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July 7, 2019

The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization.

Gonorrhoea and MDR Neisseria gonorrhoeae remain public health concerns globally. Enhanced, quality-assured, gonococcal antimicrobial resistance (AMR) surveillance is essential worldwide. The WHO global Gonococcal Antimicrobial Surveillance Programme (GASP) was relaunched in 2009. We describe the phenotypic, genetic and reference genome characteristics of the 2016 WHO gonococcal reference strains intended for quality assurance in the WHO global GASP, other GASPs, diagnostics and research worldwide.The 2016 WHO reference strains (n?=?14) constitute the eight 2008 WHO reference strains and six novel strains. The novel strains represent low-level to high-level cephalosporin resistance, high-level azithromycin resistance and a porA mutant. All strains were comprehensively characterized for antibiogram (n?=?23), serovar, prolyliminopeptidase, plasmid types, molecular AMR determinants, N. gonorrhoeae multiantigen sequence typing STs and MLST STs. Complete reference genomes were produced using single-molecule PacBio sequencing.The reference strains represented all available phenotypes, susceptible and resistant, to antimicrobials previously and currently used or considered for future use in gonorrhoea treatment. All corresponding resistance genotypes and molecular epidemiological types were described. Fully characterized, annotated and finished references genomes (n?=?14) were presented.The 2016 WHO gonococcal reference strains are intended for internal and external quality assurance and quality control in laboratory investigations, particularly in the WHO global GASP and other GASPs, but also in phenotypic (e.g. culture, species determination) and molecular diagnostics, molecular AMR detection, molecular epidemiology and as fully characterized, annotated and finished reference genomes in WGS analysis, transcriptomics, proteomics and other molecular technologies and data analysis.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

The genomic sequence of the oral pathobiont strain NI1060 reveals unique strategies for bacterial competition and pathogenicity.

Strain NI1060 is an oral bacterium responsible for periodontitis in a murine ligature-induced disease model. To better understand its pathogenicity, we have determined the complete sequence of its 2,553,982 bp genome. Although closely related to Pasteurella pneumotropica, a pneumonia-associated rodent commensal based on its 16S rRNA, the NI1060 genomic content suggests that they are different species thriving on different energy sources via alternative metabolic pathways. Genomic and phylogenetic analyses showed that strain NI1060 is distinct from the genera currently described in the family Pasteurellaceae, and is likely to represent a novel species. In addition, we found putative virulence genes involved in lipooligosaccharide synthesis, adhesins and bacteriotoxic proteins. These genes are potentially important for host adaption and for the induction of dysbiosis through bacterial competition and pathogenicity. Importantly, strain NI1060 strongly stimulates Nod1, an innate immune receptor, but is defective in two peptidoglycan recycling genes due to a frameshift mutation. The in-depth analysis of its genome thus provides critical insights for the development of NI1060 as a prime model system for infectious disease.

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July 7, 2019

Challenges, solutions, and quality metrics of personal genome assembly in advancing precision medicine.

Even though each of us shares more than 99% of the DNA sequences in our genome, there are millions of sequence codes or structure in small regions that differ between individuals, giving us different characteristics of appearance or responsiveness to medical treatments. Currently, genetic variants in diseased tissues, such as tumors, are uncovered by exploring the differences between the reference genome and the sequences detected in the diseased tissue. However, the public reference genome was derived with the DNA from multiple individuals. As a result of this, the reference genome is incomplete and may misrepresent the sequence variants of the general population. The more reliable solution is to compare sequences of diseased tissue with its own genome sequence derived from tissue in a normal state. As the price to sequence the human genome has dropped dramatically to around $1000, it shows a promising future of documenting the personal genome for every individual. However, de novo assembly of individual genomes at an affordable cost is still challenging. Thus, till now, only a few human genomes have been fully assembled. In this review, we introduce the history of human genome sequencing and the evolution of sequencing platforms, from Sanger sequencing to emerging “third generation sequencing” technologies. We present the currently available de novo assembly and post-assembly software packages for human genome assembly and their requirements for computational infrastructures. We recommend that a combined hybrid assembly with long and short reads would be a promising way to generate good quality human genome assemblies and specify parameters for the quality assessment of assembly outcomes. We provide a perspective view of the benefit of using personal genomes as references and suggestions for obtaining a quality personal genome. Finally, we discuss the usage of the personal genome in aiding vaccine design and development, monitoring host immune-response, tailoring drug therapy and detecting tumors. We believe the precision medicine would largely benefit from bioinformatics solutions, particularly for personal genome assembly.

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July 7, 2019

SAR11 bacteria linked to ocean anoxia and nitrogen loss.

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world’s largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth’s most abundant organismal group.

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July 7, 2019

Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen.

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in “arms races” with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence. © 2016 Faino et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Genomic characterization of the Atlantic cod sex-locus.

A variety of sex determination mechanisms can be observed in evolutionary divergent teleosts. Sex determination is genetic in Atlantic cod (Gadus morhua), however the genomic location or size of its sex-locus is unknown. Here, we characterize the sex-locus of Atlantic cod using whole genome sequence (WGS) data of 227 wild-caught specimens. Analyzing more than 55 million polymorphic loci, we identify 166 loci that are associated with sex. These loci are located in six distinct regions on five different linkage groups (LG) in the genome. The largest of these regions, an approximately 55?Kb region on LG11, contains the majority of genotypes that segregate closely according to a XX-XY system. Genotypes in this region can be used genetically determine sex, whereas those in the other regions are inconsistently sex-linked. The identified region on LG11 and its surrounding genes have no clear sequence homology with genes or regulatory elements associated with sex-determination or differentiation in other species. The functionality of this sex-locus therefore remains unknown. The WGS strategy used here proved adequate for detecting the small regions associated with sex in this species. Our results highlight the evolutionary flexibility in genomic architecture underlying teleost sex-determination and allow practical applications to genetically sex Atlantic cod.

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July 7, 2019

Representing genetic variation with synthetic DNA standards.

The identification of genetic variation with next-generation sequencing is confounded by the complexity of the human genome sequence and by biases that arise during library preparation, sequencing and analysis. We have developed a set of synthetic DNA standards, termed ‘sequins’, that emulate human genetic features and constitute qualitative and quantitative spike-in controls for genome sequencing. Sequencing reads derived from sequins align exclusively to an artificial in silico reference chromosome, rather than the human reference genome, which allows them them to be partitioned for parallel analysis. Here we use this approach to represent common and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements and copy-number variation. We validate the design and performance of sequin standards by comparison to examples in the NA12878 reference genome, and we demonstrate their utility during the detection and quantification of variants. We provide sequins as a standardized, quantitative resource against which human genetic variation can be measured and diagnostic performance assessed.

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July 7, 2019

The two chromosomes of the mitochondrial genome of a sugarcane cultivar: assembly and recombination analysis using long PacBio reads.

Sugarcane accounts for a large portion of the worlds sugar production. Modern commercial cultivars are complex hybrids of S. officinarum and several other Saccharum species. Historical records identify New Guinea as the origin of S. officinarum and that a small number of plants originating from there were used to generate all modern commercial cultivars. The mitochondrial genome can be a useful way to identify the maternal origin of commercial cultivars. We have used the PacBio RSII to sequence and assemble the mitochondrial genome of a South East Asian commercial cultivar, known as Khon Kaen 3. The long read length of this sequencing technology allowed for the mitochondrial genome to be assembled into two distinct circular chromosomes with all repeat sequences spanned by individual reads. Comparison of five commercial hybrids, two S. officinarum and one S. spontaneum to our assembly reveals no structural rearrangements between our assembly, the commercial hybrids and an S. officinarum from New Guinea. The S. spontaneum, from India, and one sample of S. officinarum (unknown origin) are substantially rearranged and have a large number of homozygous variants. This supports the record that S. officinarum plants from New Guinea are the maternal source of all modern commercial hybrids.

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July 7, 2019

Distinct Salmonella enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.

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July 7, 2019

The complete chloroplast genome sequence of the medicinal plant Swertia mussotii using the PacBio RS II platform.

Swertia mussotii is an important medicinal plant that has great economic and medicinal value and is found on the Qinghai Tibetan Plateau. The complete chloroplast (cp) genome of S. mussotii is 153,431 bp in size, with a pair of inverted repeat (IR) regions of 25,761 bp each that separate an large single-copy (LSC) region of 83,567 bp and an a small single-copy (SSC) region of 18,342 bp. The S. mussotii cp genome encodes 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The identity, number, and GC content of S. mussotii cp genes were similar to those in the genomes of other Gentianales species. Via analysis of the repeat structure, 11 forward repeats, eight palindromic repeats, and one reverse repeat were detected in the S. mussotii cp genome. There are 45 SSRs in the S. mussotii cp genome, the majority of which are mononucleotides found in all other Gentianales species. An entire cp genome comparison study of S. mussotii and two other species in Gentianaceae was conducted. The complete cp genome sequence provides intragenic information for the cp genetic engineering of this medicinal plant.

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July 7, 2019

DBG2OLC: Efficient assembly of large genomes using long erroneous reads of the third generation sequencing technologies.

The highly anticipated transition from next generation sequencing (NGS) to third generation sequencing (3GS) has been difficult primarily due to high error rates and excessive sequencing cost. The high error rates make the assembly of long erroneous reads of large genomes challenging because existing software solutions are often overwhelmed by error correction tasks. Here we report a hybrid assembly approach that simultaneously utilizes NGS and 3GS data to address both issues. We gain advantages from three general and basic design principles: (i) Compact representation of the long reads leads to efficient alignments. (ii) Base-level errors can be skipped; structural errors need to be detected and corrected. (iii) Structurally correct 3GS reads are assembled and polished. In our implementation, preassembled NGS contigs are used to derive the compact representation of the long reads, motivating an algorithmic conversion from a de Bruijn graph to an overlap graph, the two major assembly paradigms. Moreover, since NGS and 3GS data can compensate for each other, our hybrid assembly approach reduces both of their sequencing requirements. Experiments show that our software is able to assemble mammalian-sized genomes orders of magnitude more quickly than existing methods without consuming a lot of memory, while saving about half of the sequencing cost.

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July 7, 2019

Complete genome sequence of Bacillus oceanisediminis 2691, a reservoir of heavy-metal resistance genes.

Ocean sediments are commonly subject to the pollution of various heavy metals. Intracellular heavy metal concentrations in marine microorganisms should be kept within allowable concentrations. Here, we report redundant heavy metal resistance related genes encoding heavy metal-sensing transcriptional regulators (i.e. cadC), heavy metal efflux pumps, and detoxifying enzymes in the complete genome sequence of Bacillus oceanisediminis 2691. By comparing CadC sequences of strain 2691 with those from other bacterial genomes, we demonstrated that each cadC gene located in the chromosome or plasmid of 2691 cells are similar to those of various near or distant microbes, which might shed light on evolutionary trajectories of redundant heavy metal resistance genes. In application aspects, these diverse heavy metal sensing genes can be harnessed as synthetic biological parts, modules, and devices for the development of heavy metal-specific biosensors. Heavy metal bioremediation technologies or platform cells can be also developed based on the marine genomic information of heavy metal resistance and/or detoxification genes in a bacterial isolate from ocean sediments. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 7, 2019

Genomic insight into the host-endosymbiont relationship of Endozoicomonas montiporae CL-33(T) with its coral host.

The bacterial genus Endozoicomonas was commonly detected in healthy corals in many coral-associated bacteria studies in the past decade. Although, it is likely to be a core member of coral microbiota, little is known about its ecological roles. To decipher potential interactions between bacteria and their coral hosts, we sequenced and investigated the first culturable endozoicomonal bacterium from coral, the E. montiporae CL-33(T). Its genome had potential sign of ongoing genome erosion and gene exchange with its host. Testosterone degradation and type III secretion system are commonly present in Endozoicomonas and may have roles to recognize and deliver effectors to their hosts. Moreover, genes of eukaryotic ephrin ligand B2 are present in its genome; presumably, this bacterium could move into coral cells via endocytosis after binding to coral’s Eph receptors. In addition, 7,8-dihydro-8-oxoguanine triphosphatase and isocitrate lyase are possible type III secretion effectors that might help coral to prevent mitochondrial dysfunction and promote gluconeogenesis, especially under stress conditions. Based on all these findings, we inferred that E. montiporae was a facultative endosymbiont that can recognize, translocate, communicate and modulate its coral host.

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July 7, 2019

Complete genome sequence of thermophilic Bacillus smithii type strain DSM 4216(T).

Bacillus smithii is a facultatively anaerobic, thermophilic bacterium able to use a variety of sugars that can be derived from lignocellulosic feedstocks. Being genetically accessible, it is a potential new host for biotechnological production of green chemicals from renewable resources. We determined the complete genomic sequence of the B. smithii type strain DSM 4216(T), which consists of a 3,368,778 bp chromosome (GenBank accession number CP012024.1) and a 12,514 bp plasmid (GenBank accession number CP012025.1), together encoding 3880 genes. Genome annotation via RAST was complemented by a protein domain analysis. Some unique features of B. smithii central metabolism in comparison to related organisms included the lack of a standard acetate production pathway with no apparent pyruvate formate lyase, phosphotransacetylase, and acetate kinase genes, while acetate was the second fermentation product.

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