other genomic arrays: methylation, chip on chip …
DESCRIPTION
Other genomic arrays: Methylation, chIP on chip …. U B io Training Courses. SNP-arrays and copy number. Genotyping arrays can detect CNVs. Copy numbers from SNP arrays. Illumina SNP arrays: Hybridization to Universal IllumiCode TM. Illumina uses the same technology for methylation arrays - PowerPoint PPT PresentationTRANSCRIPT
Other genomic Other genomic arrays: Methylation, arrays: Methylation,
chIP on chipchIP on chip……UUBBioio Training Courses Training Courses
SNP-arrays and copy SNP-arrays and copy numbernumber
Genotyping arrays can detect CNVsGenotyping arrays can detect CNVs
Copy numbers from SNP arrays
Illumina SNP arrays: Hybridization to Universal IllumiCodeTM
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Illumina uses the same technology for methylation arrays(bi-sulfited nucleotides are like SNPs)
Calculation of aCGH-like ratios
Median R CEPH Individual R cell line(NCI60)
Methylation arraysMethylation arrays
BeadArrayso Until 12 samples per chip.o 27,578 CpG loci, >14.000 genes o 2 beads per locus (methylated/no methylated)o Random distribution (50 mer)o Input: Bisulphyted DNA o Includes probes for the promoter regions of miRNA 110 genes
METHYLATION MICROARRAYSIn
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Illumina Golden Gate Assay
• Until 147,456 DNA methylation measures simultaneously. • Resolution: 1 CpG•Until 96 samples simultaneously• GoldenGate Methylation Cancer Panel I 1,505 CpG loci selected from 807 gene• Allows custom designs
METHYLATION MICROARRAYS
SOFTWARE
Lumi package (Import, background correction, normalization)Beadarray package (Import, QC)Methylumi (Import, QC ,normalization, differential meth.)
Bead Studio Genome StudioMethylation modulehttp://www.illumina.com/pages.ilmn?ID=196
METHYLATION MICROARRAYS
DIFFERENTIAL METHYLATIONMETHYLATION MICROARRAYS
Bead Studio Genome StudioMethylation modulehttp://www.illumina.com/pages.ilmn?ID=196
Beta values:
β= Imethylated/Imethylated+Ino_methylated
β1 0
Hypermethylated Hypomethylated
0.7 0.3
NORMALIZATIONMETHYLATION MICROARRAYS
Methylumi normalization
1) Calculate medians for Cy3 and Cy5 at high an low betas2) Cy5 medians adjusted to Cy3 channel (dye bias)3) Recalculate betas with new intensities
DIFFERENTIAL METHYLATIONMETHYLATION MICROARRAYS
Wilcoxon rank-test (UBio)Limma (Pomelo)Permutations (Pomelo)
βs
FDR<0.05 Median βs class AMedian βs class B+
Differentially methylated genes
ChIP on chipChIP on chip
ChIP on Chip
We thank Chris Glass lab, UCSD, for the original slide
Discover protein/DNA interactions!!
ChIP on Chip
ChIP on Chip software
Chip Analytics
WORKFLOW I.
1. Pre-normalization.Background substraction: Foreground – backgroundDefault: Median blank substraction Each channel – median negative controls
2. Normalization (dye-byas and interarray normalization) Default : Median dye-byas, median interarray. Recommended: Loess
ChIP on Chip softwareChip Analytics
WORKFLOW II.
3. Error modelling To identify which probes are most representative of binding events: P(X)=P-value of a single probe matching event P(Xneighb)= Positive signals in a probe should be corroborated by the signals of probes that are its genomic neighbors,
provided they are close enough P(Xneighb) follows a Gaussian distribution Both the P(X) and the P(Xneighb) values of a probe need to satisfy significance thresholds in order for a probe to be considered as representing a binding event
ChIP on Chip softwareChip Analytics
WORKFLOW III.4. Segment identification (clusters of enriched probes)
5. Gene identification-Segment, Gene or Probe report (Gene or probe ID, Chr, Start, End, p(X)…)
bp
CoCashttp://www.ciml.univ-mrs.fr/software/cocas/index.html
Agilent platform
NormalizationQC ReportGenome VisualizationPeak Finder
Benoukraf et al. Bioinformatics 2009.
UBio training courses: See “Course on Introduction to Sequence Analysis”
Weeder: Motif discovery in sequences from co-regulated genes (single specie).
WeederH: Motif discovery in sequences from homologous genes.
Pscan: Motif discovery in sequences from co-regulated genes (JASPAR,TRANSFAC matrices)
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