Abstract：The advent of next generation sequencing (NGS) technology has shed lights on comprehensive investigation of unusual or emerging pathogens for global microbe-hunters. In this study, protocols for de novo sequencing of RNA viruses were established in our laboratory, the preliminary performance of genomic sequencing of a cultured virus pool was assessed on a GS-Junior sequencer. Viral RNAs of a hand-foot-mouth disease (HFMD) associated EV71 strain and a seasonal H1N1 subtype influenza virus were extracted, artificially pooled and reversely transcribed with random primers, an NGS library was constructed and subjected to routine pyrosequencing on the GS Junior platform. It was indicated from raw sequence reads that over 140 K reads generated approximately 46.9 M base-pairs, with an average length of 334 bp and a median read-length of 352 bp. De novo assembly produced 914 contigs, while reference mapping also showed that 99.8% of full-length EV71 genome and 96.7% of influenza virus genome were achieved in a single run. The observations demonstrated that NGS platforms are capable of rapidly and precisely de novo sequencing of multiple genomes for RNA viruses, suggesting that NGS technologies are applicable to a variety of fields in future pathogen biology.
张拥军,陈炜,翁育伟,谢剑锋,何文祥,吴冰珊,郑奎城,严延生. 高通量测序平台测定混合病毒样品基因组序列[J]. 中国人兽共患病学报, 2014, 30(9): 884-888.
ZHANG Yong-jun,CHEN Wei,WENG Yu-wei,XIE Jian-feng,HE Wen-xiang,WU Bing-shan,ZHENG Kui-cheng,YAN Yan-sheng. De novo sequencing of cultured virus pool on NGS platform. Chinese Journal of Zoonoses, 2014, 30(9): 884-888.
[1]Briese T, Paweska JT, McMullan LK, et al. Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa[J]. PLoS Pathog, 2009, 5(5): 4e1000455. DOI: 10.1371/journal.ppat.1000455 [2]Palacios G, Druce J, Du L, et al. A new arenavirus in a cluster of fatal transplant associated diseases[J]. N Engl J Med, 2008, 358: 991-998. DOI: 10.1056/NEJMoa073785 [3]Chin CS, Sorenson J, Harris JB, et al. The origin of the Haitian cholera outbreak strain[J]. N Engl J Med, 2011, 364: 33-42. DOI: 10.1056/NEJMoa1012928 [4]Rohde H, Qin J, Cui Y, et al. Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4[J]. N Engl J Med, 2011, 365: 718-724. DOI: 10.1056/NEJMoa1107643 [5]Rasko DA, Webster DR, Sahl JW, et al. Origins of the E. coli strain causing an outbreak of hemolytic uremic syndrome in Germany[J]. N Engl J Med, 2011, 365: 709-717. DOI: 10.1056/NEJMoa1106920 [6]McMullan LK, Folk SM, Kelly AJ, et al. A new phlebovirus associated with severe febrile illness in Missouri[J]. N Engl J Med, 2012, 367: 834-841. DOI: 10.1056/NEJMoa1203378 [7]Hoffmann B, Scheuch M, Hoper D, et al. Novel orthobunyavirus in cattle, Europe, 2011[J]. Emerg Infect Dis, 2012, 18: 469-472. DOI: 10.3201/eid1803.111905 [8]Assiri A, McGeer A, Perl TM, et al. Hospital outbreak of Middle East respiratory syndromecoronavirus[J].N Engl J Med. 2013, 369(5): 407-416. DOI: 10.1056/NEJMoa1306742 [9]Firth C, Lipkin WI. The genomics of emerging pathogens[J]. Annu Rev Genomics Hum Genet,2013, 14: 281-300. DOI: 10.1146/annurev-genom-091212-153446 [10]Sims D, Sudbery L, Ilott NE, et al. Sequencing depth and coverage: key consideratios in genomic analyses[J]. Nat Rev Genet, 2014, 15(2): 121-132. DOI: 10.1038/nrg3642 [11]Lennon NJ, Lintner RE, Anderson S, et al. A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454[J]. Genome Biol,2010, 11(2): R15. DOI: 10.1186/gb-2010-11-2-r15
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