This is a report of the complete genome sequence of the goatpox virus strain Gorgan, which was obtained directly from a commercial live attenuated vaccine (Caprivac, Jordan Bio-Industries Centre). Copyright © 2016 Mathijs et al.
Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under ‘priming’ conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30?kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences – but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event – with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.© 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.
Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. Here, we report the complete genome sequences of three LSDV strains obtained directly from the live attenuated vaccines: Lumpyvax (MSD Animal Health), Herbivac LS (Deltamune) and Lumpy Skin Disease Vaccine (Onderstepoort Biological Products). Copyright © 2016 Mathijs et al.
Targeted sequencing is commonly used in clinical application of NGS technology since it enables generation of sufficient sequencing depth in the targeted genes of interest and thus ensures the best possible downstream analysis. This notwithstanding, the accurate discovery and annotation of disease causing mutations remains a challenging problem even in such favorable context. The difficulty is particularly salient in the case of third generation sequencing technology, such as PacBio. We present MICADo, a de Bruijn graph based method, implemented in python, that makes possible to distinguish between patient specific mutations and other alterations for targeted sequencing of a cohort of patients. MICADo analyses NGS reads for each sample within the context of the data of the whole cohort in order to capture the differences between specificities of the sample with respect to the cohort. MICADo is particularly suitable for sequencing data from highly heterogeneous samples, especially when it involves high rates of non-uniform sequencing errors. It was validated on PacBio sequencing datasets from several cohorts of patients. The comparison with two widely used available tools, namely VarScan and GATK, shows that MICADo is more accurate, especially when true mutations have frequencies close to backgound noise. The source code is available at http://github.com/cbib/MICADo.
The human microbiota and epigenetic processes have both been shown to play a crucial role in health and disease. However, there is extremely scarce information on epigenetic modulation of microbiota members except for a few pathogens. Mainly DNA adenine methylation has been described extensively in modulating the virulence of pathogenic bacteria in particular. It would thus appear likely that such mechanisms are widespread for most bacterial members of the microbiota. This review will present briefly the current knowledge on epigenetic processes in bacteria, give examples of known methylation processes in microbial members of the human microbiota and summarize the knowledge on regulation of host epigenetic processes by the human microbiota.
Protection against enteric infections, also termed colonization resistance, results from mutualistic interactions of the host and its indigenous microbes. The gut microbiota of humans and mice is highly diverse and it is therefore challenging to assign specific properties to its individual members. Here, we have used a collection of murine bacterial strains and a modular design approach to create a minimal bacterial community that, once established in germ-free mice, provided colonization resistance against the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm). Initially, a community of 12 strains, termed Oligo-Mouse-Microbiota (Oligo-MM(12)), representing members of the major bacterial phyla in the murine gut, was selected. This community was stable over consecutive mouse generations and provided colonization resistance against S. Tm infection, albeit not to the degree of a conventional complex microbiota. Comparative (meta)genome analyses identified functions represented in a conventional microbiome but absent from the Oligo-MM(12). By genome-informed design, we created an improved version of the Oligo-MM community harbouring three facultative anaerobic bacteria from the mouse intestinal bacterial collection (miBC) that provided conventional-like colonization resistance. In conclusion, we have established a highly versatile experimental system that showed efficacy in an enteric infection model. Thus, in combination with exhaustive bacterial strain collections and systems-based approaches, genome-guided design can be used to generate insights into microbe-microbe and microbe-host interactions for the investigation of ecological and disease-relevant mechanisms in the intestine.
An ammonia-oxidizing bacterium, strain D1FHS, was enriched into pure culture from a sediment sample retrieved in Jiaozhou Bay, a hyper-eutrophic semi-closed water body hosting the metropolitan area of Qingdao, China. Based on initial 16S rRNA gene sequence analysis, strain D1FHS was classified in the genus Nitrosococcus, family Chromatiaceae, order Chromatiales, class Gammaproteobacteria; the 16S rRNA gene sequence with highest level of identity to that of D1FHS was obtained from Nitrosococcus halophilus Nc4(T). The average nucleotide identity between the genomes of strain D1FHS and N. halophilus strain Nc4 is 89.5%. Known species in the genus Nitrosococcus are obligate aerobic chemolithotrophic ammonia-oxidizing bacteria adapted to and restricted to marine environments. The optimum growth (maximum nitrite production) conditions for D1FHS in a minimal salts medium are: 50 mM ammonium and 700 mM NaCl at pH of 7.5 to 8.0 and at 37°C in dark. Because pertinent conditions for other studied Nitrosococcus spp. are 100-200 mM ammonium and <700 mM NaCl at pH of 7.5 to 8.0 and at 28-32°C, D1FHS is physiologically distinct from other Nitrosococcus spp. in terms of substrate, salt, and thermal tolerance.
Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species. It can be used to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. However, conventional MLST is costly and time consuming, which limits its power for genotyping large numbers of samples. Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in a single assay.
Haplotypes are the units of inheritance in an organism, and many genetic analyses depend on their precise determination. Methods for haplotyping single individuals use the phasing information available in next-generation sequencing reads, by matching overlapping single-nucleotide polymorphisms while penalizing post hoc nucleotide corrections made. Haplotyping diploids is relatively easy, but the complexity of the problem increases drastically for polyploid genomes, which are found in both model organisms and in economically relevant plant and animal species. Although a number of tools are available for haplotyping polyploids, the effects of the genomic makeup and the sequencing strategy followed on the accuracy of these methods have hitherto not been thoroughly evaluated.We developed the simulation pipeline haplosim to evaluate the performance of three haplotype estimation algorithms for polyploids: HapCompass, HapTree and SDhaP, in settings varying in sequencing approach, ploidy levels and genomic diversity, using tetraploid potato as the model. Our results show that sequencing depth is the major determinant of haplotype estimation quality, that 1?kb PacBio circular consensus sequencing reads and Illumina reads with large insert-sizes are competitive and that all methods fail to produce good haplotypes when ploidy levels increase. Comparing the three methods, HapTree produces the most accurate estimates, but also consumes the most resources. There is clearly room for improvement in polyploid haplotyping algorithms.
Next-generation sequencing (NGS), also known as high-throughput sequencing, is changing the field of microbial genomics research. NGS allows for a more comprehensive analysis of the diversity, structure and composition of microbial genes and genomes compared to the traditional automated Sanger capillary sequencing at a lower cost. NGS strategies have expanded the versatility of standard and widely used typing approaches based on nucleotide variation in several hundred DNA sequences and a few gene fragments (MLST, MLVA, rMLST and cgMLST). NGS can now accommodate variation in thousands or millions of sequences from selected amplicons to full genomes (WGS, NGMLST and HiMLST). To extract signals from high-dimensional NGS data and make valid statistical inferences, novel analytic and statistical techniques are needed. In this review, we describe standard and new approaches for microbial sequence typing at gene and genome levels and guidelines for subsequent analysis, including methods and computational frameworks. We also present several applications of these approaches to some disciplines, namely genotyping, phylogenetics and molecular epidemiology. Copyright © 2017 Elsevier B.V. All rights reserved.
Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules. We also highlight major applications for low-frequency mutation detection in science and medicine, describe emerging methodologies and provide our vision for the future of DNA sequencing.
The availability of genomic resources including linkage information for camelids has been very limited. Here, we describe the construction of a set of two radiation hybrid (RH) panels (5000RADand 15000RAD) for the dromedary (Camelus dromedarius) as a permanent genetic resource for camel genome researchers worldwide. For the 5000RADpanel, a total of 245 female camel-hamster radiation hybrid clones were collected, of which 186 were screened with 44 custom designed marker loci distributed throughout camel genome. The overall mean retention frequency (RF) of the final set of 93 hybrids was 47.7%. For the 15000RADpanel, 238 male dromedary-hamster radiation hybrid clones were collected, of which 93 were tested using 44 PCR markers. The final set of 90 clones had a mean RF of 39.9%. This 15000RADpanel is an important high-resolution complement to the main 5000RADpanel and an indispensable tool for resolving complex genomic regions. This valuable genetic resource of dromedary RH panels is expected to be instrumental for constructing a high resolution camel genome map. Construction of the set of RH panels is essential step toward chromosome level reference quality genome assembly that is critical for advancing camelid genomics and the development of custom genomic tools.
Streptomyces leeuwenhoekii strains C34T, C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34T.
A substantial portion of the genomes of most multicellular eukaryotes consists of large arrays of tandemly repeated sequence, collectively called satellite DNA. The processes generating and maintaining different satellite DNA abundances across lineages are important to understand as satellites have been linked to chromosome mis-segregation, disease phenotypes, and reproductive isolation between species. While much theory has been developed to describe satellite evolution, empirical tests of these models have fallen short because of the challenges in assessing satellite repeat regions of the genome. Advances in computational tools and sequencing technologies now enable identification and quantification of satellite sequences genome-wide. Here, we describe some of these tools and how their applications are furthering our knowledge of satellite evolution and function. Copyright © 2018 Elsevier Ltd. All rights reserved.
Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The “antibiotic resistance crisis” is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century. Copyright © 2018 Dumetz et al.
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