The first in-depth exploration of the genome of the engineered bacterium, Gluconobacter thailandicus.

Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.

A transcriptome atlas of silkworm silk glands revealed by PacBio single-molecule long-read sequencing.

The silk gland of the silkworm Bombyx mori is a specialized organ where silk proteins are efficiently synthesized under precise regulation that largely determines the properties of silk fibers. To understand the genes involved in the regulation of silk protein synthesis, considerable research has focused on the transcripts expressed in silk glands; however, the complete transcriptome profile of this organ has yet to be elucidated. Here, we report a full-length silk gland transcriptome obtained by PacBio single-molecule long-read sequencing technology. In total, 11,697 non-redundant transcripts were identified in mixed samples of silk glands dissected from larvae at five developmental stages. When compared with the published reference, the full-length transcripts optimized the structures of 3002 known genes, and a total of 9061 novel transcripts with an average length of 2171 bp were detected. Among these, 1403 (15.5%) novel transcripts were computationally revealed to be lncRNAs, 8135 (89.8%) novel transcripts were annotated to different protein and nucleotide databases, and 5655 (62.4%) novel transcripts were predicted to have complete ORFs. Furthermore, we found 1867 alternative splicing events, 2529 alternative polyadenylation events, 784 fusion events and 6596 SSRs. This study provides a comprehensive set of reference transcripts and greatly revises and expands the available silkworm transcript data. In addition, these data will be very useful for studying the regulatory mechanisms of silk protein synthesis.

Full-length transcriptome sequences and the identification of putative genes for flavonoid biosynthesis in safflower.

BACKGROUND: The flower of the safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study. RESULTS: High-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1-2, 2-3 and 3-6 k) library construction. Five cells were carried (2 cells for 1-2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study. CONCLUSIONS: Full-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2, CtCHS3, CtCHI3, CtF3H3, CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.

Involvement of Nucleotide Excision Repair and Rec-Dependent Pathway Genes for UV Radiation Resistance in Deinococcus irradiatisoli 17bor-2.

Strain Deinococcus irradiatisoli 17bor-2 was isolated from a soil sample exposed to γ radiation at Seoul Women's University, Republic of Korea. The genus Deinococcus is a Gram-negative, coccus-shaped, and extremophilic bacterium, well renowned as being a radiation-resistant bacterium. Therefore, the mechanism behind the resistance to radiation and the gene responsible for the resistance could be helpful for detailed experimental studies with biotechnological applications. To study the involvement of genes in UV radiation resistance in strain 17bor-2, the genomic DNA of the strain was sequenced and constructed using the Pacific Biosciences RS II system. In addition, the complete genome sequence of strain 17bor-2 was annotated and interpreted using the Genomes-Expert Review (IMG-ER) system, along with Prodigal and JGI GenePRIMP analysis. The genome analysis of strain 17bor-2 revealed evidence of excinuclease UvrABC genes, which are key enzymes in the nucleotide excision repair (NER) mechanism, as well as genes from the recA-dependent and recQ pathways. The genome of strain Deinococcus irradiatisoli 17bor-2 was a circular chromosome comprising 3,052,043 bp with a GC content of 67.0%, including 2911 coding sequences (CDs), 49 tRNA genes, and 9 rRNA genes. In addition, their complete genome sequence annotation features provided evidence that radiation resistance genes play a central part in adaptation against extreme environmental conditions. In recent decades, excision repair genes have been indicated in considerable detail for both prokaryote and eukaryote resistance against UV-C radiation.

Reference Grade Characterization of Polymorphisms in Full-Length HLA Class I and II Genes With Short-Read Sequencing on the ION PGM System and Long-Reads Generated by Single Molecule, Real-Time Sequencing on the PacBio Platform.

Although NGS technologies fuel advances in high-throughput HLA genotyping methods for identification and classification of HLA genes to assist with precision medicine efforts in disease and transplantation, the efficiency of these methods are impeded by the absence of adequately-characterized high-frequency HLA allele reference sequence databases for the highly polymorphic HLA gene system. Here, we report on producing a comprehensive collection of full-length HLA allele sequences for eight classical HLA loci found in the Japanese population. We augmented the second-generation short read data generated by the Ion Torrent technology with long amplicon spanning consensus reads delivered by the third-generation SMRT sequencing method to create reference grade high-quality sequences of HLA class I and II gene alleles resolved at the genomic coding and non-coding level. Forty-six DNAs were obtained from a reference set used previously to establish the HLA allele frequency data in Japanese subjects. The samples included alleles with a collective allele frequency in the Japanese population of more than 99.2%. The HLA loci were independently amplified by long-range PCR using previously designed HLA-locus specific primers and subsequently sequenced using SMRT and Ion PGM sequencers. The mapped long and short-reads were used to produce a reference library of consensus HLA allelic sequences with the help of the reference-aware software tool LAA for SMRT Sequencing. A total of 253 distinct alleles were determined for 46 healthy subjects. Of them, 137 were novel alleles: 101 SNVs and/or indels and 36 extended alleles at a partial or full-length level. Comparing the HLA sequences from the perspective of nucleotide diversity revealed that HLA-DRB1 was the most divergent among the eight HLA genes, and that the HLA-DPB1 gene sequences diverged into two distinct groups, DP2 and DP5, with evidence of independent polymorphisms generated in exon 2. We also identified two specific intronic variations in HLA-DRB1 that might be involved in rheumatoid arthritis. In conclusion, full-length HLA allele sequencing by third-generation and second-generation technologies has provided polymorphic gene reference sequences at a genomic allelic resolution including allelic variations assigned up to the field-4 level for a stronger foundation in precision medicine and HLA-related disease and transplantation studies.

Long-read sequencing across the C9orf72 'GGGGCC' repeat expansion: implications for clinical use and genetic discovery efforts in human disease.

BACKGROUND: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 'GGGGCC' (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences' (PacBio) and Oxford Nanopore Technologies' (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9. RESULTS: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinION was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8× coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained > 800× coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual's repeat region was > 99% G4C2 content, though we cannot rule out small interruptions. CONCLUSIONS: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

PacBio But Not Illumina Technology Can Achieve Fast, Accurate and Complete Closure of the High GC, Complex Burkholderia pseudomallei Two-Chromosome Genome.

Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68-69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201-366 contigs), sharing only 92.2-100% and 92.0-100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST). Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina datasets. Hybrid approach generated only 74 contigs, while the PacBio data alone with de novo assembly achieved complete closure of the two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing. PacBio RS II using P6-C4 chemistry is highly robust and cost-effective and should be the platform of choice in sequencing bacterial genomes, particularly for those that are well-known to be difficult-to-sequence.

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

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.

Profiling of Oral Microbiota in Early Childhood Caries Using Single-Molecule Real-Time Sequencing.

Background: Alterations of oral microbiota are the main cause of the progression of caries. The goal of this study was to characterize the oral microbiota in childhood caries based on single-molecule real-time sequencing. Methods: A total of 21 preschoolers, aged 3-5 years old with severe early childhood caries, and 20 age-matched, caries-free children as controls were recruited. Saliva samples were collected, followed by DNA extraction, Pacbio sequencing, and phylogenetic analyses of the oral microbial communities. Results: Eight hundred and seventy six species derived from 13 known bacterial phyla and 110 genera were detected from 41 children using Pacbio sequencing. At the species level, 38 species, including Veillonella spp., Streptococcus spp., Prevotella spp., and Lactobacillus spp., showed higher abundance in the caries group compared to the caries-free group (p < 0.05). The core microbiota at the genus and species levels was more stable in the caries-free micro-ecological niche. At follow-up, oral examinations 6 months after sample collection, development of new dental caries was observed in 5 children (the transitional group) among the 21 caries free children. Compared with the caries-free children, in the transitional and caries groups, 6 species, which were more abundant in the caries-free group, exhibited a relatively low abundance in both the caries group and the transitional group (p < 0.05). We conclude that Abiotrophia spp., Neisseria spp., and Veillonella spp., might be associated with healthy oral microbial ecosystem. Prevotella spp., Lactobacillus spp., Dialister spp., and Filifactor spp. may be related to the pathogenesis and progression of dental caries.

Complete genome sequence of Bacillus amyloliquefaciens subsp. plantarum S499, a rhizobacterium that triggers plant defences and inhibits fungal phytopathogens.

Bacillus amyloliquefaciens subsp. plantarum S499 is a plant beneficial rhizobacterium with a good antagonistic potential against phytopathogens through the release of active secondary metabolites. Moreover, it can induce systemic resistance in plants by producing considerable amounts of surfactins. The complete genome sequence of B. amyloliquefaciens subsp. plantarum S499 includes a circular chromosome of 3,927,922bp and a plasmid of 8,008bp. A remarkable abundance in genomic regions of putative horizontal origin emerged from the analysis. Furthermore, we highlighted the presence of genes involved in the establishment of interactions with the host plants at the root level and in the competition with other soil-borne microorganisms. More specifically, genes related to the synthesis of amylolysin, amylocyclicin, and butirosin were identified. These antimicrobials were not known before to be part of the antibiotic arsenal of the strain. The information embedded in the genome will support the upcoming studies regarding the application of B. amyloliquefaciens isolates as plant-growth promoters and biocontrol agents.

Complete genome sequence of Bacillus subtilis BSD-2, a microbial germicide isolated from cultivated cotton.

Bacillus subtilis BSD-2, isolated from cotton (Gossypium spp.), had strong antagonistic activity to Verticillium dahlia Kleb and Botrytis cinerea. We sequenced and annotated the BSD-2 complete genome to help us the better use of this strain, which has surfactin, bacilysin, bacillibactin, subtilosin A, Tas A and a potential class IV lanthipeptide biosynthetic pathways.

The genome of Pleurotus eryngii provides insights into the mechanisms of wood decay.

Pleurotus eryngii (DC.) Quél. is widely used for bioconverting lignocellulosic byproducts into biofuel and value added products. Sequencing and annotating the genome of a monokaryon strain P. eryngii 183 allows us to gain a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases for degradation of lignocellulose in white-rot fungi. The genomic data provides insights into genomic basis of degradation mechanisms of lignin and cellulose and may pave new avenues for lignocellulose bioconversion.