Genotyping Workflow

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Genotyping Workflow

• Identified candidate genes and provided
  targeted/tagging SNPs from the PAAR group PIs.
• Current genotyping methods available in our lab
• SBE-DHPLC, Taqman probe genotyping assay,
  Invader assay, GeneScan (PCR-Sizing), Sequencing,
  Snapshot Multiplex (SBE-capillary
  electrophoresis) and Quantitative-RT PCR.

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QC steps for genotyping

• Standard steps to design specific primers for
  genotyping assays using bioinformatic tools
• 1. BLAT your sequence to check if there are any
  homologous sequences present in the list of the
  search results, if so, carefully choose unique
  sequence for primers.
• 2. Link to Genome Browser to check SNPs,
  repeating elements and segmental duplications.
• 3. BLAST two PCR primers to ensure NO
  non-specific binding to homologous region in the
  human genome.
• 4. Database of Genomic Variants - TCAG DB
• http//projects.tcag.ca/variation/cgi-bin/gbrowse
  /hg17

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UGT1A9 tagging SNPs genotyping (Wanqings
project)

• This project targets the whole UGT1A9 related
  region, it spans 36kb from the upstream right
  after UGT1A10 exon1 to downstream intron1 before
  UGT1A7 exon1.
• 19 tagging SNPs (bins) were selected by high LD
  level (r2gt0.8, MAFgt0.05) and the tagger program
  using the HapMap data (114 SNPs) and the Big1A
  data (36 SNPs).
• Bin 1 and 6 have been genotyped with -118T indel,
  I399 C/T and -275 T/A, -2152 C/T previously.
• Samples to be genotyped the 83 liver DNAs.
• Plan to set up Snapshot multiplex assays.

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Identify the SNP locations

• To locate the 17 tagging SNPs in the ref. Seq -
  using Align two sequences (bl2seq) tool in NCBI
  website, entering dbSNP flanking sequence and
  UGT1A gene ref. Seq accession AF297093.
• To be cautious there are some errors in dbSNP.
  The sequences in dbSNP may not be suitable for
  designing primers.

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http//www.ncbi.nlm.nih.gov/SNP/
Bin 3
100 match to the ref. seq
bl2seq
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3 flanking seq in dbSNP
homologous region
100 match in right region
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Taqman gene copy number assays

• Go to http//www.appliedbiosystems.com/
• Click Taqman gene expression assays under
  Taqman products.
• It will show
• individual assays -
• Taqman Gene Copy Number Assays
• Taqman Gene Expression Assays
• Custom Taqman Gene Expression Assays
• (note Gene expression assays are designed with
  primers in two neighboring exons to ensure
  specificity for mRNA so it is not suitable for
  the analysis of genomic DNA.)

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UGT2B17 gene deletion genotyping(Anna DiRienzos
project)

• Gene copy number assay is not available for
  UGT2B17 gene.
• Eden has set up UGT2B17 deletion genotyping assay
  by Q-RT PCR with SYBR green and genotyped 54
  Caucasian liver DNAs.

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RT-PCR with SYBR Greenfor UGT2B17 deletion typing

• The calculated quantity per sample is based on
  its Ct and the known concentrations of each point
  on the standard curve.
• UGT2B17 copy number is expressed as the ratio of
  UGT2B17/PMP quantity for each sample
• Gene copy number discerned by value of ratio

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UGT2B17 copy number determination
Examples of ratio
Ratios defined as
Genotyping of UGT2B17 in 54 Caucasian livers
(PAAR) 2 copies 22 1 copy 27 0 copies 4 1
undetermined (high ratio, possible duplication?)
Positive controls identified from data in
McCarroll et al (2006)
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Next planning for UGT2B17

• We could design a custom Taqman assay for UGT2B17
  deletion If well genotype large-scale samples.
• - use less DNA (10ng per rxn) vs. SYBR green
  assay (20ng per rxn x 2 40ng for test gene and
  control gene).
• - more accurate as using Taqman probe and duplex
  PCR in a single tube for test gene and control
  gene.
• - also more accurate to pick up potential
  duplications.