Here we present a novel approach to address the problem of analyzing a (very) low minor contributor in an unbalanced (>1:100) two-person mixture. The method builds on: 

1. selected 1- and 2- basepair deletion markers as these markers show higher confidence basecalls with Illumina sequencing (SNV error rate: 10⁻³–10⁻² compared to 1–2 bp deletion error rate down to 10⁻⁵)
2. deep-sequencing (coverage >1000 reads per marker); 3) markers that have Minor Allele Frequencies of ≈0.5 in the global population to maximize the number of discriminating markers.

Mixed DNA traces with a dominant major contribution of the victim are often encountered in forensic rape cases and in traces related to violent crime. If the victim-to-perpetrator ratio exceeds 20:1, routine autosomal profiling often fails in detecting sufficient alleles of the minor. 

The novel IDseek® Assay consists of 148 DelSNPs and 9 Y-SNP markers and uses Reverse-Complement PCR to provide a single closed tube reaction protocol for PCR and MPS library preparation. The method is especially suited for cases with known reference profiles as no database searching is possible and can also be used for any mixture (also female-female mixtures). Data can be evaluated using statistical evaluation similar to STR-based analysis providing an advantage over lineage-based markers.

Mixtures were tested with varying major-minor ratios, different absolute DNA inputs and samples of different cellular origin. Besides analysis of the sequence data, we performed statistical interpretation to support potential application in forensic casework.

Authors

• Kristiaan J. van der Gaag (Netherlands Forensic Institute, Netherlands)
• Rachel van Leeuwen (Netherlands Forensic Institute, Netherlands)
• Joop Theelen (NimaGen B.V., Netherlands)
• Erik de Jong (NimaGen B.V., Netherlands)
• Pieter van Oers (NimaGen B.V., Netherlands)
• Titia Sijen (Netherlands Forensic Institute, Netherlands)