Novel Approaches to Develop Critical Reference Materials for Noninvasive Prenatal Testing: A Pilot Study.

BACKGROUND: Highly characterized reference materials are required to expand noninvasive prenatal testing (NIPT) for low incidence aneuploidies and microdeletions. The goal of this study was to develop reference materials for the development of next generation circulating cell-free DNA (ccfDNA) assays. METHODS: This was a prospective study of pregnancies complicated by positive prenatal genetic screening. ccfDNA was isolated from maternal plasma and amplified. Lymphoblastoid cell lines were prepared from maternal peripheral blood mononuclear cells and fetal cord blood cells. Cells were Epstein-Barr virus immortalized and expanded. Amplified DNA and to a limited extent formulated lymphoblastoid-derived ccfDNA was tested in SNP-based and chromosome counting (CC) based massively parallel sequencing assays. RESULTS: Enrolled cases included fetuses with: T21 (2), T18 (1), T18-XXX (1), XYY (1), microdeletions (1), and euploid (2). Three lymphoblastoid cells lines were prepared. Genomic DNA was extracted from cell lines and fragmented to simulate ccfDNA. ccfDNA isolation yielded about 2000 usable genome equivalents of DNA for each case for amplification. Although the sonicated genomic DNA derived from lymphoblastoid cell lines did not yield results compatible with NIPT assays, when blinded, NIPT platforms correctly identified the amplified ccfDNA isolated from blood in the majority of cases. CONCLUSIONS: This study showed that maternal blood samples from pregnancies complicated by common chromosomal abnormalities can be used to generate materials for the development and evaluation of NIPT assays.

One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation.

PURPOSE: To evaluate the impact of technically challenging variants on the implementation, validation, and diagnostic yield of commonly used clinical genetic tests. Such variants include large indels, small copy-number variants (CNVs), complex alterations, and variants in low-complexity or segmentally duplicated regions. METHODS: An interlaboratory pilot study used synthetic specimens to assess detection of challenging variant types by various next-generation sequencing (NGS)-based workflows. One well-performing workflow was further validated and used in clinician-ordered testing of more than 450,000 patients. RESULTS: In the interlaboratory study, only 2 of 13 challenging variants were detected by all 10 workflows, and just 3 workflows detected all 13. Limitations were also observed among 11 less-challenging indels. In clinical testing, 21.6% of patients carried one or more pathogenic variants, of which 13.8% (17,561) were classified as technically challenging. These variants were of diverse types, affecting 556 of 1,217 genes across hereditary cancer, cardiovascular, neurological, pediatric, reproductive carrier screening, and other indicated tests. CONCLUSION: The analytic and clinical sensitivity of NGS workflows can vary considerably, particularly for prevalent, technically challenging variants. This can have important implications for the design and validation of tests (by laboratories) and the selection of tests (by clinicians) for a wide range of clinical indications.

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

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.