Illumina Sequencing Characterises Diverse Errors in HIV-1 Resistance Testing
PHE ePoster Library. Bibby D. Apr 10, 2019; 257471; 15280
Dr. David Bibby
Dr. David Bibby
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Abstract
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Abstract Background:
PHE administers an External Quality Assurance (EQA) scheme for 17 pathology laboratories in the UK and Ireland that offer HIV-1 resistance testing (HIVResNet). These assays are complex and multi-stage, and are difficult to optimise. Substantial discordance between different labs' FASTA sequences derived from identical clinical samples reflects how targeted improvement is hampered by a lack of quantitative metrics during optimisation of component stages. Here, we show how Next Generation Sequencing (NGS) can facilitate this process.
Methods:
Panels of clinical samples are distributed annually to participants for routine testing. Returned sequence files are aligned, consensuses determined, and discordances calculated. In parallel, amplicons are requested from laboratories; those received are sequenced by NGS at PHE. Data are processed through a pipeline that includes in-house scripts to compare NGS outputs with those from the corresponding routine sequences. Each discordance is interrogated for information regarding the source of its error.
RESULTS:
The NGS data facilitated resolving of discrepancies observed using Sanger data. In descending order of impact, four broad patterns of discrepant results were observed:Inefficiencies substantially limiting the diversity of templates sequencedA wide variation in minority base frequencies at which labs were able to call mixed bases accuratelyStochastic detection errors owing to variant frequencies at some loci being at or around the 20% threshold of Sanger-based methodsSelective oversight of minority quasispecies members due to sequencing primer biasFailure to detect linkage between loci in hypervariable regionsCONCLUSIONSBy revealing factors influencing false positive and false negative antiviral resistance results, this NGS analysis allows targeted interventions in areas of assay underperformance. Coupled with anonymous sharing of protocols via HIVResNet, we have facilitated targeted improvement in assay performance, and have developed a unique perspective on such EQA schemes. Funding Work funded by PHE and the MRC HIV Drug Resistance Database.
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