Mitochondrial and nuclear disease panel (Mito-aND-Panel): Combined sequencing of mitochondrial and nuclear DNA by a cost-effective and sensitive NGS-based method

Abstract

Background: The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a robust high-throughput platform for nuclear and mitochondrial DNA (mtDNA) analyses.

Method: We developed a custom Agilent SureSelect Mitochondrial and Nuclear Disease Panel (Mito-aND-Panel) capture kit that allows parallel enrichment for subsequent NGS-based sequence analysis of nuclear mitochondrial disease-related genes and the complete mtDNA genome. Sequencing of enriched mtDNA simultaneously with nuclear genes was compared with the separated sequencing of the mitochondrial genome and whole exome sequencing (WES).

Results: The Mito-aND-Panel permits accurate detection of low-level mtDNA heteroplasmy due to a very high sequencing depth compared to standard diagnostic procedures using Sanger sequencing/SNaPshot and WES which is crucial to identify maternally inherited mitochondrial disorders.

Conclusion: We established a NGS-based method with combined sequencing of the complete mtDNA and nuclear genes which enables a more sensitive heteroplasmy detection of mtDNA mutations compared to traditional methods. Because the method promotes the analysis of mtDNA variants in large cohorts, it is cost-effective and simple to setup, we anticipate this is a highly relevant method for sequence-based genetic diagnosis in clinical diagnostic applications.

Keywords: NUMTs; heteroplasmy detection; high-throughput sequencing; mitochondrial and nuclear disease panel; mitochondrial disorders; mtDNA-server.

Figure 1

Evaluation of the capture efficiency of mtDNA when Nuclear Disease Panel (ND‐Panel) and Mito‐Panel are blended (=Mito‐aND‐Panel) at different concentrations. The validation cohort comprises 72 control samples including both mutation negative and positive distinct patients and two control DNAs of the Coriell repositories (NA12889, RM8398). The total of 72 samples was tested subdivided into three different sample preparation batches of three different molar ratios of mito:ND baits (batch A—dilution [1:50], batch B—dilution [1:100], and batch C—dilution [1:500]). Each sample batch was sequenced in independent sequencing runs. The height of the columns indicates the percentage of mtDNA covered by a minimum number of reads. Black‐colored column: minimum coverage of 100 reads, 100× coverage; gray‐colored column: minimum coverage of 1,000 reads, 1,000× coverage; white‐colored column: minimum coverage of 2,000 reads, 2,000× coverage. All data are expressed as arithmetic mean ± standard deviation of the samples

Figure 2

Bland–Altman plot for depicting the agreement between NGS (next‐generation sequencing) and Sanger sequencing/SNaPshot heteroplasmy measurements. On the x‐axis, the average of NGS and Sanger sequencing heteroplasmy is plotted. On the y‐axis, the difference between next‐generation and Sanger sequencing/SNaPshot heteroplasmy is plotted. The mean difference is indicated as green line, the 95% limits of agreement (average difference ± 1.96 standard deviation of the difference) are indicated as red dotted lines. Two of the 17 samples showed exactly the same difference between Sanger sequencing/SNaPshot and NGS heteroplasmy measurements, thus only 15 dots of 17 calculated samples are shown in the figure