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Statistics For Microarrays: Normalization

This document discusses normalization techniques for microarray data. It begins by introducing the need for normalization to correct for systematic differences between samples not due to biological variation. It then describes several common normalization methods including global adjustment, intensity-dependent normalization using LOWESS, and within print-tip group normalization. The document compares different normalization schemes and shows their impact on microarray data, noting that normalization reduces systematic effects but can increase variability. It emphasizes choosing normalization based on the experimental design and examining data before and after normalization.

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Karthi Keyan
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26 views

Statistics For Microarrays: Normalization

This document discusses normalization techniques for microarray data. It begins by introducing the need for normalization to correct for systematic differences between samples not due to biological variation. It then describes several common normalization methods including global adjustment, intensity-dependent normalization using LOWESS, and within print-tip group normalization. The document compares different normalization schemes and shows their impact on microarray data, noting that normalization reduces systematic effects but can increase variability. It emphasizes choosing normalization based on the experimental design and examining data before and after normalization.

Uploaded by

Karthi Keyan
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Statistics for Microarrays

Normalization

Class web site:


http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/
Biological question
Differentially expressed genes
Sample class prediction etc.

Experimental design

Microarray experiment
16-bit TIFF files
Image analysis
(Rfg, Rbg), (Gfg, Gbg)
Normalization
R, G
Estimation Testing Clustering Discrimination

Biological verification
and interpretation
Preprocessing: Data Visualization

• Was the experiment a success?

• Are there any specific problems?

• What analysis tools should be used?


Tools for Microarray
Normalization and Analysis

• Both commercial and free software

• R (use sma package or Bioconductor:


http://www.bioconductor.org/)
Red/Green overlay images
Co-registration and overlay offers a quick
visualization, revealing information on color
balance, uniformity of hybridization, spot
uniformity, background, and artefacts
such as dust or scratches
Bad: high bg, ghost spots, little
Good: low bg, detectable d.e.
d.e.
Scatterplots: always log*, always rotate

log2R vs log2G M=log2R/G vs A=log2√RG

* Other transformations can provide improvement


Histograms

Signal/Noise = log2(spot intensity/background intensity)


Boxplots of log2R/G

Liver samples from 16 mice: 8 WT, 8 ApoAI KO


Spatial plots: background from the two slides
Highlighting extreme log ratios

Top (black) and bottom (green) 5% of log ratios


Pin group (sub-array) effects

Lowess lines through points from pin groups Boxplots of log ratios by pin group
Boxplots and highlighting pin group
effects
Log-ratios

Print-tip groups

Clear example of spatial bias


Plate effects
Clearly visible plate effects

KO #8

Probes: ~6,000 cDNAs, including 200 related to lipid metabolism.


Arranged in a 4x4 array of 19x21 sub-arrays.
Time of printing effects

spot number

Green channel intensities (log2G). Printing over 4.5 days.


The previous slide depicts a slide from this print run.
Preprocessing: Normalization
• Why?
To correct for systematic differences
between samples on the same slide, or
between slides, which do not represent
true biological variation between samples
• How do we know it is necessary?
By examining self-self hybridizations,
where no true differential expression is
occurring.
There are dye biases which vary with spot
intensity, location on the array, plate
origin, pins, scanning parameters,…
Self-self hybridizations

False color overlay Boxplots within pin-groups Scatter (MA-)plots


Similar patterns apparent in non
self-self hybridizations

From the NCI60 data set (Stanford web site)


From Lawrence Berkeley National Laboratory
Normalization Methods (I)
• Normalization based on a global adjustment
log2 R/G -> log2 R/G - c = log2 R/(kG)
Choices for k or c = log2k are c = median or mean of log
ratios for a particular gene set (e.g. all genes, or control
or housekeeping genes). Or, total intensity
normalization, where k = ∑Ri/ ∑Gi.
• Intensity-dependent normalization
Here, run a line through the middle of the MA plot,
shifting the M value of the pair (A,M) by c=c(A), i.e.
log2 R/G -> log2 R/G - c (A) = log2 R/(k(A)G).
One estimate of c(A) is made using the LOWESS
function of Cleveland (1979): LOcally WEighted
Scatterplot Smoothing.
Normalization Methods (II)
• Within print-tip group normalization
In addition to intensity-dependent variation in log ratios,
spatial bias can also be a significant source of systematic
error.
Most normalization methods do not correct for spatial
effects produced by hybridization artefacts or print-tip or
plate effects during the construction of the microarrays.

It is possible to correct for both print-tip and intensity-


dependent bias by performing LOWESS fits to the data
within print-tip groups, i.e.
log2 R/G -> log2 R/G - ci(A) = log2 R/(ki(A)G),
where ci(A) is the LOWESS fit to the MA-plot for the ith
grid only.
Normalization: Which Spots to use?
The LOWESS lines can be run through many different
sets of points, and each strategy has its own implicit set
of assumptions justifying its applicability.
For example, the use of a global LOWESS approach can
be justified by supposing that, when stratified by mRNA
abundance, a) only a minority of genes are expected to be
differentially expressed, or
b) any differential expression is
as likely to be up-regulation as down-regulation.
Pin-group LOWESS requires stronger assumptions: that
one of the above applies within each pin-group.
The use of other sets of genes, e.g. control or
housekeeping genes, involve similar assumptions.
Normalization makes a difference

Global scale, global lowess, pin-group lowess; spatial plot after, smooth histograms of M after
Normalization by controls:
Microarray Sample Pool titration
series
Pool the
whole library

Control set to aid intensity-dependent normalization


Different concentrations in titration series
Spotted evenly spread across the slide in each pin-group
Comparison of Normalization
Schemes
(courtesy of Jason Goncalves)

• No consensus on best normalization method


• Experiment done to assess the common
normalization methods
• Based on reciprocal labeling experimental
data for a series of 140 replicate
experiments on two different arrays each
with 19,200 spots
DESIGN OF RECIPROCAL
LABELING EXPERIMENT

• Replicate experiment
with same mRNA pools
but invert fluors (dye
swap)
• Replicates are
independent experiments
• Scan, quantify,
normalize as usual
Comparison of Normalization Methods - Using 140 19K Microarrays

0.46

0.44

0.42

0.4
Average Mean Deviation Value

0.38

0.36

***
0.34

0.32

0.3
Pre Normalized Global Intensity Subarray Intensity Global Ratio Sub-Array Ratio Global LOWESS Subarray LOWESS
Normalization Method
Scale normalization: between slides

Boxplots of log ratios from 3 replicate self-self


hybridizations
Left panel: before normalization
Middle panel: after within print-tip group normalization
Right panel: after a further between-slide scale
normalization
The “NCI 60” experiments (no bg)

Some scale normalization seems desirable


Scale normalization: another data set

Log-ratios

Only small differences in spread apparent; no action


required.
One way of taking scale into account

Assumption: All slides have the same spread in M

True log ratio is mij where i represents different


slides and j represents different spots.

Observed is Mij, where


Mij = ai mij

Robust estimate of ai is

MADi = medianj { |yij - median(yij) | }


A slightly harder normalization problem

Global lowess doesn’t do the trick here


Print-tip-group normalization helps
But not completely

Still a lot of scatter in the middle in a WT vs KO comparison


Effects of previous normalization

Before normalization After print-tip-group


normalization
Within print-tip-group box plots of
M after print-tip-group
normalization
Taking scale into account, cont.

Assumption: All print-tip-groups have the same


spread in M
True log ratio is mij where i represents
different print-tip-groups and j represents
different spots.
Observed is Mij, where
Mij = ai mij
Robust estimate of ai is

MADi = medianj { |yij - median(yij) | }


Effect of location & scale
normalization

Clearly care is needed in making decisions like this


A comparison of three M v A plots

Unnormalized Print-tip normalization Print tip & scale n


The same normalization on another data set

Before

After

.
Normalization: Summary
• Reduces systematic (not random) effects
• Makes it possible to compare several arrays

• Use logratios (M vs A plots)


• Lowess normalization (dye bias)
• MSP titration series – composite normalization
• Pin-group location normalization
• Pin-group scale normalization
• Between slide scale normalization

• Control Spots
• Normalization introduces more variability
• Outliers (bad spots) are handled with replication
Affymetrix Oligo Chips

• Only one “color”


• Different technology, different
normalization issues
• Affy chip normalization is an active
research area – see
http://www.stat.berkeley.edu/users/
terry/zarray/Affy/affy_index.html
Pre-processed cDNA Gene
Expression Data
On p genes for n slides: p is O(10,000), n is O(10-100), but growing,

Slides
slide 1 slide 2 slide 3 slide 4 slide 5 …
1 0.46 0.30 0.80 1.51 0.90 ...
2 -0.10 0.49 0.24 0.06 0.46 ...
Genes 3 0.15 0.74 0.04 0.10 0.20 ...
4 -0.45 -1.03 -0.79 -0.56 -0.32 ...
5 -0.06 1.06 1.35 1.09 -1.09 ...

Gene expression level of gene 5 in slide 4


= (normalized) log2( Red / Green)

These values are conventionally displayed


on a red (>0) yellow (0) green (<0) scale.
Acknowledgments
Terry Speed (UCB and Matt Callow (LLNL)
WEHI)
Percy Luu (UCB)
Jean Yee Hwa Yang (UCB)
Sandrine Dudoit (UCB) John Ngai (UCB)
Ben Bolstad (UCB) Vivian Peng (UCB)
Natalie Thorne (WEHI)
Ingrid Lönnstedt
(Uppsala)
Dave Lin (Cornell)
Henrik Bengtsson (Lund)

Jason Goncalves (Iobion)

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