Developmental validation of the monSTR identity panel, a forensic STR multiplex assay for massively parallel sequencing.

The 21-plex STR panel monSTR was designed for high-fidelity forensic genotyping on the Illumina MiSeq platform. In this study, the panel's performance was validated according to the recommended validation guidelines of the Scientific Working Group for DNA Analysis Methods (SWGDAM). Concordance, repeatability and reproducibility, sensitivity of detection, mixture analysis, species-specificity, and the ability to analyze mock samples were assessed. Sequence data was analyzed using the genotyping software toaSTR. The assay performance was evaluated by measuring the read on-target ratio, the genotype accuracy, the inter-locus balance, the heterozygosity balance, and the signal-to-noise ratio. Results showed that profiles of NIST reference DNA samples as well as GEDNAP proficiency samples were fully concordant with CE-based methods. In addition, inter-run and intra-run variation experiments indicated high precision. Furthermore, full profiles could be obtained using 62.5 pg of DNA input amount with proper inter-locus balance and read on-target ratio; 76.4% of alleles were correctly called with 7.8 pg DNA input amount. It was demonstrated that 94.4% of minor contributor alleles were resolved accurately in a 1:49 mixture. Results suggested that the minor contribution could be precisely calculated based on the minor component allele frequency. Validation results described here demonstrate that the monSTR forensic identity panel is a valid tool for forensic STR genotyping using massively parallel sequencing.

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