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.

Ocean acidification may negatively impact the early life stages of some marine invertebrates including corals. Although reduced growth of juvenile corals in acidified seawater has been reported, coral larvae have been reported to demonstrate some level of tolerance to reduced pH. We hypothesize that the observed tolerance of coral larvae to low pH may be partly explained by reduced metabolic rates in acidified seawater because both calcifying and non-calcifying marine invertebrates could show metabolic depression under reduced pH in order to enhance their survival. In this study, after 3-d and 7-d exposure to three different pH levels (8.0, 7.6, and 7.3), we found that the oxygen consumption of Acropora digitifera larvae tended to be suppressed with reduced pH, although a statistically significant difference was not observed between pH conditions. Larval metamorphosis was also observed, confirming that successful recruitment is impaired when metamorphosis is disrupted, despite larval survival. Results also showed that the metamorphosis rate significantly decreased under acidified seawater conditions after both short (2 h) and long (7 d) term exposure. These results imply that acidified seawater impacts larval physiology, suggesting that suppressed metabolism and metamorphosis may alter the dispersal potential of larvae and subsequently reduce the resilience of coral communities in the near future as the ocean pH decreases.

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.

Abstract. Increasing the acidity of ocean waters will directly threaten calcifying marine organisms such as reef-building scleractinian corals, and the myriad of species that rely on corals for protection and sustenance. Ocean pH has already decreased by around 0.1 pH units since the beginning of the industrial revolution, and is expected to decrease by another 0.2–0.4 pH units by 2100. This study mimicked the pre-industrial, present, and near-future levels of pCO2 using a precise control system (± 5% pCO2), to assess the impact of ocean acidification on the calcification of recently settled primary polyps of Acropora digitifera, both with and without symbionts, and adult fragments with symbionts. The increase in pCO2 of ~100 μatm between the pre-industrial period and the present had more effect on the calcification rate of adult A. digitifera than the anticipated future increases of several hundreds of micro-atmospheres of pCO2. The primary polyps with symbionts showed higher calcification rates than primary polyps without symbionts, suggesting that: (i) primary polyps housing symbionts are more tolerant to near-future ocean acidification than organisms without symbionts, and (ii) corals acquiring symbionts from the environment (i.e., broadcasting species) will be more vulnerable to ocean acidification than corals that maternally acquire symbionts.

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Multi-species spawning is reported in the coral genus Acropora, but hybridization in nature rarely occurs because of the incompatibility of gametes and the timing of spawning. However, the evolutionary relationships between gamete compatibility and spawning time are obscure. Investigations of gamete compatibility in sister species that spawn at different times may provide clues to answering this question. Acropora sp. 1 has been defined as a cryptic species of Acropora digitifera, and they are morphologically similar, but spawn in different months, suggesting that they are either a cryptic species or a different species. We examined the morphology and conducted crossing experiments using cryopreserved sperm. The morphologies (branch length, branch width, and outer diameter of axial corallites) of A. digitifera and Acropora sp. 1 differed significantly. A phylogenetic tree of partial Pax-C nuclear sequences from A. digitifera and Acropora sp. 1 shows that they are monophyletic and closely related genetically, based on F ST values and P-distance. These results imply that these two species originated recently from a common ancestor. In addition, cryopreserved sperm from both A. digitifera and Acropora sp. 1 showed bidirectional inter-crossing (cryopreserved sperm of A. digitifera and eggs of Acropora sp. 1 from Sesoko: 32.1 ± 6.7 %, control-conspecific cryopreserved sperm and eggs: 46.1 ± 10.6 %; cryopreserved sperm of Acropora sp. 1 and eggs of A. digitifera from Oku: 63.3 ± 16.6 %, control: 83.6 ± 6.0 %). The results suggest that the gametes of these two species are compatible and that the pre-zygotic isolation mechanism is relaxed because their gametes do not interact. Overall, these two species should be classified as distinct species, and changes in spawning time are related to speciation in a similar gamete recognition system.

Studies using genome-wide single nucleotide polymorphisms (SNPs) have become commonplace in genetics and genomics, due to advances in high-throughput sequencing technologies. Since the numbers of required SNPs and samples vary depending on each research goal, genotyping technologies with high flexibility in the number of SNPs/samples and high repeatability have been intensively investigated. For example, the ultrahigh-multiplexed amplicon sequencing, Ion AmpliSeq, has been used as a high-throughput genotyping method mainly for diagnostic purposes. Here, we designed a custom panel targeting 3,187 genome-wide SNPs of fugu, Takifugu rubripes , and applied it for genotyping farmed fugu to test its feasibility in aquaculture studies. We sequenced two libraries consisting of different pools of individuals ( n = 326 each) on the Illumina MiSeq sequencer. Consequently, over 99% target regions (3,178 SNPs) were amplified and 2,655 SNPs were available after filtering steps. Strong correlation was observed in the mean depth of coverage of each SNP between duplicate runs ( r = 0.993). Genetic analysis using these genotype data successfully detected the known population structure and the sex determining locus of fugu. These results show the method is superior in repeatability and flexibility, and suits genetic studies including molecular breeding, such as marker assisted and genomic selection.

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.