Internal validation and improvement of mitochondrial genome sequencing using the Precision ID mtDNA Whole Genome Panel

• DOI: 10.1007/s00414-021-02686-w
• Publication Date: 2021-09-07T06:03:01Z
• Authors: Christian Faccinetto; Daniele Sabbatini; Patrizia Serventi; Martina Rigato; Cecilia Salvoro; Gianluca Casamassima; Gianluca Margiotta; Sara De Fanti; Stefania Sarno; Nicola Staiti; Donata Luiselli; Alberto Marino; Giovanni Vazza
• Keywords: 0301 basic medicine; 03 medical and health sciences; 0302 clinical medicine; Haplotypes; Genome, Mitochondrial; Method Paper; High-Throughput Nucleotide Sequencing; Humans; Reproducibility of Results; Forensic genetics; Internal validation; Ion Torrent; Massive parallel sequencing; NGS; Whole mitochondrial genome; Sequence Analysis, DNA; DNA, Mitochondrial

AbstractWith the recent advances in next-generation sequencing (NGS), mitochondrial whole-genome sequencing has begun to be applied to the field of the forensic biology as an alternative to the traditional Sanger-type sequencing (STS). However, experimental workflows, commercial solutions, and output data analysis must be strictly validated before being implemented into the forensic laboratory. In this study, we performed an internal validation for an NGS-based typing of the entire mitochondrial genome using the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific) on the Ion S5 sequencer (Thermo Fisher Scientific). Concordance, repeatability, reproducibility, sensitivity, and heteroplasmy detection analyses were assessed using the 2800 M and 9947A standard control DNA as well as typical casework specimens, and results were compared with conventional Sanger sequencing and another NGS sequencer in a different laboratory. We discuss the strengths and limitations of this approach, highlighting some issues regarding noise thresholds and heteroplasmy detection, and suggesting solutions to mitigate these effects and improve overall data interpretation. Results confirmed that the Precision ID Whole mtDNA Genome Panel is highly reproducible and sensitive, yielding useful full mitochondrial DNA sequences also from challenging DNA specimens, thus providing further support for its use in forensic practice.