Author: Malboeuf, Christine M.; Yang, Xiao; Charlebois, Patrick; Qu, James; Berlin, Aaron M.; Casali, Monica; Pesko, Kendra N.; Boutwell, Christian L.; DeVincenzo, John P.; Ebel, Gregory D.; Allen, Todd M.; Zody, Michael C.; Henn, Matthew R.; Levin, Joshua Z.
Title: Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification Document date: 2012_9_8
ID: s76c5ebd_1
Snippet: Massively parallel sequencing allows for rapid and low-cost deep sequencing of viral genomes and provides an opportunity to gain greater insight into viral evolution, fitness, emergence and transmission. Currently, reverse transcription followed by polymerase chain reaction (RT-PCR) with primers designed to amplify specific viral RNA sequences is the most common method for amplifying RNA viruses prior to sequencing and other downstream applicatio.....
Document: Massively parallel sequencing allows for rapid and low-cost deep sequencing of viral genomes and provides an opportunity to gain greater insight into viral evolution, fitness, emergence and transmission. Currently, reverse transcription followed by polymerase chain reaction (RT-PCR) with primers designed to amplify specific viral RNA sequences is the most common method for amplifying RNA viruses prior to sequencing and other downstream applications. Recent studies have used both 454 (1-11) (Newman et al., manuscript submitted) and Illumina (12) (13) (14) sequencing of RT-PCR amplicons for RNA viruses. However, standard RT-PCR methods are not adequate for the generation of templates suitable for sequencing low-copy viral RNA samples, where labor-intensive methods such as nested PCR (15) or single-genome amplification (SGA) (16, 17) are typically required. Examples of low-copy viral RNA samples include the following: HIV controllers (capable of controlling the virus in the absence of antiretroviral therapy) (18) , dengue virus (DENV) clinical samples collected after peak viremia (19) , and West Nile virus (WNV) surveillance samples (20) . Obtaining genomic sequence from such samples can provide valuable insights into viral attenuation, response to host immune pressure and drug treatment during infection, disease severity, transmission and epidemic spread.
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