MBD-Chip.

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Presentation transcript:

MBD-Chip

Workflow: Identify methylated regions

Workflow: differential methylation

Oyster DNA Tiling Array 3 x 720k design Each feature ~100bp x 720k feature =72Mb for the microarray 720k 720k 720k Genome is about 600Mb so needed to reduce it..

methylation changes in response to EE2 Day 7 Control Pool: 3 females EE2 Pool: 3 females Pooled MBD (n=3) Input EE2: red cntrl: green EE2: green cntrl: red EE2: green cntrl: red

methylation changes in response to EE2 Day 7 Control Pool: 3 females EE2 Pool: 3 females Pooled MBD (n=3) Input EE2: red cntrl: green EE2: green cntrl: red EE2: green cntrl: red

Analysis: Microarray people at Fred Hutch were not a big fan of the assay design They think the MBD v. input is more robust Note that this MBD v. MBD approach may introduce more bias (from random fragmenting; PCR) Suggest using the classic design for future assays

Analysis: Bioconductor package, "Ringo” used for analysis * Read NimbleGen pair files into R * Remove control probes * Tukey biweight mean center log2 ratios within each array (all ratios were formulated such that the estrogen treated sample is always in the numerator) * Smooth log2 ratio values by computing a running median, using a window size of 1000 bases * Within each array, use the log2 ratio distribution to calculate the threshold to be used for peak detection, then detect peaks such that at least 3 adjacent probes must be above threshold. * Identify the overlap between A02 and A03, excluding A01 using the Bioconductor package "ChIPpeakAnno."

Analysis: Region 1 Region 2 Region 3 EE2 v C C v EE2 input v input

Results: 96 regions that were hypermethylated in EE2 90 are in genes 52 of these cross exon/intron boundary another 32 are in introns 6 are just in exons 287 regions that were hypomethylated in EE2 256 are in genes 138 cross exon/intron boundary 114 are in introns only and 4 are just in exons

Functional enrichment analysis hypermethylated in EE2 treatment

Functional enrichment analysis hypomethylated in EE2 treatment

Next Steps Validation: Issues: Use targeted bisulfite sequencing to validate differentially methylated regions Issues: How to pick targets for validation? (most interesting, most different?) Tissue availability Can only validate on 2 of the 3 individuals for the EE2 sample Additional arrays? (exp date, reusing array)