Next-generation sequencing (NGS) is already established in the clinical routine of molecular diagnostics, allowing biomarker analysis and personalized treatments, especially in the oncology field [1]. However, establishing general guidelines in the genomic analysis of solid tumor specimens using NGS is an ongoing challenge due to the variety in biopsy procedures, laboratory equipment and pre-processing of samples.

Tumor samples are often preserved through the standard method of formalin-fixation and paraffin-embedding (FFPE) which facilitates histologic evaluation and maintains nucleic acid integrity at low costs. Nonetheless, FFPE compromises DNA quality as a result of fragmentation and DNA-protein crosslinking, which might result in low nucleic acid yield and sequencing artifacts [2,3].

Several pre-analytical factors are known to affect NGS success in FFPE samples, such as tissue selection, processing and storage, block age, cold ischemia time, DNA quality, and tumor cellularity [[4], [5], [6]]. Although there are studies evaluating pre-analytical characteristics, the wide institutional variation might not always be representative of a given laboratory methodology and specific scenarios [7,8]. Overall, there is still limited information about pre-analytical factors and their influence in NGS results is poorly understood and necessary for future enhancement [9].

This study aims to analyze the performance of NGS in FFPE tumor samples and to identify pre-analytical factors that could affect its outcome in molecular diagnostic laboratories.