Illumina 450k: A microarray for the study of DNA...
Transcript of Illumina 450k: A microarray for the study of DNA...
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Illumina 450k: A microarray for the study of DNAmethylation
Jean-Philippe Fortin
May 15, 2013
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Outline
DNA Methylation
Illumina Infinium 450k Human Methylation Assay
Probes Design
Statistical Challenges
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
![Page 3: Illumina 450k: A microarray for the study of DNA methylationlcolladotor.github.io/courses/misc/genomics/GWG_JPF.pdf · Illumina 450k: A microarray for the study of DNA ... After treating](https://reader033.fdocuments.net/reader033/viewer/2022051719/5a72c1cc7f8b9abb538de869/html5/thumbnails/3.jpg)
DNA methylation
DNA methylation: 5-methylcytosine residues found in CpGdinucleotides. One of the most studied epigenetics marks.
Epigenetics: study of gene expression variants caused bymechanisms that do not involve a change in the nucleotidesequence
11 http://www.invitrogen.com
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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DNA Methylation
2
2 http://missinglink.ucsf.edu/lm/genes-and-genomes/methylation.htmlJean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Bisulfite Conversion
33 http://www.diagenode.com/en/applications/bisulfite-conversion.php
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Bisulfite Conversion
After treating the DNA with bisulfite conversion, the analysis ofDNA methylation is reduced to an analysis of single nucleotidepolymorphisms (SNPs) for T’s and C’s:
C is found: original cytosine was methylated
T is found: original cytosine was non-methylated
- Can use Next Generation Sequencing or Microarrays
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Infinium HumanMethylation450 BeadChip Kit
A recent popular methylation array that allows theinterrogation of more than 485,000 methylation sites persample
Contains two chemistry technologies:
135,000 probes from Infinium I array350,000 probes from Infinium II.
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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44 Figure from http://en.wikipedia.org/wiki/Illumina-Methylation-Assay
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Probe Design
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Infinium I
Infinium I Probes:
One probe for the methylated locus
One probe for the non-methylated locus
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
![Page 11: Illumina 450k: A microarray for the study of DNA methylationlcolladotor.github.io/courses/misc/genomics/GWG_JPF.pdf · Illumina 450k: A microarray for the study of DNA ... After treating](https://reader033.fdocuments.net/reader033/viewer/2022051719/5a72c1cc7f8b9abb538de869/html5/thumbnails/11.jpg)
Unmethylated Locus
Infinium I Probes:
Hybridization of the unmethylated locus:
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Methylated Locus
Infinium I Probes:
Hybridization of the methylated locus:
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Design I
Infinium I Design
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Infinium II
Infinium II Probes:
Only one probe to interrogate both loci
The interrogated CpG site is at the end of the probe
The last base corresponding to the G is not included
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Unmethylated Locus
Infinium II Probes:
Hybridization of the unmethylated locus = RED
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Methylated Locus
Infinium II Probes:
Hybridization of the methylated locus = GREEN
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
![Page 17: Illumina 450k: A microarray for the study of DNA methylationlcolladotor.github.io/courses/misc/genomics/GWG_JPF.pdf · Illumina 450k: A microarray for the study of DNA ... After treating](https://reader033.fdocuments.net/reader033/viewer/2022051719/5a72c1cc7f8b9abb538de869/html5/thumbnails/17.jpg)
Design II
Infinium II Design
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Both designs
Infinium I + Infinium II
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Methylation measures
Beta-value:
β =M
U + M + 100
β = 0 : All cells are non-methylatedβ = 1 : All cells are methylated
M value:
Mvalue = log
(M
U
)
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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The Beta-value distributions are not the same for the two designs:
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Chip Configuration
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Background intensities
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Beta-value densities
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Statistical challenges
How to reduce technical variation? Background, dye bias andspatial effects
Presence of two types of probes
Statistical model for differential analysis
Blood mixture
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Background Effects
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Dye Bias
For Type I probes, always same color.
Not a problem if we normalize green and red probes separately.
For Type II probes
GREEN signal = methylationRED signal = no methylation
Dye bias can be a concern if inconsistent across samples
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Dye Bias
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
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Dye Bias
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation
![Page 29: Illumina 450k: A microarray for the study of DNA methylationlcolladotor.github.io/courses/misc/genomics/GWG_JPF.pdf · Illumina 450k: A microarray for the study of DNA ... After treating](https://reader033.fdocuments.net/reader033/viewer/2022051719/5a72c1cc7f8b9abb538de869/html5/thumbnails/29.jpg)
References
Figure 1: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Sequencing/Epigenetic-Sequencing/Methylation-Analysis.htmlFIgure 2: http://missinglink.ucsf.edu/lm/genes-and-genomes/methylation.htmlFigure 3: http://www.diagenode.com/en/applications/bisulfite-conversion.phpFigure 4: http://en.wikipedia.org/wiki/Illumina-Methylation-Assay
Jean-Philippe Fortin Illumina 450k: A microarray for the study of DNA methylation