Micrococcus luteus has been found in a wide range of habitats. We report the complete genome sequence and methylome analysis of strain SA211 isolated from a hypersaline, lithium-rich, high-altitude salt flat in Argentina with single-molecule real-time sequencing.
To date, clinical sequencing has focused on genomic DNA using targeted panels and exome sequencing. Sequencing of a large hypertrophic cardiomyopathy (HCM) cohort revealed that positive identification of a disease-associated variant was returned in only 32% of patients, with an additional 15% receiving inconclusive results. When genome sequencing fails to reveal causative variants, the transcriptome may provide additional diagnostic clarity. A recent study examining patients with genetically undiagnosed muscle disorders found that RNA sequencing, when used as a complement to exome and whole genome sequencing, had an overall diagnosis rate of 35%.
Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis, which is a significant cause of morbidity in China and the Philippines. A single draft genome was available for S. japonicum, yet this assembly is very fragmented and only covers 90% of the genome, which make it difficult to be applied as a reference in functional genome analysis and genes discovery.In this study, we present a high-quality assembly of the fluke S. japonicum genome by combining 20 G (~53X) long single molecule real time sequencing reads with 80 G (~ 213X) Illumina paired-end reads. This improved genome assembly is approximately 370.5 Mb, with contig and scaffold N50 length of 871.9 kb and 1.09 Mb, representing 142.4-fold and 6.2-fold improvement over the released WGS-based assembly, respectively. Additionally, our assembly captured 85.2% complete and 4.6% partial eukaryotic Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 46.80% of the genome, and 10,089 of the protein-coding genes were predicted from the improved genome, of which 96.5% have been functionally annotated. Lastly, using the improved assembly, we identified 20 significantly expanded gene families in S. japonicum, and those genes were primarily enriched in functions of proteolysis and protein glycosylation.Using the combination of PacBio and Illumina Sequencing technologies, we provided an improved high-quality genome of S. japonicum. This improved genome assembly, as well as the annotation, will be useful for the comparative genomics of the flukes and more importantly facilitate the molecular studies of this important parasite in the future.
Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B.?ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B.?ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B.?ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia?cepacia complex bacteria. Removal of the third replicon reduced B.?ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.
Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94?Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5?kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant-pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.
Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.
Construction of chromosome-level assembly is a vital step in achieving the goal of a ‘Platinum’ genome, but it remains a major challenge to assemble and anchor sequences to chromosomes in autopolyploid or highly heterozygous genomes. High-throughput chromosome conformation capture (Hi-C) technology serves as a robust tool to dramatically advance chromosome scaffolding; however, existing approaches are mostly designed for diploid genomes and often with the aim of reconstructing a haploid representation, thereby having limited power to reconstruct chromosomes for autopolyploid genomes. We developed a novel algorithm (ALLHiC) that is capable of building allele-aware, chromosomal-scale assembly for autopolyploid genomes using Hi-C paired-end reads with innovative ‘prune’ and ‘optimize’ steps. Application on simulated data showed that ALLHiC can phase allelic contigs and substantially improve ordering and orientation when compared to other mainstream Hi-C assemblers. We applied ALLHiC on an autotetraploid and an autooctoploid sugar-cane genome and successfully constructed the phased chromosomal-level assemblies, revealing allelic variations present in these two genomes. The ALLHiC pipeline enables de novo chromosome-level assembly of autopolyploid genomes, separating each allele. Haplotype chromosome-level assembly of allopolyploid and heterozygous diploid genomes can be achieved using ALLHiC, overcoming obstacles in assembling complex genomes.
Recent metagenomic analysis has revealed that our gut microbiota plays an important role in not only the maintenance of our health but also various diseases such as obesity, diabetes, inflammatory bowel disease, and allergy. However, most intestinal bacteria are considered ‘unculturable’ bacteria, and their functions remain unknown. Although culture-independent genomic approaches have enabled us to gain insight into their potential roles, culture-based approaches are still required to understand their characteristic features and phenotypes. To date, various culturing methods have been attempted to obtain these ‘unculturable’ bacteria, but most such methods require advanced techniques. Here, we have tried to isolate possible unculturable bacteria from a healthy Japanese individual by using commercially available media. A 16S rRNA (ribosomal RNA) gene metagenomic analysis revealed that each culture medium showed bacterial growth depending on its selective features and a possibility of the presence of novel bacterial species. Whole genome sequencing of these candidate strains suggested the isolation of 8 novel bacterial species classified in the Actinobacteria and Firmicutes phyla. Our approach indicates that a number of intestinal bacteria hitherto considered unculturable are potentially culturable and can be cultured on commercially available media. We have obtained novel gut bacteria from a healthy Japanese individual using a combination of comprehensive genomics and conventional culturing methods. We would expect that the discovery of such novel bacteria could illuminate pivotal roles for the gut microbiota in association with human health.
Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species’ entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or “private” gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.Copyright © 2019 Minio et al.
In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that are being applied to pathogenic microorganisms and viruses, constitutional disorders, pharmacogenomics, cancer, and more.Copyright © 2018 Elsevier Ltd. All rights reserved.
The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.
Circulating DNA in plasma consists of short DNA fragments. The biological processes generating such fragments are not well understood. DNASE1L3 is a secreted DNASE1-like nuclease capable of digesting DNA in chromatin, and its absence causes anti-DNA responses and autoimmunity in humans and mice. We found that the deletion of Dnase1l3 in mice resulted in aberrations in the fragmentation of plasma DNA. Such aberrations included an increase in short DNA molecules below 120 bp, which was positively correlated with anti-DNA antibody levels. We also observed an increase in long, multinucleosomal DNA molecules and decreased frequencies of the most common end motifs found in plasma DNA. These aberrations were independent of anti-DNA response, suggesting that they represented a primary effect of DNASE1L3 loss. Pregnant Dnase1l3-/- mice carrying Dnase1l3+/- fetuses showed a partial restoration of normal frequencies of plasma DNA end motifs, suggesting that DNASE1L3 from Dnase1l3-proficient fetuses could enter maternal systemic circulation and affect both fetal and maternal DNA fragmentation in a systemic as well as local manner. However, the observed shortening of circulating fetal DNA relative to maternal DNA was not affected by the deletion of Dnase1l3 Collectively, our findings demonstrate that DNASE1L3 plays a role in circulating plasma DNA homeostasis by enhancing fragmentation and influencing end-motif frequencies. These results support a distinct role of DNASE1L3 as a regulator of the physical form and availability of cell-free DNA and may have important implications for the mechanism whereby this enzyme prevents autoimmunity. Copyright © 2019 the Author(s). Published by PNAS.
We present reference-quality genome assembly and annotation for the stout camphor tree (Cinnamomum kanehirae (Laurales, Lauraceae)), the first sequenced member of the Magnoliidae comprising four orders (Laurales, Magnoliales, Canellales and Piperales) and over 9,000 species. Phylogenomic analysis of 13 representative seed plant genomes indicates that magnoliid and eudicot lineages share more recent common ancestry than monocots. Two whole-genome duplication events were inferred within the magnoliid lineage: one before divergence of Laurales and Magnoliales and the other within the Lauraceae. Small-scale segmental duplications and tandem duplications also contributed to innovation in the evolutionary history of Cinnamomum. For example, expansion of the terpenoid synthase gene subfamilies within the Laurales spawned the diversity of Cinnamomum monoterpenes and sesquiterpenes.
The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome-scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single-molecule real-time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi-C-based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein-coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.
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