Damage patterns observed in mtDNA control region MPS data for a range of template concentrations and when using different amplification approaches

Interpreting NUMTs in forensic genetics: Seeing the forest for the trees

“…Cytosine deamination may be more readily identified than point heteroplasmy, because it causes specific base substitutions (C → T and G → A) at the ends of natural (non-amplified), degraded DNA fragments. Variants caused by cytosine deamination have been observed in forensic samples [65][66][67][68][69], and these random variants are not reproducible across PCR or library preparation events. Mixtures may be reproducible from the same DNA sample, yet they can be readily identified when the mixed samples have differing haplogroups, producing low frequency variants at haplogroup-diagnostic sites [44,63,[70][71][72][73][74][75].…”

The time is now for ubiquitous forensicmtMPSanalysis

“…Specifically, MPS can detect and resolve mixtures, including heteroplasmy, at threshold levels as low as 1–2%, whereas STS can only detect mixed sites when the minor variant reaches 5–10%, and in some cases not until 20% depending on the noise encountered in the data (González et al, 2020; Holland et al, 2011, 2018, 2019; Irwin et al, 2009; Just, Irwin, et al, 2015; Kloss‐Brandstätter et al, 2015; McElhoe et al, 2014; Melton, 2004; Parson et al, 2015). As a result, MPS also allows for the detection and analysis of damage sites (Gorden et al, 2018; Holland et al, 2019, 2021).…”

“…Forensic samples often contain degraded and damaged DNA. Damage from cytosine deamination, depurination, and oxidation is well documented and impacts the quality of MPS data, especially when working with low template and compromised samples (C. A. Holland et al, 2021; Holland, Bonds et al, 2019; Rathbun et al, 2017). For example, as damage increases, mtDNA yield, MPS read depth, and reportable profile results decrease.…”

“…Storage temperature of DNA extracts can play a role in the accumulation of damage; for example, storing extracts at or below −20°C prevents damage (Rathbun et al, 2017). This is especially prevalent in low template samples as the accumulation of damage has a greater impact (C. A. Holland et al, 2021). While damage prior to extraction is unavoidable (e.g., treated hairs, ancient bones), with proper storage conditions, new sites of damage will accumulate at a slower rate.…”

“…Unfortunately, in some cases this results in the inability to report low‐level heteroplasmic variants. For example, the reporting threshold can be raised to 5% or higher for evidence samples exhibiting damage, but maintained at a lower threshold (2%) for samples that produce quality mtMPS data (C. A. Holland et al, 2021). Furthermore, damage typically presents itself as different types of base changes when compared to true heteroplasmy.…”