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July 7, 2019  |  

Pseudomonas syringae CC1557: a highly virulent strain with an unusually small type III effector repertoire that includes a novel effector.

Both type III effector proteins and nonribosomal peptide toxins play important roles for Pseudomonas syringae pathogenicity in host plants, but whether and how these pathways interact to promote infection remains unclear. Genomic evidence from one clade of P. syringae suggests a tradeoff between the total number of type III effector proteins and presence of syringomycin, syringopeptin, and syringolin A toxins. Here, we report the complete genome sequence from P. syringae CC1557, which contains the lowest number of known type III effectors to date and has also acquired genes similar to sequences encoding syringomycin pathways from other strains. We demonstrate that this strain is pathogenic on Nicotiana benthamiana and that both the type III secretion system and a new type III effector, hopBJ1, contribute to pathogenicity. We further demonstrate that activity of HopBJ1 is dependent on residues structurally similar to the catalytic site of Escherichia coli CNF1 toxin. Taken together, our results provide additional support for a negative correlation between type III effector repertoires and the potential to produce syringomycin-like toxins while also highlighting how genomic synteny and bioinformatics can be used to identify and characterize novel virulence proteins.


July 7, 2019  |  

A hybrid approach for the automated finishing of bacterial genomes.

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.


July 7, 2019  |  

A gapless genome sequence of the fungus Botrytis cinerea.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, we here report a gapless, near-finished genome sequence for B. cinerea strain B05.10. The assembly comprises 18 chromosomes and was confirmed by an optical map and a genetic map based on ~75 000 SNP markers. All chromosomes contain fully assembled centromeric regions, and 10 chromosomes have telomeres on both ends. The genetic map consisted of 4153 cM and comparison of genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that confer resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that UTRs of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress. This article is protected by copyright. All rights reserved. © 2016 BSPP AND JOHN WILEY & SONS LTD.


July 7, 2019  |  

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.


July 7, 2019  |  

Plasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution.

Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.


July 7, 2019  |  

De novo hybrid assembly of the rubber tree genome reveals evidence of paleotetraploidy in Hevea species.

Para rubber tree (Hevea brasiliensis) is an important economic species as it is the sole commercial producer of high-quality natural rubber. Here, we report a de novo hybrid assembly of BPM24 accession, which exhibits resistance to major fungal pathogens in Southeast Asia. Deep-coverage 454/Illumina short-read and Pacific Biosciences (PacBio) long-read sequence data were acquired to generate a preliminary draft, which was subsequently scaffolded using a long-range “Chicago” technique to obtain a final assembly of 1.26?Gb (N50?=?96.8?kb). The assembled genome contains 69.2% repetitive sequences and has a GC content of 34.31%. Using a high-density SNP-based genetic map, we were able to anchor 28.9% of the genome assembly (363?Mb) associated with over two thirds of the predicted protein-coding genes into rubber tree’s 18 linkage groups. These genetically anchored sequences allowed comparative analyses of the intragenomic homeologous synteny, providing the first concrete evidence to demonstrate the presence of paleotetraploidy in Hevea species. Additionally, the degree of macrosynteny conservation observed between rubber tree and cassava strongly supports the hypothesis that the paleotetraploidization event took place prior to the divergence of the Hevea and Manihot species.


July 7, 2019  |  

Fallacy of the unique genome: sequence diversity within single Helicobacter pylori strains.

Many bacterial genomes are highly variable but nonetheless are typically published as a single assembled genome. Experiments tracking bacterial genome evolution have not looked at the variation present at a given point in time. Here, we analyzed the mouse-passaged Helicobacter pylori strain SS1 and its parent PMSS1 to assess intra- and intergenomic variability. Using high sequence coverage depth and experimental validation, we detected extensive genome plasticity within these H. pylori isolates, including movement of the transposable element IS607, large and small inversions, multiple single nucleotide polymorphisms, and variation in cagA copy number. The cagA gene was found as 1 to 4 tandem copies located off the cag island in both SS1 and PMSS1; this copy number variation correlated with protein expression. To gain insight into the changes that occurred during mouse adaptation, we also compared SS1 and PMSS1 and observed 46 differences that were distinct from the within-genome variation. The most substantial was an insertion in cagY, which encodes a protein required for a type IV secretion system function. We detected modifications in genes coding for two proteins known to affect mouse colonization, the HpaA neuraminyllactose-binding protein and the FutB a-1,3 lipopolysaccharide (LPS) fucosyltransferase, as well as genes predicted to modulate diverse properties. In sum, our work suggests that data from consensus genome assemblies from single colonies may be misleading by failing to represent the variability present. Furthermore, we show that high-depth genomic sequencing data of a population can be analyzed to gain insight into the normal variation within bacterial strains.IMPORTANCE Although it is well known that many bacterial genomes are highly variable, it is nonetheless traditional to refer to, analyze, and publish “the genome” of a bacterial strain. Variability is usually reduced (“only sequence from a single colony”), ignored (“just publish the consensus”), or placed in the “too-hard” basket (“analysis of raw read data is more robust”). Now that whole-genome sequences are regularly used to assess virulence and track outbreaks, a better understanding of the baseline genomic variation present within single strains is needed. Here, we describe the variability seen in typical working stocks and colonies of pathogen Helicobacter pylori model strains SS1 and PMSS1 as revealed by use of high-coverage mate pair next-generation sequencing (NGS) and confirmed by traditional laboratory techniques. This work demonstrates that reliance on a consensus assembly as “the genome” of a bacterial strain may be misleading. Copyright © 2017 Draper et al.


July 7, 2019  |  

Improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data.

Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes, however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated PacBio long-read data of the genomes of three relatives of the model plant Arabidopsis thaliana and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity reached chromosome-arm-levels. We rigorously assessed the quality of contigs and scaffolds using Illumina mate-pair libraries and genetic map information. This showed that PacBio assemblies have high sequence accuracy but can contain several misassemblies, which join unlinked regions of the genome. Most, but not all of these mis-joints were removed during the integration of the optical mapping and chromosome conformation capture data. Even though none of the centromeres was fully assembled, the scaffolds revealed large parts of some centromeric regions, even including some of the heterochromatic regions, which are not present in gold standard reference sequences. Published by Cold Spring Harbor Laboratory Press.


July 7, 2019  |  

Assessment of insertion sequence mobilization as an adaptive response to oxidative stress in Acinetobacter baumannii using IS-Seq.

Insertion sequence (IS) elements are found throughout bacterial genomes and contribute to genome variation by interrupting genes or altering gene expression. Few of the more than thirty IS elements described in Acinetobacter baumannii have been characterized for transposition activity or expression effects. A targeted sequencing method, IS-seq, was developed to efficiently map the locations of new insertion events in A. baumannii genomes and was used to identify novel IS sites following growth in the presence of hydrogen peroxide, which causes oxidative stress. Serial subculture in the presence of sub-inhibitory concentrations of hydrogen peroxide led to rapid selection of cells carrying an ISAba1 element upstream of the catalase/peroxidase gene katG Several additional sites for the elements ISAba1, ISAba13, ISAba25, ISAba26, and ISAba125 were found at low abundance after serial subculture, indicating that each element is active and contributes to genetic variation that may be subject to selection. Following hydrogen peroxide exposure, rapid changes in gene expression were observed in genes related to iron homeostasis. The IS insertions adjacent to katG resulted in more than 20-fold overexpression of the gene and increased hydrogen peroxide tolerance.Importance Insertion sequences (IS) are contribute to genomic and phenotypic variation in many bacterial species, but little is known about how transposition rates vary among elements or how selective pressure influences this process. A new method, termed “IS-seq” for identifying new insertion locations that arise under experimental growth conditions in the genome was developed and tested with cells grown in the presence of hydrogen peroxide, which causes oxidative stress. Gene expression changes in response to hydrogen peroxide exposure are similar to those observed in other species and include genes that control free iron concentrations. New IS insertions adjacent to a gene encoding a catalase enzyme confirm that IS elements can rapidly contribute to adaptive variation in the presence of selection. Copyright © 2017 Wright et al.


July 7, 2019  |  

Complete genome sequence of Edwardsiella hoshinae ATCC 35051.

Edwardsiella hoshinae is a Gram-negative facultative anaerobe that has primarily been isolated from avians and reptiles. We report here the complete and annotated genome sequence of an isolate from a monitor lizard (Varanus sp.), which contains a chromosome of 3,811,650 bp and no plasmids. Copyright © 2017 Reichley et al.


July 7, 2019  |  

A pipeline for local assembly of minisatellite alleles from single-molecule sequencing data.

The advent of Next Generation Sequencing (NGS) has led to the generation of enormous volumes of short read sequence data, cheaply and in reasonable time scales. Nevertheless, the quality of genome assemblies generated using NGS technologies has been greatly affected, compared to those generated using Sanger DNA sequencing. This is largely due to the inability of short read sequence data to scaffold repetitive structures, creating gaps, inversions and rearrangements and resulting in assemblies that are, at best, draft forms. Third generation single-molecule sequencing (SMS) technologies (e.g. Pacific Biosciences Single Molecule Real Time (SMRT) system) address this challenge by generating sequences with increased read lengths, offering the prospect to better recover these complex repetitive structures, concomitantly improving assembly quality.Here, we evaluate the ability of SMS data (specifically human genome Pacific Biosciences SMRT data) to recover poorly represented repetitive sequences (specifically, GC-rich human minisatellites). To do this we designed a pipeline for the collection, processing and local assembly of single-molecule sequence data to form accurate contiguous local reconstructions. Our results show the recovery of an allele of the non-coding minisatellite MS1 (located on chromosome 1 at 1p33-35) at greater than 97% identity to reference (GRCh38) from the unprocessed sequence data of a haploid complete hydatidiform mole (CHM1) cell line. Furthermore, our assembly revealed an allele of over 500 repeat units; much larger than the reference (GRCh38), but consistent in structure with naturally occurring alleles that are segregating in human populations. This local assembly’s reconstruction was validated with the release of the whole genome assemblies GCA_001297185.1 and GCA_000772585.3, where this allele occurs. Additionally, application of this pipeline to coding minisatellites in the PRDM9 and ZNF93 genes enabled recovery of high identity allele structures for these sequence regions whose length was confirmed by PCR from cell line genomic DNA. The internal repeat structure of the PRDM9 allele recovered was consistent with common human-specific alleles.Code available at https://github.com/ndliberial/smrt_pipeline CONTACT: dno2@le.ac.uk. © The Author 2016. Published by Oxford University Press.


July 7, 2019  |  

Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses.

Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries.To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012-2015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS).Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9-36 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade.C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.


July 7, 2019  |  

Single-Molecule sequencing of the Drosophila serrata genome.

Long-read sequencing technology promises to greatly enhance de novo assembly of genomes for nonmodel species. Although the error rates of long reads have been a stumbling block, sequencing at high coverage permits the self-correction of many errors. Here, we sequence and de novo assemble the genome of Drosophila serrata, a species from the montium subgroup that has been well-studied for latitudinal clines, sexual selection, and gene expression, but which lacks a reference genome. Using 11 PacBio single-molecule real-time (SMRT cells), we generated 12 Gbp of raw sequence data comprising ~65 × whole-genome coverage. Read lengths averaged 8940 bp (NRead50 12,200) with the longest read at 53 kbp. We self-corrected reads using the PBDagCon algorithm and assembled the genome using the MHAP algorithm within the PBcR assembler. Total genome length was 198 Mbp with an N50 just under 1 Mbp. Contigs displayed a high degree of chromosome arm-level conservation with the D. melanogaster genome and many could be sensibly placed on the D. serrata physical map. We also provide an initial annotation for this genome using in silico gene predictions that were supported by RNA-seq data. Copyright © 2017 Allen et al.


July 7, 2019  |  

An improved genome assembly uncovers prolific tandem repeats in Atlantic cod.

The first Atlantic cod (Gadus morhua) genome assembly published in 2011 was one of the early genome assemblies exclusively based on high-throughput 454 pyrosequencing. Since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. The development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies.By combining data from Illumina, 454 and the longer PacBio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the Atlantic cod genome assembly. The sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. Compared to other vertebrates, the assembly contains an unusual high density of tandem repeats (TRs). Indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these TRs. We show that 21% of the TRs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual.The inclusion of PacBio reads combined with the use of multiple assembly programs drastically improved the Atlantic cod genome assembly by successfully resolving long TRs. The high frequency of heterozygous TRs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in Atlantic cod populations, which is likely of evolutionary importance.


July 7, 2019  |  

Complete genome sequence and comparative genomics of the probiotic yeast Saccharomyces boulardii.

The probiotic yeast, Saccharomyces boulardii (Sb) is known to be effective against many gastrointestinal disorders and antibiotic-associated diarrhea. To understand molecular basis of probiotic-properties ascribed to Sb we determined the complete genomes of two strains of Sb i.e. Biocodex and unique28 and the draft genomes for three other Sb strains that are marketed as probiotics in India. We compared these genomes with 145 strains of S. cerevisiae (Sc) to understand genome-level similarities and differences between these yeasts. A distinctive feature of Sb from other Sc is absence of Ty elements Ty1, Ty3, Ty4 and associated LTR. However, we could identify complete Ty2 and Ty5 elements in Sb. The genes for hexose transporters HXT11 and HXT9, and asparagine-utilization are absent in all Sb strains. We find differences in repeat periods and copy numbers of repeats in flocculin genes that are likely related to the differential adhesion of Sb as compared to Sc. Core-proteome based taxonomy places Sb strains along with wine strains of Sc. We find the introgression of five genes from Z. bailii into the chromosome IV of Sb and wine strains of Sc. Intriguingly, genes involved in conferring known probiotic properties to Sb are conserved in most Sc strains.


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