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Nucleic Acids Res. 2012 Sep 8. [Epub ahead of print]
Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification.
Malboeuf CM, Yang X, Charlebois P, Qu J, Berlin AM, Casali M, Pesko KN, Boutwell CL, Devincenzo JP, Ebel GD, Allen TM, Zody MC, Henn MR, Levin JZ.
Source
Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA Ragon Institute of MGH, MIT and Harvard, Boston, MA, 02129 USA, Department of Pediatrics, University of Tennessee School of Medicine, LeBonheur Children's Medical Center, The Children's Foundation Research Center and Department of Molecular Sciences, University of Tennessee Graduate School of Health Sciences, Memphis, TN, 38103 USA.
Abstract
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.
PMID:
22962364
[PubMed - as supplied by publisher]
Free full text
Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification.
Malboeuf CM, Yang X, Charlebois P, Qu J, Berlin AM, Casali M, Pesko KN, Boutwell CL, Devincenzo JP, Ebel GD, Allen TM, Zody MC, Henn MR, Levin JZ.
Source
Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, 87131, USA Ragon Institute of MGH, MIT and Harvard, Boston, MA, 02129 USA, Department of Pediatrics, University of Tennessee School of Medicine, LeBonheur Children's Medical Center, The Children's Foundation Research Center and Department of Molecular Sciences, University of Tennessee Graduate School of Health Sciences, Memphis, TN, 38103 USA.
Abstract
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.
PMID:
22962364
[PubMed - as supplied by publisher]
Free full text
