Introduction to Bioinformatics Microarrays1: Microarray Technology

1
Introduction to Bioinformatics Microarrays1
Microarray Technology

• Course 341
• Department of Computing
• Imperial College, London
• Moustafa Ghanem

2
Aims for the 2nd part of Course Microarray
Bioinformatics

• Appreciate the bigger picture of bioinformatics
• Bioinformatics is more than nucleotide sequence
  analysis
• Functional Genomics and Drug Discovery
• Understand basic microarray technology and its
  use in gene expression analysis.
• Learn basic data analysis methods and how to
  apply them in the analysis of gene expression
  data
• Data Clustering
• Data Classification
• Statistical Analysis

3
Recommended Texts

• For this part of the course
• Lecture Notes
• Handouts
• General overview of microarray data analysis
• Microarray Gene Expression Data Analysis A
  Beginners Guide (Causton, Quakenbush and
  Brazma)
• Microarray Bioinformatics (Stekel)
• Data Mining
• Data Mining Concepts and Techniques (Han)

4
Microarray TechnologyLecture Overview

• Aims, Motivation and Overview of 2nd Part of
  Course
• Biology Background
• Basic Idea of Microarrays
• Types of Microarray technologies and how they
  work
• Outputs of Microarrays
• Image Analysis required to transform output to
  gene expression matrices
• Generating Gene Expression Matrices

5
BackgroundFunctional Genomics

• Functional Genomics
• Systematic analysis of gene activity in healthy
  and diseased tissues.
• The study of obtaining an overall picture of
  genome functions, including the expression
  profiles at the mRNA level and the protein level.
  
• Functional Genome Analysis
• used to understand the functions of genes and
  proteins in an organism. This is typically known
  as genome annotation.
• used in integrative biology and systems biology
  studies aiming to understand health and disease
  states (e.g. cancer, obesity, etc)
• Used as an important step in the search for new
  target molecules in the drug discovery process.

6
BackgroundThe Drug Discovery Pipeline

• Drug Discovery is a lengthy process that takes
  years and requires the use of bioinformatics,
  chemoinformatics and clinical-informatics tools.
• Functional genomics plays an important role in
  speeding up the pipeline and also in allowing us
  to try new therapeutic methods.

7
BackgroundDrug Discovery

• Functional genomics plays an important role in
  identifying functions of potential therapeutic
  targets such as encoded proteins. Gene expression
  studies plays an important role in most stages
• Target Identification
• Understand disease states, identify genetics
  changes that cause disease (genes, proteins,
  tissues, environmental conditions, etc)
• Target Validation
• Understand the role of a target and the effects
  of manipulating a target candidate (e.g. what if
  I knock a gene out)
• Compound Screening
• Understand compounds effect on target and its
  risk profile
• Pre-clinical and clinical trials
• Prioritise studies

8
BackgroundBiology, Cells and DNA

• All living organisms consist of cells. Humans
  have trillions of cells Yeast - one cell.
• Cells are of many different types (blood, skin,
  nerve), but all arose from a single cell (the
  fertilized egg)
• Each cell contains a complete copy of the genome
  (the program for making the organism), encoded in
  DNA.
• A gene is a segment of DNA that specifies how to
  make a protein. Human DNA has about 30-35,000
  genes Rice has about 50-60,000, but shorter
  genes.

9
What is?

• Gene Expression
• The process by which the information encoded in a
  gene is converted into an observable phenotype
  (most commonly production of a protein).
• The degree to which a gene is active in a certain
  tissue of the body, measured by the amount of
  mRNA in the tissue.
• Microarrays
• Tools used to measure the presence and abundance
  of gene expression in tissue.
• microarray technologies provide a powerful tool
  by which the expression patterns of thousands of
  genes can be monitored simultaneously

10
BackgroundGene Expression

• Cells are different because of differential gene
  expression.
• About 40 of human genes are expressed at one
  time.
• Gene is expressed by transcribing DNA into
  single-stranded mRNA
• mRNA is later translated into a protein
• Microarrays measure the level of mRNA expression

11
A Dynamic ViewGene expression depends on
environment!
Interactions
Environment
DNA
Protein
Growth rate
Expression
12
A Dynamic ViewGene expression varies with time !
13
Microarray Technology Quantitative Measurement
of Gene Expression

• Also known as DNA microarrays, DNA arrays, DNA
  chips, gene chips, Whatever the name, their use
  is effectively transforming a living from a black
  box into a transparent box.

14
Applications of Microarray Technology
15
Data Analysis over microarray data

• What type of data analysis is required to
• Identify Genes expressed in different cell types
  (e.g. Liver vs finger)
• Learn how expression levels change in different
  developmental stages (embryo vs. adult)
• Learn how expression levels change in different
  developmental stages (cancerous vs non-cancerous)
• Learn how groups of genes inter-relate (gene-gene
  interactions)
• Identify cellular processes that genes
  participate in (structure, repair, metabolism,
  replication, etc)
• Applications covered only as example contexts,
  emphasis is on analysis methods

16
MicroarraysBasic Idea
Affymetrix Inc. is the leading provider of
Microarray technology (GeneChip
) http//www.affymetrix.com/

• A Microarray is a device that detects the
  presence and abundance of labelled nucleic acids
  in a biological sample.
• In the majority of experiments, the labelled
  nucleic acids are derived from the mRNA of a
  sample or tissue.
• The Microarray consists of a solid surface onto
  which known DNA molecules have been chemically
  bonded at special locations.
• Each array location is typically known as a probe
  and contains many replicates of the same
  molecule.
• The molecules in each array location are
  carefully chosen so as to hybridise only with
  mRNA molecules corresponding to a single gene.

17
Basic Idea
Several companies sell equipment to make DNA
chips, including spotters to deposit the DNA on
the surface and scanners to detect the
fluorescent or radioactive signals.

• A Microarray works by exploiting the ability of a
  given mRNA molecule to bind specifically to, or
  hybridize to, the DNA template from which it
  originated.
• By using an array containing many DNA samples,
  scientists can determine, in a single experiment,
  the expression levels of hundreds or thousands of
  genes within a cell by measuring the amount of
  mRNA bound to each site on the array.
• With the aid of a computer, the amount of mRNA
  bound to the spots on the Microarray is precisely
  measured, generating a profile of gene expression
  in the cell.

18
BackgroundDNA/RNA Hybridization

• DNA molecules
• DNA molecules are long double-stranded chains 4
  types of bases are attached to the backbone
  adenine (A), guanine (G), cytosine (C), and
  thymine (T). A pairs with T, C with G.
• DNA-RNA hybridization
• When a mixture of DNA and RNA is heated to
  denaturation temperatures to form single strands
  and then cooled, RNA can hybridize (form a double
  helix) with DNA that has a complementary
  nucleotide sequence.

19
The Array
The technology for making DNA chips has become so
well-defined that it is even possible to
construct all of the equipment for under 50,000
using directions on the Internet from Professor
Pat Browns laboratory at Stanford.
http//cmgm.stanford.edu/pbrown/
20
Applying a Labelled Sample

• The molecules in the target biological sample are
  labelled using a fluorescent dye before sample is
  applied to array
• If a gene is expressed in the sample, the
  corresponding mRNA hybridises with the molecules
  on a given probe (array location).
• If a gene is not expressed, no hybridisation
  occurs on the corresponding probe.
• Reading the array output
• After the sample is applied, a laser light source
  is applied to the array.
• The fluorescent label enables the detection of
  which probes have hybridised (presence) via the
  light emitted from the probe.
• If gene is highly expressed, more mRNA exists and
  thus more mRNA hybridises to the probe molecules
  (abundance) via the intensity of the light
  emitted.

21
The Process
Chemistry Basics Surface Chemistry is used to
attach the probe molecules to the glass
substrate. Chemical reactions are used to attach
the florescent dyes to the target molecules Probe
and Target hybridise to form a double helix
22
The array
23
Steps of a Microarray Experiment

• Prepare DNA chip(s) by choosing probes and
  attaching them to glass substrate. Note location
  and properties of each probe.
• Generate a hybridization solution containing a
  mixture of fluorescently labelled targets. 
• Incubate hybridization mixture.
• Detect probe hybridization using laser technology
  
• Scan the arrays and store output as images
• Quantify each spot
• Subtract background
• Normalize
• Export a table of fluorescent intensities for
  each gene in the array
• Analyze data using computational methods.

24
Types of Microarrays

• How are Microarrays are made?
• What molecules make the probes?
• cDNA (PCR products) vs Oligos
• How are the probes added to the chip?
• Spotting vs. In-situ synthesis
• Output type
• Single label vs. Dual label
• Why ? Appreciation of some of the concepts of the
  technology.
• Helps us understand and choose between available
  technology.
• Helps us design our experiments.
• Helps understand sources of errors in array
  outputs and compensate for them.

25
Designing the Probes
Each probe represents the measurement for a
single gene An array represents measurements for
many genes

• The probes need to be of high specificity to
  avoid hybridization with wrong target molecules.
• The probes need to generate an output that is
  easy to read (spots lie in defined positions and
  be of regular size and shape and even spacing).
• The probes have to have high sensitivity to
  detect the mRNA and the intensity of the spot
  light must be differentiable from background
  noise.
• The intensity of a spot light also needs to
  correlate with the abundance of the target
  molecule in the sample.
• Results must be reproducible across multiple
  experiments.

26
Probe Types
Different chip manufacturers use different
technologies As an end user you will use the
probe types recommended for the chips, but would
have to select the sequences for the probes to be
used in your experiments Affymetrix technology
is based on oligos (20 bases per probe)

• The DNA probes used on a an array can either be
  polymerase chain reaction (PCR) products (cDNAs)
  or Oligonucleotides.
• In the first case (cDNA), highly parallel PCR is
  used to amplify DNA from a clone library, and the
  amplified DNA is purified, the clones are
  typically long sequences (Complete genes or
  ESTs).
• In the second case, DNA oligonucleotides are
  presynthesised for use on the array --- An
  oligonucleotide, or oligo as it is commonly
  called, is a short fragment of a single-stranded
  DNA that is typically 5 to 50 nucleotides long.
  This can achieve a higher density of probes per
  chip.
• In both cases the probes are attached (fixed or
  immobilized) to a glass (or nylon) surface using
  special surface chemical techniques (Beyond this
  course).

27
Spotting vs. In-situ SynthesisSpotting

• Spotting works for both cDNA probes and oligo
  probes
• The Spotting Process
• The DNA probes are produced and stored in wells.
• A Spotting robot is used to deposit them onto
  individual locations on the glass slide
• The glass slide is post-processed so no further
  DNA can attach to it.
• Spotting is easy to automate but may generate
  poor quality spots (irregular spots of different
  shapes and sizes)

28
The Spotting Robot

• The Operation of the Spotting Robot
• The pins are dipped into the wells to collect the
  first batch of DNA.
• This DNA is spotted onto a number of different
  arrays, depending on the number of arrays being
  made and the amount of liquid the pins can hold.
• The pins are washed to remove any residual
  solution and ensure no contamination of the next
  sample.
• The pins are dipped into the next set of wells.
• Return to step 2 and repeat until the array is
  complete.

29
Spotting Process
30
Spotting vs. In-situ SynthesisIn-situ Synthesis
Affymetrix technology is based on in-situ
synthesis in a series of addition steps separated
by mask addition and then photo-deprotection.

• Since oligos are synthesized short sequences,
  their bases can be added to the glass surface one
  at a time.
• Using high tech processes this can generate best
  quality (regular even spots).
• Different patented technologies are used to
  enable this to happen while not allowing more
  than one base to be added at a time, including
• Photodeprotection technology (Affymetrix)
• Inkjet Array Synthesis

31
In-situ SynthesisAffymetrix
32
Comparison of Probe Types
Many other variations of the technology exist,
such as the use of longer oligos, the use of
fibre optics, etc.
In-situ Synthesis / Oligos
PCR Products / cDNA Probes

• Advantages
• Flexibility to study cDNAs from any source.
• cDNAs do not require any a priori information
  about the corresponding genes.
• Longer sequences increase hybridization
  specificity, which reduces false positives.

• Advantages
• No need to isolate and purify cDNAs because
  oligonucleotides can be synthesized.
• Short oligonucleotides are less likely to have
  cross-reactivity with other sequences in the
  target DNA.
• Density of chips is higher than with cDNAs.

• Limitations
• Isolation of individual cDNAs to immobilize on
  each spot can be cumbersome.
• Density is lower than synthesizing
  oligonucleotides on the surface of the chip.
• cDNAs are longer sequences and are more likely to
  randomly contain sequences found in target DNA,
  which results in cross-reactivity.

• Limitations
• The sequence has to be known.
• Synthesis can be expensive and time-consuming.
• The short sequences are not as specific for
  target DNA, so appropriate controls must be
  added.

33
Single Label vs. Dual LabelSingle Channel vs
Dual Channel
Affymetrix technology is based on the use of
single labels

• Most laboratories use fluorescent labelling, with
  the two dyes Cy3 (excited by a green laser) and
  Cy5 (excited by a red laser).
• In Dual label experiments, two samples are
  hybridised to the arrays, one labelled with each
  dye this allows the simultaneous measurement of
  two samples (e.g. for differential analysis)
• In Single label experiments, only one sample is
  hybridised to the arrays labelled with one dye.
  (in which case control needs to be measured using
  a separate chip).
• Choice between single and dual label is governed
  by array technology and underlying chemistry.

34
Dual Label Experiments
Typically used in custom made cDNA chips
Typically used to study one sample (e.g.
diseased tissue) vs. a control sample (e.g.
normal tissue) Separate images are obtained for
each channel, and then combined
35
Qualitative Interpretation of Double Label
Experiments

• GREEN represents High Control hybridization
•  RED represents High Sample hybridization
•  YELLOW represents a combination of Control and
  Sample where both hybridized equally. BLACK
  represents areas where neither the Control nor
  Sample hybridized.
• Main issue is to quantify the results
• How green is green?
• What is the ratio of the signal to background
  noise?
• How to compare multiple experiments using
  different chips?
• How to quantify cross hybridization (if any)?

36
Affymetrix GeneChipExample of Single Label Chips

• Hundreds of thousands of oligonucleotide probes
  packed at extremely high densities. The probes
  designed to maximize sensitivity, specificity,
  and reproducibility, allowing consistent
  discrimination between specific and background
  signals, and between closely related target
  sequences.
• RNA labeled and scanned in a single color one
  sample per chip

37
Interpreting Affymetrix OutputPerfect
Match/Mismatch Strategy

• GeneChips use a Perfect Match/Mismatch probe
  strategy
• Each probe designed to be perfectly complementary
  to a target sequence, a partner probe is
  generated that is identical except for a single
  base mismatch in its centre.
• These probe pairs, called the Perfect Match probe
  (PM) and the Mismatch probe (MM), allow the
  quantitation and subtraction of signals caused by
  non-specific cross-hybridization.
• The difference in hybridization signals between
  the partners, as well as their intensity ratios,
  serve as indicators of specific target abundance.

38
PM to maximize hybridization MM to
ascertain the degree of cross-hybridization
Affymetrix GeneChipsPerfect Matches and
Mismatches
39
Other Image Processing Problems Spot Quality
Problems
Various Image processing techniques may be
applied to read and interpret the outputs of
Microarrays Commercial Microarray (e.g.
Affymetrix) systems use proprietary
software Image Analysis software packages exist
for the analysis of the output of custom made
chips (e.g. GenePix Pro, Array Vision, TIGR Spot
Finder, etc)
40
From Microarray images to Gene Expression
Matrices
41
From Microarray images to Gene Expression
Matrices

• In spot quantitation matrices, rows typically
  represent all the measurements made from
  individual spots on the array. These can include
  mean and median pixel intensities of the spot and
  local background, etc.
• An experiment typically consists of one or more
  spot quantitation matrices representing all
  arrays used in the study.
• In the gene expression matrix, rows represent
  genes (as opposed to features/spots on the array)
  and columns represent measurements from different
  experimental conditions measured on individual
  arrays.
• An example is each column representing
  measurements at different time points (to, t1,
  t2, ) in time course experiments
• A second example is each column representing
  different tissue type
• A third is each column representing a different
  individual
• A fourth is having groups of columns representing
  measurements from diseased cells, and other
  groups representing measurements from health
  cells,
• etc,
• Each of the above matrices requires the
  application of data normalisation technuiques as
  discussed in the next lecture.

42
SummaryMicroarrays

• Basic Concept
• Based on Crick-Watson Hybridization
• Different Microarray technologies exist.
• Probe type (cDNA vs oligo)
• Spotting vs in-situ synthesis
• Single vs. dual channel
• Output is a typically an image
• Sources of errors
• Image processing is required
• Images are converted into gene expression
  matrices for further analysis