Abstract In recent years, in depth exploration of genomes structure and function has revealed a central role for non-coding RNAs (ncRNAs) in orchestrating key biological and cellular processes through the fine tuning of gene expression regulation. Most importantly a role for ncRNAs has also started to emerge in human disease pathogenesis. This further speaks to the importance of an in-depth characterization of ncRNA involvement in human diseases, including cancer. In this work, we have built a comprehensive atlas of gene expression across the human transcriptome containing over 100,000 genes by leveraging two publicly available resources: the FANTOM CAGE Associated Transcriptome (FANTOM-CAT), and recount2. The FANTOM-CAT is a comprehensive meta-assembly of the human transcriptome encompassing coding and non-coding genes, including promoters, enhancers, and lncRNAs. recount2 is the largest, available collection of human RNA-seq data processed and quantified using a unified pipeline, containing over 4.4 trillion reads from over 70,000 human samples from the SRA, GTEx and TCGA projects. Using FANTOM-CAT/recount2 gene expression summaries across human tissue samples from the GTEx project, we validated our approach by reproducing key findings recently described by the FANTOM consortium. In particular, we could identify and confirm the reported tissue specific transcription patterns for the distinct classes of coding and non-coding genes, such as messenger RNAs, intergenic long ncRNAs, and expressed divergent promoters and enhancers. Most importantly, we have leveraged the FANTOM-CAT/recount2 atlas to perform differential gene expression analysis across 13 cancer types in TCGA, identifying a comprehensive list of mRNAs, promoters, enhancers, and other ncRNAs differentially expressed between cancer and normal samples. Overall, this analysis revealed a set of molecular signatures encompassing coding and non-coding genes that are shared across cancer types or restricted to specific tissues of origin. In summary, our findings establish the FANTOM-CAT/recount2 gene expression atlas as a reliable resource to study the role ncRNAs play in cancer and other human diseases, providing important cues to understand their biological function. Finally, we make the FANTOM-CAT/recount2 atlas available as a public tool to empower other researchers to study important biological and clinical phenotypes and identify new candidate ncRNAs for further investigation. Citation Format: Eddie Luidy-Imada, Tejasvi Matam, Leonardo Collado-Torres, Wikum Dinalankara, Aleksei Stupnikov, Christopher Wilks, Andrew E. Jaffe, Ben Langmead, Jeffrey T. Leek, Alexander Favorov, Luigi Marchionni. Differential analysis of gene expression across the human genome using recount2 and FANTOM-CAT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2297.