DNA Methylation mapping Reduced Representation Bisulfite Sequencing (RRBS) 1. Dynamic DNA...

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.

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

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.

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

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.

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

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.

Profile of the libraries analyzed on Transgenomics HPLC

Libraries

MW Markers

Profile of the libraries run on Bioanalyzer