microarray(dataanalysis( - uio
TRANSCRIPT
Microarray data analysis
MBV-‐INFX410 26th Nov, 2012
Ståle Nygård, BioinformaCcs core facility, OUS/UiO [email protected]
Gene expression Gene expression is the process by which informaCon from a gene is used in the synthesis of a funcConal gene product.
Microarrays
• Measure the expression of several thousand genes simultaneously
• Are oQen used to find differenCally expressed genes – Between groups of individuals (with different phenotypes, e.g. disease/healthy, long/short survival etc)
– Over Cme (e.g as disease develop, as Cssue develop)
4
Development of microarrays • MulCple Northern blots • Macroarrays • cDNA microarrays • OligonucleoCde microarrays • Todays technology: High
density arrays • High througput sequencing
(”Next generaCon sequencing”)
• High througput sequencing
1977
1987
1995
1996
2003
2005
Future
5
Development of microarrays • MulCple Northern blots 1977
6
• MulCple Northern blots • Macroarrays (spo`ed cDNAs, nylon filter, ~ 1000
gener) 1987
Development of microarrays 1977
7
Development of microarrays • MulCple Northern blots • Macroarrays • cDNA microarrays (cDNA probes >200 nt, PCR
produced)
1977
1987
1995
8
Development of microarrays • MulCple Northern blots • Macroarrays • cDNA microarrays • OligonucleoCde microarrays (oligos ~50-‐80 nt,
more than 10 000 genes)
1977
1987
1995
1996
9
Development of microarrays • MulCple Northern blots • Macroarrays • cDNA microarrays • OligonucleoCde microarrays • Todays technology: High
density arrays (e.g Illumina BeadArrays: 50 nt probes, 1 000 000s of probes)
1977
1987
1995
1996
2003
10
Development of microarrays • MulCple Northern blots • Macroarrays • cDNA microarrays • OligonucleoCde microarrays • Todays technology: High
density arrays • Next generaCon sequencing
(RNA-‐Seq)
1977
1987
1995
1996
2003
2005
11
Development of microarrays • MulCple Northern blots • Macroarrays • cDNA microarrays • OligonucleoCde microarrays • Todays technology: High density
arrays • Next generaCon sequencing (RNA-‐
seq) • Next-‐next generaCon sequencing:
True single molecule sequencing. E.g NanoPore technology (h`p://www.nanoporetech.com)
1977
1987
1995
1996
2003
2005
Future
Microarray technology vs RNAseq • Main caveats microarrays:
– Problem with alternaCve splicing; Probes on the microarray might not represent all the (alternaCvely spliced) RNAs
– Problem with degradaCon (less of a problem for RNAseq)
• Main caveats RNA-‐Seq: – Highly expressed genes can take up very much of the space on the slide, giving low accuracy to lowly expressed genes
– RNAseq technology is sCll more expensive than microarrays (but the prices are dropping)
13
The experiment pipeline
Biological question
Experimental design
QC of samples
Microarray experiment Preprocessing
of data
Statistical analysis
Biological verification & interpretation
1
2
3
4
QC of data
56
7
8
14
Microarray pipline (simplified)
AmplificaCon and
Labelling
RNA/DNA Nucleic acid
purificaCon
Labeled RNA/DNA
HybridisaCon, washing
Bioinformaticanalysis
Scan, QuanCtate
Raw data
E B E`B E pBEBLE ÐB@E @B@E àB@E BhEpBHE °BPE pB‚E`B`EðBE BHE PB$E �BE B�E B@E(E BEBPE €B8E àB$E àB$E PB E#°BLE `B`E àBPE °B E ÐBDE B8E B���B B���E B$E�ÀBLE BE �B`E`B@E"�BTE °B E �B€E @B,E ���ÀB8E%BªE ÀB\E °BHE �B8E @B\E �BLE €B4E àB$E `B E ÀB8E @B4E ðB@E B E àB$E �BDE B<E ÐBTE ���°B,E B$E PB E B@E ðB,E B<E 0BHE €B4E B E @BE B(E €B,E BXE!@BXE `BDE àBdEpBHE B(E#ÀB4E `B4E €B4E °B4E)`B E @B4E 0BDE pBdE`BHE PB E @B E @B�E ÀBE!PB0E pB E"°B E pB,EàBPE B`E��BHE ��� B8EpB���E pB@E B
Pre-‐processing
Sample
15
The experiment pipeline
Biological question
Experimental design
QC of samples
Microarray experiment Preprocessing
of data
Statistical analysis
Biological verification & interpretation
1
2
3
4
QC of data
56
7
8
16
Experimental design: general strategy
• Ensure that you will not have any systemaCcs biases: – Distribute the biological groups in a balanced way. – Divide into batches of the same sizes, limited b the capacity on each step.
– Tip: In Excel (or similar program) color code sample name according to biological group, and in next column color code by batch.
• Randomize and balance according to the biology your are interested in.
17
Experimental plan: an example Biology
A1
A2
A3
A4
A5
A6
B1
B2
B3
B4
B5
B6
C1
C2
C3
C4
C5
C6
Biology Sample prepara3on
order
A1 1 B4 2 C2 3 A3 4 B6 5 C4 6 A5 7 B2 8 C6 9 A2 10 B3 11 C1 12 A4 13 B5 14 C3 15 A6 16 B1 17 C5 18
Biology Sample
prepara3on order
Extrac3on
order
A2 10 1 B6 5 2 C1 12 3 A5 7 4 B5 14 5 C6 9 6 A6 16 7 B4 2 8 C5 18 9 A3 4 10 C3 15 11 B2 8 12 A4 13 13 C4 6 14 B1 17 15 A1 1 16 B3 11 17 C2 3 18
18
Experimental design: Batch effect (1)
Samples color coded according to biology
19
Exp. design: Batch effect (2)
Samples color coded according to labeling date
20
Image analysis of microarray data • Main steps
– Address spots – Separate foreground from background – Quality check: Localize and remove bad quality spots
– Quality check of the microarray as a whole
• AutomaCzaCon ü Commercial plamorms
• Today image analysis is basically an automated procedure performed by the soQware
• Manual quality check is relevant for protein arrays and tailor-‐made microarrays
NormalizaCon
• Goal: remove technical arCfacts, which can be due to – Different amounts of input material – Different degrees of degradaCon – Dust, scratches etc on the arrays – ++
• Most normalizaCon methods assume that the overall intensity is the same for different samples (e.g quanCle normlizaCon).
22
QuanCle normalizaCon • Enforce equal distribuCon between the
microarrays. Procedure – Sort the expression values for each
microarray from highest to lowest – Calculate the mean value for each rank – For every array
• let the highest ranked gene have the mean value of the highest ranked genes (of all arrays)
• Let the second highest ranked gene have the mean value of the second highest ranked genes (of all arrays)
• and so on for all ranks
NormalizaCon using TMM (Trimmed Mean of M-‐values)
Highly expressed genes having big influence on library size
.
(a)
log2(Kidney1 NK1) − log2(Kidney2 NK2)
Den
sity
-6 -4 -2 0 2 4 6
0.0
0.4
0.8
log2(Liver NL) - log2(Kidney NK)
Den
sity
-6 -4 -2 0 2 4 6
0.0
0.2
0.4(b)
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-20 -15 -10
-50
5
A = log2( Liver NL Kidney NK)
M=
log 2
(Liv
erN
L)-
log 2
(Kid
ney
NK)
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Housekeeping genesUnique to a sample
(c)
In TMM the genes with the smallest and largest raCos (i. e 40% of the genes) are not used in the normalizaCon.
24
DistribuCon of microarray data • Ordinary scale: noise proporConal to signal, data not normally distributed
• Log2 scale: noise less proporConal to signal, distribuCon closer to normal (a prerequisit for many tests)
Normal scale
Log2 scale
TesCng for differenCal expression – microarray data
Ordinary t-‐test: Variance esCmates can be improved by ”borrowing strength” across genes in a technique called variance shrinkage Many methods use this technique, e.g SAM and limma. NB! This technique is relevant only for small sample sizes.
ti =xi − yiσ i
t 'i =xi − yi
B*σ i + (1−B)*σ all
DistribuCon of RNAseq data
• What is the distribuCon of counts for a parCcular RNA – Counts from technical replicates are approximately Poisson distributed.
– Biological replicates exhibit more variance, for which the negaCve binomial distribuCon gives a be`er fit. (Ballard et al, 2010)
Poisson vs negaCve binomial distribuCon
0 5 10 15 20
0.00
0.10
Mean=5
Count
Prob
abilit
y
PoissonNeg.binom (phi=0.01)Neg.binom (phi=0.1)
0 50 100 150 200
0.00
0.02
0.04
Mean=100
Count
Prob
abilit
y
PoissonNeg.binom (phi=0.01)Neg.binom (phi=0.1)
• Counts are normalized using TMM (Trimmed mean of M-‐values)
• A negaCve binomial distribuCon is assumed and the extra dispersion parameter is esCmated. The parameter can be common to all genes, gene-‐specific, or a combinaCon
The edgeR procedure
CorrecCon for mulCple tesCng
In ordinary microarray studies (looking at all genes), use false discovery rates instead of ordinary p-‐values
30
Hierarchical clustering • Genes and samples can
be clustered at the same Cme
• AgglomeraCve: start with one element as a cluster (bo`om-‐up). Most common
• Divisive: start with all elements in one large cluster (top-‐down)
• Dendrogram: a cluster tree
• Why cluster genes? ü Reduce complexity ü Generate hypothesis, e.g.
hypothesize that a group of genes with similar expression profiles interact or are involved in the same process
• Why cluster samples? ü IdenCfy known sub-‐
groups ü Find new or more
detailed subgroups ü Quality check (detect
outliers)
31
Distance measures • In clustering algorithms two similar elements should be placed in the same cluster
What profiles are most similar?
-‐ Dependent on the distance measure used
X If
Eucledian distance measure is
used
If correlaCon is used as distance measure
X
Network construcCon based on microarray data
• Network construcCon from genomic data is difficult. Many possible combinaCons of interacCons. • Network construcCon could be guided by including external informaCon about interacCons. • Seeded Bayesian Networks (Djebbari and Quackenbush, 2008) guide the network construcCon by including interacCons reported in literature and protein-‐protein interacCon databases. • The R package Bionet connects regulated genes using a protein-‐protein interacCon database.
PaCent focused analysis (predicCon/classificaCon)
ClassificaCon/predicCon approach • Instead of looking at each gene’s correlation to the phenotype
one by one (gene focused analysis), the optimal classification/prediction rule looks at the effect of all genes simultaneously. We then answer the question: what is the effect of gene i when we account for the effect of all other genes.
• Best prediction rule picks out genes with orthogonal (independent) information about the phenotype.
• Methodological problem: How to fit a model with a much larger number (p) of explanatory variables (the genes) than the number of individuals (n). This is called the p > n (p larger than n) problem.
• The solution is to reduce the number of dimensions
Variance bias trade-‐off • Such methods are in fact biased, i.e underesCmaCng the effect of each gene.
• But they have reduced variance, leading to smaller predicCon error.
• PredicCon error=bias^2 +variance
Dealing with survival data
• Survival or time to event data have the problem of censoring. Event (e.g. death) does not always occur before end of study.
• The Cox model is the most common model dealing with censoring. In the Cox model the hazard rate , i.e. the instantaneous risk of failure at Cme t, is modeled by
where t is Cme and x is the gene expressions of a specific gene and β is the effect of the gene on survival.