Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

The overall goal of this procedure is to generate sequencing libraries for base pair resolution D-N-A-C-P-G, methylation analysis based on restriction enzyme digestion combined with cytosine by sulfite conversion. This is accomplished by first performing MSP one restriction enzyme digest of high quality DNA, followed by end repair, a tailing and ligation of methylated adapters. The next step is to perform by sulfite conversion on size selected fragments after by sulfite conversion.

The fragments are amplified using PCR. The final step is performing quality control and sequencing, followed by data visualization and analysis. Ultimately, enhanced reduced representation of bi sulfite sequencing detects quantitative base pair resolution of cytidine methylation patterns at CG rich genomic loci.

The main advantage of this technique over other DNA methylation methods, such as microarrays, is that it can utilize small input material quantities to generate high coverage D-N-A-C-P-G methylation data at base pair resolution and biologically relevant sites. We first had the idea for this method when we were interested in exploring epigenetic patterns beyond regions covered in traditional assays. We were interested in developing this technique to transition from microarrays to the generation of base pair resolution DNA methylation data that could be integrated with data generated from other next generation techniques.

This method can help answer key questions in the field of epigenetics. For example, epigenetic patterning and heterogeneity in biological samples compared to normal controls or other disease states. Generally, individuals new to this method will find it challenging due to the length of the procedure, the numerous specific requirements and the unique sequencing characteristics of the libraries generated Mamie fall.

A research specialist in our lab will be assisting in demonstrating the procedure To begin this protocol. First, prepare purified aliquots of genomic DNA samples that have undergone an MSP one restriction enzyme digest, a blunt end repair reaction, and a single a nucleotide addition at the three prime ends. At this point, the DNA products will be ready for adapter ligations to start transfer 10 microliters of the A-A-L-D-N-A sample into a 0.2 milliliter PCR tube, and place the tube on ice.

In addition, place an aliquot of the adapter molecules on ice. Next, prepare the ligation reagent mix on ice. Add both the ligation reagent and the adapter molecules to the DNA sample and pipette up and down a few times to mix.

Place a PCR tube into a thermal cycler and allow the ligation reaction to proceed overnight at 16 degrees Celsius on the following day. Purify the ligation products using a magnetic bead or silica column based solid phase DNA extraction kit. At the last step of the purification process.

Elute the DNA by adding 30 microliters of DNAs free water. The purified DNA product can be stored at negative 20 degrees Celsius until needed for samples, which started with the total DNA of 25 nanograms or greater. Use an automated gel extraction apparatus such as the PI and prep to select for ligated products with lengths between 150 base pairs and 400 base pairs.

Be sure to exclude DNA visualization dyes such as Ethereum bromide or cyber green from the agros cassette because they can alter the DNA migration properties of the forked adapter bound fragments. Standard agros gel-based size selection protocols can also be used for this step of the protocol to set up DNA electrophoresis on a pippin 2%dye-free aros cassette. Create a new protocol in the Pippin Prep console and select the 2%DF marker L option as the cassette of choice.

Then enable the use internal standards option and verify that the reference lane number matches with the lane numbers for isolating DNA fragments ranging from 150 base pairs and 400 base pairs. Select the range option as the collection mode and enter the following limit parameters 135 for base pair start 410 for base pair end, and 240 for base pair paws. This instructs the apparatus to align the electrophoretic field to the electro elution output port.

When DNA fragments within that range are traveling near the vicinity of the outlet. After saving the protocol file, prepare the gel cassette and the sample loading wells of the instrument by following the standard operating procedures from Pippen Prep upon instrument setup. Prepare the DNA samples for electrophoresis by adding 10 microliters of the DNA marker to a 30 microliter aliquot of a given post ligation sample to load the mixtures onto the gel cassette.

First, remove 40 microliters of the electrophoresis buffer from each sample. Well then replace the volume by pipetting each 40 microliters sample marker mixture into individual wells. Returning to the console, load the saved protocol and press start to begin electrophoresis.

When the program reaches the 240 base pair, pause step manually collect 40 microliters of the ouit from each output port with a pipette. Label these fractions as the lower library fraction. After collecting the lower library fractions, immediately wash the outlet ports by adding 40 microliters of fresh electrophoresis buffer.

Pipetting the buffer up and down at least three times, and then aspirating the snet. Repeat this wash two more times to remove all traces of short length DNA species from the outlets. Now add 40 microliters of electrophoresis buffer to the sample.

Well seal the elucian ports and press the run button to resume the elucian process. Upon completion of the Ellucian program at the 410 base pair setting, collect the 40 microliters of Inuit from all outlet ports and label these fractions as the upper library fraction. At this point, both library fractions are gel purified and already for bi sulfite conversion.

Using a commercially available kit, perform the bi sulfite conversion assay on both library fractions. At the final step of the conversion process, elute the DNA samples in 40 microliters of DNAs free water. After bi sulfite conversion, the resulting DNA library fragments will undergo an enrichment PCR amplification using primers that hybridize at the adapter ends of each DNA molecule.

To prepare for enrichment PCR first, add 160 microliters of the PCR master mix to every 40 microliter aliquot of a given by sulfide converted sample. Then divide the resulting 200 microliter mixture into four 50 microliter aliquots. In separate PCR tubes.

Place the tubes in a thermal cycler and amplify the converted DNA template. After enrichment, pull the four post PCR products into a single 1.5 milliliter tube and purify the DNA with a commercial PCR cleanup kit. If a magnetic bead solid phase extraction kit is used to purify the PCR products, modify the standard operating procedures by first mixing the combined lower library PCR products with 1.7 times their total volume in beads.

Then mix the combined upper library PCR products with 1.1 times their total volume in beads. Then follow the manufacturer's protocol until the 70%ethanol wash steps where each of the wash volumes should be changed to 800 microliters at the last step of the purification process. Elute the DNA with 50 microliters of DNA free elution buffer.

Store the purified libraries at negative 20 degrees Celsius until they are needed using a fluorescence based assay selective for double stranded DNA quantitate the amount of post enrichment DNA content for each library fraction. Furthermore, assess the post enrichment library size and quality with a bioanalyzer. Calculate the effective DNA molarity of a particular library fraction by using the average DNA length expressed in base pairs.

Once the molarity is known, use DNA's free water to dilute each corresponding upper and lower library fraction down to two nanomolar final concentration. Combine the lower and upper library pairs into a single tube to create a two nanomolar hole library mixture at 20 microliters each. The pooled sample is now ready for a sequencing run.

In sequencing related assays, including E-R-R-B-S, the quality and size distribution of the upper and lower library molecules are key factors in determining the quality of the final sequencing data. When compared to traditional manual gel extraction protocols, the automated gel extraction apparatus used in the E-R-R-B-S protocol will produce a consistent size distribution and minimize the potential for cross sample contamination. Next, upon sending the BIS sulfite converted library molecules for sequencing the raw data from all DNA strands show that for any given strand, the data quality for each nucleotide position increases dramatically right after the first three nucleotides.

Since the consensus sequence for this three nucleotides set with CGG for the vast majority of library molecules, the data suggests the E-R-R-B-S protocol has produced a library collection that contains a desirable set of genomic fragments that are predominantly flanked. With MSP one restriction digest ends from a bird's eye point of view. The E-R-R-B-S protocol can yield base pair resolution data of cytidine methylation sites across the entire genome.

For example, chromosome 12 is shown here. The protocol covers a reduced representation of genome-wide CPGs. After watching this video, you should have a good understanding of how to prepare E-R-R-B-S libraries for the generation of base resolution D-N-A-C-P-G methylation data once mastered, this technique can be done in four days if performed properly.

While attempting this procedure, it's important to remember to start with high quality DNA, include all steps described, validate the efficiency of bi sulfate conversion and sequence using the recommended parameters. Don't forget that working with phenyl and chloroform can be extremely hazardous. Common precautions, such as working in a chemical hood and having proper waste disposal should always be taken while performing this procedure.

Following this procedure. Other methods such as spyro sequencing or mass array epitype can be performed to validate the findings. Furthermore, gene expression profiling can be performed to determine the biological significance of the DNA methylation patterns detected.

The ability to generate base pair resolution DNA, methylation patterns from limited amounts of input materials has enabled the profiling of rare cell populations never before possible. It has also made feasible the profiling of large cohorts of clinical samples for the exploration of epigenetic mediated regulation and heterogeneity of disease.