The broad variety of next-generation sequencing methodologies and the impact on biomarker testing

Objective: Next-generation sequencing (NGS) is the next best thing for oncology biomarker testing in molecular diagnostics. A comparative study with different NGS panels and bio-informatics pipelines gives more insight in the efficiency of the panels and the quality control (QC) metrics used in practice. Methods: Twenty-six institutes reported on identified variants in 10 patient samples of the ESP EGFR external quality assessment (EQA) and 2 synthetic DNA samples containing more than 300 variants. The raw data is further analysed and used QC metrics are questioned. Results: Comparison of performance scores in the ESP EGFR EQA show an advantage for NGS users versus non-NGS users, respectively 91,8% and 87,1%. Less false positive and false negative genotyping errors are identified, however, additional SNPs are more often reported as actual variants in NGS using laboratories. Most laboratories were not able to identify all variants in the two synthetic DNA samples. There is a great variety in QC of the NGS analysis, both in number and type. Up to 15 different QC metrics are reported in 1 laboratory. Conclusion: Even though NGS is a promising technique in molecular diagnostics, laboratories must be aware of the pitfalls when implementing the technique for the first time.status: publishe