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We have never halted the exploration of pathogenic microorganisms. With the improvement of genome-sequencing technology and the continuous reduction of sequencing costs, an increasing number of complete genome sequences of pathogenic microorganisms have become available. Genome annotation of this massive sequence information has become a daunting task in biological research. This paper summarizes the approaches to the genome annotation of pathogenic microorganisms and the available popular genome annotation tools for prokaryotes, eukaryotes and viruses. Furthermore, real-world comparisons of different annotation tools using 12 genomes from prokaryotes, eukaryotes and viruses were conducted. 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