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July 19, 2019

Genetic variation and the de novo assembly of human genomes.

The discovery of genetic variation and the assembly of genome sequences are both inextricably linked to advances in DNA-sequencing technology. Short-read massively parallel sequencing has revolutionized our ability to discover genetic variation but is insufficient to generate high-quality genome assemblies or resolve most structural variation. Full resolution of variation is only guaranteed by complete de novo assembly of a genome. Here, we review approaches to genome assembly, the nature of gaps or missing sequences, and biases in the assembly process. We describe the challenges of generating a complete de novo genome assembly using current technologies and the impact that being able to perfectly sequence the genome would have on understanding human disease and evolution. Finally, we summarize recent technological advances that improve both contiguity and accuracy and emphasize the importance of complete de novo assembly as opposed to read mapping as the primary means to understanding the full range of human genetic variation.

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July 19, 2019

Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine.

The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus.We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination.In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy.These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

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July 19, 2019

Variable genetic architectures produce virtually identical molecules in bacterial symbionts of fungus-growing ants.

Small molecules produced by Actinobacteria have played a prominent role in both drug discovery and organic chemistry. As part of a larger study of the actinobacterial symbionts of fungus-growing ants, we discovered a small family of three previously unreported piperazic acid-containing cyclic depsipeptides, gerumycins A-C. The gerumycins are slightly smaller versions of dentigerumycin, a cyclic depsipeptide that selectively inhibits a common fungal pathogen, Escovopsis. We had previously identified this molecule from a Pseudonocardia associated with Apterostigma dentigerum, and now we report the molecule from an associate of the more highly derived ant Trachymyrmex cornetzi. The three previously unidentified compounds, gerumycins A-C, have essentially identical structures and were produced by two different symbiotic Pseudonocardia spp. from ants in the genus Apterostigma found in both Panama and Costa Rica. To understand the similarities and differences in the biosynthetic pathways that produced these closely related molecules, the genomes of the three producing Pseudonocardia were sequenced and the biosynthetic gene clusters identified. This analysis revealed that dramatically different biosynthetic architectures, including genomic islands, a plasmid, and the use of spatially separated genetic loci, can lead to molecules with virtually identical core structures. A plausible evolutionary model that unifies these disparate architectures is presented.

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July 19, 2019

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems.

Bifidobacterial genome analysis has provided insights as to how these gut commensals adapt to and persist in the human GIT, while also revealing genetic diversity among members of a given bifidobacterial (sub)species. Bifidobacteria are notoriously recalcitrant to genetic modification, which prevents exploration of their genomic functions, including those that convey (human) health benefits.PacBio SMRT sequencing was used to determine the whole genome seqeunces of two B. longum subsp. longum strains. The B. longum pan-genome was computed using PGAP v1.2 and the core B. longum phylogenetic tree was constructed using a maximum-likelihood based approach in PhyML v3.0. M.blmNCII was cloned in E. coli and an internal fragment if arfBarfB was cloned into pORI19 for insertion mutagenesis.In this study we present the complete genome sequences of two Bifidobacterium longum subsp. longum strains. Comparative analysis with thirty one publicly available B. longum genomes allowed the definition of the B. longum core and dispensable genomes. This analysis also highlighted differences in particular metabolic abilities between members of the B. longum subspecies infantis, longum and suis. Furthermore, phylogenetic analysis of the B. longum core genome indicated the existence of a novel subspecies. Methylome data, coupled to the analysis of restriction-modification systems, allowed us to substantially increase the genetic accessibility of B. longum subsp. longum NCIMB 8809 to a level that was shown to permit site-directed mutagenesis.Comparative genomic analysis of thirty three B. longum representatives revealed a closed pan-genome for this bifidobacterial species. Phylogenetic analysis of the B. longum core genome also provides evidence for a novel fifth B. longum subspecies. Finally, we improved genetic accessibility for the strain B. longum subsp. longum NCIMB 8809, which allowed the generation of a mutant of this strain.

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July 19, 2019

The pineapple genome and the evolution of CAM photosynthesis.

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ? duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.

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July 19, 2019

Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships.

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33-35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.

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July 19, 2019

A supergene determines highly divergent male reproductive morphs in the ruff.

Three strikingly different alternative male mating morphs (aggressive ‘independents’, semicooperative ‘satellites’ and female-mimic ‘faeders’) coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

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July 19, 2019

Detection and screening of chromosomal rearrangements in uterine leiomyomas by long-distance inverse PCR.

Genome instability is a hallmark of many tumors and recently, next-generation sequencing methods have enabled analyses of tumor genomes at an unprecedented level. Studying rearrangement-prone chromosomal regions (putative “breakpoint hotspots”) in detail, however, necessitates molecular assays that can detect de novo DNA fusions arising from these hotspots. Here we demonstrate the utility of a long-distance inverse PCR-based method for the detection and screening of de novo DNA rearrangements in uterine leiomyomas, one of the most common types of human neoplasm. This assay allows in principle any genomic region suspected of instability to be queried for DNA rearrangements originating there. No prior knowledge of the identity of the fusion partner chromosome is needed. We used this method to screen uterine leiomyomas for rearrangements at genomic locations known to be rearrangement-prone in this tumor type: upstream HMGA2 and within RAD51B. We identified a novel DNA rearrangement upstream of HMGA2 that had gone undetected in an earlier whole-genome sequencing study. In more than 30 additional uterine leiomyoma samples, not analyzed by whole-genome sequencing previously, no rearrangements were observed within the 1,107 bp and 1,996 bp assayed in the RAD51B and HMGA2 rearrangement hotspots. Our findings show that long-distance inverse PCR is a robust, sensitive, and cost-effective method for the detection and screening of DNA rearrangements from solid tumors that should be useful for many diagnostic applications. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

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July 19, 2019

Lifespan of restriction-modification systems critically affects avoidance of their recognition sites in host genomes.

Avoidance of palindromic recognition sites of Type II restriction-modification (R-M) systems was shown for many R-M systems in dozens of prokaryotic genomes. However the phenomenon has not been investigated systematically for all presently available genomes and annotated R-M systems. We have studied all known recognition sites in thousands of prokaryotic genomes and found factors that influence their avoidance.Only Type II R-M systems consisting of independently acting endonuclease and methyltransferase (called ‘orthodox’ here) cause avoidance of their sites, both palindromic and asymmetric, in corresponding prokaryotic genomes; the avoidance takes place for?~?50 % of 1774 studied cases. It is known that prokaryotes can acquire and lose R-M systems. Thus it is possible to talk about the lifespan of an R-M system in a genome. We have shown that the recognition site avoidance correlates with the lifespan of R-M systems. The sites of orthodox R-M systems that are encoded in host genomes for a long time are avoided more often (up to 100 % in certain cohorts) than the sites of recently acquired ones. We also found cases of site avoidance in absence of the corresponding R-M systems in the genome. An analysis of closely related bacteria shows that such avoidance can be a trace of lost R-M systems. Sites of Type I, II?/G, IIM, III, and IV R-M systems are not avoided in vast majority of cases.The avoidance of orthodox Type II R-M system recognition sites in prokaryotic genomes is a widespread phenomenon. Presence of an R-M system without an underrepresentation of its site may indicate that the R-M system was acquired recently. At the same time, a significant underrepresentation of a site may be a sign of presence of the corresponding R-M system in this organism or in its ancestors for a long time. The drastic difference between site avoidance for orthodox Type II R-M systems and R-M systems of other types can be explained by a higher rate of specificity changes or a less self-toxicity of the latter.

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July 19, 2019

AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences.

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.

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July 19, 2019

The epigenomic landscape of prokaryotes.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

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July 19, 2019

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle.

Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or “pinkeye” in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK.

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July 19, 2019

Genome analysis of the fruiting body forming myxobacterium Chondromyces crocatus reveals high potential for natural product biosynthesis.

Here we report the first complete genome sequence of the type strain of the myxobacterial genus Chondromyces – Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters is in line with the capability of Cm c5 to produce an arsenal of anti-bacterial, anti-fungal and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-body forming type-strain (Cm c5 = DSM 14714) to an accustomed laboratory strain which has lost this ability (Cm c5 fr-) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication, and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3 Mbp prokaryotic genome. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

The complete genome sequence of the murine pathobiont Helicobacter typhlonius.

Immuno-compromised mice infected with Helicobacter typhlonius are used to model microbially inducted inflammatory bowel disease (IBD). The specific mechanism through which H. typhlonius induces and promotes IBD is not fully understood. Access to the genome sequence is essential to examine emergent properties of this organism, such as its pathogenicity. To this end, we present the complete genome sequence of H. typhlonius MIT 97-6810, obtained through single-molecule real-time sequencing.The genome was assembled into a single circularized contig measuring 1.92 Mbp with an average GC content of 38.8%. In total 2,117 protein-encoding genes and 43 RNA genes were identified. Numerous pathogenic features were found, including a putative pathogenicity island (PAIs) containing components of type IV secretion system, virulence-associated proteins and cag PAI protein. We compared the genome of H. typhlonius to those of the murine pathobiont H. hepaticus and human pathobiont H. pylori. H. typhlonius resembles H. hepaticus most with 1,594 (75.3%) of its genes being orthologous to genes in H. hepaticus. Determination of the global methylation state revealed eight distinct recognition motifs for adenine and cytosine methylation. H. typhlonius shares four of its recognition motifs with H. pylori.The complete genome sequence of H. typhlonius MIT 97-6810 enabled us to identify many pathogenic features suggesting that H. typhlonius can act as a pathogen. Follow-up studies are necessary to evaluate the true nature of its pathogenic capabilities. We found many methylated sites and a plethora of restriction-modification systems. The genome, together with the methylome, will provide an essential resource for future studies investigating gene regulation, host interaction and pathogenicity of H. typhlonius. In turn, this work can contribute to unraveling the role of Helicobacter in enteric disease.

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July 19, 2019

Genome Update. Let the consumer beware: Streptomyces genome sequence quality.

A genome sequence assembly represents a model of a genome. This article explores some tools and methods for assessing the quality of an assembly, using publicly available data for Streptomyces species as the example. There is great variability in quality of assemblies deposited in GenBank. Only in a small minority of these assemblies are the raw data available, enabling full appraisal of the assembly quality. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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