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.

We used both the Ovation RNA-Seq version 1 and 2 systems for HIV NL4-3 clone and clinical samples. As the input amounts for all these viral reactions was below the minimum recommended amount of 500 pg of RNA, we evaluated the success of the amplification reactions using HIV-specific qPCR assay (Supplementary Table S1 ). For WNV clone samples, Ovation RNA-Seq version 2 system reactions were performed using 100-10 000 copies of input RNA with technical duplicates. The success of each amplification reaction was determined using WNV-specific qPCR assay (Supplementary Table S1 ). For RSV clinical samples, Ovation RNA-Seq version 2 system reactions were performed using 1795 and 30 000 copies of input RNA. The success of each amplification reaction was determined using RSV-specific qPCR assay. All amplification reactions generated sufficient final products to make Illumina libraries. Illumina libraries were generated, pooled, and sequenced. We obtained 5.2-86.9 million reads per library (Supplementary Table S1 ).