Resolution of mitochondrial DNA mixtures using a probe capture next generation sequencing system and phylogenetic-based software. (July 2021)

• Record Type: Journal Article
• Authors: Wisner, Mary; Erlich, Henry; Shih, Shelly; Calloway, Cassandra
• Is Part Of: Forensic science international. Volume 53(2021)
• Publication Date: 2021-07
• Subjects: Forensic DNA -- Mitochondrial DNA -- Mixture interpretation -- Next-generation sequencing -- Phylogenetic software; Forensic genetics -- Periodicals; Génétique légale -- Périodiques; Forensic genetics; Electronic journals; Periodicals; 614.1
• DOI: 10.1016/j.fsigen.2021.102531
• Languages: English
• ISSNs: 1872-4973

Abstract

Abstract: Interpreting mixtures with nuclear genetic markers remains one of the persisting challenges in forensic DNA analysis, particularly when the DNA is degraded or present in trace amounts. In these scenarios, analyzing mitochondrial (mt) DNA can be useful due to the higher copy number per cell compared to nuclear DNA. However, until the emergence of Next-Generation Sequencing (NGS) with its clonal sequencing capability, analysis of mtDNA mixtures was very challenging. A mitochondrial genome probe capture Next-Generation Sequencing (NGS) system was used to sequence complex mtDNA mixtures and two different software programs to analyze the sequence data. Analysis of contrived mixtures of two contributors in 50:50 and 95:5 ratios as well as three-person mixtures ranging from near equal proportions (~33:33:33 ratio) to low amounts of the minor contributors (e.g., a 90:5:5 ratio) is reported. This system was also applied to the analysis of mtDNA mixtures from forensically relevant samples. For data analysis, both the variant frequency-based software program GeneMarker®HTS and the phylogenetic-based software program Mixemt was used to de-convolute the mixtures. Using the massively parallel, clonal features of NGS, one can bioinformatically separate and count the individual sequence reads to calculate the proportions of individual contributors using phylogenetically informative polymorphisms. GeneMarker®HTS allows us to detect all mutations, including "private" mutations (non-phylogenetically informative polymorphisms) and assign them to individual contributors based on the frequency of the sequence reads, provided that the proportions of the various contributors are sufficiently different. Using a probe capture NGS system and both GeneMarker®HTS and Mixemt software programs, the interpretation of complex mixtures of equal proportion contributors, trace amount contributors, and more than two contributors in contrived mixtures, as well as interpretation of challenging forensic specimens is demonstrated. Highlights: Complex mitochondrial DNA mixtures are difficult to resolve using Sanger sequencing. Sequencing the mitochondrial genome improves discrimination power. Together frequency and phylogenetic-based software programs can resolve mixtures. Mixtures were created to present the most challenging proportions. Mixtures of two and three individuals, as well as case-type mixtures, were resolved.