DNA Methylation: Methods and Protocols
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When analyzed by conventional methodologies this modularity is destroyed. Our lab has modified a previously described footprinting strategy 9,10 , which now allows us to study the chromatin structure of individual molecules. Sssi , followed by genomic bisulfite sequencing of individual progeny DNA molecules This gives single molecule resolution over the promoter and allows for the physical linkage between binding sites on individual promoter molecules to be maintained.
Our lab has successfully used this method to study the difference in nucleosomal positioning in the p16 promoters in two human cell lines 11 , to identify transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress 12 , and to study the changes in nucleosome occupancy silencing of the three transcription start sites in the bidirectional MLH1 promoter CpG island in cancer cells Bisulfite sequencing allows for precise identification of methylated cytosines within DNA Frommer et al.
This method is based on different rate of chemical conversion of methylated and non-methylated cytosines to uracil where non-methylated cytosines are converted efficiently while methylated cytosines remain non-reactive. This method was further developed by embedding analyzed DNA into an agarose bead Olek et al. The protocol presented here was further optimized for bisulfite sequencing of small samples where the starting material was a small number of cells Fedoriw et al.
The smallest amount of material from which several unique clones were recovered was 25 oocytes, which corresponds to DNA molecules in the initial material Svoboda et al. This protocol can be also used for analyzing up to ng purified genomic DNA in one sample. Email feedback to: This email address is being protected from spambots. Sequencing of sodium-bisulfite modified genomic DNA originally introduced by M. Frommer Frommer et al. In the meantime several commercial kits are available for this procedure which work reasonably well when starting with large amounts of DNA. Here we describe a protocol for small numbers of cells and little DNA which requires some specific handling.
The protocol is based on a strategy originally introduced by our lab Hajkova et al. This physical trapping helps to avoid DNA loss during the various incubation steps while maintaining a good bisulphite conversion rate. We will introduce two alternative procedures to perform bisulphite treatment of agarose embedded small DNA aliquots or cells and guide through some generally critical points in the bisulphite reaction and primer design.
We also include tips for the process of data processing after sequencing which is facilitated by a new and very useful software tool BiQ Analyzer. This tool allows rapid and reproducible processing and evaluation of bisulphite sequencing data. It generates standardized table output formats allowing direct database integration.
Saarland University, FR 8. View table of content. DNA Methylation. Every sample was converted in duplicate, and after conversion, the duplicates were pooled to provide sufficient material for subsequent testing. Based on the first analysis, the Epitect Bisulfite kit Qiagen, was selected as the least fragmenting kit.
Subsequently, this kit was used to perform the protocol identically as described above on the five PBMC samples, except for the conversion time or temperature which were modified S7 and S8 Tables. To evaluate the amount of intact DNA copies of increasing length after bisulfite treatment, the converted DNA was amplified using qPCR with 6 different primer pairs, resulting in amplicons of increasing length 88— bp. Every qPCR was performed in triplicate on all five bisulfite treated samples for each kit.
DNA methylation —Epigenomics —Systems Biology —BIO-PROTOCOL
The geometric mean of the Cq values among all replicates, which corresponds with the amount of intact fragments, was used to rank all the kits. S5 and S6 Figs [ 52 ]. To eliminate the potential inhibitory effect of elution buffers or impurities on the qPCR reaction, fragmentation was also analyzed using dPCR. Similar as in the qPCR, several CF primers resulting in amplicons of different lengths 88 to bp , were used to investigate the difference in fragmentation between the twelve kits.
Each sample was analyzed in duplicate. Moreover, 8. Based on the DNA concentration absolute amount of intact copies per ng bisulfite treated input DNA , a ranking of the 12 kits was made for each primer pair. The conversion efficiency was calculated as the amount of converted to non-converted non-CpG Cs, which are usually not methylated in human PBMCs [ 43 — 48 ].
Almost all these Cs should thus be converted during bisulfite treatment, providing us with a method to calculate the conversion efficiency. Based on the results from the DNA recovery and the fragmentation analysis, we selected the eight least fragmenting kits for subsequent sequencing. The second primer pair can be used to assess the overall conversion efficiency, while analysis with CCP3 can potentially result in an overestimation of this efficiency. To assess the variability between the different donors, converted DNA samples of a second donor donor 3 obtained by three of the kits were also analyzed.
The sequencing reads were aligned using the Bismark package version 0. Two aliquots of all five DNA samples were treated two independent times, yielding in ten bisulfite treated samples, and subsequently, the duplicate samples were pooled. First the average of the three technical replicates was calculated for all five samples. The data given is the geometric mean of the average values from the five donor samples.
Genomic DNA is the measurement of untreated DNA, which is only conducted for the cytosine free primers since they have the same efficiency before and after treatment. The different kits are ranked by the Cq values for every primer pair Rank in the table. Subsequently, the median of these rankings is calculated to assess a final ranking. This is the ranking given in Table 1. First, the average of the two technical replicates was calculated and normalized to the DNA input for all five samples. The data given is the geometric mean of the average values from all five donor samples.
The different kits are ranked by the amount of copies per ng bisulfite treated DNA for every primer pair Rank in the table. This is the final ranking given in Table 1. This dPCR measures the intact copies of specific lengths present in the samples. To obtain the averages given in the table, the average of the two technical replicates was calculated both before and after bisulfite treatment.
These averages were used to calculate the average loss per kit of all the five donor samples. Subsequently, the geometric means and standard deviations from these averages were calculated. The data is obtained by sequencing of the 8 kits that performed best in the previous fragmentation assessments. The different protocols followed a fixed time schedule as provided by the manual from the manufacturer.
Temperature protocol 3 is the same as the protocol provided by the manufacturer. The different protocols always followed the temperature scheme as provided by the manual from the manufacturer. Time protocol 3 is the same as the protocol provided by the manufacturer. In the end of the protocol, 2 elutions of the same sample were performed as recommended by the manufacturer to maximize DNA yield.
The data given is a single measurement of the donor used in these time and temperature experiments. Bioanalyzer upper plots and gel electrophoresis lower plots analysis of the different kits. The plots shown in this supplement show the electrophoresis data of the five donor samples. Upper panel: protocol changed in conversion temperature S7 Table.
Targeted DNA Methylation Analysis Methods
Lower panel: protocol changed in conversion time S8 Table. Fastq files of the sequencing data that were used for the conversion efficiency calculation. We would like to thank Johan Vandersmissen and Hendrik Van de Voorde for the provided help and efforts during the development of the assays and during the practical work. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract DNA methylation is one of the most important epigenetic modifications in the regulation of gene transcription.
Introduction DNA methylation 5-methylcytosine is an epigenetic modification that is typically associated with stable transcriptional silencing [ 1 — 4 ]. Download: PPT. Fig 1. Principle of bisulfite-mediated methylcytosine mC mapping. Evaluation of fragmentation Gel electrophoresis. Table 1. Overview over the kits and their main characteristics and performance results.
Fig 3. Digital PCR. Fig 4. Visual representation of the rankings of the dPCR experiments. Evaluation of conversion efficiency and inappropriate conversion Since correct estimation of the methylation in the samples is the main goal after bisulfite treatment, both appropriate conversion i. Evaluation of effect of conversion time and temperature To analyze the effect of increasing temperature and time of the bisulfite conversion on the fragmentation, the degree of fragmentation of the different kits was linked with the time and conversion temperature of each kit Fig 5.
Discussion The importance of DNA methylation in different biological processes and the need for an easy-to-use technology have resulted in a wide range of commercially available bisulfite conversion kits. Material and methods Human blood samples Blood samples from healthy individuals were purchased from the Belgian Red Cross. Bisulfite treatment First analysis—selection of the best kits. Second analysis—effect of time and temperature on fragmentation. Fragmentation Gel electrophoresis.
Conversion efficiency The conversion efficiency was calculated as the amount of converted to non-converted non-CpG Cs, which are usually not methylated in human PBMCs [ 43 — 48 ]. Supporting information. S1 Table. Concentration of the DNA samples before bisulfite treatment. S2 Table. S3 Table. Cq values and ranking of the qPCR experiments from the six used primer pairs. S4 Table. Results and ranking of the dPCR experiments from the three primer pairs that were used.
S5 Table. The percentages of overall DNA loss in the samples.
Table of contents
S6 Table. Conversion efficiencies and overall methylation percentage for the different kits. S7 Table. Conversion protocol for different temperatures for Epitect kit S8 Table. Conversion protocol for different time schedules for Epitect kit S9 Table. Concentration after elution with Epitect kit 10 with the different time and temperature protocols as depicted in S7 and S8 Tables.
S10 Table. S1 Fig. S2 Fig. Comparison of different recovery analyzes red: Qubit vs black: dPCR. S3 Fig. S4 Fig. S5 Fig. S6 Fig. S1 Dataset. Sequencing data used for the conversion efficiency. Acknowledgments We would like to thank Johan Vandersmissen and Hendrik Van de Voorde for the provided help and efforts during the development of the assays and during the practical work.
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