We evaluated the abilities of three second-generation sequencers, Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq), as well as a third-generation sequencer, Pacific Biosciences RS sequencer (PacBio), to determine the nucleotide sequence of the genome from Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes.