Genotyping console

Raw image files were converted into CEL-files by Affymetrix® genotyping console, which were then processed with the Affymetrix Power Tools (APT) apt-copynumber-workflow v 1.67. The values for contrastQC (based on Affymetrix® GTC 3.0.1 User Manual) and MAPD were extracted and samples that failed default QC values were discarded (MAPD > 0.4 and/or contrastQC < 0.4). For the remaining samples an identity by state (IBS) and principal component analysis (PCA) was performed as described previously [17 (link)]. The output of apt-copynumber-workflow was used as the input file for our in-house developed CNV data mining tool “CNVineta” [27 (link)]. A preliminary batch-wise filtering was performed based on the number of called CNVs per samples. Outliers were defined as samples which had more CNVs than the 75 % quantile plus 1.5 fold of the interquantile range. A rigorous manual raw data inspection for identifying false-negative and -positive CNVs was done subsequently. For the whole data mining process, the predicted CNVs with less than five supporting probes per CNV and mean probe set distance less than one kilobase, were ignored.

DNA from tumor and peripheral blood was processed using Affymetrix Genome-Wide Human SNP 6.0 (Affymetrix, CA, USA) according to manufacturer’s protocols. Briefly, DNA was digested with NspI and StyI enzymes (New England Biolabs, USA), ligated to the respective Affymetrix adapters using T4 DNA ligase (New England Biolabs, USA) amplified (Clontech-Takara Bio, USA), purified using magnetic beads (Agencourt, Beckman, USA), labeled, fragmented, and hybridized to the arrays. Following hybridization, the arrays were washed and stained with streptavidin-phycoerythrin (Invitrogen, USA). Arrays preparation and scanning was performed at the genotyping core laboratory of INMEGEN. Background correction and extraction of raw fluorescence intensity were performed with the Affymetrix Genotyping Console. Raw microarray data was deposited in GEO under the number GSE87048.

The intensity (*.CEL) files of 222 individuals from the above QC protocol were extracted for copy number analysis using Affymetrix® Genotyping Console. Copy number segments were filtered to regions (minimum genomic size of 2 kbps) with 10 marker per segment [26 ]. The copy number data was exported as tab-delimited file for copy number association in CNVRuler [27 (link)]. CNV regions were significantly associated at FDR p-value < 0.05. The significant CNV regions were visualized using Phenogram [28 (link)] then mapped to genes using UCSC browser for GRCh37/hg19 assembly (https://genome.ucsc.edu/). The genes within CNV regions were analyzed for functional and diseases processes and visualized using Ingenuity Pathway Analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

SNP microarray analysis was used to determine chromosomal aberrations. Two types of SNP microarray chips were used, the Affymetrix 250K_NSP-chip, with ~250,000 probes across the genome and the Affymetrix Cytoscan HD chip, with ~750,000. The first 28 samples were analyzed with the Affymetrix 250K_NSP chip, and the remaining 36 samples with the Affymetrix Cytoscan HD chip. Analysis of the Affymetrix 250K_NSP chips was performed with the ‘Genotyping Console’ to determine the copy number values and the ‘GCT Browser’ to visualize the data (both from Affymetrix, Santa Clara, USA). Affymetrix Cytoscan HD chips were analyzed with ‘ChAS’. Different loci per chromosome were evaluated to adjust for partial gains or deletions. ~200 probes per gene locus were averaged to determine eventual copy number.

Affymetrix GeneChip Gene 2.0 expression arrays (Affymetrix) were used to profile alterations in global mRNA expression after silencing of S6K1, S6K2 or S6K1+S6K2 in triplicates. The assay was performed according to manufacturer’s protocols with 250 ng total RNA as starting material. Briefly, RNA was reverse transcribed into first and second strand cDNA, thereafter converted to cRNA and finally sscDNA, using Ambion, Life Technologies, WT Expression Kit (Ambion, Life Technologies). The sscDNA was fragmented and labelled with GeneChip WT Terminal labelling and hybridisation kit (Affymetrix), and hybridised to the arrays. Affymetrix Scanner 3000 7G was used to scan the arrays and generated data were converted to cell intensity files in Affymetrix Genotyping Console. Cell intensity data were processed by the software Genespring (Agilent Technologies). Raw data was normalised to the median of the control samples using the Robust Multi-array Average (RMA) summarisation algorithm [28 (link)]. Paired two-sided t-tests were used to estimate significant differences in gene expression after downregulation of S6K1 and/or S6K2 in comparison to scrambled siRNA. Only results from annotated genes were reported. As a significance limit p<0.05 was used.