Slide 1
Made by - SHRUTI DABRALMeasuring dissimilarity of expression
pattern
NORMALIZATION There are three widely used techniques that can be
used to normalize gene-expression data from a single array
hybridization. All of these assume that all (or most) of the genes
in the array, some subset of genes, or a set of exogenous controls
that have been spiked into the RNA before labelling, should have an
average expression ratio equal to one. The normalization factor is
then used to adjust the data to compensate for experimental
variability and to balance the fluorescence signals from the two
samples being compared. Total intensity normalization Total
intensity normalization data relies on the assumption that the
quantity of initial mRNA is the same for both labeled samples.
Furthermore, one assumes that some genes are upregulated in the
query sample relative to the control and that others are
downregulated. For the hundreds or thousands of genes in the array,
these changes should balance out so that the total quantity of RNA
hybridizing to the array from each sample is the same. Under this
assumption, a normalization factor can be calculated and used to
re-scale the intensity for each gene in the array.
Normalization using regression techniques For mRNA derived from
closely related samples, a significant fraction of the assayed
genes would be expected to be expressed at similar levels.
Normalization of these data is equivalent to calculating the
best-fit slope using regression techniques and adjusting the
intensities so that the calculated slope is one. In many
experiments, the intensities are nonlinear, and local regression
techniques are more suitable, such as LOWESS (LOcally WEighted
Scatterplot Smoothing) regression.
Normalization using ratio statistics A third normalization
option is a method based on the ratio statistics described by Chen
et al.20. They assume that although individual genes might be up-
or downregulated, in closely related cells, the total quantity of
RNA produced is approximately the same for essential genes, such as
housekeeping genes. Using this assumption, they develop an
approximate probability density for the ratio Tk = Rk /Gk (where Rk
and Gk are, respectively, the measured red and green intensities
for the kth array element). They then describe how this can be used
in an iterative process that normalizes the mean expression ratio
to one and calculates confidence limits that can be used to
identify differentially expressed genes.
Gene expression matrix that contains rows representing genes and
columns representing particular conditions. Each cell contains a
value, given in arbitrary units, that refl ects the expression
level of a gene under a corresponding condition. B: Condition C4 is
used as a reference and all other conditions are normalized with
respect to C4 to obtain expression ratios. C: In this table all
expression ratios were converted into the log2 (expression ratio)
values. This representation has an advantage of treating
up-regulation and down-regulation on comparable scales. D: Discrete
values for the elements in Table 1.C. Genes with log2 (expression
ratio) values greater than 1 were changed to 1, genes with values
less than 1 were changed to 1. Any value between 1 and 1 was
changed to 0.
Distance measures Analysis of gene expression data is primarily
based on comparison of gene expression profiles or sample
expression profiles. In order to compare expression profiles, we
need a measure to quantify how similar or dissimilar are the
objects that are being considered. A variety of distance measures
can be used to calculate similarity in expression profiles and
these are discussed below.
Euclidean distance Euclidean distance is one of the common
distance measures used to calculate similarity between expression
profiles. The Euclidean distance between two vectors of dimension
2, say A=[a1, a2] and B=[b1, b2] can be calculated as: D(A,B)=
In other words, the Euclidean distance between two genes is the
square root of the sum of the squares of the distances between the
values in each condition (dimension).(a1-b1)(a1-b1) +
(a2-b2)(a2-b2)
Pearson correlation coefficient =One of the most commonly used
metrics to measure similarity between expression profiles is the
Pearson correlation coeffi cient (PCC) (Eisen et al. 1998). Given
the expression ratios for two genes under three conditions A=[a1,
a2, a3] and B=[b1, b2, b3], PCC can be computed as follows: Step1:
Compute mean of A and B Step2: Mean centre expression profiles
Step3: Calculate PCC as the cosine of the angle between the
mean-centred profi les
Step2: Mean centre expression profiles
Step3: Calculate PCC as the cosine of the angle between the
mean-centred profiles
\The reason why we mean centre the expression profiles is to
make sure that we compare shapes of the expression profiles and not
their magnitude. Mean centring maintains the shape of the profile,
but it changes the magnitude of the profile as shown in . A PCC
value of 1 essentially means that the two genes have similar
expression profiles and a value of 1 means that the two genes have
exactly opposite expression profiles. A value of 0 means that no
relationship can be inferred between the expression profiles of
genes. In reality, PCC values range from 1 to +1. A PCC value 0.7
suggests that the genes behave similarly and a PCC value -0.7
suggests that the genes have opposite behavior. The value of 0.7 is
an arbitrary cut-off, and in real cases this value can be chosen
depending on the dataset used.
CLUSTER ANALYSIS The term cluster analysis actually encompasses
several different classification algorithms that can be used to
develop taxonomies (typically as part of exploratory data
analysis). Note that in this classification, the higher the level
of aggregation, the less similar are members in the respective
class.
Clustering methods can be hierarchical (grouping objects into
clusters and specifying relationships among objects in a cluster,
resembling a phylogenetic tree) or non-hierarchical (grouping into
clusters without specifying relationships between objects in a
cluster) as schematically represented in Figure 5. Remember, an
object may refer to a gene or a sample, and a cluster refers to a
set of objects that behave in a similar manner.
Hierarchical methods Hierarchical clustering methods produce a
tree ordendrogram. They avoid specifying how many clusters are
appropriateby providing a partition for each k obtained from
cuttingthe tree at some level. The tree can be built in two
distinct ways bottom-up: agglomerative clustering; top-down:
divisive clustering.
Single-linkage clustering The distance between two clusters, i
and j, is calculated as the minimum distance between a member of
cluster i and a member of cluster j. Consequently, this technique
is also referred to as the minimum, or nearestneighbour, method.
This method tends to produce clusters that are loose because
clusters can be joined if any two members are close together. In
particular, this method often results in chaining, or the
sequential addition of single samples to an existing cluster. This
produces trees with many long, single-addition branches
representing clusters that have grown by accretion.
Complete-linkage clustering. Complete-linkage clustering is also
known as the maximum or furthest-neighbour method. The distance
between two clusters is calculated as the greatest distance between
members of the relevant clusters. Not surprisingly, this method
tends to produce very compact clusters of elements and the clusters
are often very similar in size. Average-linkage clustering The
distance between clusters is calculated using average values. There
are, in fact, various methods for calculating averages. The most
common is the unweighted pair-group method average (UPGMA). The
average distance is calculated from the distance between each point
in a cluster and all other points in another cluster. The two
clusters with the lowest average distance are joined together to
form a new cluster. Related methods substitute the CENTROID or the
median for the average.
Centroid linkage clustering In centroid linkage clustering, an
average expression profile (called a centroid) is calculated in two
steps. First, the mean in each dimension of the expression profiles
is calculated for all objects in a cluster. Then, distance between
the clusters is measured as the distance between the average
expression profiles of the two clusters.
Distances between clusters used forhierarchical clustering
Calculation of the distance between two clustersis based on the
pairwise distances betweenmembers of the clusters Mean-link:
average of pairwise dissimilarities Single-link: minimum of
pairwise dissimilarities. Complete-link: maximum& of pairwise
dissimilarities. Distance between centroids Complete linkage gives
preference to compact/sphericalclusters. Single linkage can produce
long stretched clusters
Hierarchical clustering divisive Hierarchical divisive
clustering is the opposite of the agglomerative method, where the
entire set of objects is considered as a single cluster and is
broken down into two or more clusters that have similar expression
profiles. After this is done, each cluster is considered separately
and the divisive process is repeated iteratively until all objects
have been separated into single objects. The division of objects
into clusters on each iterative step may be decided upon by
principal component analysis which determines a vector that
separates given objects. This method is less popular than
agglomerative clustering, but has successfully been used in the
analysis of gene expression data by Alon et al. (1999).
Non-hierarchical clustering One of the major criticisms of
hierarchical clustering is that there is no compelling evidence
that a hierarchical structure best suits grouping of the expression
profi les. An alternative to this method is a non-hierarchical
clustering, which requires predetermination of the number of
clusters. Non-hierarchical clustering then groups existing objects
into these predefi ned clusters rather than organizing them into a
hierarchical structure.
Example using R
RELATING EXPRESSION DATA TO OTHER BIOLOGICAL
INFORMATIONPredicting binding sitesPredicting protein interactions
and protein functionsPredicting functionally conserved
modulesReverse-engineering of gene regulatory networks
Study
materialhttp://www.mrc-lmb.cam.ac.uk/genomes/madanm/microarray/chapter-final.pdfhttp://www.dbbm.fiocruz.br/class/Lecture/d17/array_2/NatureReviews_JQ.pdf
