Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq^® 46GY...

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• Forensic DNA analysis relies on accurate and efficient methods for DNA profiling.
• Optimizing library preparation and quantification is crucial for maximizing data quality in next-generation sequencing (NGS) based forensic applications.
• The PowerSeq 46GY System is a prototype NGS platform for forensic DNA analysis.

Purpose of the Study:

• To evaluate the prototype PowerSeq 46GY System for forensic DNA profiling.
• To assess the impact of protocol modifications on read coverage and sample analysis.
• To compare different library preparation kits and quantification methods.

Main Methods:

• Evaluation of the PowerSeq 46GY System using donor DNA and casework samples.
• Preparation of buccal and casework-type libraries using TruSeq DNA PCR-Free HT and KAPA HyperPrep kits.
• Comparison of PowerSeq Quant MS System and KAPA Library Quantification Kit, alongside a KAPA size-adjustment workbook.
• Sequencing on the MiSeq FGx platform and data analysis with STRait Razor.

Main Results:

• All evaluated quantification methods overestimated library concentration; PowerSeq Quant MS System showed the highest accuracy.
• The TruSeq library kit yielded superior results, including higher coverage and fewer allelic dropouts, compared to the KAPA kit.
• Bone and hair samples showed complete profile recovery, with bone samples exhibiting higher average coverage.

Conclusions:

• The manufacturer's protocol for the PowerSeq 46GY System provided the best overall results compared to alternative library preparation methods.
• Optimized library preparation and accurate quantification are key to successful forensic DNA profiling using NGS technologies.
• The PowerSeq 46GY System demonstrates potential for reliable forensic DNA analysis, even with challenging sample types.