Ion Torrent ™ Genexus ™ Integrated Sequencer and ForeNGS Analysis Software-An automatic NGS-STR workflow from DNA to...

Area of Science:

• Forensic Science
• Genomics
• Molecular Biology

Background:

• The Ion Torrent Genexus Sequencer integrates library construction, templating, and sequencing.
• Forensic DNA analysis traditionally involves multiple manual steps.
• Automating the DNA-to-profile workflow is crucial for efficiency.

Purpose of the Study:

• To validate the Ion Torrent Genexus Sequencer and ForeNGS Analysis Software (FNAS) for a fully automated forensic DNA analysis workflow.
• To determine optimal assay parameters for the Genexus system using the Precision ID Globalfiler NGS STR Panel v2.
• To assess the performance, sensitivity, and reproducibility of the automated workflow for forensic casework.

Main Methods:

• Utilized the Ion Torrent Genexus Sequencer for automated library construction, templating, and sequencing.
• Employed ForeNGS Analysis Software (FNAS) for automated analysis of sequencing data, including allele calling according to ISFG guidelines.
• Validated optimal assay parameters including target amplification cycles, library and template size, sequencing flows, and read depth.
• Tested the system with the Precision ID Globalfiler NGS STR Panel v2 on various forensic samples.

Main Results:

• Established optimal assay parameters for Genexus, including 24 cycles amplification, 40 μL library, 400 bp template, 852 flows, and minimum 750,000 reads/sample.
• Achieved competitive performance with repeatable and reproducible results for forensic STR markers.
• Demonstrated sensitivity down to 100 pg input DNA, suitability for low copy number and degraded samples.
• Successfully detected minor contributors in mixtures at ratios between 4:1 and 1:4 with a 50x threshold.

Conclusions:

• The Genexus system combined with FNAS offers a fully automated, robust, and labor-effective DNA-to-profile workflow for forensic applications.
• The system provides accurate and reproducible STR profiles, enabling rapid analysis within a single day.
• This automated NGS workflow is well-suited for various forensic casework, particularly for challenging samples like low quantity or degraded DNA.