Computational protocol: Genome-Wide and Locus Specific Alterations in CDC73/HRPT2-Mutated Parathyroid Tumors

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Protocol publication

[…] After scanning, features intensities were extracted using GenePix Pro 6.0 software (Axon Instrument, CA, USA). The generated gene pix result (GPR) files were uploaded to the web-based a-CGH analytic tool Bio Array Software Environment version 2 (BASE2) (http://base.thep.lu.se/). After importing the data, background correction was performed by subtracting median foreground from median background of Cy3 and Cy5 intensities respectively. Data was filtered for flagged bad or null spots. The samples were then normalized using BASE 1 LOWES plugin and transformed using smooth default parameters with 3 clones sliding window size.CNAs were visualized using CGH plotter applying cut-offs values for log2 ratio of 0.25 for gains, −0.25 for losses, +1 for amplifications and −0.8 for homozygous losses. Whole genome frequency plots and individual chromosome profiles were generated using BEFC viewer plugin in BASE2. Sex chromosomes were excluded from the final analysis. [...] Genotype files (CHP) were generated by the Genotype console (Affymetrix Inc. Santa Clara, CA, USA) using the Bayesian Robust Linear Model with Mahalanobis (BRLMM) algorithm. The CHP files were then imported into Partek Genomic Suite V6.5 software (Partek Incorporated. St. Louis, MO) using the Hidden Markov Model (HMM). The default Partek parameters were used for further LOH analysis including a heterozygosity call rate of 0.3 and the minimum number of consecutive probes required to a make call set as 10 probes. All samples were checked for quality and as both T5a and T1 did not pass the QC they were excluded from further analysis. Similar to a-CGH, sex chromosomes were excluded from the final analysis. Based on all the genomic alterations detected, unsupervised hierarchical clustering of all the CDC73-mutated tumors was performed using Euclidean algorithm with average linkage and zero distance using Partek Genome Suite 6.5 (Partek inc. MI, U.S.A). [...] Fisher exact test was used to estimate any significant differences in the distribution of the CNAs between the adenoma and the carcinoma groups. Considering the sample size and at power of 80%, a p value of <0.05 was regarded as statistically significant. Statistical analyses were performed using SigmaPlot (SigmaPlot for windows v11.0, SYSTAT software inc, Hamburg, Germany). […]

Pipeline specifications

Software tools GenePix Pro, BRLMM, SigmaPlot
Applications Miscellaneous, aCGH data analysis
Diseases Adenoma, Carcinoma, Neoplasms, Parathyroid Neoplasms