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 don’t 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