Next-generation sequencing has produced a large quantity of DNA or RNA sequences related to the processes occurring within tumors and their microenvironment in a reasonable time and cost. These data have been used to guide the identification of neoantigens and to determine their specific T-cell receptors. Furthermore, adoptive T-cell therapy targeting neoantigens is under development for cancer treatment. In this review, we first provide an overview of sequencing technologies and the updated findings concerning neoantigens related to adoptive T-cell therapy and then summarize the methods and principles underlying the development of next-generation sequencing-based neoantigen-reactive T-cell therapy for cancer.