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September 22, 2019

A multiplex homology-directed DNA repair assay reveals the impact of more than 1,000 BRCA1 missense substitution variants on protein function.

Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Comparative genomics of Czech vaccine strains of Bordetella pertussis.

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness.

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September 22, 2019

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

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September 22, 2019

Deletions linked to PROG1 gene participate in plant architecture domestication in Asian and African rice.

Improving the yield by modifying plant architecture was a key step during crop domestication. Here, we show that a 110-kb deletion on the short arm of chromosome 7 in Asian cultivated rice (Oryza sativa), which is closely linked to the previously identified PROSTRATE GROWTH 1 (PROG1) gene, harbors a tandem repeat of seven zinc-finger genes. Three of these genes regulate the plant architecture, suggesting that the deletion also promoted the critical transition from the prostrate growth and low yield of wild rice (O. rufipogon) to the erect growth and high yield of Asian cultivated rice. We refer to this locus as RICE PLANT ARCHITECTURE DOMESTICATION (RPAD). Further, a similar but independent 113-kb deletion is detected at the RPAD locus in African cultivated rice. These results indicate that the deletions, eliminating a tandem repeat of zinc-finger genes, may have been involved in the parallel domestication of plant architecture in Asian and African rice.

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September 22, 2019

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

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September 22, 2019

Whole genome sequencing for investigations of meningococcal outbreaks in the United States: a retrospective analysis.

Although rare in the U.S., outbreaks due to Neisseria meningitidis do occur. Rapid, early outbreak detection is important for timely public health response. In this study, we characterized U.S. meningococcal isolates (N?=?201) from 15 epidemiologically defined outbreaks (2009-2015) along with temporally and geographically matched sporadic isolates using multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), and six whole genome sequencing (WGS) based methods. Recombination-corrected maximum likelihood (ML) and Bayesian phylogenies were reconstructed to identify genetically related outbreak isolates. All WGS analysis methods showed high degree of agreement and distinguished isolates with similar or indistinguishable PFGE patterns, or the same strain genotype. Ten outbreaks were caused by a single strain; 5 were due to multiple strains. Five sporadic isolates were phylogenetically related to 2 outbreaks. Analysis of 9 outbreaks using timed phylogenies identified the possible origin and estimated the approximate time that the most recent common ancestor emerged for outbreaks analyzed. U.S. meningococcal outbreaks were caused by single- or multiple-strain introduction, with organizational outbreaks mainly caused by a clonal strain and community outbreaks by divergent strains. WGS can infer linkage of meningococcal cases when epidemiological links are uncertain. Accurate identification of outbreak-associated cases requires both WGS typing and epidemiological data.

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September 22, 2019

Multi-population genomic analysis of malaria parasites indicates local selection and differentiation at the gdv1 locus regulating sexual development.

Parasites infect hosts in widely varying environments, encountering diverse challenges for adaptation. To identify malaria parasite genes under locally divergent selection across a large endemic region with a wide spectrum of transmission intensity, genome sequences were obtained from 284 clinical Plasmodium falciparum infections from four newly sampled locations in Senegal, The Gambia, Mali and Guinea. Combining these with previous data from seven other sites in West Africa enabled a multi-population analysis to identify discrete loci under varying local selection. A genome-wide scan showed the most exceptional geographical divergence to be at the early gametocyte gene locus gdv1 which is essential for parasite sexual development and transmission. We identified a major structural dimorphism with alternative 1.5?kb and 1.0?kb sequence deletions at different positions of the 3′-intergenic region, in tight linkage disequilibrium with the most highly differentiated single nucleotide polymorphism, one of the alleles being very frequent in Senegal and The Gambia but rare in the other locations. Long non-coding RNA transcripts were previously shown to include the entire antisense of the gdv1 coding sequence and the portion of the intergenic region with allelic deletions, suggesting adaptive regulation of parasite sexual development and transmission in response to local conditions.

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September 22, 2019

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance.

Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology.Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0-3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7.The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates.

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September 22, 2019

Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai.

Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

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September 22, 2019

Assembling the genome of the African wild rice Oryza longistaminata by exploiting synteny in closely related Oryza species.

The African wild rice species Oryza longistaminata has several beneficial traits compared to cultivated rice species, such as resistance to biotic stresses, clonal propagation via rhizomes, and increased biomass production. To facilitate breeding efforts and functional genomics studies, we de-novo assembled a high-quality, haploid-phased genome. Here, we present our assembly, with a total length of 351?Mb, of which 92.2% was anchored onto 12 chromosomes. We detected 34,389 genes and 38.1% of the genome consisted of repetitive content. We validated our assembly by a comparative linkage analysis and by examining well-characterized gene families. This genome assembly will be a useful resource to exploit beneficial alleles found in O. longistaminata. Our results also show that it is possible to generate a high-quality, functionally complete rice genome assembly from moderate SMRT read coverage by exploiting synteny in a closely related Oryza species.

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September 22, 2019

Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producing Escherichia coli

Carbapenem-resistant Enterobacteriaceae are considered by WHO as critical priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates, as well as the genetic bases for the emergence and global dissemination of specific lineages, remain largely unknown. Here, by combining a thorough genomic and evolutionary analysis of Ec ST410 isolates with a broad analysis of 12,398 E. coli and Shigella genomes, we showed that the fixation of carbapenemase genes depends largely on a combination of mutations in ftsI encoding the penicillin binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele spread across the species by recombination. Those mutations were in most cases selected prior to carbapenemase gene acquisition. The selection of CP-Ec lineages able to disseminate is more complex than the mere acquisition of carbapenemase genes and might be largely triggered by beta-lactams other than carbapenems.

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September 22, 2019

Recurrent loss of HMGCS2 shows that ketogenesis is not essential for the evolution of large mammalian brains.

Apart from glucose, fatty acid-derived ketone bodies provide metabolic energy for the brain during fasting and neonatal development. We investigated the evolution of HMGCS2, the key enzyme required for ketone body biosynthesis (ketogenesis). Unexpectedly, we found that three mammalian lineages, comprising cetaceans (dolphins and whales), elephants and mastodons, and Old World fruit bats have lost this gene. Remarkably, many of these species have exceptionally large brains and signs of intelligent behavior. While fruit bats are sensitive to starvation, cetaceans and elephants can still withstand periods of fasting. This suggests that alternative strategies to fuel large brains during fasting evolved repeatedly and reveals flexibility in mammalian energy metabolism. Furthermore, we show that HMGCS2 loss preceded brain size expansion in toothed whales and elephants. Thus, while ketogenesis was likely important for brain size expansion in modern humans, ketogenesis is not a universal precondition for the evolution of large mammalian brains.© 2018, Jebb et al.

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September 22, 2019

Recovery of novel association loci in Arabidopsis thaliana and Drosophila melanogaster through leveraging INDELs association and integrated burden test.

Short insertions, deletions (INDELs) and larger structural variants have been increasingly employed in genetic association studies, but few improvements over SNP-based association have been reported. In order to understand why this might be the case, we analysed two publicly available datasets and observed that 63% of INDELs called in A. thaliana and 64% in D. melanogaster populations are misrepresented as multiple alleles with different functional annotations, i.e. where the same underlying variant is represented by inconsistent alignments leading to different variant calls. To address this issue, we have developed the software Irisas to reclassify and re-annotate these variants, which we then used for single-locus tests of association. We also integrated them to predict the functional impact of SNPs, INDELs, and structural variants for burden testing. Using both approaches, we re-analysed the genetic architecture of complex traits in A. thaliana and D. melanogaster. Heritability analysis using SNPs alone explained on average 27% and 19% of phenotypic variance for A. thaliana and D. melanogaster respectively. Our method explained an additional 11% and 3%, respectively. We also identified novel trait loci that previous SNP-based association studies failed to map, and which contain established candidate genes. Our study shows the value of the association test with INDELs and integrating multiple types of variants in association studies in plants and animals.

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September 22, 2019

Discovery of mcr-1-mediated colistin resistance in a highly virulent Escherichia coli lineage.

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum ß-lactamase blaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCEEscherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage. Copyright © 2018 Forde et al.

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September 22, 2019

An introduced crop plant is driving diversification of the virulent bacterial pathogen Erwinia tracheiphila.

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrpT3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber (Cucumis sativus), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens.IMPORTANCEErwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways. Copyright © 2018 Shapiro et al.

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