The widespread use of high-throughput sequencing technologies provides unprecedented insights into the human genome and cancer biology. Next-generation sequencing (NGS) technologies have become the standard approaches to explore gene expression profiles and detect oncogenic mutations. However, NGS methods are limited by short reads, which hamper the detection of complex mutations like high repetitive genomic regions and larger structure variations (SVs). Nanopore sequencing generates ultra-long reads of target sequences, which could help capture the entire structure and modification of DNA and transcript molecules. Nanopore technology enables real-time, ultra-long read sequencing for direct RNA sequencing and detection of methylation information. In addition, nanopore sequencing can be combined with emerging technologies like CRISPR and Pore-C to overcome some of the shortcomings of NGS technologies, especially in the detection of tumor mutations.