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

Biosens Bioelectron. 2023 Oct 15:238:115548. doi: 10.1016/j.bios.2023.115548. Epub 2023 Jul 27.

Abstract

Circulating tumor DNA (ctDNA) was short and rare, making the detection performance of the current targeted sequencing methods unsatisfying. We developed the One-PrimER Amplification (OPERA) system and examined its performance in detecting mutations of low variant allelic frequency (VAF) in various samples with short-sized DNA fragments. In cell line-derived samples containing sonication-sheared DNA fragments with 50-150 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. OPERA can effectively detect mutations in rare and highly-fragmented DNA.

Keywords: Cell-free DNA; Library preparation; Liquid biopsy; Mutation; Next-generation sequencing.

MeSH terms

  • Biosensing Techniques*
  • Cell-Free Nucleic Acids*
  • Circulating Tumor DNA* / genetics
  • ErbB Receptors / genetics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lung Neoplasms* / diagnosis
  • Lung Neoplasms* / genetics
  • Mutation
  • Protein Kinase Inhibitors

Substances

  • ErbB Receptors
  • Cell-Free Nucleic Acids
  • Protein Kinase Inhibitors
  • Circulating Tumor DNA