Simona Serratì, Simona De Summa, Brunella Pilato, Daniela Petriella, Rosanna Lacalamita, Stefania Tommasi, Rosamaria Pinto Molecular Genetics Laboratory, IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy Abstract: Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment. Keywords: hereditary breast cancer, melanoma, prostate cancer, thyroid cancer, lung cancer, colorectal cancer, hematologic cancer