Development and validation of a next-generation sequencing assay with open-access analysis software for detecting resistance-associated mutations in CMV.

Cytomegalovirus (CMV) resistance testing by targeted next-generation sequencing (NGS) allows for the simultaneous analysis of multiple genes. We developed and validated an amplicon-based Ion Torrent NGS assay to detect CMV resistance mutations in UL27, UL54, UL56, and UL97 and compared the results to standard Sanger sequencing. NGS primers were designed to generate 83 overlapping amplicons of four CMV genes (~10 kb encompassing 138 mutation sites). An open-access software plugin was developed to perform read alignment, call variants, and interpret drug resistance. Plasmids were tested to determine NGS error rate and minor variant limit of detection. NGS limit of detection was determined using the CMV WHO International Standard and quantified clinical specimens. Reproducibility was also assessed. After establishing quality control metrics, 185 patient specimens previously tested using Sanger were reanalyzed by NGS. The NGS assay had a low error rate (<0.05%) and high accuracy (95%) for detecting CMV-associated resistance mutations present at ≥5% in contrived mixed populations. Mutation sites were reproducibly sequenced with 40× coverage when plasma viral loads were ≥2.6 log IU/mL. NGS detected the same resistance-associated mutations identified by Sanger in 68/69 (98.6%) specimens. In 16 specimens, NGS detected 18 resistance mutations that Sanger failed to detect; 14 were low-frequency variants (<20%), and six would have changed the drug resistance interpretation. The NGS assay showed excellent agreement with Sanger and generated high-quality sequence from low viral load specimens. Additionally, the higher resolution and analytic sensitivity of NGS potentially enables earlier detection of antiviral resistance.

HIV-1 Drug Resistance Assay Using Ion Torrent Next Generation Sequencing and On-Instrument End-to-End Analysis Software.

HIV-1 antiretroviral therapy management requires sequencing the protease, reverse transcriptase, and integrase portions of the HIV-1 pol gene. Most resistance testing is performed with Sanger sequencing, which has limited ability to detect minor variants. Next generation sequencing (NGS) platforms enable variant detection at frequencies as low as 1% allowing for earlier detection of resistance and modification of therapy. Implementation of NGS assays in the clinical laboratory is hindered by complicated assay design, cumbersome wet bench procedures, and the complexity of data analysis and bioinformatics. We developed a complete NGS protocol and companion analysis and reporting pipeline using AmpliSeq multiplex PCR, Ion Torrent S5 XL sequencing, and Stanford's HIVdb resistance algorithm. Implemented as a Torrent Suite software plugin, the pipeline runs automatically after sequencing. An optimum variant frequency threshold of 10% was determined by comparing Sanger sequences of archived samples from ViroSeq testing, resulting in a sensitivity of 98.2% and specificity of 99.0%. The majority (91%) of drug resistance mutations were detected by both Sanger and NGS, with 1.7% only by Sanger and 7.3% only by NGS. Variant calls were highly reproducible and there was no cross-reactivity to VZV, HBV, CMV, EBV, and HCV. The limit of detection was 500 copies/mL. The NGS assay performance was comparable to ViroSeq Sanger sequencing and has several advantages, including a publicly available end-to-end analysis and reporting plugin. The assay provides a straightforward path for implementation of NGS for HIV drug resistance testing in the laboratory setting without additional investment in bioinformatics infrastructure and resources.

Evaluation of the Abbott RealTime HCV genotype II plus RUO (PLUS) assay with reference to core and NS5B sequencing.

BACKGROUND: HCV genotyping remains a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. Current commercial genotyping assays may have difficulty identifying 1a, 1b and genotype 6. OBJECTIVE: To evaluate the concordance for identifying 1a, 1b, and genotype 6 between two methods: the PLUS assay and core/NS5B sequencing. STUDY DESIGN: This study included 236 plasma and serum samples previously genotyped by core/NS5B sequencing. Of these, 25 samples were also previously tested by the Abbott RealTime HCV GT II Research Use Only (RUO) assay and yielded ambiguous results. The remaining 211 samples were routine genotype 1 (n=169) and genotype 6 (n=42). Genotypes obtained from sequence data were determined using a laboratory-developed HCV sequence analysis tool and the NCBI non-redundant database. RESULTS: Agreement between the PLUS assay and core/NS5B sequencing for genotype 1 samples was 95.8% (162/169), with 96% (127/132) and 95% (35/37) agreement for 1a and 1b samples respectively. PLUS results agreed with core/NS5B sequencing for 83% (35/42) of unselected genotype 6 samples, with the remaining seven "not detected" by the PLUS assay. Among the 25 samples with ambiguous GT II results, 15 were concordant by PLUS and core/NS5B sequencing, nine were not detected by PLUS, and one sample had an internal control failure. CONCLUSIONS: The PLUS assay is an automated method that identifies 1a, 1b and genotype 6 with good agreement with gold-standard core/NS5B sequencing and can aid in the resolution of certain genotype samples with ambiguous GT II results.

Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing.

BACKGROUND: HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. OBJECTIVE: To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. STUDY DESIGN: The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. RESULTS: Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. CONCLUSIONS: The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method.

Application of a new informatics tool for contamination screening in the HIV sequencing laboratory.

BACKGROUND: Current HIV-1 sequencing-based methods for detecting drug resistance-associated mutations are open and susceptible to contamination. Informatic identification of clinical sequences that are nearly identical to one another may indicate specimen-to-specimen contamination or another laboratory-associated issue. OBJECTIVES: To design an informatic tool to rapidly identify potential contamination in the clinical laboratory using sequence analysis and to establish reference ranges for sequence variation in the HIV-1 protease and reverse transcriptase regions among a U.S. patient population. STUDY DESIGN: We developed an open-source tool named HIV Contamination Detection (HIVCD). HIVCD was utilized to make pairwise comparisons of nearly 8000 partial HIV-1 pol gene sequences from patients across the United States and to calculate percent identities (PIDs) for each pair. ROC analysis and standard deviations of PID data were used to determine reference ranges for between-patient and within-patient comparisons and to guide selection of a threshold for identifying abnormally high PID between two unrelated sequences. RESULTS: The PID reference range for between-patient comparisons ranged from 83.8 to 95.7% while within-patient comparisons ranged from 96 to 100%. Interestingly, 48% of between-patient sequence pairs with a PID>96.5 were geographically related. The selected threshold for abnormally high PIDs was 96 (AUC=0.993, sensitivity=0.980, specificity=0.999). During routine use, HIVCD identified a specimen mix-up and the source of contamination of a negative control. CONCLUSIONS: In our experience, HIVCD is easily incorporated into laboratory workflow, useful for identifying potential laboratory errors, and contributes to quality testing. This type of analysis should be incorporated into routine laboratory practice.

Development and validation of a hepatitis B virus DNA sequencing assay for assessment of antiviral resistance, viral genotype and surface antigen mutation status.

The objective of this study was to develop a DNA sequencing assay that examines sensitively and reliably all conserved domains of the reverse transcriptase-encoding region of the HBV genome for antiviral resistance-associated mutations while simultaneously producing ample information for precise genotyping and determination of HBsAg mutation. This assay was used to examine 1000 de-identified HBV DNA positive samples with known viral loads from a broad-based, unselected patient population from across the United States. Of these, 946 were assayed successfully. Antiviral resistance-associated mutations were identified in 104 samples. The escape mutation sG145R in the surface antigen was identified in 0.8% of patient samples. Infections with genotypes A, B, C, D, E, F, G and H were observed in 36.6%, 19.6%, 21.7%, 13.5%, 3.6%, 0.7%, 2.2%, and 0.5% of patient samples respectively. Fifteen samples (1.6%) appeared to harbor infections with multiple genotypes as shown by the presence of double peaks throughout sequence electropherograms. The limit of detection of this assay was approximately 150IU/mL.