P206 – Internal Validation of the Precision ID mtDNA Whole Genome Panel and Ion Torrent Sequencing for Forensic Casework Applications

10:25

Thursday August 20^th

Station 10

Duration: 12 minutes

03. Human identification

Zena Mankal

The application of Massively Parallel Sequencing (MPS) in forensic laboratories has become increasingly common due to its utility in analyzing low-quality and highly degraded DNA samples. Recent availability of commercial kits has made whole mitochondrial genome (mtgenome) sequencing more accessible and operationally feasible for casework laboratories. Whole mtgenome sequencing using the Precision ID™ (PID) mtDNA Whole Genome Panel on the Ion Chef™ and Ion S5™ instrument was internally validated at the Centre of Forensic Sciences (CFS) in accordance with Standards Council of Canada (SCC) requirements for DNA analysis methods and SWGDAM validation guidelines. System accuracy and precision were established by evaluating haplotype concordance using NIST SRM 2391d reference material, sample replicates, and familial sample sets. Inter-laboratory concordance was assessed using a small number of externally generated samples (n=4). Complete haplotype concordance with reference data was observed across all studies, except in instances of amplicon dropout attributable to poor sample quality and variants in which there is no degenerate primer present in the panel. Contamination studies demonstrated minimal background signal with an instrument baseline of 0.18% ± 0.11%. Sensitivity studies were conducted with multiple tissue types (blood, buccal, & teeth) to address the absence of a mtDNA QPCR system at the CFS. The Precision ID mtDNA system generated full and concordant haplotypes down to 3.75 pg of genomic DNA. Multiplexing strategies, with respect to the number and types of samples pooled for sequencing, had a greater impact on sample profiling success in comparison to DNA input amount. Compatibility of the Precision ID system with different extraction methods ranging from manual and automated extraction methods to sample type-specific chemistries, was also demonstrated. Challenging samples commonly encountered at the CFS, such as formalin-fixed tissue and bone samples, were successfully sequenced using mtgenome with success rates improving by up to 75% compared to samples that generated partial to no STR profile. Two-person mixtures (ratios 1:1 to 1:20) were created to assess the system’s ability to detect mixed haplotypes in a contamination context and secondary haplotypes were reliably detected in mixture ratios ranging from 1:1 to 1:5. Overall, this validation supports the robustness and suitability of whole mtgenome sequencing in forensic casework laboratories. Validation results and implementation considerations will be presented with a particular focus for first time users, including the establishment of analytical thresholds for technical review of mtgenome data.

Authors

Zena Mankal (Centre of Forensic Sciences, Canada)
Matthew Butchart (Centre of Forensic Sciences, Canada)
Jessica Lim (Centre of Forensic Sciences, Canada)
Soulbee Jin (Centre of Forensic Sciences, Canada)

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P206 – Internal Validation of the Precision ID mtDNA Whole Genome Panel and Ion Torrent Sequencing for Forensic Casework Applications

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