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

Christian Faccinetto,Gianluca Casamassima,Patrizia Serventi,Nicola Staiti,Martina Rigato,Donata Luiselli,Giovanni Vazza,Stefania Sarno,Cecilia Salvoro,Gianluca Margiotta,Sara De Fanti,Alberto Marino,Daniele Sabbatini

doi:10.1007/s00414-021-02686-w

Christian Faccinetto, Gianluca Casamassima + Show 11 more

Open Access

https://doi.org/10.1007/s00414-021-02686-w

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Abstract

With 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.

Highlights

Portions of the nuclear genome, such as autosomal short tandem repeat (STR) loci, are more often utilized during forensic investigations, as their typing results are much more informative than mitochondrial DNA
We describe the internal validation study for the next-generation sequencing (NGS)-based typing of the mitochondrial DNA (mtDNA) genome, conducted using the Precision ID mtDNA Whole Genome Panel v.2.2 (Thermo Fisher Scientific, Waltham, MA, USA) on the Ion S5 system (Thermo Fisher Scientific), in accordance with the Validation Guidelines for Forensic DNA Analysis Methods of the Scientific Working Group on DNA Analysis Methods (SWGDAM) [13] and the European Network of Forensic Science Institute (ENFSI, Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process) [14]
Negative controls were distributed across the 5 runs as follows: one per run for runs 1, 2, and 3; four in run 4; and two in run 5

Summary

Introduction

Portions of the nuclear genome, such as autosomal short tandem repeat (STR) loci, are more often utilized during forensic investigations, as their typing results are much more informative than mitochondrial DNA (mtDNA). International Journal of Legal Medicine (2021) 135:2295–2306 of robust sequencing protocols, the growth of high-quality databases, and the publication of guidelines for typing, annotating, and interpreting results from the International Society for Forensic Genetics allowed the scientific community to validate and rationalize the analysis of mtDNA in forensic casework [3, 4]. The recent advances in next-generation sequencing (NGS) technologies have generated an increasing interest in the use of mtDNA in forensic sciences, because of the potential capability of NGS to capture variants along the entire mitochondrial genome and to detect heteroplasmy at very low levels [5,6,7]

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