Added Value of Next Generation over Sanger Sequencing in Kenyan Youth with Extensive HIV-1 Drug Resistance

Novitsky, Vlad; Nyandiko, Winstone; Vreeman, Rachel C.; DeLong, Allison; Manne, Akarsh; Scanlon, Michael; Ngeresa, Anthony; Aluoch, Josephine; Sang, Festus; Ashimosi, Celestine; Jepkemboi, Eslyne; Orido, Millicent; Hogan, Joseph W.; Kantor, Rami

doi:10.1128/spectrum.03454-22

Cited by 3 publications

(3 citation statements)

References 58 publications

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“…Notably, consensus genomes assembled from NGS data can be used as input data for all tools developed for Sanger sequences [23], while minority variant calling (from reads mapped to the consensus sequence) enhances drug resistance prediction [24][25][26][27], and enables more precise assessments of infection recency [28] or transmission patterns [29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Comparative Evaluation of Bioinformatic Pipelines for Full-Length Viral Genome Assembly

Zsichla,

Zeeb,

Fazekas

et al. 2024

Preprint

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The increasingly widespread application of next-generation sequencing (NGS) in clinical diagnostics and epidemiological research has generated a demand for robust, fast, automated, and user-friendly bioinformatic workflows. To guide the choice of tools for the assembly of full-length viral genomes from NGS datasets, we assessed the performance and applicability of four widely adopted bioinformatic pipelines (shiver - for which we created a user-friendly Dockerized version, referred to as dshiver; SmaltAlign, viral-ngs, and V-pipe) using both simulated datasets and real-world HIV-1 paired-end short-read sequences and default settings. All four pipelines produced high-quality consensus genome assemblies and minority variant calls when the reference sequence used for assembly had high similarity to the analyzed sample. However, while shiver and SmaltAlign showed robust performance also with more divergent samples (non-matching subtypes), viral-ngs and V-Pipe proved to be sensitive to genetic distance from the reference sequence. With empirical datasets, SmaltAlign and viral-ngs exhibited substantially shorter runtime compared to V-Pipe and shiver. In terms of applicability, V-Pipe provides the broadest functionalities; SmaltAlign and dshiver combine user-friendliness with robustness; while the use of viral-ngs requires a less computational resources compared to other tools. To conclude, all four pipelines can perform well in terms of quality metrics; however, the reference sequence needs to be adjusted to closely match the sample data for viral-ngs and V-Pipe. Differences in user-friendliness and runtime may guide the choice of the pipeline in a particular setting. The new Dockerized version of shiver offers ease of use in addition to the accuracy and robustness of the original pipeline.

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Section: Introductionmentioning

confidence: 99%

Comparative Evaluation of Bioinformatic Pipelines for Full-Length Viral Genome Assembly

Zsichla,

Zeeb,

Fazekas

et al. 2024

Preprint

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“…This technology has been validated for HIVDR determination; it is generally limited to the detection of nucleotide variants and variant haplotype signatures present at 20% prevalence [ 1 , 2 ]. Several studies have clearly demonstrated that HIVDR variants detection between 1% and 20% could improve treatment outcomes [ 3 , 4 , 5 , 6 , 7 ]. It is therefore important to detect mutations at 20% but also minor variants that occur below 20% frequency, using a Next-Generation Sequencing (NGS) method.…”

Section: Introductionmentioning

confidence: 99%

From Capillary Electrophoresis to Deep Sequencing: An Improved HIV-1 Drug Resistance Assessment Solution Using In Vitro Diagnostic (IVD) Assays and Software

Mohamed¹,

Boulmé²,

Sayada

2023

Viruses

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Background: Drug-resistance mutations were mostly detected using capillary electrophoresis sequencing, which does not detect minor variants with a frequency below 20%. Next-Generation Sequencing (NGS) can now detect additional mutations which can be useful for HIV-1 drug resistance interpretation. The objective of this study was to evaluate the performances of CE-IVD assays for HIV-1 drug-resistance assessment both for target-specific and whole-genome sequencing, using standardized end-to-end solution platforms. Methods: A total of 301 clinical samples were prepared, extracted, and amplified for the three HIV-1 genomic targets, Protease (PR), Reverse Transcriptase (RT), and Integrase (INT), using the CE-IVD DeepChek® Assays; and then 19 clinical samples, using the CE-IVD DeepChek® HIV Whole Genome Assay, were sequenced on the NGS iSeq100 and MiSeq (Illumina). Sequences were compared to those obtained by capillary electrophoresis. Quality control for Molecular Diagnostics (QCMD) samples was added to validate the clinical accuracy of these in vitro diagnostics (IVDs). Nineteen clinical samples were then tested with the same sample collection, handling, and measurement procedure for evaluating the use of NGS for whole-genome HIV-1. Sequencing analyzer outputs were submitted to a downstream CE-IVD standalone software tailored for HIV-1 analysis and interpretation. Results: The limits of range detection were 1000 to 106 cp/mL for the HIV-1 target-specific sequencing. The median coverage per sample for the three amplicons (PR/RT and INT) was 13,237 reads. High analytical reproducibility and repeatability were evidenced by a positive percent agreement of 100%. Duplicated samples in two distinct NGS runs were 100% homologous. NGS detected all the mutations found by capillary electrophoresis and identified additional resistance variants. A perfect accuracy score with the QCMD panel detection of drug-resistance mutations was obtained. Conclusions: This study is the first evaluation of the DeepChek® Assays for targets specific (PR/RT and INT) and whole genome. A cutoff of 3% allowed for a better characterization of the viral population by identifying additional resistance mutations and improving the HIV-1 drug-resistance interpretation. The use of whole-genome sequencing is an additional and complementary tool to detect mutations in newly infected untreated patients and heavily experienced patients, both with higher HIV-1 viral-load profiles, to offer new insight and treatment strategies, especially using the new HIV-1 capsid/maturation inhibitors and to assess the potential clinical impact of mutations in the HIV-1 genome outside of the usual HIV-1 targets (RT/PR and INT).

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“…Recently, we assessed the potential added value of using NGS over Sanger sequencing in detecting DRMs in this population. Despite good overall agreement between sequencing technologies at high NGS thresholds, even in this resistance-saturated cohort, 12% of participants had higher, potentially clinically relevant predicted resistance detected only by NGS, suggesting potential benefits of the more sensitive NGS over existing technology [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

Added Value of Next Generation Sequencing in Characterizing the Evolution of HIV-1 Drug Resistance in Kenyan Youth

Novitsky

Nyandiko

Vreeman

et al. 2023

Viruses

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Drug resistance remains a global challenge in children and adolescents living with HIV (CALWH). Characterizing resistance evolution, specifically using next generation sequencing (NGS) can potentially inform care, but remains understudied, particularly in antiretroviral therapy (ART)-experienced CALWH in resource-limited settings. We conducted reverse-transcriptase NGS and investigated short-and long-term resistance evolution and its predicted impact in a well-characterized cohort of Kenyan CALWH failing 1st-line ART and followed for up to ~8 years. Drug resistance mutation (DRM) evolution types were determined by NGS frequency changes over time, defined as evolving (up-trending and crossing the 20% NGS threshold), reverting (down-trending and crossing the 20% threshold) or other. Exploratory analyses assessed potential impacts of minority resistance variants on evolution. Evolution was detected in 93% of 42 participants, including 91% of 22 with short-term follow-up, 100% of 7 with long-term follow-up without regimen change, and 95% of 19 with long-term follow-up with regimen change. Evolving DRMs were identified in 60% and minority resistance variants evolved in 17%, with exploratory analysis suggesting greater rate of evolution of minority resistance variants under drug selection pressure and higher predicted drug resistance scores in the presence of minority DRMs. Despite high-level pre-existing resistance, NGS-based longitudinal follow-up of this small but unique cohort of Kenyan CALWH demonstrated continued DRM evolution, at times including low-level DRMs detected only by NGS, with predicted impact on care. NGS can inform better understanding of DRM evolution and dynamics and possibly improve care. The clinical significance of these findings should be further evaluated.

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Added Value of Next Generation over Sanger Sequencing in Kenyan Youth with Extensive HIV-1 Drug Resistance

Cited by 3 publications

References 58 publications

Comparative Evaluation of Bioinformatic Pipelines for Full-Length Viral Genome Assembly

Comparative Evaluation of Bioinformatic Pipelines for Full-Length Viral Genome Assembly

From Capillary Electrophoresis to Deep Sequencing: An Improved HIV-1 Drug Resistance Assessment Solution Using In Vitro Diagnostic (IVD) Assays and Software

Added Value of Next Generation Sequencing in Characterizing the Evolution of HIV-1 Drug Resistance in Kenyan Youth

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