Development and validation of a multiplex targeted PCR-sequencing assay for opioid pharmacogenes genotyping

Interindividual variability in opioid analgesic efficacy and susceptibility to adverse effects is substantially influenced by genetic polymorphisms in pharmacokinetic and pharmacodynamic pathways. However, simultaneous detection of multiple predefined opioid-related variants within a single workflow remains technically challenging, particularly when some targets are located in highly homologous genomic regions. We developed and validated a multiplex targeted PCR-sequencing assay to simultaneously genotype 23 predefined variants across 11 opioid-related pharmacogenes. To overcome homology-related nonspecific amplification, target loci were stratified into two independent sets. Set 1 (six homologous loci) utilized a dedicated three-step nested PCR featuring an initial long-range amplification (2.3–3.8 kb), while Set 2 (17 conventional loci) underwent a streamlined two-step multiplex PCR. Primer ratios within each multiplex pool were iteratively optimized via quantitative PCR to achieve a maximum inter-locus Ct difference of < 5 cycles. The assay was applied to genomic DNA from 50 healthy individuals and sequenced on the Illumina NovaSeq platform. Orthogonal Sanger sequencing was performed on 58 representative genotype instances covering all observed alleles for validation. The optimized assay generated highly balanced sequencing libraries, yielding an average of 224,440 reads per sample with a mapping rate of 99.44% and a Q30 fraction > 96.34%. The mean target depth was 7,120× with a coverage uniformity of 97.30%. When applied to the 50 healthy individuals, the assay achieved a 100% genotyping call rate across all 23 predefined variants at the prespecified quality thresholds (depth ≥ 30×, variant allele frequency ≥ 20%). Sanger sequencing validation demonstrated 100.0% (58/58) concordance with the assay’s genotype calls. These findings demonstrate the analytical accuracy and feasibility of the proposed workflow, offering a scalable methodological platform to facilitate broader pharmacogenetic profiling in opioid translational research.