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Quality assurance for clinical next-generation sequencing (NGS)–based assays is difficult given the complex methods and the range of sequence variants such assays can detect. As the number and range of mutations detected by clinical NGS assays has increased, it is difficult to apply standard analyte-specific proficiency testing (PT). Most current proficiency testing challenges for NGS are methods-based PT surveys that use DNA from reference samples engineered to harbor specific mutations that test both sequence generation and bioinformatics analysis. These methods-based PTs are limited by the number and types of mutations that can be physically introduced into a single DNA sample. In silicoproficiency testing, which evaluates only the bioinformatics component of NGS assays, is a recently introduced PT method that allows for evaluation of numerous mutations spanning a range of variant classes. In silicoPT data sets can be generated from simulated or actual sequencing data and are used to test alignment through variant detection and annotation steps. In silicoPT has several advantages over the use of physical samples, including greater flexibility in tested variants, the ability to design laboratory-specific challenges, and lower costs. Herein, we review the use of in silicoPT as an alternative to traditional methods-based PT as it is evolving in oncology applications and discuss how the approach is applicable more broadly.

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (1000-fold) of FLT3and 26 other genes from 22 FLT3ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3ITDs can be reliably detected in panel-based, next-generation sequencing assays.