Transcript RExPrimer: an integrated primer designing tool increases

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?
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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
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Continuous genomic
DNA resequencing
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Combining two short
exons as one template
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Overlapping primers for
large exons
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Sequence information
retrievals and
annotations
Promoter resequencing
Redesign
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++ : 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)
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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
DNA Resequencing
SNP Genotyping
Case Study: CYP2D6 Resequencing
DNA Resequencing
SNP Genotyping
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
pseudogenes
Case Study: CYP2D6 Resequencing
Example Scenario: CYP2D6 Resequencing
CYP2D6 gene
CNVs
pseudogenes
Example Scenario: CYP2D6 Resequencing
21912690..219
12920
21918000..2
1919500
Example Scenario: CYP2D6 Resequencing
CYP2D6 gene
CNVs
pseudogenes
RExPrimer Results
Experimental Validation
Name
Sequence 5'→ 3'
CYP2D6_1F
GGCCTACCCTGGGTAAGGGCCTGGAGCAGGA
CYP2D6_1R
CTCAGCCTCAACGTACCCCTGTCTCAAATGCG
CYP2D6_2F
GAGACTCCTCGGTCTCTCG
CYP2D6_2R
TAATGCCTTCATGGCCACGCG
CYP2D6_3F
AGGCCTTCCTGGCAGAGATGAAG
CYP2D6_3R
CCCCTGCACTGTTTCCCAGA
CYP2D6_4F
CCAGCCACCATGGTGTCTTTG
CYP2D6_4R
GCCTCAACGTACCCCTGTCTC
CYP2D6_5F
GTACCCCTGTCTCAAATGCG
CYP2D6_5R
GTAAGGGCCTGGAGCAGGA
Purpose
whole gene
amplification
exon 1-2
amplification
exon 3-5 PCR
amplification
exon 6-8
amplification
exon 9
amplification
Amplification Results
4.6 kb
1
1
2
3
4
5
100 bp M
2
Exon 1-2
Exon 3-5
Exon 6-8
Exon 9
1180 bp
860 bp
624 bp
468 bp
Whole gene amplicon of
CYP2D6 gene amplification
from 5 diff samples
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
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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
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Bovine
Porcine
Mouse
Canine
Chicken
Horse
Chimpanzee
Acknowledgements
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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?