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Auto Tag: De novo assembly

July 19, 2019

TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

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July 19, 2019

A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae contains an N(6)-adenine DNA-methyltransferase (ModA) that is subject to phase-variable expression (random ON/OFF switching). Five modA alleles, modA2, modA4, modA5, modA9 and modA10, account for over two-thirds of clinical otitis media isolates surveyed. Here, we use single molecule, real-time (SMRT) methylome analysis to identify the DNA-recognition motifs for all five of these modA alleles. Phase variation of these alleles regulates multiple proteins including vaccine candidates, and key virulence phenotypes such as antibiotic resistance (modA2, modA5, modA10), biofilm formation (modA2) and immunoevasion (modA4). Analyses of a modA2 strain in the chinchilla model of otitis media show a clear selection for ON switching of modA2 in the middle ear. Our results indicate that a biphasic epigenetic switch can control bacterial virulence, immunoevasion and niche adaptation in an animal model system.

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July 19, 2019

Single-Molecule Real-Time Sequencing combined with optical mapping yields completely finished fungal genome.

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio-generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes.Studying whole-genome sequences has become an important aspect of biological research. The advent of next-generation sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBio-generated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome analyses to facilitate functional studies into an organism’s biology. Copyright © 2015 Faino et al.

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July 19, 2019

SMRT Sequencing of long tandem nucleotide repeats in SCA10 reveals unique insight of repeat expansion structure.

A large, non-coding ATTCT repeat expansion causes the neurodegenerative disorder, spinocerebellar ataxia type 10 (SCA10). In a subset of SCA10 patients, interruption motifs are present at the 5′ end of the expansion and strongly correlate with epileptic seizures. Thus, interruption motifs are a predictor of the epileptic phenotype and are hypothesized to act as a phenotypic modifier in SCA10. Yet, the exact internal sequence structure of SCA10 expansions remains unknown due to limitations in current technologies for sequencing across long extended tracts of tandem nucleotide repeats. We used the third generation sequencing technology, Single Molecule Real Time (SMRT) sequencing, to obtain full-length contiguous expansion sequences, ranging from 2.5 to 4.4 kb in length, from three SCA10 patients with different clinical presentations. We obtained sequence spanning the entire length of the expansion and identified the structure of known and novel interruption motifs within the SCA10 expansion. The exact interruption patterns in expanded SCA10 alleles will allow us to further investigate the potential contributions of these interrupting sequences to the pathogenic modification leading to the epilepsy phenotype in SCA10. Our results also demonstrate that SMRT sequencing is useful for deciphering long tandem repeats that pose as “gaps” in the human genome sequence.

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July 19, 2019

HLA Class-II associated HIV polymorphisms predict escape from CD4+ T Cell responses.

Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-? ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p<0.0001). However, regardless of the group, the magnitude of responses to AE was lower as compared to NAE (p<0.0001). CD4+ T cell responses in patients with acute HIV infection (AHI) demonstrated poor immunogenicity towards AE as compared to NAE encoded by their transmitted founder virus. Longitudinal data in AHI off antiretroviral therapy demonstrated sequence changes that were biologically confirmed to represent CD4+ escape mutations. These data demonstrate an innovative application of HLA-associated polymorphisms to identify biologically relevant CD4+ epitopes and suggests CD4+ T cells are active participants in driving HIV evolution.

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July 19, 2019

Whole genome?

The reference human genome assembly is remarkable in its completeness and usefulness in research. However, the range of allelic variation in the human population is not well described by a haploid assembly with a profusion of alternative loci. Homozygous regions and the use of multiple sequencing technologies increasingly have roles in strategies for identifying regulatory and trait-associated variation.

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July 19, 2019

Selections that isolate recombinant mitochondrial genomes in animals.

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germ line and revealed somatic recombination. When the recombination partner was a diverged D. melanogaster genome or a genome from a different species such as D. yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome.

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July 19, 2019

SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome.

Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base.We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly.SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.

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July 19, 2019

Birth of a new gene on the Y chromosome of Drosophila melanogaster.

Contrary to the pattern seen in mammalian sex chromosomes, where most Y-linked genes have X-linked homologs, the Drosophila X and Y chromosomes appear to be unrelated. Most of the Y-linked genes have autosomal paralogs, so autosome-to-Y transposition must be the main source of Drosophila Y-linked genes. Here we show how these genes were acquired. We found a previously unidentified gene (flagrante delicto Y, FDY) that originated from a recent duplication of the autosomal gene vig2 to the Y chromosome of Drosophila melanogaster. Four contiguous genes were duplicated along with vig2, but they became pseudogenes through the accumulation of deletions and transposable element insertions, whereas FDY remained functional, acquired testis-specific expression, and now accounts for ~20% of the vig2-like mRNA in testis. FDY is absent in the closest relatives of D. melanogaster, and DNA sequence divergence indicates that the duplication to the Y chromosome occurred ~2 million years ago. Thus, FDY provides a snapshot of the early stages of the establishment of a Y-linked gene and demonstrates how the Drosophila Y has been accumulating autosomal genes.

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July 19, 2019

Biosynthesis of the novel macrolide antibiotic anthracimycin.

We report the identification of the biosynthetic gene cluster for the unusual antibiotic anthracimycin (atc) from the marine derived producer strain Streptomyces sp. T676 isolated off St. John’s Island, Singapore. The 53?253 bps atc locus includes a trans-acyltransferase (trans-AT) polyketide synthase (PKS), and heterologous expression in Streptomyces coelicolor resulted in anthracimycin production. Analysis of the atc cluster revealed that anthracimycin is likely generated by four PKS gene products AtcC-AtcF without involvement of post-PKS tailoring enzymes, and a biosynthetic pathway is proposed. The availability of the atc cluster provides a basis for investigating the biosynthesis of anthracimycin and its subsequent bioengineering to provide novel analogues with improved pharmacological properties.

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July 19, 2019

Mind the gap; seven reasons to close fragmented genome assemblies.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 19, 2019

An improved genome reference for the African cichlid, Metriaclima zebra.

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

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July 19, 2019

Heterogeneous composition of key metabolic gene clusters in a vent mussel symbiont population.

Chemosynthetic symbiosis is one of the successful systems for adapting to a wide range of habitats including extreme environments, and the metabolic capabilities of symbionts enable host organisms to expand their habitat ranges. However, our understanding of the adaptive strategies that enable symbiotic organisms to expand their habitats is still fragmentary. Here, we report that a single-ribotype endosymbiont population in an individual of the host vent mussel, Bathymodiolus septemdierum has heterogeneous genomes with regard to the composition of key metabolic gene clusters for hydrogen oxidation and nitrate reduction. The host individual harbours heterogeneous symbiont subpopulations that either possess or lack the gene clusters encoding hydrogenase or nitrate reductase. The proportions of the different symbiont subpopulations in a host appeared to vary with the environment or with the host’s development. Furthermore, the symbiont subpopulations were distributed in patches to form a mosaic pattern in the gill. Genomic heterogeneity in an endosymbiont population may enable differential utilization of diverse substrates and confer metabolic flexibility. Our findings open a new chapter in our understanding of how symbiotic organisms alter their metabolic capabilities and expand their range of habitats.

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July 19, 2019

Genome-directed lead discovery: biosynthesis, structure elucidation, and biological evaluation of two families of polyene macrolactams against Trypanosoma brucei.

Marine natural products are an important source of lead compounds against many pathogenic targets. Herein, we report the discovery of lobosamides A-C from a marine actinobacterium, Micromonospora sp., representing three new members of a small but growing family of bacterially produced polyene macrolactams. The lobosamides display growth inhibitory activity against the protozoan parasite Trypanosoma brucei (lobosamide A IC50 = 0.8 µM), the causative agent of human African trypanosomiasis (HAT). The biosynthetic gene cluster of the lobosamides was sequenced and suggests a conserved cluster organization among the 26-membered macrolactams. While determination of the relative and absolute configurations of many members of this family is lacking, the absolute configurations of the lobosamides were deduced using a combination of chemical modification, detailed spectroscopic analysis, and bioinformatics. We implemented a “molecules-to-genes-to-molecules” approach to determine the prevalence of similar clusters in other bacteria, which led to the discovery of two additional macrolactams, mirilactams A and B from Actinosynnema mirum. These additional analogs have allowed us to identify specific structure-activity relationships that contribute to the antitrypanosomal activity of this class. This approach illustrates the power of combining chemical analysis and genomics in the discovery and characterization of natural products as new lead compounds for neglected disease targets.

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July 19, 2019

Recurrent methicillin-resistant Staphylococcus aureus cutaneous abscesses and selection of reduced chlorhexidine susceptibility during chlorhexidine use.

We describe the selection of reduced chlorhexidine susceptibility during chlorhexidine use in a patient with two episodes of cutaneous USA300 methicillin-resistant Staphylococcus aureus abscess. The second clinical isolate harbors a novel plasmid that encodes the QacA efflux pump. Greater use of chlorhexidine for disease prevention warrants surveillance for resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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