A rapid and robust method for Mitochondrial DNA profiling

Human mitochondrial DNA (mtDNA) variations, particularly the presence of both mutated and wild type mtDNA known as mtDNA heteroplasmies, have garnered increasing attention due to their clinical relevance to numerous diseases. Nonetheless, the absence of a feasible and economical approach has hampered large-scale population studies on mtDNA variations. Here, we present a novel human mtDNA sequencing method called 123-seq, which is based on the 123-multiplex PCR and completed in consecutive three-step biochemical reactions, with experimental validation of its reliability. The entire library construction process takes only about 90 minutes, enabling efficient capture, amplification, and library construction of mtDNA at the bulk genomic DNA level or single-cell level in a streamlined workflow. We then applied our method to assess single-cell mtDNA mutations in a human sample, profiling over 286 T lymphocytes from a 79-year-old female. We discovered that over 90% of cells carried at least one mtDNA mutation with variant allele frequencies (VAFs) exceeding 20%. Furthermore, 83.78% of these mutation sites were enriched in the protein-coding regions of mtDNA. Our findings suggest that mtDNA mutations with functional significance may be widespread in advanced age, indicating a potential link to T cell aging. This emphasizes the need for further investigation into the dynamics of age-related mtDNA mutations at the single-cell level. Genomic DNA from bulk cells or the lysate of single cells can serve as the initial template, and the mtDNA library construction process can be completed through a consecutive three-step method. In the 1st step, the entire mitochondrial genome is selectively amplified using 123 multiple primers targeting mtDNA, with each primer pair containing a shared sequence of 20 nucleotides at the 5' end. The first round of PCR involves a low-temperature, low-cycle phase for targeted amplification of mtDNA to address specificity issues between targets, and a high-temperature phase for exponential amplification of the shared sequence to address compatibility issues between targets. In the 2nd step, Exonuclease I (Exo I) is added to the reaction system to remove residual multiple PCR primers from the first round of PCR. In the 3rd step, library construction is completed by adding sequencing adapter primers containing the shared sequence and cell barcode (sample index) into the reaction system, followed by the second round of PCR.