Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

被引:65
|
作者
Malboeuf, Christine M. [1 ]
Yang, Xiao [1 ]
Charlebois, Patrick [1 ]
Qu, James [1 ]
Berlin, Aaron M. [1 ]
Casali, Monica [1 ]
Pesko, Kendra N. [2 ]
Boutwell, Christian L. [3 ]
DeVincenzo, John P. [4 ,5 ,6 ,7 ]
Ebel, Gregory D. [2 ]
Allen, Todd M. [3 ]
Zody, Michael C. [1 ]
Henn, Matthew R. [1 ]
Levin, Joshua Z. [1 ]
机构
[1] Broad Inst MIT & Harvard, Cambridge, MA 02142 USA
[2] Univ New Mexico, Sch Med, Dept Pathol, Albuquerque, NM 87131 USA
[3] Ragon Inst MGH MIT & Harvard, Boston, MA 02129 USA
[4] Univ Tennessee, Sch Med, Dept Pediat, Memphis, TN 38103 USA
[5] Univ Tennessee, Grad Sch Hlth Sci, Lebonheur Childrens Med Ctr, Memphis, TN 38103 USA
[6] Univ Tennessee, Grad Sch Hlth Sci, Childrens Fdn Res Ctr, Memphis, TN 38103 USA
[7] Univ Tennessee, Grad Sch Hlth Sci, Dept Mol Sci, Memphis, TN 38103 USA
基金
美国国家卫生研究院;
关键词
IMMUNODEFICIENCY-VIRUS TYPE-1; WEST-NILE-VIRUS; REVERSE-TRANSCRIPTASE; SECONDARY STRUCTURES; INTRAHOST DIVERSITY; CONTROLLERS; LIBRARIES; MULTIPLE; CHILDREN; IMPACT;
D O I
10.1093/nar/gks794
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.
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页数:10
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