ADVANCED LABORATORY PROTOCOLS FOR ULTRASENSITIVE DETECTION OF RARE SOMATIC MUTATIONS

Abstract

Background: Rare somatic mutations are emerging as important biomarkers for early detection of cancer, minimal residual disease, and precision oncology. However, sequencing artifacts, polymerase errors and limited abundance of circulating tumor DNA challenge the detection of ultra-low-frequency variants.
Objective: The objective of this work was to develop and assess advanced laboratory protocols for the ultrasensitive detection of rare somatic mutations by integrated molecular and computational approaches.
Methodology: We developed an optimized workflow comprising circulating free DNA extraction, ultra-deep next-generation sequencing, unique molecular identifiers (UMIs), duplex error correction and digital PCR validation. Sequencing was performed to an average depth of >100,000× coverage to improve low frequency variant calling.
Findings: We show that the proposed protocol with duplex sequencing can detect variants with allele frequencies below 0.01% with 99% sensitivity and 99.8% specificity. We reduce false-positive rates to 0.2% compared with conventional sequencing approaches. “We saw better reproducibility and better analytical performance across liquid biopsy samples.
Conclusion: The integrated protocol demonstrated highly accurate and reproducible detection of ultra-rare somatic mutations supporting its potential application in clinical diagnostics, translational genomics and personalized cancer therapy.