Analytical Validation of the Thermo Fisher ABI HIV‑1 Genotyping Kit for Oxford Nanopore Sequencing Against the Sanger Gold Standard Using the Exatype Platform

Dr Cathrine Scheepers 28/11/2025

Executive Summary

Exatype, a cloud-based DNA-sequence analysis platform, developed by Hyrax Biosciences, underpins HIV drug-resistance testing for programmes worldwide, supporting more than 1,100 users across 70+ organisations in 103 countries and processing over 150,000 HIV sequences to date. Its broad adoption - particularly across Global Health Equity settings - has established Exatype as a trusted platform for routine Sanger-based HIV-1 genotyping and reporting.

Given this widespread adoption, Hyrax Biosciences evaluated whether data generated using the Thermo Fisher ABI HIV-1 Genotyping Kit with Integrase, when sequenced on Oxford Nanopore Technologies (ONT), delivers drug-resistance results equivalent to those generated from Sanger sequencing, with both datasets analysed using the Exatype platform. These findings show that the assay performs consistently across both technologies, with strong concordance in consensus sequences and drug-resistance calls. Exatype served as the shared analytical workflow, enabling direct comparison and simplifying cross-platform interpretation.

Key outcomes:

Specificity and repeatability: 100% concordance with Sanger and between ONT replicates.
Sensitivity: Consistent detection of resistance mutations at ≥20% prevalence.
Native vs Rapid ONT workflows validated: both equivalent to Sanger, with trade-offs in depth (Native) and speed (Rapid).
Exatype-driven standardisation: A unified analytical layer enabling QC, consensus generation, and HIVDB reporting across sequencing technologies.

Background and Rationale

As HIV antiretroviral therapy coverage expands - reaching 29.8 million people by 2022 (WHO, 2025)¹ - selective pressure has increased the emergence of HIV drug resistance, driving demand for scalable, reliable genotyping (WHO, HIV Drug Resistance)². With laboratories now incorporating flexible platforms such as ONT alongside traditional Sanger sequencing, a single, standardised workflow becomes increasingly valuable.

This study validates the use of the Applied Biosystems HIV-1 Genotyping Kit with Integrase from Thermo Fisher Scientific, sequenced with ONT and benchmarked against Sanger. Both datasets were analysed through Hyrax Biosciences’ Exatype platform, providing a like-for-like comparison and confirming that the assay can be deployed reliably across multiple sequencing technologies.

Validation Results

Study design: included 2 samples × 3 replicates for protease (PR) and reverse transcriptase (RT) - as a single amplicon - and integrase (IN) amplified with Thermo Fisher ABI HIV-1 Genotyping Kit with Integrase, sequenced on ONT and Sanger, and analysed using Exatype. Two separate barcoding strategies were tested on the ONT platform, the standard long-read barcoding (termed Native) and a short-read barcoding (termed Rapid).

Quantitative Outcomes

ONT (Native and Rapid) vs Sanger

Metric	Result	Benchmark / Notes
Specificity	100% concordance between ONT and Sanger consensus sequences	Validates assay equivalence
Repeatability	100% concordance across replicates	Demonstrates assay robustness
Sensitivity	Matching variant detection at ≥20% threshold (industry standard)	Confirms equivalence with Sanger
Sensitivity threshold	Sanger threshold 15 - 20%³; ONT threshold ≥1%	Improved detection of minor variants with ONT
Drug-resistance calls	Consistent across Sanger and ONT	Reliable HIV-DR detection

Native vs Rapid ONT Strategies

Metric	Native ONT	Rapid ONT	Benchmark / Notes
Average Read Length	1,042 nt	353 nt	Both read lengths are sufficient for HIV-DR genotyping, demonstrating ONT’s versatility
Average Quality Score	Q=28	Q=24	Quality exceeded the ONT minimum (Q=8)
Average Read Depth / Coverage across pol	111k / 80k	37k / 12k	Both datasets exceed the minimum coverage (15 reads) needed to reliably detect variants present at ≥20%*
Average Runtime on Exatype	21 min	5 min	Shorter runtime for shorter read lengths (Rapid ONT)
Concordance with Sanger	100%	100%	Consensus sequences were concordant despite variation in sequencing metrics

Key Takeaways:

Specificity: ONT (Native & Rapid) matches Sanger; identical consensus sequences and HIV-1 drug-resistance calls.
Sensitivity: Detects variants at ≥20% prevalence; ONT also captures lower-prevalence variants.
Speed vs Coverage: Native = longer reads, higher depth; Rapid = faster analysis with shorter reads.
Practical Impact: Both strategies support reliable, high-throughput HIV-DR testing.

*Sequencing depth of the Rapid ONT dataset may not represent all rapid-barcoding strategies.

Analytical and Applied Implications enabled by Hyrax Biosciences’ Exatype Platform

This validation demonstrates that the Thermo Fisher ABI HIV-1 Genotyping Kit with Integrase performs consistently when sequenced on ONT, producing drug-resistance results that align with those obtained from Sanger sequencing. Exatype served as the shared analytical environment, applying the same interpretation rules, thresholds, and reporting structure across both datasets to enable a direct, like-for-like comparison of assay performance.

Exatype retains compatibility with ≥20% reporting thresholds while also accommodating the richer variant signal available from ONT, supporting programmes moving toward higher-throughput or decentralised sequencing. Automated QC, consensus generation, and standardised reporting remove the need for platform-specific bioinformatics, enabling laboratories to increase capacity and adopt ONT without changing analytical workflows.

Conclusion

This validation confirms that data generated using the Thermo Fisher ABI HIV-1 Genotyping Kit with Integrase on ONT, when analysed through Exatype, produces HIV-1 drug-resistance results equivalent to Sanger. Both Native and Rapid ONT workflows demonstrate reliable analytical performance while offering the operational flexibility associated with decentralised sequencing.

Together, these findings position Exatype as a consistent, cross-platform analytical solution that supports ONT adoption in routine testing, surveillance, and decentralised HIV-DR applications while preserving full comparability with established Sanger workflows.

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