The overall goal of this procedure is to quantify CPG methylation in targeted regions of interest out of genomic DNA, isolated from tissue or cells of interest. This is accomplished by first designing by sulfite specific PCR primers to amplify regions of interest from bi sulfite converted genomic DNA. In the second step, the bi sulfite converted region of interest is amplified, and next generation sequencing or NGS libraries are generated from the amplicons cons.
Next, the libraries are sequenced and FASTQ files are generated for analysis. In the final step, the sequencing reads are aligned to the bi sulfite converted reference target sequence, and the five mc percent is calculated ultimately by sulfite amplicon sequencing or BS a s can be performed to precisely and accurately quantify the CPG methylation in any region of interest from any model organism. The main advantage of this technique over existing methods like Sanger sequencing, is that BS A S provides accurate and precise CPG methylation quantitation in a rapid high throughput manner, providing the opportunity to assay multiple regions in a high number of samples.
After identifying the target and designing the primer, assemble the reaction for the target amplification optimization of a single amplicon, and then sealed a PCR plate with heat sealed film to visualize the amplicons by page electrophoresis. Next, load the appropriate wells with 30 microliters of PCR reactions or ladder, and then run the gel at 200 volts for approximately 45 minutes. When the first dye reaches the end of the gel, stop the reaction and then cover the entire gel with five micrograms per milliliter of freshly prepared AUM bromide.
After five minutes, image the gel via a UV transluminator at an excitation wavelength of 482 nanometers. Use the ladder to size the PCR AMPLICONS to determine specificity of the reaction. Look for multiple bands other than the expected amplicon size to determine the average size of the detected library peaks and an estimation of the molarity.
Next, run the libraries on a capillary electrophoresis DNA sizing chip with a high sensitivity DNA assay according to the manufacturer's instructions. To quantify the libraries by QPCR. Use primers designed against the adapter sequences in the generated libraries and standards with a known concentration on a real-time instrument, using the absolute quantification settings according to manufacturer's instructions.
Then use the size of the libraries and the molar quantitation from the QPCR to calculate the molar concentrations of the libraries. After sequencing, import the zipped fast Q files into the appropriate sequencing analysis pipeline, select import, and then select the sequencing method used to generate the reads to retain the Q scores for the read trimming applications. Import the appropriate zipped fast Q read files, and then under general options, deselect discard quality scores.
Select a location for the imported reads and click finish. Then under NGS core tools, select trim sequences. Highlight the reads to be trimmed, retaining only the reads containing greater than or equal to Q 30 scores.
Then select the trim using quality scores and set the limit to 0.001. To further select only the reeds containing less than or equal to one ambiguous nucleotide. Select trim ambiguous nucleotides and set the maximum number of ambiguities to one.
Then select a location to save the trimmed reads and click finish to map the trimmed res to the in silico by sulfite converted gene reference. Under NGS core tools, select map reads. To reference, select the trimmed reads to be mapped and click next.
Then under reference masking, select the converted reference sequence and select no masking. Click next and then assign a score of three for mismatches, insertions, and deletions. Assign a score of one for the minimum fraction of read length map to the reference and assign a score of 0.9 for the minimum fraction of identity between the map, read and reference under nonspecific match handling, select, ignore, and click Next under output options.
Next, select Create standalone read mappings. Then under result handling, click save. Select a location to save the read mapping and click finish to run the variant calling on the mapped reads.
Under re-sequencing analysis, select the low frequency variant detection and choose the read mapping to be analyzed. Click next and then under low frequency variant parameter, enter 0.01%and click next. Under general filters, set the minimum coverage to 1000 to ensure that the sequence is at a depth of greater than or equal to 1000 x.
Finally, click next, and then under output options select create annotated table and select a location to save the variant table file, click finish and use the variant frequencies at the annotated CPG sites in the region of interest from the variant table file to calculate the frequency of the cytosine methylation at a reference cytosine in CG context by sulfite amplicon sequencing reads properly aligned to the converted reference sequence will resemble those illustrated in the figure. The CPG dinucleotides can clearly be identified and the methylation states can be estimated by observing the base calls at the CPG sites. In the mapped reads, 0%methylation will result in mapped reads containing thymine mapped to the CPG sites.
100%methylation controls will result in mapped reads containing cytosines mapped to CPG sites. In this experiment, RNA and DNA were co isolated from a mouse, cerebellum and retina. The opsin expression selectively expressed in retinal tissue was then measured using QPCR as expected.
The opsin RNA was detected only in the retina after quantification of the CPG methylation levels in the Opsin promoter region by bi sulfite amplification sequencing. However, the cumulative methylation levels across the promoter region were greater than 80%in the cerebellum compared to less than 15%in the retina. B SAS methylation quantitation benefits from site-specific methylation quantitation, allowing the comparison of the methylation levels on a CPG specific basis across any given genomic region.
Indeed, in this experiment, the CPG methylation levels across the opsin promoter were found to be significantly higher in the cerebellar samples when compared to the retina samples. After watching this video, you should have a good understanding of how to quantify CPG methylation in targeted regions of interest out of genomic DNA, isolated from either tissues or cells.
