PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

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Deep‐sea hydrothermal vents are unique light‐independent ecosystems that are sustained by chemosynthetic bacteria. For many of the invertebrates inhabiting in such environments, bacteria play essential roles in both energy acquisition and detoxification of potentially toxic gases such as H2S. In this study, the bacterial flora present on the gills of Alvinocaris longirostris (Bresiliidae: Caridea), a shrimp inhabiting hydrothermal vents (1532 m depth) at the Hatoma Knoll of the Okinawa Trough, was investigated. Bacterial 16S rDNA fragments were successfully amplified from the gills and 70% of these fragments showed an identical pattern in the restriction fragment length polymorphism analysis. These fragments were assigned to the ribotype AL‐1. Phylogenetic analyses suggest that AL‐1 forms a monophyletic clade with Sulfurovum spp. (ε‐Proteobacteria). Fluorescence in situ hybridization for AL‐1 and electron microscopy showed the presence of short‐rod bacteria lining up on the cuticular layer of the surface of the gill filaments. These results suggest that bacterial association with gills also occurs in bresiliid shrimps.

Here, we report the complete genome sequences of low-passage virulent and high-passage avirulent variants of pathogenic Leptospira interrogans serovar Manilae strain UP-MMC-NIID, a major causative agent of leptospirosis. While there were no major differences between the genome sequences, the levels of base modifications were higher in the avirulent variant.

Deep-sea hydrothermal vents and methane seeps are extreme environments that have a high concentration of hydrogen sulphide. However, abundant unique invertebrates including shrimps of the family Bresiliidae have been found in such environments. The bresiliid shrimps are believed to have radiated in the Miocene (less than 20 Myr); however, the period when and the mechanisms by which they dispersed across the hydrothermal vents and cold seeps in oceans worldwide have not been clarified. In the present study, we collected the deep-sea blind shrimp Alvinocaris longirostris from the hydrothermal vent site in the Okinawa Trough and carried out the first investigation of the 18S rRNA gene of a bresiliid shrimp. The phylogenetic analysis revealed that the bresiliid shrimp is situated at an intermediate lineage within the infraorder Caridea and shows monophyly with palaemonid shrimps, which live in shallow sea and freshwater. Furthermore, the mitochondrial cytochrome oxidase I (COI ) gene sequences were analysed to determine the phylogenetic relationship with known bresiliid shrimps. A. longirostris of the Okinawa Trough had two haplotypes of the COI gene, one of which was identical to the Alvinocaris sp. of the cold seeps in Sagami Bay. These results indicate that a longdistance dispersal of A. longirostris occurred possibly within the last 100 000 years.

We report the complete genome sequences of eight Helicobacter pylori strains isolated from patients with gastrointestinal diseases in Okinawa, Japan. Whole-genome sequencing and DNA methylation detection were performed using the PacBio platform. De novo assembly determined a single, complete contig for each strain. Furthermore, methylation analysis identified virulence factor genotype-dependent motifs.

The first complete genome sequence of Lactobacillus curvatus was determined by PacBio RS II. The single circular chromosome (1,848,756 bp, G+C content of 42.1%) of L. curvatus FBA2, isolated from fermented vegetables, contained low G+C regions (26.9% minimum) and 43 sets of >1,000-bp identical sequence pairs. No plasmids were detected.

The first complete genome sequences of Staphylococcus aureus subsp. aureus Rosenbach 1884 strain DSM 20231T, the type strain of the bacterium causing staphylococcal disease, were determined using PacBio RS II. The sequences represent the chromosome (2,755,072 bp long; G+C content, 32.86%) and a plasmid (27,490 bp long; G+C content, 30.69%).