AUTHOR=Fragkouli Styliani-Christina , Pechlivanis Nikos , Anastasiadou Anastasia , Karakatsoulis Georgios , Orfanou Aspasia , Kollia Panagoula , Agathangelidis Andreas , Psomopoulos Fotis 
  
TITLE=Synth4bench: generating synthetic data for benchmarking tumor-only somatic variant calling algorithms
  
JOURNAL=Frontiers in Bioinformatics
  
VOLUME=Volume 6 - 2026
  
YEAR=2026
  
URL=https://www.frontiersin.org/journals/bioinformatics/articles/10.3389/fbinf.2026.1858375
  
DOI=10.3389/fbinf.2026.1858375
  
ISSN=2673-7647
  
ABSTRACT=BackgroundSomatic variant calling is a key activity towards identifying genomic alterations; yet, the evaluation of the respective tools remains challenging due to the scarcity of high quality ground truth datasets. To overcome this limitation, we developed synth4bench, a synthetic data generation pipeline, which utilizes the NEAT simulator, for robust benchmarking. Using a systematic process to create distinct synthetic datasets, we thoroughly evaluated five variant callers (Mutect2, FreeBayes, VarDict, VarScan2 and LoFreq). We compared tool outputs against our synthetic ground truth across key sequencing aspects (such as depth and read length) to assess their capacities and shed light on their underlying algorithmic principles.ResultsSynth4bench is an approach for evaluating tumor-only somatic variant callers that relies on a systematic definition of fully controlled ground-truth datasets. Our analysis revealed significant inconsistencies among the tool outputs and a strong dependence of caller performance on sequencing parameters. Indels remain the hardest-to-call variant type, driven by errors at low allele frequencies. Algorithmic choice is also critical; the most robust callers displayed the highest Precision in allele frequency estimation, while the most sensitive caller was best for maximizing true positive recovery. Conversely, the least suitable caller exhibited systematic errors along with the poorest overall performance.ConclusionThese findings indicate that there is not a one-size-fits-all approach; sequencing optimization together with caller selection are necessary to maximize sensitivity and reliability. Furthermore, the pronounced inconsistencies suggest that current algorithms are not yet able to capture all mutational mechanisms adequately, with the modeling of the underlying processes remaining an open challenge.