Development of a Multiplex Amplification System Using Oxford Nanopore Sequencing for STRs and InDels

Nanopore sequencing has emerged as a promising technology due to its real-time data acquisition, portability, and high throughput. However, the genotyping capabilities of the latest flow cell, R10.4.1, for short tandem repeats (STRs) remain insufficiently validated, and its accuracy in typing insertions or deletions (InDels) in earlier versions has been rarely investigated. To bridge these gaps, we developed the NanoID panel, a multiplex amplification system incorporating 29 autosomal STRs, 29 Y-chromosome STRs, 61 autosomal InDels, 2 Y-chromosome InDels, and amelogenin. We conducted genotyping on 112 unrelated individual samples and screened 114 loci with 100% accuracy. Subsequently, we evaluated these 114 loci for reproducibility, sensitivity, kinship inference, and species specificity. All loci were consistently and accurately genotyped across triplicate experiments. NanoID achieved an accuracy rate exceeding 99.12% (based on the 2-out-of-3 rule) when the DNA input was ≥ 50 pg. The combined power of discrimination and the cumulative probability of exclusion were 1 - 7.990 × 10^-57 and 1 - 2.299 × 10^-16, respectively. For full-sibling kinship testing, the sensitivity, specificity, and accuracy reached 100% at likelihood ratio (LR) cutoff values of 0.0001 and 10000. Nonhuman samples were clearly distinguishable from human samples. These findings strongly support the NanoID system's effectiveness for individual identification and full-sibling kinship analysis using nanopore sequencing.