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Improved detection of rare HIV-1 variants using 454 pyrosequencing
Brendan B Larsen1, Lennie Chen, Brandon S Maust
1Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
Plos One
|October 8, 2013
Summary
Detecting rare HIV variants is challenging due to PCR errors. This study optimized PCR methods and developed an amplicon-based approach to improve detection sensitivity for HIV genetic variation analysis.
Area of Science:
- Virology
- Genetics
- Molecular Biology
Background:
- 454 pyrosequencing (a massively parallel sequencing technology) is crucial for studying HIV genetic variation.
- The polymerase chain reaction (PCR) step in sample preparation introduces significant mismatch errors, limiting rare variant detection to ~1%.
Purpose of the Study:
- To optimize PCR enzymes and conditions for high sensitivity and low error rates in HIV variant detection.
- To develop an amplicon-based method for consistent read coverage across the HIV-1 genome.
Main Methods:
- Systematic variation of PCR enzymes and conditions to assess substitution error rates and amplification sensitivity.
- Design of 22 primers for 11 overlapping amplicons covering 4.7 kb of HIV-1 clade B gag-pol and env gp120 regions.
Main Results:
- Substitution errors varied up to 3-fold among different PCR enzymes.
- Enzyme sensitivity varied, impacting template amplification for pyrosequencing.
- The amplicon-based method achieved detection sensitivities as low as 0.01-0.2% for rare HIV variants.
Conclusions:
- Optimized PCR protocols and an amplicon-based strategy significantly enhance the detection of rare HIV variants.
- This improved methodology is vital for comprehensive HIV genetic variation studies and understanding viral evolution.
