Mitochondrial DNA Genome Sequencing
The TDMM Core offers targeted mtDNA sequencing (mtDNA-Seq) for identification of the mitochondrial mutations and deletions and their relative abundance. mtDNA enrichment from total DNA by PCR followed by NGS of the entire mtDNA genome at depths of ≥1,000x. This approach permits identification of 1) large-scale deletions and breakpoints, 2) homozygous or inherent variants, and 3) heterozygous or low frequency somatic mutations arising from damaging hyperglycemia. We have validated this method with in vitro derived standards and can accurately identify and quantitate specific large-scale deletions and single nucleotide variants.
The advantages of our approach are: 1) it does not require mitochondrial isolation, 2) it provides extremely high levels of sequencing depth (1,000-20,000X) using only a benchtop next-generation sequencer, 3) it requires very limited amout of sample (1ng DNA), and 4) the assay can be readily adapted to other model organisms of interest to the aging community. This approach allows a relatively inexpensive and high sample throughput analysis workflow for mtDNA sequencing with high levels of accuracy. Currently assays for human, mouse, and rat are available.
mtDNA sequencing workflow. A total of 1ng genomic DNA is subjected to long-range PCR of two overlapping mtDNA regions to selectively amplify mitochondrial genome. Long-range amplicons are subjected to transposome-mediated library construction, which simultaneously fragments and ligates sequencing adapters. Libraries are then amplified using primers with specific dual index sequences for multiplexed sequencing reactions. Libraries are then sequenced using paired-end 250 cycle benchtop sequencing). Low frequency variant detection is used to identify both protein coding and non-protein coding variants with frequencies > 0.5%.
Quantitative accuracy of mt-DNA seq method. In vitro generated synthetic mtDNA with mutations at four sites was mixed with unmutated DNA at percentages ranging from 0.1% to 100%. At all levels each of the four mutated sites was detected and accurately quantified.
Targeted mitochondrial sequencing is performed by amplifying and thereby enriching mtDNA by long-range PCR. These amplicons are then purified and sequencing libraries will be generated by transposome methods (Nextera XT). Libraries are then sequenced (Illumina MiSeq, paired end). Bioinformatic analysis will be carried out using CLC Genomics Workbench in a somatic mutation workflow, and using adaptations of procedures including the generation of intra-sample consensus sequences for reference genome mapping, allowing both identification of somatic and germline mutations.
Data provided to core users include the FASTQ sequencing files and data files with frequency and location of mutations and deletions.
Total genomic DNA (both nuclear and mitochondrial) should be extracted from tissues using a standard column preparation (e.g., Qiagen AllPrep DNA/RNA) or using solution (Trizol/TriReagent) methods. Core users should prepare their own DNA according their experience as isolation techniques vary according to sample type (tissues, cells, blood). DNA quality should be determined by spectrophotometry with attention to organic contamination (absorbance at 230nm) as high levels of organic contamination can inhibit PCR amplification. As little as 1ng of template DNA can be analyzed. However, 100ng is the preferred starting amount.