A screen for the presence of structural alterations was performed on PBL DNA from MLH1 epimutation carriers using a custom high-definition CGH array designed with eArray Software (Agilent Technologies) and manufactured by Agilent’s SurePrint oligo technology. This comprised 15,000 probes encompassing the MLH1 locus (region Chr3:36,450,000–37,900,000 within cytoband 3p22.2), with an average probe spacing of 100 bp intervals. Bioinformatics analysis was performed in R using the 2.15.12 Bioconductor statistical packages. Results were visualized on CytoGenomics Software (Agilent Technologies).
Cytogenomics software
Manufactured by Agilent Technologies
Sourced in United States
CytoGenomics is a software solution designed for analysis and interpretation of cytogenetic and genomic data. It provides comprehensive tools for visualization, analysis, and reporting of chromosomal aberrations, copy number variations, and other genomic changes.
Automatically generated - may contain errors
Lab products found in correlation
Feature extraction software, by Agilent Technologies (5 mentions)
Suretag complete dna labeling kit, by Agilent Technologies (4 mentions)
Dna microarray scanner, by Agilent Technologies (4 mentions)
Sureprint g3 human cgh microarray 4 180k, by Agilent Technologies (3 mentions)
Qiaamp dna micro kit, by Qiagen (3 mentions)
Suretag dna labeling kit, by Agilent Technologies (3 mentions)
Agilent genomic workbench software v7, by Agilent Technologies (2 mentions)
Genomic dna high throughput uls labeling kit, by Agilent Technologies (2 mentions)
Humanomni2, by Illumina (2 mentions)
Iscan array scanner, by Illumina (2 mentions)
Human omni5, by Illumina (2 mentions)
Earray online tool 4 180k microarray, by Agilent Technologies (2 mentions)
Genomestudio, by Illumina (2 mentions)
Surescan microarray scanner, by Agilent Technologies (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Qiasymphony automatic extractor, by Qiagen (2 mentions)
Genomic dna and human reference dna, by Promega (2 mentions)
Earray software, by Agilent Technologies (1 mentions)
Sureprint oligo technology, by Agilent Technologies (1 mentions)
Agilent suredesign, by Agilent Technologies (1 mentions)
43 protocols using cytogenomics software
1
Screening MLH1 Epimutation Structural Alterations
2
FFPE Tissue DNA Profiling Using CGH
3
CNV Detection and Analysis of Hearing Loss Genes
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We performed a CNV detection method with Ion AmpliSeq sequencing and multiplex PCR-based targeted genome enrichment. The detailed protocol has been described elsewhere [40 (link)]. The read depth data was used for copy number analysis. From the results of the CNVs analysis of the 2262 probands, we picked up 14 patients with OTOA gene CNVs.
We designed a custom aCGH for 68 genes previously reported as genetic causes of non-syndromic hearing loss using the Agilent web software (Agilent SureDesign, Agilent Technologies, Santa Clara, CA, USA), with the probes covering specific chromosomal regions of those genes at 150–200 bp intervals as a design-setting on the Agilent 8 × 60 K platform (Agilent Technologies, Santa Clara, CA, USA) [41 (link)]. There were 235 probes laid across the OTOA region (chr16:21,740,000–21,772,500). We used the same DNA samples as used for the amplicon resequencing, with quality assessment also performed. Five micrograms of genomic DNA were fragmented, and labeled with cyanine-3 for reference DNA samples and cyanine-5 for subjects, and then hybridized. We performed scanning of the array with a G2600D SureScan Microarray Scanner (Agilent Technologies) according to the manufacturer’s recommended protocols, and analyzed scanned aCGH data using CytoGenomics software version 3.0.6.6 (Agilent Technologies).
We designed a custom aCGH for 68 genes previously reported as genetic causes of non-syndromic hearing loss using the Agilent web software (Agilent SureDesign, Agilent Technologies, Santa Clara, CA, USA), with the probes covering specific chromosomal regions of those genes at 150–200 bp intervals as a design-setting on the Agilent 8 × 60 K platform (Agilent Technologies, Santa Clara, CA, USA) [41 (link)]. There were 235 probes laid across the OTOA region (chr16:21,740,000–21,772,500). We used the same DNA samples as used for the amplicon resequencing, with quality assessment also performed. Five micrograms of genomic DNA were fragmented, and labeled with cyanine-3 for reference DNA samples and cyanine-5 for subjects, and then hybridized. We performed scanning of the array with a G2600D SureScan Microarray Scanner (Agilent Technologies) according to the manufacturer’s recommended protocols, and analyzed scanned aCGH data using CytoGenomics software version 3.0.6.6 (Agilent Technologies).
4
Genomic DNA Profiling and CNV Analysis
5
aCGH Profiling for Chromosomal Anomalies
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aCGH profiling was performed with the Human Agilent Sureprint G3 8 × 60 K Microarray Kit (Agilent Technologies, Santa Clara, CA, USA). Tumor DNA was labeled with cyanine 5 (Cy5), while reference DNA from an individual of the same sex as the patient was labeled with Cy3. Sample and reference DNAs were pooled and hybridized for 24 h at 67°C on the arrays. The fluorescence was read by an Agilent SureScan Microarray scanner, and the Cy5/Cy3 fluorescence ratios were converted into log2-transformed values with Cytogenomics software (Agilent).
The threshold of the absolute value of the log2 fluorescence ratio retained to define a chromosomal anomaly was 0.25. A mean log2 ratio was calculated when, for at least three probes located on contiguous positions on the chromosome, a log2 ratio absolute value greater than 0.25 and of the same sign was measured. The minimum size of the anomalies considered in the interpretation of the results was set at 1 Mb.
The different chromosomal anomalies were defined by the Cytogenomics software according to the mean log2 ratio values, as follows: homozygous deletion for a value <−1, loss of one gene copy for a value between −0.25 and −1, gain of one gene copy for a value between 0.25 and 1, and amplification (gain of at least five copies) for a value >2.
The threshold of the absolute value of the log2 fluorescence ratio retained to define a chromosomal anomaly was 0.25. A mean log2 ratio was calculated when, for at least three probes located on contiguous positions on the chromosome, a log2 ratio absolute value greater than 0.25 and of the same sign was measured. The minimum size of the anomalies considered in the interpretation of the results was set at 1 Mb.
The different chromosomal anomalies were defined by the Cytogenomics software according to the mean log2 ratio values, as follows: homozygous deletion for a value <−1, loss of one gene copy for a value between −0.25 and −1, gain of one gene copy for a value between 0.25 and 1, and amplification (gain of at least five copies) for a value >2.
6
Cytogenetic and Molecular Karyotyping of BMMSCs
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Cytogenetic stability of cultured BMMSCs was confirmed via GTG banding. Metaphases were analyzed under an Olympus BX51 microscope with a camera (Olympus Corporation, Tokyo, Japan, https://www.olympus-global.com ) to document photomicrographs. A Cytovision program (Leica Microsystem, Inc., Buffalo Grove, IL, https://www.leica-microsystems.com ) was used to arrange chromosomes into a karyogram.
Molecular karyotyping was performed through aCGH with the SurePrint G3 CGH ISCA v2 Microarray Kit 8 × 60K (Agilent Technologies, Santa Clara, CA,https://www.agilent.com ) according to the manufacturer's protocol. DNA for aCGH analysis was isolated from samples of cells prepared for autologous transplantations. DNA was extracted using the machine and isolation QuickGene DNA tissue S kit (Kurabo Industries, Osaka, Japan, http://www.kurabo.co.jp /). Arrays were analyzed through the Agilent scanner and the Feature Extraction software (v8.1). Graphical overview was obtained using the Agilent CytoGenomics software (v2.9.2.4).
Molecular karyotyping was performed through aCGH with the SurePrint G3 CGH ISCA v2 Microarray Kit 8 × 60K (Agilent Technologies, Santa Clara, CA,
7
High-resolution aSNP/aCGH Analysis of Wilms Tumor
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The SNP/CGH array (aSNP/aCGH) analysis of the primary tumor and the tumor cell culture from patient Wilms10 was performed using a 2x400K oligonucleotide microarray (Sure Print G3 Human Genome CGH + SNP Microarray; Agilent Technologies Alto, CA, USA). With this array format, copy number changes, as well as copy neutral aberrations, such as uniparental disomy (UPD) can be detected on the same array. The samples were prepared and labeled as described by the manufacturer (Protocol Version 7.3 March 2014). To detect uniparental disomy (UPD), a female gender reference DNA (NA12878, Coriell Institute, USA) has to be used as control. To quantify the array data the feature extraction module of Agilent's CytoGenomics software (Version 1.5.1.0) was used and, for visualization, CytoGenomics Version 2.0.6.0.
To determine the breakpoints of the homozygous WT1 and heterozygous 11p13 deletion, a high-resolution array was designed (http://earray.chem.agilent.com/earray/ ). The region of the homozygous WT1 alteration was covered with oligonucleotides at a distance of 100 bp, whereas the probes for the heterozygous deletion had a spacing of 300 bp.
To determine the breakpoints of the homozygous WT1 and heterozygous 11p13 deletion, a high-resolution array was designed (
8
Comparative Genomic Hybridization Analysis of Tumor DNA
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The comparative genomic hybridization (CGH)-array technique was performed using tumor DNA from frozen tumor samples to assess differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. All samples were histologically checked for the presence of more than 50% tumor cells. Commercial DNA was chosen as the control DNA. After a DNA extraction (DNeasy® Blood & Tissue Qiagen® kit), the DNA was treated with RNase, then purified and quantified (spectrophotometer, Nanodrop®). After a digestion step by two restriction enzymes to obtain DNA fragments from 100 to 500 bp (SureTag DNA Labeling Kit, Agilent®), the tumor DNA and control DNA were labeled with two distinct fluorochromes (Cy5 and Cy3). They were co-hybridized on oligonucleotide sequences fixed on a solid support (4 × 180k chip, Agilent® with a resolution of 13 kb). The chips were read on a scanner (G2565CA, Agilent Technologies) that measures the fluorescence ratio for each locus. Data interpretation was performed using Cytogenomics software (version 2.0.6.0, Agilent Technologies), Hg19 database.
9
Comparative Genomic Analysis Protocol
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Resource limitations at times dictated the first intention genetic test, despite recommendations. For 169 patients, karyotyping was used as the first choice. All 371 cases, irrespective of karyotyping results, were run though CMA as soon as it became available.
High-resolution aCGH was performed using testing options from Agilent Technologies, Santa Clara, CA, USA: Agilent SurePrint G3 CGH ISCA v2 8 × 60K (141 patients), 4 × 180K (37 patients), and Oxford Gene Technology Operations Ltd.: CytoSure ISCA V2 CGH 8 × 60K microarrays (193 patients), following the protocols provided by the manufacturer [28 (link)]. A feature extraction program was used to obtain post-hybridization data. Subsequent analysis was performed with the recommended software: CytoGenomics software from Agilent and Cytosure Interpret Software from OGT, respectively.
CMA, standard karyotyping and/or multiplex ligation-dependent probe amplification (MLPA) were used to confirm the CMA findings and perform segregation analysis where commercial kits to cover the region of interest were available, and/or parents consented.
High-resolution aCGH was performed using testing options from Agilent Technologies, Santa Clara, CA, USA: Agilent SurePrint G3 CGH ISCA v2 8 × 60K (141 patients), 4 × 180K (37 patients), and Oxford Gene Technology Operations Ltd.: CytoSure ISCA V2 CGH 8 × 60K microarrays (193 patients), following the protocols provided by the manufacturer [28 (link)]. A feature extraction program was used to obtain post-hybridization data. Subsequent analysis was performed with the recommended software: CytoGenomics software from Agilent and Cytosure Interpret Software from OGT, respectively.
CMA, standard karyotyping and/or multiplex ligation-dependent probe amplification (MLPA) were used to confirm the CMA findings and perform segregation analysis where commercial kits to cover the region of interest were available, and/or parents consented.
10
Array CGH for Comprehensive Copy Number Variation Detection
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We performed aCGH experiments on SurePrint G3 Human CGH Microarray, 2x400K KIT (Agilent) to detect CNVs. The experimental and analysis protocol were as advised by the manufacturer. The purified, labelled DNA was checked on a Nanodrop with specific Activity of Cyanine 3 and Cyanine 5 Labeled Sample (pmol/ μg) from 20 to 60. Each specimen sample was mixed together with the corresponding commercial reference sample provided in the Kit. Then, samples were hybridized with Agilent slides at 67°C for 40 hours. After washing, the slide was scanned by Agilent Microarray Scanner. Agilent Feature Extraction Software 11.5.1.1 was used to assess the quality of image data file. The first-pass quality control (QC) filter of <0.23 derivative log-ratio threshold was applied.
The data-quality was checked in 4 steps, (i) input DNA quality in agarose gel (ii) digestion of DNA after restriction digestion by agarose gel, (iii) labeling efficiency by Nanodrop, and (iv) the quality of aCGH data after analysis by the Cytogenomics software provided by Agilent.
The data-quality was checked in 4 steps, (i) input DNA quality in agarose gel (ii) digestion of DNA after restriction digestion by agarose gel, (iii) labeling efficiency by Nanodrop, and (iv) the quality of aCGH data after analysis by the Cytogenomics software provided by Agilent.
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