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 next compared the consensus assembly between technical replicates for HIV clone and clinical samples (Table 2) . First, we compared the nucleotide differences between the replicates (Assembly identity (%) in Table 2 ). For HIV clone replicates, the assemblies were 99.94% identical. For HIV clinical sample B, the assemblies were 99.26% and 98.55% identical for the 200 and 100 copy replicates, respectively. For WNV clone replicates, the assemblies were 99.98% and 100% identical for the 250 and 100 copy replicates, respectively. The discrepancies between assembled consensus sequences may be either caused by the differences in data generation or by the assembly process. To further evaluate the reproducibility of the method, we examined the nucleotide composition differences (Table 2 ) between any two assemblies. At each mismatch residue, the consensus base in the first assembly was considered to be consistent with the consensus base in the second assembly if the mismatch was supported by read alignments in the second assembly (Composition mismatches in Table 2 ). For HIV clone replicates, the assemblies were 99.98% identical when we include read support for any differences in the assembly. For HIV clinical sample B, the assemblies were 99.79% and 99.73% identical for the 200 and 100 copy replicates, respectively. For WNV clone replicates, the assemblies were 100% identical for both 250 and 100 copy replicates. Thus assemblies of both HIV and WNV consensus sequences are very reproducible.