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Asset Tag: Whole Genome Sequencing,

July 19, 2019

De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii.

Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species.We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm.The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.

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July 19, 2019

Genome sequence of the progenitor of the wheat D genome Aegilops tauschii.

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat (Triticum aestivum, genomes AABBDD) and an important genetic resource for wheat. The large size and highly repetitive nature of the Ae. tauschii genome has until now precluded the development of a reference-quality genome sequence. Here we use an array of advanced technologies, including ordered-clone genome sequencing, whole-genome shotgun sequencing, and BioNano optical genome mapping, to generate a reference-quality genome sequence for Ae. tauschii ssp. strangulata accession AL8/78, which is closely related to the wheat D genome. We show that compared to other sequenced plant genomes, including a much larger conifer genome, the Ae. tauschii genome contains unprecedented amounts of very similar repeated sequences. Our genome comparisons reveal that the Ae. tauschii genome has a greater number of dispersed duplicated genes than other sequenced genomes and its chromosomes have been structurally evolving an order of magnitude faster than those of other grass genomes. The decay of colinearity with other grass genomes correlates with recombination rates along chromosomes. We propose that the vast amounts of very similar repeated sequences cause frequent errors in recombination and lead to gene duplications and structural chromosome changes that drive fast genome evolution.

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July 19, 2019

The diversity, structure, and function of heritable adaptive immunity sequences in the Aedes aegypti genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions. Copyright © 2017 Elsevier Ltd. All rights reserved.

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July 19, 2019

The first near-complete assembly of the hexaploid bread wheat genome, Triticum aestivum.

Common bread wheat, Triticum aestivum, has one of the most complex genomes known to science, with 6 copies of each chromosome, enormous numbers of near-identical sequences scattered throughout, and an overall haploid size of more than 15 billion bases. Multiple past attempts to assemble the genome have produced assemblies that were well short of the estimated genome size. Here we report the first near-complete assembly of T. aestivum, using deep sequencing coverage from a combination of short Illumina reads and very long Pacific Biosciences reads. The final assembly contains 15 344 693 583 bases and has a weighted average (N50) contig size of 232 659 bases. This represents by far the most complete and contiguous assembly of the wheat genome to date, providing a strong foundation for future genetic studies of this important food crop. We also report how we used the recently published genome of Aegilops tauschii, the diploid ancestor of the wheat D genome, to identify 4 179 762 575 bp of T. aestivum that correspond to its D genome components.© The Author 2017. Published by Oxford University Press.

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July 19, 2019

Long-read genome sequence assembly provides insight into ongoing retroviral invasion of the koala germline.

The koala retrovirus (KoRV) is implicated in several diseases affecting the koala (Phascolarctos cinereus). KoRV provirus can be present in the genome of koalas as an endogenous retrovirus (present in all cells via germline integration) or as exogenous retrovirus responsible for somatic integrations of proviral KoRV (present in a limited number of cells). This ongoing invasion of the koala germline by KoRV provides a powerful opportunity to assess the viral strategies used by KoRV in an individual. Analysis of a high-quality genome sequence of a single koala revealed 133 KoRV integration sites. Most integrations contain full-length, endogenous provirus; KoRV-A subtype. The second most frequent integrations contain an endogenous recombinant element (recKoRV) in which most of the KoRV protein-coding region has been replaced with an ancient, endogenous retroelement. A third set of integrations, with very low sequence coverage, may represent somatic cell integrations of KoRV-A, KoRV-B and two recently designated additional subgroups, KoRV-D and KoRV-E. KoRV-D and KoRV-E are missing several genes required for viral processing, suggesting they have been transmitted as defective viruses. Our results represent the first comprehensive analyses of KoRV integration and variation in a single animal and provide further insights into the process of retroviral-host species interactions.

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July 19, 2019

The Aegilops tauschii genome reveals multiple impacts of transposons.

Wheat is an important global crop with an extremely large and complex genome that contains more transposable elements (TEs) than any other known crop species. Here, we generated a chromosome-scale, high-quality reference genome of Aegilops tauschii, the donor of the wheat D genome, in which 92.5% sequences have been anchored to chromosomes. Using this assembly, we accurately characterized genic loci, gene expression, pseudogenes, methylation, recombination ratios, microRNAs and especially TEs on chromosomes. In addition to the discovery of a wave of very recent gene duplications, we detected that TEs occurred in about half of the genes, and found that such genes are expressed at lower levels than those without TEs, presumably because of their elevated methylation levels. We mapped all wheat molecular markers and constructed a high-resolution integrated genetic map corresponding to genome sequences, thereby placing previously detected agronomically important genes/quantitative trait loci (QTLs) on the Ae. tauschii genome for the first time.

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July 19, 2019

Single-molecule sequencing reveals the chromosome-scale genomic architecture of the nematode model organism Pristionchus pacificus.

The nematode Pristionchus pacificus is an established model for integrative evolutionary biology and comparative studies with Caenorhabditis elegans. While an existing genome draft facilitated the identification of several genes controlling various developmental processes, its high degree of fragmentation complicated virtually all genomic analyses. Here, we present a de novo genome assembly from single-molecule, long-read sequencing data consisting of 135 P. pacificus contigs. When combined with a genetic linkage map, 99% of the assembly could be ordered and oriented into six chromosomes. This allowed us to robustly characterize chromosomal patterns of gene density, repeat content, nucleotide diversity, linkage disequilibrium, and macrosynteny in P. pacificus. Despite widespread conservation of synteny between P. pacificus and C. elegans, we identified one major translocation from an autosome to the sex chromosome in the lineage leading to C. elegans. This highlights the potential of the chromosome-scale assembly for future genomic studies of P. pacificus. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Genome and methylome variation in Helicobacter pylori with a cag pathogenicity island during early stages of human infection.

Helicobacter pylori is remarkable for its genetic variation. Yet little isknown about its genetic changes during early stages of human infection, as the bacteria adapt to their new environment. We analyzed genome and methylome variations in a fully virulent strain of H pylori strain during experimental infection.We performed a randomized Phase 1 and 2, observer-blind, placebo-controlled, study of 12 healthy, H pylori-negative adults in Germany from October 2008 through March 2010. The volunteers were given a prophylactic vaccine candidate (n=7) or placebo (n=5) and then challenged with H pylori strain BCM-300. Biopsy samples were collected and H pylori were isolated. Genomes of the challenge strain and 12 re-isolates, obtained 12 weeks after (or in 1 case, 62 weeks after) infection were sequenced by single-molecule, real-time technology, which, in parallel, permitted determination of genome-wide methylation patterns for all strains. Functional effects of genetic changes observed in H pylori strains during human infection were assessed by measuring release of interleukin 8 from AGS cells (to detect cag PAI function), neutral red uptake (to detect vacuolating cytotoxin activity), and adhesion assays.The observed mutation rate was in agreement with rates previously determined from patients with chronic H pylori infections, without evidence of a mutation burst. A loss; of cag PAI function was observed in 3 re-isolates. In addition, 3 re-isolates from the vaccine; group acquired mutations in the vacuolating cytotoxin gene vacA, resulting in loss of; vacuolization activity from gastric epithelial cells. We observed inter-strain variation in; methylomes due to phase variation in genes encoding methyltransferases.We analyzed adaptation of a fully virulent strain of H pylori to 12 differentvolunteers to obtain a robust estimate of the frequency of genetic and epigenetic changes inthe absence of inter-strain recombination. Our findings indicate that the large amount of; genetic variation in H pylori poses a challenge to vaccine development. ClinicalTrials.gov no: NCT00736476. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

The evolution of dark matter in the mitogenome of seed beetles.

Animal mitogenomes are generally thought of as being economic and optimized for rapid replication and transcription. We use long-read sequencing technology to assemble the remarkable mitogenomes of four species of seed beetles. These are the largest circular mitogenomes ever assembled in insects, ranging from 24,496 to 26,613?bp in total length, and are exceptional in that some 40% consists of non-coding DNA. The size expansion is due to two very long intergenic spacers (LIGSs), rich in tandem repeats. The two LIGSs are present in all species but vary greatly in length (114-10,408?bp), show very low sequence similarity, divergent tandem repeat motifs, a very high AT content and concerted length evolution. The LIGSs have been retained for at least some 45 my but must have undergone repeated reductions and expansions, despite strong purifying selection on protein coding mtDNA genes. The LIGSs are located in two intergenic sites where a few recent studies of insects have also reported shorter LIGSs (>200?bp). These sites may represent spaces that tolerate neutral repeat array expansions or, alternatively, the LIGSs may function to allow a more economic translational machinery. Mitochondrial respiration in adult seed beetles is based almost exclusively on fatty acids, which reduces the need for building complex I of the oxidative phosphorylation pathway (NADH dehydrogenase). One possibility is thus that the LIGSs may allow depressed transcription of NAD genes. RNA sequencing showed that LIGSs are partly transcribed and transcriptional profiling suggested that all seven mtDNA NAD genes indeed show low levels of transcription and co-regulation of transcription across sexes and tissues.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 19, 2019

Comparative genomic analyses of Clavibacter michiganensis subsp. insidiosus and pathogenicity on Medicago truncatula.

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,930 genes, with 401 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.

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July 19, 2019

Centromere evolution and CpG methylation during vertebrate speciation.

Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here, we perform de novo long-read genome assembly of three inbred medaka strains that are derived from geographically isolated subpopulations and undergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain three chromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource of twenty-two centromeric regions of length 20-345kbp. Centromeres are positionally conserved among the three strains and even between four pairs of chromosomes that were duplicated by the teleost-specific whole-genome duplication 320-350 million years ago. The centromeres do not all evolve at a similar pace; rather, centromeric monomers in non-acrocentric chromosomes evolve significantly faster than those in acrocentric chromosomes. Using methylation sensitive SMRT reads, we uncover centromeres are mostly hypermethylated but have hypomethylated sub-regions that acquire unique sequence compositions independently. These findings reveal the potential of non-acrocentric centromere evolution to contribute to speciation.

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July 19, 2019

Methylation in Mycobacterium tuberculosis is lineage specific with associated mutations present globally.

DNA methylation is an epigenetic modification of the genome involved in regulating crucial cellular processes, including transcription and chromosome stability. Advances in PacBio sequencing technologies can be used to robustly reveal methylation sites. The methylome of the Mycobacterium tuberculosis complex is poorly understood but may be involved in virulence, hypoxic survival and the emergence of drug resistance. In the most extensive study to date, we characterise the methylome across the 4 major lineages of M. tuberculosis and 2 lineages of M. africanum, the leading causes of tuberculosis disease in humans. We reveal lineage-specific methylated motifs and strain-specific mutations that are abundant globally and likely to explain loss of function in the respective methyltransferases. Our work provides a set of sixteen new complete reference genomes for the Mycobacterium tuberculosis complex, including complete lineage 5 genomes. Insights into lineage-specific methylomes will further elucidate underlying biological mechanisms and other important phenotypes of the epi-genome.

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July 19, 2019

Genomic analysis of hospital plumbing reveals diverse reservoir of bacterial plasmids conferring carbapenem resistance.

The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization ofblaKPC/blaNDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections withblaKPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission ofLeclerciasp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings.IMPORTANCECarbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and mortality associated with these resistant Gram-negative bacteria. Horizontal plasmid transfer spreads the resistance mechanism to new bacteria, and understanding the plasmid ecology of the hospital environment can assist in the design of control strategies to prevent nosocomial infections. A 5-year genomic and epidemiological survey was undertaken to study the CPOs in the patient-accessible environment, as well as in the plumbing system removed from the patient. This comprehensive survey revealed a vast, unappreciated reservoir of CPOs in wastewater, which was in contrast to the low positivity rate in both the patient population and the patient-accessible environment. While there were few patient-environmental isolate associations, there were plasmid backbones common to both populations. These results are relevant to all hospitals for which CPO colonization may not yet be defined through extensive surveillance.

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July 19, 2019

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American Photinus pyralis and Japanese Aquatica lateralis. To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.© 2018, Fallon et al.

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July 19, 2019

Linking secondary metabolites to gene clusters through genome sequencing of six diverse Aspergillus species.

The fungal genus ofAspergillusis highly interesting, containing everything from industrial cell factories, model organisms, and human pathogens. In particular, this group has a prolific production of bioactive secondary metabolites (SMs). In this work, four diverseAspergillusspecies (A. campestris,A. novofumigatus,A. ochraceoroseus, andA. steynii) have been whole-genome PacBio sequenced to provide genetic references in threeAspergillussections.A. taichungensisandA. candidusalso were sequenced for SM elucidation. ThirteenAspergillusgenomes were analyzed with comparative genomics to determine phylogeny and genetic diversity, showing that each presented genome contains 15-27% genes not found in other sequenced Aspergilli. In particular,A. novofumigatuswas compared with the pathogenic speciesA. fumigatusThis suggests thatA. novofumigatuscan produce most of the same allergens, virulence, and pathogenicity factors asA. fumigatus, suggesting thatA. novofumigatuscould be as pathogenic asA. fumigatusFurthermore, SMs were linked to gene clusters based on biological and chemical knowledge and analysis, genome sequences, and predictive algorithms. We thus identify putative SM clusters for aflatoxin, chlorflavonin, and ochrindol inA. ochraceoroseus,A. campestris, andA. steynii, respectively, and novofumigatonin,ent-cycloechinulin, andepi-aszonalenins inA. novofumigatusOur study delivers six fungal genomes, showing the large diversity found in theAspergillusgenus; highlights the potential for discovery of beneficial or harmful SMs; and supports reports ofA. novofumigatuspathogenicity. It also shows how biological, biochemical, and genomic information can be combined to identify genes involved in the biosynthesis of specific SMs.

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