Genomic annotation and pathway analysis

Genomic annotation of smoking-DMPs was carried out initially using the EPIC Illumina manifest, for the purpose of identifying novel CpG-sites that are specific to the EPIC-array, for allocating CpG-sites to genes, and relative to CpG-density, including CpG island (CGI), CGI-shore, CGI-shelf, and open sea. Further genomic annotations took into account data from the ENCODE project [20]. We explored if smoking-DMPs mapped within ChromHMM [11] categories to assess enrichment or depletion in smoking-DMPs relative to different functional genomic domains, including insulators, enhancers and specific transcription factors binding sites. In the enrichment analysis, we considered all smoking-DMP probes mapping to a specific annotation category compared to the total number of probes tested that mapped to that category. For each genome annotation category, the results show the log fold change for smoking-DMP probes compared to total probes tested, and the significance of the difference is based on a Fisher’s exact test. Pathway analysis was carried out for genes annotated to smoking-DMPs using Ingenuity Pathway Analysis (IPA; QIAGEN Inc. https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis).