An ultrasensitive method for detecting mutations from short and rare cell-free DNA

Abstract

AbstractBackgroundCell-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.MethodsThrough 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.ResultsIn 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 5.9×10−5errors 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 inEGFRT790M 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.ConclusionsOPERA can effectively detect mutations in rare and highly-fragmented DNA.Trial registrationThis study has been registered on ChiCTR (ChiCTR1900024028) at 23rdJune 2019. Keywords: cell-free DNA; library preparation; liquid biopsy; mutation; next-generation sequencing.