Computational protocol: Identifying breast cancer risk loci by global differential allele-specific expression (DASE) analysis in mammary epithelial transcriptome

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Protocol publication

[…] Genomic and mRNA sequences flanking selected SNPs were retrieved from NCBI and primers were designed accordingly using the web-based Primer3 software (http://frodo.wi.mit.edu/primer3/). The sequences of primers are available upon request. PCR amplification was performed using GoTaq® Green Master Mix (Promega) and relevant gDNA and cDNA samples on a thermal cycler (Applied Biosystems, Model 2720) using the following program: 94°C 3 minutes for initial denaturing, followed by 10 cycles touchdown PCR (94°C 30 seconds, 65°C −55°C <−1°C / cycle> 30 seconds, 72°C 30 seconds) and 35 cycles regular PCR (94°C 30 seconds, 60°C 30 seconds, 72°C 30 seconds), final extension for 5 minutes at 72°C and then hold at 4°C. PCR product purification and Sanger sequencing were performed by Beckman Coulter Genomic Services (Danvers, MA). Sequencing trace files were analyzed using Sequencher software (v4.1.4., Gene Codes, MI). The DASE value between two alleles X and Y in a sample of a given SNP was then calculated using the peak height of each allele in the chromograms originated from cDNA samples, justified by that originated from genomic DNA samples. A positive DASE event by sequencing is defined when the height of the peak representing one allele is less than half of the peak height of the other allele. The fact that we chose a different threshold (DASE=1) for DASE validation by Sanger sequencing is justified by the different data dynamic ranges between these two platforms. The SNP array gives numeric results with a dynamic range of 216. On the other hand, Sanger sequencing gives graphic trace files, and the usable peak-heights for quantification are usually within a few dozen pixels. Based on this dissimilarity, we chose different thresholds for DASE calling for each method. […]

Pipeline specifications

Software tools Primer3, Sequencher
Application qPCR
Organisms Homo sapiens