Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Abstract

Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.

Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels

“…We have recently demonstrated, albeit in a smaller sample size, a 100% sensitivity on the BRCA targeted panel compared with the parallel MLPA analysis. 12 However, no molecular technique, including our approach, can claim to have 100% sensitivity to all possible structural rearrangements, including rare balanced rearrangements that may not be detectable by standard molecular techniques, such as MLPA, microarrays, or NGS CNV algorithms.…”

“…*BRCA panel description given by Schenkel et al 12 jmd.amjpathol.org -The Journal of Molecular Diagnostics probe sets used, and false-positive results attributable to polymorphism-induced allele dropouts. 8 Taken together, these assays have their unique advantages and disadvantages, related to limited coverage, low resolution, and high cost.…”

“…In contrast, recent advancements in next-generation sequencing (NGS) technologies have provided more costeffective, rapid, and high-throughput methods to allow identification of sequence variants and CNVs. 12,13 Clinical use of NGS enables simultaneous assessment of targeted gene panels and entire exomes or genomes using a limited quantity of biological samples. 14 Some of the characteristics of using NGS for the detection of CNVs include a high resolution, the ability to detect novel small CNVs and balanced genomic rearrangements, and accurate estimation of copy number.…”

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

“…The technical validity of gene-panel/NGS in comparison with the reference technology was thoroughly assessed in different studies [29,36,42,66,67,68,69,70]. In particular, one of these assessed the sensitivity and specificity of NGS compared with standard Sanger sequencing and MLPA (multiplex ligation-probe amplification) and also confirmed the performance of three different panel designs for HBOC (Custom Design by Castera et al [29]: Table 1 and Table 2).…”

Current companion diagnostics in advanced colorectal cancer; getting a bigger and better piece of the pie

“…By limiting the size of a panel, increasing depth at each mutation is possible with similar cost. Mutant allele frequency required for detection is reported in the range of 3-5% and with significant depth of coverage or paired reads that span breakpoints, copy number variation (CNV) can be detected with sensitivities nearing 100% (44,45,86,119,120). This does require that the break point and adjacent territory both have adequate coverage which may not always be present depending on the particular panel.…”