Cancer is caused by a vast number of recurrent genetic alterations important for disease initiation, progression and relapse (Hanahan and Weinberg, 2011). The presence of specific genetic alterations in many cancer types today provide a critical basis for the establishment of a correct diagnosis, prognostication, treatment selection, and as markers to monitor treatment response (Edsjö et al., 2023a). A number of technologies are available to detect genetic alterations in cancer diagnostics. Originally, Sanger sequencing was used to identify individual variants (point mutations) at the DNA-level, whereas chromosome and fluorescence in situ hybridization (FISH) analyses were applied to detect chromosomal alterations, and later on array-based methods to assess copy-number variants (CNVs) at high resolution (Rosenquist et al., 2023). However, despite the broad initial use of Sanger sequencing in routine diagnostics, the restricted efficiency of this method to assess alterations in multiple genes became a limiting factor. This drawback became even more obvious as a growing number of genetic alterations in cancer were described and the list of targeted cancer treatments, coupled to treatment predictive genetic aberrations, started to increase (Stenzinger et al., 2022). During the last decade, next-generation sequencing (NGS) (also called massive parallel sequencing) (Rodriguez and Krishnan, 2023) has instead rapidly replaced Sanger sequencing as well as other methods for precision cancer diagnostics. The shift has been driven not only by the need to assess a growing number of genomic biomarkers from each patient but also by the rapid reduction in sequencing costs (Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) Available at: www.genome.gov/sequencingcostsdata. Accessed [2023-11-13]). Today, NGS can be employed for a range of clinical purposes and sample types, calling for different types of NGS-based analyses, all with their pros and cons. The currently used clinical analyses span from single genes to global analyses and from low to extreme sequencing depth to achieve the needed sensitivity (Fig. 1).

In this review, we first discuss the use of targeted gene panel sequencing in precision cancer diagnostics, ranging from small panels focusing on treatment prediction to larger panels for more comprehensive genomic profiling (Donoghue et al., 2020). The second part describes the application of global whole-genome and whole-transcriptome sequencing analyses (WGS and WTS) (Jobanputra et al., 2022), as well as whole-exome sequencing (WES); the latter sharing features with both global and gene panel analyses (Menzel et al., 2023). We will also exemplify the implementation of WGS/WTS by current nationwide efforts in hematological and pediatric malignancies (Rosenquist et al., 2022). The third part features current and potential future clinical utility of mutation signatures, a group of biomarkers that has fundamentally changed the amount of information needed to gather from each tumor sample to allow for treatment decisions (Koh et al., 2021). The fourth part will shift from global analyses and highlight the need of longitudinal, sensitive disease monitoring in malignant disease. Finally, the method-oriented sections will be complemented by a closer look at what is needed to implement and upscale NGS-based analyses in a routine clinical setting, and we end by discussing future directions in developing sequencing-based methods and our overarching conclusions.