Simultaneous sequencing of 102 Y-STRs on Ion Torrent ™ GeneStudio ™ S5 System

Area of Science:

• Forensic Science
• Genetics
• Molecular Biology

Background:

• Next-generation sequencing (NGS) platforms are widely used in forensic laboratories.
• Current NGS systems often lack comprehensive Y-chromosomal short tandem repeat (Y-STR) analysis capabilities.
• There is a need for validated Y-STR panels compatible with automated NGS workflows.

Purpose of the Study:

• To develop a Y-STR panel for the Thermo Fisher Scientific Precision ID NGS System.
• To evaluate the panel's performance according to SWGDAM guidelines.
• To establish a combined autosomal and Y-STR (AY-STR) NGS workflow.

Main Methods:

• Design of the GrandFiler Y-STR Panel using 'separating' and 'merging' strategies.
• Developmental validation including species specificity, reproducibility, concordance, sensitivity, degraded samples, and mixtures.
• Establishment of an AY-STR NGS workflow by combining the Y-STR panel with a commercial autosomal STR panel.

Main Results:

• The GrandFiler Y-STR Panel successfully detected 102 Y-STRs and Amelogenin, showing high performance metrics (e.g., 92.47% bases ≥ Q20, 91.80% effective reads, 2106× depth of coverage).
• The panel demonstrated high specificity for human males, 99.40% concordance, and sensitivity down to 0.1 ng DNA.
• The AY-STR NGS workflow proved stable and provided enhanced genetic information from degraded samples compared to capillary electrophoresis (CE).

Conclusions:

• The GrandFiler Y-STR Panel is the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System.
• The panel is robust, reliable, and sensitive, comparable to existing CE/NGS kits, and suitable for real forensic cases, especially with degraded DNA.
• The combined AY-STR NGS workflow offers forensic scientists expanded options for diverse case scenarios.