Nexus copy number software

Raw CMA data were processed using Nexus Copy Number Software (Biodiscovery Inc., El Segundo, CA, USA) to identify any deletion that was covered by at least three consecutive probes. The sequences were aligned to the GRCh37/hg19 reference genome. To determine the minimum number of probes necessary to detect a microdeletion reliably, we compared the CMAs from two pairs of monozygotic twins and two singletons whose tests were inadvertently performed twice. There were, in essence, four replicate pairs of CMAs. Each twin pair was tested concurrently using either the SNP6.0 or Cytoscan HD platform. Each singleton had two blood samples obtained 3–4 weeks apart, and the replicate test was performed using the same SNP6.0 or Cytoscan HD platform. The fraction of reproducibly detected microdeletions was calculated as a function of probe number. A microdeletion was included for subsequent analysis based on a 20-probe size threshold, as explained in the results.

Locus-specific loss of heterozygosity (LOH) analyses were performed using DNA extracted from tumour material and adjacent normal tissue. The respective DNA fractions were used to perform PCRs generating small amplicons covering the patient-specific mutations, which were sequenced by Sanger sequencing (primers and conditions available upon request). Genome-wide SNP array analysis of tumour DNA was performed using the OncoScan FFPE Express service (Affymetrix, Santa Clara, CA, USA). The SNP array data were analysed using the Nexus Copy Number software package (Biodiscovery, Hawthorne, CA, USA).

In order to evaluate whether different expression levels of Gal-4 were determined by copy number variation of this gene, genomic DNAs were extracted from PDAC-1 and PDAC-2 cells using Ambion®-RecoverAll kit (Life Technologies). The quantity and purity of extracted DNAs were determined as described above. DNAs were subjected to aCGH, using Agilent 4×180K platform (Agilent, Santa Clara, CA), as described previously [21 (link)]. The slides were scanned on Agilent Microarray Scanner, followed by data extraction and normalization by Feature Extraction v10.5 software (Agilent). Nexus Copy Number™ software was used to analyze the DNA copy number variations (BioDiscovery, Hawthorne, CA).

The Infinium Global Screening Array-24 v1.0 BeadChip from Illumina containing ~ 700.000 SNPs to determine copy number variation was used for genome-wide SNP genotyping of the different generations PDX tumours of TC1, TC4 and TC5. DNA processing, tagging and hybridization to the CHIP were performed according to the manufacturer's protocol (Illumina). Primary assessment and SNP call rate quality control of SNP intensity output files were performed using GenomeStudio software (Illumina). Samples passed the inclusion quality control criteria, including call rates > 95%. Further analysis was performed with Nexus Copy number software (BioDiscovery, El Segundo, CA, USA) to generate copy number alteration (CNA) profiles. Quantitative CNA correlative analysis of the different PDX passages were performed as described previously^{12 (link)}.

Microarray-based genomic profiling was carried out in a blinded fashion using two different platforms; the CytoSan HD array platform (Affymetrix, Inc., Santa Clara, CA, USA) and the HumanOmniExpress12v1.0 array platform (Illumina Inc., San Diego, CA, USA). Hybridizations were performed according to the manufacturer’s protocols. The data obtained by the CytoScan HD array platform were analyzed using the Chromosome Analysis Suite software package (Affymetrix), and for the HumanOmniExpress12v1.0 platform data were analyzed using Nexus copy number software (Biodiscovery Inc., Hawthorne, CA, USA) using annotations of genome version GRCh37 (hg19).