We would like to acknowledge Silvia Marchet and Constanza Lamperti (Istituto Neurologico &#34;Carlo Besta&#34;, Fondazione IRCCS, Milan) for preparing and providing the m.3243A&#62;G cybrid cells used as illustrative examples for this protocol. We would also like to acknowledge the members of the Mitochondrial Genetics Group (MRC-MBU, University of Cambridge) for useful discussion during the course of this research. This work was supported by core funding from the Medical Research Council UK (MC_UU_00015/4 and MC_UU_00028/3). P.A.N. and P.S.-P. are additionally supported by The Lily Foundation and The Champ Foundation, respectively.

Informed consent was provided for the use of the human 3243A&#62;G cybrid cells and the immortalized m.5024C&#62;T MEFs used in this study. Ethical approval was not required in this instance as the patient cells were not collected at the University of Cambridge. The use of human fibroblasts may, however, require ethical approval. It is highly recommended to follow best practices for PCR setup when preparing the sample DNA for pyrosequencing. Frequent amplification using identical primers can lead to amplicon contaminatio...

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A., Minczuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+manipulation+of+human+mitochondrial+DNA+heteroplasmy+in+vitro+using+tunable+mtZFN+technology.">Enhanced manipulation of human mitochondrial DNA heteroplasmy in vitro using tunable mtZFN technology.</a> Methods in Molecular Biology. 1867, 43-56 (2018).</li><li>Pickett, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+in+m.3243A&gt;G+mitochondrial+disease:+The+role+of+nuclear+factors.">Phenotypic heterogeneity in m.3243A&gt;G mitochondrial disease: The role of nuclear factors.</a> Annals of Clinical and Translational Neurology. 5 (3), 333-345 (2018).</li><li>Nunes, J. 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M.M. is a co-founder, shareholder, and member of the Scientific Advisory Board of Pretzel Therapeutics, Inc. P.S.-P. and P.A.N. provide consultancy services for Pretzel Therapeutics, Inc.

A critical aspect for the success of the protocol is avoiding contaminations, particularly when using low amounts of starting material. It is recommended to use a UV hood and filtered pipette tips when preparing the samples wherever possible, as well as to keep the preamplification and post-amplification areas separate. Blank measurements and samples of known heteroplasmy (such as wild-type DNA) should always be included to be used as benchmarks to check for technical or biological bias. 
A no...

The mitochondrial genome exists in the form of small (16.5 kb) circular molecules (mtDNA) present in the innermost compartment of the mitochondria named the matrix and encodes 13 subunits of the mitochondrial respiratory chain, as well as the tRNAs and rRNAs necessary for their translation in situ by the mitochondrial ribosome1. This genome represents approximately 1% of all the proteins necessary for mitochondrial function, the remainder of which are encoded by the nuclear DNA (nDNA). It is commonly assumed that mitochondria are derived from an endosymbiotic fusion event between an alpha-proteobacterial ancestor and an ancestral eukaryotic cell. Once this hypothetical symbiosis took place, the genetic information of the mitochondria was gradually transferred to the nucleus over eons, which explains the aforementioned compactness of the mtDNA when compared to the genomes of modern cyanobacteria2. Such a transfer of genes is most strongly evidenced by the existence of long stretches of nDNA that are highly homologous to the sequences found in mtDNA. These nuclear mitochondrial sequences (NUMTs) are a common source of misinterpretation during genotyping, and certain precautions must be taken to avoid nuclear biases when genotyping mtDNA3 (Figure 1A).
Another distinctive feature of mtDNA is that its copy number varies depending on the cell type, numbering anywhere from tens to thousands of copies per cell4. Owing to this multi-copy nature, mtDNA can harbor a wide range of genotypes within a single cell, which can result in a more continuous distribution of alleles in contrast to the discrete alleles associated with nuclear genes when considering the zygosity of chromosomes. This heterogeneity of mitochondrial alleles is referred to as mitochondrial heteroplasmy, which is typically expressed in the percent prevalence of a given mutation as a proportion of the total mtDNA in a given cell. Heteroplasmy can be contrasted with homoplasmy, which refers to a unique species of mtDNA being present across a cell.
Measuring mitochondrial heteroplasmy is of particular interest when quantifying the proportion of mtDNA molecules harboring pathogenic variants. Such variants come in the form of single nucleotide polymorphisms (SNPs), small indels, or large-scale deletions5. Most humans are heteroplasmic for pathogenic variants; however, they do not exhibit any clinical phenotypes, which often only manifest at higher heteroplasmy levels of pathogenic mtDNA in a phenomenon referred to as the threshold effect6. While the values associated with pathogenicity are highly dependent on the nature of the pathogenic mutation and the tissue in which it occurs, they typically lie above 60% heteroplasmy7.
There are several research areas in which mitochondrial genotyping is common. In the medical field, testing for or quantifying mtDNA mutations can serve as a diagnostic criterion for mitochondrial diseases, many of which have mtDNA aberrations as their origin5. In addition to the study of human pathogenic mutations, the prevalence of animal models harboring pathogenic SNPs in the mtDNA is likely to increase, given the recent advent of mitochondrial base editing enabled by mitochondrially targeted DddA-derived cytosine base editors (DdCBEs)8 and TALE-based deaminases (TALEDs) for adenine base editing9. This approach will be instrumental in understanding the interplay between aberrant mitochondrial genotypes and the resulting dysfunctions. There is also ongoing scientific research into remodeling the mitochondrial genome for ultimate use as a therapeutic strategy in human mitochondrial diseases via an approach known as heteroplasmy shifting. This field of research primarily involves directing mutation-specific nucleases to the mitochondrial matrix; this results in the preferential degradation of pathogenic mtDNA, leading to rescues in phenotype10,11,12,13. Any experiments involving the remodeling of the mitochondrial genotype require a robust quantitative method to assess heteroplasmy shifts.
A wide variety of methods are used to genotype mtDNA, and these vary according to the nature of the mutation. Next-generation sequencing (NGS) methods are more precise when it comes to quantifying SNPs in mtDNA; however, these methods remain prohibitively expensive for the routine quantification of mitochondrial heteroplasmy, particularly if the number of samples is small. Sanger sequencing can also allow for the detection of SNPs; however, this approach is not quantitative and often fails to detect low levels of heteroplasmy or can be inaccurate when estimating high heteroplasmies. Pyrosequencing, as an assay that involves minimal preparation and enables the rapid quantification of heteroplasmy for any mtDNA sample, is proposed as an apt compromise between these two extremes. This method has been used routinely to quantify mitochondrial SNPs by numerous researchers in various contexts, including forensic analysis14,15, clinical diagnosis16, or the genotyping of mtDNA from single cells17.
This assay involves a first PCR preamplification step of a region flanking the SNP in the mtDNA, which is followed by a sequencing-by-synthesis assay using one strand of the previously generated amplicon. One of the two primers used in the preamplification step must be biotinylated on the 5&#39; end, which will enable the pyrosequencing apparatus to isolate the single strand of DNA to be used as template for the sequencing reaction. A third sequencing primer is then annealed onto the retained biotinylated strand, which allows for nascent DNA synthesis as deoxynucleotides to be dispensed in a predefined order into the reaction chamber. The pyrosequencer records the amount of each base incorporated based on a luminescent readout, allowing the relative quantification of mutant and wild-type mitochondrial alleles upon DNA synthesis (Figure 1B). The luminescence is generated by a luciferase enzyme, which emits light in the presence of ATP that an ATP sulfurylase synthesizes de novo at each incorporation event from the pyrophosphates released by each nucleotide. These two reactions can be summarized as follows:
1. PPi (from nucleotide incorporation) + APS &#8594; ATP + sulphate (ATP sulfurylase)
2. ATP + luciferin + O2 &#8594; AMP + PPi + oxyluciferin + CO2 + light (luciferase)
Detecting adenine bases by the pyrosequencer without ATP cross-reacting with the luciferase in the second reaction is a challenge. However, this is solved by using an adenine analog for DNA synthesis, namely dATP&#945;S. Despite not being a perfect substrate for luciferase, it produces a stronger luminescence compared with the three other nucleotides, which is digitally adjusted by the pyrosequencer and set to a factor of 0.9. Due to this inherent variability, it is suggested to avoid sequencing adenine at the SNP position (see the discussion for further details).
The following protocol details the method of mtDNA heteroplasmy assessment by pyrosequencing and outlines the necessary precautions in designing the amplification primers to avoid biological or technical bias when genotyping SNPs in mtDNA. The latter involves digitally surveying and selecting the primer sets, optimizing the preamplification PCR, and finally, sequencing and refining the assay. Two applied example assays are demonstrated: first, the optimization of the most common human pathogenic variant m.3243A&#62;G18, and second, the genotyping of mouse embryonic fibroblast (MEF) cells that have undergone heteroplasmy shifting using technologies developed at the Minczuk laboratory in Cambridge10,11,12,19,20,21,22.

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		<tr>
			<td>KOD Hot Start DNA Polymerase</td>
			<td>Sigma-Aldrich</td>
			<td>71086</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Assay Design&#160;2.0</td>
			<td>QIAGEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Absorber Strips&#160;</td>
			<td>QIAGEN</td>
			<td>974912</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Q48 Advanced CpG Reagents (4 x 48)</td>
			<td>QIAGEN</td>
			<td>974022</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Autoprep&#160;</td>
			<td>QIAGEN</td>
			<td>9002470</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Q48 Cartridge Set</td>
			<td>QIAGEN</td>
			<td>9024321</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Disks</td>
			<td>QIAGEN</td>
			<td>974901</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyromark Q48 Magnetic beads&#160;</td>
			<td>QIAGEN</td>
			<td>974203</td>
			<td></td>
		</tr>
		<tr>
			<td>PyroMark Q48 Software License (1)</td>
			<td>QIAGEN</td>
			<td>9024325</td>
			<td></td>
		</tr>
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</table>

genotyping single nucleotide polymorphisms in the mitochondrial genome by pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

This section presents an example optimization of a pyrosequencing assay for a human pathogenic mtDNA mutation, as well as sequencing data from the genotyping of heteroplasmic (m.5024C&#62;T) mouse embryonic fibroblasts (MEFs) treated with mitochondrial zinc finger nucleases (mtZFNs). Optimizing the assay for human cells and comparing two different assays demonstrates how to select the most accurate one, whereas genotyping genetically modified MEF cells in the second example serves as an applied example of detecting heter...

Watch this Scientific Journal Video about Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing at JoVE.com

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

Pyrosequencing is a sequencing by synthesis technique which can be used to genotype single nucleotide polymorphisms in mitochondrial DNA. This method is advantageous in its ease of implementation and cost-effectiveness when compared to other sequencing methods. It can easily be used as a diagnostic method for certain mitochondrial diseases by determining heteroplasmy values of pathogenic variants in mitochondrial DNA.To begin, obtain a mitochondrial DNA sequence file for the species being genotyped and identify the position of the single nucleotide polymorphism, or SNP. Ensure that the reference sequence used employs the appropriate base numbering for the mutation studied so that the position of the SNP can be easily identified. Copy 1, 000 base pairs upstream and downstream of the SNP site and paste the truncated sequence into the pyrosequencing primer design software.Highlight the polymorphic base, right-click on it, and then select Set Target Region to set the analyzed SNP base as the target of the pyrosequencing assay. Press the Play icon in the top right-hand corner of the interface to launch the primer selection, and wait for the software to automatically generate the primer trios, two primers for preamplification of the template DNA, and the third primer for sequencing by the synthesis in the pyrosequencing machine. Right-click and select Copy All Primer Sets to retain all the primer sets.Open the run software provided with the pyrosequencer, select New Assay, then select the allele quantification assay template. Input the sequence to be analyzed into the corresponding box by typing in the nucleotides directly downstream of the sequencing primer. For the variable position, denote the two possible bases, separated by a slash.Press Generate Dispensation Order, and let the software automatically determine a suitable order for the nucleotides to be dispensed in the sequencing by synthesis reaction. Save, and provide a name for the assay. Next, execute the run by diluting the DNA to be analyzed to 5 nanograms per microliter.In the first run, ensure to include samples of known heteroplasmy as a reference and wild-type samples. Then perform a presequencing PCR of 5 microliters of diluted DNA and 25 microliters reactions, using the amplification primers and parameters. To run the file setup, select New Run in the pyrosequencer software.The pyrosequencer can simultaneously sequence 48 separate preamplified reactions with an empty square representing a single sequencing well on a pyrosequencing disc. Load the assays by right-clicking on a square and selecting Load Assay. If required, sequence up to four separate assays with different sequencing primers.Set the primer dispensation mode to automatic to automatically assign an injection chamber for each sequencing primer used for the run. Set run mode to standard, unless running four different types of assays on one sequencing disc. Ensure the number of assays on the run template file matches the number of PCR reactions being amplified.Then save the run files to a USB drive. To prime the pyrosequencer, press the cleaning button on the main touchscreen of the device and clean all injectors with high-purity water, following the instructions on the screen. When inserting the absorber strip into the machine, ensure that the ends meet in the 9 o'clock position.After plugging in the USB stick, press the Sequence button to load the run files prepared earlier. Follow the instructions on the device to load and prime the reagents, as required, into the corresponding injectors on the machine. Load 3 microliters of beads into the wells, and then load 10 microliters of each PCR reaction to be sequenced into the corresponding sample.Pipette up and down to mix the sample with the magnetic beads. Look for the indication in the primed pyrosequencer that the disc can be loaded into the machine. Unscrew the plate holding nut, and align the loaded sample plate with the metal pin in the disc compartment.Once the plate is firmly screwed into the plate compartment, launch the sequencing run by pressing the start button on the touchscreen interface. Once the run is complete, remove the USB drive from the machine, and plug it back into the computer running the pyrosequencer software. After the newly generated run file appear on the USB drive, double-click on the Run Result file.This automatically analyzes the luciferase output of each well upon nucleotide incorporation, and quantifies the mitochondrial allele of interest. Look for the color scores shown by the software based on the quality of the reads. To save the results, select Reports in the top window, followed by Full Report to generate a PDF file containing the pyrograms and results from each well of the run.Alternatively, download the results in other formats under the Reports tab. Agarose gel electrophoresis of a PCR amplification using the amplification primers from both sets selected for the m. 3243A>G mutation as shown in this figure.Here, Het denotes the nearly homoplasmic m. 3243A>G sample to be analyzed. A comparison of the performance of both primer sets using molar standards for calibration is shown in this figure.The measured values are denoted by the vertical dotted lines. An X=Y linear function is displayed to illustrate how close each of the two tested assays is to an ideal measurement. The names of the cell lines on the X-axis are the names of the various cybrid clones that were genotyped using this assay.Genotyping results on four cybrid clones of unknown m. 3243A>G heteroplasmy using both assays are shown here. Sequencing was performed in technical triplicate.Genotyping results of mitochondrial zinc finger nuclease-treated cells, along with untreated and mock-treated controls, and the pyrograms from two MEF cell sample PCR replicates used to generate the results as well as the wild-type genotyping control are shown here. Pyrosequencing can be used as a readout for gene therapy experiments for assessing various gene therapy technologies in mitochondrial DNA.

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1.2K Views.  University of Cambridge, Cambridge Biomedical Campus. Pyrosequencing is a sequencing by synthesis technique which can be used to genotype single nucleotide polymorphisms in mitochondrial DNA. This method is advantageous in its ease of implementation and cost-effectiveness when compared to other sequencing methods. It can easily be used as a diagnostic method for certain mitochondrial diseases by determining heteroplasmy values of pathogenic variants in mitochondrial DNA.To begin, obtain a mitochondrial DNA sequence file for the species be...