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TrueAllele® Casework validation of the Verogen ForenSeq DNA Signature Prep Kit Primer Set B and the MiSeq FGX
M. Legler, J. Bracamontes, M. Van Buren, "TrueAllele® Casework validation of the Verogen ForenSeq DNA Signature Prep Kit Primer Set B and the MiSeq FGX", Promega's Thirty Fourth International Symposium on Human Identification, Denver, CO, 20-Sep-2023.
Next Generation Sequencing (NGS) is an exciting technology that will allow forensic labs to extract more DNA information from a single sample. Currently, DNA data interpretation is becoming an increasingly complex task as sample types (like touch DNA samples) and newer testing methods (like NGS) become more common. As new DNA technology becomes available, a lab will look to extend their current, established tools for new data interpretation. Toward this end, Cybergenetics, the creators of the TrueAllele® probabilistic genotyping software, updated their system to support NGS STR data produced by the Verogen ForenSeq DNA Signature Prep Kit Primer Set B and the MiSeq FGX.
The Kern Regional Crime Laboratory along with Cybergenetics validated the updated TrueAllele software to establish the sensitivity, specificity, and reproducibility of the process. The validation includes testing across different axes, including:
For single source data, three studies were performed:
- Sensitivity & Stochasticity - consisting of two dilution series.
- Reproducibility & Repeatability - two users set up the same data.
- Known references - 45 known donors with unique genotypes.
For mixture data, two studies were performed:
- Mock Casework - 21 mock casework samples of known composition involving varying sample types and contributor number.
- Known Mixtures - samples of known composition involving varying contributor number and mixture weight (MW) ratios.
Finally, for concordance, three studies were performed:
- Capillary Electrophoresis (CE) - A set of 45 single source samples prepared using two sequencing methods (CE and NGS).
- Plate Setup - samples of varying contributor number prepared using two plate setup methods.
- Kit Autosomal STR – Comparison of STR markers of 57 samples of varying contributor number prepared using two NGS kits (Primer Set A & Primer Set B).
This validation showed that the system produced sensitive, specific, and reproducible results for DNA data containing one to five contributors produced by the Kern Regional Crime Laboratory using the ForenSeq DNA Signature Prep kit Primer Set B with the MiSeq FGx Next Generation Sequencer.
- Promega's Thirty Fourth International Symposium on Human Identification - Site