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

Wei Han1, Qingzhen Zhang1, Xiaochang Zhang1

  • 1Laboratory of Biotechnology, Bioinformatics Center of AMMS, Beijing, China.

Human Mutation
|April 13, 2026
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Summary

The NanoID panel effectively uses nanopore sequencing for accurate genetic identification. This system demonstrates high accuracy for short tandem repeats (STRs) and insertions/deletions (InDels), proving reliable for kinship analysis.

Keywords:
InDelsSTRsforensic DNA analysiskinship testingnanopore sequencing

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Area of Science:

  • Genomics
  • Forensic Science
  • Molecular Biology

Background:

  • Nanopore sequencing offers real-time data, portability, and high throughput.
  • Validation of R10.4.1 flow cell for short tandem repeat (STR) genotyping is limited.
  • Accuracy of insertion/deletion (InDel) typing with earlier nanopore versions is understudied.

Purpose of the Study:

  • To develop and validate the NanoID panel for comprehensive genetic analysis using nanopore sequencing.
  • To assess the accuracy, reproducibility, and sensitivity of nanopore-based genotyping for STRs and InDels.
  • To evaluate the NanoID system's utility in individual identification and kinship analysis.

Main Methods:

  • Developed the NanoID panel: a multiplex system for 29 autosomal STRs, 29 Y-chromosome STRs, 61 autosomal InDels, 2 Y-chromosome InDels, and amelogenin.
  • Genotyped 112 individuals across 114 loci, evaluating reproducibility, sensitivity, kinship inference, and species specificity.
  • Assessed accuracy using the 2-out-of-3 rule with varying DNA input levels (≥50 pg).

Main Results:

  • Screened 114 loci with 100% accuracy; all loci showed consistent genotyping across triplicate experiments.
  • Achieved >99.12% accuracy for NanoID with ≥50 pg DNA input.
  • Demonstrated high discriminatory power (1 - 7.990 × 10-57) and cumulative probability of exclusion (1 - 2.299 × 10-16).
  • Achieved 100% sensitivity, specificity, and accuracy for full-sibling kinship testing.
  • Successfully distinguished human from nonhuman samples.

Conclusions:

  • The NanoID panel is a highly accurate and reproducible system for nanopore sequencing-based genetic analysis.
  • NanoID is effective for individual identification and full-sibling kinship analysis.
  • This system expands the application of nanopore sequencing in forensic and genetic identification contexts.