TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">One remarkable issue in mitochondrial DNA sequencing and variant detection is heavy nuclear DNA contamination persistent throughout the commonly used mitochondria enrichment protocols including differential and gradient centrifugations [1,2]. The chromosomes contain nuclear mitochondrial sequences [3], which might cause false positive or negative results in variant detection. This problem is often overcome by enrichment of the mtDNA by long-range PCR [4], however, such methods are prone to early-cycle PCR errors. Here, we improved existing mtDNA isolation protocols (e.g. Kennedy et al. (2013) [11]) and describe a detailed step-by-step guide to obtain high-quality, highly enriched mtDNA suitable for sequencing and low-frequency variant detection as well as for other sensitive applications.</p>

[BEFORE_START]
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">The protocol here is presented for a single tissue sample, but it can be scaled up for simultaneous preparation of several samples. However, significantly prolonged incubation times at any step due to increased amount of samples should be avoided.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">The protocol has been mainly optimized for soft tissues, such as liver and brain (as in [11]). However, it also works for heart, although more extensive yet gentle homogenization is required. The mtDNA yield from heart is also much lower than from the bigger tissues and nDNA contamination levels are often higher.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p> </p>
<p><strong>REAGENT SETUP</strong></p>
<p>CRITICAL: Reagent storage is at room temperature unless mentioned otherwise.</p>
<p> </p>
<p> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>STOCK SOLUTIONS<br /></strong></p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Acetic acid, 2 M</strong> Combine 11.48 ml glacial acetic acid with 88.52 ml distilled H<sub>2</sub>O to obtain 100 ml 2 M acetic acid solution.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>DNase I, 5 mg/ml in 5 mM CaCl<sub>2</sub></strong> Dissolve DNase I according to the weight into suitable volume of 5 mM CaCl<sub>2</sub> (diluted from 1 M CaCl<sub>2</sub> stock solution, e.g. 7.5 µl 1 M CaCl<sub>2</sub> in 1.5 ml distilled H<sub>2</sub>O) to obtain 5 mg/ml DNase I solution. Dispense into aliquots and store at -20 °C.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> DNase I activity might decrease if the storage period exceeds several months.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>dNTPs, 1.25 mM each</strong> Combine 250 µl of each dNTP (100 mM stocks) and add 19 ml distilled H<sub>2</sub>O to obtain a solution containing 1.25 mM each dNTP. Dispense into aliquots and store at -20 °C.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>EDTA, 0.5 M, pH 8.0</strong> Dissolve 18.1 g EDTA into 80 ml distilled H<sub>2</sub>O. Adjust the pH to 8.0 with NaOH and bring the volume to 100 ml with distilled H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>EtOH, 70-%</strong> Measure 35 ml of absolute EtOH and 15 ml distilled H<sub>2</sub>O separately and combine to obtain ca. 48 ml of 70-% EtOH.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> 70-% EtOH is hygroscopic i.e. it will evaporate and absorb water over time, which will lower the concentration. Thus, always use rather freshly prepared 70-% EtOH solution.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>NaOH, 15 M</strong> Dissolve 600 g NaOH into 600 ml distilled H<sub>2</sub>O. Bring the volume to 1 liter with distilled H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CAUTION!</strong> The reaction is exothermic. The solution can be kept on ice during preparation.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Potassium acetate, 6 M</strong> Dissolve 58.92 g potassium acetate into 40 ml distilled H<sub>2</sub>O. Adjust the pH to 7.5 with 2 M acetic acid. Bring the volume to 100 ml with H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> Long-term storage is recommended at -20 °C.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Proteinase K, 10 mg/ml</strong> Dissolve 100 mg Proteinase K into 10 ml of distilled H<sub>2</sub>O. Dispense into aliquots and store at -20 °C.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>RNase A (free of DNase), 9.09 mg/ml</strong> Dissolve RNase A at a concentration of 10 mg/ml into 10 mM sodium acetate pH 5.2 (e.g. dissolve 100 mg of RNase A into 1 ml of distilled H<sub>2</sub>O, and dilute 100 µl of that RNase A solution with 890 µl of distilled H<sub>2</sub>O and 10 µl of 1 M sodium acetate). Incubate the aliquot tubes in a heat block at 100 °C for 15 min. Allow the tubes to slowly cool down to room temperature. Adjust the pH by adding 100 µl (i.e. 0.1 volumes) of 1 M Tris (pH 7.4) to obtain 9.09 mg/ml RNase A solution. Dispense into aliquots and store at -20 °C.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> RNase A precipitates if the concentrated solution is boiled at neutral pH.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>SDS, 10-%</strong> Dissolve 10 g SDS into 80 ml distilled H<sub>2</sub>O. Bring the volume to 100 ml with distilled H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Sodium acetate, 1 M, pH 5.2</strong> Dissolve 24.61 g sodium acetate into 50 ml distilled H<sub>2</sub>O. Adjust the pH to 5.2 with glacial acetic acid. Allow the solution to cool down to room temperature and adjust the pH again to 5.2 with glacial acetic acid. Bring the volume to 100 ml with distilled H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Tris, 1 M, pH 7.4</strong> Dissolve 12.11 g Tris into 80 ml distilled H<sub>2</sub>O. Adjust the pH to 7.4 with HCl. Allow the solution to cool down to room temperature and adjust the pH again with HCl. Bring the volume to 100 ml with distilled H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Tris, 1 M, pH 8.0</strong> Prepare as Tris, 1 M, pH 7.4, but adjust the pH to 8.0.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p><strong>BUFFERS</strong></p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Lysis buffer: 20 mM Tris, 150 mM NaCl, 20 mM EDTA, 1 % SDS, pH 8.75</strong> Dissolve 0.877 g NaCl into 60 ml distilled H<sub>2</sub>O. Add 2 ml Tris (1 M, pH 8.0), 4 ml EDTA (0.5 M, pH 8.0) and 10 ml SDS (10-%). Adjust the pH to 8.75 with NaOH at room temperature. Bring the volume to 100 ml with distilled H<sub>2</sub>O.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> High concentrations of NaCl and SDS precipitate.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> Tris pH is temperature dependent: pH 8.75 at room temperature corresponds to pH 7.8 at the usage temperature of 56 °C.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Mitochondria isolation buffer (MIB), 2x concentrate: 640 mM sucrose, 40 mM Tris, 2 mM EGTA, pH 7.2</strong> Dissolve 219 g sucrose, 4.58 g Tris, 0.76 g EGTA into 950 ml distilled H<sub>2</sub>O. Adjust the pH to 7.2 with HCl and bring the solution to 1 liter with distilled H<sub>2</sub>O. Aliquot and store the 2x concentrate MIB at -20 ℃ until use.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> Tris pH is temperature dependent: pH 7.2 at room temperature corresponds to pH ca. 7.8 at the usage temperature of 0-4 ℃.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> MIB 2x concentrate solution can be stored frozen for very long periods of time (&gt;12 months).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Mitochondria isolation buffer (MIB02): 320 mM sucrose, 20 mM Tris, 1 mM EGTA, 0.2 % BSA, pH 7.2</strong> Thaw 2x concentrate MIB and dilute it with equal volume of distilled H<sub>2</sub>O to obtain 1x MIB solution. Add 0.2 % (w/v) BSA to obtain MIB02 solution.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> Store thawed MIB at 4 ℃ only short periods of time (1-2 days).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> BSA-containing MIB solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Mitochondria isolation buffer (MIB1): 320 mM sucrose, 20 mM Tris, 1 mM EGTA, 1 % BSA, pH 7.2</strong> Thaw 2x concentrate MIB and dilute it with equal volume of distilled H<sub>2</sub>O to obtain 1x MIB solution. Add 1 % (w/v) BSA to obtain MIB1 solution.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> Store thawed MIB at 4 ℃ only short periods of time (1-2 days).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> BSA-containing MIB solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Mito-DNase buffer base: 300 mM sucrose, 10 mM MgCl~2~, 20 mM Tris, pH 7.5</strong> Dissolve 10.27 g sucrose, 0.203 g MgCl<sub>2</sub> and 0.243 g Tris into 90 ml distilled H<sub>2</sub>O. Adjust the pH to 7.5 with HCl and bring the solution to 100 ml with distilled H<sub>2</sub>O. Aliquot and store the Mito-DNase buffer base at -20 ℃ until use.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> Tris pH is temperature dependent: pH 7.5 at room temperature corresponds to pH ca. 7.2 and 8.1 at the usage temperatures of 37 and 0-4 ℃, respectively.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• <strong>Mito-DNase buffer: Mito-DNase buffer base, 0.15 % (w/v) BSA, 0.03 mg/ml DNase I, 0.02 mg/ml RNase A</strong> Thaw Mito-DNase buffer base and add 0.15 % (w/v) BSA. Just before use, add 0.03 and 0.02 mg/ml DNase I and RNase A, respectively.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>CRITICAL:</strong> BSA-containing Mito-DNase solution should always be prepared freshly on the day of usage and stored on ice or at 4 ℃.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; line-height: 100%;"><strong>EQUIPMENT</strong></p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• 1.5-ml microcentrifuge tubes</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• 250-µl low-bind, wide-orifice pipette tips (VWR, cat. no. 613-0370)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• 50-ml polypropylene Falcon tubes</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Gel electrophoresis system (e.g. Bio-Rad Sub-Cell(TM) GT Cell)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Gel imaging system (e.g. Syngene U:Genius)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Heat block for 1.5 ml microcentrifuge tubes (e.g. Grant QB-H2)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Motor-driven glass/Teflon Potter Elvehjem homogenizer (e.g. Sartorius Potter S)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• PCR tubes (e.g. VWR® 8-well tube strips with bubble cap)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• PCR thermocycler (e.g. The Applied Biosystems® Veriti® 96-Well Thermal Cycler)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Qubit® Assay tubes (Thermo Fisher Scientific, cat.no. Q32856)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Qubit® 3.0 Fluorometer (Thermo Fisher Scientific, cat.no. Q33216)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Refrigerated centrifuge for 1.5 ml microcentrifuge tubes (e.g. Eppendorf 5417R)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">• Refrigerated centrifuge for 50-ml Falcon tubes, swing- and fixed rotors (e.g. Eppendorf 5804R)</p>
<p> </p>

[GUIDELINES]
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><strong>INTRODUCTION</strong></p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">Although a cell harbors several thousands of mtDNA molecules, only &lt;1 % of a total genomic DNA extraction is mtDNA. Thus, experiments focusing on mtDNA analysis often include an mtDNA enrichment step. Traditionally mitochondria are enriched from the cellular debris with differential or gradient centrifugation methods [1,2] or by amplification-based enrichment of the mtDNA [4,5]. The former methods will only partially enrich mtDNA, but samples still contain high nuclear DNA contamination levels [6]. Amplification-based methods suffer from errors caused by DNA polymerases or unintended amplification of nuclear sequences of mitochondrial origin (NuMTs) [7].</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">In the mouse genome, 172 chunks ranging from 33 bp to 4.7 kbp in length with 66–100 % identity were identified [8]. Others estimated that &gt;95 % of the mouse mtDNA genome is present in the nuclear chromosomes [9]. NuMTs may be present or absent in different individuals and mtDNA is constantly being transferred to the nucleus, thus, NuMT content varies between individuals [7,8]. All these characteristics of NuMTs complicate the design of primers for mtDNA enrichment, and thus, might affect amplification- or blotting-based mtDNA studies [4,10]. Furthermore, ever increasing deep sequencing approaches to detect mtDNA mutations are especially prone for false results if a large amount of nuclear DNA is present in the sample. As NuMTs may be fully homologous to true mtDNA sequences, results would be biased towards wild-type reads [8]. On the other hand, polymorphic NuMTs would be observed as false-positive mutations which are impossible to distinguish from true mtDNA mutations [8].</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">Computational approaches [10] or fast plasmid preparations in combination with PCR-amplification [12] have been suggested as solutions to avoid NuMT reads in mtDNA mutation detection or to highly enrich mtDNA. Computational approaches, however, are very challenging to implement, whereas fast DNA extraction procedures and amplification step may decrease the quality of the DNA and expose it to damage. DNA damage may also appear as false-positive mutations in sequencing applications, as DNA polymerase, for example, might bypass the damaged site by incorporating a wrong nucleotide or damage might increase the polymerase jumping increasing the amount of chimeric DNA molecules [14]. Furthermore, artefacts caused by oxidative damage to the DNA, often observed as G&gt;T/C&gt;A mutations, are a remarkable issue in reliable variant detection [15,16]. Hence, we reasoned that mtDNA analyses, especially variant detection by deep sequencing, should begin with high-quality, highly enriched mtDNA sample. Here, we describe a step-by-step protocol to extract mtDNA from mouse tissues suitable for highly-sensitive mtDNA experiments. The extremely high enrichment of mtDNA from nuclear DNA contamination is based on gentle tissue homogenization, extensive DNase I treatment of the isolated, intact mitochondria (modified from the protocol described by Kennedy et al. [2013] [11]) and DNA extraction by chloroform:isoamyl alcohol and ethanol precipitation.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; line-height: 100%;"><strong>References</strong></p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">1. Frezza, C. et al. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroplasts. <em>Nat. Protoc.</em> <strong>2</strong>, 287-295 (2007).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">2. Wieckowski, M.R. et al. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. <em>Nat. Protoc.</em> <strong>4</strong>, 1582-1590 (2008).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">3. Hazkani-Covo, E. et al. Molecular poltergeist: Mitochondrial DNA copies (numts) in sequenced nuclear genomes. <em>PLoS Genet.</em> <strong>6</strong>, e1000834 (2010)</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">4. Payne, B.A.I. et al. Deep Resequencing of Mitochondrial DNA. <em>Methods Mol. Biol</em>. <strong>1264</strong>, 59-66 (2015).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">5. Ni, T. et al. MitoRCA-seq reveals unbalanced cytocine to thymine transition in Polg mutant mice. <em>Sci. Reports</em> <strong>5</strong>, 12049 (2015).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">6. Ameur, A. et al. Ultra-deep sequencing of mouse mitochondrial DNA: mutational patterns and their origins. <em>PLoS Genet.</em> <strong>7</strong>, e1002028 (2011).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">7. Hazkani-Covo, E. et al. Molecular poltergeists: mitochondrial DNA copies (numts) in sequenced nuclear genomes. <em>PLoS Genet.</em> <strong>6</strong>, e1000834 (2010).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">8. Calabrese, F.M., et al. Primates and mouse NumtS in the UCSC Genome Browser.</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"><em>BMC Bioinformatics</em> <strong>13</strong>, S15 (2012).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">9. Malik, A.N. et al. Accurate quantification of mouse mitochondrial DNA without co-amplification of nuclear mitochondrial insertion sequences. <em>Mitochondrion</em> <strong>29</strong>, 59–64 (2016).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">10. Li, M. et al. Fidelity of capture-enrichment for mtDNA genome sequencing: Influence of NUMTs. <em>Nucleic Acids Res.</em> <strong>40</strong>, e137 (2012).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">11. Kennedy, S.R. et al. Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage. <em>PLoS Genet.</em> <strong>9</strong>, e1003794 (2013).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">12. Samuels, D.C. et al. Finding the lost treasures in exome sequencing data. <em>Trends Genet.</em> <strong>29</strong>, 593–599 (2013).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">13. Quispe-tintaya, W. et al. Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing. <em>Biotechniques</em> <strong>55</strong>, 133–136 (2015).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">14. Eckert, K.A. &amp; Kunkel, T.A. DNA polymerase fidelity and the polymerase chain reaction. <em>PCR Methods Appl.</em> <strong>1</strong>, 17–24 (1991).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">15. Costello, M. et al.. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. <em>Nucleic Acids Res.</em> <strong>41</strong>, 1–12 (2013).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">16. Chen, L. et al. DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification. <em>Science</em> <strong>355</strong>, 752–756 (2017).</p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;"> </p>
<p style="margin-bottom: 0in; line-height: 100%;"><strong>Acknowledgements</strong></p>
<p style="margin-bottom: 0in; font-weight: normal; line-height: 100%;">This work was supported by Max Planck Society.</p>

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