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).

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 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.

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.

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.

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.

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.