RExPrimer Pongsakorn Wangkumhang, M.Sc. Biostatistics and Informatics Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand
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)
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
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 )
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
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.)
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
RExPrimer Workflow
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
Case Study: CYP2D6 Resequencing SNP Genotyping DNA Resequencing
SNP Genotyping DNA Resequencing CYP2D6 Case Study: CYP2D6 Resequencing
Gene location of CYP2D6 and its pseudogenes Case Study: CYP2D6 Resequencing
Gene location of CYP2D6 and its pseudogenes Chromosome mapping of CYP2D6 and its pseudogenes Case Study: CYP2D6 Resequencing
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
Case Study: CYP2D6 Resequencing
Example Scenario: CYP2D6 Resequencing CYP2D6 gene pseudogenes CNVs
Example Scenario: CYP2D6 Resequencing
Example Scenario: CYP2D6 Resequencing CYP2D6 gene pseudogenes CNVs
RExPrimer Results
Experimental Validation
Amplification Results Whole gene amplicon of CYP2D6 gene amplification from 5 diff samples 4.6 kb 468 bp 624 bp 860 bp 1180 bp Exon 1-2 Exon 3-5 Exon 6-8 Exon bp M 1 2 Nested PCR yields exon 1-9 amplicon
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
Future Work Currently adding more organisms to the RExPrimer platform Bovine Porcine Mouse Canine Chicken Horse Chimpanzee
Acknowledgements Jittima Piriyapongsa Chumpol Ngamphiw Anunchai Assawamakin Payiarat Suwannasri Uttapong Ruangrit Sissades Tongsima Philip J. Shaw BIOTEC Siriraj Hospital, Mahidol University
Thank you for your attention Questions?