RExPrimer

1. RExPrimer Pongsakorn Wangkumhang, M.Sc. Biostatistics and Informatics Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand

2. Motivations • de facto Primer3 program has limitations • Graphical user interface for visualizing gene structure is crucial for the resequencing primer design • Existing primer designing tools do not consider • SNP-in-Primer • mis-matching problem • Pseudogene form • mis-priming problem • Structural variation (CNV) • mismatching (deletion) and mis-priming (duplication)

3. Generic steps for primer design • Identify target sequence • Genome sequence, intron/exon boundaries • Design primers • Use de facto Primer3 • Check for Specificity • Use BLAST, primerBLAST or UCSC In-Silico PCR

4. What are the tools out there? • PrimerZ [Tsai et al, 2007 ] • EasyExonPrimer [Wu and Munroe, 2006] • MutScreener [Yao et al, 2006 ] • VariantSEQr [Applied Biosystem, 2005] • ELXR [Schageman et al, 2004] • SNPbox [Weckx , 2004] • ExonPrimer (ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html) • Genomic Primer (www-fgg.eur.nl/kgen/primer/Genomic_Primers.html)

5. Software Functionality ++ : Very good + : Available with limitation - : Not available

6. Problems to be solved!! • Mis-matching (No amplification due to variation in primer) • SNP-in-primer • Insertion/deletion (indel) polymorphisms • Copy number variation (CNV) • Mis-priming (Non-target homology seq. binding) • Structural complexity of the genome • Pseudogene forms • Chromosome segmental duplications • Repetitive elements (e.g. SINES, LINES, satellite sequences, etc.)

7. Why RExPrimer (features must have) • DNA Resequencing/SNP genotyping primer design • Integrated to Human genome database, variation databases • Intuitive and comprehensive visualization for avoiding unwanted regions at first • Re-designable to escape previous unwanted regions

8. RExPrimer Workflow

9. Case Study : CYP2D6 Resequencing • Cytochrome P450 2D6 gene • Important drug metabolizing enzyme • Ethnic-specific resequencing/genotyping CYP2D6 is crucial for medical treatments • Highly polymorphic i.e. SNPs, CNVs and Pseudogenes • Pseudogenes, CYP2D7P and CYP2D8P, share 98% homology with CYP2D6

10. Case Study: CYP2D6 Resequencing DNA Resequencing SNP Genotyping

11. Case Study: CYP2D6 Resequencing DNA Resequencing CYP2D6 SNP Genotyping

12. Gene location of CYP2D6 and its pseudogenes Case Study: CYP2D6 Resequencing

13. Chromosome mapping of CYP2D6 and its pseudogenes Gene location of CYP2D6 and its pseudogenes Case Study: CYP2D6 Resequencing

14. Chromosome mapping of CYP2D6 and its pseudogenes Gene location of CYP2D6 and its pseudogenes Multiple sequence alignment of CYP2D6 and its pseudogenes Case Study: CYP2D6 Resequencing CYP2D6 gene pseudogenes

15. Case Study: CYP2D6 Resequencing

16. Example Scenario: CYP2D6 Resequencing CYP2D6 gene CNVs pseudogenes

17. Example Scenario: CYP2D6 Resequencing 21912690..21912920 21918000..21919500

18. Example Scenario: CYP2D6 Resequencing CYP2D6 gene CNVs pseudogenes

19. RExPrimer Results

20. Experimental Validation

21. 1 2 3 4 5 100 bp M 1180 bp Exon 1-2 860 bp Exon 3-5 624 bp Exon 6-8 468 bp Exon 9 Amplification Results 4.6 kb Whole gene amplicon of CYP2D6 gene amplification from 5 diff samples 1 Nested PCR yields exon 1-9 amplicon 2

22. Conclusions RExPrimer is successful at : • designing resequencing primers; specific to the target gene • Guiding/assisting users to make the PCR experimental design • All-in-one graphic user-interface of • gene structure view • alignment true/pseudogenes • SNP from several ethnics • CNVs • www4a.biotec.or.th/rexprimer

23. Future Work Currently adding more organisms to the RExPrimer platform • Bovine • Porcine • Mouse • Canine • Chicken • Horse • Chimpanzee

24. Acknowledgements • Jittima Piriyapongsa • Chumpol Ngamphiw • Anunchai Assawamakin • Payiarat Suwannasri • Uttapong Ruangrit • Sissades Tongsima • Philip J. Shaw • BIOTEC • Siriraj Hospital, Mahidol University

25. Thank you for your attention  Questions?