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July 7, 2019

Strategies for complete plastid genome sequencing.

Plastid sequencing is an essential tool in the study of plant evolution. This high-copy organelle is one of the most technically accessible regions of the genome, and its sequence conservation makes it a valuable region for comparative genome evolution, phylogenetic analysis and population studies. Here, we discuss recent innovations and approaches for de novo plastid assembly that harness genomic tools. We focus on technical developments including low-cost sequence library preparation approaches for genome skimming, enrichment via hybrid baits and methylation-sensitive capture, sequence platforms with higher read outputs and longer read lengths, and automated tools for assembly. These developments allow for a much more streamlined assembly than via conventional short-range PCR. Although newer methods make complete plastid sequencing possible for any land plant or green alga, there are still challenges for producing finished plastomes particularly from herbarium material or from structurally divergent plastids such as those of parasitic plants.© 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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July 7, 2019

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2016;9999: 1-13. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

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July 7, 2019

The genetic basis of anoxygenic photosynthetic arsenite oxidation.

‘Photoarsenotrophy’, the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H2 S, H2 and NO2-. Photoarsenotrophy was recently identified from Paoha Island’s (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019

NOVOPlasty: de novo assembly of organelle genomes from whole genome data.

The evolution in next-generation sequencing (NGS) technology has led to the development of many different assembly algorithms, but few of them focus on assembling the organelle genomes. These genomes are used in phylogenetic studies, food identification and are the most deposited eukaryotic genomes in GenBank. Producing organelle genome assembly from whole genome sequencing (WGS) data would be the most accurate and least laborious approach, but a tool specifically designed for this task is lacking. We developed a seed-and-extend algorithm that assembles organelle genomes from whole genome sequencing (WGS) data, starting from a related or distant single seed sequence. The algorithm has been tested on several new (Gonioctena intermedia and Avicennia marina) and public (Arabidopsis thaliana and Oryza sativa) whole genome Illumina data sets where it outperforms known assemblers in assembly accuracy and coverage. In our benchmark, NOVOPlasty assembled all tested circular genomes in less than 30 min with a maximum memory requirement of 16 GB and an accuracy over 99.99%. In conclusion, NOVOPlasty is the sole de novo assembler that provides a fast and straightforward extraction of the extranuclear genomes from WGS data in one circular high quality contig. The software is open source and can be downloaded at https://github.com/ndierckx/NOVOPlasty.

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July 7, 2019

Draft genome assembly and annotation of Glycyrrhiza uralensis, a medicinal legume.

Chinese liquorice/licorice (Glycyrrhiza uralensis) is a leguminous plant species whose roots and rhizomes have been widely used as a herbal medicine and natural sweetener. Whole-genome sequencing is essential for gene discovery studies and molecular breeding in liquorice. Here, we report a draft assembly of the approximately 379-Mb whole-genome sequence of strain 308-19 of G. uralensis; this assembly contains 34 445 predicted protein-coding genes. Comparative analyses suggested well-conserved genomic components and collinearity of gene loci (synteny) between the genome of liquorice and those of other legumes such as Medicago and chickpea. We observed that three genes involved in isoflavonoid biosynthesis, namely, 2-hydroxyisoflavanone synthase (CYP93C), 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase/isoflavone 4′-O-methyltransferase (HI4OMT) and isoflavone-7-O-methyltransferase (7-IOMT) formed a cluster on the scaffold of the liquorice genome and showed conserved microsynteny with Medicago and chickpea. Based on the liquorice genome annotation, we predicted genes in the P450 and UDP-dependent glycosyltransferase (UGT) superfamilies, some of which are involved in triterpenoid saponin biosynthesis, and characterised their gene expression with the reference genome sequence. The genome sequencing and its annotations provide an essential resource for liquorice improvement through molecular breeding and the discovery of useful genes for engineering bioactive components through synthetic biology approaches.© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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July 7, 2019

LoRTE: Detecting transposon-induced genomic variants using low coverage PacBio long read sequences.

Population genomic analysis of transposable elements has greatly benefited from recent advances of sequencing technologies. However, the short size of the reads and the propensity of transposable elements to nest in highly repeated regions of genomes limits the efficiency of bioinformatic tools when Illumina or 454 technologies are used. Fortunately, long read sequencing technologies generating read length that may span the entire length of full transposons are now available. However, existing TE population genomic softwares were not designed to handle long reads and the development of new dedicated tools is needed.LoRTE is the first tool able to use PacBio long read sequences to identify transposon deletions and insertions between a reference genome and genomes of different strains or populations. Tested against simulated and genuine Drosophila melanogaster PacBio datasets, LoRTE appears to be a reliable and broadly applicable tool to study the dynamic and evolutionary impact of transposable elements using low coverage, long read sequences.LoRTE is an efficient and accurate tool to identify structural genomic variants caused by TE insertion or deletion. LoRTE is available for download at http://www.egce.cnrs-gif.fr/?p=6422.

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July 7, 2019

Turkey meat as source of CC9/CC398 methicillin-resistant Staphylococcus aureus in humans?

Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) of clonal complex (CC) 398 were first reported to cause severe infections in humans in 2005 [1]. Direct animal exposure is considered the most effective means of MRSA CC398 transmission from livestock to humans. However, about 20%–38% of MRSA CC398 cases among humans cannot be epidemiologically linked to direct livestock contact, indicating other transmission pathways [2]. As recently reported in this journal by Larsen et al [3], poultry meat may serve as a vehicle for livestock-to-human transmission. Here, we present similar findings for CC9/CC398 MRSA (displaying spa type t899 and related), which shares unique characteristics with human clinical isolates in Denmark as shown by Larsen et al [3], strongly supporting the implication of poultry, especially turkey meat, as the source of CC9/CC398.

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July 7, 2019

Modified low-salt CTAB extraction of high-quality DNA from contaminant-rich tissues.

The increasing use of high-throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant-rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in-house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open-source MoLSC protocol provides a cost-effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant-rich samples. © 2016 Her Majesty the Queen in Right of Canada. Molecular Ecology Resources © 2016 John Wiley & Sons Ltd.

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July 7, 2019

Methods for genome-wide methylome profiling of Campylobacter jejuni.

Methylation has a profound role in the regulation of numerous biological processes in bacteria including virulence. The study of methylation in bacteria has greatly advanced thanks to next-generation sequencing technologies. These technologies have expedited the process of uncovering unique features of many bacterial methylomes such as characterizing previously uncharacterized methyltransferases, cataloging genome-wide DNA methylations in bacteria, identifying the frequency of methylation at particular genomic loci, and revealing regulatory roles of methylation in the biology of various bacterial species. For instance, methylation has been cited as a potential source for the pathogenicity differences observed in C. jejuni strains with syntenic genomes as seen in recent publications. Here, we describe the methodology for the use of Pacific Biosciences’ single molecule real-time (SMRT) sequencing for detecting methylation patterns in C. jejuni and bioinformatics tools to profile its methylome.

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July 7, 2019

Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff.

Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction.© 2016 Blackwell Verlag GmbH.

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July 7, 2019

The comparative landscape of duplications in Heliconius melpomene and Heliconius cydno.

Gene duplications can facilitate adaptation and may lead to interpopulation divergence, causing reproductive isolation. We used whole-genome resequencing data from 34 butterflies to detect duplications in two Heliconius species, Heliconius cydno and Heliconius melpomene. Taking advantage of three distinctive signals of duplication in short-read sequencing data, we identified 744 duplicated loci in H. cydno and H. melpomene and evaluated the accuracy of our approach using single-molecule sequencing. We have found that duplications overlap genes significantly less than expected at random in H. melpomene, consistent with the action of background selection against duplicates in functional regions of the genome. Duplicate loci that are highly differentiated between H. melpomene and H. cydno map to four different chromosomes. Four duplications were identified with a strong signal of divergent selection, including an odorant binding protein and another in close proximity with a known wing colour pattern locus that differs between the two species. Heredity advance online publication, 7 December 2016; doi:10.1038/hdy.2016.107.

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July 7, 2019

Draft genome sequence of Mentha longifolia (L.) and development of resources for mint cultivar improvement.

The genus Mentha encompasses mint species cultivated for their essential oils, which are formulated into a vast array of consumer products. Desirable oil characteristics and resistance to the fungal disease Verticillium wilt are top priorities for the mint industry. However, cultivated mints have complex polyploid genomes and are sterile. Breeding efforts, therefore, require the development of genomic resources for fertile mint species. Here, we present draft de novo genome and plastome assemblies for a wilt-resistant South African accession of Mentha longifolia (L.) Huds., a diploid species ancestral to cultivated peppermint and spearmint. The 353 Mb genome contains 35 597 predicted protein-coding genes, including 292 disease resistance gene homologs, and nine genes determining essential oil characteristics. A genetic linkage map ordered 1397 genome scaffolds on 12 pseudochromosomes. More than two million simple sequence repeats were identified, which will facilitate molecular marker development. The M. longifolia genome is a valuable resource for both metabolic engineering and molecular breeding. This is exemplified by employing the genome sequence to clone and functionally characterize the promoters in a peppermint cultivar, and demonstrating the utility of a glandular trichome-specific promoter to increase expression of a biosynthetic gene, thereby modulating essential oil composition. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Comparative genomics of extrachromosomal elements in Bacillus thuringiensis subsp. israelensis.

Bacillus thuringiensis subsp. israelensis is one of the most important microorganisms used against mosquitoes. It was intensively studied following its discovery and became a model bacterium of the B. thuringiensis species. Those studies focused on toxin genes, aggregation-associated conjugation, linear genome phages, etc. Recent announcements of genomic sequences of different strains have not been explicitly related to the biological properties studied. We report data on plasmid content analysis of four strains using ultra-high-throughput sequencing. The strains were commercial product isolates, with their putative ancestor and type B. thuringiensis subsp. israelensis strain sequenced earlier. The assembled contigs corresponding to published and novel data were assigned to plasmids described earlier in B. thuringiensis subsp. israelensis and other B. thuringiensis strains. A new 360 kb plasmid was identified, encoding multiple transporters, also found in most of the earlier sequenced strains. Our genomic data show the presence of two toxin-coding plasmids of 128 and 100 kb instead of the reported 225 kb plasmid, a co-integrate of the former two. In two of the sequenced strains, only a 100 kb plasmid was present. Some heterogeneity exists in the small plasmid content and structure between strains. These data support the perception of active plasmid exchange among B. thuringiensis subsp. israelensis strains in nature. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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July 7, 2019

Detection, isolation and characterization of Fusobacterium gastrosuis sp. nov. colonizing the stomach of pigs.

Nine strains of a novel Fusobacterium sp. were isolated from the stomach of 6-8 months old and adult pigs. The isolates were obligately anaerobic, although they endured 2h exposure to air. Phylogenetic analysis based on 16S rRNA and gyrase B genes demonstrated that the isolates showed high sequence similarity with Fusobacterium mortiferum, Fusobacterium ulcerans, Fusobacterium varium, Fusobacterium russii and Fusobacterium necrogenes, but formed a distinct lineage in the genus Fusobacterium. Comparative analysis of the genome of the type strain of this novel Fusobacterium sp. confirmed that it is different from other recognized Fusobacterium spp. DNA-DNA hybridization, fingerprinting and genomic %GC determination further supported the conclusion that the isolates belong to a new, distinct species. The isolates were also distinguishable from these and other Fusobacterium spp. by phenotypical characterization. The strains produced indole and exhibited proline arylamidase and glutamic acid decarboxylase activity. They did not hydrolyse esculin, did not exhibit pyroglutamic acid arylamidase, valine arylamidase, a-galactosidase, ß-galactosidase, ß-galactosidase-6-phosphate or a-glucosidase activity nor produced acid from cellobiose, glucose, lactose, mannitol, mannose, maltose, raffinose, saccharose, salicin or trehalose. The major fatty acids were C16:0 and C18:1?9c. The name Fusobacterium gastrosuis sp. nov. is proposed for the novel isolates with the type strain CDW1(T) (=DSM 101753(T)=LMG 29236(T)). We also demonstrated that Clostridium rectum and mortiferum Fusobacterium represent the same species, with nomenclatural priority for the latter. Copyright © 2016 Elsevier GmbH. All rights reserved.

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July 7, 2019

Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

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