SCHOOL OF BIO AND CHEMICAL ENGINEERING

DEPARTMENT OF BIOINFORMATICS

UNIT – 1- SBIA5304 MICROARRAY DATAANALYSIS

 SBIA5304- MICROARRAY DATA ANALYSIS

UNIT I GENE EXPRESSION

Basics of Gene expression- definition , gene expression studies, gene expression patterns-
Applications of gene expression studies

Microarrays – definition , discovery, technique, making microarrays, spotted microarrays, In-situ

synthesized oligonucleotide arrays, inkjet array synthesis, Affymetrix techniques, DNA CHIP technology,
photolithography, spot quality, sample preparation and labelling, washing, image acquisition Sequencing
by Hybridization Arrays- DNA MassArray™ Technology- Printing DNA Microarrays-Types of
microarrays - Designing a microarray experiment.

• Gene, unit of hereditary information that occupies a fixed position (locus) on a chromosome. Genes
achieve their effects by directing the synthesis of proteins.

• Gene, unit of hereditary information that occupies a fixed position (locus) on a chromosome. Genes
achieve their effects by directing the synthesis of proteins.

•
In eukaryotes (such as animals, plants, and fungi), genes are contained within the cell nucleus.
• The mitochondria (in animals) and the chloroplasts (in plants) also contain small subsets of genes
distinct from the genes found in the nucleus.

• In prokaryotes (organisms lacking a distinct nucleus, such as bacteria), genes are contained in a
single chromosome that is free-floating in the cell cytoplasm.

• Many bacteria also contain plasmids—extrachromosomal genetic elements with a small number of
genes.

• The number of genes in an organism’s genome (the entire set of chromosomes) varies significantly
between species.

• For example, whereas the human genome contains an estimated 20,000 to 25,000 genes, the
genome of the bacterium Escherichia coli O157:H7 houses precisely 5,416 genes.

• Arabidopsis thaliana—the first plant for which a complete genomic sequence was recovered—has
roughly 25,500 genes; its genome is one of the smallest known to plants.

• Among extant independently replicating organisms, the bacterium Mycoplasma genitalium has the
fewest number of genes, just 517.

• Basics of Gene expression:

• Gene expression

• the phenotypic manifestation of a gene or genes by the processes of genetic transcription and
genetic translation.

• Gene expression analysis

• the determination of the pattern of genes expressed at the level of genetic transcription, under
specific circumstances or in a specific cell.

• When genes are expressed, the genetic information (base sequence) on DNA is first copied to a
molecule of mRNA (transcription).

• The mRNA molecules then leave the cell nucleus and enter the cytoplasm, where they participate
in protein synthesis by specifying the particular amino acids that make up individual proteins
(translation).

• At any given time, the amount of a particular protein in a cell reflects the balance between that
protein's synthetic and degradative biochemical pathways.
 • On the synthetic side of this balance, recall that protein production starts at transcription (DNA to
RNA) and continues with translation (RNA to protein).

• Thus, control of these processes plays a critical role in determining what proteins are present in a
cell and in what amounts.

• In addition, the way in which a cell processes its RNA transcripts and newly made proteins also
greatly influences protein levels.

How Is Gene Expression Regulated?

• The amounts and types of mRNA molecules in a cell reflect the function of that cell. In fact,
thousands of transcripts are produced every second in every cell. Given this statistic, it is not
surprising that the primary control point for gene expression is usually at the very beginning of the
protein production process — the initiation of transcription. RNA transcription makes an efficient
control point because many proteins can be made from a single mRNA molecule.

Transcript processing provides an additional level of regulation for eukaryotes, and the
presence of a nucleus makes this possible.

• In prokaryotes, translation of a transcript begins before the transcript is complete, due to

the proximity of ribosomes to the new mRNA molecules.
•
In eukaryotes, however, transcripts are modified in the nucleus before they are exported to the
cytoplasm for translation.

•
Eukaryotic transcripts are also more complex than prokaryotic transcripts.
• For instance, the primary transcripts synthesized by RNA polymerase contain sequences that will
not be part of the mature RNA.

• These intervening sequences are called introns, and they are removed before the mature mRNA
leaves the nucleus.

• The remaining regions of the transcript, which include the protein-coding regions, are called
exons, and they are spliced together to produce the mature mRNA.

• Eukaryotic transcripts are also modified at their ends, which affects their stability and translation.

• Of course, there are many cases in which cells must respond quickly to changing environmental
conditions.
• In these situations, the regulatory control point may come well after transcription.

• For example, early development in most animals relies on translational control because very little
transcription occurs during the first few cell divisions after fertilization. Eggs therefore contain
many maternally originated mRNA transcripts as a ready reserve for translation after fertilization.

• On the degradative side of the balance, cells can rapidly adjust their protein levels through the
enzymatic breakdown of RNA transcripts and existing protein molecules.

• Both of these actions result in decreased amounts of certain proteins. Often, this breakdown is
linked to specific events in the cell.

• The eukaryotic cell cycle provides a good example of how protein breakdown is linked to cellular
events. This cycle is divided into several phases, each of which is characterized by distinct cyclin
proteins that act as key regulators for that phase.

• Before a cell can progress from one phase of the cell cycle to the next, it must degrade the cyclin
that characterizes that particular phase of the cycle. Failure to degrade a cyclin stops the cycle
from continuing.
How Do Different Cells Express the Genes They Need?

• Only a fraction of the genes in a cell are expressed at any one time.

• The variety of gene expression profiles characteristic of different cell types arise because these cells
have distinct sets of transcription regulators.

• Some of these regulators work to increase transcription, whereas others prevent or suppress it.

• Normally, transcription begins when an RNA polymerase binds to a so-called promoter

sequence on the DNA molecule.

• This sequence is almost always located just upstream from the starting point for transcription (the
5' end of the DNA), though it can be located downstream of the mRNA (3' end).

• In recent years, researchers have discovered that other DNA sequences, known
as enhancer sequences, also play an important part in transcription by providing binding sites for
regulatory proteins that affect RNA polymerase activity.

• Binding of regulatory proteins to an enhancer sequence causes a shift in chromatin structure that
either promotes or inhibits RNA polymerase and transcription factor binding. A more open
chromatin structure is associated with active gene transcription.

• In contrast, a more compact chromatin structure is associated with transcriptional inactivity

 • Some regulatory proteins affect the transcription of multiple genes.

• This occurs because multiple copies of the regulatory protein binding sites exist within the
genome of a cell.

• Consequently, regulatory proteins can have different roles for different genes, and this is one
mechanism by which cells can coordinate the regulation of many genes at once.

How Is Gene Expression Increased or Decreased in Response to Environmental Change?

• In prokaryotes, regulatory proteins are often controlled by nutrient availability.

• This allows organisms such as bacteria to rapidly adjust their transcription patterns in response to
environmental conditions.

• In addition, regulatory sites on prokaryotic DNA are typically located close to transcription
promoter sites — and this plays an important part in gene expression.

• For an example of how this works, imagine a bacterium with a surplus of amino acids that signal
the turning "on" of some genes and the turning "off" of others.

• In this particular example, cells might want to turn "on" genes for proteins that metabolize amino
acids and turn "off" genes for proteins that synthesize amino acids.

• Some of these amino acids would bind to positive regulatory proteins called activators.
• Activator proteins bind to regulatory sites on DNA nearby to promoter regions that act as on/off
switches. This binding facilitates RNA polymerase activity and transcription of nearby genes.

• At the same time, however, other amino acids would bind to negative regulatory proteins
called repressors, which in turn bind to regulatory sites in the DNA that effectively block RNA
polymerase binding

• The control of gene expression in eukaryotes is more complex than that in prokaryotes. In general,
a greater number of regulatory proteins are involved, and regulatory binding sites may be located
quite far from transcription promoter sites. Also, eukaryotic gene expression is usually regulated
by a combination of several regulatory proteins acting together, which allows for greater flexibility
in the control of gene expression.

• Different cell types express characteristic sets of transcriptional regulators. In fact, as multicellular
organisms develop, different sets of cells within these organisms turn specific combinations of
regulators on and off. Such developmental patterns are responsible for the variety of cell types
present in the mature organism

• Transcriptional regulators can determine cell types

• The wide variety of cell types in a single organism can depend on different transcription factor
activity in each cell type. Different transcription factors can turn on at different times during
successive generations of cells. As cells mature and go through different stages (arrows),
transcription factors (colored balls) can act on gene expression and change the cell in different
ways. This change affects the next generation of cells derived from that cell. In subsequent
generations, it is the combination of different transcription factors that can ultimately determine
cell type.

• Technologies

• Real Time quantitative RT-PCR

• In situ hybridization

• Microarrays

• Massively Parallel Signature Sequencing (MPSS)

• Gene expression analysis is most simply described as the study of the way genes are transcribed
to synthesize functional gene products — functional RNA species or protein products.

• The study of gene regulation provides insights into normal cellular processes, such as
differentiation, and abnormal or pathological processes.

• In 1941, Beadle and Tatum published experiments that would explain the basis of the central
dogma of molecular biology, whereby the DNA through an intermediate molecule, called RNA,
results proteins that perform the functions in cells.

• Currently, biomedical research attempts to explain the mechanisms by which develops a

particular disease, for this reason, gene expression studies have proven to be a great resource.

• Strictly, the term “gene expression” comprises from the gene activation until the mature protein is
located in its corresponding compartment to perform its function and contribute to the expression
of the phenotype of cell.

• The expression studies are directed to detect and quantify messenger RNA (mRNA) levels of
a specific gene.

• The development of the RNA-based gene expression studies began with the Northern Blot
by Alwine et al. in 1977.
• In 1969, Gall and Pardue and John et al. independently developed the in situ hybridization,
but this technique was not employed to detect mRNA until 1986 by Coghlan. Today, many of
the techniques for quantification of RNA are deprecated because other new techniques provide
more information. Currently the most widely used techniques are qPCR, expression microarrays,
and RNAseq for the transcriptome analysis. In this chapter, these techniques will be reviewed.

• Gene expression workflow.

• Researchers may perform gene expression analysis at any one of several different levels at which
gene expression is regulated: transcriptional, post-transcriptional, translational, and post-
translational

• protein modification.

• Transcription, the process of creating a complementary RNA copy of a DNA sequence, can be
regulated in a variety of ways. Transcriptional regulation processes are the most commonly
studied and manipulated in typical gene expression analysis experiments.

•
As previously mentioned, enhancer sequences are DNA sequences that are bound by an activator
protein, and they can be located thousands of base pairs away from a promoter, either upstream or
downstream from a gene. Activator protein binding is thought to cause DNA to loop out, bringing
the activator protein into physical proximity with RNA polymerase and the other proteins in the
complex that promote the initiation of transcription
 •
The binding of regulatory proteins to DNA binding sites is the most direct method by which
transcription is naturally modulated. Alternatively, regulatory processes can also interact with the
transcriptional machinery of a cell. More recently, the influence of epigenetic regulation, such as
the effect of variable DNA methylation on gene expression, has been uncovered as a powerful tool
for gene expression profiling. Varying degrees of methylation are known to affect chromatin
folding and strongly affect accessibility of genes to active transcription.

• Following transcription, eukaryotic RNA is typically spliced to remove noncoding intron sequences
and capped with a poly(A) tail. At this post-transcriptional level, RNA stability has a significant
effect on functional gene expression, that is, the production of functional protein. Small interfering
RNA (siRNA) consists of double-stranded nucleic acid molecules that are participants in the RNA
interference pathway, in which the expression of specific genes is modulated (typically by
decreasing activity). Precisely how this modulation is accomplished is not yet fully understood. A
growing field of gene expression analysis is in the area of microRNAs (miRNAs), short RNA
molecules that also act as eukaryotic post-transcriptional regulators and gene silencing agents

Researchers studying gene expression employ a wide variety of molecular biology techniques and
experimental methods.

Gene expression analysis studies can be broadly divided into four areas:

1. RNA expression,

2. promoter analysis,

3. protein expression, and

4. post-translational modification.
RNA Expression

• Northern blotting — steady-state levels of mRNA are directly quantitated by electrophoresis and
transfer to a membrane followed by incubation with specific probes.

• The RNA-probe complexes can be detected using a variety of different chemistries or radionuclide
labeling.

• This relatively laborious technique was the first tool used to measure RNA levels
•
 The reverse northern blot is a method by which gene expression patterns may be analyzed by
comparing isolated RNA molecules from a tester sample to samples in a control cDNA library.

• It is a variant of the northern blot in which the nucleic acid immobilized on a membrane is a
collection of isolated DNA fragments rather than RNA, and the probe is RNA extracted from a
tissue and radioactively labelled.
 • A reverse northern blot can be used to profile expression levels of particular sets of RNA sequences
in a tissue or to determine presence of a particular RNA sequence in a sample.

• Although DNA Microarrays and newer next-generation techniques have generally supplanted
reverse northern blotting, it is still utilized today and provides a relatively cheap and easy means of
defining expression of large sets of genes.

Procedure

• In order to prepare the reverse northern membrane, cDNA sequences for transcripts of interest are
immobilized on nylon membranes, which can be accomplished by use of dot blots or bidirectional
agarose gel blotting and UV fixation of the DNA to the membranes.

• In many cases, cDNA probes may be preferred over RNA probes in order to mitigate problems of
RNA degradation by RNAses or tissue metabolites.

• Prepared reverse northern blot membranes are pre-hybridized in Denhardt's solution with SSC
buffer and labeled cDNA probes are denatured at 100 °C and added to the pre-hybridization
solution.

• The membrane is incubated with the probes for at least 15 hours at 65 °C, then washed and exposed.

DNA microarrays:

• an array of oligonucleotide probes bound to a chip surface enables gene expression profiling of
many genes in response to a condition.

• Labeled cDNA from a sample is hybridized to complementary probe sequences on the chip, and
strongly associated complexes are identified optically.

• Gene expression profiling is often a first step in a gene expression analysis workflow, investigating
changes in the expression profile of a whole system or examining the effects of mutations in
biological systems
Real-Time PCR

• Steady-state levels of mRNA are quantitated by reverse transcription of the RNA to cDNA
followed by quantitative PCR (qPCR) on the cDNA.

• The amount of each specific target is determined by measuring the increase in fluorescence signal
from DNA-binding dyes or probes during successive rounds of enzyme-mediated amplification.

• This precise, versatile tool is used to investigate mutations (including insertions, deletions, and
single-nucleotide polymorphisms (SNPs)), identify DNA modifications (such as methylation),
confirm results from northern blotting or microarrays, and conduct gene expression profiling.
 • Expression levels can be measured relative to other genes (relative quantification) or against a
standard (absolute quantification). Real-time PCR is the gold standard in nucleic acid quantification
because of its accuracy and sensitivity.

• Real-time PCR can be used to quantitate mRNA or miRNA expression following conversion to
cDNA or to quantitate genomic DNA directly to investigate transcriptional activity

Promoter Analysis

• Expression of reporter genes/promoter fusions in host cells — promoter activity (transcription rate)
is measured in vivo by introducing fusions of various promoter sequences with a gene encoding a
product that can be readily measured to monitor activity levels

Most commonly used reporter gene that fits the definition, widely available and commonly used are:

• β-galactosidase [β-Galactosidase Assay (CPRG), Fluorescent β-Galactosidase Assay (MUG)]

• β-glucuronidase (GUS assay used mostly for expression in plants)

• Luciferase (Lumino™ Firefly Luciferase Assay)

• Green fluorescent protein (GFP)

• Secreted Placental Alkaline Phosphatase

• In vitro transcription (nuclear run-on assays) — transcription rates are measured by

incubating isolated cell nuclei with labeled nucleotides, hybridizing the resultant product to a
membrane (slot blot), and then exposing this to film or other imaging media

•
Gel shift assays — also called electrophoretic mobility shift assays, these are used to study
protein-DNA or protein-RNA interactions.

• DNA or RNA fragments that are tightly associated with proteins (such as transcription factors)
migrate more slowly in an agarose or polyacrylamide gel (showing a positional shift). Identifying
the associated sequences provides insight into gene regulation
 • Chromatin immunoprecipitation (ChIP) —

• protein-binding regions of DNA can be identified in vivo. In living cells, DNA and protein are
chemically cross-linked, and the resulting complex is precipitated by antibody-coated beads
(immunoprecipitation). Following protein digestion and DNA purification, the sequences of the
precipitated DNA are determined

Protein Expression

• Western blotting — quantification of relative expression levels for specific proteins is

accomplished by electrophoretically separating extracted cell proteins, transferring them to a
membrane, and then probing the bound proteins with antibodies (targeted to antigens of interest)
that are subsequently detected using various chemistries or radiolabelling

• 2-D Gel Electrophoresis — protein expression profiling is achieved by separating a complex

mixture of proteins in two dimensions and then staining to detect differences at the whole-proteome
level

• Immunoassays — proteins are quantitated in solution using antibodies that are bound to color-
coded beads (as in the Bio-Plex supension array system) or immobilized to a surface (ELISA),
 which is subsequently probed with an antibody suspension and is typically detected using a
chromogenic or fluorogenic reporter

Posttranslational Modification Analysis

• Immunoassays — levels of protein phosphorylation and other post-translational modifications are

detected using antibodies that are specific for these adducts

• Mass spectrometry — proteins and their modifications are identified based on their mass

• What is RNA-seq?

• RNA-seq (RNA-sequencing) is a technique that can examine the quantity and sequences of RNA
in a sample using next generation sequencing (NGS). It analyzes the transcriptome of gene
expression patterns encoded within our RNA. Here, we look at why RNA-seq is useful, how the
technique works, and the basic protocol which is commonly used today1.

•
What are the applications of RNA-seq?

• RNA-seq lets us investigate and discover the transcriptome, the total cellular content of RNAs
including mRNA, rRNA and tRNA. Understanding the transcriptome is key if we are to connect
the information on our genome with its functional protein expression.
 • RNA-seq can tell us which genes are turned on in a cell, what their level of expression is, and at
what times they are activated or shut off. This allows scientists to more deeply understand the
biology of a cell and assess changes that may indicate disease.

• Some of the most popular techniques that use RNA-seq are transcriptional profiling, SNP
identification, RNA editing and differential gene expression analysis.

This can give researchers vital information about the function of genes. For example, the
transcriptome can highlight all the tissues in which a gene of unknown function is expressed, which
might indicate what its role is.

• It also captures information about alternative splicing events (Figure 1), which produce different
transcripts from one single gene sequence. These events would not be picked up by DNA
sequencing. It can also identify post-transcriptional modifications that occur during mRNA
processing such as polyadenylation and 5’ capping.

How does RNA-seq work?

• Early RNA-seq techniques used Sanger sequencing technology, a technique that although
innovative at the time, was also low-throughput, costly, and inaccurate. It is only recently, with the
advent and proliferation of NGS technology, have we been able to fully take advantage of RNA-
seq’s potential4.

The first step in the technique involves converting the population of RNA to be sequenced into
cDNA fragments (a cDNA library). This allows the RNA to be put into an NGS workflow. Adapters
are then added to each end of the fragments. These adapters contain functional elements which
permit sequencing; for example, the amplification element and the primary sequencing site. The
cDNA library is then analyzed by NGS, producing short sequences which correspond to either one
or both ends of the fragment. The depth to which the library is sequenced varies depending on
techniques which the output data will be used for. The sequencing often follows either single-read
or paired-end sequencing methods. Single-read sequencing is a cheaper and faster technique (for
reference, about 1% of the cost of Sanger sequencing) that sequences the cDNA from just one end,
whilst paired-end methods sequence from both ends, and are therefore more expensive and time-
consuming5,6.
•
A further choice must be made between strand-specific and non-strand-specific protocols. The
former method means the information about which DNA strand was transcribed is retained. The
value of extra information obtained from strand-specific protocols make them the favorable option.

These reads, of which there will be many millions by the end of the workflow, can then be aligned
to a genome of reference and assembled to produce an RNA sequence map that spans the
transcriptome7.

• RNA-seq vs microarrays: Why RNA-seq is considered superior

• RNA-seq is widely regarded as superior to other technologies, such as microarray hybridization.

There are several reasons for RNA-seq’s well-regarded status

• Not limited to genomic sequences – unlike hybridization-based approaches, which may require
species-specific probes, RNA-seq can detect transcripts from organisms with previously
undetermined genomic sequences. This makes it fundamentally superior for the detection of novel
transcripts, SNPs or other alterations.

• Low background signal – the cDNA sequences used in RNA-seq can be mapped to targeted regions
on the genome, which makes it easy to remove experimental noise. Furthermore, issues with cross-
hybridization or sub-standard hybridization, which can plague microarray experiments, are not an
issue in RNA-seq experiments.

• More quantifiable - Microarray data is only ever displayed as values relative to other signals
detected on the array, whilst RNA-seq data is quantifiable. RNA-seq also avoids the issues
microarrays have in detecting very high or very low expression levels.

• Applications of gene expression studies

Microarrays – definition , discovery, technique, making microarrays

• Microarray technology is a developing technology used to study the expression of many genes at
once.

• It involves placing thousands of gene sequences in known locations on a glass slide called a gene
chip.

• A sample containing DNA or RNA is placed in contact with the gene chip. Complementary base
pairing between the sample and the gene sequences on the chip produces light that is measured.

• Areas on the chip producing light identify genes that are expressed in the sample.
Microarrays – definition , discovery

Early DNA arrays

• After the first description of the double helix DNA structure by Watson and Crick in 1953, the
process of separating the two strands was soon reversed with methods of DNA molecular
hybridization quickly explored.

• Molecular hybridization is the occurrence of single-stranded DNA binding to complimentary DNA.

The complimentary base pairs that form the structure of the opposite strands of DNA are the
foundation for all analysis methods involving DNA sequences.

• In 1975, Grunstein and Hogness applied the process of molecular hybridization to DNA released
from blotted microbial colonies, a useful process for screening bacteria clones.
The colony hybridization method was formed by randomly cloning Escherichia coli (E. coli) DNA onto
agar petri plates covered with nitrocellulose filters. A radioactively labeled probe was then added which
would bind to complimentary DNA within the sample.

The aforementioned method formed a random orientation of sample DNA spots representing the cloned
fragments of DNA. This is an early example of a labeled probe being utilized in order to identify
complimentary base pair binding. It can therefore be considered as one of the first examples of a DNA
array.

• Gergan et al. adapted this methodology to produce arrays in 1979. Multiple plates on agar were
replicated to produce arrays through the use of a mechanical 144 pin device for placing samples in
the corresponding amount of well microplates. This allowed for the production of arrays for over a
thousand different bacterial colonies.
• The colonies could then be easily transferred to paper filters for the necessary lysis, denaturation
and fixing steps for producing hybridized DNA. The technology of filter based arrays was used in
research that led to the identification of single nucleotide polymorphisms (SNPs) and the ability to
clone specific genes of interest.

• The ability to analyze multiple hybridization targets was automated in the late 1980s and early
1990s. Robotic technology was used to quickly array clones from microtiter plates onto filters. The
arrays created a defined pattern allowing parallel hybridizations to be produced.

• Efficiency was increased with the errors that occur during repetitive procedures being reduced
through the automated placing of samples on the array. The increased speed and accuracy from
automation was an important step in the development towards microarrays.

• A further development of complimentary DNA (cDNA) cloning was also an important foundation
for the microarray, as it led to the creation of reference sets of cDNA and corresponding filter arrays
for whole genomes.

• In 1995, the first study that used the word 'microarray' was published which explained how the
expression of many genes could be monitored in parallel through the use of this new technology.
The sample array was constructed through high-speed robotic printing of cDNA on glass.

• The small size of spots on the array and high density of the arrays produced hybridization volumes
of two microliters, which was the volume that enabled the detection of rare transcripts within the
probe samples.

• The microarray was a technical advancement that meant a broader examination of gene expression
could be accomplished. In 1997, the researchers from Stanford University published the first whole-
genome microarray study of gene expression by placing the whole yeast genome on a microarray.

• History
Earliest form of microarray is the Southern blot, developed in 1975 by Dr. Edward Southern of
Edinburgh University

• In this technique, fragmented DNA is bound to a substrate (often a nitrocellulose or nylon

membrane), denatured, dried and then exposed to a labeled hybridization probe in an appropriate
buffer

• Blot is then extensively washed and analyzed by Xray film, autoradiography or membrane
chromogen detection, depending on the type of probe label employed
• Southern blotting has been largely replaced by newer molecular techniques but it has value in
analyzing several trinucleotides repeat syndromes (Fragile X syndrome, Huntington chorea), where
the length of the expanded DNA is greater than the usual amplification ability of PCR

• Array technology was used by Augenlicht et al. in 1984 to analyze retroviral long terminal repeat
(LTR element expression in murine colon tumors (J Biol Chem 1984; 259:1842)

• In 1987, Kulesh et al. used arrays to analyze the expression of more than 2,000 different genes
constructed from a human fibrosarcoma cell line, with and without interferon treatment (Proc Natl
Acad Sci USA 1987;84:8453)

• Different mRNA derived cDNAs were spotted onto filter paper and analyzed

• 29 sequences were induced by interferon treatment

• Miniaturized microarrays were introduced in 1995 (Science 1995; 270:467)

• First complete eukaryotic genome was placed on microarray in 1997, when Lashkari et al. placed
a maximum of 2,470 open reading frames on a glass slide and analyzed total mRNA expression
(cDNA) in S. cerevisia, examining the effects of heat and cold shock and culture in glucose vs
galactose on global gene expression profiles (Proc Natl Acad Sci USA 1997;94:13057)

• Since its first research use in the 1980s, the development of better surface technologies, more
powerful robots for arraying, better nucleic acid dye labeling techniques and improved
computational power and automated analyzers have vastly improved the power and efficiency of
microarray, while also lowering the cost of these analyses

• Microarray is currently used to analyze many different systems, including the classification of
microbes and human microbial pathogens, cellular responses to pathogens, drug and toxic
exposures, tumor classification, single nucleotide polymorphism detection, the detection of gene
fusions, comparative genomic hybridization, alternative splicing detection (exon junction array /
exon arrays) and gene expression profiling via analyzing global mRNA levels

• Most microarray protocols use reverse transcriptase to convert mRNA into cDNA, as DNA is more
stable with RNA

• DNA microarrays, also called DNA chips, gene chips, DNA arrays, gene arrays and biochips, are
microscopic slides of glass or silicon printed with thousands of small spots in grid fashion with
each containing known DNA or gene.
 • Each slide acts as probe to detect gene expression.

• Basically, it ‘s been evolved from southern blotting .It is different from Southern blotting as here
the probe is fixed/attached and sample DNA is labeled rather than probe.

PRINCIPLE

• DNA mICROARRAY

• The basic principle behind DNA microarray lies on Nucleic acid hybridization.

• During this method, two complementary strands of DNA are joined together by hydrogen bond to
make a double stranded molecule by hydrogen bond.

• Restriction endonuclease is employed to cut the unknown DNA molecules into small fragments.
Fluorescent markers are attached to the fragments and these get react with probes in DNA chips.

• DNA probes are then binds with the target DNA with complementary sequences and unbounded
DNA fragments are washed away.

• Identification of the target pieces of DNA is done by their fluorescene emission passing through a
laser beam and computer recorded the pattern of emission as well as DNA identification.

TYPES OF DNA MICROARRAY

There are four types of DNA microarray:

1. Oligo DNA microarray: It uses oligonucleotides of 20-50 nucleotides long. Oligonucleotides are
synthesized directly on the slide. Single color hybridization used for each probe. It has good
specifcity but poor sensitivity.

2. cDNA microarray: It is usually referred to as spotted microarray in within which DNA

fragments of any length (500bp-1kb) or oligos of 20-100 nts are stuck to the glassslides. It uses
two colors hybridization for every probe.

3. BAC Microarray: It uses the template which is amplified by polymerase chain reaction as the
probe.

4. SNA Microarray: It is used to detect polymorphisms within a population.

REQUIREMENTS:

1. DNA Chip

2. Target sample

3. Sample

4. Enzymes

5. Fluorescent dyes

6. Probes

• DNA microarrays are solid supports, usually of glass or silicon, upon which DNA is attached in an
organized pre-determined grid fashion.

• Each spot of DNA, called a probe, represents a single gene.

• DNA microarrays can analyze the expression of tens of thousands of genes simultaneously.

• There are several synonyms of DNA microarrays such as DNA chips, gene chips, DNA arrays,
gene arrays, and biochips.
Principle of DNA Microarray Technique

❑ The principle of DNA microarrays lies on the hybridization between the nucleic acid strands.

❑ The property of complementary nucleic acid sequences is to specifically pair with each other by
forming hydrogen bonds between complementary nucleotide base pairs.

❑ For this, samples are labeled using fluorescent dyes.

❑ At least two samples are hybridized to chip.

❑ Complementary nucleic acid sequences between the sample and the probe attached on the chip
get paired via hydrogen bonds.

❑ The non-specific bonding sequences while remain unattached and washed out during the washing
step of the process.

❑ Fluorescently labeled target sequences that bind to a probe sequence generate a signal.

❑ The signal depends on the hybridization conditions (ex: temperature), washing after hybridization
etc while the total strength of the signal, depends upon the amount of target sample present.

❑ Using this technology, the presence of one genomic or cDNA sequence in 1,00,000 or more
sequences can be screened in a single hybridization.
There are 2 types of DNA Chips/Microarrays:

1. cDNA based microarray

2. Oligonucleotide based microarray

Spotted DNA arrays (“cDNA arrays”)

• Chips are prepared by using cDNA.

• Called cDNA chips or cDNA microarray or probe DNA.

• The cDNAs are amplified by using PCR.

• Then these immobilized on a solid support made up of nylon filtre of glass slide (1 x 3 inches).
The probe DNA are loaded into a a spotting spin by capillary action.

• Small volume of this DNA preparation is spotted on solid surface making physical contact
between these two.

• DNA is delivered mechanically or in a robotic manner.

Oligonucleotide arrays (Gene Chips)

• In oligonucleotide microarrays, short DNA oligonucleotides are spotted onto the array.

• Small number of 20-25mers/gene.

• The main feature of oligonucleotide microarray is that each gene is normally represented by more
than one probe.

• Enabled by photolithography from the computer industry

• Off the shelf

Some key issues involved in microarray analysis

Parameter Issue

• Experimental design

❑ Consider the biological question(s) and the ability to achieve statistical significance

❑ Seek expert statistical advice during the early planning stages

❑ Microarray experiments have multiple sources of variation and must be carefully controlled

❑ Biological and technical replication are essential

❑ Sample pooling should be avoided if accurate sample synchronisation is not possible

❑ Microarray analysis of purified cells will only reveal genes expressed by these cells, but removal
from the in vivo microenvironment may alter gene expression

❑ There are limitations in the use of both whole tissue and purified cells, which may necessitate the
use of microdissection and RNA amplification techniques

❑ When using clinical samples, detailed patient history and tissue histopathology are critical to the
interpretation of gene expression profiles

Target RNA preparation

❑ The quality of the target RNA is one of the most important factors in the success or failure of a
microarray experiment

Data analysis
 ❑ While critical to the outcome of a microarray experiment, statistical analysis of microarray data is
not well understood by many biologists and expert advice should be sought

Data validation

❑ The biomedical research community does not yet accept that microarray data can stand alone
without independent validation

❑ The investigator must decide which genes to examine further, and those with larger fold changes
and statistical significance are often the best candidates

❑ To describe a biological event or system, gene expression data obtained by microarray analysis
must be extended to the study of protein products

Spotted DNA Microarrays

• The first DNA microarrays were spotted with probes that were made by oligonucleotide chemical
synthesis and then attached to the array.

• These probes have to be “spotted” or “printed” using a robot onto a very fine grid by a sort of
specialized inkjet-like printer, which uses the same technology as computer printers to expel
nanoliter to picoliter volume droplets of probe solution, instead of ink, onto the slide.

• Alternatively, these probes can be applied with a pin directly onto a specific location on the surface.
The number of spots (aka features) applied onto the DNA microarray is limited to prevent cross-
contamination problems.

•
Affymetrix platform include Agilent arrays that use an inkjet spotting process for in-situ
oligonucleotide synthesis, using five “ink” printing of the 4 nucleotide precursors plus catalyst,
combined with coupling and deprotection steps that do not require use of photolithographic masks.
 This technology relies on printing picoliter volumes of nucleotides on the array surface in repeated
rounds of base-by-base printing that extends the length of specific oligonucleotide probes.

This approach therefore allows synthesis of longer molecules (60-mer length oligos) for their probes.
Increased length improves specificity of probes but at increased complexity of design, which reduces the
number of features (Affymetrix chips usually feature >106 spots per microarray, compared to
0.24×106features for Agilent). In Situ-Synthesized DNA Microarrays

• In situ-synthesized arrays are high-density oligonucleotide probe DNA microarrays, with

Affymetrix GeneChip arrays being the most common.

• These are made using photolithography, which literally means to use light to create a pattern.

• The method relies on UV masking and light-directed combinatorial chemical synthesis on a solid
support to selectively synthesize probes directly on the surface of the array, one nucleotide at a time
per spot, for many spots simultaneously.

• This process works in the following way: a solid support contains covalent linker molecules that
have a protecting group on the free end that can be removed by light.

Affymetrix’s proprietary photolithography process for creating DNA microarrays uses a series of
photolithographic masks, light deprotection reactions and nucleotide coupling. During each
deprotection step, a specific mask is used with particular transparent “windows” to allow the light from
a single UV source to deprotect spots, or features on the array to receive a nucleotide.
 • UV light is directed through a photolithographic mask to deprotect and activate selected sites with
hydroxyl groups that initiate coupling with incoming protected nucleotides that attach to the
activated sites.

• The mask is designed in such a way that you can choose the exposure sites, and thus specify the
coordinates on the array where each nucleotide will be attached. The process is repeated, a new
mask is applied activating different sets of sites and coupling different bases, allowing arbitrary
DNA probes to be constructed at each site. This process is used to synthesize hundreds of thousands
of different oligonucleotides.

• However, it is the length of oligos, not their number, that determines the number of steps required,
since many different sites could be synthesized simultaneously.

• Each probe on the chip requires four masks per round of synthesis: one mask to allow addition of
the required base and three other masks to prevent light from deprotecting the same spot while the
other three nucleotides are being added. On average, each probe is 25 nucleotides long, requiring
about 100 masks per chip!!! These microarrays generally employ multiple probes for each gene to
improve specificity and feature a match/mismatch probe pair that enable the discrimination of
single mismatched base pairs.

High-Density Bead DNA microarrays

• Another type of high-density DNA microarray are the BeadArrays manufactured by Illumina.

• Illumina’s Bead Array Technology is based on color-coded 3-micron silica beads that randomly
self assemble in either a fiber-optic bundle substrate that then themselves assemble into arrays, or
a silica slide substrate.
• When randomly assembled on one of these two substrates, the beads have a uniform spacing of
approximately 5.7 microns, with a packing density of about 40,000 array elements per square
millimeter.

• This gives the Bead Array platform about 400 times the information density of a typical spotted
array.

• Each bead is covered with hundreds of thousands of copies of a specific oligonucleotide that act
as the capture sequences in one of Illumina’s assays. Each bead has a 23-mer oligo “address”
attached to it, which then anchors a 50-mer sequence-specific oligo probe.

• The beads are randomly scattered across etched substrates during the array production process,
with each array bundle containing about 50,000 beads.

• With this platform design, a specific oligonucleotide sequence is assigned to each bead type, but
is replicated about 30 times on the array at random positions.

• Each gene is represented by two probe sequences. A series of decoding hybridizations are used to
determine which oligos are present at each matrix coordinate for every array.
•
Affymetrix GeneChip® System for Gene Expression Analysis

The Affymetrix GeneChip system is a commercial microarray platform that allows whole genome gene
expression analysis for a wide variety of experimental organisms
([Link]
This system has three major advantages over other array systems: it is easy to get rapid results; it has the
capability to monitor the expression of every gene in the genome; and it is the most widely used
commercial microarray platform.

Affymetrix GeneChip® miRNA Array

The GeneChip® miRNA Array is a powerful and cost effective tool for studying the role of microRNAs
(miRNAs). The array provides comprehensive miRNA coverage (Sanger miRNA database V20 content
and additional human small nuclear RNAs (snoRNAs and scaRNAs)) with multiple organisms (Human,
mouse, rat, canine, and monkey) on a single array.

Input amounts: 0.13 – 3µg of total RNA or low molecular weight RNA enriched from 0.1-3 ug of total
RNA (for new users we recommend to start with 1 ug of total RNA or LMW RNA enriched from 1 ug of
total RNA).

Affymetrix microarray solutions are now branded Applied Biosystems and include all necessary
components for a microarray experiment, from arrays and reagents to instruments and software. Our
solutions enable scientists and clinicians to understand underlying disease mechanisms, identify
biomarkers for personalized medicine, create novel molecular diagnostic tests, and improve genetic
marker-assisted breeding programs in agriculture, thereby translating research results into biology for a
better world.

Key applications

Transcriptome Analysis

Phenotypic abnormalities are rarely a result of expression changes in single genes, so generating a
comprehensive expression profile is critical when studying normal biology and disease processes. Profile
all known coding and non-coding splice variants.
Human Genotyping for Precision Medicine Research

Large-scale genotyping studies aimed at improving understanding disease risk and drug response are
helping to pave the way toward precision medicine. To be successful, these studies require affordable,
high-density genotyping arrays with accurate imputation and assurance that every marker will be on every
array, every time.

Cytogenetics Analysis

Microarray-based assays provide a genome-wide approach that enables high-solution DNA copy number
analysis to detect gains, losses, loss/absence of heterozygosity (LOH/AOH), copy-neutral LOH (cnLOH),
regions identical-by-descent, and mosaicism in a single assay.

miRNA Profiling

Perform comprehensive miRNA profiling from as little as 130 ng and start exploring the role of miRNA
in.24 hours—no bioinformatics resources required.

Large-scale Biobank Genotyping

Our Axiom Biobank Genotyping Arrays feature imputation-aware modular designs that enable scientists
to conduct large-scale, state-of-the-art traits and population studies that help us understand how complex
interactions between genes, environment, and lifestyle relate to health.

Plant and Animal Genotyping

Agrigenomics research is growing as climate change, population growth, and urbanization threaten
farmers’ ability to meet the world’s food demands. To address these needs, breeders and farmers are
employing new genomic strategies. Our powerful, flexible array-based genotyping solutions can help.

The goal of microarray image analysis is to extract intensity descriptors from each spot that represent
gene expression levels and input features for further analysis. Biological conclusions are then drawn
based on the results from data mining and statistical analysis of all extracted features.

Components of DNA Microarray image analysis are (1) Grid Alignment Problem, (2) Foreground
Separation, (3) Quality Assurance, (4) Quantification and (5) Normalization. Additionally, the data
management must conform with the Minimal Information About Microarray Experiments (MIAME)
standard.

Input: Laser image scans (data) and underlying experiment hypotheses or experiment designs (prior
knowledge).
Output: Conclusions about statistical behavior of measurements and thus the the test of the hypotheses or
knowledge. The results are derived automatically from data (machine learning perspective) for
subsequent model fitting.

Applications of Microarrays

• Gene Discovery: DNA Microarray technology helps in the identification of new genes, know about
their functioning and expression levels under different conditions.

• Disease Diagnosis: DNA Microarray technology helps researchers learn more about different
diseases such as heart diseases, mental illness, infectious disease and especially the study of cancer.
Until recently, different types of cancer have been classified on the basis of the organs in which the
tumors develop. Now, with the evolution of microarray technology, it will be possible for the
researchers to further classify the types of cancer on the basis of the patterns of gene activity in the
tumor cells. This will tremendously help the pharmaceutical community to develop more effective
drugs as the treatment strategies will be targeted directly to the specific type of cancer.

• Drug Discovery: Microarray technology has extensive application in Pharmacogenomics.

Pharmacogenomics is the study of correlations between therapeutic responses to drugs and the
genetic profiles of the patients. Comparative analysis of the genes from a diseased and a normal
cell will help the identification of the biochemical constitution of the proteins synthesized by the
diseased genes. The researchers can use this information to synthesize drugs which combat with
these proteins and reduce their effect.

• Toxicological Research: Microarray technology provides a robust platform for the research of the
impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes
correlation between responses to toxicants and the changes in the genetic profiles of the cells
exposed to such toxicants.
UNIT – 2- SBIA5304-MICROARRAY DATAANALYSIS
UNIT II IMAGE PROCESSING

Image processing, feature extraction, identifying positions of features- Normalization – data cleaning and
transformation, within array normalization, between array normalization, measuring and quantifying
microarray variability –variability between replicate features on an array-, variability between
hybridizations to different arrays. Analysis of differentially expressed genes- significance analysis of
microarrays.

Microarray Image Analysis

• Microarray image processing leads to the characterization of gene expression levels

simultaneously, for all cellular transcripts (mRNAs) in a single experiment.

• The calculation of expression levels for each microarray spot/gene is a crucial step to extract
valuable information.

• By measuring the mRNA levels for the whole genome, the microarray experiments are capable to
study functionality, pathological phenotype, and response of cells to a pharmaceutical treatment.
The processing of the extensive number of non-homogeneous data contained in microarray
images is still a challenge.
• Typically, the microarray images are stored in the Tagged Image File Format (TIFF) as a two-
dimensional array of intensities.

• In a two colour microarray experiment, two microarray images are available, each image being
recorded from a specific cyanine dye.

• The images are denoted by ICy3 and ICy5, corresponding to Cy3 and Cy5 dyes, respectively.

• Microarray grid alignment and foreground separation are the basic processing steps of DNA
microarray images that affect the quality of gene expression information, and hence impact our
confidence in any data-derived biological conclusions.

• Thus, understanding microarray data processing steps becomes critical for performing optimal
microarray data analysis.

• The workflow of microarray data processing starts with raw image data acquired with laser
scanners and ends with the results of data mining that have to be interpreted by biologists.

• The microarray data processing workflow includes issues related to

• data management (e.g., MIAME compliant database,

• (2) image processing (grid alignment, foreground separation, spot quality assessment, data
quantification and normalization,
• (3) data analysis (identification of differentially expressed genes, data mining, integration with
other knowledge sources, and quality and repeatability assessments of results, and

• (4) biological interpretation (visualization).

• The main objective of this project is related to image processing, namely grid alignment,
foreground separation, spot quality assessment, data quantification, normalization and
visualization.

• Microarray data processing workflow: Fluorescent DNA microarray images obtained from laser
scanners containing a 2D array of dots with two channels of 532nm (red) and 632nm (green)
wavelengths.

• The grid alignment is performed producing a set of lines intersecting at each dot.

• Dots define a valid foreground.

• Quality assurance screening eliminates grid cells with unreliable microarray information.

• Finally, image of sample mean values extracted at each grid cell using particular mask is
extracted and colored in a red-green-blue space with color assigned to each cluster/pixel.
• Statistics of each cluster can be viewed in the text area

• Microarray images represent a collection of microarray spots arranged in one or more sub-grids,
each grid representing a two dimensional array of spots.

• Image processing technique are used further on in order to determine spot location within each
subgrid, spot sizes, spot intensities and background intensities values which are typically delivered
as raw data parameters for microarray image analysis and interpretation

• A typical microarray image is generated from an array of cDNA probes which is hybridized
to two samples, one being red fluor-tagged and the other green fluor-tagged. The composite
color image is constructed by placing each monochrome image into the appropriate color
channel.

• The tasks of microarray image analysis can be further-divided into following tasks:

1. Array target segmentation

2. Background intensity extraction

3. Target detection

4. Target intensity extraction

5. Normalization and Ratio analysis

6. Measurement quality assessment

7. software package based interpretation

Segmentation is the method of segregating a spitting an image into multiple fundamental fragments. The
segmentation phase of the image study shows a key role in the statistical analysis, a step where the data is
produced.

Four categories of methods for microarray image segmentation are

(a) fixed /adaptive circle segmentation

(b) Histogram based techniques

(c) adaptive shape segmentation

(d) Machine learning techniques.

Thus the integration of machine learning in Image processing will contribute a better analysis of medical
and biological data

• The fourth category is based on machine learning techniques.

• There are two categories in this method.

• They are

• (a) supervised segmentation techniques

• (b) unsupervised segmentation technique.

• More specifically, methods in unsupervised category employ clustering algorithms, such as k-

means, hybrid k-means, fuzzy c-means,expectation-maximization and partitioning method for
segmentation of microarray images.

• An important first step of any microarray experiment is the normalization of the samples.

• Although the relative impacts differ from platform to platform and sample preparation, non-
biological differences in microarray signals can stem from a variety of factors, such as: global
constant background noise, non-specific binding signal, non-linear signal response between
samples, bad spots on the chip due to dust or bubbles or rare manufacturing defects, labeling
efficiency, hybridization efficiency, and RNA quality.

• Statistical analysis of microarray data is started through software programs using CEL files
defined as raw data.

• Prior to the start of the analysis, quality assessment of raw data is performed as the first step.
 • In order to evaluate the homogeneity of the arrays and to compare the density distribution
between the arrays, box graphs are plotted for each array using the densities of the logarithm2
base of the raw data.

• Images of the CEL files are obtained to observe the dimensional distributions of the densities on
each array and to detect dimensional artifacts.

• CEL file: Cell intensity file, probe level PM and MM values.

Each gene or portion of a gene is represented by 1 to 20 oligonucleotides of 25 base-pairs.

Probe: an oligonucleotide of 25 base-pairs, i.e., a 25-mer

• Perfect match (PM): A 25-mer complementary to a reference sequence of interest (e.g., part of a
gene).

• Mismatch (MM): same as PM but with a single base change for the middle (13th) base
(transversion purine <-> pyrimidine, G <->C, A <->T). Used to measure non-specific binding and
background noise.

• Probe-pair: a (PM,MM) pair. Probe-pair set: a collection of probe-pairs (1 to 20) related to a

common gene or fraction of a gene.

• Affy ID: an identifier for a probe-pair set.

• MA-plots are used to compare the expression values for all possible pair of arrays with a
probeset-wise median array.

• The MA plots are generated by plotting M values which are obtained by logarithmic ratios versus
A values which are average logarithmic intensity values.

• The pre-normalization quality control step can be complemented by histograms drawn to assess
the density distributions of each array

• After quality control of raw data, background correction and normalization should be applied to
the data using background correction methods such as RMA (Robust Multiple-Array Average)
method.

• With the RMA method, the probe-level signal is removed from the background signal.

• Quantile normalization is performed by the RMA method and it is ensured that all the arrays have
the same quantile.

• Using the RMA method, the expression set to be used in the analysis is generated by normalized
and the background corrected intensities.

• After the background correction and the normalization methods are performed, box charts related
to each array are drawn to re-evaluate the quality control.
 • Following normalization and background correction, a list of genes that differ between two
different conditions can be obtained by applying various statistical tests to the expression dataset
to be used for analysis

Preprocessing of microarray data

• Measurement values may have undergone various adjustments in the device system, such as
calibration.

• Thus, in the presentation of gene expression data, it must be explained how the values are
generated by the device system.

• These expression measures always contain a component called “background noise.”

• Local background noise levels are measured from the areas of the glass slide that do not contain
probes.

• The background correction tries to remove non-specific background noise and local variations of
the overall signal level on each chip.

• The most common method to remove the background effect is to remove the measured
fluorescence intensity around the spots.
The most common methods used for background correction in microarray analysis are; The “Robust
Multi-Array Average (RMA) Background Correction” method and the “MAS 5.0 Background
Extraction” methods
 • RMA background correction has been one of the most commonly used pre-processing method in
the recent literature.

• Performed assessments of the measure’s precision, consistency of fold change, and specificity
and sensitivity of the measure’s ability to detect differential expression and demonstrated the
substantial benefits of using the RMA measure to users of the Gene Chip technology.

• They used data from spike-in and dilution experiments to conduct various assessments on the
MAS 5.0, dChip and RMA expression measures.

• Irizarry have demonstrated that RMA has similar accuracy but better precision than the other two
summaries and RMA provides more consistent estimates of fold change

Quality assessment:

• It is necessary to evaluate the quality of the data before the normalization of the arrays. Quality
control assessment should be carried out to determine whether the quality of experimental data is
acceptable and whether any hybridization should be repeated.
• Various descriptive data plots are drawn to identify potential problems with hybridization or other
experimental structures in the quality control evaluation process.

• Quality control plots are basically divided into diagnostic and spot statistics .

• Diagnostic Plots: The diagnostic plots include various plots such as MA-plots for evaluating
intensity bias and histograms for examining signal-to-noise ratios for each channel.

• Diagnostic plots are usually used to observe non-linear trends between two channels
Histograms:

• In microarray designs, it is very important to obtain the histograms of the p-values of tests
conducted to identify different gene expression.

• Histograms are graphs that are easy to interpret and contain considerable information.

• A histogram is an indication of whether there is a signal in the gene and whether the genes are
differently expressed. Histograms also allow for estimation of how many genes are differentially
expressed in reality.

Spot statistics plots:

• Spot Statistics help to predict the structures of spot and hybridizations.

 • The main plots that can be obtained with spot statistics are spatial plots, box plots, scatter
diagrams and volcanic plots .

Spatial plots:

• Spatial plots are used to reveal irregular spot and hybridization structures.

• Spatial plots are used to observe the spatial distributions of the intensities on each array and to
detect the artifacts.

• Spatial plots play a fundamental role in determining the background correction, depending on
whether there are any dimensional artifacts on the arrays
Box plots:

Box plots are one of the most commonly used plots for displaying spot and hybridization structures. At the
same time, box plots can be drawn to understand the scale differences between different arrays. It is
necessary to evaluate the box plots to see if between-array normalization is required. The homogeneity of
the arrays can be observed quite clearly from the box plots .

Scatter diagrams:
• Scatter diagrams used to compare the expression values of two samples are the most commonly
used plots for visualizing microarray data.

• In the first step of the microarray data analysis, a scatter diagram is drawn between the two
intensity channels to view the general structures and variability.

• Scatter diagrams are also commonly used to find out slides lying away from the center, which
have abnormal expression structures .

Volcano plots:

• Volcano Plots are used to summarize fold change and t-test criteria.

• A volcano plot is constructed by plotting the negative log of the p-value on the y-axis and log of
the fold change between the two conditions on the x-axis.

• For each gene, there is a point on the graph that represents the t-statistic and the fold change

• A volcano plot of the genes in microarray. The Log 2 fold changes and their corresponding-log 10
p-value of all genes were taken for construction of the volcano plot in the microarray. The genes
 with p < 0.05 are depicted in blue dots. All other genes that were not found to be significant
altered are in black dots in this array

• Normalization

• The aim of normalization methods for large scale expression data, including microarray and
RNA-seq, is to eliminate systematic experimental bias and technical variation while preserving
biological variation.

• Dozens of normalization methods for correcting non-linear experimental differences between

arrays have been developed during the last two decades. Among them, quantile and lowess are
well-adopted for analyzing microarray expression data.

• Expression ratios: the primary comparison

• Most microarray experiments investigate relationships between

• related biological samples based on patterns of expression, and

• the simplest approach looks for genes that are differentially

• expressed.

• If we have an array that has N array distinct elements,and compare a query and a reference
sample, which for convenience we will call R and G, respectively (for the red and green colors
commonly used to represent array data), then the ratio (T) for the ith gene (where i is an index
running over all the arrayed genes from 1 to Narray) can be written as

Normalization:

• The purpose of the normalization phase is to adjust the data according to the technical variation.
Variations can cause measurement differences between general fluorescence intensity levels of
various arrays. The normalization process is necessary to make the measured values obtained
from different arrays comparable.
• Normalization methods depend on which microarray technology is used. Generally,
logarithmically transformed data are used for further analysis.

• The most commonly used methods of normalization are as follows

1. Scaling Normalization Method

2. Nonlinear Normalization Methods

3. Quantile Normalization

4. Cyclic Loess Normalization

5. Contrast Normalization

• Normalization

• Typically, the first transformation applied to expression data,referred to as normalization, adjusts

the individual hybridization.

• (Note that this definition does not limit us to any particular array technology: the measures Ri and
Gi can be made on either a single array or on two replicate arrays. Furthermore, all the
transformations described below can be applied to data from any microarray platform.)

• Although ratios provide an intuitive measure of expression changes, they have the disadvantage
of treating up- and downregulated genes differently. Genes upregulated by a factor of 2 have an
expression ratio of 2, whereas those downregulated by the same factor have an expression ratio of
(–0.5).

• The most widely used alternative transformation of the ratio is the logarithm base 2, which has
the advantage of producing a continuous spectrum of values and treating up- and downregulated
genes in a similar fashion.

• Recall that logarithms treat numbers and their reciprocals symmetrically: log2(1) = 0, log2(2) = 1,
log2(1⁄ 2) = −1, log2(4) = 2, log2(1⁄ 4) = −2, and so on.

• The logarithms of the expression ratios are also treated symmetrically, so that a gene upregulated
by a factor of 2 has a log2(ratio) of 1, a gene downregulated by a factor of 2 has a log2(ratio) of
−1, and a gene expressed at a constant level (with a ratio of 1) has a log2(ratio) equal to zero.

• For the remainder of this discussion, log2(ratio) will be used to represent expression levels

• There are three major normalization methods that are commonly employed:
• linear scaling (MAS5), quantile normalization (RMA), and pair-wise rank-invariant
normalization (dChip).

• Linear normalization is the simplest of the methods, which applies a global scaling factor to each
chip (at the probeset level in MAS5) in order to scale all chips to the same trimmed mean
intensity.

• Quantile normalization ranks the intensities for each chip, then replaces the intensities at each
rank with the mean intensity for all probes of that rank across all chips, effecting a non-linear
rank-dependent normalization.

• Pair-wise rank-invariant normalization normalizes all chips against a single reference chip by
identifying a different subset of rank-invariant genes for each sample/reference chip pair, fitting a
curve through the training set, then adjusting the intensities of the target chip in an intensity-
dependent manner so that the fit curve will lie on the sample vs. reference diagonal of the
scatterplot.

• Linear normalization is unable to correct for non-linear, intensity-dependent differences in gene

expression between chips, but can be applied to a single chip, independently of other chips.

• Quantile normalization assumes that differential gene expression is symmetric, in that there will
be a roughly equal number of up and down regulated genes with equal magnitude distributions.
Due to its population-based signal, it requires a moderately large number of chips in order to
work well, and may introduce unexpected artifacts, particularly in outlier samples, in small
experiments, or experiments in which different cell/tissue types are represented.

• Rank-invariant normalization makes similar assumptions to those of quantile normalization,

since both are rank based, but can be applied to as few as two chips.

• The three most commonly used software packages for processing Affymetrix microarrays, as
evidenced by recently querying the GEO and ArrayExpress microarray repositories, are: RMA,
MAS5 , and dChip

• Each of these employs different methods for background subtraction, signal normalization, and
probeset summarization (an issue unique to Affymetrix arrays, where multiple probes for the
same transcript are condensed into a single representative signal).

• MAS5.0
• A significant challenge with Affymetrix expression data is to provide an algorithm that combines
the signals from the multiple Perfect-Match (PM) and Mismatch (MM) probes that target
each transcript into a single value that sensitively and accurately represents its concentration.

• MAS5.0 does this by calculating a robust average of the (logged) PM-MM values; increased
variation is observed at low signal strengths and is at least in part due to the extra noise generated
by subtracting the MM values from their PM partners.

• Many gene expression normalization algorithms exist for Affymetrix GeneChip microarrays.

• The most popular of these is RMA, primarily due to the precision and low noise produced during
the process.

• A significant strength of this and similar approaches is the use of the entire set of arrays during
both normalization and model-based estimation of signal.

•
Quantile normalization is an important normalization technique commonly used in high-
dimensional data analysis.

• However, it is susceptible to class-effect proportion effects (the proportion of class-correlated

variables in a dataset) and batch effects (the presence of potentially confounding technical
variation) when applied blindly on whole data sets, resulting in higher false-positive and false-
negative rates.
• Pair-wise rank-invariant normalization (dchip)

• Pair-wise rank-invariant normalization normalizes all chips against a single reference chip by
identifying a different subset of rank-invariant genes for each sample/reference chip pair, fitting a
curve through the training set, then adjusting the intensities of the target chip in an intensity-
dependent manner so that the fit curve will lie on the sample vs. reference diagonal of the
scatterplot.

Differential gene expression analysis

• Differential expression analysis means taking the normalised read count data and performing
statistical analysis to discover quantitative changes in expression levels between experimental
groups.

• For example, we use statistical testing to decide whether, for a given gene, an observed difference
in read counts is significant, that is, whether it is greater than what would be expected just due to
natural random variation.

• Methods for differential expression analysis

• There are different methods for differential expression analysis such as edgeR and DESeq based
on negative binomial (NB) distributions or baySeq and EBSeq which are Bayesian approaches
based on a negative binomial model.

• It is important to consider the experimental design when choosing an analysis method.

• While some of the differential expression tools can only perform pair-wise comparison, others
such as edgeR,

limma-voom, DESeq and maSigPro can perform multiple comparisons.

•
GEO2R performs comparisons on original submitter-supplied processed data tables using
the GEOquery and limma R packages from the Bioconductor project.

• Bioconductor is an open source software project based on the R programming language that
provides tools for the analysis of high-throughput genomic data.
• The GEOquery R package parses GEO data into R data structures that can be used by other R
packages. The limma (Linear Models for Microarray Analysis) R package has emerged as one of
the most widely used statistical tests for identifying differentially expressed genes.

• It handles a wide range of experimental designs and data types and applies multiple-testing
corrections on P-values to help correct for the occurrence of false positives. Thus, GEO2R
provides a simple interface that allows users to perform R statistical analysis without command
line expertise.
UNIT – 3- SBIA5304-MICROARRAY DATAANALYSIS
UNIT III PREDICTION

• Prediction of cross hybridization to related genes,

• Thermodynamics of nucleic acid duplexes,

• Prediction of Tm- probe

• Dimensionality reduction, principal component analysis,

• Machine learning methods for cluster analysis; Hierarchical clustering

• Analysis of relationships between genes, tissues or treatments- similarity of gene or sample

profiles –Classification of tissues and samples – validation.

• Cross-hybridization is the tendency for chains of nucleic acids to bind to other chains of nucleic
acids that have similar but not identical sequences.

• This has the potential to make the interpretation of microarray experiments difficult since
intensity at a spot on the array does not simply depend on the quantity of target in the sample.
CrossHybDetector: detection of cross-hybridization events in DNA microarray experiments

• DNA microarrays contain thousands of different probe sequences represented on their surface.

• These are designed in such a way that potential cross-hybridization reactions with non-target
sequences are minimized.

• However, given the large number of probes, the occurrence of cross hybridization events cannot
be excluded.

• This problem can dramatically affect the data quality and cause false positive/false negative
results.

• CrossHybDetector is a software package aimed at the identification of cross-hybridization events

occurred during individual array hybridization, by using the probe sequences and the array
intensity values.

• As output, the software provides the user with a list of array spots potentially 'corrupted' and their
associated p-values calculated by Monte Carlo simulations.

• Graphical plots are also generated, which provide a visual and global overview of the quality of
the microarray experiment with respect to cross-hybridization issues.

• CrossHybDetector is a software package aimed at the identification of cross-hybridization events

occurred during individual array hybridization, by using the probe sequences and the array
intensity values.

• As output, the software provides the user with a list of array spots potentially 'corrupted' and their
associated p-values calculated by Monte Carlo simulations.

• Graphical plots are also generated, which provide a visual and global overview of the quality of
the microarray experiment with respect to cross-hybridization issues.
R is ‘GNU S’, a freely available language and environment for statistical computing and graphics which
provides a wide variety of statistical and graphical techniques: linear and nonlinear modelling, statistical
tests, time series analysis, classification, clustering, etc.

CRAN is a network of ftp and web servers around the world that store identical, up-to-date, versions of
code and documentation for R.

CrossHybDetector is implemented as a package within the statistical computing environment R.

Functions of marray and methods R packages are internally utilized and are required
by CrossHybDetector to work.

Data formats

CrossHybDetector algorithm uses as input data

i) the array probe sequences, ii) the spot intensities and array layout, iii) the spot type information
(i.e. for each spot, whether it is "standard probe", "negative control", "spike-in").

This information is respectively contained into three separated text files.

Thermodynamics of nucleic acid duplexes

• Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of
double-stranded DNA (dsDNA).

• The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are
in the random coil or single-stranded (ssDNA) state.
• Tm depends on the length of the DNA molecule and its specific nucleotide sequence.

• DNA, when in a state where its two strands are dissociated (i.e., the dsDNA molecule exists as
two independent strands), is referred to as having been denatured by the high temperature.

DNA hybridization

• DNA is a nucleic acid that contains the genetic instructions monitoring the biological
development of all cellular forms of life, and many viruses.

• DNA is a long polymer of nucleotides and encodes the sequence of the amino-acid residues in
proteins using the genetic code, a triplet code of nucleotides. DNA it is organized as two
complementary strands, head-to-toe, with the hydrogen bonds between them.

• Each strand of DNA is a chain of chemical “building blocks”, called nucleotides, of which there
are four types: adenine (A), cytosine (C), guanine (G) and thymine (T).

• Between the two strands, each base can only bond with one single predetermined other base: A
with T, T with A, C with G, and G with C, being the only possible combination.

• Hybridization refers to the annealing of two nucleic acid strands following the base pairing rule.

• As shown in Fig at high temperatures approximately 90°C to 100°C the complementary strands
of DNA separate, denature, yielding single-stranded molecules.

• Two single strands under appropriate conditions of time and temperature e.g. 65°C, will re-
naturate to form the double stranded molecule.

• Nucleic acid hybrids can be formed between two strands of DNA, two strands of RNA or one
strand of DNA and one of RNA.

• Nucleic acids hybridization is useful in detecting DNA or RNA sequences that are
complementary to any isolated nucleic acid.
• The nucleic acid duplex stability can be endangered by the interaction between the nucleotide bases.
Thermodynamics for double helix formation of DNA/DNA, RNA/RNA or DNA/RNA can be
estimated with nearest neighbour parameters.

• Enthalpy change, ΔH∘, entropy change, ΔS∘, free energy change, ΔG∘, and melting temperature,
Tm, were obtained on the basis of the nearest-neighbour model.

• The nearest-neighbour model for nucleic acids, known as the NN model, assumes that the stability
of a given base pair depends on the identity and orientation of neighbouring base pairs

• In the NN model, sequence dependent stability is considered in terms of nearest-neighbour

doublets.

• In duplex DNA there are 10 such unique internal nearest-neighbour doublets.

• Listed in the 5’-3’ direction, these are AT/AT TA/TA AA/TT AC/GT CA/TG TC/GA CT/AG
CG/CG GC/GC and GG/CC.

• Dimmer duplexes are represented with a slash separating strands in antiparallel orientation e.g.
AC/TG means 5’-AC-3’ Watson–Crick base-paired with 3’-TG-5’.

• The total difference in the free energy of the folded and unfolded states of a DNA duplex can be
approximated at 37o, with a nearest-neighbour model:
•
• The nearest-neighbour parameters of Delcourt et al., SantaLucia et al., Sugimoto et al. and
Allawi et [Link] evaluated from the analysis of optical melting curves of a variety of short
synthetic DNA duplexes in 1 M Na+.

• The observed trend in nearest-neighbor stabilities at 37°C is GC/CG = CG/GC > GG/CC >
CA/GT = GT/CA = GA/CT = CT/GA > AA/TT > AT/TA > TA/AT, as in Table 2.

• This trend suggests that both sequence and base composition are important determinants of DNA
duplex stability. It has long been recognized that DNA stability depends of the percent G-C
content.

Prediction of Tm- probe

Dimensionality reduction in microarray

• The number of input variables or features for a dataset is referred to as its dimensionality.

• Dimensionality reduction refers to techniques that reduce the number of input variables in a
dataset.

• More input features often make a predictive modeling task more challenging to model, more
generally referred to as the curse of dimensionality.

• High-dimensionality statistics and dimensionality reduction techniques are often used for data
visualization. Nevertheless these techniques can be used in applied machine learning to simplify a
classification or regression dataset in order to better fit a predictive model.
• Traditionally manual management of the high dimensional data set is more challenging.

• With the advent of data mining and machine learning techniques, knowledge discovery and
recognition of patterns from these data can be done automatically.

• However, the data in the database is filled with a high level of noise and redundancy.

• One of the reasons causing noise in these data is an imperfection in the technologies that collected
the data and the source of the data itself is another reason.

• Dimensionality reduction is one of the famous techniques to remove noisy (i.e. irrelevant) and
redundant features.

• For data mining techniques such as classification and clustering dimensionality reduction is
treated as preprocessing task for better performance of the model.

• Dimensionality reduction techniques can be classified mainly into feature extraction and
feature selection.

• Feature extraction approaches set features into a new feature space with lower dimensionality and
the newly constructed features are usually combinations of original features.

• On the other hand, the objective of feature selection approaches is to select a subset of features
that minimize redundancy and maximize relevance to the target such as the class labels in
classification.

• Therefore, both feature extraction and feature selection are capable of improving learning
performance, lowering computational complexity, building better-generalized models, and
decreasing required storage.

• Fig shows the classification of dimension reduction process and the data set in which these are
generally applied in the literature.
• Feature selection selects a group of features from the original feature set without any changeover
and maintains the physical meanings of the original features.

• Therefore, feature selection is superior in terms of better readability and interpretability.

• One of the applications would be in gene microarray data analysis.

• Feature selection has its significance in many real-world applications such as finding relevant
genes to a specific disease in Microarray data, analysis of written text, and analysis of medical
images, analysis of the image for face recognition and for weather forecasting.

There are four basic stages in feature selection method:

Generation Procedure (GP), to select candidate feature subset

 Evaluation Procedure (EP), to evaluate the generated candidate feature subset and output, a
relevancy value

Stopping Criteria (SC): To determine when to stop

Validation Procedure (VP): To determine whether it is the optimal feature subset or not.

Generation Procedure (GP)

This procedure generates a subset of features that is relevant to the target concept.

GP are of two types

Individual Ranking

Measures the relevance of each feature. The feature relevance is measured based on some
evaluation function. In this case, each individual feature is evaluated by assigning some weight or score.

Subset Selection

A subset of features is selected based on some search strategy. If the size of the data set is N×M,
then a total number of features in the data set is N. The possible number of subsets of features is 2N. This
is even very large for a medium sized feature set. Therefore suitable search strategy is applied to this
process.

The search is classified as:

A. Complete: It traverses all the feasible solutions. This procedure does an exhaustive search for
the best possible subset pertaining to the evaluation function. Example of complete search is a branch and
bound best first search.

B Heuristic Deterministic: uses a greedy strategy to select features according to local change.
There are many alternatives to this straightforward method, but the creation of subset is basically
incremental. Examples of this procedure are sequential forward selection, sequential backward selection,
sequential floating forward selection, and sequential floating backward selection.

C. Nondeterministic (Random): It attempts to find an optimal solution in a random fashion. This

procedure is new in the field of feature selection methods compared to the above two categories.
Optimality of the selected subset depends on the resources available.

Evaluation Procedure (EP)

 An optimal subset is always relative to a certain evaluation function. An evaluation function tries
to measure the discriminating ability of a feature or a subset to distinguish the different class labels.

The evaluation function is categorized as distance, information (or uncertainty), dependence,

consistency, and classifier error rate.

Distance Measures

For a two-class problem say A and B are two features, then A and B are selected on the basis of
their distance (e.g. Euclidian distance). If the distance is zero then the features are said to be redundant
and ignored. The higher the distance the more the features are discriminating.

Information Measures

This determines the information gain for the feature. Feature A is preferred over feature B if the
information gain of A is more than B (e.g. entropy measure).

Dependence Measures

Dependence or correlations of the ability to predict the value of one variable from the value of
another. If the correlation of feature A with class C is higher than the correlation of feature B with class C
then feature A is preferred to B. This measure finds the minimally sized subset that satisfies the
acceptable inconsistency rate that is usually set by the user.

Consistency Measure

This measure finds the minimally sized subset that satisfies the acceptable inconsistency rate that
is usually set by the user.

Classifier Error Rate

The evaluation function is the classifier itself. It measures the accuracy of the classifier for
different subsets of feature set and measures the error rate for the different subset. We have classified the
feature selection method as non-soft computing based and soft computing based. Based on the generation
procedure and evaluation function, the feature selection methods are classified, where the generation
procedure and evaluation functions are two dimensions.

Stopping Criteria

It indicates the end of the process. Commonly used stopping criteria are:

(i) When the search completes

(ii) When some given bound (minimum number of features or a maximum number of iterations) is
reached.

(iii) When a subsequent addition (or deletion) of any feature does not produce a better subset and

(iv) When a sufficiently good subset (e.g. a subset if its classification error rate is less than the
allowable error rate for a given task) is selected.

Feature selection approaches are primarily categorized as a filter, wrapper, and embedded
method.

Recently other feature selection methods are gaining popularity i.e., hybrid and ensemble
methods (Fig).

•
Filter method deals with individual ranking as well as subset selection.

• The individual ranking is based on the evaluation functions such as distance, information,
dependence, and consistency excluding the classifier (Fig).

• Filter techniques judge the relevance of genes by looking only at the intrinsic properties of the
data. In microarray data, a gene relevance score is calculated, and low-scoring genes are removed.
Afterward, this subset of genes is presented as input to the classification algorithm.

• The filtering technique can be used as a pre-processing step to reduce space dimensionality and
overcome overfitting.
• The filter approach is commonly divided into two different sub-classes:

• Individual evaluation and subset evaluation.

• In individual evaluation method, the gene expression dataset is given as input. The method has an
inbuilt evaluation process according to which a rank is provided to each individual gene based on
which the selection is done.

• Different criteria can be adopted, like setting a threshold for the scores and selecting the genes
which satisfy the threshold criteria, or sometimes the threshold can be chosen in such a way that a
maximum number of genes can be selected.

• Then, the subset selected can be the final subset which is used as the input to the classifiers. In
subset selection, all GP and evaluation function excluding the classifier can be taken for the
combination

• However, methods in this framework may suffer from an inevitable problem, which is caused by
searching through

• the possible feature subsets.

• The subset generation process usually increases the computational time but gives more relevant
feature subset.

• In literature, it is found that the subset evaluation approach outperformed the ranking methods

• The filter method is again classified into the ranking method and space search method.

• [Link] the taxonomy of filter feature selection method.

• Taxonomy of filter FS methods: Pros of Filter Feature Selection Method.

• The method is simple and fast.

• It scales well to high dimensional data.

• It is independent of classifiers.

• Cons of Filter Feature Selection Method

• The method is generally univariate or low variate.

Wrapper Method

• In the wrapper approach, all GP can be taken in combination with the classifier as evaluation
function and generates the relevant feature subset.

• Wrappers are feedback methods, which incorporate the machine-learning algorithm in the feature
selection process, i.e., they rely on the performance of a specific classifier to evaluate the quality
of a set of features.

• Wrapper methods search through the space of feature subsets and calculate the estimated
accuracy of a single learning algorithm for each feature that can be added to or removed from the
feature subset.

• The search may be a GP and the evaluation function is a classifier.

 Wrapper Method

1. How to find all possible feature subsets for evaluation?

2. How to satisfy oneself with the classification performance of the chosen classifier in order to
guide the search and what should be the stopping criteria?

Which predictor to use?

The wrapper approach of feature selection is classified as sequential search based and Heuristic
search based.

The taxonomy of the wrapper model is given in Fig

• Usually, an exhaustive search is too expensive, and thus non-exhaustive, heuristic search
techniques like genetic algorithms, greedy stepwise, best first or random search are often used.

• Here, feature selection occurs externally to the induction method using the method as a
subroutine rather than as a post-processor.

• In this process, the induction algorithm is called for every subset of feature consequently inducing
high computational cost

• Embedded Method

• Despite the lower time consumption of the filter method, a major limitation of the filter approach
is that it is independent of the classifier, usually resulting in worse performance than the
wrappers.

• However, the wrapper model comes with an expensive computational cost, which is particularly
aggravated by the high dimensionality of microarray data.

• An intermediate solution for researchers is the use of hybrid or embedded methods, which use the
core of the classifier to establish criteria to rank features.

• Embedded methods are more tractable and efficient in comparison to wrapper approach.

• This method has a lower risk of overfitting compared to wrapper approach. Probably the most
famous embedded method is Support Vector Machine based on Recursive Feature Elimination
(SVM-RFE).
The embedded method is classified into three different categories.

The taxonomy of embedded method is shown in Fig.

Hybrid Method

• It is the combination of any number of same or different classical methods of feature selection
such as filter and wrapper methods.

• The combination can be a filter-filter, filter-wrapper, and filter-filter-wrapper where the gene
subset obtained from one method is served as the input to another selection algorithm.

• Generally, filter is used to select the initial gene subset or help to remove redundant genes.
• Any combination of several filter techniques can be applied vertically to select the preliminary
feature subset.

• In the next phase, the selected features are given to the wrapper method for the optimal feature
selection. This method uses different evaluation criteria.

• Therefore, it manages to improve the efficiency and prediction accuracy with the better
computational cost for high dimensional data.

Clustering Techniques Analysis for Microarray Data

• There are two major types of microarray experiments: cDNA microarray and oligonucleotide
arrays .
 • Both the experiments consist of basic three steps: first is chip manufacturing, second are target
preparation, labelling and hybridization and third is the scanning process.

• Gene expression data is expressed in form of expression matrix having real values showing the
protein level of a particular gene.

• Gene expression data contains thousands of genes but less number of samples.

• There are various problems with microarray data such as:

(a) Microarray data is high dimensional data characterized by thousands of genes for small sample
size, which grounds significant problems such as irrelevant and noise genes, complexity in
constructing classifiers, and multiple gene-expression values are missing due to inappropriate
scanning.

(b) Another drawback is mislabeled sample data or doubtful sample results by experts.

(c) Biological relevancy result is another integral criterion that should be taken into account in analyzing
microarray data rather than only focusing on accuracy of cancer classification.

Clustering techniques:

• In gene expression data, it is worth to cluster both genes and samples. There are three types of
clustering that can be applied on microarray data: gene based clustering, sample based clustering
and subspace clustering where genes and samples are treated in same manner.

• In case of gene clustering, the clustering is used to reduce the search dimension of the dataset.

• In case of sample based clustering, the clustering is used to group the samples of same kind
whereas in subspace based clustering both the tasks are performed.

• Gene based clustering can be applied on the supervised dataset where the samples are already
classified.

• The distinctive characteristic of gene expression data allows clustering both gene and samples.
The clustering analysis of sampled data is to find new biological classes or to refine the existing
ones

Hierarchical Clustering:

(a) Agglomerative hierarchical clustering –In this each object initially represents a cluster of its own.
Then clusters are recursively merged until the desired cluster formation is obtained.
(b) Divisive hierarchical clustering - All objects initially belong to one cluster. Then the cluster is
divided into sub-clusters which are successively divided into sub clusters. This process continues until the
desired cluster structure is obtained.

Some commonly used metrics for hierarchical clustering are: Euclidean distance, Squared Euclidean
distance, Manhattan distance, Maximum distance, Mahalanobis distance and cosine similarity
 Partitioning Algorithms:

They are iterative relocation algorithm.

They are non hierarchical or flat methods.

This method divides the data objects into non overlapping clusters such that each data object is in exactly
one subset.

There are several methods which are used to implement partitioning clustering such as:

(a) K-medoids,

(b) K-means,

(c) Probabilistic

(d) K-means is one of the simplest unsupervised learning algorithms that solve the well known
clustering problem. It performs the division of objects into clusters which are similar between
them and dissimilar to the objects belonging to another cluster.

(e) The procedure follows a simple and easy way to classify a given data set through a certain
number of clusters (assume k clusters) fixed a priority. The main idea is to define k centroids, one
for each cluster. These centroids should be placed in a cunning way because of different location
causes different result.

(f) So, the better choice is to place them as much as possible far away from each other. The next step
is to take each point belonging to a given data set and associate it to the nearest centroid.

(g) When no point is pending, the first step is completed and an early groupage is done. At this
point we need to re-calculate k new centroids as barycenters of the clusters resulting from the
previous step.
 (h) After we have these k new centroids, a new binding has to be done between the same data set
points and the nearest new centroid. A loop has been generated. As a result of this loop we may
notice that the k centroids change their location step by step until no more changes are done.

Working of K-medoid Algorithm

K-Medoids Method:

• It is one of the important method of partitioning. K-medoid is based on medoids calculating by

minimizing the absolute

• distance between the points and the selected centroid, rather than minimizing the square distance.
As a result, it's more robust to noise and outliers than k-means. In k-medoids clustering, each
cluster is represented by one of the data point in the cluster.

• These points are named cluster medoids. Here, k data objects are selected randomly as medoids to
represent k cluster and remaining all data objects are placed in a cluster having medoid nearest (or
most similar) to that data object. After processing all data objects, new medoid is determined
which can represent cluster in a better way and the entire process is repeated.
 • Again all data objects are bound to the clusters based on the new medoids. In each iteration,
medoids change their location step by step. This process is continued until no any medoid move.
As a result, k clusters are found representing a set of n data objects

. Density based clustering:

The clusters in this are dense regions of objects in space that are separated by low density regions where
cluster density is defined as each point must have a minimum number of points in its neighborhood.

(i) Based on density based connectivity e.g. DBSCAN

(ii) Based on density distribution functions e.g. DENCLUE

4. Constraint based clustering:

• Constraints are strong background information that should be satisfied.

• Constraints also reduce the search space and all the data in dataset has common property.

• e.g. in gene expression data set we have a constraint of low and high expressed genes.

5. Evolutionary Clustering:

• It is used to process time stamped data to produce a series of clustering.

• The similarity among existing data points varies along with time. Present clusters mainly depend
on the current data features.

• Data is likely to change not too rapidly.

• Evolutionary clustering is useful for the following reasons: (i) consistency, (ii) noise removal (iii)
smoothing (iv) cluster correspondence.

Mostly used for online document clustering

Graph Partitioning based Algorithms:

It depends on finding the minimum cut or minimum cliques in the proximity graph

Many other graph partitioning algorithms depends on eigen vectors and eigen values also.

It consists of three steps:

(i) preprocessing i.e. to covert data into graph and finding similarity between the nodes.

(ii) partitioning of the graph.

(iii) performing clustering until required number of clusters are not obtained.

Each clustering algorithms belongs to one of the clustering types listed above.

So that, Partitioning method is exclusive clustering, Fuzzy C-means is an overlapping clustering

algorithm, Hierarchical clustering is obvious and lastly Mixture of Gaussian is a probabilistic clustering
algorithm.

• Once gene expression data is obtained, one typically wishes to compare one experimental group
versus a second one (or more) in order to find out which genes/transcripts change significantly
between conditions.

• The process is called differential expression analysis.

• The goal of differential expression analysis is to perform statistical analysis to discover changes
in expression levels of defined features (genes, transcripts, exons) between experimental groups
with replicated samples.

• Essentially, it aims at comparing the average expression of a gene in group A with the average
expression of this gene in group B.
Many tools exist that will perform differential expression analysis. The output of such tools is similar, and
essentially revolves around interpreting:

• Fold change:
For a given comparison, a positive fold change value indicates an increase of expression, while a
negative fold change indicates a decrease in expression.
This value is typically reported in logarithmic scale (base 2). For example, log2 fold change of
1.5 for a specific gene in the “WT vs KO comparison” means that the expression of that gene is
increased in WT relative to KO by a multiplicative factor of 2^1.5 ≈ 2.82.

• P-value: Indicates whether the gene analysed is likely to be differentially expressed in that
comparison.
This applies to each gene individually, assuming that the gene was tested on its own without
consideration that all other genes were also tested. More on P-value will follow!

• Adjusted (or, corrected for multiple genes testing) p-value: The p-value obtained for each
gene above is re-calculated to correct for running many statistical tests (as many as the number of
genes). In the result, we can say that all genes with adjusted p-value < 0.05 are significantly
differentially expressed in these two samples.
How to interpret P-value ?

First, we need to talk about statistical hypothesis testing.

Two hypotheses should be described upon designing an experiment.

• The NULL hypothesis H0: that there is NO difference, for example, of means of weight between
two populations of subjects.

• The alternative hypothesis H1: there is difference.

• Once the hypothesis are drawn and the significance level set, we perform a statistical test… and
we obtain a p-value.
• If the p-value is below the significance level (for example, 0.05), we can reject the null
hypothesis in favor of the alternative hypothesis, i.e. we conclude that the observed difference is
the result of a real effect.
• Once the hypothesis are drawn and the significance level set, we perform a statistical test… and
we obtain a p-value.
 • If the p-value is below the significance level (for example, 0.05), we can reject the null
hypothesis in favor of the alternative hypothesis, i.e. we conclude that the observed difference is
the result of a real effect.
• Example: You study some bacteria and it appears to you that their colonies don’t live more than
65 days.
• But you want to check if it is true.
• And you want to test your hypothesis at the significance level of 0.05.
• So you take a sample of 157 colonies with known life span for each.
• You calculate the mean (65.12 days) and the standard deviation (9).
• H0: life spam mean = 65
• HA: life spam mean > 65
• It is well known that to test such a hypothesis on the mean of a population there is the z-test.
• The test statistic for this z-test is calculated as (each test uses its own formula to calculate the test
statistic): z = (65.12 - 65) / (9 * sqrt(157)) = 0.167

Each test statistics has a known distribution:

which allows to calculate the p-value, that is, to find the probability of observing a test statistic at
least this extreme when assuming the null hypothesis.

In our case the p-value to obtain the values of z-test statistic greater than 0.167 is equal 0.3936.

Since this is greater than our significance level, 0.05, we fail to reject the null hypothesis (we are
NOT in the green zone of the distribution above).
 This means that the data does not support the claim that the mean is greater than 65.

Errors can happen in hypothesis testing:

Type II errors (false negatives): you are missing some real changes!

Type I errors (false positives): some changes appear to be the result of a real effect while they are
not!

Type I errors (false positives) are the most dangerous as they can lead to wrong conclusions.
UNIT – 4- SBIA5304-MICROARRAY DATAANALYSIS
UNIT IV CARBOHYDRATE MICROARRAYS

Carbohydrate microarrays- Carbohydrate sources- Synthesis of oligosaccharides - Isolation of

oligosaccharides from natural sources- Arrays of monosaccharides and disaccharides- Arrays of
polysaccharides- Arrays of oligosaccharides- immunological applications.

• Carbohydrate microarray technologies are new developments at the frontiers of glycomics.

• Results of ‘proof of concept’ experiments with carbohydrate-binding proteins of the immune

system — antibodies, selectins, a cytokine and a chemokine and several plant lectins indicate that
microarrays of carbohydrates (glycoconjugates, oligosaccharides and monosaccharides) will
greatly facilitate not only surveys of proteins for carbohydrate-binding activities but also
elucidation of their ligands.

• It is predicted that both naturally occurring and synthetic carbohydrates will be required for the
fabrication of microarrays that are sufficiently comprehensive and representative of entire
glycomes.

• New leads to biological pathways that involve carbohydrate–protein interactions and new
therapeutic targets are among biomedically important outcomes anticipated from applications of
carbohydrate microarrays.

• Unlike proteins and nucleic acids, oligosaccharides are difficult to synthesize chemically.

• This is because some oligosaccharide chains are linear, others are branched, the monosaccharide
building blocks are in alpha or beta anomeric configurations, and adjacent monosaccharides are
linked via different carbon atoms in their sugar rings.

• For these reasons, multiple selective protection and deprotection steps are required for the hydroxyl
groups of monosaccharides during chemical synthesis of oligosaccharides; the manual synthesis of
oligosaccharides is a major undertaking

• The solid-phase synthesis approach has the advantage of avoiding intermediate isolation and
purification steps.

• An automated solid-phase method that includes selective protection and deprotection steps has been
introduced and applied to the synthesis of several glucose- and mannose-containing
oligosaccharides
• An alternative approach to the synthesis of oligosaccharides is a programmable ‘one-pot’
approach, in which an oligosaccharide of interest is generated by the sequential addition of
building blocks (thioglycosides) that are either fully protected or have one hydroxyl group
exposed.

•
It has been shown that the relative reactivity value (RRV) of a thioglycoside building block in the
glycosidation reaction can be tuned in the presence of protecting groups; more than 200 building
blocks, with RRVs ranging from 1 to 105 , have been designed and synthesized.

• A computer programme called ‘Optimer’ has been developed to guide the selection of building
blocks for the one-pot synthesis of a given oligosaccharide.

• If RRVs differ by more than 102 , the desired glycosidic bonds will be formed by the sequential
addition of building blocks in the order of the RRV values.
• Once the required building blocks with protecting groups are prepared, oligosaccharides can be
synthesized in a short period of time (in minutes or hours, instead of days or months using
traditional methods) using this programmable one-pot approach

• Glycans are one of the major biological polymers found in the mammalian body.

• They play a vital role in a number of physiologic and pathologic conditions.

• Glycan microarrays allow a plethora of information to be obtained on protein–glycan binding

interactions.

• Overview of a typical glycan microarray workflow

•
Glycans are chemically or enzymatically synthesized, or isolated and purified from either source
materials, and then conjugated with a linker which is appropriate for the printing surface.
• The glycoconjugates are then printed upon appropriately functionalized slides, followed by
blocking; the printed slides are stored under ideal conditions prior to experiments.

• Many arrays can be printed on a single slide, termed sub-arrays. The slides can then be used in
a glycan microarray experiment where they are incubated with a glycan binding protein (GBP),
such as lectin, antibody, or serum, virus, etc., Followed by addition of a detection reagent, if the
primary analyte was not fluorescently labeled, for example a fluorescent secondary antibody or
streptavidin.

• After washing the slide to remove unbound material, the bound material is then identified and
measured by scanning using a fluorescence microarray scanner. The image produced can then be
analyzed using automated or manual methods to generate the array results. These results can in turn
be stored in a database, or interpreted either manually or by automatic algorithms.

• Isolation of oligosaccharides from natural sources

• Oligosaccharides with reducing termini are ideal for derivatization so that they can be immobilized.

• Free reducing oligosaccharides may be isolated from human or animal milk and urine, or they may
be in the form of N-linked glycoprotein oligosaccharides released by the enzymes peptide-N-(N-
acetyl-b-glucosaminyl)asparagine amidase (PNGase F) and endo-b-N-acetylglucosaminidase F
(Endo F) or by hydrazinolysis.

• O-linked glycoprotein oligosaccharides may be released by mild alkaline hydrolysis or

hydrazinolysis.

• Oligosaccharides may, if desired, be released from glycolipids by endoceramidase .

• Oligosaccharide fragments can be obtained from proteoglycans and glycosaminoglycans by lyase

digestion or nitrous acid degradation , and, in the case of hyaluronic acid, also by hydrolase
digestion .

• Various chemical methods may be used to obtain oligosaccharide fragments from bacterial and
plant polysaccharides; these include acid or alkaline hydrolysis, acetolysis and Smith degradation.

• Reduced oligosaccharides (oligosaccharide alditols) can be manipulated chemically at the reduced

end after mild periodate oxidation to cleave the terminal open chain monosaccharide residue and
create a reactive aldehyde for derivatization.
• Reduced oligosaccharides are typically obtained when O-linked glycans are released from
glycoproteins by reductive alkaline hydrolysis.

• Oligosaccharide alditols are also available when reduction is carried out, for example, before HPLC
separation, to eliminate double peaks resulting from the resolution of a and b anomers at their
reducing ends.

• Multiple chromatographic steps are often necessary for the isolation/purification of

oligosaccharides.

• These include gel filtration, weak and strong anion-exchange chromatography, thin-layer
chromatography (TLC),normal-phase HPLC with an amine or amide column, and reversed-phase
HPLC using a C18 or graphitized porous carbon column.

• Arrays of monosaccharides and disaccharides

• The monosaccharides were covalently immobilized by conjugation to self-assembling monolayers

of alkenethiols on the gold surface.

• The first step was to prepare monolayers consisting of two alkenethiols, one of which has a
benzquinone group exposed.

• The monosaccharides, in the form of diene conjugates, are then applied as 1 ml spots (2 mM in
water) and attached to the slides through the Diels–Alder cycloaddition reaction.

• This is a very high yielding process, often reagent free, and moisture and solvent tolerant, and is
therefore ideal for the microarray format
• The covalently immobilized monosaccharides were evaluated by profiling the binding
specificities of five plant lectins, concanavalin A (Con A), Benderia simplicifolia, Erythrinia
cristalli, Ulex europeeus and Galanthus nivalis, that are known to bind to different
monosaccharides.

• Specific monosaccharide binding was observed for the five lectins, which were labeled
fluorescently with rhodamine. Specific binding of Con A to arrayed mannose was also shown by
surface plasmon resonance spectroscopy.

• In further experiments, the monosaccharide array was probed with the glycosyltransferase b-1,4-
galactosyltransferase; it was shown that enzyme-mediated glycosylation of immobilized N-
acetylglucosamine occurred in the presence of the donor substrate, UDP-galactose.
• Shin’s group has reported another approach to carbohydrate microarray fabrication. They used
glass slides modified by thiol groups as solid supports.

• One monosaccharide, N-acetylglucosamine, and three disaccharides, lactose, cellobiose and

maltose, in the form of glycosylamines, were converted into maleimide conjugates and then
covalently bound to the glass surface by hetero-Michael addition reaction between the thiol group
on the solid surface and the maleimide moiety of the sugar derivative .

• The maleimide-conjugated carbohydrates (from 0.1 to 5.0 mM) were printed with a pin-type
microarrayer on the slides at a spot size of 100 mm and a pitch of 200 mm. Carbohydrate–protein
interaction studies were performed with fluoresce in labeled plant lectins.

• The binding of the three plant lectins examined, Con A, Erythrina cristagalli and Triticum vulgaris,
to the monosaccharide and the disaccharides was in accord with their known specificities.

• Arrays of polysaccharides

• Wang et al. described microarrays of polysaccharides and glycoproteins on nitrocellulose-coated

glass slides.

• They used a high-precision robotic arrayer that was developed for cDNA and the spots were
generated without derivatization.

• The spot sizes were 150 mm with a pitch of 375 mm. These were air dried to allow adsorption
(noncovalent immobilization) onto the hydrophobic surface.
UNIT – 5- SBIA5304-MICROARRAY DATAANALYSIS
UNIT V DATABASES AND TOOLS FOR MICROARRAYS

• Bioinformatics in Arrays- Databases and tools for microarrays- Bioconductor, expression

profiler, EST databases- Assessing levels of gene expression using EST’s, TIGR gene
indices, STACK, SAGE, CGAP, Xprofiler, ARRAY DB, cluster, tree view, Scanalyze,
gene cluster, informatics aspects of microarray production- MGED and gene-ontology,
description of MIAME ((Minimum Information About a Microarray Experiment),
Business Aspects of Biochip Technologies- Microarray Technology in Treating Disease.

Bioconductor

What is Bioconductor used for?

• Bioconductor provides tools for the analysis and comprehension of high-throughput genomic data.

• Bioconductor uses the R statistical programming language, and is open source and open
development.
• It has two releases each year, and an active user community.

limma powers differential expression analyses for RNA-sequencing and microarray studies

limma is an R/Bioconductor software package that provides an integrated solution for analysing
data from gene expression experiments. It contains rich features for handling complex experimental
designs and for information borrowing to overcome the problem of small sample sizes. Over the
past decade, limma has been a popular choice for gene discovery through differential expression
analyses of microarray and high-throughput PCR data. The package contains particularly strong
facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have
been significantly expanded in two important directions. First, the package can now perform both
differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All
the downstream analysis tools previously restricted to microarray data are now available for RNA-
seq as well. These capabilities allow users to analyze both RNA-seq and microarray data with very
similar pipelines. Second, the package is now able to go past the traditional gene-wise expression
analyses in a variety of ways, analyzing expression profiles in terms of co-regulated sets of genes
or in terms of higher-order expression signatures. This provides enhanced possibilities for
biological interpretation of gene expression differences.

ne
The limma package is a core component of Bioconductor, an R-based open-source software
development project in statistical genomics. It has proven a popular choice for the analysis of data
from experiments involving microarrays, high-throughput polymerase chain reaction (PCR),
protein arrays and other platforms. The package is designed in such a way that, after initial pre-
processing and normalization, the same analysis pipeline is used for data from all technologies.

Recently, the capabilities of limma have expanded significantly in two important directions. First,
the package can now perform both differential expression (DE) and differential splicing analyses
of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to
microarray data are now available for RNA-seq as well. These capabilities allow users to analyse
both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to
go past the traditional gene-wise expression analyses in a variety of ways, analysing expression
profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures.
This provides enhanced possibilities for biological interpretation of gene expression differences.

EST databases- Assessing levels of gene expression using EST’s

• EST expression profiling has by now become well-established high-throughput method for
acquiring quantitative information on a sample's transcriptome and for studying differential
gene expression, inferred from the differences in the relative numbers of EST tags between
two libraries.
• To facilitate gene discovery, the EST content of a library can be altered to reduce the
abundance of transcripts representing genes with high expression.
• To achieve this a library can be either normalised by removing the most abundant
transcripts in order to reduce or eliminate the differences in the relative transcript
abundances to a narrow range , or subtracted to enrich the library for rare novel transcripts
.
• Ideally this should create a library containing the same or similar tag counts for the low
abundance sequences as before, but with vastly reduced counts for abundant or unwanted
cDNAs.
• Neither normalised nor subtracted libraries are suitable for studying differential mRNA
expression because of the significantly changed representation or removal of the original
transcripts

The TIGR Gene Indices

• The TIGR Gene Indices ([Link] are a collection of 77 species-

specific databases that use a highly refined protocol to analyze gene and EST
sequences in an attempt to identify and characterize expressed transcripts and to
present them on the Web in a user-friendly, consistent fashion.
• A Gene Index database is constructed for each selected organism by first clustering,
then assembling EST and annotated cDNA and gene sequences from GenBank.
This process produces a set of unique, high-fidelity virtual transcripts, or tentative
consensus (TC) sequences. The TC sequences can be used to provide putative genes
with functional annotation, to link the transcripts to genetic and physical maps, to
provide links to orthologous and paralogous genes, and as a resource for
comparative and functional genomic analysis.
Construction of the Gene Indices
The process used to assemble each Gene Index is similar to that described previously, although
some modifications have been made to improve the efficiency and accuracy of the process.
mgBLAST, a modified version of the Megablast program, is now used for the pairwise sequence
comparisons that are the basis for defining the sequence clusters which form the basis for assembly.
For large clusters containing hundreds or thousands of sequences (e.g. highly expressed genes such
as actin), sequence representation is reduced prior to assembly using a variety of multilayer
approaches, including transitive clustering, containment clustering and seeded clustering with
known genes. Following clustering, the Paracel Transcript Assembler (PTA), a modified version
of CAP3 assembly program, is used to assemble each TC. An open source set of software tools
that embody this process, TGICL, is available ([Link] with other
open-source utilities for users interested in performing a similar analysis on their own datasets.

SAGE
Serial Analysis of Gene Expression (SAGE) is a transcriptomic technique used by molecular
biologists to produce a snapshot of the messenger RNA population in a sample of interest in the
form of small tags that correspond to fragments of those transcripts.
Serial analysis of gene expression (SAGE) uses mRNA from a particular sample to create complementary
DNA (cDNA) fragments which are then amplified and sequenced using high-throughput sequencing
technology.

The mechanism behind SAGE is based on tags which can identify the original transcript, and rapid
sequencing of chains of tags linked together. The procedure essentially simplifies sequencing by linking
the cDNA segments together in a long chain.

The resulting analysis gives a snapshot of the transcriptome of the sample, including the identity and
abundance of each mRNA.

Steps of SAGE
SAGE is a complex protocol with many steps.
Step 1: mRNA is isolated from the sample and reverse transcribed using biotinylated primers to generate
cDNA

Step 2: cDNA is bound via biotin to streptavidin microbeads

Step 3: cDNA is cleaved with restriction enzymes freeing it from the beads

Step 4: Cleaved DNA is washed out, leaving truncated cDNA bound to the beads

Step 5: Two oligonucleotides with sticky ends are added to the remaining truncated cDNA, in separate
samples

Step 6: Cleaved DNA is “tagged” enzymatically, removing it from the beads

Step 7: Sticky ends are repaired with DNA polymerase

Step 8: Blunt ended tags from the two separate samples are ligated together, generating ditags with two
different oligonucleotide adapter ends

Step 9: Ditags are cleaved to remove the oligonucleotides. Ditags will form long cDNA chains, or
concatemers

Step 10: Transform concatemers into bacteria for replication

Step 11: Isolate concatemers from bacteria and sequence

Challenges when using SAGE

One challenge is that the tags are only about 13 or 14 base pairs. It can be difficult to identify such a short
tag if it’s from an unknown gene.

The flip side of that problem is that SAGE can be used to find unknown genes, and in some studies it’s an
advantage to be able to measure gene expression quantitatively without prior sequence information.

Tags may also have issues with specificity; multiple genes could share the same tag if there is an overlap
in sequence. There also can be inconsistencies with the restriction enzymes, and incompatibilities for certain
species.

SAGE and DNA microarray

SAGE is similar in many ways to a DNA microarray; however, in a DNA microarray, the mRNAs hybridize
to cDNA probes on the array. In SAGE, the data output is based on sequencing. That means SAGE analysis
is more quantitative and it does not depend on the use of known genes.

Microarray experiments are generally less costly, and so are used more often in larger-scale studies.

Application
• A study of new markers in cancer illustrates how SAGE can be used in biomedical
research.
• Researchers compared gene expression levels in cancerous tissues with those in non-
cancerous tissues to search for markers that could diagnose the pancreatic cancer at an
early stage.
• Because the results of a SAGE analysis of many representative tissues had already been
published online, the scientists were able to search the database for genes preferentially
expressed in pancreatic cancer.
• From this, they were able to identify a gene calledprostate stem cell antigen (PCSA), that
had previously not been associated with pancreatic cancer.

• CGAP- Cancer Genome Anatomy Project

• What you can do:

• Find the information and technological tools needed to decipher the molecular anatomy of
the cancer cell from a annotated index of the genes that are important in cancer.

• Highlights:

• The goal of the NCI's Cancer Genome Anatomy Project is to determine the gene expression
profiles of normal, precancer, and cancer cells, leading eventually to improved detection,
diagnosis, and treatment for the patient.

• CGAP offers technological, informational (data and analysis tools), resource (clones and
libraries) and methodological infrastructure for the cancer research community.

• The current CGAP program has expanded to include in addition to the Tumor Gene Index
(TGI), a Genetic Annotation Initiative (GAI) and the Cancer Chromosome Aberration
Project (cCAP).

• The TGI and GAI are focused towards building a catalog of annotated genes.