As next-generation sequencing (NGS) and liquid biopsy become more prevalent in research and clinic, there is an increasing need for better methods to reduce cost and improve sensitivity and specificity of low-frequency mutation detection (Alternative Allele Frequency, AAF < 1%). Here we propose a likelihood-based approach, called Low-Frequency Mutation Detector (LFMD), which combines the advantages of duplex sequencing (DS) and bottleneck sequencing system (BotSeqS) to maximize the utilization of duplicate sequence reads. Compared with the existing state-of-the-art method DS, our method achieves higher sensitivity (improved by ~16%), comparable specificity (< 4 × 10−10 errors per base sequenced) and lower cost (reduced by ~70%) without involving additional experimental steps, customized adapters or molecular tags. LFMD is useful in areas where high precision is required, such as drug resistance prediction and cancer screening. In addition, this method can also be used to improve power of other variant calling algorithms by making it unnecessary to remove polymerase chain reaction (PCR) duplicates.

LFMD: detecting low-frequency mutations in high-depth genome sequencing data without molecular tags