The Novo Nordisk Foundation Centre for Basic Metabolic Research is an independent research center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation.

1. Restriction Enzyme Treatment
<ol>
	<li>Linearize mtDNA by treating total human DNA with the restriction enzyme BamHI, that cuts at position 14258 in the human mitochondrial DNA. 
	NOTE: For mouse DNA, use the restriction enzyme BglII under identical conditions as below.</li>
	<li>For each sample where mtDNA methylation is to be assessed, prepare one 0.2 mL reaction tube and add the following mix:3 &#956;g genomic DNA (quantified by fluorometry), 15 &#956;L Buffer 3, 3 &#956;L BamHI and water up to of 150 &#956;L</li>
	<li>Place tubes in a thermal cycler for 4 h at 37 &#176;C.</li>
</ol>
2. Bisulfite Conversion
<ol>
	<li>Convert BamHI-treated DNA using sodium bisulfite as described elsewhere29.</li>
	<li>Use 200-500 ng BamHI-treated DNA for each conversion reaction in a total volume of 20 &#956;L. The DNA recovery is 50-70%.</li>
	<li>Elute the Bisulfite-converted DNA in 10 &#956;L elution buffer.</li>
	<li>Quantify the single-stranded DNA by fluorometry.</li>
	<li>Optional: Store bisulfite-converted DNA at -20 &#176;C before proceeding the remaining of the protocol. For long-term storage, store bisulfite-converted DNA at -80 &#176;C</li>
</ol>
3. Design of Bisulfite Sequencing Primers
<ol>
	<li>Design primers for bisulfite sequencing using the online tools Methprimer30 and BiSearch31,32. 
	NOTE: Methprimer and BiSearch allow to search for primer binding sequences on genomic sequences where cytosines are converted to thymines, similarly to post-bisulfite treatment.</li>
	<li>For optimal and unbiased amplification, avoid CpG dinucleotides in the primers and design the amplicon size between 100-300 bp.</li>
	<li>Ensure that designed primers are specific to the mitochondrial DNA using BiSearch&#39;s function Primer Search. Insert the already designed bisulfite converted primers, tick the bisulfite box and include a reference genome and PCR parameters. 
	NOTE: A list of possible PCR products will be displayed.</li>
	<li>Copy the top 1-5 primer sequences and paste them into an ordering form for oligonucleotide synthesis. 
	NOTE: A list of human and mouse mtDNA primer sequences that has been validated in the lab is displayed in Table 1.</li>
	<li>Test primers using bisulfite converted PCR following agarose gel electrophoresis as described in step 4. Test and optimize all primer pairs separately and ensure the correct amplicon size appears on the agarose gel. 
	NOTE: If multiplexing primers are needed, add multiple primers to one single PCR reaction and visualize the PCR products on agarose gel electrophoresis or, a more resolutive method such as polyacrylamide gel electrophoresis, to distinguish products of similar size.</li>
</ol>
4. Bisulfite Converted PCR and Gel Extraction
<ol>
	<li>To amplify the regions of interest, perform PCR using a hot start Taq polymerase. Briefly, mix 100 ng converted DNA, 500 &#181;M sense and anti-sense primers,&#160;1 &#181;L dNTP mix (10 &#181;M of each dNTP), and polymerase at 7.5 U/reaction in a total volume of 50 &#181;L. Run PCR with the following conditions: 5 min at 95 &#176;C (60 s 94 &#176;C; 60 s 55 &#176;C*; 60 s 72 &#176;C) <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg" /> 35 cycles; 10 min 72 &#176;C. Use primers either separately or multiplexed.</li>
	<li>Optional: When multiplexing primers, use 200 ng converted DNA and the following cycling conditions: 5 min 95 &#176;C (60 s 94 &#176;C; 90 s 55 &#176;C*; 90 s 72 &#176;C) <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg" /> 35 cycles; 10 min 72 &#176;C. 
	NOTE: Temperature may vary depending on the melting temperature of primers.</li>
	<li>Subject PCR products to gel electrophoresis on 2% agarose at 100 volts.</li>
	<li>Under gentle UV light, extract PCR products with a scalpel and add to microtubes. Do not expose PCR products to excessive UV light to avoid DNA damage. Avoid exposure time over 30 s.</li>
	<li>Purify the PCR products using gel purification as described previously3.</li>
	<li>Quantify the purified DNA from step 4.5 using fluorometry. Proceed to step 5 or store samples at -20&#176;C.</li>
</ol>
5. Bisulfite Sequencing Library Preparation
<ol>
	<li>Perform library preparation of bisulfite-converted PCR products. 
	Optional: multiplex PCR products at this stage.</li>
	<li>Perform end-repair using up to 100 ng of PCR product. Add 3 &#181;L End prep enzyme and 6.5 &#181;L End repair reaction buffer (10x) to 55.5 &#181;L PCR product in a 0.2 mL tube. Place and incubate tube in a thermal cycler for 30 min at 20 &#176;C followed by 30 min at 65 &#176;C.</li>
	<li>Ligate sequencing adaptors by mixing the end-repaired DNA with 15 &#181;L Blunt/TA ligase mix, 2.5 &#181;L Adaptor and 1 &#181;L Ligation enhancer. If using &#60;100 ng PCR product as input, dilute adaptor 10 times.</li>
	<li>Place the mix (step 5.3) in a thermal cycler and incubate 15 min at 20 &#176;C. Pause the thermal cycler and add 3 &#181;l USER Enzyme to the tube. Mix and place the tube back in the Thermal cycler and incubate 15 min at 37 &#176;C.</li>
	<li>Size select the adaptor-ligated DNA by using Solid Phase Reversible Immobilization (SPRI) beads with varying ratios of beads to DNA depending on PCR product size.</li>
	<li>Add 13.5 &#181;L H2O to adaptor-ligated DNA from step 5.4 and 55 &#181;L resuspended SPRI beads. Incubate at room temperature for 5 min and place tube on a magnetic stand. When the solution is clear, transfer the supernatant to a new tube and discard tube containing the beads. 
	NOTE: The supernatant contains the adaptor-ligated DNA and large unwanted fragments are bound to the discarded beads.</li>
	<li>Add 25 &#181;L resuspended SPRI beads to the supernatant from step 5.6, mix and incubate for 5 min at room temperature. Place the tube on the magnetic stand and discard the supernatant when the solution is clear. 
	NOTE: The discarded supernatant contains unwanted DNA, whereas the adaptor-ligated DNA is bound to beads.</li>
	<li>While the tube is on the magnetic stand, add 200 &#181;L 80% ethanol (freshly prepared) to wash the beads. Incubate 30 s and remove and discard the supernatant. Repeat this step for a total of 2 washes.</li>
	<li>Air dry beads for 5 min while the tube is on magnetic stand with an open lid.</li>
	<li>Remove the tube from the magnet, elute the DNA in 23 &#181;L elution buffer and mix. Incubate at room temperature for 2 min.</li>
	<li>Place the tube on a magnetic stand and when the solution is clear, transfer 2 &#181;L to a 96-well PCR plate for the pre-PCR amplification (step 5.14).</li>
	<li>Transfer the rest approximately 21 &#181;L to a new 0.2 mL tube for PCR amplification (step 5.17). 
	NOTE: Be sure NOT to transfer any beads as beads can inhibit enzymatic reactions of next steps.</li>
	<li>Proceed to step 5.14 or store samples at -20&#176;C.</li>
	<li>Perform pre-PCR amplification by RT-qPCR to estimate the number of cycles needed to amplify the adaptor-ligated DNA. Per reaction, add the following in the 96-well PCR plate containing 2 &#181;L DNA (step 5.11): 0.4 &#181;L Index primer, 0.4 &#181;L universal PCR primer, 7.2 &#181;L H2O, 10 &#181;L RT-qPCR master mix.</li>
	<li>Place the plate in a real-time PCR machine and subject the plate to the following conditions: 30 s at 98 &#176;C (10 s 98 &#176;C; 75 s 65 &#176;C) <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg" /> 20 cycles; 5 min 65 &#176;C.</li>
	<li>Estimate the number of amplification cycles needed for each sample by subtracting one Ct-value to the Ct-value of the pre-amplification RT-qPCR corresponding to the end of the linear phase of the PCR reaction.</li>
	<li>Amplify the adaptor-ligated DNA from step 5.12 by mixing: 21 &#181;L DNA, 25 &#181;L PCR master mix, 1 &#181;L Index primer, 1 &#181;L Universal PCR primer and 2 &#181;L H2O. In a thermal cycler, subject each sample to the following conditions: 30 s at 98 &#176;C (10 s 98 &#176;C; 75 s 65 &#176;C) <img alt="arrow" src="/files/ftp_upload/57772/57772eq2.jpg" /> [calculated Ct from step 5.16] cycles; 5 min 65 &#176;C. 
	NOTE: Remember to only use one Index primer per sample to allow for multiplexing. The index is inserted during the PCR.</li>
	<li>Purify the amplified DNA SPRI beads and elute in 22 &#181;L elution buffer.</li>
	<li>Control library quality by gel electrophoresis or alternative method. Look for correct library peak sizes (PCR product(s) size plus adaptor).</li>
	<li>Estimate the average base-pair size of the library product(s) by smear analysis: In advanced global settings, double-click on &#34;Table&#34; under smear analysis. Define the region from 100-1000 bp and click OK. Under Region Table, the defined region appears and library average size (bp) is calculated.</li>
	<li>Quantify libraries by fluorometry. Proceed to step 6 or store libraries at -20 &#176;C.</li>
</ol>
6. Next Generation Sequencing
<ol>
	<li>Calculate the nM concentration of libraries using the formula: 
	<img alt="Equation" src="/files/ftp_upload/57772/57772eq1.jpg" /></li>
	<li>Dilute libraries to the same nM concentration e.g. 2 nM (choose 4 nM, 2 nM, 1 nM, or 0.5 nM depending on library concentrations). Pool all libraries to achieve a pool of 2 nM libraries.</li>
	<li>Denature and dilute libraries following the sequencing instrument guide. In short: Denature the 2 nM pool by adding 0.2 N NaOH and incubate at room temperature for 5 min. Dilute the denatured pool to a final dilution of 10 pM. Denature and dilute a commercially available Control library to a final dilution of 12.5 pM. To a new tube, mix the 10 pM pool with 20% 12.5 pM Control library. 
	NOTE: Bisulfite conversion introduces very low complexity. 20% Control library spike-in will result in better sequencing quality.</li>
	<li>Run a 150 bp paired-end sequencing using a deep sequencing instrument.</li>
</ol>
7. Computational Analysis - Estimate Methylation Levels
<ol>
	<li>Generate .fastq files (here called sample1_R1.fastq.gz and sample1_R2.fastq.gz), generate a bismark index of the entire genome of interest (here in a folder called bismarkIndex) and make sure that the genomic sequence of chrM is available in fasta format (here in a folder called chrM).</li>
	<li>Pre-process reads to remove adapters and bias introduced by the primers: Use the tool Trim_galore33. Remove two nucleotides from the 5&#39; end of both the forward and reverse reads. 
	trim_galore --clip_R1 2 --clip_R2 2 -o ./ --trim1 --paired sample1_R1.fastq.gz sample1_R2.fastq.gz</li>
	<li>Align pre-processed reads to the entire genome using Bismark34 
	bismark --dovetail --bam -o ./ ./bismarkIndex / -1 ./sample1_R1_val_1.fq.gz -2 ./sample1_R2_val_2.fq.gz 
	NOTE: If another aligner is used, make sure that reads that cannot be uniquely aligned are discarded.</li>
	<li>Extract reads mapping to the chromosome M, here using Samtools35 
	samtools sort -l 0 -O BAM -o sample1_sorted.bam 
	sample1_R1_val_1_bismark_bt2_pe.bam 
	samtools index sample1_sorted.bam 
	samtools view -u sample1_sorted.bam chrM | samtools sort -n -O BAM -o sample1_chrM.bam</li>
	<li>Extract the methylation information 
	bismark_methylation_extractor -p --CX --cytosine_report --comprehensive --genome_folder ./chrM ./sample1_chrM.bam</li>
	<li>Use the .bedGraph, the .cov or the CX_report.txt files for further analysis. Use the sample1_chrM.CX_report.txt file for visualization and testing.</li>
	<li>Perform further clipping in the preprocessing step, if multiple regions are interrogated and a primer bias may be visible in the M-bias plots generated during the mapping. Refer to the Bismark documentation for more information.</li>
</ol>
8. Computational Analysis - Test of differences
<ol>
	<li>Ensure that the CX_report.txt file contains 7 columns, chromosome (here chrM), position, strand, number of methylated reads, number of unmethylated reads, C context and surrounding sequence, for every C on chromosome M.</li>
	<li>As the CX_report covers chrM in its entirety, check the subset to the region(s) which is amplified.</li>
	<li>Use non-parametric tests for differences between samples, such as a Fishers&#39;s exact test for individual C&#39;s or a sign-test for an entire region. 
	NOTE: Quantification will often be close to 0% methylation, which is the reason why assuming normality is not appropriate.</li>
	<li>Similarly, for visualization, either visualize quantiles or calculate binomial proportion confidence intervals.</li>
</ol>

<ol>
	<li>Falkenberg, M., Larsson, N. -. G., Gustafsson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+and+transcription+in+mammalian+mitochondria.">DNA replication and transcription in mammalian mitochondria.</a> Annu Rev Biochem. 76, 679-699 (2007).</li><li>Kolesar, J. E., Wang, C. Y., Taguchi, Y. V., Chou, S. -. H., Kaufman, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+intact+mitochondrial+DNA+agarose+electrophoresis+reveals+the+structural+complexity+of+the+mammalian+mitochondrial+genome.">Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome.</a> Nucleic Acids Res. 41 (4), e58 (2013).</li><li>Mechta, M., Ingerslev, L. R., Fabre, O., Picard, M., Barres, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+Suggesting+Absence+of+Mitochondrial+DNA+Methylation.">Evidence Suggesting Absence of Mitochondrial DNA Methylation.</a> Front Genet. 8, 166 (2017).</li><li>Ehrlich, M., Gama-Sosa, M. A., 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=Amount+and+distribution+of+5-methylcytosine+in+human+DNA+from+different+types+of+tissues+of+cells.">Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells.</a> Nucleic Acids Res. 10 (8), 2709-2721 (1982).</li><li>Lister, R., Pelizzola, M., 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=Human+DNA+methylomes+at+base+resolution+show+widespread+epigenomic+differences.">Human DNA methylomes at base resolution show widespread epigenomic differences.</a> Nature. 462 (7271), 315-322 (2009).</li><li>Yan, J., Zierath, J. R., Barres, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+non-CpG+methylation+in+mammals.">Evidence for non-CpG methylation in mammals.</a> Exp Cell Res. 317 (18), 2555-2561 (2011).</li><li>Patil, V., Ward, R. L., Hesson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evidence+for+functional+non-CpG+methylation+in+mammalian+cells.">The evidence for functional non-CpG methylation in mammalian cells.</a> Epigenetics. 9 (6), 823-828 (2014).</li><li>Nass, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+methylation+of+mitochondrial+and+nuclear+DNA+in+cultured+mouse,+hamser+and+virus+trasnforemd+hamser+cells+in+vivo+and+in+vitro+methylation.">Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamser and virus trasnforemd hamser cells in vivo and in vitro methylation.</a> J Mol Biol. 80 (1), 155-175 (1973).</li><li>Shock, L. S., Thakkar, P. V., Peterson, E. J., Moran, R. G., Taylor, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methyltransferase+1,+cytosine+methylation,+and+cytosine+hydroxymethylation+in+mammalian+mitochondria.">DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria.</a> PNAS. 108 (9), 3630-3635 (2011).</li><li>Bellizzi, D., D'Aquila, P., 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=The+Control+Region+of+Mitochondrial+DNA+Shows+an+Unusual+CpG+and+Non-CpG+Methylation+Pattern.">The Control Region of Mitochondrial DNA Shows an Unusual CpG and Non-CpG Methylation Pattern.</a> DNA Res. 20 (6), 537-547 (2013).</li><li>Jia, Y., Li, R., 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=Maternal+Low-Protein+Diet+Affects+Epigenetic+Regulation+of+Hepatic+Mitochondrial+DNA+Transcription+in+a+Sex-Specific+Manner+in+Newborn+Piglets+Associated+with+GR+Binding+to+Its+Promoter.">Maternal Low-Protein Diet Affects Epigenetic Regulation of Hepatic Mitochondrial DNA Transcription in a Sex-Specific Manner in Newborn Piglets Associated with GR Binding to Its Promoter.</a> PLoS ONE. 8 (5), e63855 (2013).</li><li>Jia, Y., Song, H., Gao, G., Cai, D., Yang, X., Zhao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Betaine+Supplementation+during+Gestation+Enhances+Expression+of+mtDNA-Encoded+Genes+through+D-Loop+DNA+Hypomethylation+in+the+Skeletal+Muscle+of+Newborn+Piglets.">Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.</a> J Agric Food Chem. 63 (46), 10152-10160 (2015).</li><li>Infantino, V., Castegna, A., Iacobazzi, F., Spera, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+methyl+cycle+affects+mitochondrial+methyl+availability+and+glutathione+level+in+Down's+syndrome.">Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome.</a> Mol Genet Metab. 102 (3), 378-382 (2011).</li><li>Chen, H., Dzitoyeva, S., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+valproic+acid+on+mitochondrial+epigenetics.">Effect of valproic acid on mitochondrial epigenetics.</a> Eur J Pharmacol. 690 (1-3), 51-59 (2012).</li><li>Dzitoyeva, S., Chen, H., Manev, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+aging+on+5-hydroxymethylcytosine+in+brain+mitochondria.">Effect of aging on 5-hydroxymethylcytosine in brain mitochondria.</a> Neurobiol Aging. 33 (12), 2881-2891 (2012).</li><li>Menga, A., Palmieri, E. M., 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=SLC25A26+overexpression+impairs+cell+function+via+mtDNA+hypermethylation+and+rewiring+of+methyl+metabolism.">SLC25A26 overexpression impairs cell function via mtDNA hypermethylation and rewiring of methyl metabolism.</a> FEBS J. 284 (6), 967-984 (2017).</li><li>Frommer, M., McDonald, L. E., 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=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> PNAS. 89 (5), 1827-1831 (1992).</li><li>Byun, H. -. M., Panni, T., 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=Effects+of+airborne+pollutants+on+mitochondrial+DNA+Methylation.">Effects of airborne pollutants on mitochondrial DNA Methylation.</a> Part Fibre Toxicol. 10 (1), 1 (2013).</li><li>Janssen, B. G., Byun, H. -. M., Gyselaers, W., Lefebvre, W., Baccarelli, A. A., Nawrot, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+mitochondrial+methylation+and+exposure+to+airborne+particulate+matter+in+the+early+life+environment:+An+ENVIRONAGE+birth+cohort+study.">Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study.</a> Epigenetics. 10 (6), 536-544 (2015).</li><li>Zheng, L. D., Linarelli, L. E., 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=Insulin+resistance+is+associated+with+epigenetic+and+genetic+regulation+of+mitochondrial+DNA+in+obese+humans.">Insulin resistance is associated with epigenetic and genetic regulation of mitochondrial DNA in obese humans.</a> Clin Epigenetics. 7 (1), 1739 (2015).</li><li>Byun, H. -. M., Colicino, E., Trevisi, L., Fan, T., Christiani, D. C., Baccarelli, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Air+Pollution+and+Blood+Mitochondrial+DNA+Methylation+on+Markers+of+Heart+Rate+Variability.">Effects of Air Pollution and Blood Mitochondrial DNA Methylation on Markers of Heart Rate Variability.</a> J Am Heart Assoc. 5 (4), (2016).</li><li>Bianchessi, V., Vinci, M. C., 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=Mitochondrion.">Mitochondrion.</a> MITOCH. 27 (C), 40-47 (2016).</li><li>Wijst, M. G. P., van Tilburg, A. Y., Ruiters, M. H. J., Rots, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mitochondria-targeted+DNA+methylation+identifies+GpC+methylation,+not+CpG+methylation,+as+potential+regulator+of+mitochondrial+gene+expression.">Experimental mitochondria-targeted DNA methylation identifies GpC methylation, not CpG methylation, as potential regulator of mitochondrial gene expression.</a> Sci Rep. 7 (1), 177 (2017).</li><li>Wong, M., Gertz, B., Chestnut, B. A., Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+DNMT3A+and+DNA+methylation+in+skeletal+muscle+and+CNS+of+transgenic+mouse+models+of+ALS.">Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS.</a> Front cellr Neurosci. 7, 279 (2013).</li><li>Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-methylcytidylic+acid:+absence+from+mitochondrial+DNA+of+frogs+and+HeLa+cells.">5-methylcytidylic acid: absence from mitochondrial DNA of frogs and HeLa cells.</a> Science. 184 (4132), 80-81 (1974).</li><li>Gama-Sosa, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Levels and distribution of 5-methylcytosine in Chordate DNA. , 1-201 (1985).</li><li>Hong, E. E., Okitsu, C. Y., Smith, A. D., Hsieh, C. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionally+specific+and+genome-wide+analyses+conclusively+demonstrate+the+absence+of+CpG+methylation+in+human+mitochondrial+DNA.">Regionally specific and genome-wide analyses conclusively demonstrate the absence of CpG methylation in human mitochondrial DNA.</a> Mol Cell Biol. 33 (14), 2683-2690 (2013).</li><li>Liu, B., Du, Q., 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=CpG+methylation+patterns+ofhuman+mitochondrial+DNA.">CpG methylation patterns ofhuman mitochondrial DNA.</a> Nature Publishing Group. , 1-10 (2016).</li><li>Donkin, I., Versteyhe, S., 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=Obesity+and+Bariatric+Surgery+Drive+Epigenetic+Variation+of+Spermatozoa+in+Humans.">Obesity and Bariatric Surgery Drive Epigenetic Variation of Spermatozoa in Humans.</a> Cell Metab. 23 (2), 369-378 (2016).</li><li>Li, L. C., Dahiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MethPrimer:+designing+primers+for+methylation+PCRs.">MethPrimer: designing primers for methylation PCRs.</a> Bioinformatics. 18 (11), 1427-1431 (2002).</li><li>Tusn&#225;dy, G. E., Simon, I., V&#225;radi, A., Ar&#225;nyi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BiSearch:+primer-design+and+search+tool+for+PCR+on+bisulfite-treated+genomes.">BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes.</a> Nucleic Acids Res. 33 (1), e9 (2005).</li><li>Ar&#225;nyi, T., V&#225;radi, A., Simon, I., Tusn&#225;dy, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BiSearch+web+server.">The BiSearch web server.</a> BMC bioinformatics. 7, 431 (2006).</li><li>Krueger, F., Andrews, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bismark:+a+flexible+aligner+and+methylation+caller+for+Bisulfite-Seq+applications.">Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.</a> Bioinformatics. 27 (11), 1571-1572 (2011).</li><li>Li, H., Handsaker, B., 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=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25 (16), 2078-2079 (2009).</li><li>Ramos, A., Barbena, E., 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=Nuclear+insertions+of+mitochondrial+origin:+Database+updating+and+usefulness+in+cancer+studies.">Nuclear insertions of mitochondrial origin: Database updating and usefulness in cancer studies.</a> Mitochondrion. 11 (6), 946-953 (2011).</li><li>Warnecke, P. M., Stirzaker, C., Song, J., Grunau, C., Melki, J. R., Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+resolution+of+artifacts+in+bisulfite+sequencing.">Identification and resolution of artifacts in bisulfite sequencing.</a> Methods (San Diego, Calif). 27 (2), 101-107 (2002).</li></ol>

The authors have no competing financial interests to declare.

Here, we provide a bisulfite-sequencing protocol which is specifically designed to interrogate mtDNA methylation. The differences with bisulfite-sequencing protocols used for genomic DNA lies in the utilization of a prior restriction enzyme digestion step and a bioinformatic analysis ruling out false positive arising from NUMT sequences.
We provide a protocol to avoid bisulfite-sequencing artifacts when investigating mtDNA methylation. Bisulfite sequencing artifacts leading to false-positives ...

The mitochondrial genome is a circular, double-stranded structure of approximately 16.5-kilo base (kb) long, constituting of a heavy and a light strand. The mitochondrial genome is present in multiple copies within each cell, maternally-inherited, and encodes essential components of the respiratory chain complexes1. Similar to bacterial genomes and unlike the nuclear genome, the mitochondrial genome is organized in numerous secondary and tertiary structures, such as in coiled and supercoiled structures2, which can render access difficult during sequencing experiments3.
In the nucleus, methylation of the DNA is an extensively studied epigenetic mark that plays a role in numerous processes, notably in the regulation of gene expression. In mammalian genomes, DNA methylation occurs primarily on the 5-position of the pyrimidine ring of deoxycytidines, mostly on CG dinucleotides (or CpG). Cytosine methylation is found at 70% of all CpG in the genome of somatic cells and accounts for ~1% of total DNA bases4. DNA methylations has also been described in non-CpG contexts, such as CpA, CpT, and CpC and exist in various amounts in nuclear DNA, with values up to 25% of all methylated cytosines in embryonic stem cells5,6,7.
While cytosine methylation of the nuclear genome is widely accepted, the existence of mitochondrial DNA (mtDNA) methylation is still controversial. The first study investigating mtDNA methylation was performed in cultured cells where mtDNA methylation was readily detected, although at lower levels compared to nuclear DNA8. In both human and murine cells, mtDNA methylation was also detected at low levels (2-5%). Using assays relying on 5 methylcytosine capture such as methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR, mtDNA methylation was also detected in various mouse and human and cells lines9,10,11,12. Using antibodies against 5-methylcytosine in an ELISA assay or mass spectrometry, substantial levels of DNA methylation were detected from purified mitochondrial fractions13,14,15,16. However, most of the assays in the aforementioned studies used techniques that were not designed to provide absolute quantification of DNA methylation at the single base-resolution.
Quantitative and resolutive DNA methylation analysis can be achieved by a technique named &#34;bisulfite sequencing&#34;, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil in single-stranded DNA context17. Using bisulfite sequencing, a constellation of studies has detected the presence of cytosine methylation at various levels. Methylation mtDNA in the D-loop region, the 12S or the 16S region was readily detected in human18,19,20,21,22,23 and mouse24 tissues and cells, however, with an intriguing variability, of 1-20% of total cytosines across studies.
In comparison to these numerous studies, only a few studies, including from our group, have disputed the presence of mtDNA methylation3,25,26,27 or questioned the biological relevance of very low levels of mtDNA levels (below 2%)28. Recently, we reported the observation of a potential bisulfite-sequencing artifact in whole mitochondrial bisulfite sequencing3. We provided evidence that the secondary structure of mitochondrial DNA could lead to false positives in bisulfite sequencing, thereby overestimating methylation levels. We provide here a protocol to prevent an artifact of bisulfite-conversion of mtDNA. This protocol uses a simple enzymatic digestion of DNA to disrupt mtDNA secondary structures and allow full access to bisulfite following a bisulfite sequencing protocol. In addition, we provide an accompanying bioinformatic pipeline for the analysis of bisulfite sequencing.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">BamHI</td>
			<td style="width:164px;">New England BioLabs</td>
			<td style="width:113px;"># R0136</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ DNA methylation-lightning kit</td>
			<td>Zymo Research</td>
			<td># D5030&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit ssDNA assay</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q10212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit assay tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStarTaq plus DNA polymerase kit</td>
			<td>Qiagen</td>
			<td># 203603&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td># 28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEBNext Ultra DNA Library Kit for Illumina</td>
			<td>New England BioLabs</td>
			<td># E7370S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEBNext Multiplex Oligoes for Illumina</td>
			<td>New England BioLabs</td>
			<td># E7335S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td># A63881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Sensitivity DNA chip</td>
			<td>Agilent</td>
			<td># 5067-4626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit high sensitivity dsDNA assay</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q33230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiSeq reagent kit v2 300 cycles</td>
			<td>Illumina</td>
			<td># MS-102-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PhiX control v3</td>
			<td>Illumina</td>
			<td># FC-110-3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide&#160;</td>
			<td>Sigma</td>
			<td># S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermal cycler C1000</td>
			<td>Biorad</td>
			<td># 1851148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX96 Real-Time PCR detection system</td>
			<td>Biorad&#160;</td>
			<td>#1855195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td># Q33226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td># G2939BA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiSeq instrument</td>
			<td>Illumina</td>
			<td># SY-410-1003</td>
			<td></td>
		</tr>
	</tbody>
</table>

methodology for accurate detection of mitochondrial dna methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Two steps in this protocol are crucial when investigating mtDNA methylation. 1) The opening of the secondary structure and 2) the design of mitochondrial DNA-specific primers.
By digesting the human genomic DNA with the restriction enzyme BamHI (Figure 1), the mitochondrial DNA structure will be cut at nucleotide position 14,258 and the secondary structure will be opened.
<p class="jove_content"...

Watch this Scientific Journal Video about Methodology for Accurate Detection of Mitochondrial DNA Methylation at JoVE.com

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

methodology-for-accurate-detection-of-mitochondrial-dna-methylation

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

13.2K Views.  University of Copenhagen. Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Video: Methodology for Accurate Detection of Mitochondrial DNA Methylation

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

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Methyl-binding DNA capture Sequencing for Patient Tissues

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Detection of Copy Number Alterations Using Single Cell Sequencing

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

Protocols for C-Brick DNA Standard Assembly Using Cpf1

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms