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Microarrays.Rpres
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Bioinformatics for Big Omics Data: Introduction to gene expression microarrays
========================================================
width: 1440
height: 900
transition: none
font-family: 'Helvetica'
css: my_style.css
author: Raphael Gottardo, PhD
date: `r format(Sys.Date(), format="%B %d, %Y")`
<a rel="license" href="http://creativecommons.org/licenses/by-sa/3.0/deed.en_US"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-sa/3.0/88x31.png" /></a><br /><tiny>This work is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by-sa/3.0/deed.en_US">Creative Commons Attribution-ShareAlike 3.0 Unported License</tiny></a>.
Outline
=======
- Motivation
- Reverse transcription and hybridization cDNA microarrays
- Oligonucleotide arrays (Affymetrix)
- Illumina beadarrays
Motivation
==========
- Why are microarrays so important?
- First technology to enable the genomewide quantification of gene-expression
- Characterize and classify diseases, design new drugs, etc.
- "Recent" technology (1995,1996)
- Fast moving field
Microarrays
===========
- Isolate mRNA from cells
- From RNA we can get DNA using an enzyme called reverse transcriptase (e.g. Retrovirus)
- The derived "copy" DNA (cDNA) is hybridized to known DNA targets (genes) to quantify gene expression
Reverse transcription
=====================
Hybridization
==============
cDNA-microarray
===============
cDNA-microarray (suite)
===============
Oligonucleotide arrays
======================
------
- Fabricated by placing short cDNA sequences (oligonucleotides) on a small silicon chip by a photolithographic process
- Each gene is represented by a set of distinct probes, cDNA segments of length 25 nucleotides (11-20)
- Probes chosen based on uniqueness criteria and empirical rules
- Single color
Affymetrix arrays - probes
=================
-----------
- ~20 probes that “perfectly” represent the gene (Perfect Match)
- ~20 probes that do not match the gene sequence (Mismatch)
- Probeset
Affymetrix arrays - probes (suite)
=================
-------------------
For a valid gene expression measurement
the Perfect Match sticks and the Mistach does not!
Probe synthesis
===============
Let's watch a small video
http://www.youtube.com/watch?v=ui4BOtwJEXs
Affymetrix arrays
=================
Affymetrix protocol
=================
Affymetrix probesets
====================
Affymetrix image
====================
Illumina bead arrays
====================
<small>(Source: http://www.illumina.com/)</small>
-----
<small>(Source: http://www.mouseclinic.de/)</small>
Illumina bead arrays - image
===================
-------
- Illumina bead arrays can be applied to different problems
- Can use one or two colors depending on the application
- Gene expression arrays use one color
Illumina bead arrays
====================
- High density arrays
- HumanHT-12 v4.0 Expression BeadChip, about 47,000 probes
- A specific oligonucleotide sequence is assigned to each bead type, which is replicated about 30 times on an array
- Bead level data can be relavitely large
Summary of technologies
=======================
cDNA microarray | Affymetrix | Illumina
---|---|---
1 probe/gene | 1-20 probes/gene | 30 beads(probes)/gene
Probe of variable lengths | 25-mer probes | 50-mer probes
Two colors | One color | One color
Flexible, choice of probes | High density/Replication | High density/Replication
- | Cross hybridization | -
Analysis pipeline
==================
1. Biological question
2. Experimental design
3. Experiment
4. Image analysis
5. Normalization, Batch effect removal
6. Estimation, Testing, Clustering, Prediction, Classification, Feature selection, etc.
7. Validate finding generate new hypothesis $\rightarrow$ New experiments
**Most of these steps require the use of statistical methods and/or computational tools**
**Note:** Even though microarrays were designed to study gene expression, they can be used to study many other things (DNA-protein binding, methylation, splicing, etc). Though they are slowly being replaced by sequencing-based assays.