CLAmp-seq: A Novel Amplicon-Based NGS Assay with Concatemer Error Correction for Improved Detection of Actionable Mutations in Plasma cfDNA from Patients with NSCLC

High-throughput sequencing of circulating cell-free DNA (cfDNA) is critical for targeted therapy selection and monitoring in non-small cell lung cancer (NSCLC) patients. However, low quantities of cfDNA in the blood and assay artifacts limit the sensitivity and specificity of next-generation sequencing (NGS) test. This study develops a novel amplicon-based NGS technology, CLAmp-seq (circular ligation amplification and sequencing), with a concatemer-based error correction strategy, which effectively removes polymerase errors from the first round of amplification. Analytical validation shows median detection rate of 100% at a mutant allele frequency (MAF) of 0.1% for 20 ng of input. The assay achieves 99.8% and 99.5% concordance of repeatability and reproducibility for 0.1% MAF, respectively, and both 100% for 0.5% MAF cfDNA standards. A comparative analysis of 134 NSCLC patient plasma samples demonstrates strong concordance (97.4%) and linearity (R-2 = 0.95) between the CLAmp-seq NGS readouts and droplet digital PCR (ddPCR) results. The detection of mutants in cfDNA obtained from pretreatment plasma samples shows 94.8% concordance with tissue genotyping results. No mutation is detected in noncancerous control samples. It shows that CLAmp-seq NGS assay can offer an easy-to-use, fast, and accurate molecular diagnostic tool with multiplex capacity that is well-suited for clinical in vitro diagnosis.