High throughput sequencing of full length HIV-1 genomes
Chopra, A., Deganaar, J., Rushton, B., Maiden, T., Keller, J., Park, P.L., Gaudieri, S., John, M. and Mallal, S. (2007) High throughput sequencing of full length HIV-1 genomes. In: 4th International AIDS Conference on HIV Pathogenesis, Treatment and Prevention, 22 - 25 July, Sydney, Australia.
Objectives: To develop novel approaches to deliver cost effective, high throughput full genome HIV sequencing that best captures the full extent of within-host and between-host HIV genetic diversity at a population level.
Methods: Methods for each step of sample preparation, including reverse transcription, PCR reactions, and sequencing were developed on high throughput automated liquid handling instruments (Biomek FX, Beckman Coulter) with 96/384 well capacity. Protocols for RNA extraction were optimised based on plasma viral load. Rather than using multiple small fragments, large (6kb) fragments were amplified and sequenced using a set of standard sequencing primers. Alternate primers were then used where necessary to fill gaps remaining in the sequence. A software program “Primer Sleeper” was developed to automate selection of alternative primers based on the sequence already generated. We incorporated all the above into an iterative workflow system in which a ‘first pass’ nearly complete full-length sequence could be generated for majority of samples and made available for analysis, while mixtures, insertions/deletions and persistent gaps in a minority of problematic sequences were resolved with alternative strategies, including cloning.
Results: We generated 900 full and partial genomes in four study populations with an optimal throughput rate of 44 genomes per week. In the most recently sequenced project of 311 samples, the rate of successful amplification of two fragments spanning the full genome using standard primers was 75%, increasing to 91% by second reactions using alternate primers. Samples from different HIV clades and with a range of viral loads, including those with <2,000 copies/ml have been successfully sequenced.
Conclusions: High throughput HIV sequencing relies on innovations in all aspects of the sequencing assay and laboratory workflow. Our system can deliver sequence data with the turnaround, cost and quality required for investigators involved in contemporary HIV vaccinology, immunology and virology research.
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|Murdoch Affiliation:||Institute for Immunology and Infectious Diseases|
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