A sensitive and robust approach for calling single-nucleotide variants (SNVs) from high-coverage sequencing datasets, based on a formal model for biases in sequencing error rates. LoFreq adapts automatically to sequencing run and position-specific sequencing biases and can call SNVs at a frequency lower than the average sequencing error rate in a dataset. LoFreq’s robustness, sensitivity and specificity were validated using several simulated and real datasets (viral, bacterial and human) and on two experimental platforms (Fluidigm and Sequenom).