Genome-wide sequencing has enabled modern biomedical research to relate more and more events in healthy as well as disease-affected cells and tissues to the genomic sequence. Now next generation sequencing (NGS) extends that reach into multiple almost complete genomes of the same species, revealing more and more details about how individual genomes as well as individual aspects of their regulation differ from each other. The inclusion of NGS-based transcriptome sequencing, chromatin-immunoprecipitation (ChIP) of transcription factor binding and epigenetic analyses (usually based on DNA methylation or histone modification ChIP) completes the picture with unprecedented resolution enabling the detection of even subtle differences such as alternative splicing of individual exons. Functional genomics aims at the elucidation of the molecular basis of biological functions and requires analyses that go far beyond the primary analysis of the reads such as mapping to a genome template sequence. The various and complex interactions between the genome, gene products and metabolites define biological function, which necessitates inclusion of results other than sequence tags in quite elaborative approaches. However, the extra efforts pay off in revealing mechanisms as well as providing the foundation for new strategies in systems biology and personalized medicine. This review emphasizes the particular contribution NGS-based technologies make to functional genomics research with a special focus on gene regulation by transcription factor binding sites.