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 PrimerZ Mut Screener Easy ExonPrimer Variant SEQr ELXR SNP box Exon Primer Genomic Primer Sequence information retrievals and annotations +++-++-- Promoter resequencing ++ - - -- Continuous genomic DNA resequencing -++--- -- Combining two short exons as one template -+----++- Overlapping primers for large exons ++ - - Redesign +------- ++ : 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 SNP Genotyping DNA Resequencing

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

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

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

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

15. Case Study: CYP2D6 Resequencing

16. Example Scenario: CYP2D6 Resequencing CYP2D6 gene pseudogenes CNVs

17. 21918000..2 1919500 Example Scenario: CYP2D6 Resequencing 2191 2690..2 1912920

18. Example Scenario: CYP2D6 Resequencing CYP2D6 gene pseudogenes CNVs

19. RExPrimer Results

20. Experimental Validation

21. Amplification Results Whole gene amplicon of CYP2D6 gene amplification from 5 diff samples 4.6 kb 468 bp 624 bp 860 bp 1180 bp 12345 Exon 1-2 Exon 3-5 Exon 6-8 Exon 9 100 bp M 1 2 Nested PCR yields exon 1-9 amplicon

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?