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.

Due to the challenges of large amounts of host contamination, highly variable coverage and the presence of SPIA primer found in our sequence data, we used a newly developed assembler, VICUNA, to generate de novo assemblies for HIV, RSV and WNV low input samples (Yang et al., manuscript submitted). VICUNA is designed to assemble genetically heterogeneous viral genomes in the presence of large amounts of host contamination. To enable a direct comparison among all our viral libraries, we randomly sampled 5 million reads for each sample. This choice reflects a realistic sequencing process in production and allowed us to obtain a high average coverage (e.g. over 1000-fold) of the target region as well as sufficient coverage across the genome, given that up to 99% of the reads are non-viral sequences and coverage is uneven. For each HIV clinical sample, we generated consensus sequence that covers over 96% of the target region (Table 1 ). In 7 out of 10 clinical samples, we generated consensus sequence that covers 100% of the target region (Supplementary Table S2 ). For WNV clone samples, we generated a consensus sequence that covers over 99% of the target region (Table 1 ). In 15 out of 16 WNV samples, we generated a consensus sequences that covers 100% of the target region (Supplementary Table S2 ). For both RSV clinical samples, we generated a consensus sequence that covers 100% of the target region (Table 1) .