Probe selection for Microarrays

1
Probe selection for Microarrays

• Considerations and pitfalls

2
Probe selection wish list

• Probe selection strategy should ensure
• Biologically meaningful results (The truth...)
• Coverage, Sensitivity (... The whole truth...)
• Specificity (... And nothing but the truth)
• Annotation
• Reproducibility

3
Technology

• Probe immobilization
• Oligonucleotide coupling Synthesis with
  linker, covalent coupling to surface
• Oligonucleotide photolithography
• ds-cDNA coupling cDNA generated by PCR,
  nonspecific binding to surface
• ss-cDNA coupling PCR with one modified primer,
  covalent coupling, 2nd strand removal
• Spotting
• With contact (pin-based systems)
• Without contact (ink jet technology)

4
Technology-specific requirements

• General
• Not too short (sensitivity, selectivity)
• Not too long (viscosity, surface properties)
• Not too heterogeneous (robustness)
• Degree of importance depends on method
• Single strand methods (Oligos, ss-cDNA)
• Orientation must be known
• ss-cDNA methods are not perfect
• ds-cDNA methods dont care

5
Probe selection approaches
Accuracy
Throughput
SelectedGenes
ESTs
Selected GeneRegions
ClusterRepresentatives
Anonymous
6
Non-Selective Approaches

• Anonymous (blind) spotting
• Using clones from a library without prior
  sequencing
• Only clones with interesting expression pattern
  are sequenced
• Normalization of library highly recommended
• Typical uses
• HT-arrays of exotic organisms or tissues
• Large-scale verification of Differential Display
  clones
• EST spotting
• Using clones from a library after sequencing
• Little justification since sequence availability
  allow selection

7
Spotting of cluster representatives

• Sequence Clustering
• For human/mouse/rat EST clones public cluster
  libraries
• Unigene (NCBI)
• THC (TIGR)
• For custom sequence clustering tools
• STACK_PACK (SANBI)
• JESAM (HGMP)
• PCP (Paracel, commercial)

8
A benign clustering situation
9
In the absence of 5-3 links
Two clusters corresponding to one gene
10
Overlap too short
Three clusters corresponding to one gene
11
Chimeric ESTs
One cluster corresponding to two genes
12
Chimeric ESTs ... continued

• Chimeric ESTs are quite common
• Chimeric ESTs are a major nuisance for array
  probe selection
• One of the fusion partners is usually a highly
  expressed mRNA
• Double-picking of chimeric ESTs can fool even
  cautious clustering programs.
• Unigene contains several chimeric clusters
• The annotation of chimeric clusters is erratic
• Chimeric ESTs can be detected by genome
  comparison
• There is one particularly bad class of chimeric
  sequences that will be subject of the exercises.

13
How to select a cluster representative

• If possible, pick a clone with completely known
  sequence
• Avoid problematic regions
• Alu-repeats, B1, B2 and other SINEs
• LINEs
• Endogenous retroviruses
• Microsatellite repeats
• Avoid regions with high similarity to
  non-identical sequences
• In many clusters, orientation and position
  relative to ORF are unknown and cannot be
  selected for.
• Test selected clone for sequence correctness
• Test selected clone for chimerism
• Some commercial providers offer sequence verified
  UNIGENE cluster representatives

14
Selection of genes

• If possible, use all of them
• Biased selection
• Selection by tissue
• Selection by topic
• Selection by visibility
• Selection by known expression properties
• Selection from unbiased pre-screen
• Use sources of expression information
• EST frequency
• Published array studies
• SAGE data

15
Selection of gene regions
3 UTR
ORF
5 UTR
16
Alternative polyadenylation
17
Alternative polyadenylation

• Constitutive polyA heterogeneity
• 3-Fragments reduced sensitivity
• no impact on expression ratio
• Regulated polyA heterogeneity
• Fragment choice influences expression ratio
• Multiple fragments necessary
• Detection of cryptic polyA signals
• Prediction (AATAAA)
• Polyadenylated ESTs
• SAGE tags

18
Alternative splicing
19
Alternative splicing

• Constitutive splice form heterogeneity
• Fragment in alternative exon reduced sensitivity
• No impact on expression ratio
• Regulated splice form heterogeneity
• Fragment choice influences expression ratio
• Multiple fragments necessary
• Detection of alternative splicing events
• Hard/Impossible to predict
• EST analysis (beware of pre-mRNA)
• Literature

20
Alternative promoter usage
21
Alternative promoter usage

• What is the desired readout?
• If promoter activity matters most multiple
  fragments
• If overall mRNA level matters most downstream
  fragment
• Detection of alternative promoter usage
• Prediction difficult (possible?)
• EST analysis
• Literature

22
UDP-Glucuronosyltransferases
UGT1A8
UGT1A7
23
Selection of gene regions

• Coding region (ORF)
• Annotation relatively safe
• No problems with alternative polyA sites
• No repetitive elements or other funny sequences
• danger of close isoforms
• danger of alternative splicing
• might be missing in short RT products

• 3 untranslated region
• Annotation less safe
• danger of alternative polyA sites
• danger of repetitive elements
• less likely to cross-hybridize with isoforms
• little danger of alternative splicing
• 5 untranslated region
• close linkage to promoter
• frequently not available

24
A checklist

• Pick a gene
• Try get a complete cDNA sequence
• Verify sequence architecture (e.g. cross-species
  comparison)
• Mask repetitive elements (and vector!)
• If possible, discard 3-UTR beyond first polyA
  signal
• Look for alternative splice events
• Use remaining region of interest for similarity
  searches
• Mask regions that could cross-hybridize
• Use the remaining region for probe amplification
  or EST selection
• When working with ESTs, use sequence-verified
  clones