Background Incorporation of next-generation sequencing (NGS) technology into clinical utility in targeted and immunotherapies requires stringent validation, including the assessment of tumor mutational burden (TMB) and microsatellite instability (MSI) status by NGS as important biomarkers for response to immune checkpoint inhibitors. Materials and Methods We designed an NGS assay, Cancer Sequencing YS panel (CSYS), and applied algorithms to detect five classes of genomic alterations and two genomic features of TMB and MSI. Results By stringent validation, CSYS exhibited high sensitivity and predictive positive value of 99.7% and 99.9%, respectively, for single nucleotide variation; 100% and 99.9%, respectively, for short insertion and deletion (indel); and 95.5% and 100%, respectively, for copy number alteration (CNA). Moreover, CSYS achieved 100% specificity for both long indel (50-3,000 bp insertion and deletion) and gene rearrangement. Overall, we used 33 cell lines and 208 clinical samples to validate CSYS's NGS performance, and genomic alterations in clinical samples were also confirmed by fluorescence in situ hybridization, immunohistochemistry, and polymerase chain reaction (PCR). Importantly, the landscape of TMB across different cancers of Chinese patients (n = 3,309) was studied. TMB by CSYS exhibited a high correlation (Pearson correlation coefficient r = 0.98) with TMB by whole exome sequencing (WES). MSI measurement showed 98% accuracy and was confirmed by PCR. Application of CSYS in a clinical setting showed an unexpectedly high occurrence of long indel (6.3%) in a cohort of tumors from Chinese patients with cancer (n = 3,309), including TP53, RB1, FLT3, BRCA2, and other cancer driver genes with clinical impact. Conclusion CSYS proves to be clinically applicable and useful in disclosing genomic alterations relevant to cancer target therapies and revealing biomarkers for immune checkpoint inhibitors. Implications for Practice The study describes a specially designed sequencing panel assay to detect genomic alterations and features of 450 cancer genes, including its overall workflow and rigorous clinical and analytical validations. The distribution of pan-cancer tumor mutational burden, microsatellite instability, gene rearrangement, and long insertion and deletion mutations was assessed for the first time by this assay in a broad array of Chinese patients with cancer. The Cancer Sequencing YS panel and its validation study could serve as a blueprint for developing next-generation sequencing-based assays, particularly for the purpose of clinical application.