Recent years have seen the introduction of next-generation sequencing (NGS) technologies and their use in the fields of bioscience and biotechnology. Not only has the availability of NGS technologies revolutionized the way genome research is carried out, the massive sequence data produced by NGS technologies have also been driving the advancement in bioinformatics required for downstream analysis, storage, and accessibility. However, the processing of NGS data is still challenging owing to such properties as the shortness of reads, the sheer amount of data, and the low base qualities. For high-quality de novo genome sequencing, we used a hybrid approach that utilizes Sanger end sequencing of fosmid libraries for the scaffolding of NGS-derived contigs. This strategy was successfully applied to the genome sequencing of microbial cell factories requiring a complete gene list for the metabolic pathway, and a regulatory network for the design and development of industrial strains. Recently, software developments have facilitated de novo genome assembly which can produce genome scaffolds from short reads only. Automatic gap closing, which incorporates paired-end short reads into preexisting scaffolds, is also feasible. High-throughput multiplexed genome sequencing based on the Illumina platform has become a routine task for genome prospecting and the comparative genomic analysis of useful microbial strains. We also carried out the resequencing of E. coli strains generated from a combined approach of a long-term experimental evolution and proton beam-induced mutagenesis. NGS-based resequencing allows the identification of genetic variations from multiple samples at a lower cost, and the tracing of evolutionary pathways that are engraved in the genomic sequences. NGS-based transcriptome sequencing (RNA-seq), which is becoming popular as a substitute for traditional microarray experiments, also provides evidence for the identification of genes from novel genomes. Recent achievements in genome sequencing and analysis of eukaryotic microbes will also be introduced.