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.

During the assembly process, we observed in several cases that by invoking the optional reference guided merging component in VICUNA after the initial assembly, we could improve the final consensus. The majority of the clinical samples were assembled in only one or two contigs prior to reference guided merging. A significant portion of the viral genome was assembled into a single large contig (Supplementary Table S4 ) without the Coverage was computed as the total number of reads covering a given residue and was normalized by the total coverage summed across all residues; at each residue, the coverage was divided by the total coverage and sum of normalized coverage equals one. need for reference guided merging, but the reference guided merging increased the coverage of the final CDS to >96% of the viral genome (Table 1 and Supplementary Table S2 ). In addition, upon further investigation, we discovered an artifact that produced chimeric fragments, which might be caused by stem loops of RNA secondary structure (Supplementary Figure S4) . This artifact complicated the de novo assembly by increasing the number of contigs and required reference guided merging of contigs.