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.

By further analysing the read data, we identified several types of artifacts of the Ovation RNA-Seq system. First, the majority of reads (>95%) contained a partial (>9 bp) Ovation RNA-Seq SPIA (single primer isothermal amplification) primer, which often appears in the start of the read. Second, a substantial fraction (up to 25%) of the reads did not align to the viral or host references. With a fraction of the unaligned reads (50 000), we used MEGAN to identify the composition of unaligned reads for the HIV and WNV clones (Supplementary Figure S3) . For the HIV clone, the read composition was: 12% bacterial, 3% eukaryotic, 4% retrovirus, 33% not assigned and 48% no hit. The retrovirus hits were unaligned HIV sequences. The majority of the not assigned reads were bacterial rRNA and HIV hits that could not be classified to specific taxonomical group. The no hit reads were mostly simple sequence repeats and SPIA primer sequences. The high abundance of these reads might be due to the low input amounts, which led to the greater abundance of these non-specific by-products. For the WNV clone, the read composition was: 0.5% bacterial, 3% eukaryotic, 0.5% flavivirus, 2% not assigned and 95% no hit. The eukaryotic hits were mostly rat and mouse. The flavivirus hits were unaligned WNV sequences. Some of the no hits for WNV clone were simple sequence repeats and SPIA primer, but the majority of the no hit reads for WNV could not be identified as similar to known sequences. It is possible that these reads could be hamster-specific sequences that were not aligned to the preliminary golden hamster assembly.