MBD-Chip. Workflow: Identify methylated regions Workflow: differential methylation.

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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

Oyster DNA Tiling Array Reducing the genome to most ‘informative’ parts (100Mb) 28k oyster genes (227Mb) 13k with evalue <1e-20 (total: 138Mb) 12k on scaffolds that cover 90% of genome (132kb) Generate intervals that include the gene and 2kb upstream (promoter) Randomly select 6500 (of the ~12k) intervals =100Mb

QC: – Dye swap – Input v. Input (CNV) Validation – MBD-PCR – pyrosequencing Example..

Guerrero-Bosagna et al 2010 (vinclozolin treated sperm F3 gen) Experiment 1 Treated v Control rep1 Pooled DNA treatment* R:G G:R Treated v Control rep2 R:G G:R Pooled DNA control*

Guerrero-Bosagna et al 2010 (vinclozolin treated sperm F3 gen) Experiment 1 Experiment 2 Treated v Control rep1 R:G G:R Treated v Control rep2 R:G G:R Treated v Control rep1 R:G G:R Treated v Control rep2 R:G G:R Pooled DNA treatment* Pooled DNA control*

Skinner paper

Chip #1: methylation changes after heat shock

Animal A (MBD) Pre: red Post: green Animal B (MBD) Pre: green Post: red Animal B (input) Pre: green Post: red

Chip #2: methylation changes in response to EE2 Day 7 Control Pool: 4 females (across 3 tanks) EE2 Pool: 8 females (select 4 across 3 tanks) EE2: red cntrl: green EE2: green cntrl: red EE2: green cntrl: red Pooled MBD (n=4) Input