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. 2023 Jun;22(6):e13842.
doi: 10.1111/acel.13842. Epub 2023 May 3.

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Amy R Vandiver  1   2 , Austin N Hoang  3 , Allen Herbst  4 , Cathy C Lee  2   3 , Judd M Aiken  5 , Debbie McKenzie  6 , Michael A Teitell  7   8   9   10   11 , Winston Timp  12   13   14 , Jonathan Wanagat  2   3
Affiliations

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

Amy R Vandiver et al. Aging Cell. 2023 Jun.

Abstract

Mitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next-generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. We employed nanopore Cas9-targeted sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. NCATS-mtDNA sequencing allows the identification of mtDNA deletions on a single-molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.

Keywords: DNA sequencing; aging; human; mitochondrial DNA; skeletal muscle; substantia nigra.

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Conflict of interest statement

Dr. Timp holds two patents (US 8,748,091 and US 8,394,584) which have been licensed by Oxford Nanopore Technologies.

Figures

FIGURE 1
FIGURE 1
Optimization of mtDNA deletion calling algorithms using in silico test data. (a) Schematic of algorithms used for identifying large mtDNA deletions. (b) Fraction of expected deletions identified versus size of expected deletion in simulated data set. Results of primary alignment algorithm shown in blue, supplemental alignment algorithm in yellow, total MiniMap2 alignment in green, and BLAST alignment algorithm in orange.
FIGURE 2
FIGURE 2
Large mtDNA deletions in primary alignments. (a) Localization of deletions >2000 bp identified in CIGAR sequence of primary alignments in a random subset of 30,000 reads per sample. The mitochondrial genome is depicted in a clockwise orientation with lines linking the start and end of each identified deletion. The human mitochondrial “common” deletion is highlighted in red where identified. (b) R 2 correlation coefficient for log‐transformed number of identified deletions per mitochondrial read versus age plotted versus the minimum deletion size cutoff used in calculating correlation. (c) Log‐transformed frequency of deletions >3 kbp per mitochondrial read versus age. (d) R 2 correlation coefficient for log‐transformed number of identified deletions per mitochondrial read versus log‐transformed deletion frequency by ddPCR plotted versus the minimum deletion size cutoff used in calculating correlation. (e) Log‐transformed frequency of deletions >3 kbp per mitochondrial read versus log‐transformed ddPCR deletion frequency. The red dashed line is the line of identity.
FIGURE 3
FIGURE 3
Large mtDNA deletions mapped using combined Minimap2 alignments. (a) Localization of deletions >2000 bp identified using the Minimap2 algorithm in a random subset of 30,000 reads per sample. The mitochondrial genome is depicted in a clockwise orientation with lines linking the start and end of each identified deletion. The human mitochondrial “common” deletion is highlighted in red where it was detected. (b) R 2 correlation coefficient for log‐transformed number of identified deletions per mitochondrial read versus age plotted versus the minimum deletion size cutoff used in calculating correlation. (c) Log‐transformed frequency of deletions >2 kbp per mitochondrial read versus age. (d) R 2 correlation coefficient for log‐transformed number of identified deletions per mitochondrial read versus log‐transformed deletion frequency by ddPCR plotted versus the minimum deletion size cutoff used in calculating correlation. (e) Log‐transformed frequency of deletions >2 kbp per mitochondrial read versus log‐transformed ddPCR deletion frequency. Red dashed line is the line of identity.
FIGURE 4
FIGURE 4
Distribution of mtDNA deletions across the mitochondrial genome. (a) Boxplots depicting the distribution of mtDNA deletion size versus age of sample. (b) Density of deletion breakpoints identified using primary alignment algorithm (blue), combined Minimap2 deletions (green) and blast alignment deletions (dark orange) as compared to all deletion breakpoints reported in MitoMap (Lott et al., 2013) and MitoBreak (Damas et al., 2014) databases. Bottom panel shows annotation of the mitochondrial genome. D‐loop regions shown in red, rRNA shown in dark blue, tRNA shown in light blue, coding regions shown in green. Cas9 cut site shown in dotted lines. Location of human common deletion breakpoints shown in light red rectangles. (c) Log‐transformed frequency of deletions >2 kbp per mitochondrial read versus age, plotted separately for deletions contained entirely within the minor arc (dark blue) entirely within the major arc (orange), or involving both arcs (green).
FIGURE 5
FIGURE 5
Mapping of large mtDNA deletions in human substantia nigra. (a) Localization of deletions >2000 bp identified using minimap2 alignments in a random subset of 12,000 reads per sample. The mitochondrial genome is depicted in a clockwise orientation with lines linking the start and end of each identified deletion. The human mitochondrial “common” deletion is highlighted in red where identified. (b) Density of deletion breakpoints identified in human substantia nigra (top panel), as compared to those identified in human muscle tissue (second panel), the MitoMap database (third panel) and the MitoBreak database (fourth panel). Bottom panel shows the annotation of the mitochondrial genome. D‐loop regions shown in red, rRNA shown in dark blue, tRNA shown in light blue, coding regions shown in green. Cas9 cut site shown in dotted lines. Location of human common deletion breakpoints shown in light red rectangles. (c) Log 10‐transformed frequency of mtDNA deletions >2000 bp in total chromosome M reads vs age for muscle (green) and substantia nigra (purple) samples.
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