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Friday, February 6, 2009

Kevin McKernan, Applied Biosystems - "The whole Methylome: Sequencing a Bisulfite Converted Genome using SOLiD"

Background on methylation. It's not rare, but it is clustered. This is begging for enrichment. You can use Restriction Enzymes. Uses Mate Pairs to set this up. People can also use MeDIP and a new third method: methyl binding protein from Invitrogen. (Seems to be more sensitive.)

MeDIP doesn't grab CpG, tho... just leaving single stranded DNA, which is a pain for making libraries. Using only 90ng. There is a slight bias on adaptors, tho. Not yet optimized. If they're bisulfite converting, it has issues (protecting adaptors, requires densely methylated DNA, etc). They get poor alignment because methylation areas tend to be repetitive. Stay tuned, though, for more developments

MethylMiner workflow: Sheer genomic DNA, and put adaptors on it, and then biotin bind methyls? You can titrate methyl fractions off the solid support, so you can then sequence and know how many methyls you'll have. Thus, mapping becomes much easier, and sensitivity is better.

When you start getting up to 10-methyls in a 50mer, bisulfite treating + mapping is a problem. It's also worth mentioning that methylation is not binary when you have a large cell population.

The methyl miner system was tested on A. thaliana, SOLiD fragments generated... good results obtained, and salt titration seems to have worked well, and mapping reads show that you get the right number (aprox) of methyl Cs. - but mapping is easy, since you don't need to bisulfite.

Showed examples where some of genes are missed by MeDIP, but found by MethylMiner.

(Interesting note, even though they only have 3 bases after conversion (generally), it's still 4 colour.)

Do you still get the same number of reads on both strands? Yes...

Apparently methylation is easier to align in colourspace. [Not sure I caught why.] Doing 50mers with 2MM. (Seems to keep % align-able in colourspace, but bisulfite treated base space libraries can only be aligned about 2/3rds as well.

When bisulfite converted, 5mCTP will appear as a SNP in alignment. To approach that, you can do fractionation in MethylMiner kit, which gives you a more rational approach to alignments.

You can also make a LMP library, and then treat with 5mCTP when extending, so you get two tags, then separate tags – (they keep a barcode) and then pass over methylminer kit... etc etc... barcoded mapping to detect methyl C's better.

Also have a method in which you do something the same way, but ligate hairpins on the ends... then put on adaptors, and then sequence the ends, to get mirror imaged mate pairs. (Stay tuned for this too.)

There are many tools to do Methylation mapping: colourspace, lab kits and techniques.



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