The overall goal of this bisulfite sequencing protocol and bioinformatic pipeline is to achieve an accurate quantification of mitochondrial DNA methylation. This method can help answer key questions in the mitoepigenetic field such as is mitochondrial DNA methylation changing human disease? The main advantage of this technique is that it allows for accurate quantification of mitochondrial DNA methylation.
Crucial step when investigating mitochondrial DNA methylation is to linearize the mitochondrial DNA by BamH1 digestion before treatment with sodium bisulfite. The BamH1 restriction enzyme site is displayed in this map of the human mitochondrial genome. Also shown are the five regions of the mitochondrial genome that will be investigated by bisulfite sequencing.
Displacement loop, tRNA phenylalanine, and 12S ribosome, 16S ribosome, NADH dehydrogenase 5, cytochrome B mRNA encoding gene. After designing the primers for bisulfite sequencing, using online tools as described in the text protocol, primer specificity toward the mitochondrial genome is verified using BiSearch. Primer sequences are added to the BiSearch function primer search.
The bisulfite box is checked and a reference genome is selected. The search results will show potential PCR products generated on sense and antisense chains. To amplify the regions of interest from the bisulfite converted mitochondrial genome, perform PCR using a Hot-start Taq polymerase.
If using primers separately, mix 100 nanograms of converted DNA, 500 micromolar of sense and antisense primers, one microliter dNTP mix and polymerase at 7.5 units per reaction in a total volume of 50 microliters. Run the PCR with the following conditions:five minutes at 95 degrees Celsius, one minute at 94 degrees Celsius, one minute at 55 degrees Celsius, one minute at 72 degrees Celsius for 35 cycles, followed by 10 minutes at 72 degrees Celsius. For multiplexing primers, use 200 nanograms of converted DNA in the following cycling conditions:five minutes at 95 degrees Celsius, one minute at 94 degrees Celsius, 1.5 minutes at 55 degrees Celsius, and 1.5 minutes at 72 degrees Celsius for 35 cycles, followed by 10 minutes at 72 degrees Celsius.
When the PCR is complete, perform gel electrophoresis on the PCR products, using a 2%agarose at 100 volts. Extraction of the PCR products from the agarose gel must be done in a way that minimizes UV exposure and the risk of DNA damage. Take a photo of the gel under UV light.
Then, very quickly, mark around the PCR product with a scalpel and turn off the UV light. Next, cut the PCR product slowly without exposure to UV light. Place the gel slices in microcentrifuge tubes and purify the PCR products using gel purification as described in the text protocol.
Quantify the purified DNA using fluorometry. Store the samples at 20 degrees Celsius, if not proceeding immediately to bisulfite sequencing library preparation. Begin the library preparation of bisulfite converted PCR products by performing end repair using up to 100 nanograms of PCR product.
To each 0.2 milliliter tube, add 55.5 microliters of PCR product, three microliters of end prep enzyme, and 6.5 microliters of 10X end repair reaction buffer. Incubate the tubes in a thermocycler at 20 degrees Celsius for 30 minutes, followed by 65 degrees Celsius for 30 minutes. Next, ligate sequencing adapters by mixing the end repaired DNA with 15 microliters of Blunt/TA ligase mix, 2.5 microliters of adapter, and one microliter of ligation enhancer.
Place the mix in a thermocycler, and incubate at 20 degrees Celsius for 15 minutes. Pause the thermocycler and add 3 microliters of USER enzyme to each tube. Mix and place the tubes back in the thermocycler and incubate at 37 degrees Celsius for 15 minutes.
Size select the adaptor-ligated DNA using solid phase reversible immobilization or SPRI. Add to the adaptor-ligated DNA 13.5 microliters of water and 55 microliters of resuspended SPRI beads. Incubate at room temperature for five minutes.
Place the tubes on a magnetic stand. When the solution is clear, transfer the supernatant, containing the adaptor-ligated DNA, to a new tube, and discard the tube containing the beads. Add 25 microliters of resuspended SPRI beads to the supernatant;mix and incubate at room temperature for five minutes.
Place the tubes on the magnetic stand and when the solution is clear, discard the supernatant. The adaptor-ligated DNA is now bound to the beads. While the tubes are on the magnetic stand, add 200 microliters of freshly prepared 80%ethanol to each tube and incubate for 30 seconds to wash the beads.
Remove and discard the supernatant. Repeat this ethanol wash step for a total of two washes. Remove the ethanol from the second wash and air dry the beads by leaving the tubes on the magnetic stand, with the lid open, for five minutes.
After five minutes, remove the tubes from the magnetic stand, add 23 microliter of DNA elution buffer to the beads and mix. Incubate at room temperature for two minutes. Place the tubes on the magnetic stand until the solution is clear.
Then, transfer two microliter of the solution to a 96-Well PCR plate for the pre-PCR amplification. While carefully avoiding the beads, transfer the rest of the solution to a new 0.2 milliliter tube for PCR amplification. Perform pre-PCR amplification by RT-qPCR to estimate the number of cycles needed to amplify the adaptor-ligated DNA.
Make a master mix of the following per reaction used. 0.4 microliters of index primer, 0.4 microliters of universal PCR primer, 7.2 microliters of water, and 10 microliters of RT-qPCR master mix. Then, mix the master mix and add 18 microliters of master mix to the two microliters of DNA in the 96-Well PCR plate.
Place the plate in a real-time PCR machine and perform amplification under the following conditions:30 seconds at 98 degrees Celsius, 10 seconds at 98 degrees Celsius, and 75 seconds at 65 degrees Celsius for 20 cycles, followed by five minutes at 65 degrees Celsius. After estimating the number of amplification cycles needed for each sample, as described in the text protocol, amplify the adaptor-ligated DNA. For each sample, mix 21 microliters of DNA, 25 microliters of PCR master mix, one microliter of index primer, one microliter of universal PCR primer, and two microliters of water.
In the thermocycler, subject each sample to the following conditions:30 seconds at 98 degrees Celsius, 10 seconds at 98 degrees Celsius, and 75 seconds at 65 degrees Celsius for the calculated number of cycles, followed by five minutes at 65 degrees Celsius. Purify the amplified DNA using SPRI beads, as demonstrated earlier and elute in 22 microliters of elution buffer. Control the library quality by using a Bioanalyzer.
Look for correct library peak sizes. Estimate the average base pair size of the library products by smear analysis. In Advanced Global Settings, under Smear Analysis, double-click on Table.
Find the region from 100 to 1000 base pair and click OK.Under Region Table, the defined region will appear and the library average size is calculated. Quantify the library by fluorometry. Store the library at 20 degrees Celsius until ready for next generation sequencing and computational analyses.
By bisulfite sequencing, mitochondrial DNA methylation was investigated in undigested and digested total DNA from human skeletal muscle cells at five different regions of the mitochondrial genome. A full square represents cytosine guanine dinucleotide sites. The open square represents non-cytosine guanine dinucleotide sites.
For undigested mitochondrial DNA, methylation levels ranged from 0-15.1%across all investigated regions. However, when the DNA was digested prior to bisulfite conversion, the uncoversion rate dropped to a maximum of 1%across all regions. This illustrates the importance of BamH1 digestion prior to bisulfite conversion to avoid overestimation of mitochondrial DNA methylation levels.
After watching this video, you should have a good understanding on how to perform mitochondrial bisulfite sequencing, and by following our bioinformatic pipeline, you can achieve accurate quantification of mitochondrial DNA methylation.
