A multiplex droplet digital PCR (ddPCR) panel was requested to detect six actionable non-small-cell lung cancer driver alterations — KRAS G12C and G12D, EGFR L858R and T790M, BRAF V600E, and the EML4-ALK variant-1 fusion — from circulating cell-free DNA on the Bio-Rad QX200/QX600 platform, with full multiplex-compatibility QC, channel-layout plans for both instruments, in-silico specificity and common-SNP screening, orderable synthetic control sequences, and a limit-of-blank/limit-of-detection validation plan. The delivery provides 20 genome-verified oligos (competitive wild-type/mutant hydrolysis probes for the five point mutations plus a junction-spanning probe for the fusion), 10 synthetic gBlock controls, QX200 and QX600 channel layouts, a full-panel cross-dimer/Tm/homopolymer compatibility screen, genome and paralog specificity checks, a gnomAD common-SNP screen, restriction-enzyme compatibility, and a CLSI-EP17 validation plan, packaged as a PDF report, an Excel workbook, a FASTA file, and figures. Every primer pair is genome-specific — the BRAFP1 pseudogene was deliberately designed out so it cannot dilute the BRAF allele fraction — no two assays' primers cross-dimer below -5 kcal/mol, and a very common SNP under the EGFR T790M probe (rs1050171, ~56% population frequency) was neutralised with a degenerate base so the assay works in either genotype. The most important caveat is one intra-assay liability for wet-lab attention: the two competitive EGFR L858R probes form a strong cross-dimer arising from a palindrome intrinsic to that locus, so the L858R assay should be empirically validated (and likely run with an LNA-shortened probe) before use; separately, the EML4-ALK assay requires an RNA/cDNA input arm (one-step RT-ddPCR) rather than cfDNA, because the fusion's genomic breakpoints are patient-specific. This panel was designed and screened in silico and must be confirmed at the bench before clinical use.
