |
| 2 | Intra-host Ebola viral adaption during human infection显示文摘The onsite next generation sequencing(NGS)of Ebola virus(EBOV)genomes during the 2013–2016 Ebola epidemic in Western Africa provides an opportunity to trace the origin,transmission,and evolution of this virus.Herein,we have diagnosed a cohort of EBOV patients in Sierra Leone in 2015,during the late phase of the outbreak.The surviving EBOV patients had a recovery process characterized by decreasing viremia,fever,and biochemical parameters.EBOV genomes sequenced through the longitudinal blood samples of these patients showed dynamic intra-host substitutions of the virus during acute infection,including the previously described short stretches of 13 serial TNC mutations.Remarkably,within individual patients,samples collected during the early phase of infection possessed Ts at these nucleotide sites,whereas they were replaced by Cs in samples collected in the later phase,suggesting that these short stretches of TNC mutations could emerge independently.In addition,up to a total of 35 nucleotide sites spanning the EBOV genome were mutated coincidently.Our study showed the dynamic intra-host adaptation of EBOV during patient recovery and gave more insight into the complex EBOV-host interactions. | William JLiu Weifeng Shi Wuyang Zhu Cong Jin Shumei Zou Ji Wang Yuehua Ke Xiaofeng Li Mi Liu Tao Hu Hang Fan Yigang Tong Xiang Zhao Wenbin Chen Yuhui Zhao Di Liu Gary Wong Chengchao Chen Chunyu Geng Weiwei Xie Hui Jiang Idrissa Laybor Kamara Abdul Kamara Matt Lebby Brima Kargbo Xiangguo Qiu Yu Wang Xiaofeng Liang Mifang Liang Xiaoping Dong Guizhen Wu George F.Gao Yuelong Shu | 2019 | Biosafety and Health2019，1，1： | 1 |
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