Background: Cell-free DNA (cfDNA) promises to serve as surrogate biomarkers for non-invasive molecular diagnostics. Disease-specific cfDNA, such as circulating tumor DNA (ctDNA), was short and rare, making the detection performance of the current targeted sequencing methods unsatisfying. Methods: Through introducing a linear pre-amplification process and optimizing the adapter ligation with customized reagents, we developed the One-PrimER Amplification (OPERA) system. In this study, we examined its performance in detecting mutations of low variant allelic frequency (VAF) in various samples with short-sized DNA fragments. Results: In cell line-derived samples containing sonication-sheared DNA fragments with 50-150 bp (peak at 70-80 bp), OPERA was capable of detecting mutations as low as 0.0025% VAF, while CAPP-Seq only detected mutations of >0.03% VAF. Both single nucleotide variant and insertion/deletion can be detected by OPERA. In synthetic fragments as short as 80 bp with low VAF (0.03%-0.1%), the detection sensitivity of OPERA was significantly higher compared to that of droplet digital polymerase chain reaction. The error rate was 5.9x10-5 errors per base after de-duplication in plasma samples collected from healthy volunteers. By suppressing single-strand errors, the error rate can be further lowered by >5 folds in EGFR T790M hotspot. In plasma samples collected from lung cancer patients, OPERA detected mutations in 57.1% stage I patients with 100% specificity and achieved a sensitivity of 30.0% in patients with tumor volume of less than 1 cm3. Conclusions: OPERA can effectively detect mutations in rare and highly-fragmented DNA. Trial registration: This study has been registered on ChiCTR (ChiCTR1900024028) at 23rd June 2019.

The authors have declared no competing interest.

This study was supported by the National Science Foundation of China (No. 82002224 and 81902453), Shanghai Municipal Health Commission (No. 2019CXJQ03), Shanghai Hospital Development Center (No. SHDC2022CRD020), and 511 Taking-off Project of JSPH (No. JSPH-511C-2018-1).

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