Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

Area of Science:

• Forensic genetics
• Molecular biology
• Genomics

Background:

• Next generation sequencing (NGS) enables high-throughput analysis of forensic DNA.
• The HID-Ion AmpliSeq™ Identity Panel is a prototype commercial kit for forensic SNP genotyping.
• Validation of new forensic technologies is crucial for reliable casework.

Purpose of the Study:

• To evaluate the performance of a prototype forensic NGS kit (HID-Ion AmpliSeq™ Identity Panel) on the Ion PGM™ system.
• To assess sequence coverage, genotyping precision, sensitivity, and mixed DNA profiles.
• To determine the suitability of the panel for forensic DNA analysis.

Main Methods:

• Evaluation across three laboratories using standardized Ion PGM™ protocols and DNA controls.
• Assessment of sequence coverage, SNP genotyping concordance against known standards (1000 Genomes, Complete Genomics).
• Sensitivity testing with varying amounts of input DNA (25-100pg).

Main Results:

• Extensive sequence coverage was achieved for most targeted SNPs.
• High SNP genotyping concordance (99.8%) was observed.
• Sensitivity studies demonstrated 90-95% SNP genotype success from 25-100pg DNA.
• Specific loci (rs1979255, rs1004357, rs938283, rs2032597, rs2399332) showed discordant genotypes and should be excluded.
• Varied sequence coverage and genotyping reliability issues were noted with low-level DNA and mixed samples.

Conclusions:

• The HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system offer a sensitive and accurate forensic SNP genotyping assay.
• Calibration of sample loading and optimization of analysis parameters are necessary for reliable forensic application, especially with low-level and mixed DNA.
• Further optimization is required before routine implementation in forensic casework.