Evaluation of ArmedXpert software tools, MixtureAce and Mixture Interpretation, to analyze MPS-STR data

Area of Science:

• Forensic Genetics
• Bioinformatics

Background:

• Massively parallel sequencing (MPS) is crucial for genomic variation studies.
• Distinguishing sequencing artifacts from true polymorphisms is vital in MPS assays.
• PCR-based methods for microsatellite markers can introduce artifacts like stutter and mis-incorporation.

Purpose of the Study:

• To evaluate the performance of ArmedXpert software (MixtureAce™ and Mixture Interpretation) for analyzing short tandem repeat (STR) profiles from single-sourced and mixed DNA samples.
• To assess the software's ability to differentiate true alleles from artifacts in forensic DNA analysis.

Main Methods:

• Evaluated ArmedXpert tools on 240 samples using the ForenSeq™ DNA Signature Prep kit.
• Tested sensitivity, degraded samples, various DNA sources, and mixtures (2-6 contributors).
• Optimized stutter and non-stutter artifact thresholds based on benchmark data for improved accuracy.

Main Results:

• Initial settings led to misclassification of artifacts as alleles.
• Adjusting stutter thresholds significantly reduced false positive allele calls from 435 to 57.
• ArmedXpert accurately classified artifacts and alleles with optimized, laboratory-specific thresholds.
• Mixture Interpretation tool successfully deconvoluted two- and three-person mixtures.

Conclusions:

• ArmedXpert software, particularly MixtureAce, effectively analyzes MPS-generated STR profiles.
• Optimized, laboratory-specific threshold settings are critical for accurate artifact and allele classification.
• The software aids in resolving complex forensic DNA mixtures.