Lambrinos, M. R.; Rahimi, T.; Mehra, T.; Schmid, M. P.; Miglino, N.; Romanens, A.; Mertz, K. D.; Krauthammer, M.; Nienhold, R.; Wicki, A.; Balazs, Z.

The analysis of cell-free DNA (cfDNA) offers a non-invasive approach for cancer detection and monitoring. Nucleosome positioning, inferred from cfDNA coverage data, serves as a proxy for gene expression, but existing peak callers fail to resolve overlapping peaks, limiting their utility in identifying precise nucleosome configurations. Here, we introduce SAUNA (Simulated Annealing for Unique Nucleosome Arrangements), a novel algorithm that leverages simulated annealing to optimize nucleosome positions in cfDNA sequencing data. SAUNA ensures non-overlapping nucleosome configurations by employing energy-based optimization with Monte Carlo moves, overcoming sterical incompatibilities inherent in conventional methods. SAUNA outperformed all tested peak callers, especially in scenarios involving phase-shifted or randomly placed nucleosomes. Biological validation using high-coverage cfDNA samples confirmed expected nucleosome patterns, such as distinct interpeak distances at transcription start sites and different chromatin compartments. Moreover, SAUNA demonstrated its clinical relevance by differentiating high and low tumor fraction samples in a cohort of colorectal cancer patients, showcasing its potential for non-invasive cancer monitoring. By combining computational efficiency with biological accuracy, SAUNA provides a versatile tool for high-resolution nucleosome positioning, with applications in cancer diagnostics, epigenetic profiling, and broader cfDNA-based studies. The software is available at https://github.com/uzh-dqbm-cmi/SAUNA, facilitating reproducibility and integration into existing workflows.