Representation of the genome generated by digestion with either MspI (methylation-insensitive) or HpaII (methylation-sensitive) and amplification by ligation-mediated PCR according to the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) protocol (See PMID 16809668: B. Khulan, et al. Genome Res. 2006 Aug;16(8):1046-55).
Representation of the genome generated by digestion with either MspI (methylation-insensitive) or HpaII (methylation-sensitive) and amplification by ligation-mediated PCR according to the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) protocol (See PMID 16809668: 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
Hybridizations were carried out for 18 hr at 42C in the NimbleGen Service Laboratory. See Roche NimbleGen website and PMID 16075461 (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) at 5-um resolution, and data were extracted from scanned images using NimbleScan 2.0 extraction software, as previously described in PMID 16075461 (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
AM3042
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). After normalization, background noise was calculated for each channel by determining a threshold of 2.5 median absolute deviations from the median of the random probes’ signal. Each normalized channel was subsequently centered by subtracting its noise threshold from the array’s signal. The HpaII/MspI (unmethylated/whole genome reference) ratio was then determined for each probe set on array.
