MAGE-TAB Version	1.1										
Investigation Title	TARGET Analysis of Methylation In Childhood AML										
Experimental Design	disease state design										
Experimental Design Term Source REF	EFO										
Experimental Factor Name											
Experimental Factor Type											
Experimental Factor Term Source REF											
Person Last Name	NCI Office of Cancer Genomics (OCG)	NCI Center for Biomedical Informatics and Information Technology (CBIIT)	Arceci	Meshinchi	Farrar	Wai					
Person First Name			Robert	Soheil	Jason	Daniel					
Person Mid Initials			J		E	H					
Person Email	ocg@mail.nih.gov	ncicbiit@mail.nih.gov	rarceci@phoenixchildrens.com	smeshinc@fhrc.org	jefarrar@uams.edu	dwai@phoenixchildrens.com					
Person Phone	+1 301 451 8027	+1 888 478 4423	602-827-2508	206-667-4077	501-603-1224	602-827-2534					
Person Fax	+1 301 480 4368		602-271-0264	206-667-4310	501-526-5813	602-271-0264					
Person Address	31 Center Dr, Rm 10A07, Bethesda, MD 20892	9609 Medical Center Dr, Rockville, MD 20850	445 N. 5th St, Tgen Building Room 322, Phoenix, AZ 85004	1100 Fairview Avenue North, D5-380, POB 19024, Seattle, WA  98109	1 Children's Way, MS 512-10, Little Rock, AR 72202	445 N. 5th St, Tgen Building Room 322, Phoenix, AZ 85004					
Person Affiliation	National Cancer Institute	National Cancer Institute	University of Arizona College of Medicine	Fred Hutchinson Cancer Research Center	University of Arkansas for Medical Sciences	University of Arizona College of Medicine					
Person Roles	funder	data coder;curator	investigator	investigator	submitter;investigator;data analyst	submitter;investigator;data analyst					
Person Roles Term Source REF	EFO	EFO;EFO	EFO	EFO	EFO	EFO					
Quality Control Type											
Quality Control Term Source REF											
Replicate Type											
Replicate Term Source REF											
Normalization Type											
Normalization Term Source REF											
Date of Experiment											
Public Release Date											
PubMed ID											
Publication DOI											
Publication Author List											
Publication Title											
Publication Status											
Publication Status Term Source REF											
Experiment Description	TARGET Analysis of Methylation In Childhood AML										
Protocol Name	phoenixchildrens.com:Protocol:DNA-Extraction-Qiagen:01	jhu.edu:Protocol:MethylationArray-Labeling-Illumina-27k:01	tgen.org:Protocol:MethylationArray-Labeling-Illumina-450k:01	jhu.edu:Protocol:MethylationArray-Hybridization-Illumina-27k:01	tgen.org:Protocol:MethylationArray-Hybridization-Illumina-450k:01	jhu.edu:Protocol:MethylationArray-Scanning-Illumina-27k:01	tgen.org:Protocol:MethylationArray-Scanning-Illumina-450k:01	uams.edu:Protocol:MethylationArray-DataNormalization-Illumina-27k:01	uams.edu:Protocol:MethylationArray-DataNormalization-Illumina-450k:01	uams.edu:Protocol:MethylationArray-GeneAnnotation:01	uams.edu:Protocol:MethylationArray-GeneAnnotation:02
Protocol Type	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol 	nucleic acid hybridization to array protocol 	array scanning and feature extraction protocol	array scanning and feature extraction protocol	normalization data transformation protocol	normalization data transformation protocol	data transformation protocol	data transformation protocol
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Protocol Description	"Starting with one vial of cryopreserved cells derived from Bone Marrow (or if Bone Marrow unavailable, Peripheral Blood), cells undergo a quick thaw and are adding to a tube containing PBS (to dilute the residual DMSO).  Cells are spun at 1000RPM and pelleted.  The pellet is vortexed and resuspended in lysis buffer from the Qiagen AllPrep DNA/RNA mini kit.  The lysate is added to an RNeasy spin column to isolate RNA while the flow through is added to the DNA spin column for subsequent DNA isolation.  After a series of washes on each column, DNA or RNA is eluted into a tube using elution buffer or RNase-free H20 respectively.  Both nucleic acids are evaluated on the NanoDrop for initial sample concentration and quality.  DNA quantification is then assessed using the Picogreen assay for better accuracy.  RNA quality and quantity is also assessed using the Agilent Bioanalyzer nanochip."	"See hybridization protocol description since  for Illumina Infinium 27k labeling is done after hybridization."	"See hybridization protocol description since  for Illumina Infinium 450k labeling is done after hybridization."	"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 37oC 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 95oC for 30 seconds and 50oC 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 37oC. Samples were fragmented at 37oC for 60 minutes and precipitated in isopropanol. Re-suspended samples were denatured in a 96-well plate heat block at 95oC 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 48oC 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."	"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 37oC 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 95oC for 30 seconds and 50oC 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 37oC. Samples were fragmented at 37oC for 60 minutes and precipitated in isopropanol. Re-suspended samples were denatured in a 96-well plate heat block at 95oC 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 48oC 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"	"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). "	"Methylated and unmethylated signal intensity and detection p-values were extracted after background correction and dye-bias equalization by normal-exponential convolution ('noob') as implemented in the minfi package. "	"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 &gt;  0.01 are masked as NA for individual samples."
Protocol Parameters											
Protocol Hardware											
Protocol Software											
Protocol Contact											
SDRF File	TARGET_AML_MethylationArray_20160812.sdrf.txt										
Term Source Name	NCBITaxon	NCIt	MO	EFO	OBI						
Term Source File	http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://ncit.nci.nih.gov/	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo	http://purl.obolibrary.org/obo/obi						
Term Source Version											