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September 22, 2019

Analysis of structural variants in four African cichlids highlights an association with developmental and immune related genes

African Lakes Cichlids are one of the most impressive example of adaptive radiation. Independently in Lake Victoria, Tanganyika, and Malawi, several hundreds of species arose within the last 10 million to 100,000 years. Whereas most analyses in cichlids focused on nucleotide substitutions across species to investigate the genetic bases of this explosive radiation, to date, no study has investigated the contribution of structural variants (SVs) to speciation events (through a reduction of gene flow) and adaptation to different ecological niches. Here, we annotate and characterize the repertoires and evolutionary potential of different SV classes (deletion, duplication, inversion, insertions and translocations) in five cichlid species (Astatotilapia burtoni, Metriaclima zebra, Neolamprologus brichardi, Pundamilia nyererei and Oreochromis niloticus). We investigate the patterns of gain/loss evolution across the phylogeny for each SV type enabling the identification of both lineage specific events and a set of conserved SVs, common to all four species in the radiation. Both deletion and inversion events show a significant overlap with SINE elements, while inversions additionally show a limited, but significant association with DNA transposons. Genes lying inside inverted regions are enriched for genes regulating behaviour, or involved in skeletal and visual system development. Moreover, we find that duplicated genes show enrichment for textquoterightantigen processing and presentationtextquoteright (GO:0019882) and other immune related categories. Altogether, we provide the first, comprehensive overview of rearrangement evolution in East African Cichlids, and some initial insights into their possible contribution to adaptation.

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September 22, 2019

Genome mining for fungal polyketide-diterpenoid hybrids: discovery of key terpene cyclases and multifunctional P450s for structural diversification

A biosynthetic gene cluster for chevalone E (1) and its oxidized derivatives have been identified within the genome of the endophytic fungus Aspergillus versicolor 0312, by a mining strategy targeting a polyke- tide-diterpenoid hybrid molecule. The biosynthetic pathway has been successfully reconstituted in the heterologous fungus Aspergillus oryzae. Interestingly, two P450 monooxygenases, Cle2 and Cle4, were found to transform 1 into seven new analogues including 7 and 8 that possess a unique five-membered lactone ring. Furthermore, the replacement of the terpene cyclase gene with that from another fungus led to the production of sartorypyrone D (11), which has a monocyclic terpenoid moiety. Finally, some of the compounds obtained in this study synergistically enhanced the cytotoxicity of doxorubicin (DOX) in breast cancer cells.

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September 22, 2019

Complete Genome Sequence of Massilia oculi sp. nov. CCUG 43427T (=DSM 26321T), the Type Strain of M. oculi, and Comparison with Genome Sequences of Other Massilia Strains.

Massilia oculi sp. nov. of type strain CCUG 43427T is a Gram-negative, rod-shaped, nonspore-forming bacterium, which was recently isolated from the eye of a patient suffering from endophthalmitis and was described as novel species in Massilia genus. In this study, we present the complete genome sequence of this strain by using Pacbio SMRT cell platform and compare this sequence with the genomes of 30 Massilia representative strains. Also, a comprehensive search was conducted for genes and proteins involved in antibiotic resistance and pathogenicity. The genome of CCUG 43427T is 5,844,653 bp with 65.55% GC content. This genome contains four prophages and four genomic islands (GIs). The cobalt/zinc/cadmium transporter locus CzcABCD is included in these GIs. This GI was predicted to play important role in bacterial heavy-metal tolerance. The in silico genome analysis also revealed that this strain contains a lot of antibiotic resistance and pathogenicity related genes. This result suggested that this strain may has evolved a wide arsenal of weapons for pathogenicity and survival. Genome comparison among CCUG 43427T and other 30 Massilia strains revealed that more than 400 genes are unique in CCUG 43427T. Among these, one gene cluster, which was annotated to be important for LOS biosynthesis, catalytic mechanism and the substrate specificity of the enzyme, was predicted to be horizontally transferred by using phylogenies and biased GC content.

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September 22, 2019

Genome sequences of two diploid wild relatives of cultivated sweetpotato reveal targets for genetic improvement

Sweetpotato [Ipomoea batatas (L.) Lam.] is a globally important staple food crop, especially for sub-Saharan Africa. Agronomic improvement of sweetpotato has lagged behind other major food crops due to a lack of genomic and genetic resources and inherent challenges in breeding a heterozygous, clonally propagated polyploid. Here, we report the genome sequences of its two diploid relatives, I. trifida and I. triloba, and show that these high-quality genome assemblies are robust references for hexaploid sweetpotato. Comparative and phylogenetic analyses reveal insights into the ancient whole-genome triplication history of Ipomoea and evolutionary relationships within the Batatas complex. Using resequencing data from 16 genotypes widely used in African breeding programs, genes and alleles associated with carotenoid biosynthesis in storage roots are identified, which may enable efficient breeding of varieties with high provitamin A content. These resources will facilitate genome-enabled breeding in this important food security crop.

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September 22, 2019

Molecular characteristics and comparative genomics analysis of a clinical Enterococcus casseliflavus with a resistance plasmid.

The aim of this work was to investigate the molecular characterization of a clinical Enterococcus casseliflavus strain with a resistance plasmid.En. casseliflavus EC369 was isolated from a patient in a hospital in southern China. The minimum inhibitory concentration was found by means of the agar dilution method to determine the antimicrobial susceptibilities of the strains. Whole-genome sequencing and comparative genomics analysis were performed to analyze the mechanism of antibiotic resistance and the horizontal gene transfer of the resistance gene-related mobile genetic elements.En. casseliflavus EC369 showed resistance to erythromycin, kanamycin, and streptomycin, but was susceptible to vancomycin, ampicillin, and streptothricin and other antimicrobials. There were six resistance genes (aph3′, ant6, bla, sat4, and two ermBs) carried by a transposon identified on the plasmid pEC369 and a complete resistance gene cluster of vancomycin and a tet (M) gene encoded on the chromosome. This is the first complete plasmid sequence reported in clinically isolated En. casseliflavus. The plasmid with the greatest sequence identity with pEC369 was the plasmid of Enterococcus sp. FDAARGOS_375, followed by the plasmids of Enterococcus faecium strains F12085 and pRE25, whereas the sequence with the greatest identity to the resistance genes carrying a transposon of pEC369 was on the chromosome of Staphylococcus aureus strain GD1677.The resistance profiles of En. casseliflavus EC369 might contribute to the resistance genes encoded on the plasmid. The fact that the most similar sequence to the transposon carrying resistance genes of pEC369 was encoded in the chromosome of a S. aureus strain provides insights into the mechanism of dissemination of multidrug resistance between bacteria of different species or genera through horizontal gene transfer.

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September 22, 2019

Identification of DNA base modifications by means of Pacific Biosciences RS Sequencing technology.

Whole phage genomes can be sequenced readily using one or a combination of next generation sequencing (NGS) technologies. One of the most recently developed NGS platforms, the so-called Single-Molecule Real-Time (SMRT) sequencing approach provided by the PacBio RS platform, is particularly useful in providing complete (i.e., un-gapped) genome sequences, but differs from other technologies in that the platform also allows for downstream analysis to identify nucleotides that have been modified by DNA methylation. Here, we describe the methodological approach for the detection of genomic methylation motifs by means of SMRT sequencing.

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September 22, 2019

Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp. Enterica serovar Gaminera, S. Mbandaka, S. Tennesse, S. Minnesota, S. Senftenberg and S. Typhimurium. These new strains fluoresce when irradiated with UV light and maintain this phenotype in absence of antibiotic selection. Recombinants were phenotypically equivalent to the parent strain, except for S. Tennessee Sal66 that appeared Lac- on Xylose Lysine Deoxycholate (XLD) agar plates and Lac+ on Mac Conkey and Hektoen Enteric agar plates. Analysis of closed whole genome sequences revealed that Sal66 had lost one lactose operon; slower rates of lactose metabolism may affect lactose fermentation on XLD agar. These fluorescent enteric control strains were challenging to develop and should provide an easy and effective means of identifying cross-contamination. Published by Elsevier Ltd.

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September 22, 2019

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

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September 22, 2019

Three New Genome Assemblies Support a Rapid Radiation in Musa acuminata (Wild Banana).

Edible bananas result from interspecific hybridization between Musa acuminata and Musa balbisiana, as well as among subspecies in M. acuminata. Four particular M. acuminata subspecies have been proposed as the main contributors of edible bananas, all of which radiated in a short period of time in southeastern Asia. Clarifying the evolution of these lineages at a whole-genome scale is therefore an important step toward understanding the domestication and diversification of this crop. This study reports the de novo genome assembly and gene annotation of a representative genotype from three different subspecies of M. acuminata. These data are combined with the previously published genome of the fourth subspecies to investigate phylogenetic relationships. Analyses of shared and unique gene families reveal that the four subspecies are quite homogenous, with a core genome representing at least 50% of all genes and very few M. acuminata species-specific gene families. Multiple alignments indicate high sequence identity between homologous single copy-genes, supporting the close relationships of these lineages. Interestingly, phylogenomic analyses demonstrate high levels of gene tree discordance, due to both incomplete lineage sorting and introgression. This pattern suggests rapid radiation within Musa acuminata subspecies that occurred after the divergence with M. balbisiana. Introgression between M. a. ssp. malaccensis and M. a. ssp. burmannica was detected across the genome, though multiple approaches to resolve the subspecies tree converged on the same topology. To support evolutionary and functional analyses, we introduce the PanMusa database, which enables researchers to exploration of individual gene families and trees.

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September 22, 2019

Extensive and deep sequencing of the Venter/HuRef genome for developing and benchmarking genome analysis tools.

We produced an extensive collection of deep re-sequencing datasets for the Venter/HuRef genome using the Illumina massively-parallel DNA sequencing platform. The original Venter genome sequence is a very-high quality phased assembly based on Sanger sequencing. Therefore, researchers developing novel computational tools for the analysis of human genome sequence variation for the dominant Illumina sequencing technology can test and hone their algorithms by making variant calls from these Venter/HuRef datasets and then immediately confirm the detected variants in the Sanger assembly, freeing them of the need for further experimental validation. This process also applies to implementing and benchmarking existing genome analysis pipelines. We prepared and sequenced 200?bp and 350?bp short-insert whole-genome sequencing libraries (sequenced to 100x and 40x genomic coverages respectively) as well as 2?kb, 5?kb, and 12?kb mate-pair libraries (49x, 122x, and 145x physical coverages respectively). Lastly, we produced a linked-read library (128x physical coverage) from which we also performed haplotype phasing.

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September 22, 2019

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous insertions and deletions of virulence genes. In particular, among the inserted genes, there is a cluster of eight connected genes associated with the synthesis of capsular polysaccharide. This cluster encodes a transferase and capsular polysaccharide synthase, promotes the formation of capsules and causes changes in pathogenicity. Electron microscopy revealed a distinct capsule surrounding the SDLY strain. The pathogenicity test showed that the SDLY strain could cause significant clinical symptoms and pathological changes in both SPF chickens and mice. In addition, these clinical symptoms and pathological changes were the same as those observed in field cases. Furthermore, the anti-microbial susceptibility test demonstrated that the SDLY strain exhibits multiple-antibiotic resistance. The emergence of pathogenic M. caseolyticus indicates that more attention should be paid to the effects of this micro-organism on both poultry and public health.© 2018 Blackwell Verlag GmbH.

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September 22, 2019

Hybrid correction of highly noisy long reads using a variable-order de Bruijn graph.

The recent rise of long read sequencing technologies such as Pacific Biosciences and Oxford Nanopore allows to solve assembly problems for larger and more complex genomes than what allowed short reads technologies. However, these long reads are very noisy, reaching an error rate of around 10-15% for Pacific Biosciences, and up to 30% for Oxford Nanopore. The error correction problem has been tackled by either self-correcting the long reads, or using complementary short reads in a hybrid approach. However, even though sequencing technologies promise to lower the error rate of the long reads below 10%, it is still higher in practice, and correcting such noisy long reads remains an issue.We present HG-CoLoR, a hybrid error correction method that focuses on a seed-and-extend approach based on the alignment of the short reads to the long reads, followed by the traversal of a variable-order de Bruijn graph, built from the short reads. Our experiments show that HG-CoLoR manages to efficiently correct highly noisy long reads that display an error rate as high as 44%. When compared to other state-of-the-art long read error correction methods, our experiments also show that HG-CoLoR provides the best trade-off between runtime and quality of the results, and is the only method able to efficiently scale to eukaryotic genomes.HG-CoLoR is implemented is C++, supported on Linux platforms and freely available at https://github.com/morispi/HG-CoLoR.Supplementary data are available at Bioinformatics online.

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September 22, 2019

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass.

A number of Pyricularia species are known to infect different grass species. In the case of Pyricularia oryzae (syn. Magnaporthe oryzae), distinct populations are known to be adapted to a wide variety of grass hosts, including rice, wheat and many other grasses. The genome sizes of Pyricularia species are typical for filamentous ascomycete fungi [~?40 Mbp for P. oryzae, and ~?45 Mbp for P. grisea]. Genome plasticity, mediated in part by deletions promoted by recombination between repetitive elements [Genome Res 26:1091-1100, 2016, Nat Rev Microbiol 10:417-430,2012] and transposable elements [Annu Rev Phytopathol 55:483-503,2017] contributes to host adaptation. Therefore, comparisons of genome structure of individual species will provide insight into the evolution of host specificity. However, except for the P. oryzae subgroup, little is known about the gene content or genome organization of other Pyricularia species, such as those infecting Pennisetum grasses.Here, we report the genome sequence of P. penniseti strain P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. Phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species indicated that P1609 belongs to a Pyricularia subclade, which is genetically distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires had diverged between P1609 and the P. oryzae strain 70-15, including the known avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements relative to P. oryzae.Our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species and provide new insight into evolution of pathogen genomes during host adaptation.

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September 22, 2019

Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio.

Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4 Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact of these errors and bias on resulting genotype calls from low-coverage sequencing.We used a dataset of 26 pigs sequenced both at 2× with multiplexing and at 30× without multiplexing to show that index hopping and bias towards the reference allele due to alignment had little impact on genotype calls. However, pruning of alternative haplotypes supported by a number of reads below a predefined threshold, which is a default and desired step of some variant callers for removing potential sequencing errors in high-coverage data, introduced an unexpected bias towards the reference allele when applied to low-coverage sequence data. This bias reduced best-guess genotype concordance of low-coverage sequence data by 19.0 absolute percentage points.We propose a simple pipeline to correct the preferential bias towards the reference allele that can occur during variant discovery and we recommend that users of low-coverage sequence data be wary of unexpected biases that may be produced by bioinformatic tools that were designed for high-coverage sequence data.

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