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.

For point of comparison with our newly developed methods, we attempted to generate RT-PCR amplicons for HIV clone and clinical samples A and B and sequence them by 454 using previously described methods (10) . For clinical sample B, we were not able to generate all four-overlapping PCR amplicons spanning the CDS after four attempts. With our newly developed methods, this sample was successful in all six attempts to generate consensus assembly covering >98% of the CDS (Supplementary Table S1 ). We did generate all four-overlapping PCR amplicons, sequence by 454 and built consensus assemblies for HIV clone and clinical sample A. For HIV clone, assemblies were 100% identical between the two methods (Table 3 ). For clinical sample A, the assemblies were 98.25 and 99.41 % identical at the nucleotide and composition levels, respectively (Table 3) . Our newly developed methods generated assemblies for HIV clone and clinical samples that were highly similar to those generated using standard RT-PCR methods.