We thank all the authors of the original ERRBS report. We thank Mame Fall for technical assistance. We acknowledge the Weill Cornell Medical College Epigenomics Core for technical services and assistance. The work was supported by a Sass Foundation Judah Folkman Fellowship, an NCI K08CA169055 and ASH-AMFDP12005 to FGB, NIH R01HG006798 and R01NS076465, funding from the Irma T. Hirschl and Monique Weill-Caulier Charitable Trusts and STARR Consortium (I7-A765) to CEM, and an LLS SCORE grant (7006-13) to AMM.

NOTE: Institutional review board approval was obtained at Weill Cornell Medical College (protocol number 0805009783) and this study was performed in accordance with the Helsinki protocol.
NOTE: Please consult material safety data sheets for relevant materials before use (indicated throughout the protocol with &#8220;CAUTION&#8221;). Several of the reagents used are toxic and appropriate safety measures are advised (personal protective equipment and fume hood).
NOTE: All steps are performed at room temperature unless otherwise indicated throughout the protocol
1. Preparation and digestion of genomic DNA
<ol>
	<li>Prepare 50 ng of high quality genomic DNA (&#62; 40 kilobases in size for human DNA) as starting material in 50 microliter (&#181;l) of DNase-free water. Quantify the DNA using a fluorescence-based quantitation assay per manufacturer&#8217;s recommendations.</li>
	<li>Mix 50 ng of DNA with 2 &#181;l of MspI (100,000 units/milliliter), 10 &#181;l of appropriate 10X reaction buffer, and DNase-free water to bring the reaction to a total volume of 100 &#181;l.</li>
	<li>Incubate the reaction at 37 &#176;C in a thermal cycler or water bath for at least 18 hours.</li>
	<li>DNA extraction and precipitation
	<ol>
		<li>Purify the digested DNA using a phenol-chloroform extraction.
		<ol>
			<li>Add 200 &#956;l of 10 millimolar (mM) tris(hydroxymethyl)aminomethane (Tris-Cl) pH 8.0 buffer to each reaction from step 1.3 to bring the volume to 300 &#956;l.</li>
			<li>Add 150 &#181;l of Tris- Ethylenediaminetetraacetic acid (TE) -saturated phenol and 150 &#181;l of chloroform mix (1:1; CAUTION) to the DNA in a chemical hood and vortex briefly.</li>
			<li>Centrifuge at 20,800 x g in a microcentrifuge for 10 min at room temperature.</li>
			<li>Transfer top aqueous phase (approximately 300 &#181;l) from last step into a new tube.</li>
		</ol>
		</li>
		<li>Precipitate the digested DNA using ethanol precipitation.
		<ol>
			<li>Add 1 &#181;l of glycogen (20 milligrams/milliliter), 1/10 volume (30 &#181;l) of 3 M pH 5.2 sodium acetate and 2.5 volumes (750 &#181;l) of room temperature 100% ethanol. Mix by vortexing at high speed.</li>
			<li>Centrifuge at 4 &#176;C for 45-60 min at 20,800 x g in a microcentrifuge.</li>
			<li>Remove ethanol quickly by inverting and dragging each tube over a paper towel with a quick flick of the wrist. Ensure that the DNA pellet remains on the side wall of the tube.</li>
			<li>Add 600 &#181;l of room temperature 70% ethanol to each tube. Visualize the pellet as it dislodges from the tube side. Mix gently by inverting the tube five times.</li>
			<li>Centrifuge at 4 &#176;C for 45-60 min at 20,800 x g in a microcentrifuge.</li>
			<li>Remove the ethanol by quickly inverting and dragging each tube over a paper towel with a quick flick of the wrist.</li>
			<li>Remove as much ethanol as possible by carefully removing any residual volume. Use a vacuum apparatus with a DNase-free 10 &#181;l pipette tip at the end, and allow the pellet to air dry if necessary to fully eliminate any further residual ethanol. 
			NOTE: Avoid over drying the pellet since that will reduce the solubility upon resuspension in the next step.</li>
			<li>Resuspend the DNA pellet into 30 &#181;l of 10 mM Tris-Cl, pH 8.5 (Buffer EB). 
			NOTE: To avoid re-annealing of the &#8220;sticky&#8221; CG overhangs created by the MspI digestion, it is necessary to complete the protocol through the ligation step on the same day (protocol step 4).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. End-repair
<ol>
	<li>Transfer the 30 &#181;l of MspI digested DNA (from step protocol 1.4.2.8) into a PCR tube on ice (4 &#176;C) and add the reagents from Table 1.</li>
	<li>Incubate the end-repair reaction for 30 min at 20 &#176;C in a thermal cycler with the heat lid on.</li>
	<li>Purify the DNA products to remove unincorporated dNTPs and other reaction reagents using a commercial column-based kit that binds of DNA in high-salt buffer and elutes DNA in low-salt buffer conditions per manufacturer&#8217;s recommendations. Elute the DNA products in 32 &#181;l of Buffer EB.</li>
</ol>
3. A-tailing
<ol>
	<li>Transfer the 32 &#181;l of DNA solution from step 2.3 into a PCR tube on ice (4 &#176;C) and add the reagents listed in Table 2.</li>
	<li>Incubate the A-tailing reaction for 30 min at 37 &#176;C in a thermal cycler with the heat lid on.</li>
	<li>Purify the DNA products to remove unincorporated dATPs and other reaction reagents using a commercial column-based kit that binds of DNA in high-salt buffer and elutes DNA in low-salt buffer conditions per manufacturer&#8217;s recommendations. Elute in 10 &#181;l of Buffer EB.</li>
</ol>
4. Adapter ligation
<ol>
	<li>Transfer the 10 &#181;l of A-tailed DNA into a PCR tube on ice (4 &#176;C)</li>
	<li>Add the ligation reaction reagents to the DNA as detailed in Table 3 and adapters as detailed in Table 4.</li>
	<li>Incubate the ligation reaction overnight at 16 &#176;C in a thermal cycler with the heat lid on.</li>
	<li>Purify the ligation products using a solid-phase reversible immobilization (SPRI) bead isolation protocol per manufacturer&#8217;s recommendations. For example, with Agencourt AMPure XP beads, use a 1.8X ratio of bead volume to sample volume (90 &#181;l of beads for the 50 &#181;l ligation reaction). Elute into 30 &#181;l of DNase-free water.
	<ol>
		<li>Ligated DNA can be stored at -20 &#176;C before proceeding with size selection.</li>
	</ol>
	</li>
</ol>
5. Size Selection
NOTE: Follow either section 5.1 for an automated size selection protocol or section 5.2 for a manual gel extraction size selection protocol. For samples with 25 ng or more of input DNA, an automated size selection protocol (using an instrument such as the Pippin Prep) can be used. Manual gel extraction is necessary for low DNA input amounts of 5-10 ng.
<ol>
	<li>Size selection using Pippin Prep for samples with 25 ng or higher of input DNA (Figure 2A) .
	<ol>
		<li>Create a new protocol on the Pippin Prep for size selection.</li>
		<li>Select &#8220;2% DF Marker L&#8221; as the Cassette. Click the &#8220;Use internal standards&#8221; button. Verify that the &#8220;Ref lane&#8221; numbers match the lane numbers.</li>
		<li>Select &#8220;Range&#8221; as the collection mode for each lane. Enter 135 under &#34;BP Start&#34;, 410 under &#34;BP End&#34;, and 240 under &#34;BP Pause&#34; for each lane.</li>
		<li>Save the protocol.</li>
		<li>Add 10 &#181;l of marker L to each 30 &#181;l sample from the ligation reaction (step 4.3), bringing the total volume to 40 &#181;l. Follow standard Pippin Prep protocol to prepare the gel cassette and instrument.</li>
		<li>Remove 40 &#181;l of electrophoresis buffer from the sample wells, and load a 40 &#181;l sample from step 5.1.5 into each of the 5 wells of the dye-free gel cassette. 
		NOTE: Dye-free, agarose gel cassettes are critical to size selecting adapter-ligated DNA fragments as the presence of ethidium bromide can alter the migration properties of the forked-adapter bound fragments.</li>
		<li>Select the protocol created in step 5.1.1, and start the run.</li>
		<li>For each lane used collect 40 &#181;l from the elution module when the run pauses at the 240 base pair (bp) point (lower library fraction: 135-240 bp). The instrument will pause individually for each lane. Repeat steps 5.1.5-5.1.7 for each lane as it pauses at 240 bp.</li>
		<li>Wash the elution module with 40 &#181;l of fresh electrophoresis buffer by pipetting up and down three times. Discard the 40 &#181;l of wash buffer. Repeat the wash step two additional times and remove all residual liquid from the elution module. Washing the elution module will decrease the amount of lower fraction DNA carried over into the higher fraction collection.</li>
		<li>Add 40 &#181;l of fresh electrophoresis buffer and re-seal the elution module and resume the run.</li>
		<li>Collect 40 &#181;l from each elution module into a new tube when the instrument indicates that the elution is complete. This elution contains the higher library fraction (240-410 bp). If running multiple gel cassettes, store size-selected samples at 4 &#176;C before proceeding with bisulfite conversion.</li>
	</ol>
	</li>
	<li>Size selection usingmanual gel extraction (Figure 2B)
	<ol>
		<li>Prepare a 1.5% agarose gel with 0.2 &#181;g/ml ethidium bromide (CAUTION).</li>
		<li>Load 50 bp and 100 bp ladders prepared with loading buffer in adjacent wells on both sides of the gel.</li>
		<li>Add 2 &#956;l of 6X Orange G loading dye to the cleaned-up ligation products. Load the entire volume of each sample into individual wells, skipping at least one well between samples to avoid cross-contamination.</li>
		<li>Run the gel at 3.5 volts per centimeter until ladder is fully separated and the Orange G dye runs to the bottom of the gel (minimum of one hour)</li>
		<li>Slice off the ladders using a clean razor blade and visualize them on a UV transilluminator (CAUTION). Do not expose the samples to UV light. Mark the 150 bp, 250 bp, and 400 bp bands of the ladders using a razor blade or pipette tips. Marking the ladder bands will allow the excision of samples without exposing them to UV light.</li>
		<li>Align the ladder slices with the rest of the gel. Using the marked bands as reference, excise one slice containing the 150-250 bp library fraction (lower) and another slice containing the 250-400 bp library fraction (higher). Place each of the two slices into different tubes.</li>
		<li>Repeat the excision for all samples prepared using a clean razor blade for each gel lane used.</li>
		<li>Purify the lower and higher library fractions using a gel extraction kit following manufacturer&#8217;s protocols. Elute each sample extracted into 40 &#181;l of EB. To ensure efficient PCR amplification of both the lower and higher library fractions in step 7, maintain the two fractions as independent samples for the bisulfite conversion step.</li>
	</ol>
	</li>
</ol>
6. Bisulfite Conversion
<ol>
	<li>Set up a bisulfite conversion control per manufacturer&#8217;s recommendations (e.g., Universal Methylated Human DNA Standard kit). Treat the control in the same manner as the samples throughout the bisulfite conversion protocol.</li>
	<li>Perform bisulfite conversion using a commercial kit protocol per manufacturer&#8217;s recommendations. Use a commercial kit that has limited template degradation and DNA loss during treatment and cleanup, while resulting in high bisulfite conversion rates of the DNA. If using the EZ DNA Methylation Kit, perform the protocol per manufacturer&#8217;s recommendations with the following exception: incubate the samples in a thermal cycler using the following protocol: 55 cycles: 95 &#176;C for 30 sec, 50 &#176;C for 15 min. Hold at 4 &#176;C.</li>
	<li>Elute the samples in 40 &#181;l of DNase free water. Proceed with the enrichment PCR (step 7) on the same day that the bisulfite conversion is completed. Bisulfite-treated DNA is AU-rich and single-stranded, which reduces its stability.</li>
	<li>Confirm efficient bisulfite conversion of the control used via Sanger sequencing with non-methylated cytosines greater than 99% converted.</li>
</ol>
7. Enrichment PCR
<ol>
	<li>Prepare a PCR master mix using the reagents in Table 5 per library fraction (optimized for the use of FastStart Taq DNA Polymerase).</li>
	<li>Add the PCR master mix to each 40 &#181;l bisulfite-converted library fraction. Mix by pipetting. Divide the 200 &#181;l reaction into four PCR tubes with 50 &#181;l each.</li>
	<li>Amplify the reactions in a thermal cycler with the following protocol: Set the heat lid to 100 oC. Initialize with a step of 94 &#176;C for 5 min. Run 18 cycles of denaturing, annealing and extension/elongation steps: 94 &#176;C for 20 seconds followed by 65 &#176;C for 30 seconds followed by 72 &#176;C for 1 min. Run a final extension/elongation step of 72 &#176;C for 3 min and hold at 4 &#176;C. 
	NOTE: 18 cycles of PCR is recommended for low input material quantities (less than 10 ng) and for first time users of the protocol. The protocol can be adjusted for a lower number of PCR cycles (as low as 14 cycles for 50 ng of input DNA; see Table 6).</li>
</ol>
8. Purify PCR reactions
<ol>
	<li>Combine each 4-reaction set (four 50 &#181;l PCR reactions per library fraction).</li>
	<li>Purify the PCR products using a SPRI bead approach (or other approach which can remove unincorporated primers and other PCR reaction reagents) per manufacturer&#8217;s recommendations. The following steps have been optimized for Agencourt AMPure XP.
	<ol>
		<li>Add 1.7X volume of SPRI beads (340 &#956;l for 200 &#956;l of PCR product) to the lower fraction amplification products.</li>
		<li>Add 1.1X volume of SPRI beads (220 &#956;l for 200 &#956;l of PCR product) to the higher fraction amplification products.</li>
		<li>Purify PCR products per manufacturer&#8217;s recommendations using 800 &#181;l of 70% ethanol for wash steps.</li>
		<li>Elute into 50 &#181;l of DNase-free water and add 1M Tris buffer to bring each library to 10 mM Tris-Cl, pH 8.5. Store libraries at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
9. Library Quality Control
<ol>
	<li>Quantify libraries using a fluorescence-based quantitation assay selective for double-stranded DNA per manufacturer&#8217;s recommendations. Spectrophotometry-based measurements are not reliable. Expected concentrations measure 10 - 50 ng/&#181;l for the lower library fraction and 3 - 15 ng/&#181;l for the higher library fraction.</li>
	<li>Assess library sizes and quality using a bioanalyzer instrument and the High Sensitivity DNA Kit. Visualize library products and determine the average size of each library fraction. The lower library fraction typically has an average size between 180 and 210 bp. The higher fraction typically has an average size between 280 and 310 bp.</li>
</ol>
10. Prepare libraries for sequencing
<ol>
	<li>Calculate the molarity of each library fraction as follows:
	<ol>
		<li>Size each library fraction using the trace obtained in step 9.2. For example, if using a bioanalyzer, use the &#8220;region&#8221; feature, and cover the beginning and end of the library fraction assessed.</li>
		<li>Record the average size of each library fraction in bp.</li>
		<li>Calculate molarity of each library fraction (Nanomolar concentration of DNA) using the following formula: Nanomolar concentration of DNA = [(ng/1000)/(bp*660)]*10^9 where ng is the concentration expressed in ng/&#956;l (as measured in step 9.1) , bp is the average size of the library fraction, and 660 is the weight of a standard double stranded DNA base pair. For example: with a library that is 326 bp in size, at a concentration of 14.2 ng/&#956;l, the Nanomolar concentration of DNA is 66 nM.</li>
	</ol>
	</li>
	<li>Prepare 10 &#181;l of a 2 nM solution of each library fraction. Dilute the library with DNase-free water.</li>
	<li>Pool the library fractions by combining 10 &#181;l of the 2 nM lower fraction with 10 &#181;l of the 2 nM higher fraction for each sample library prepared. This pool is the final ERRBS library for sequencing.</li>
	<li>Sequence the ERRBS library
	<ol>
		<li>Load the libraries with a goal of optimal densities of 600,000 &#8211; 650,000 clusters per millimeter squared (suggested loading concentration of 7-8 picomolar).</li>
		<li>Sequence the libraries using a minimum of single-read 51 cycles sequencing on a HiSeq 2500 sequencer in high output mode.</li>
		<li>Use a designated control lane (see discussion for rationale).</li>
	</ol>
	</li>
</ol>
11. Data Analysis
NOTE: Please refer to the Supplemental code files 1 and 2 for full details of commands and scripts recommended for use.
<ol>
	<li>Convert the base call files (.bcl files) to individual FASTQ files for each sample using the software provided by the sequencer&#8217;s manufacturer (example: CASAVA v1.8.2 for Illumina). If a sequencing core facility is used, this step may be provided and/or performed as an automated step on-board the sequencing computer.</li>
	<li>Filter the sequencing reads for reads that pass quality filtering (see Supplemental code file 1 for details). The CASAVA FASTQ file contains both reads that pass quality filtering and reads that do not pass quality filtering. Use a custom script that utilizes the &#60;is filtered&#62; element of the sequence identifier and keeps reads that have passed quality filtering.</li>
	<li>Trim adapter sequences from 3&#39; end of the sequence reads in the filtered FASTQ files using a software tool that can remove adapter sequences, such as Flexbar 37. The minimum overlap length (-ao) set to 6, the minimum read length to keep after adapter removal (--m) set to 21, the cutoff for the number of allowed mismatches (-at) set to 2, and defaults for all other parameters. As an alternate to Flexbar, any software which can remove adapters can be used in step 11.3 (examples: cutadapt38, trimmomatic39).</li>
	<li>Use Bismark41 to align the filtered, adapter trimmed sequence reads to the bisulfite converted reference human genome hg19 (whole genome alignment approach) and determine the methylation context for each cytosine. Bismark is a customized short read mapping tool that aligns bisulfite treated reads to a bisulfite converted genome (where all &#8216;C&#8217;s are converted to &#8216;T&#8217;s) and returns methylation calls for cytosines in CpG, CHG, and CHH context. Typically set the seed length (-l) to the read length for alignment accuracy and use defaults for all other parameters.</li>
	<li>Sort aligned reads first by chromosome, then start position, and finally strand, after alignment is complete.</li>
	<li>Use custom scripts (see Supplemental code file 2 for script commands) to iterate over the sorted methylation calls output by Bismark to compute the percent methylation scores of bisulfite converted Cytosines (T&#8217;s; representing unmethylated C&#8217;s) and non-converted C&#8217;s (representing methylated C&#8217;s) for each cytosine methylation context (CpG, CHG, CHH), retaining only the cytosines that have at least phred quality score of 20 and have at least 10x coverage. 
	NOTE: The outputs are a methylation score file for each cytosine context with columns corresponding to: the position, strand, coverage, percent cytosine, and percent thymine, and for CHG and CHH context, a column for the next base (the H).</li>
	<li>Use a custom script (see Supplemental code file 2 for script commands) to compute the conversion rates, from the methylation scores, and output the total of other C&#8217;s considered (CHG and CHH context), average conversion rate, and median conversion rate. Compute conversion rates for both strands independently as well as summarized over the entire library. Mean conversion rate is the fraction of C&#8217;s (converted and unconverted) in non-CpG context of the total number of C&#8217;s. Cytosines in CHG or CHH context are typically unmethylated and therefore present as thymine in the sequencing data.</li>
	<li>Generate a BAM file of the aligned reads using Bismark&#8217;s bismark2SAM_v5_xm.pl41 and SAMTOOLS42, and generate a wiggle format file using a custom script to convert the CpG methylation calls. The post-processed output can be converted to other formats, such as a bedgraph, and viewed in a genome browser such as the UCSC Genome Browser43 or IGV44.</li>
</ol>

The authors have no conflicts of interest to disclose.

The protocol presented yields base-pair resolution data of cytosine methylation at biologically-relevant genomic regions. The protocol as written is optimized for 50 ng of starting material, however, it can be adapted to handle a range of input material (5 ng or more)26. This will require adjustments of some of the protocol steps as seen in Table 6. The ERRBS libraries are amenable to paired end sequencing and further genomic coverage can also be accomplished by sequencing reads longer than 51 cycles. Multiplexed sequencing will offer a lower cost protocol per sample, however, this will result in reduced coverage per CpG site represented in the data (Figure 5 and Table 8), and will not yield sufficient depth of coverage to perform analyses which require high coverage per CpG site (e.g. as described by Landan et al.33). Finally, this protocol (or any bisulfite-based protocol) cannot distinguish between methyl-cytosine and hydroxymethyl-cytosine46,47. However, the data generated can be integrated with other protocol results48,49 to delineate the different modifications, and other cytosine modifications recently reported50, should they be of interest.
High quality libraries will appear as shown in Figure 3A-C, and once pooled for sequencing yields a trace as shown in Figure 3G (red trace) representing equal molar contributions from both library fractions. Library preparation failure can result from any step during the procedure. If degraded DNA is processed it will result in libraries that are not enriched in MspI fragments and hence in low CpG coverage using the sequencing parameters described in this protocol. If an enzyme is non-functional or inadvertently excluded from one of the reactions, the protocol will not yield the expected library. If the ligation reaction is inefficient, adaptors are at a higher concentration than expected, and/or the primers concentration used is a limiting reagent for the final amplification steps, library failure can occur. Excess adapters (seen as a peaks at ~150 bp in bioanalyzer results; Figure 3D-F) in the library will also interfere with sequencing due to the indiscriminate clustering of both the library and excess adapters. While such a library may sequence apparently normally, a significant portion of the reads will be merely adapter sequences. If excess adapters are observed in a library, it is best to repeat the library preparation if material is available using optimal input material to adapter quantity ratios. Finally, to ensure efficient PCR amplification of the libraries, the lower and higher library fractions are maintained as separate samples throughout the bisulfite conversion and PCR enrichment steps. Failure to do so yields differential efficiency of amplification during the PCR reaction of higher and lower fractions (as seen in Figure 3G blue trace) and the potential for unequal representation of the respective genomic loci covered in each library fraction during sequencing. The user may opt to include a quantitative PCR step immediately after the bisulfite conversion for further titration of optimal PCR cycles needed to amplify the libraries being generated.
ERRBS library preparation protocol has several key steps in which specific reagents are recommended. At the end-repair step, the use of a four-nucleotide dNTP mix allows for end-repair of any products not containing the CG overhang, such as those resulting from MspI enzymatic star activity and sheared DNA fragments present in the original DNA sample. This results in improved CpG representation in the results. At the ligation step it is critical to use a high concentration ligase (2,000,000 units/ml) and methylated adapters to ensure that the ligation reaction is efficient and that the bisulfite conversion does not influence the adapter sequences essential for accurate data alignment. At the PCR step, using a polymerase capable of amplifying bisulfite-treated GC-rich DNA fragments is necessary for high specificity. Finally, to ensure elimination of excess adapters and primers, SPRI bead purification (for example: Agencourt AMPure XP) is recommended rather than column based assays for ligation and PCR product isolations.
In order to generate high quality data, it is important to ensure efficient bisulfite conversion. The control presented offers the user the ability to determine conversion efficiency prior to sequencing. As an alternative, a non-human DNA such as lambda DNA can be used as an internal control (spike-in). Due to the differences in species, this type of a control can be directly included in downstream sequencing (e.g. as used by Yu, et al.34). However, if the spike-in is utilized, it cannot be used to determine conversion efficiency prior to library sequencing unless uniquely amplified and independently sequenced prior to library sequencing. The conversion rates determined are based on the methylation status at non-CpG sites. This may not be appropriate for use in the context of high cytosine methylation in non-CpG context (for example embryonic stem cells) and parallel samples or other means of assessing for conversion efficiency can be utilized for this purpose.
There are a few caveats to address that are unique to the sequencing of ERRBS libraries. The first three bases of the library fractions sequenced are nearly uniformly non-random due to the MspI recognition cut site (C^CGG; see Figure 4B, C). This results in the potential for significant data loss due to low quality reads resulting from poor cluster localization in spite of apparent high cluster density during sequencing. To overcome this barrier, include a high complexity library in an independent lane (PhiX control or other library type) as a dedicated control lane. High complexity libraries have ends containing a balanced representation of A, C, T and G in the first four bases sequenced. Suitable control lanes include libraries such as RNA-seq, ChIP-seq, whole genome sequencing, or a control offered by the sequencing machine manufacturer (e.g. PhiX Control v3). When designated as a control lane for the respective sequencing run, it can serve as the basis for the matrix generation which is utilized during the first four bases of sequencing to detect cluster positions. The higher quality reads captured will raise the mean coverage per CpG site by 5.2 (n = 4). Alternatively, this technical difficulty can also be overcome using a dark sequencing approach as previously described23. Other sequencing criteria follow standard operating procedures per manufacturer&#8217;s protocols. Finally, the coverage per CpG chosen for data analysis will be guided by the user and in part by the biological questions of interest. 10x coverage threshold affords a high coverage analysis approach, however this threshold can be lowered should that be of interest.
A full discussion of ERRBS data analysis is beyond the scope of this article, however, differentially methylated cytosines and regions can be determined using open source tools 31,51-53. Additional analysis considerations and approaches have been well-described54,55, and the reader is encouraged to search the literature for tools most appropriate to the analysis planned.
Compared to other published methods, ERRBS offers a four-day protocol which when performed as described yields high rates of reproducibility. It has been validated compared to the gold standard MassARRAY EpiTYPER26, is cost-effective for high coverage data, and is adaptable for various input material amounts (favorable for clinical sample processing and other cell types of low frequency) and sequencing approaches. It offers base-pair resolution at biologically relevant loci and can be used in integrative analyses with other techniques profiling genome-wide transcription factor binding, chromatin remodeling, epigenetic marks and other cytosine modifications of interest. ERRBS data use in such studies can contribute to a comprehensive molecular approach and allow for high dimensional analyses in the study of biological models and human disease.

DNA methylation at cytosine (5-methylcytosine) is an epigenetic mark critical in mammalian cells for a variety of biological processes, including but not limited to imprinting, X chromosome inactivation, development, and regulation of gene expression1-8. The study of DNA methylation patterns in malignant and other disorders has determined disease specific patterns and contributed to the understanding of disease pathogenesis and potential biomarker discoveries9-17.&#160;There are many protocols that interrogate the epigenome for DNA methylation status. These can be divided into affinity-based, restriction enzyme-based, and bisulfite conversion-based assays that utilize microarray or sequencing platforms downstream. Furthermore, there are a few protocols that bridge these general categories including, but not limited to, Combined Bisulfite Restriction Analysis18 and Reduced Representation Bisulfite Sequencing (RRBS19).
RRBS was originally described by Meissner et al.19,20. The protocol introduced a step to enrich GC-rich genomic regions followed by bisulfite sequencing, which resulted in quantitative base-pair resolution data that is cost effective21,22. The GC-rich regions are targeted by the MspI (C^CGG) restriction enzyme, and cytosine methylation is resolved by bisulfite conversion of cytosines (deamination of unmodified cytosines to uracil), followed by polymerase chain reaction (PCR) amplification. RRBS covered the majority of gene promoters and CpG islands in a fraction of the sequencing required for a whole genome; however RRBS had limited coverage of CpG shores and other intergenic regions of biological relevance. Several groups have published updated RRBS protocols since the original report that improve upon the methodology and resultant coverage of these genomic regions23-25. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) includes library preparation modifications and an alternate data alignment approach26 when compared to RRBS. ERRBS resulted in a higher number of CpGs represented in the data generated and increased coverage of all genomic regions interrogated26. This method has been used to resolve DNA methylation patterns in human patient and other animal specimens26-30.
The ERRBS protocol described offers details on all steps needed for completion and data was generated using representative human DNA (samples were obtained from previously reported, de-identified patient samples31, and a CD34+ bone marrow sample from a normal human donor). The protocol includes an automated size selection process, which reduces the processing time per sample and allows for increased accuracy in library size selection. The protocol combines a series of established molecular biology techniques. High molecular weight DNA is digested with a methylation-insensitive restriction enzyme (MspI) followed by end-repair, A-tailing, and ligation of methylated adapters. Size selection of the GC-rich fragments is followed by bisulfite conversion and PCR amplification prior to sequencing. Bisulfite conversion has been previously described32 and detailed review of data analysis and applications is beyond the scope of this paper, however recommendations and references are included for the readers&#8217; use. The protocol can be performed over four days and is amenable to small input (50 ng or less) material amounts. The protocol as described yields data with high coverage per CpG site sufficient not only for differential methylation site and region determinations but also for epigenetic polymorphism detection as described by Landan, et al.33.

<table><tbody><tr><td>MspI</td><td>New England Biolabs</td><td>R0106M</td><td>100,000 units/ml</td></tr><tr><td>NEBuffer 2</td><td>New England Biolabs</td><td>B7002S</td><td>Reaction buffer for MspI enzyme; protocol step 1.2</td></tr><tr><td>Phenol solution</td><td>Sigma-Aldrich</td><td>P4557</td><td>Equilibrated with 10&amp;nbsp;mM Tris HCl, pH&amp;nbsp;8.0; see safety and handling instructions at http://www.sigmaaldrich.com/catalog/product/sigma/p4557</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>C2432</td><td>See safety and handling instructions at http://www.sigmaaldrich.com/catalog/product/sial/c2432</td></tr><tr><td>Glycogen</td><td>Sigma-Aldrich</td><td>G1767</td><td>19-22 mg/ml</td></tr><tr><td>NaOAc</td><td>Sigma-Aldrich</td><td>S7899</td><td>3 M, pH 5.2</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>200 proof, for molecular biology</td></tr><tr><td>Buffer EB</td><td>Qiagen</td><td>19086</td><td>10 mM Tris-Cl, pH 8.5</td></tr><tr><td>tris(hydroxymethyl)aminomethane (Tris)</td><td>Sigma-Aldrich</td><td>T1503</td><td>prepare a 1 M, pH 8.5 solution</td></tr><tr><td>Tris- Ethylenediaminetetraacetic acid (TE)</td><td>Sigma-Aldrich</td><td>T9285</td><td>Dilute to 1x buffer solution per manufacturer&#039;s recommendations</td></tr><tr><td>T4 DNA Ligase Reaction Buffer</td><td>New England Biolabs</td><td>B0202S</td><td>10x concentration</td></tr><tr><td>Deoxynucleotide triphosphate (dNTP) Solution Mix</td><td>New England Biolabs</td><td>N0447L</td><td>10 mM each nucleotide</td></tr><tr><td>T4 DNA Polymerase</td><td>New England Biolabs</td><td>M0203L</td><td>3,000 units/ml</td></tr><tr><td>DNA Polymerase I, Large (Klenow) Fragment</td><td>New England Biolabs</td><td>M0210L</td><td>5,000 units/ml</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England Biolabs</td><td>M0201L</td><td>10,000 units/ml</td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td>Used for DNA product purification in protocol step 2.3</td></tr><tr><td>2&#039;-deoxyadenosine 5&#039;-triphosphate (dATP)</td><td>Promega</td><td>U1201</td><td>100 mM</td></tr><tr><td>Klenow Fragment (3&#039;&amp;rarr;5&#039; exo-)</td><td>New England Biolabs</td><td>M0212L</td><td>5,000 units/ml</td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td>Used for DNA product purification in protocol step 3.3</td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202M</td><td>2,000,000 units/ml</td></tr><tr><td>Methylation Adapter Oligo Kit</td><td>Illumina</td><td>ME-100-0010</td><td></td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>Used in protocol sections that implement magnetic bead purification steps (steps 4.3 and 8.2). Equilibrate to room temperature before use.</td></tr><tr><td>Pippin Prep Gel Cassettes, 2% Agarose, dye-free</td><td>Sage Science</td><td>CDF2010</td><td>with internal standards</td></tr><tr><td>Certified Low Range Ultra Agarose</td><td>Bio-Rad</td><td>161-3106</td><td></td></tr><tr><td>Tris-Borate-EDTA (TBE) buffer</td><td>Sigma-Aldrich</td><td>T4415</td><td></td></tr><tr><td>Ethidium bromide solution</td><td>Sigma-Aldrich</td><td>E1510</td><td>10 mg/ml</td></tr><tr><td>50 bp DNA Ladder</td><td>NEB</td><td>N3236S</td><td></td></tr><tr><td>100 bp DNA Ladder</td><td>NEB</td><td>N3231S</td><td></td></tr><tr><td>Gel Loading Dye, Orange (6x)</td><td>NEB</td><td>B7022S</td><td></td></tr><tr><td>Scalpel&amp;nbsp;Blade&amp;nbsp;No.&amp;nbsp;11</td><td>Fisher Scientific</td><td>3120030</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>EZ DNA Methylation Kit</td><td>Zymo Research</td><td>D5001</td><td>Used in protocol step 6.2</td></tr><tr><td>EZ DNA Methylation-Lightning Kit</td><td>Zymo Research</td><td>D5030</td><td>Alternative for step 6.2</td></tr><tr><td>Universal Methylated Human DNA Standard</td><td>Zymo Research</td><td>D5011</td><td>Used as bisulfite conversion control</td></tr><tr><td>FastStart High Fidelity PCR System</td><td>Roche</td><td>03553426001</td><td></td></tr><tr><td>Qubit dsDNA High Sensitivity Assay Kit</td><td>Life Technologies</td><td>Q32854</td><td>A fluorescence-based DNA quantitation assay; used in protocol steps 1.1, 9.1 and 10.1</td></tr><tr><td>DynaMag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>PhiX Control v3</td><td>Illumina</td><td>FC-110-3001</td><td></td></tr><tr><td>HiSeq 2500</td><td>Illumina</td><td></td><td></td></tr><tr><td>Pippin Prep</td><td>Sage Science</td><td></td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Life Technologies</td><td>Q32872</td><td></td></tr><tr><td>TruSeq SR Cluster Kit v3-cBot-HS</td><td>Illumina</td><td>GD-401-3001</td><td></td></tr><tr><td>TruSeq SBS Kit v3-HS</td><td>Illumina</td><td>FC-401-3002</td><td></td></tr><tr><td>TruSeq RNA Sample prep</td><td>Illumina</td><td>RS-122-2001</td><td>Barcoded adapters used for multiplexing libraries; See Supplemental file for multiplexing protocol.</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Vortex Mixer</td><td></td><td></td><td></td></tr><tr><td>Dry Block Heater</td><td></td><td></td><td></td></tr><tr><td>Thermal Cycler</td><td></td><td></td><td></td></tr><tr><td>Water Bath</td><td></td><td></td><td></td></tr><tr><td>Gel electrophoresis system</td><td></td><td></td><td></td></tr><tr><td>Electrophoresis power supply</td><td></td><td></td><td></td></tr><tr><td>Gel doc</td><td></td><td></td><td></td></tr><tr><td>UV or blue light transilluminator</td><td></td><td></td><td></td></tr></tbody></table>

enhanced reduced representation bisulfite sequencing for assessment of dna methylation at base pair resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Watch this Scientific Journal Video about Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution at JoVE.com

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

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

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25.5K Views.  Weill Cornell Medical College. Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

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

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 ...

Genetics

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 ...

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.

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 ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Biochemistry

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neuroscience

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid storage processes contributing to the development of the disease. We demonstrate a complete protocol from selection to epigenetic data analysis of patients with and without obesity. All steps from the protocol were tested and validated in a pilot study. 32 women participated in the study, in which 15 individuals were classified with obesity according to Body Mass Index (BMI) (45.1 &#177; 5.4 kg/m2); and 17 individuals were classified without obesity according to BMI (22.6 &#177; 1.8 kg/m2). In the group with obesity, 564 CpG sites related to fat mass were identified by linear regression analysis. The CpG sites were in the promoter regions. The differential analysis found 470 CpGs hypomethylated and 94 hypermethylated sites in individuals with obesity. The most hypomethylated enriched pathwayswere in the RUNX, WNT signaling, and response to hypoxia. The hypermethylated pathways were related to insulin secretion, glucagon signaling, and Ca2+. We conclude that the protocol effectively identified DNA methylation patterns and trait-related DNA methylation. These patterns could be associated with altered gene expression, affecting adipogenesis and lipid storage. Our results confirmed that an obesogenic lifestyle could promote epigenetic changes in human DNA.

The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

DNA Methylation Analysis

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the methylation of clusters of methylation sites, called &#8220;CpG islands&#8221;, near promoters and other gene regulatory elements may contribute to the stable silencing of genes, for example, during epigenetic processes such as genomic imprinting and X-chromosome inactivation. At the same time, aberrant CpG methylation has been shown to be associated with cancer.In this video, the biological functions and mechanisms of DNA methylation will be presented, along with various techniques used to identify methylation sites in the genome. We will then examine the steps of bisulfite analysis, one of the most commonly used methods for detecting DNA methylation, as well as several applications of this technique.

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the ...

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Bisulfite Conversion of Genomic DNA: A Method to Study DNA Methylation in Genomic DNA Samples from Gastrointestinal Cancer Tissue

Source: Bonetto, A. et al. <a href="https://www.jove.com/video/54893" target="_blank">The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure of bisulfite modification of genomic DNA from gastrointestinal cancer samples. The technique is beneficial for investigating the relationship between genes&#8217; methylation patterns and establishing their contribution to carcinogenesis.

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

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