Cytoscan hd chip

The Leiden University Medical Centre (LUMC) cohort includes clinical, histopathological, and genetic information on 64 UM cases, enucleated between 1999 and 2008. Clinical information was collected from the Integral Cancer Center West patient records and updated in 2019. For each sample, part of the tumor was snap frozen with 2-methyl butane and used for mRNA and DNA isolation, while the remainder was embedded in paraffin after 48 hours of fixation in 4% neutral-buffered formalin and sent for histological analysis. Chromosome status was determined with the Affymetrix 250K_NSP-chip and Affymetrix Cytoscan HD chip (Affymetrix, Santa Clara, California, United States of America). RNA was isolated with the RNeasy mini kit (Qiagen, Venlo, The Netherlands) and mRNA expression was determined with the HT-12 v4 chip (Illumina, San Diego, California, United States of America). Statistical analyses of the LUMC cohort were carried out in SPSS, version 25 (IBM Corp). For survival analysis, Kaplan-Meier and log-rank test were performed with death due to metastases as endpoint. Cases that died of another or unknown cause were censored. The two subpopulations that were compared in each analysis were determined by splitting the total cohort along the median value of mRNA expression for each analyzed gene.

Fetal genomic DNA was extracted from cord blood (1 mL) and amniotic fluid (10 mL) using DNA extraction kits from Qiagen (USA) and BioChain (USA), respectively. The DNA of fetuses was then subjected to CMA. The genomic DNA samples were purified, hybridized with microarrays, and analyzed in accordance with the standard procedures provided by Affymetrix. A genome-wide CytoScan™ HD chip (Affymetrix, USA) with a single-nucleotide polymorphism (SNP) probe was used. The corresponding ChAS software and related bioinformatics methods were used to analyze the CMA results. Copy number variations (CNVs) were determined using the scatter plot distribution of the DNA fragment copy number. We compared the CNVs using reference databases, including the Database of Genomic Variants (DGV) (http://projects.tcag.ca/variation), DECIPHER (http://www.sanger.ac.uk/PostGenomics/decipher), Online Mendelian Inheritance in Man (OMIM) (http://www.omim.org), and University of California Santa Cruz (UCSC) databases (http://www.genome.UCSC.edu/). CNVs were classified as pathogenic, likely pathogenic, benign, likely benign, and those with a variant of uncertain significance [18 (link)].

DNA was extracted from PDX cells at low (p3), middle (p15) and high (p30) in vitro passages using DNeasy Blood and Tissue Kit (Qiagen). For analysis, genomic DNA was hybridized to an Affymetrix CytoScan HD chip (Affymetrix) containing approximately 2.6 million markers of which almost 750,000 are SNPs. Constitutional copy number variants were removed by filtering against the Database of Genomic Variants (Oct. 2016). Copy number variation analyses were performed using ChAS software. The analyses of xenografts and the corresponding patient tumors were reported previously^{10 (link)}.

To confirm our WES results, we performed chromosomal microarray testing using the CytoScan HD chip (Affymetrix, Santa Clara, CA, USA) in accordance with the manufacturer's instructions. Moreover, we analyzed data with ChAS software (Affymetrix), which had a calling threshold of 20 consecutive probes encompassing at least 25 kb in length. Genomic DNA was extracted from the peripheral blood samples. The first strand of cDNA was synthesized with this DNA sample as a template and designed primers. Then, the cDNA fragments were amplified by PCR with the primers, labeled, and hybridized. We analyzed pathogenic CNVs using Agilent Cytogenomics software (Agilent Technologies).
All the reported CNVs were subject to the build 37 of human genome/hg19 on NCBI. The screened CNVs for comparative analysis had to meet the following conditions: (1) deletions ≥50 kb/25 markers; duplications ≥100 kb/50 markers; (2) <50% overlap with known segmental duplications (SD); and (3) not found in the control populations cataloged in the Database of Genomic Variants (DGV). We selected 178 individuals without heart disease from our local database as controls. Other controls were selected from the SNP database (https://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 Genomes Project (1000G, https://1000genomes.org), and the DGV (https://dgv.tcag.ca/dgv/app/home).

DNA was extracted from saliva samples collected using the Oragene 250 saliva collection kit (DNA Genotek; Ottawa, Ontario, Canada). DNA was extracted using an automated extraction instrument, AutoPure LS (Qiagen; Valencia, CA), and samples were genotyped using the Illumina Human OmniExpress Chip (Illumina; San Diego, CA) according to protocols provided by the manufacture's. Technical replication was performed using the Affymetrix CytoScan HD chip according to the manufacturer's protocol (Affymetrix; Santa Clara, CA).