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![Page 1: DNA Methylation mapping Reduced Representation Bisulfite Sequencing (RRBS) 1. Dynamic DNA methylation across diverse human cell lines and tissues; Katherine.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e705503460f94b6db90/html5/thumbnails/1.jpg)
DNA Methylation mapping
Reduced Representation Bisulfite Sequencing(RRBS)
1. Dynamic DNA methylation across diverse human cell lines and tissues;Katherine E. Varley et al, Genome Res. 2013 23: 555-567 originally published online January 16,
2013.
2. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling;
Hongcang Gu1, Zachary D Smith1–3, Christoph Bock1–4, Patrick Boyle1, Andreas Gnirke1 & Alexander Meissner1–3; Nature Protocols, VOL.6 NO.4 | 2011.
3. SOLiD™ Bisulfite-Converted Fragment Library Preparation Protocol.
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Bisulfite conversion reaction
1. cytosine → cytosine-6- sulfonate; 2. cytosine-6-sulfonate → uracil-6-sulfonate; 3. uracil-6-sulfonate → uracil. 5-methylcytosines is non-reactive to bisulfite ions - therefore remains unchanged.
Bisulfite Sequencing
![Page 3: DNA Methylation mapping Reduced Representation Bisulfite Sequencing (RRBS) 1. Dynamic DNA methylation across diverse human cell lines and tissues; Katherine.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e705503460f94b6db90/html5/thumbnails/3.jpg)
Advantages: - Quantitative method directly measures C/T percentage as all BS approaches.
- CpG enrichment: RRBS uniquely uses a specific restriction enzyme as Msp1 that cut in CpG rich areas.
- Decreases the amount of sequencing required as well as the cost relative to WGBS.
- Only a low sample concentration, between 10-300 ng, is required for accurate data analysis.
- Formalin-fixed and paraffin-embedded inputs can also be used.
- Allows for the sequencing of methylated areas that are unable to be properly profiled using conventional bisulfite sequencing techniques.
- MethylC-seq and MeDip-seq have a greater genome-wide coverage of CpGs compared to RRBS, but RRBS has a greater coverage on CpG islands is consider quantitative and has single CpG resolution.
- The data obtained on RRBS and the Illumina Infinium methylation are highly comparable as both use an absolute measurement of DNA.
Limitations:
- Restriction Enzyme limitations: MspI digestion covers the majority, but not all the CG regions in the genome. Most CpG island and promoters are covered.
- Non-proofreading polymerase must be used as a proof-reading enzyme would stop at uracil residues found in the ssDNA template.
- Complete denaturation and conversion is a must as in all BS methods.
![Page 4: DNA Methylation mapping Reduced Representation Bisulfite Sequencing (RRBS) 1. Dynamic DNA methylation across diverse human cell lines and tissues; Katherine.](https://reader036.fdocuments.in/reader036/viewer/2022062718/56649e705503460f94b6db90/html5/thumbnails/4.jpg)
Flowchart of RRBS library construction for Solid
• Genomic DNA isolation
• Msp1 digestion (C/CGG): CpG islands enrichment
• Filling in and A-taililing: to facilitate ligation of adapters
• Adapter ligation: use 10x less than original protocol
• Nick-translation: to fill the ligation gap and to replace C with metC
• Gel sizing: select for fragments 40 -220bp (plus the length of the adapters); carrier DNA added if small amount od DNA
• Bisulfite conversion: Zymo Research gold kit; 50o for 60min x 16 cycles
• PCR amplification: test PCR to select number of cycles; amplify the whole library• Sequencing: MGL
• Analysis: MGL
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Msp digestion and ligation
MSP digestion of genomic DNA:Msp repeats are detected
The top sequence of P1 is the one that is ordered with methylated Cs
After ligation Nick Translation : - to replace C with 5-metC - covalent bond between adapter and A on the insert.
For small amounts of DNA: - 10 times less adapters - ligation 20 hours at 16o.
From Hongcang Gu et al, ; Nature Protocols, VOL.6 NO.4 ,2011.
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Size selection
Size selection on 2% agarose gel:Cut fragments 160 -350bp
Add sonicated dephosphorylated e. coli DNA as carrier
Bisulfite conversion:
Zymo Research gold kit; 95 for 30”o, 50o for 60min x 16 cycles
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Test PCR
Cycles: 12 15 18 21 24
To minimize the bias that might be introduced by PCR
• For final library amplification use the lowest number • that generates enough PCR products
• 200 ml reaction aliquote in 8 wells of 96 well plate.• After PCR combine all reactions and purify the library.
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Profile of the libraries analyzed on Transgenomics HPLC
Libraries
MW Markers
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Profile of the libraries run on Bioanalyzer