Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

Abstract

RNA viruses are the causative agents for AIDS, influenza, SARS, and other serious health threats. Development of rapid and broadly applicable methods for complete viral genome sequencing is highly desirable to fully understand all aspects of these infectious agents as well as for surveillance of viral pandemic threats and emerging pathogens. However, traditional viral detection methods rely on prior sequence or antigen knowledge. In this study, we describe sequence-independent amplification for samples containing ultra-low amounts of viral RNA coupled with Illumina sequencing and de novo assembly optimized for viral genomes. With 5 million reads, we capture 96 to 100% of the viral protein coding region of HIV, respiratory syncytial and West Nile viral samples from as little as 100 copies of viral RNA. The methods presented here are scalable to large numbers of samples and capable of generating full or near full length viral genomes from clone and clinical samples with low amounts of viral RNA, without prior sequence information and in the presence of substantial host contamination.

Figure 1.

Complete sequence coverage of viral coding region. (A) Reproducibility of read coverage for technical replicates for HIV clone and clinical and WNV clone samples. (B) Comparison of read coverage for HIV clone and clinical samples between Ovation RNA-Seq version 1 (red) and version 2 (blue) systems. Reads were aligned to the CDS of the relevant viral reference using Mosaik. 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.