Development and Validation of a Template-Independent Next-Generation Sequencing Assay for Detecting Low-Level Resistance-Associated Variants of Hepatitis.

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Presentation transcript:

Development and Validation of a Template-Independent Next-Generation Sequencing Assay for Detecting Low-Level Resistance-Associated Variants of Hepatitis C Virus Bo Wei, John Kang, Miho Kibukawa, Lei Chen, Ping Qiu, Fred Lahser, Matthew Marton, Diane Levitan The Journal of Molecular Diagnostics Volume 18, Issue 5, Pages 643-656 (September 2016) DOI: 10.1016/j.jmoldx.2016.04.001 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 HCV whole genome next-generation sequencing (NGS) read coverage for serially diluted four commercial HCV samples with template-independent NGS assay. Comparison of read coverage (number of NGS reads with Q20 or higher base quality scores at each nucleotide position across the HCV genome) of four commercial HCV samples with 1:10 serially diluted viral loads. Even though different samples have distinct read coverage patterns across the HCV genome, each sample has a consistent coverage pattern across different viral loads. (For the GT1a sample, the highest viral load is 4.8 × 106 IU/mL; for GT1b sample, 7.0 × 106 IU/mL; for GT2b sample, 1.2 × 107 IU/mL; and for GT6a sample, 1.7 × 107 IU/mL). UTR, untranslated region. The Journal of Molecular Diagnostics 2016 18, 643-656DOI: (10.1016/j.jmoldx.2016.04.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 HCV whole genome next-generation sequencing read coverage for clinical samples collected at multiple time points from two patients with HCV direct-acting antiviral treatment failure. For each patient, the earliest collection date indicates the baseline screening sample and the subsequent dates are the samples collected after treatment. The Journal of Molecular Diagnostics 2016 18, 643-656DOI: (10.1016/j.jmoldx.2016.04.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Measured GT1a/1b ratios versus expected GT1a/1b ratios for HCV mixed infection detection. Commercial GT1a and 1b HCV samples were mixed at the specified percentages using the viral load information provided by the tissue vendors. Measured GT1a/1b ratios were calculated using the number of next-generation sequencing (NGS) reads that best matched 1a or 1b subtypes through BLAST against NS3, NS5A, NS5B, and the three genes combined. Highly correlated ratios of measured GT1a/1b versus expected 1a/1b show that template-independent NGS assay can accurately detect the mixed HCV infection. Data are presented in log2 scale. The Journal of Molecular Diagnostics 2016 18, 643-656DOI: (10.1016/j.jmoldx.2016.04.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Correlation between measured variant allele fraction (VAF) versus expected VAFs at positions with expected variants. Two in vitro–transcribed (IVT) HCV RNA samples differing at 60 nucleotide positions were mixed at various percentages to mimic different expected VAFs. The expected VAFs are the percentages of one IVT RNA of the mixed RNA samples. Measured VAFs at the indicated 12 nucleotide positions were selected as examples to demonstrate that the template-independent next-generation sequencing assay can accurately estimate the VAFs of expected variants. The Journal of Molecular Diagnostics 2016 18, 643-656DOI: (10.1016/j.jmoldx.2016.04.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Confirmation of variant allele fraction (VAF) measured by template-independent next-generation sequencing (TI-NGS) assay with VAFs estimated by custom-designed template-dependent NGS assays. VAFs of the baseline screening sample of patient P4 estimated by TI-NGS were compared to VAFs measured by template-dependent (TD)-NGS custom-designed for the patient at NS3, NS4A, NS5A, and NS5B. The Journal of Molecular Diagnostics 2016 18, 643-656DOI: (10.1016/j.jmoldx.2016.04.001) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions