Computational protocol: Systematic Identification of Placental Epigenetic Signatures for the Noninvasive Prenatal Detection of Edwards Syndrome

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Protocol publication

[…] MeDIP-identified locus was analyzed by a quantitative DNA methylation assay, the Epityper (Sequenom) (). Briefly, the genomic locus of interest in a bisulfite-converted DNA sample was subjected to PCR amplification by primers listed in , in vitro transcription into RNA, and uracil-specific cleavage on the complementary strand. The product derived from PCR amplicons would therefore be fragmented. Fragments derived from the methylated and unmethylated DNA molecules would have different masses due to the difference in nucleotide sequence at the CpG site caused by bisulfite conversion. The mass differences were readily resolved and quantified as distinct peaks by matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry. In the Epityper, the DNA methylation level of one or more CpG sites in any one cleaved fragment was reported as an integral unit, namely a CpG unit. For each CpG unit, a methylation index (MI) was calculated as the ratio of the methylated peak height to the sum of the methylated and unmethylated peak heights.Priority for Epityper analysis was given for MeDIP-identified loci that would (i) allow appropriate PCR design, including a high annealing temperature of the primers; (ii) result in Epityper assays with the maximum number of detectable fragments (CpG units), the masses of which were within the detection range of the mass spectrometer; and (iii) allow specific PCR amplification from bisulfite-converted DNA. (i)-(ii) were mainly calculated by the Epidesigner 2.0 program (Sequenom); and (iii) was checked by the BiSearch search tool . Occurrence of genomic variations in the analyzed regions were checked using the Database of Genomic Variants ( . We made sure that there was no reported variant of higher than 1% frequency in the regions we analyzed. […]

Pipeline specifications

Software tools EpiDesigner, BiSearch
Databases DGV
Application BS-seq analysis