Simultaneous Detection of Human Mitochondrial DNA and Nuclear‐Inserted Mitochondrial‐origin Sequences (NumtS) using Forensic mtDNA Amplification Strategies and Pyrosequencing Technology

Abstract

Next-generation sequencing technologies enable the identification of minor mitochondrial DNA variants with higher sensitivity than Sanger methods, allowing for enhanced identification of minor variants. In this study, mixtures of human mtDNA control region amplicons were subjected to pyrosequencing to determine the detection threshold of the Roche GS Junior(®) instrument (Roche Applied Science, Indianapolis, IN). In addition to expected variants, a set of reproducible variants was consistently found in reads from one particular amplicon. A BLASTn search of the variant sequence revealed identity to a segment of a 611-bp nuclear insertion of the mitochondrial control region (NumtS) spanning the primer-binding sites of this amplicon (Nature 1995;378:489). Primers (Hum Genet 2012;131:757; Hum Biol 1996;68:847) flanking the insertion were used to confirm the presence or absence of the NumtS in buccal DNA extracts from twenty donors. These results further our understanding of human mtDNA variation and are expected to have a positive impact on the interpretation of mtDNA profiles using deep-sequencing methods in casework.

The genomic landscape of polymorphic human nuclear mitochondrial insertions

“…Although many recent studies of mitochondrial heteroplasmy have taken NumtS into consideration (22,24,25,33,46–48), they have all been limited to those insertions present in the reference sequence. To assess the potential impact of more recent insertions, we compared our set of sequenced polymorphic NumtS to these studies by identifying single nucleotide differences in the Numt insertions relative to the mtDNA reference and comparing the allelic changes to those reported (Table 2).…”

“… a Largest Numt allele frequency is listed from among sites with multiple overlapping insertions. b Studies included are 1:(24), 2:(48), 3:(25), 4:(46), 5:(47), 6:(33), and 7:(22). It should be noted that (5) identified heteroplasmy in RNA sequences while the remaining studies were from DNA.…”

“…The identified variants were then annotated using Haplogrep (45) to identify potential haplotypes of origin. These SNP profiles were also used to assess potential heteroplasmy artifacts by direct comparison with reported events (22,24,25,33,46–48). To be considered a match, both the position and the Numt allele must match what was reported and sample specific information was also indicated where available (22).…”

The genomic landscape of polymorphic human nuclear mitochondrial insertions

“…Although many recent studies of mitochondrial heteroplasmy have taken NumtS into consideration (He et al 2010; Li et al 2010; Ramos et al 2013; Bintz et al 2014; Diroma et al 2014; Hodgkinson et al 2014; Jayaprakash et al 2014), they have all been limited to those insertions present in the reference sequence. To assess the potential impact of more recent insertions, we compared our set of sequenced polymorphic NumtS to these studies by identifying single nucleotide differences in the Numt insertions relative to the mtDNA reference and comparing the allelic changes to those reported (Table 2).…”

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples

“…However, as predicted nearly five years ago [10], massively parallel sequencing technologies (MPS) are now finding their way into the forensic arena [8,[11][12][13][14][15][16][17][18][19][20]. These technologies largely remove the greatest existing technical and cost barriers to mtGenome sequencing of low DNA quality and quantity specimens.…”