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

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 5.9×10−5 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. Keywords: cell-free DNA; library preparation; liquid biopsy; mutation; next-generation sequencing. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement 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). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics Committee of the First Affiliated Hospital of Nanjing Medical University gave ethical approval for this work (Approval no.: 2019-SR-156). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All the data associated with this study are present in the paper or the Supplementary Materials. * (ASSER) : Average single strand error rate (cfDNA) : Cell-free DNA (ctDNA) : Circulating tumor DNA (ddPCR) : Droplet digital PCR (DL primer) : Dualistic primer (FFPE) : Formalin-fixed paraffin-embedded (gDNA) : Genomic DNA (Indel) : Insertion/deletion (NGS) : Next-generation sequencing (NSCLC) : Non-small cell lung cancer (OPERA) : One-PrimER Amplification (SNV) : Single nucleotide variant (ssDNA) : Single-stranded DNA (SD) : Standard deviation (UMI) : Unique molecular identifier (VAF) : Variant allelic frequency