The authors sequenced virus from an Indian rhesus macaque experimentally infected with SIVmac239 and coding regions from 11 HIV-positive patients. Overlapping RT-PCR amplicons were used to cover the virus genome sequences, and the RT-PCR-amplified genomes were simultaneously fragmented and tagged using the Nextera Roche 454-Compatible Enzyme Mix, followed by pyrosequencing. An average of 41,826 sequence reads per SIV genome was obtained, with an average coverage depth of 380 sequences. For HIV samples, an average of 29,000 sequence reads per genome with a sequencing depth range of 208-846 was obtained. Full or near-full coverage was obtained with Nextera libraries prepared from 50 ng of DNA.
The authors conclude that, using Nextera technology, they are able to demonstrate:
“...a new and highly practical approach to study the complexity of the viral population within a host and identify minor variants on a genome-wide scale. While this manuscript applies pyrosequencing to immunodeficiency viruses, this approach could be applied to any viral pathogen.”
Bimber, B. et al. (2010). Whole genome characterization of HIV/SIV intra-host diversity by ultra-deep pyrosequencing Journal of Virology DOI: 10.1128/JVI.01378-10