Ggplot2

We downloaded the level 3 HTseq-FPKM RNA sequencing (RNA-seq) data of PRAD from TCGA. The RNA-seq data in FPKM format was converted into TPM format, and log2 transformation was performed to compare the expression among samples. The R package “ggplot2” in R version 3.6.3 (The R Foundation for Statistical Computing, Vienna, Austria) was also employed to draw the boxplots and line plots. The statistical analysis was conducted using the Wilcoxon rank -um test and Wilcoxon signed rank test for unpaired and paired samples, respectively. A P value <0.05 was considered to be statistically significant.

Only non derived strains were included in the phylogeographic analysis. Metadata corresponding to the country of origin and the year of sampling were considered in the analysis, using Beast 2.5 software (Bouckaert et al., 2019 ). Substitution model averaging was estimated using boMdelTest package on Beast 2.5 (Bouckaert and Drummond, 2017 (link)). Phylogeographic analysis was carried out considering country of origin as a discrete trait and year of sampling as sampling date. GTR was used as substitution model. Uncorrelated relaxed lognormal clock was used as a molecular clock model (Drummond et al., 2006 (link)). Bayesian skyline was used as population model for this analysis (Drummond et al., 2005 (link)). The analysis was set to run for 30 million iterations. Tracer software was used to assess for Effective Sampling Size (ESS). Maximum credibility tree was generated and annotated using Tree Annotator. The resulting tree was edited and visualized using ggmap (Kahle and Wickham, 2013 ) and ggplot2 (Wickham, 2016 ) packages on R 3.5 Software (R Foundation for Statistical Computing, Vienna, Austria);
All results were rendered to KML format using SpreadD3 software and visualized on Google Earth (Bielejec et al., 2016 (link)).