Report of the 2019 NIST-FDA workshop on standards for next generation sequencing detection of viral adventitious agents in biologics and biomanufacturing.

Adventitious virus testing assures product safety by demonstrating the absence of viruses that could be unintentionally introduced during the manufacturing process. The capabilities of next-generation sequencing (NGS) for broad virus detection in biologics have been demonstrated by the detection of known and novel viruses that were previously missed using the recommended routine assays for adventitious agent testing. A meeting was co-organized by the National Institute of Standards and Technology and the U.S. Food and Drug Administration on September 18-19, 2019 in Gaithersburg, Maryland, USA, to facilitate standardization of NGS technologies for applications of adventitious virus testing in biologics. The goal was to assess the currently used standards for virus detection by NGS and their public availability, and to identify additional needs for different types of reference materials and standards (natural and synthetic). The meeting focused on the NGS processes from sample preparation through sequencing but did not thoroughly cover bioinformatics, since this was considered to be the topic of a separate meeting.

Use of Biosynthetic Controls as Performance Standards for Next-Generation Sequencing Assays of Somatic Tumors: A Multilaboratory Study.

BACKGROUND: Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test. METHODS: Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory. RESULTS: Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed. CONCLUSION: This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications.

Multiplexed Reference Materials as Controls for Diagnostic Next-Generation Sequencing: A Pilot Investigating Applications for Hypertrophic Cardiomyopathy.

Diagnostic next-generation sequencing (NGS)-based gene panels are increasingly used for prevalent disorders with genetic and clinical heterogeneity. Clinical development, validation, and quality management of these panels ideally includes reference samples containing prevalent pathogenic variants; however, clinical domain expertise to select appropriate variants may not be present, samples are often not publicly available, and their inclusion is associated with added cost. Expert-designed, multiplexed controls can remedy some of these challenges. One approach relies on spiking biosynthetic fragments carrying desired variants into human genomic DNA. We piloted the utility of this approach for hypertrophic cardiomyopathy. Data from >3000 previously sequenced probands were used to select 10 common pathogenic and/or technically challenging variants in the top hypertrophic cardiomyopathy genes. Multiplexed controls were constructed across a range of ideal and realistic allelic fractions for heterozygous germline variants. NGS was performed in quadruplicate, and results were compared with diagnostic NGS data for the source patient samples. Overall, results were indistinguishable from patient-derived data with variants being detected at or reasonably close to the targeted allelic fraction ratios. The exception was a common 25-bp deletion in MYBPC3, underscoring the importance of including such variants in test development. These controls may be an attractive addition to the repertoire of materials for development, validation, and quality monitoring of clinical NGS assays.

Pilot studies for development of an HIV subtype panel for surveillance of global diversity.

The continued global spread and evolution of HIV diversity pose significant challenges to diagnostics and vaccine strategies. NIAID partnered with the FDA, WRAIR, academia, and industry to form a Viral Panel Working Group to design and prepare a panel of well-characterized current and diverse HIV isolates. Plasma samples that had screened positive for HIV infection and had evidence of recently acquired infection were donated by blood centers in North and South America, Europe, and Africa. A total of 80 plasma samples were tested by quantitative nucleic acid tests, p24 antigen, EIA, and Western blot to assign a Fiebig stage indicative of approximate time from initial infection. Evaluation of viral load using FDA-cleared assays showed excellent concordance when subtype B virus was tested, but lower correlations for subtype C. Plasma samples were cocultivated with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from normal donors to generate 30 viral isolates (50-80% success rate for samples with viral load >10,000 copies/ml), which were then expanded to 10(7)-10(9) virus copies per ml. Analysis of env sequences showed that sequences derived from cultured PBMCs were not distinguishable from those obtained from the original plasma. The pilot collection includes 30 isolates representing subtypes B, C, B/F, CRF04_cpx, and CRF02_AG. These studies will serve as a basis for the development of a comprehensive panel of highly characterized viral isolates that reflects the current dynamic and complex HIV epidemic, and will be made available through the External Quality Assurance Program Oversight Laboratory (EQAPOL).