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July 7, 2019

Whole genome DNA sequence analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks. Using WGS can delimit contamination sources for foodborne illnesses across multiple outbreaks and reveal otherwise undetected DNA sequence differences essential to the tracing of bacterial pathogens as they emerge.

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July 7, 2019

Extensive sequencing of seven human genomes to characterize benchmark reference materials.

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

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July 7, 2019

Accelerated dysbiosis of gut microbiota during aggravation of DSS-induced colitis by a butyrate-producing bacterium.

Butyrate-producing bacteria (BPB) are potential probiotic candidates for inflammatory bowel diseases as they are often depleted in the diseased gut microbiota. However, here we found that augmentation of a human-derived butyrate-producing strain, Anaerostipes hadrus BPB5, significantly aggravated colitis in dextran sulphate sodium (DSS)-treated mice while exerted no detrimental effect in healthy mice. We explored how the interaction between BPB5 and gut microbiota may contribute to this differential impact on the hosts. Butyrate production and severity of colitis were assessed in both healthy and DSS-treated mice, and gut microbiota structural changes were analysed using high-throughput sequencing. BPB5-inoculated healthy mice showed no signs of colitis, but increased butyrate content in the gut. In DSS-treated mice, BPB5 augmentation did not increase butyrate content, but induced significantly more severe disease activity index and much higher mortality. BPB5 didn’t induce significant changes of gut microbiota in healthy hosts, but expedited the structural shifts 3 days earlier toward the disease phase in BPB5-augmented than DSS-treated animals. The differential response of gut microbiota in healthy and DSS-treated mice to the same potentially beneficial bacterium with drastically different health consequences suggest that animals with dysbiotic gut microbiota should also be employed for the safety assessment of probiotic candidates.

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July 7, 2019

Complete genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, two phylogenetically distinct probiotics.

Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

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July 7, 2019

Efficient cardinality estimation for k-mers in large DNA sequencing data sets

We present an open implementation of the HyperLogLog cardinality estimation sketch for counting fixed-length substrings of DNA strings (k-mers). The HyperLogLog sketch implementation is in C++ with a Python interface, and is distributed as part of the khmer software package. khmer is freely available from urlhttps://github.com/dib-lab/khmer under a BSD License. The features presented here are included in version 1.4 and later.

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July 7, 2019

Atypical Salmonella enterica serovars in murine and human infection models: Is it time to reassess our approach to the study of salmonellosis?

Nontyphoidal Salmonella species are globally disseminated pathogens and the predominant cause of gastroenteritis. The pathogenesis of salmonellosis has been extensively studied using in vivo murine models and cell lines typically challenged with Salmonella Typhimurium. Although serovars Enteritidis and Typhimurium are responsible for the most of human infections reported to the CDC, several other serovars also contribute to clinical cases of salmonellosis. Despite their epidemiological importance, little is known about their infection phenotypes. Here, we report the virulence characteristics and genomes of 10 atypical S. enterica serovars linked to multistate foodborne outbreaks in the United States. We show that the murine RAW 264.7 macrophage model of infection is unsuitable for inferring human relevant differences in nontyphoidal Salmonella infections whereas differentiated human THP-1 macrophages allowed these isolates to be further characterised in a more relevant, human context.

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July 7, 2019

Characterization of the first cultured representative of Verrucomicrobia subdivision 5 indicates the proposal of a novel phylum.

The recently isolated strain L21-Fru-AB(T) represents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-AB(T) represents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate. On the basis of the analyses of phenotypic, genomic and environmental data, it is proposed that strain L21-Fru-AB(T) and related bacteria are specifically adapted to the utilization of sulfated glycopolymers produced in microbial mats or biofilms.

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July 7, 2019

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture.The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90.Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

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July 7, 2019

Ploidy influences the functional attributes of de novo lager yeast hybrids.

The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae × Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 °P) and very high gravity wort (25 °P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (ATF1, ATF2¸ EHT1, EEB1, and BAT1) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the S. cerevisiae- and S. eubayanus-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.

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July 7, 2019

Amino acid sequence repertoire of the bacterial proteome and the occurrence of untranslatable sequences.

Bioinformatic analysis of Escherichia coli proteomes revealed that all possible amino acid triplet sequences occur at their expected frequencies, with four exceptions. Two of the four underrepresented sequences (URSs) were shown to interfere with translation in vivo and in vitro. Enlarging the URS by a single amino acid resulted in increased translational inhibition. Single-molecule methods revealed stalling of translation at the entrance of the peptide exit tunnel of the ribosome, adjacent to ribosomal nucleotides A2062 and U2585. Interaction with these same ribosomal residues is involved in regulation of translation by longer, naturally occurring protein sequences. The E. coli exit tunnel has evidently evolved to minimize interaction with the exit tunnel and maximize the sequence diversity of the proteome, although allowing some interactions for regulatory purposes. Bioinformatic analysis of the human proteome revealed no underrepresented triplet sequences, possibly reflecting an absence of regulation by interaction with the exit tunnel.

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July 7, 2019

Complete genome sequence of Vibrio alginolyticus ATCC 33787(T) isolated from seawater with three native megaplasmids.

Vibrio alginolyticus, an opportunistic pathogen, is commonly associated with vibriosis in fish and shellfish and can also cause superficial and ear infections in humans. V. alginolyticus ATCC 33787(T) was originally isolated from seawater and has been used as one of the type strains for exploring the virulence factors of marine bacteria and for developing vaccine against vibriosis. Here we sequenced and assembled the whole genome of this strain, and identified three megaplasmids and three Type VI secretion systems, thus providing useful information for the study of virulence factors and for the development of vaccine for Vibrio. Copyright © 2016. Published by Elsevier B.V.

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July 7, 2019

Evaluation of an optimal epidemiologic typing scheme for Legionella pneumophila with whole genome sequence data using validation guidelines.

Sequence-based typing (SBT), analogous to multi-locus sequence typing (MLST), is the current gold-standard typing method for investigation of legionellosis outbreaks caused by Legionella pneumophila However, as common sequence types (STs) cause many infections, some investigations remain unresolved. Here, various whole genome sequencing (WGS)-based methods were evaluated according to published guidelines, including: i) single nucleotide polymorphism (SNP)-based; ii) extended multi-locus sequence typing (MLST) using different numbers of genes; iii) gene presence/absence, and iv) kmer-based. L. pneumophila serogroup 1 isolates (n=106) from the standard “typing panel”, previously used by the European Society for Clinical Microbiology Study Group on Legionella Infections (ESGLI) were tested together with another 229 isolates.Over 98% isolates were considered typable using the mapping- and kmer-based methods. Percentages of isolates with complete extended MLST profiles ranged from 99.1% (50-gene) to 86.8% (1455-gene) whilst only 41.5% produced a full profile with the gene presence/absence scheme. Replicates demonstrated that all methods offer 100% reproducibility. Indices of discrimination range from 0.972 (ribosomal MLST) to 0.999 (SNP-based), and all values are higher than that achieved with SBT (0.940). Epidemiological concordance is generally inversely related to discriminatory power. We propose that an extended MLST scheme with ~50 genes provides optimal epidemiological concordance whilst substantially improving the discrimination offered by SBT, and can be used as part of a hierarchical typing scheme that should maintain backwards compatibility and increase discrimination where necessary. This analysis will be useful for the ESGLI to design a scheme that has the potential to become the new gold standard typing method for L. pneumophila. Copyright © 2016 David et al.

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July 7, 2019

Suppressed recombination and unique candidate genes in the divergent haplotype encoding Fhb1, a major Fusarium head blight resistance locus in wheat.

Fine mapping and sequencing revealed 28 genes in the non-recombining haplotype containing Fhb1 . Of these, only a GDSL lipase gene shows a pathogen-dependent expression pattern. Fhb1 is a prominent Fusarium head blight resistance locus of wheat, which has been successfully introgressed in adapted breeding material, where it confers a significant increase in overall resistance to the causal pathogen Fusarium graminearum and the fungal virulence factor and mycotoxin deoxynivalenol. The Fhb1 region has been resolved for the susceptible wheat reference genotype Chinese Spring, yet the causal gene itself has not been identified in resistant cultivars. Here, we report the establishment of a 1 Mb contig embracing Fhb1 in the donor line CM-82036. Sequencing revealed that the region of Fhb1 deviates from the Chinese Spring reference in DNA size and gene content, which explains the repressed recombination at the locus in the performed fine mapping. Differences in genes expression between near-isogenic lines segregating for Fhb1 challenged with F. graminearum or treated with mock were investigated in a time-course experiment by RNA sequencing. Several candidate genes were identified, including a pathogen-responsive GDSL lipase absent in susceptible lines. The sequence of the Fhb1 region, the resulting list of candidate genes, and near-diagnostic KASP markers for Fhb1 constitute a valuable resource for breeding and further studies aiming to identify the gene(s) responsible for F. graminearum and deoxynivalenol resistance.

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July 7, 2019

A detailed analysis of the recombination landscape of the button mushroom Agaricus bisporus var. bisporus.

The button mushroom (Agaricus bisporus) is one of the world’s most cultivated mushroom species, but in spite of its economic importance generation of new cultivars by outbreeding is exceptional. Previous genetic analyses of the white bisporus variety, including all cultivars and most wild isolates revealed that crossing over frequencies are low, which might explain the lack of introducing novel traits into existing cultivars. By generating two high quality whole genome sequence assemblies (one de novo and the other by improving the existing reference genome) of the first commercial white hybrid Horst U1, a detailed study of the crossover (CO) landscape was initiated. Using a set of 626 SNPs in a haploid offspring of 139 single spore isolates and whole genome sequencing on a limited number of homo- and heterokaryotic single spore isolates, we precisely mapped all COs showing that they are almost exclusively restricted to regions of about 100kb at the chromosome ends. Most basidia of A. bisporus var. bisporus produce two spores and pair preferentially via non-sister nuclei. Combined with the COs restricted to the chromosome ends, these spores retain most of the heterozygosity of the parent thus explaining how present-day white cultivars are genetically so close to the first hybrid marketed in 1980. To our knowledge this is the first example of an organism which displays such specific CO landscape. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

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July 7, 2019

deBWT: parallel construction of Burrows-Wheeler Transform for large collection of genomes with de Bruijn-branch encoding.

With the development of high-throughput sequencing, the number of assembled genomes continues to rise. It is critical to well organize and index many assembled genomes to promote future genomics studies. Burrows-Wheeler Transform (BWT) is an important data structure of genome indexing, which has many fundamental applications; however, it is still non-trivial to construct BWT for large collection of genomes, especially for highly similar or repetitive genomes. Moreover, the state-of-the-art approaches cannot well support scalable parallel computing owing to their incremental nature, which is a bottleneck to use modern computers to accelerate BWT construction.We propose de Bruijn branch-based BWT constructor (deBWT), a novel parallel BWT construction approach. DeBWT innovatively represents and organizes the suffixes of input sequence with a novel data structure, de Bruijn branch encoding. This data structure takes the advantage of de Bruijn graph to facilitate the comparison between the suffixes with long common prefix, which breaks the bottleneck of the BWT construction of repetitive genomic sequences. Meanwhile, deBWT also uses the structure of de Bruijn graph for reducing unnecessary comparisons between suffixes. The benchmarking suggests that, deBWT is efficient and scalable to construct BWT for large dataset by parallel computing. It is well-suited to index many genomes, such as a collection of individual human genomes, with multiple-core servers or clusters.deBWT is implemented in C language, the source code is available at https://github.com/hitbc/deBWT or https://github.com/DixianZhu/deBWTContact: ydwang@hit.edu.cnSupplementary data are available at Bioinformatics online.© The Author 2016. Published by Oxford University Press.

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