Next-generation sequencing (NGS) has transformed genomics research by providing cost-effective and unprecedented capacities for analyzing large volumes of DNA and RNA molecules. This breakthrough technique has rapidly advanced genomics studies across various research fields. With the advent of NGS techniques, scientists can now examine and comprehend bacterial genomes at a finer level and get a deeper understanding of their genetic variants, regulation of genes, and epigenetic alterations. To speed up biological research, these high-throughput sequencing technologies have been widely applied in various ways, including small RNA sequencing, target sequencing, whole-genome sequencing, and gene expression profiling. Moreover, the functional characterization of the intricate relationships among various microbial communities within a single microbiome has been aided by metatranscriptomic techniques. Despite the significant technological advances in bioinformatics and NGS, advanced bioinformatic algorithms are still necessary for understanding complex samples and environments. This chapter highlights the advancement in NGS analysis, its application, and the workflows and tools employed for NGS analysis.