Adapting an established Ampliseq microhaplotype panel to nanopore sequencing through direct PCR

Area of Science:

• Forensic Genetics
• Genomic Technologies
• Molecular Biology

Background:

• Established Ampliseq microhaplotype panels are crucial for forensic genetics.
• Oxford Nanopore Technologies (ONT) offers scalable sequencing solutions.
• Direct PCR amplification from unextracted DNA presents a significant challenge.

Purpose of the Study:

• To adapt an Ampliseq microhaplotype panel for ONT nanopore sequencing.
• To develop a cost-effective and scalable workflow for forensic genetics.
• To evaluate the performance and forensic utility of the developed nanopore workflow.

Main Methods:

• Direct PCR amplification from unextracted DNA.
• ONT library construction and sequencing using the MinION device.
• Analysis of reference samples, dilution series, and forensic pedigrees.

Main Results:

• Stable coverage, allele balance, and strand bias achieved with 5-10 ng DNA.
• Profile completeness and concordance rates of ~95% demonstrated.
• High sensitivity down to 62.5 pg DNA, with informative profiles.
• Significantly higher likelihood ratios in pedigree analysis compared to STR data.

Conclusions:

• The adapted Ampliseq panel for ONT nanopore sequencing is a viable, cost-effective solution for forensic genetics.
• The workflow demonstrates high sensitivity, accuracy, and forensic utility.
• This method enhances kinship analysis and offers improved discriminatory power in forensic investigations.