Male, 45 years, FAB M1, 46,XY,add(2)(q35)[13]/46,XY[19], CEBPA silenced MspI representation of genomic DNA
Extracted molecule
genomic DNA
Extraction protocol
Representation of the genome generated by digestion with either MspI or HpaII and amplification by ligation-mediated PCR according to the HELP protocol (See B. Khulan, et al. Genome Res. 2006 Aug;16(8):1046-55)
Male, 45 years, FAB M1, 46,XY,add(2)(q35)[13]/46,XY[19], CEBPA silenced HpaII representation of genomic DNA
Extracted molecule
genomic DNA
Extraction protocol
Representation of the genome generated by digestion with either MspI or HpaII and amplification by ligation-mediated PCR according to the HELP protocol (See B. Khulan, et al. Genome Res. 2006 Aug;16(8):1046-55)
Label
Cy5
Label protocol
Random 9-mers pre-labeled with either Cy3 or Cy5
Hybridization protocol
See Roche NimbleGen website and Selzer RR, Richmond TA, Pofahl NJ, Green RD, Eis PS, et al. (2005) Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319. for details
Scan protocol
Scanning was performed using a GenePix 4000B scanner (Axon Instruments) as previously described in Selzer RR, Richmond TA, Pofahl NJ, Green RD, Eis PS, et al. (2005) Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319.
Description
none
Data processing
Signal intensities at each HpaII amplifiable fragment were calculated as a robust (25% trimmed) mean of their component probe-level signal intensities. Any fragments found within the level of background MspI signal intensity, measured as 2.5 mean-absolute-differences (MAD) above the median of random probe signals, were categorized as “failed.” These “failed” loci therefore represent the population of fragments that did not amplify by PCR, whatever the biological (e.g. genomic deletions and other sequence errors) or experimental cause. On the other hand, “Methylated” loci were so designated when the level of HpaII signal intensity was similarly indistinguishable from background. PCR-amplifying fragments (those not flagged as either “methylated” or “failed”) were normalized using an intra-array quantile approach wherein HpaII/MspI ratios are aligned across density-dependent sliding windows of fragment size-sorted data (described in detail in Thompson et al, Bioinformatics 2008;24:1161-1167.
