Methylation

Infinium HumanMethylation27 Bead Chip (Illumina) for Acute Myeloid Leukemia (AML)

*Protocols performed at the Johns Hopkins University.

Bisulfite conversion of genomic DNA was performed with EZ DNA methylation Kit (Zymo Research, Irvine, CA, #D5002) following the manufacturer’s protocol with modifications for the Infinium Methylation Assay. Briefly, one microgram of genomic DNA was mixed with 5 µl of Dilution Buffer and incubated at 37°C for 15 minutes and then mixed with 100 µl of conversion reagent prepared as instructed in the protocol. Mixtures were incubated in a thermocycler for 16 cycles at 95°C for 30 seconds and 50°C for 60 minutes. Bisulfite-converted DNA samples were loaded onto the provided 96-column plates for desulphonation, washing and elution. The concentration of bisulfite-converted, eluted DNA was measured by UV-absorbance using a NanoDrop-1000 (Thermo Fisher Scientific, Waltham, MA). Bisulfite-converted genomic DNA was analyzed using the Infinium Human Methylation27 Beadchip Kit (Illumina, San Diego, CA, #WG-311-1202). DNA amplification, fragmentation, array hybridization, extension and staining were performed with reagents provided in the kit according to the manufacturer’s protocol (Illumina Infinium II Methylation Assay, #WG-901-2701). Briefly, 4 µl of bisulfite-converted genomic DNA at a minimum concentration of 20 ng/µL) was added to 0.8 ml 96-well storage plate (Thermo Fisher Scientific), denatured in 0.014N sodium hydroxide, neutralized and then amplified for 20-24 hours at 37°C. Samples were fragmented at 37°C for 60 minutes and precipitated in isopropanol. Re-suspended samples were denatured in a 96-well plate heat block at 95°C for 20 minutes. 15 µl of each sample was loaded onto a 12-sample BeadChip, assembled in the hybridization chamber as instructed by the manufacturer and incubated at 48°C for 16-20 hours.  Following hybridization, the BeadChips were washed and assembled in a fluid flow-through station for primer-extension reaction and staining with reagents and buffers provided.

Polymer-coated BeadChips were scanned in an iScan scanner (Illumina) using Inf Methylation mode.

Signal intensity and Beta value data were extracted using the Methylation module of GenomeStudio (Illumina, v2011.1) software following the Methylation analysis pipeline without normalization or background subtraction using BeadChip content descriptors provided by the manufacturer (HumanMethylation27_270596_v.1.2.bpm).

Summary beta values for each locus with annotations for Illumina probe name, gene symbol, chromosome and CpG position (UCSC hg18).

Data were normalized using functional normalization (funnorm) as implemented in the minfi package and summarized as beta values [M /(M+U)] with annotation at each locus for Illumina probe name, gene symbol, chromosome and CpG position (UCSC hg19).  Probes having an annotated SNV within the CpG or SBE site are masked as NA across all samples.  Probes where the non-detection probability was >  0.01 are masked as NA for individual samples.

Infinium HumanMethylation450 Bead Chip (Illumina) for Clear Cell Sarcoma of the Kidney (CCSK)

*Protocol performed at Ann & Robert H. Lurie Children's Hospital.

DNA was extracted from tumor samples at Nationwide Children's BioPathology Center (BPC) by using the standard BPC protocol. The DNA samples were analyzed by Pico green to verify gDNA concentration, spectrophotometry to verify DNA purity, and gel electrophoresis to verify DNA quality.  DNA samples (1.5 ug) diluted in nuclease-free water were provided to the Northwestern University Genomics Core in 96-well plate format for Illumina 450K DNA methylation analysis. Randomly selected samples were tested by Northwestern University to verify that the correct concentration had been provided.                

Nucleic acid hybridization and labeling were performed according to the manufacturer's protocol for the Illumina 450K array at the Northwestern University Genomics Core Facility. Nucleic acid labeling is completed after the hybridization step with Illumina Infinium 450K arrays.   

The array scanning protocol was performed according to the manufacturer's protocol for the Illumina 450K array at the Northwestern University Genomics Core Facility.                 

Raw data files (1 red and 1 green .idat file per sample and 1 .sdf file from each array, which included 12 samples per array) were processed at the Northwestern University Genomics Core Facility by BeadStudio software. The following subtables were generated by BeadStudio and were downloaded in .txt format (Level 2 data): (1) the Sample Methylation Profile .txt, (2) the Control Profile .txt, and (3) the Control Probe Profile .txt. Several quality control steps were used for these data. Samples were subjected to the internal quality controls in the Bioconductor lumi package. Samples were subjected to a color balance check using the Bioconductor lumi package. Gender analysis was performed to increase our confidence that the data correctly corresponded to the expected sample. Because one of the X chromosomes is heavily methylated in females, the density of X-chromosome methylation is a good indicator of gender. Unsupervised hierarchical clustering based on all methylated regions on the X-chromosome was performed using average-linkage clustering with CLUSTER and the results were displayed with TREEVIEW. The tumors were assigned a gender based on their clustering in the tumor dendogram, which was checked against the known gender of the sample.               

The Sample Methylation Profile text, which included information for all of the samples, was broken down at the DCC into a single .txt file (level 2 data) per sample containing the following columns: (1) the sample ID, (2) probe name, (3) AVG_Beta value, (4) gene Symbol, (5) chromosome, and (6) position.

Infinium HumanMethylation450 Bead Chip (Illumina) for Osteosarcoma (OS)

*Protocols performed at the Phoenix Children’s Hospital and Baylor College of Medicine.

Bisulfite conversion of genomic DNA was performed with EZ-96 DNA methylation Kit (Zymo Research, Irvine, CA, #D5002) following the manufacturer’s protocol with modifications for the Infinium Methylation Assay. Briefly, one microgram of genomic DNA was mixed with 5 µl of Dilution Buffer and incubated at 37°C for 15 minutes and then mixed with 100 µl of conversion reagent prepared as instructed in the protocol. Mixtures were incubated in a thermocycler for 16 cycles at 95°C for 30 seconds and 50°C for 60 minutes. Bisulfite-converted DNA samples were loaded onto the provided 96-column plates for desulphonation, washing and elution. Bisulfite-converted genomic DNA was analyzed using the Infinium Human Methylation450K Beadchip Kit (Illumina, San Diego, CA, #WG-314-1001). DNA amplification, fragmentation, array hybridization, extension and staining were performed with reagents provided in the kit according to the manufacturer’s protocol (Illumina Infinium II Methylation Assay, #WG-901-2701). Briefly, 4 µl of bisulfite-converted genomic DNA was added to 0.8 ml 96-well storage plate (Thermo Fisher Scientific), denatured in 0.014N sodium hydroxide, neutralized and then amplified for 20-24 hours at 37°C. Samples were fragmented at 37°C for 60 minutes and precipitated in isopropanol. Re-suspended samples were denatured in a 96-well plate heat block at 95°C for 20 minutes. 15 µl of each sample was loaded onto a 12-sample BeadChip, assembled in the hybridization chamber as instructed by the manufacturer and incubated at 48°C for 16-20 hours.  Following hybridization, the BeadChips were washed and assembled in a fluid flow-through station for primer-extension reaction and staining with reagents and buffers provided.

Polymer-coated BeadChips were scanned using Illumina iScan technology which outputs data in the format of IDAT files.  These are then used retrieve the probe intensities and calculate the beta-values. Raw unmethylated and methylated intensities were background corrected using out-of-band correction.

Probe intensities were then color corrected using Lumi's dye bias correction algorithm.  Beta-values were calculated from probe intensities and corrected for probe bias using the beta mixture quantile dilation (BMIQ) normalization method.