Fig. 1

Ageing increases the load of both SNVs and deletions in mtDNA across all brain regions. a Schematic illustration of the workflow from mouse to prepared library. Briefly, brain regions of interest were rapidly sampled and total DNA was extracted. Linear DNA was enzymatically degraded by exonuclease (ExoV), and non-linear DNA is purified and used for library preparation. FL: full-length mtDNA molecule, ∆: mtDNA molecule with deletion. b Overview of the analysis workflow to optimise mtDNA variant detection. Shortly, after quality filtering, reads were mapped to mm10 without the mitochondrial chromosome (MT). Unmapped reads were then re-mapped to a modified MT reference (dMT: two MT references in tandem) and variants called. c Overview of mouse mtDNA. Green: rRNA encoding genes; blue: protein-coding genes; red: tRNA-encoding genes; orange: non-coding region (NCR). d Schematic showing the areas isolated as the cortex (COR), caudate putamen (CP), dorsal raphe (DR), nucleus accumbens (NAc), paraventricular nucleus of the thalamus (PVT), and substantia nigra (SN). e DNA stored before and after ExoV digestion was subjected to qPCR to determine the relative levels of three mtDNA and three nuclear targets before and after digestion (shown for two different mice, A and B). Mouse C was treated as A and B but without the addition of ExoV. Bars show the mean of target signals and the standard deviation is indicated. †: nDNA after ExoV treatment was not detected or only detected at a very low level by qPCR and may not be visible in the bar plot. f Dot plot illustrating the age-dependent increase in the load of SNVs (left) and deletions (right) across the investigated brain regions (as indicated by the colour legend). All samples have been normalised to the mean of the variants at 10 weeks. Grey diamonds indicate the mean of all regions at the indicated age, and the 95% confidence interval is shown. Three-way ANOVA showed age, not region or animal, significantly (p < 0.01) contributed to SNV and deletion levels. Tukey’s test was used post hoc to determine p values between each age group