GBS-MeDIP: A protocol for parallel identification of genetic and epigenetic variation in the same reduced fraction of genomes across individuals

Abstract

The GBS-MeDIP protocol combines two previously described techniques, Genotype-by-Sequencing (GBS) and Methylated-DNA-Immunoprecipitation (MeDIP). Our method allows for parallel and cost-efficient interrogation of genetic and methylomic variants in the DNA of many reduced genomes, taking advantage of the barcoding of DNA samples performed in the GBS and the subsequent creation of DNA pools, then used as an input for the MeDIP. The GBS-MeDIP is particularly suitable to identify genetic and methylomic biomarkers when resources for whole genome interrogation are lacking.

Keywords: Cancer; Clinical Protocol; Genomics; High Throughput Screening; Molecular Biology; Sequencing.

Graphical abstract

Figure 1

Schematic representation of the structure of each fragment bound to the common and the barcode adapters The figure represents the disposition of barcodes on the final fragment constructs after ligation, and the locations where the primers bind in order to amplify the desired fragment.

Figure 2

Agarose gel electrophoresis showing genomic digestion and PCR amplification of the libraries (A–C) The figure represents the agarose gel electrophoresis of the PstI digestion reaction after 16 h PstI digestion (A); PCR amplifications of the genomic (G) and methylomic (M) libraries before (B) and after (C) purification by AMPure XP magnetic beads (representation of two pools of 24 samples each), which eliminates small fragments, including unbound adapters and primer-dimers.

Figure 3

Real time amplification curve of GBS MeDIP-PCR Green line represents the number of cycles chosen; red lines represent the qPCR amplification curve of the library in triplicate, and the black line is the Ct threshold, which is the point where fluorescence of the PCR product can be detected above the background signal. The x and y axes represent number of cycles and fluorescence, respectively.

Figure 4

High-sensitivity chip electropherogram of the purified libraries after PCR The fragments pattern after GBS and GBS-MeDIP PCR purifications using AMpure XP magnetic beads. Green and purple lines show lower and upper markers, respectively. X and Y axes demonstrate the migration time (s) and fluorescence units (FU), respectively. Each number represents one fragment length in bps.