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July 7, 2019

Genome sequencing reveals the origin of the allotetraploid Arabidopsis suecica.

Polyploidy is an example of instantaneous speciation when it involves the formation of a new cytotype that is incompatible with the parental species. Because new polyploid individuals are likely to be rare, establishment of a new species is unlikely unless polyploids are able to reproduce through self-fertilization (selfing), or asexually. Conversely, selfing (or asexuality) makes it possible for polyploid species to originate from a single individual-a bona fide speciation event. The extent to which this happens is not known. Here, we consider the origin of Arabidopsis suecica, a selfing allopolyploid between Arabidopsis thaliana and Arabidopsis arenosa, which has hitherto been considered to be an example of a unique origin. Based on whole-genome re-sequencing of 15 natural A. suecica accessions, we identify ubiquitous shared polymorphism with the parental species, and hence conclusively reject a unique origin in favor of multiple founding individuals. We further estimate that the species originated after the last glacial maximum in Eastern Europe or central Eurasia (rather than Sweden, as the name might suggest). Finally, annotation of the self-incompatibility loci in A. suecica revealed that both loci carry non-functional alleles. The locus inherited from the selfing A. thaliana is fixed for an ancestral non-functional allele, whereas the locus inherited from the outcrossing A. arenosa is fixed for a novel loss-of-function allele. Furthermore, the allele inherited from A. thaliana is predicted to transcriptionally silence the allele inherited from A. arenosa, suggesting that loss of self-incompatibility may have been instantaneous.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4 and KPC-2 carbapenemases.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that blaNDM-1 was carried on an IncX3 plasmid. The blaIMP-4 and blaKPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Rapid and consistent evolution of colistin resistance in XDR Pseudomonas aeruginosa during morbidostat culture.

Colistin is a last resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa To investigate the potential for in-situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous culture device known as morbidostat. As a result, colistin resistance reproducibly increased 10-fold within ten days, and 100-fold within 20 days, along with highly stereotypic, yet strain specific mutation patterns. The majority of mutations hit the pmrAB two component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA We tracked the frequencies of all arising mutations by whole genome deep sequencing every 3-4 days to provide a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant sub-populations. In seven out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Whole genome sequence of the heterozygous clinical isolate Candida krusei 81-B-5.

Candida krusei is a diploid, heterozygous yeast that is an opportunistic fungal pathogen in immunocompromised patients. This species also is utilized for fermenting cocoa beans during chocolate production. One major concern in the clinical setting is the innate resistance of this species to the most commonly used antifungal drug fluconazole. Here we report a high-quality genome sequence and assembly for the first clinical isolate of C. krusei, strain 81-B-5, into 11 scaffolds generated with PacBio sequencing technology. Gene annotation and comparative analysis revealed a unique profile of transporters that could play a role in drug resistance or adaptation to different environments. In addition, we show that while 82% of the genome is highly heterozygous, a 2.0 Mb region of the largest scaffold has undergone loss of heterozygosity. This genome will serve as a reference for further genetic studies of this pathogen. Copyright © 2017 Author et al.

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July 7, 2019

ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments.

Single molecule sequencing (SMS) platforms enable base sequences to be read directly from individual strands of DNA in real-time. Though capable of long read lengths, SMS platforms currently suffer from low throughput compared to competing short-read sequencing technologies. Here, we present a novel strategy for sequencing library preparation, dubbed ConcatSeq, which increases the throughput of SMS platforms by generating long concatenated templates from pools of short DNA molecules. We demonstrate adaptation of this technique to two target enrichment workflows, commonly used for oncology applications, and feasibility using PacBio single molecule real-time (SMRT) technology. Our approach is capable of increasing the sequencing throughput of the PacBio RSII platform by more than five-fold, while maintaining the ability to correctly call allele frequencies of known single nucleotide variants. ConcatSeq provides a versatile new sample preparation tool for long-read sequencing technologies.

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July 7, 2019

Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all ß-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the ß-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to ß-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of ß-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.

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July 7, 2019

Chromosomal 16S ribosomal RNA methyltransferase RmtE1 in Escherichia coli sequence type 448.

We identified rmtE1, an uncommon 16S ribosomal methyltransferase gene, in an aminoglycoside- and cephalosporin-resistant Escherichia coli sequence type 448 clinical strain co-harboring blaCMY-2. Long-read sequencing revealed insertion of a 101,257-bp fragment carrying both resistance genes to the chromosome. Our findings underscore E. coli sequence type 448 as a potential high-risk multidrug-resistant clone.

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July 7, 2019

Evolution of the wheat blast fungus through functional losses in a host specificity determinant.

Wheat blast first emerged in Brazil in the mid-1980s and has recently caused heavy crop losses in Asia. Here we show how this devastating pathogen evolved in Brazil. Genetic analysis of host species determinants in the blast fungus resulted in the cloning of avirulence genes PWT3 and PWT4, whose gene products elicit defense in wheat cultivars containing the corresponding resistance genes Rwt3 and Rwt4 Studies on avirulence and resistance gene distributions, together with historical data on wheat cultivation in Brazil, suggest that wheat blast emerged due to widespread deployment of rwt3 wheat (susceptible to Lolium isolates), followed by the loss of function of PWT3 This implies that the rwt3 wheat served as a springboard for the host jump to common wheat. Copyright © 2017, American Association for the Advancement of Science.

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July 7, 2019

Repeated divergent selection on pigmentation genes in a rapid finch radiation.

Instances of recent and rapid speciation are suitable for associating phenotypes with their causal genotypes, especially if gene flow homogenizes areas of the genome that are not under divergent selection. We study a rapid radiation of nine sympatric bird species known as capuchino seedeaters, which are differentiated in sexually selected characters of male plumage and song. We sequenced the genomes of a phenotypically diverse set of species to search for differentiated genomic regions. Capuchinos show differences in a small proportion of their genomes, yet selection has acted independently on the same targets in different members of this radiation. Many divergent regions contain genes involved in the melanogenesis pathway, with the strongest signal originating from putative regulatory regions. Selection has acted on these same genomic regions in different lineages, likely shaping the evolution of cis-regulatory elements, which control how more conserved genes are expressed and thereby generate diversity in classically sexually selected traits.

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July 7, 2019

Virulence and genomic feature of a virulent Klebsiella pneumoniae sequence type 14 strain of serotype K2 harboring blaNDM-5 in China.

The objective of this study was to reveal the molecular mechanism involved in carbapenem resistance and virulence of a K2 Klebsiella pneumoniae clinical isolate 24835. The virulence of the strain was determined by in vitro and in vivo methods. The de novo whole-genome sequencing technology and molecular biology methods were used to analyze the genomic features associated with the carbapenem resistance and virulence of K. pneumoniae 24835. Strain 24835 was highly resistant to carbapenems and belonged to ST14, exhibited hypermucoviscous and unique K2-aerobactin-kfu-rmpA positive phenotype. As the only carbapenemase gene in strain 24835, blaNDM-5 was located on a 46-kb IncX3 self-transmissible plasmid, which is a very close relation of pNDM-MGR194 from India. Genetic context of blaNDM-5 in strain 24835 was closely related to those on IncX3 plasmids in various Enterobacteriaceae species in China. The combination of multiple virulence genes may work together to confer the relative higher virulence in K. pneumoniae 24835. Significantly increased resistance to serum killing and mice mortality were found in the virulent New Delhi metallo-ß-lactamase (NDM)-producing K. pneumoniae strain compared to the other NDM-producing K. pneumoniae strain. Our study provides basic information of phenotypic and genomic features of K. pneumoniae 24835, a strain displaying carbapenem resistance and relatively high level of virulence. These findings are concerning for the potential of NDM-like genes to disseminate among virulent K. pneumoniae isolates.

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July 7, 2019

CLOVE: classification of genomic fusions into structural variation events.

A precise understanding of structural variants (SVs) in DNA is important in the study of cancer and population diversity. Many methods have been designed to identify SVs from DNA sequencing data. However, the problem remains challenging because existing approaches suffer from low sensitivity, precision, and positional accuracy. Furthermore, many existing tools only identify breakpoints, and so not collect related breakpoints and classify them as a particular type of SV. Due to the rapidly increasing usage of high throughput sequencing technologies in this area, there is an urgent need for algorithms that can accurately classify complex genomic rearrangements (involving more than one breakpoint or fusion).We present CLOVE, an algorithm for integrating the results of multiple breakpoint or SV callers and classifying the results as a particular SV. CLOVE is based on a graph data structure that is created from the breakpoint information. The algorithm looks for patterns in the graph that are characteristic of more complex rearrangement types. CLOVE is able to integrate the results of multiple callers, producing a consensus call.We demonstrate using simulated and real data that re-classified SV calls produced by CLOVE improve on the raw call set of existing SV algorithms, particularly in terms of accuracy. CLOVE is freely available from http://www.github.com/PapenfussLab .

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July 7, 2019

Genome graphs

There is increasing recognition that a single, monoploid reference genome is a poor universal reference structure for human genetics, because it represents only a tiny fraction of human variation. Adding this missing variation results in a structure that can be described as a mathematical graph: a genome graph. We demonstrate that, in comparison to the existing reference genome (GRCh38), genome graphs can substantially improve the fractions of reads that map uniquely and perfectly. Furthermore, we show that this fundamental simplification of read mapping transforms the variant calling problem from one in which many non-reference variants must be discovered de-novo to one in which the vast majority of variants are simply re-identified within the graph. Using standard benchmarks as well as a novel reference-free evaluation, we show that a simplistic variant calling procedure on a genome graph can already call variants at least as well as, and in many cases better than, a state-of-the-art method on the linear human reference genome. We anticipate that graph-based references will supplant linear references in humans and in other applications where cohorts of sequenced individuals are available.

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July 7, 2019

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis.

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

The MHC locus and genetic susceptibility to autoimmune and infectious diseases.

In the past 50 years, variants in the major histocompatibility complex (MHC) locus, also known as the human leukocyte antigen (HLA), have been reported as major risk factors for complex diseases. Recent advances, including large genetic screens, imputation, and analyses of non-additive and epistatic effects, have contributed to a better understanding of the shared and specific roles of MHC variants in different diseases. We review these advances and discuss the relationships between MHC variants involved in autoimmune and infectious diseases. Further work in this area will help to distinguish between alternative hypotheses for the role of pathogens in autoimmune disease development.

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July 7, 2019

The origin, diversification and adaptation of a major mangrove clade (Rhizophoreae) revealed by whole-genome sequencing

Mangroves invade some very marginal habitats for woody plants—at the interface between land and sea. Since mangroves anchor tropical coastal communities globally, their origin, diversification and adaptation are of scientific significance, particularly at a time of global climate change. In this study, a combination of single-molecule long reads and the more conventional short reads are generated from Rhizophora apiculata for the de novo assembly of its genome to a near chromosome level. The longest scaffold, N50 and N90 for the R. apiculata genome, are 13.3 Mb, 5.4 Mb and 1.0 Mb, respectively. Short reads for the genomes and transcriptomes of eight related species are also generated. We find that the ancestor of Rhizophoreae experienced a whole-genome duplication ~70 Myrs ago, which is followed rather quickly by colonization and species diversification. Mangroves exhibit pan-exome modifications of amino acid (AA) usage as well as unusual AA substitutions among closely related species. The usage and substitution of AAs, unique among plants surveyed, is correlated with the rapid evolution of proteins in mangroves. A small subset of these substitutions is associated with mangroves’ highly specialized traits (vivipary and red bark) thought to be adaptive in the intertidal habitats. Despite the many adaptive features, mangroves are among the least genetically diverse plants, likely the result of continual habitat turnovers caused by repeated rises and falls of sea level in the geologically recent past. Mangrove genomes thus inform about their past evolutionary success as well as portend a possibly difficult future.

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