Standardization of Sequencing Coverage Depth in NGS: Recommendation for Detection of Clonal and Subclonal Mutations in Cancer Diagnostics

Frontiers in Oncology

Tomas Novosad¹  Michal Vasinek¹  Tomas Papajik²  Anna Petrackova³  Tereza Dyskova³  Petra Schneiderova³  Eva Kriegova³  Lenka Sedlarikova³ 

[1] Department of Computer Science, Faculty of Electrical Engineering and Computer Science, Technical University of Ostrava, Ostrava, Czechia;Department of Hemato-Oncology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czechia;Department of Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czechia;

关键词: next-generation sequencing;    variant allele frequency (VAF);    coverage depth calculator;    sequencing error;    small subclones;    TP53 gene;   

DOI  :  10.3389/fonc.2019.00851

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown