Development of a multiplex forensic identity panel for massively parallel sequencing and its systematic optimization...

Area of Science:

• Forensic genetics
• Molecular biology
• Biotechnology

Background:

• Massively parallel sequencing (MPS) enhances forensic short tandem repeat (STR) genotyping.
• Optimizing multiplex PCR assays for MPS is challenging due to labor-intensive conventional methods.
• Efficient assay development requires systematic optimization of primer design and PCR conditions.

Purpose of the Study:

• To develop and optimize a novel multiplex PCR assay (monSTR) for simultaneous amplification of 21 forensic STR markers for MPS.
• To apply Design of Experiments (DOE) methodology for systematic optimization of thermocycling conditions.
• To ensure compatibility with existing forensic DNA databases by adapting the European Standard Set (ESS).

Main Methods:

• Development of the monSTR multiplex PCR assay for 21 forensic STR markers.
• Utilized bioinformatics tools for multiplex-compatible primer design and evaluation.
• Employed Design of Experiments (DOE) with a central composite face design to optimize PCR thermocycling conditions.
• Sequencing performed on the Illumina MiSeq platform.

Main Results:

• Achieved specific, balanced, and low-noise amplification of all 21 forensic targets.
• DOE methodology enabled efficient simultaneous investigation of critical process parameters and their interactions.
• Obtained fully concordant STR profiles, even with low input DNA concentrations, demonstrating reproducibility and reduced artefacts.

Conclusions:

• The monSTR assay provides a practical and economically justifiable approach for forensic STR genotyping using MPS.
• DOE principles significantly streamline assay development and optimization in forensic genetics.
• This optimized assay enhances the characterization of biological evidence for forensic applications.