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Genotyping Workflow

. . 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 Ge

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Genotyping Workflow

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    1. 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.

    2. 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

    3. UGT1A9 tagging SNPs genotyping (Wanqing’s 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 (r2>=0.8, MAF>0.05%) 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.

    6. 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.

    9. 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.)

    11. UGT2B17 gene deletion genotyping (Anna DiRienzo’s 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.

    12. RT-PCR with SYBR Green for UGT2B17 deletion typing

    13. UGT2B17 copy number determination

    14. Next planning for UGT2B17 We could design a custom Taqman assay for UGT2B17 deletion If we’ll 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.

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