High throughput long amplicon sequencing, assembly and reference mapping of HIV genome using the GS FLX sequencer
Chopra, A., Watson, M., Cooper, D., Leary, S., John, M. and Mallal, S. (2009) High throughput long amplicon sequencing, assembly and reference mapping of HIV genome using the GS FLX sequencer. In: 21st Annual Conference of the Australasian Society for HIV Medicine (ASHM), 9 - 11 September, Brisbane, Australia.
Enhanced sequencing capacity may be of particular value in studies of pathogens with extreme levels of intra- and inter-sample polymorphism such as HIV and HCV. Current bulk sequencing methods rely on iterative primer selection and primer-walking to combat polymorphism efficiently, however malalignment due to complex polymorphisms with insertions and/or deletions in quasi-species often cannot be resolved easily without more laborious cloning of multiple sequences. We therefore wished to conduct a pilot study to test and develop methods for sequencing HIV using the GS FLX system that are high throughput, cost-effective and resolve complexity associated with diversity. A proof of principle run was undertaken in collaboration with Roche at the 454 centre Branford, Connecticut.
Nebulized PCR Amplicon spanning the entire HIV genome from 4, 8 or 12 plasma samples were barcoded, pooled into a single sequencing library and sequenced on a region of the 8 lane 454 picotitre plate. In the same run, a replicate experiment was carried out using the same samples to assess the reproducibility.
The number of reads per lane on the picotitre plate ranged from 41,000-54,000 with an average read length of 230bases. High quality reads from the samples were de-multiplexed and mapped, using 454 mapper software, to the reference sequence HXB2. On average 75-95% of the reference sequence was covered by the mapped reads. The depth of coverage across the genome ranged between 50-400 fold. With the most polymorphic region (env) showing lowest depth coverage. More detailed analysis of the data and the comparison of the sequences generated using the current sequencing protocol will be presented.
|Publication Type:||Conference Item|
|Murdoch Affiliation:||Institute for Immunology and Infectious Diseases|
|Item Control Page|
Downloads per month over past year