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

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.

The methods described here have several promising applications. The sequence-independent amplification methods coupled with our de novo assembly of viral genomes provides a new methodology for sequencing viral genomes from many different sample types. These methods can generate next-generation sequencing data of pathogens allowing for greater insight into pathogen evolution, fitness, emergence and transmission as well as factors that are important for the development of new therapeutics and vaccines. In addition, these methods could be used to discover known or unknown pathogens in clinical samples, not limited to viruses. Likewise, these methods could also be used for surveillance to detect viral RNA from environmental sources, such as WNV in mosquito samples. This surveillance ability could provide valuable insight into what pathogens are circulating and provide an ''early warning'' of future Nucleotide % identity between Ovation RNA-Seq-Illumina and RT-PCR-454 assemblies. b Number of mismatches between assemblies that were not supported by read data (see Materials and Methods). c Number outside the parentheses was the total number of indel bases. Number within the parentheses refers to size of individual indels with a hyphen between each occurrence.