Browsing by Author "Ji, Hezhao"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Open Access External Quality Assessment Program for Next-Generation Sequencing-Based HIV Drug Resistance Testing: Logistical Considerations.(Viruses, 2020-05-18) Ji, Hezhao; Parkin, Neil; Gao, Feng; Denny, Thomas; Jennings, Cheryl; Sandstrom, Paul; Kantor, RamiNext-generation sequencing (NGS) is likely to become the new standard method for HIV drug resistance (HIVDR) genotyping. Despite the significant advances in the development of wet-lab protocols and bioinformatic data processing pipelines, one often-missing critical component of an NGS HIVDR assay for clinical use is external quality assessment (EQA). EQA is essential for ensuring assay consistency and laboratory competency in performing routine biomedical assays, and the rollout of NGS HIVDR tests in clinical practice will require an EQA. In September 2019, the 2nd International Symposium on NGS HIVDR was held in Winnipeg, Canada. It convened a multidisciplinary panel of experts, including research scientists, clinicians, bioinformaticians, laboratory biologists, biostatisticians, and EQA experts. A themed discussion was conducted on EQA strategies towards such assays during the symposium. This article describes the logistical challenges identified and summarizes the opinions and recommendations derived from these discussions, which may inform the development of an inaugural EQA program for NGS HIVDR in the near future.Item Open Access Genetic Characterization of a Panel of Diverse HIV-1 Isolates at Seven International Sites.(PLoS One, 2016) Hora, Bhavna; Keating, Sheila M; Chen, Yue; Sanchez, Ana M; Sabino, Ester; Hunt, Gillian; Ledwaba, Johanna; Hackett, John; Swanson, Priscilla; Hewlett, Indira; Ragupathy, Viswanath; Vikram Vemula, Sai; Zeng, Peibin; Tee, Kok-Keng; Chow, Wei Zhen; Ji, Hezhao; Sandstrom, Paul; Denny, Thomas N; Busch, Michael P; Gao, Feng; REDS-III and EQAPOL programsHIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic comparison of results from multiple sites is needed to determine whether a standardized protocol is required for consistent and accurate data analysis. A panel of well-characterized HIV-1 isolates (N = 50) from the External Quality Assurance Program Oversight Laboratory (EQAPOL) was assembled for evaluation at seven international sites. This virus panel included seven subtypes, six circulating recombinant forms (CRFs), nine unique recombinant forms (URFs) and three group O viruses. Seven viruses contained 10 major drug resistance mutations (DRMs). HIV-1 isolates were prepared at a concentration of 107 copies/ml and compiled into blinded panels. Subtypes and DRMs were determined with partial or full pol gene sequences by conventional Sanger sequencing and/or Next Generation Sequencing (NGS). Subtype and DRM results were reported and decoded for comparison with full-length genome sequences generated by EQAPOL. The partial pol gene was amplified by RT-PCR and sequenced for 89.4%-100% of group M viruses at six sites. Subtyping results of majority of the viruses (83%-97.9%) were correctly determined for the partial pol sequences. All 10 major DRMs in seven isolates were detected at these six sites. The complete pol gene sequence was also obtained by NGS at one site. However, this method missed six group M viruses and sequences contained host chromosome fragments. Three group O viruses were only characterized with additional group O-specific RT-PCR primers employed by one site. These results indicate that PCR protocols and subtyping tools should be standardized to efficiently amplify diverse viruses and more consistently assign virus genotypes, which is critical for accurate global subtype and drug resistance surveillance. Targeted NGS analysis of partial pol sequences can serve as an alternative approach, especially for detection of low-abundance DRMs.