RExPrimer

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RExPrimer

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

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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)

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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

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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/ExonPri
  mer.html)
• Genomic Primer (www-fgg.eur.nl/kgen/primer/Genomic
  _Primers.html)

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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
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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.)

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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

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RExPrimer Workflow
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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

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Case Study CYP2D6 Resequencing
DNA Resequencing
SNP Genotyping
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Case Study CYP2D6 Resequencing
DNA Resequencing
CYP2D6
SNP Genotyping
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Case Study CYP2D6 Resequencing
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Case Study CYP2D6 Resequencing
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Case Study CYP2D6 Resequencing
CYP2D6 gene
pseudogenes
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Case Study CYP2D6 Resequencing
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Example Scenario CYP2D6 Resequencing
CYP2D6 gene
CNVs
pseudogenes
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Example Scenario CYP2D6 Resequencing
21912690..21912920
21918000..21919500
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Example Scenario CYP2D6 Resequencing
CYP2D6 gene
CNVs
pseudogenes
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RExPrimer Results
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Experimental Validation
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Amplification Results
4.6 kb
Whole gene amplicon of CYP2D6 gene
amplification from 5 diff samples
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Nested PCR yields exon 1-9 amplicon
2
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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

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Future Work

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

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Acknowledgements

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

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Thank you for your attention ?
Questions?