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July 7, 2019

Genomic innovation for crop improvement.

Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genomes and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access the large and complex genomes of crops and their wild relatives. These have helped to identify a wide spectrum of genetic variation and permitted the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems.

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July 7, 2019

AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15(T), isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.

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July 7, 2019

Efficient CNV breakpoint analysis reveals unexpected structural complexity and correlation of dosage-sensitive genes with clinical severity in genomic disorders.

Genomic disorders are the clinical conditions manifested by submicroscopic genomic rearrangements including copy number variants (CNVs). The CNVs can be identified by array-based comparative genomic hybridization (aCGH), the most commonly used technology for molecular diagnostics of genomic disorders. However, clinical aCGH only informs CNVs in the probe-interrogated regions. Neither orientational information nor the resulting genomic rearrangement structure is provided, which is a key to uncovering mutational and pathogenic mechanisms underlying genomic disorders. Long-range polymerase chain reaction (PCR) is a traditional approach to obtain CNV breakpoint junction, but this method is inefficient when challenged by structural complexity such as often found at the PLP1 locus in association with Pelizaeus-Merzbacher disease (PMD). Here we introduced ‘capture and single-molecule real-time sequencing’ (cap-SMRT-seq) and newly developed ‘asymmetry linker-mediated nested PCR walking’ (ALN-walking) for CNV breakpoint sequencing in 49 subjects with PMD-associated CNVs. Remarkably, 29 (94%) of the 31 CNV breakpoint junctions unobtainable by conventional long-range PCR were resolved by cap-SMRT-seq and ALN-walking. Notably, unexpected CNV complexities, including inter-chromosomal rearrangements that cannot be resolved by aCGH, were revealed by efficient breakpoint sequencing. These sequence-based structures of PMD-associated CNVs further support the role of DNA replicative mechanisms in CNV mutagenesis, and facilitate genotype-phenotype correlation studies. Intriguingly, the lengths of gained segments by CNVs are strongly correlated with clinical severity in PMD, potentially reflecting the functional contribution of other dosage-sensitive genes besides PLP1. Our study provides new efficient experimental approaches (especially ALN-walking) for CNV breakpoint sequencing and highlights their importance in uncovering CNV mutagenesis and pathogenesis in genomic disorders.© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

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July 7, 2019

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

The human reference genome assembly plays a central role in nearly all aspects of today’s basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health. © 2017 Schneider et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

HySA: a Hybrid Structural variant Assembly approach using next-generation and single-molecule sequencing technologies.

Achieving complete, accurate, and cost-effective assembly of human genomes is of great importance for realizing the promise of precision medicine. The abundance of repeats and genetic variations in human genomes and the limitations of existing sequencing technologies call for the development of novel assembly methods that can leverage the complementary strengths of multiple technologies. We propose a Hybrid Structural variant Assembly (HySA) approach that integrates sequencing reads from next-generation sequencing and single-molecule sequencing technologies to accurately assemble and detect structural variants (SVs) in human genomes. By identifying homologous SV-containing reads from different technologies through a bipartite-graph-based clustering algorithm, our approach turns a whole genome assembly problem into a set of independent SV assembly problems, each of which can be effectively solved to enhance the assembly of structurally altered regions in human genomes. We used data generated from a haploid hydatidiform mole genome (CHM1) and a diploid human genome (NA12878) to test our approach. The result showed that, compared with existing methods, our approach had a low false discovery rate and substantially improved the detection of many types of SVs, particularly novel large insertions, small indels (10-50 bp), and short tandem repeat expansions and contractions. Our work highlights the strengths and limitations of current approaches and provides an effective solution for extending the power of existing sequencing technologies for SV discovery.© 2017 Fan et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Genome sequence of Plasmopara viticola and insight into the pathogenic mechanism.

Plasmopara viticola causes downy mildew disease of grapevine which is one of the most devastating diseases of viticulture worldwide. Here we report a 101.3?Mb whole genome sequence of P. viticola isolate ‘JL-7-2’ obtained by a combination of Illumina and PacBio sequencing technologies. The P. viticola genome contains 17,014 putative protein-coding genes and has ~26% repetitive sequences. A total of 1,301 putative secreted proteins, including 100 putative RXLR effectors and 90 CRN effectors were identified in this genome. In the secretome, 261 potential pathogenicity genes and 95 carbohydrate-active enzymes were predicted. Transcriptional analysis revealed that most of the RXLR effectors, pathogenicity genes and carbohydrate-active enzymes were significantly up-regulated during infection. Comparative genomic analysis revealed that P. viticola evolved independently from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. The availability of the P. viticola genome provides a valuable resource not only for comparative genomic analysis and evolutionary studies among oomycetes, but also enhance our knowledge on the mechanism of interactions between this biotrophic pathogen and its host.

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July 7, 2019

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids.

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.

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July 7, 2019

Complex routes of nosocomial vancomycin-resistant Enterococcus faecium transmission revealed by genome sequencing.

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of nosocomial infection. Here, we describe the utility of whole-genome sequencing in defining nosocomial VREfm transmission.A retrospective study at a single hospital in the United Kingdom identified 342 patients with E. faecium bloodstream infection over 7 years. Of these, 293 patients had a stored isolate and formed the basis for the study. The first stored isolate from each case was sequenced (200 VREfm [197 vanA, 2 vanB, and 1 isolate containing both vanA and vanB], 93 vancomycin-susceptible E. faecium) and epidemiological data were collected. Genomes were also available for E. faecium associated with bloodstream infections in 15 patients in neighboring hospitals, and 456 patients across the United Kingdom and Ireland.The majority of infections in the 293 patients were hospital-acquired (n = 249) or healthcare-associated (n = 42). Phylogenetic analysis showed that 291 of 293 isolates resided in a hospital-associated clade that contained numerous discrete clusters of closely related isolates, indicative of multiple introductions into the hospital followed by clonal expansion associated with transmission. Fine-scale analysis of 6 exemplar phylogenetic clusters containing isolates from 93 patients (32%) identified complex transmission routes that spanned numerous wards and years, extending beyond the detection of conventional infection control. These contained both vancomycin-resistant and -susceptible isolates. We also identified closely related isolates from patients at Cambridge University Hospitals NHS Foundation Trust and regional and national hospitals, suggesting interhospital transmission.These findings provide important insights for infection control practice and signpost areas for interventions. We conclude that sequencing represents a powerful tool for the enhanced surveillance and control of nosocomial E. faecium transmission and infection.

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July 7, 2019

The Nephila clavipes genome highlights the diversity of spider silk genes and their complex expression.

Spider silks are the toughest known biological materials, yet are lightweight and virtually invisible to the human immune system, and they thus have revolutionary potential for medicine and industry. Spider silks are largely composed of spidroins, a unique family of structural proteins. To investigate spidroin genes systematically, we constructed the first genome of an orb-weaving spider: the golden orb-weaver (Nephila clavipes), which builds large webs using an extensive repertoire of silks with diverse physical properties. We cataloged 28 Nephila spidroins, representing all known orb-weaver spidroin types, and identified 394 repeated coding motif variants and higher-order repetitive cassette structures unique to specific spidroins. Characterization of spidroin expression in distinct silk gland types indicates that glands can express multiple spidroin types. We find evidence of an alternatively spliced spidroin, a spidroin expressed only in venom glands, evolutionary mechanisms for spidroin diversification, and non-spidroin genes with expression patterns that suggest roles in silk production.

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July 7, 2019

Population and clinical genetics of human transposable elements in the (post) genomic era.

Recent technological developments-in genomics, bioinformatics and high-throughput experimental techniques-are providing opportunities to study ongoing human transposable element (TE) activity at an unprecedented level of detail. It is now possible to characterize genome-wide collections of TE insertion sites for multiple human individuals, within and between populations, and for a variety of tissue types. Comparison of TE insertion site profiles between individuals captures the germline activity of TEs and reveals insertion site variants that segregate as polymorphisms among human populations, whereas comparison among tissue types ascertains somatic TE activity that generates cellular heterogeneity. In this review, we provide an overview of these new technologies and explore their implications for population and clinical genetic studies of human TEs. We cover both recent published results on human TE insertion activity as well as the prospects for future TE studies related to human evolution and health.

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July 7, 2019

Antibodyomics: bioinformatics technologies for understanding B-cell immunity to HIV-1.

Numerous antibodies have been identified from HIV-1-infected donors that neutralize diverse strains of HIV-1. These antibodies may provide the basis for a B cell-mediated HIV-1 vaccine. However, it has been unclear how to elicit similar antibodies by vaccination. To address this issue, we have undertaken an informatics-based approach to understand the genetic and immunologic processes controlling the development of HIV-1-neutralizing antibodies. As DNA sequencing comprises the fastest growing database of biological information, we focused on incorporating next-generation sequencing of B-cell transcripts to determine the origin, maturation pathway, and prevalence of broadly neutralizing antibody lineages (Antibodyomics1, 2, 4, and 6). We also incorporated large-scale robotic analyses of serum neutralization to identify and quantify neutralizing antibodies in donor cohorts (Antibodyomics3). Statistical analyses furnish another layer of insight (Antibodyomics5), with physical characteristics of antibodies and their targets through molecular dynamics simulations (Antibodyomics7) and free energy perturbation analyses (Antibodyomics8) providing information-rich output. Functional interrogation of individual antibodies (Antibodyomics9) and synthetic antibody libraries (Antibodyomics10) also yields multi-dimensional data by which to understand and improve antibodies. Antibodyomics, described here, thus comprise resolution-enhancing tools, which collectively embody an information-driven discovery engine aimed toward the development of effective B cell-based vaccines.© 2017 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.

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July 7, 2019

Resequencing array for gene variant detection in malignant hyperthermia and butyrylcholinestherase deficiency.

Malignant hyperthermia (MH) and butyrylcholinestherase (BCHE) deficiency are two relevant pharmacogenetic disorders in anesthetic practice linked with sequence variants, the former in the RyR1 and CACNA1S genes, the latter in the BCHE gene. Genotyping for known pathogenic variants in these genes is useful to help identify susceptible individuals, and others may exist but remain unknown, because full-length sequence of these genes is, in general, not investigated. To facilitate this task, we developed a resequencing DNA array, the perioperative patient safety (POPS) array, to be able to screen the entire coding sequences of the RyR1, CACNA1S and BCHE genes. MH-susceptible individuals (n?=?121) identified with the in vitro contracture test, the standard diagnostic tool for MH susceptibility, were genotyped with the arrays. Compared with capillary sequencing, call rates with the arrays could achieve 100% at maximal sensitivity, although to reduce false positive rates, sensitivity was adjusted to 0.85, 0.87 and 0.66 for RyR1, CACNA1S and BCHE respectively, with overall base call specificity exceeding 99%. Detection of 29 predetermined RyR1 variants in 44 individuals was successful in 97% of the cases, among them all 16 variants of established diagnostic value. In a trial application of the arrays, 21 MH-susceptible subjects with no known RyR1 or CACNA1S variants were screened, resulting in the discovery of new variants, all confirmed by capillary sequencing. In conclusion, arrays offer an efficient high-throughput alternative for diagnostic genotyping of candidate genes affecting MH susceptibility, BCHE deficiency and other neuromuscular disorders, simultaneously enabling a comprehensive search for rare variants in these genes. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Mistranslation can enhance fitness through purging of deleterious mutations.

Phenotypic mutations are amino acid changes caused by mistranslation. How phenotypic mutations affect the adaptive evolution of new protein functions is unknown. Here we evolve the antibiotic resistance protein TEM-1 towards resistance on the antibiotic cefotaxime in an Escherichia coli strain with a high mistranslation rate. TEM-1 populations evolved in such strains endow host cells with a general growth advantage, not only on cefotaxime but also on several other antibiotics that ancestral TEM-1 had been unable to deactivate. High-throughput sequencing of TEM-1 populations shows that this advantage is associated with a lower incidence of weakly deleterious genotypic mutations. Our observations show that mistranslation is not just a source of noise that delays adaptive evolution. It could even facilitate adaptive evolution by exacerbating the effects of deleterious mutations and leading to their more efficient purging. The ubiquity of mistranslation and its effects render mistranslation an important factor in adaptive protein evolution.

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July 7, 2019

The evolutionary life cycle of the polysaccharide biosynthetic gene cluster based on the Sphingomonadaceae.

Although clustering of genes from the same metabolic pathway is a widespread phenomenon, the evolution of the polysaccharide biosynthetic gene cluster remains poorly understood. To determine the evolution of this pathway, we identified a scattered production pathway of the polysaccharide sanxan by Sphingomonas sanxanigenens NX02, and compared the distribution of genes between sphingan-producing and other Sphingomonadaceae strains. This allowed us to determine how the scattered sanxan pathway developed, and how the polysaccharide gene cluster evolved. Our findings suggested that the evolution of microbial polysaccharide biosynthesis gene clusters is a lengthy cyclic process comprising cluster 1???scatter???cluster 2. The sanxan biosynthetic pathway proved the existence of a dispersive process. We also report the complete genome sequence of NX02, in which we identified many unstable genetic elements and powerful secretion systems. Furthermore, nine enzymes for the formation of activated precursors, four glycosyltransferases, four acyltransferases, and four polymerization and export proteins were identified. These genes were scattered in the NX02 genome, and the positive regulator SpnA of sphingans synthesis could not regulate sanxan production. Finally, we concluded that the evolution of the sanxan pathway was independent. NX02 evolved naturally as a polysaccharide producing strain over a long-time evolution involving gene acquisitions and adaptive mutations.

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