Analysis of allelic imbalance in JMML sample D003. (a) For SNP array analysis, granulocytes were purified by density centrifugation on a Ficoll gradient. DNA was extracted using the Trifast kit (Peqlab, Erlangen, Germany) and checked for integrity by agarose gel electrophoresis. The oligonucleotide arrays (GeneChip Mapping 100K array set, Affymetrix, Santa Clara, CA, USA) were prepared according to the manufacturer's instructions. Briefly, genomic DNA was digested using HindIII or XbaI, before ligation to adapters and PCR-based amplification of adapter-ligated fragments. Amplified products were fragmented, end-labeled and hybridized to the probe arrays. Signals corresponding to a total of 116 204 probe sets were read using a confocal scanner (Affymetrix). DNA copy numbers were calculated from raw signal values using dChip (Zhao et al., 2004). A reference data set of 60 acute myeloid leukemia remission samples processed at the same facility was used as standard for diploidy at each locus. Genomic copy numbers at 3955 SNP loci on chromosome 3 and 3959 SNP loci on chromosome 6 are displayed. Values were inferred by median smoothing with window size 50. The data indicate gain of a 63-Mbp segment comprised of chromosome bands 3q22–q29 and heterozygous deletion of a 72-Mbp segment corresponding to bands 6q16–q27. (b) Confirmation of copy number abnormalities by array-based comparative genomic hybridization. We used a DNA chip containing more than 8000 bacterial artificial chromosome/P1-derived artificial chromosome clones (manufactured at DKFZ, Heidelberg, Germany). Clone selection, spotting, labeling and hybridization were performed as described previously (Zielinski et al., 2005). Image data were acquired using a dual laser scanner and GenePix Pro 4.0 software (Axon Instruments, Union City, CA, USA). Further analysis was carried out using R software (‘marray’ and ‘aCGH’, http://www.r-project.org). Raw fluorescence intensity values were normalized with the print-tip LOESS function, and spot quality criteria were set as foreground to background >3.0 and standard deviation of triplicates <0.2. GLAD software was used for breakpoint calling (Hupe et al., 2004). Signal ratios of 388 probes on chromosome 3 and 318 probes on chromosome 6 are displayed.