Genomic workbench 7

We analyzed by array-CGH the genome wide copy number variations in the DNA extracted from the 10 NB samples here considered coming from pre-chemotherapy surgical resection using Human Genome CGH Microarray 4X 180K Kit (Agilent Technologies, Santa Clara, CA, USA), with a mean resolution of approximately 25 kb, as previously described [93 (link)]. Tumor DNAs were extracted using the QIAamp DNA Extraction Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Images of the array were acquired with the Agilent C Scanner (Agilent Technologies), which were processed using the Agilent Feature Extraction 10.5 Software. The data were analyzed using the Genomic Workbench 7.0.40 software (Agilent), the altered chromosomal regions and breakpoints events were detected using the algorithm ADM-1 (threshold 10) with 0.5 Mb window size to reduce false positives [93 (link)]. The raw data are stored in the BIT-NB Bio Bank of IRCCS Gaslini.

All samples were profiled on CGH oligonucleotide array platform (Agilent Technologies, Santa Clara, California, USA) with a mean resolution of ~25 kb. Oligo-array data were analyzed with Genomic Workbench 7.0.40 software (Agilent). Chromosome positions were determined using GRCh37/hg19 (UCSC Genome Browser, http://genome.ucsc.edu, Feb. 2009 release). To make aberration calls, an aberration detection algorithm ADM-1 was used at threshold 10, and an aberration filter was set at 2 for the minimum number of probe region, and 1 for minimum absolute average log2 ratio to reduce false positives. The quality of the test was assessed on the strength of the QCmetrics values. Polymorphisms (http://projects.trag.ca/variation/) were not included because considered normal variants. As in discovery set, gains and losses were set at 0.2 and −0.3, respectively whereas amplification and homozygous deletions were set at 2 and −2, respectively.

DNAs from 260 NB primary samples and 12 NB cell lines were tested by high-resolution oligonucleotide array-CGH and Single Nucleotide Polymorphism (SNP) array using a 4 × 180 K platform (Agilent Technologies, Santa Clara, CA, USA) with a mean resolution of approximately 25 kb. The copy number variants were defined as a displacement of the normal value of at least 8 consecutive probes and the mapping positions refer to the Genome Assembly GRCh19/hg19 (UCSC genome browser, http://genome.ucsc.edu, accessed on 20 October 2022). Images of the array were acquired by the Agilent C Scanner, and visualized by the Agilent Feature Extraction 10.5 Software. Data were examined by the genomic Workbench 7.0.40 software (Agilent), to detect chromosomal aberrations and breakpoints by ADM-1 (threshold 10), with 0.5 Mb window size in order to minimize potential false positives. The cutoffs to call aberrations were: 0.2 = gains, −0.3 = losses, 2 = amplifications, and −2 = homozygous deletions. The quality of the test was assessed by the strength of the QC metrics values. Polymorphisms (http://projects.trag.ca/variation/, accessed on 20 October 2022) were not included since they were considered normal variants.

TIFF multiscan images from SureScan Microarray Scanner (Agilent Technologies, USA) were processed using Feature Extraction Software 11.5 (Agilent Technologies, USA). In this software also, the image processing was performed and acquired files with spot intensities for every microarray field (corresponding to one condition). The raw data underwent quality control, normalization, and statistical analysis in GeneSpring 14.9 GX software for gene expression analysis and Agilent Genomic Workbench 7.0.4.0 for DNA methylation analysis.

EDTA blood-derived genomic DNA (average DLRS value 0,1) of F16 and F17, carrying the GT198 truncating mutation p.(Trp173*) (Table 1), was further screened for copy number changes and structural rearrangements affecting GT198 using a custom-made 60k eArray (Design:081270, brca1-2region, Agilent Technologies) with high resolution in the genomic regions on chromosome 17 (chr17:40,600,000-41,756,000, 1.2 Mb, GRCh37/hg19) and chromosome 13 (chr13:32,414,344-33,490,000, 1.1 Mb, GRCh37/hg19), with an average probe spacing of 300 bp. The GT198 gene was covered by 85 probes.
Selected index cases (n=8) carrying putative pathogenic GT198 variants were also screened for HBOC predisposing copy number variations by a customized high resolution 60k eArray (Design:069100, HBOC-2, Agilent technologies) [32 (link)]. Array CGH analysis was performed as recommended by the manufacturer. The female human DNA EA-100F was used as control (Kreatech Biotechnology, Amsterdam, The Netherlands). Fluorescence signals were scanned using a Dual Laser Scanner G2565CA (Agilent Technologies). Raw data analysis was performed using Feature extraction version 11.0.1.1 (Agilent Technologies). For further data analysis, Genomic Workbench 7.0.4.0 (Agilent Technologies) was used: ADM-2 algorithm, threshold 6, and no aberration filter for the brca1-2region, while a 2log0.2 filter was used for the HBOC-2 design.

For 32 of a total of 51 patients, CGH array data was collected. DNA was extracted using the DNeasy kit. CGH arrays were applied using Agilent SurePrint G3 Human CGH 4 × 180 k arrays, according to the manufacturer's instructions. CGH FE files were processed and normalized, using the Agilent Genomic WorkBench 7.0.4.0. The DNAcopy R package was used to estimate DNA copy number for genomic segments. From the segmentation data, the copy number of each gene was calculated, by averaging copy numbers of all exonic segments of each gene.

Array-based comparative genomic hybridization (array CGH) was performed using a 400 k SurePrint G3 Custom CGH Human Genome Microarray (e-Array design 84704, Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s protocol by hybridizing 500 ng of patient DNA isolated from EDTA material against sex-matched Human Reference DNA (Megapool Reference DNA, Kreatech, Amsterdam, The Netherlands). Regions of genomic copy number change were analyzed and interpreted with Agilent Genomic Workbench 7.0 software under the following analysis setting: aberration detection algorithm ADM-2, threshold 6.0, aberration filter of at least four aberrant probes (resulting in an average genome-wide resolution of ~ 30 kb), and average log2 ratios of + 0.3 for gains and − 0.3 for losses.
The original description of the IKZF1^plus gene deletion profile by Stanulla et al. has been based on MLPA analyses targeting IKZF1, CDKN2A, CDKN2B, PAX5, and CRLF2-P2RY8, as well as multiplex PCR for the detection of ERG deletions. Prior to this study, reproducibility of IKZF1^plus profiles in our high-resolution array CGH was verified for selected cases (data not shown).
Cases indicative of masked hypodiploidy (i.e., presence of multiple tetrasomies) were analyzed on CGH+SNP array (e-Array design 85320, Agilent Technologies) using sex-matched reference DNA (Human Reference DNA, Agilent Technologies).