Genechip system 3000

Total RNA was extracted from formalin-fixed, paraffin embedded samples using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Invitrogen, Carlsbad, CA, USA), quantified on a Qubit fluorometer (Thermo Fisher, Waltham, MA, USA) and assessed for integrity on a 4200 TapeStation, (Agilent Technologies, Santa Clara, CA, USA). Gene expression profiles were generated using GeneChip Human Transcriptome Array 2.0 microarrays (Thermo Fisher). RNA labeling, processing, and hybridization were performed according to manufacturer’s instructions, and microarrays were scanned with the GeneChip System 3000 (Thermo Fisher) scanner. Quality controls were performed on raw and preprocessed data to control for batch effects and outliers.

CMA was carried out using CytoScan™ Optima array, GeneChip™ System 3000 and Affymetrix platform (Thermo Fisher Scientific, USA) as per the manufacturer’s instructions. Chromosome Analysis Suite Software (ChAS) (Thermo Fisher Scientific, USA) was used to carry out the analysis of the data as per the manufacturer’s recommendations which suggested a minimum resolution of 1 Mb for losses, 2 Mb for gains and 5 Mb for copy neutral loss of heterozygosity. For all candidate CNVs, variants were primarily screened for population frequency and known disease associations using publicly available databases like gnomAD database [20 (link)], DGV [21 (link)] and DECIPHER [22 (link)] and OMIM [23 (link)]. Pathogenicity of CNVs were classified in accordance with ACMG and ClinGen classification system [24 (link)]. All candidate CNVs were validated in proband and parents using SYBR Green based quantitative PCR (Q-PCR) using ABI’s StepOne Real Time PCR system (Thermo Fisher Scientific, USA) (Supplementary Table 1).

Cytogenetic analysis was performed with DNA from tumor xenograft (passage 1) using a CytoScan 750K Array (Affymetrix, Thermo Fisher Scientific) with genome-wide coverage (750,000). The sample was processed in the GeneChip System 3000 platform from Affymetrix (Thermo Fisher Scientific). The Chromosome Analysis Suite program was used for the analysis (v.3.2, Thermo Fisher Scientific) with version 33.1 that uses the UCSC hg19 version of the human genome for the annotations of genes. The alterations were analyzed with a minimum of 25 affected markers and an average resolution of 110 kb.