The insufficient standardization of diagnostic next-generation sequencing (NGS) still limits its implementation in clinical practice, with the correct detection of mutations at low variant allele frequencies (VAF) facing particular challenges. We address here the standardization of sequencing coverage depth in order to minimize the probability of false positive and false negative results, the latter being underestimated in clinical NGS. There is currently no consensus on the minimum coverage depth, and so each laboratory has to set its own parameters. To assist laboratories with the determination of the minimum coverage parameters, we provide here a user-friendly coverage calculator. Using the sequencing error only, we recommend a minimum depth of coverage of 1,650 together with a threshold of at least 30 mutated reads for a targeted NGS mutation analysis of ≥3% VAF, based on the binomial probability distribution. Moreover, our calculator also allows adding assay-specific errors occurring during DNA processing and library preparation, thus calculating with an overall error of a specific NGS assay. The estimation of correct coverage depth is recommended as a starting point when assessing thresholds of NGS assay. Our study also points to the need for guidance regarding the minimum technical requirements, which based on our experience should include the limit of detection (LOD), overall NGS assay error, input, source and quality of DNA, coverage depth, number of variant supporting reads, and total number of target reads covering variant region. Further studies are needed to define the minimum technical requirements and its reporting in diagnostic NGS.

• Publication type
• Journal Article MeSH

• MeSH
• Physical Phenomena MeSH
• Magnetic Resonance Imaging MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• radiologie, nukleární medicína a zobrazovací metody
• NML Publication type
• kolektivní monografie

• MeSH
• Nursing MeSH
• Research MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• ošetřovatelství

• MeSH
• Echocardiography MeSH
• Cardiovascular Diseases diagnosis   MeSH
• Publication type
• Monograph MeSH
• Textbook MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• kardiologie
• angiologie
• radiologie, nukleární medicína a zobrazovací metody

• MeSH
• Biomedical Research methods   MeSH
• Biometry * methods   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Statistika
• NML Fields
• biologie
• statistika, zdravotnická statistika