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

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Circulating tumor DNA (ctDNA) is often short and rare, limiting current targeted sequencing sensitivity.
• Detecting low variant allelic frequency (VAF) mutations in fragmented DNA is challenging for existing methods.

Purpose of the Study:

• To develop and evaluate the One-PrimER Amplification (OPERA) system for enhanced detection of low VAF mutations in short DNA fragments.
• To assess OPERA's performance against established methods like CAPP-Seq and droplet digital PCR.

Main Methods:

• OPERA system development for amplifying short DNA fragments.
• Performance evaluation using cell line-derived and synthetic DNA samples with varying fragment sizes and VAFs.
• Analysis of plasma samples from healthy volunteers and lung cancer patients.

Main Results:

• OPERA detected mutations down to 0.0025% VAF in 50-150 bp fragments, outperforming CAPP-Seq (>0.03% VAF).
• Significantly higher sensitivity than droplet digital PCR for 80 bp fragments at 0.03%-0.1% VAF.
• Achieved a low error rate of 5.9×10^-5 per base in plasma, further reduced by suppressing single-strand errors.
• Detected mutations in 57.1% of stage I lung cancer patients with 100% specificity and 30.0% sensitivity in patients with tumors <1 cm³.

Conclusions:

• The OPERA system effectively detects mutations in rare and highly fragmented ctDNA.
• OPERA offers a significant advancement in sensitivity and accuracy for liquid biopsy-based cancer detection.
• This technology holds promise for earlier and more precise cancer diagnosis and monitoring.