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July 19, 2019

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms.

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ~3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

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July 19, 2019

Separate F-type plasmids have shaped the evolution of the H30 subclone of Escherichia coli sequence type 131.

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

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July 19, 2019

Rapid sequencing of complete env genes from primary HIV-1 samples

The ability to study rapidly evolving viral populations has been constrained by the read length of next-generation sequencing approaches and the sampling depth of single-genome amplification methods. Here, we develop and characterize a method using Pacific Biosciences Single Molecule, Real-Time (SMRT) sequencing technology to sequence multiple, intact full-length human immunodeficiency virus-1 env genes amplified from viral RNA populations circulating in blood, and provide computational tools for analyzing and visualizing these data.

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July 19, 2019

The mitochondrial genome map of Nelumbo nucifera reveals ancient evolutionary features.

Nelumbo nucifera is an evolutionary relic from the Late Cretaceous period. Sequencing the N. nucifera mitochondrial genome is important for elucidating the evolutionary characteristics of basal eudicots. Here, the N. nucifera mitochondrial genome was sequenced using single molecule real-time sequencing technology (SMRT), and the mitochondrial genome map was constructed after de novo assembly and annotation. The results showed that the 524,797-bp N. nucifera mitochondrial genome has a total of 63 genes, including 40 protein-coding genes, three rRNA genes and 20 tRNA genes. Fifteen collinear gene clusters were conserved across different plant species. Approximately 700 RNA editing sites in the protein-coding genes were identified. Positively selected genes were identified with selection pressure analysis. Nineteen chloroplast-derived fragments were identified, and seven tRNAs were derived from the chloroplast. These results suggest that the N. nucifera mitochondrial genome retains evolutionarily conserved characteristics, including ancient gene content and gene clusters, high levels of RNA editing, and low levels of chloroplast-derived fragment insertions. As the first publicly available basal eudicot mitochondrial genome, the N. nucifera mitochondrial genome facilitates further analysis of the characteristics of basal eudicots and provides clues of the evolutionary trajectory from basal angiosperms to advanced eudicots.

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July 19, 2019

High-quality assembly of an individual of Yoruban descent

De novo assembly of human genomes is now a tractable effort due in part to advances in sequencing and mapping technologies. We use PacBio single-molecule, real-time (SMRT) sequencing and BioNano genomic maps to construct the first de novo assembly of NA19240, a Yoruban individual from Africa. This chromosome-scaffolded assembly of 3.08 Gb with a contig N50 of 7.25 Mb and a scaffold N50 of 78.6 Mb represents one of the most contiguous high-quality human genomes. We utilize a BAC library derived from NA19240 DNA and novel haplotype-resolving sequencing technologies and algorithms to characterize regions of complex genomic architecture that are normally lost due to compression to a linear haploid assembly. Our results demonstrate that multiple technologies are still necessary for complete genomic representation, particularly in regions of highly identical segmental duplications. Additionally, we show that diploid assembly has utility in improving the quality of de novo human genome assemblies.

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July 19, 2019

Emergence of a Homo sapiens-specific gene family and chromosome 16p11.2 CNV susceptibility.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates, including more recently the genomes of archaic hominins. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for approximately 1% of cases of autism and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. Here we reconstruct the evolutionary history of the locus and identify bolA family member 2 (BOLA2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95-kilobase-pair segment containing BOLA2 duplicated across the critical region approximately 282 thousand years ago (ka), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carried one or more copies of the duplication, which nearly fixed early in the human lineage-a pattern unlikely to have arisen so rapidly in the absence of selection (P?

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July 19, 2019

Long read sequencing technology to solve complex genomic regions assembly in plants

Background: Numerous completed or on-going whole genome sequencing projects have highlighted the fact that obtaining a high quality genome sequence is necessary to address comparative genomics questions such as structural variations among genotypes and gain or loss of specific function. Despite the spectacular progress that has been made in sequencing technologies, obtaining accurate and reliable data is still a challenge, both at the whole genome scale and when targeting specific genomic regions. These problems are even more noticeable for complex plant genomes. Most plant genomes are known to be particularly challenging due to their size, high density of repetitive elements and various levels of ploidy. To overcome these problems, we have developed a strategy to reduce genome complexity by using the large insert BAC libraries combined with next generation sequencing technologies. Results: We compared two different technologies (Roche-454 and Pacific Biosciences PacBio RS II) to sequence pools of BAC clones in order to obtain the best quality sequence. We targeted nine BAC clones from different species (maize, wheat, strawberry, barley, sugarcane and sunflower) known to be complex in terms of sequence assembly. We sequenced the pools of the nine BAC clones with both technologies. We compared assembly results and highlighted differences due to the sequencing technologies used. Conclusions: We demonstrated that the long reads obtained with the PacBio RS II technology serve to obtain a better and more reliable assembly, notably by preventing errors due to duplicated or repetitive sequences in the same region.

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July 19, 2019

Variation and evolution in the glutamine-rich repeat region of Drosophila argonaute-2.

RNA interference pathways mediate biological processes through Argonaute-family proteins, which bind small RNAs as guides to silence complementary target nucleic acids . In insects and crustaceans Argonaute-2 silences viral nucleic acids, and therefore acts as a primary effector of innate antiviral immunity. Although the function of the major Argonaute-2 domains, which are conserved across most Argonaute-family proteins, are known, many invertebrate Argonaute-2 homologs contain a glutamine-rich repeat (GRR) region of unknown function at the N-terminus . Here we combine long-read amplicon sequencing of Drosophila Genetic Reference Panel (DGRP) lines with publicly available sequence data from many insect species to show that this region evolves extremely rapidly and is hyper-variable within species. We identify distinct GRR haplotype groups in Drosophila melanogaster, and suggest that one of these haplotype groups has recently risen to high frequency in a North American population. Finally, we use published data from genome-wide association studies of viral resistance in D. melanogaster to test whether GRR haplotypes are associated with survival after virus challenge. We find a marginally significant association with survival after challenge with Drosophila C Virus in the DGRP, but we were unable to replicate this finding using lines from the Drosophila Synthetic Population Resource panel. Copyright © 2016 Palmer and Obbard.

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July 19, 2019

Living apart together: crosstalk between the core and supernumerary genomes in a fungal plant pathogen.

Eukaryotes display remarkable genome plasticity, which can include supernumerary chromosomes that differ markedly from the core chromosomes. Despite the widespread occurrence of supernumerary chromosomes in fungi, their origin, relation to the core genome and the reason for their divergent characteristics are still largely unknown. The complexity of genome assembly due to the presence of repetitive DNA partially accounts for this.Here we use single-molecule real-time (SMRT) sequencing to assemble the genome of a prominent fungal wheat pathogen, Fusarium poae, including at least one supernumerary chromosome. The core genome contains limited transposable elements (TEs) and no gene duplications, while the supernumerary genome holds up to 25 % TEs and multiple gene duplications. The core genome shows all hallmarks of repeat-induced point mutation (RIP), a defense mechanism against TEs, specific for fungi. The absence of RIP on the supernumerary genome accounts for the differences between the two (sub)genomes, and results in a functional crosstalk between them. The supernumerary genome is a reservoir for TEs that migrate to the core genome, and even large blocks of supernumerary sequence (>200 kb) have recently translocated to the core. Vice versa, the supernumerary genome acts as a refuge for genes that are duplicated from the core genome.For the first time, a mechanism was determined that explains the differences that exist between the core and supernumerary genome in fungi. Different biology rather than origin was shown to be responsible. A “living apart together” crosstalk exists between the core and supernumerary genome, accelerating chromosomal and organismal evolution.

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July 19, 2019

Biosynthesis and function of modified bases in bacteria and their viruses.

Naturally occurring modification of the canonical A, G, C, and T bases can be found in the DNA of cellular organisms and viruses from all domains of life. Bacterial viruses (bacteriophages) are a particularly rich but still underexploited source of such modified variant nucleotides. The modifications conserve the coding and base-pairing functions of DNA, but add regulatory and protective functions. In prokaryotes, modified bases appear primarily to be part of an arms race between bacteriophages (and other genomic parasites) and their hosts, although, as in eukaryotes, some modifications have been adapted to convey epigenetic information. The first half of this review catalogs the identification and diversity of DNA modifications found in bacteria and bacteriophages. What is known about the biogenesis, context, and function of these modifications are also described. The second part of the review places these DNA modifications in the context of the arms race between bacteria and bacteriophages. It focuses particularly on the defense and counter-defense strategies that turn on direct recognition of the presence of a modified base. Where modification has been shown to affect other DNA transactions, such as expression and chromosome segregation, that is summarized, with reference to recent reviews.

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July 19, 2019

High frequency of mitochondrial DNA mutations in HIV-infected treatment-experienced individuals.

We recently observed a decrease in deoxyribonucleotide (dNTP) pools in HIV-infected individuals on antiretroviral therapy (ART). Alterations in dNTPs result in mutations in mitochondrial DNA (mtDNA) in cell culture and animal models. Therefore, we investigated whether ART is associated with mitochondrial genome sequence variation in peripheral blood mononuclear cells (PBMCs) of HIV-infected treatment-experienced individuals.In this substudy of a case-control study, 71 participants were included: 22 ‘cases’, who were HIV-infected treatment-experienced patients with mitochondrial toxicity, 25 HIV-infected treatment-experienced patients without mitochondrial toxicity, and 24 HIV-uninfected controls. Total DNA was extracted from PBMCs and purified polymerase chain reaction (PCR) products were subjected to third-generation sequencing using the PacBio Single Molecule Real-Time (SMRT) sequencing technology. The sequences were aligned against the revised Cambridge reference sequence for human mitochondrial DNA (NC_012920.1) for detection of variants.We identified a total of 123 novel variants, 39 of them in the coding region. HIV-infected treatment-experienced patients with and without toxicity had significantly higher average numbers of mitochondrial variants per participant than HIV-uninfected controls. We observed a higher burden of mtDNA large-scale deletions in HIV-infected treatment-experienced patients with toxicity compared with HIV-uninfected controls (P = 0.02). The frequency of mtDNA molecules containing a common deletion (mt.d4977) was higher in HIV-infected treatment-experienced patients with toxicity compared with HIV-uninfected controls (P = 0.06). There was no statistically significant difference in mtDNA variants between HIV-infected treatment-experienced patients with and without toxicity.The frequency of mtDNA variants (mutations and large-scale deletions) was higher in HIV-infected treatment-experienced patients with or without ART-induced toxicity than in uninfected controls.© 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

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July 19, 2019

Condition-dependent co-regulation of genomic clusters of virulence factors in the grapevine trunk pathogen Neofusicoccum parvum.

The ascomycete Neofusicoccum parvum, one of the causal agents of Botryosphaeria dieback, is a destructive wood-infecting fungus and a serious threat to grape production worldwide. The capability to colonize woody tissue, combined with the secretion of phytotoxic compounds, is thought to underlie its pathogenicity and virulence. Here, we describe the repertoire of virulence factors and their transcriptional dynamics as the fungus feeds on different substrates and colonizes the woody stem. We assembled and annotated a highly contiguous genome using single-molecule real-time DNA sequencing. Transcriptome profiling by RNA sequencing determined the genome-wide patterns of expression of virulence factors both in vitro (potato dextrose agar or medium amended with grape wood as substrate) and in planta. Pairwise statistical testing of differential expression, followed by co-expression network analysis, revealed that physically clustered genes coding for putative virulence functions were induced depending on the substrate or stage of plant infection. Co-expressed gene clusters were significantly enriched not only in genes associated with secondary metabolism, but also in those associated with cell wall degradation, suggesting that dynamic co-regulation of transcriptional networks contributes to multiple aspects of N. parvum virulence. In most of the co-expressed clusters, all genes shared at least a common motif in their promoter region, indicative of co-regulation by the same transcription factor. Co-expression analysis also identified chromatin regulators with correlated expression with inducible clusters of virulence factors, suggesting a complex, multi-layered regulation of the virulence repertoire of N. parvum.© 2016 BSPP AND JOHN WILEY & SONS LTD.

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July 19, 2019

Winding paths to simplicity: genome evolution in facultative insect symbionts.

Symbiosis between organisms is an important driving force in evolution. Among the diverse relationships described, extensive progress has been made in insect-bacteria symbiosis, which improved our understanding of the genome evolution in host-associated bacteria. Particularly, investigations on several obligate mutualists have pushed the limits of what we know about the minimal genomes for sustaining cellular life. To bridge the gap between those obligate symbionts with extremely reduced genomes and their non-host-restricted ancestors, this review focuses on the recent progress in genome characterization of facultative insect symbionts. Notable cases representing various types and stages of host associations, including those from multiple genera in the family Enterobacteriaceae (class Gammaproteobacteria), Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), are discussed. Although several general patterns of genome reduction associated with the adoption of symbiotic relationships could be identified, extensive variation was found among these facultative symbionts. These findings are incorporated into the established conceptual frameworks to develop a more detailed evolutionary model for the discussion of possible trajectories. In summary, transitions from facultative to obligate symbiosis do not appear to be a universal one-way street; switches between hosts and lifestyles (e.g. commensalism, parasitism or mutualism) occur frequently and could be facilitated by horizontal gene transfer. © FEMS 2016.

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July 19, 2019

Multiple origins of the pathogenic yeast Candida orthopsilosis by separate hybridizations between two parental species.

Mating between different species produces hybrids that are usually asexual and stuck as diploids, but can also lead to the formation of new species. Here, we report the genome sequences of 27 isolates of the pathogenic yeast Candida orthopsilosis. We find that most isolates are diploid hybrids, products of mating between two unknown parental species (A and B) that are 5% divergent in sequence. Isolates vary greatly in the extent of homogenization between A and B, making their genomes a mosaic of highly heterozygous regions interspersed with homozygous regions. Separate phylogenetic analyses of SNPs in the A- and B-derived portions of the genome produces almost identical trees of the isolates with four major clades. However, the presence of two mutually exclusive genotype combinations at the mating type locus, and recombinant mitochondrial genomes diagnostic of inter-clade mating, shows that the species C. orthopsilosis does not have a single evolutionary origin but was created at least four times by separate interspecies hybridizations between parents A and B. Older hybrids have lost more heterozygosity. We also identify two isolates with homozygous genomes derived exclusively from parent A, which are pure non-hybrid strains. The parallel emergence of the same hybrid species from multiple independent hybridization events is common in plant evolution, but is much less documented in pathogenic fungi.

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July 19, 2019

Short tandem repeats, segmental duplications, gene deletion, and genomic instability in a rapidly diversified immune gene family.

Genomic regions with repetitive sequences are considered unstable and prone to swift DNA diversification processes. A highly diverse immune gene family of the sea urchin (Strongylocentrotus purpuratus), called Sp185/333, is composed of clustered genes with similar sequence as well as several types of repeats ranging in size from short tandem repeats (STRs) to large segmental duplications. This repetitive structure may have been the basis for the incorrect assembly of this gene family in the sea urchin genome sequence. Consequently, we have resolved the structure of the family and profiled the members by sequencing selected BAC clones using Illumina and PacBio approaches.BAC insert assemblies identified 15 predicted genes that are organized into three clusters. Two of the gene clusters have almost identical flanking regions, suggesting that they may be non-matching allelic clusters residing at the same genomic locus. GA STRs surround all genes and appear in large stretches at locations of putatively deleted genes. GAT STRs are positioned at the edges of segmental duplications that include a subset of the genes. The unique locations of the STRs suggest their involvement in gene deletions and segmental duplications. Genomic profiling of the Sp185/333 gene diversity in 10 sea urchins shows that no gene repertoires are shared among individuals indicating a very high gene diversification rate for this family.The repetitive genomic structure of the Sp185/333 family that includes STRs in strategic locations may serve as platform for a controlled mechanism which regulates the processes of gene recombination, gene conversion, duplication and deletion. The outcome is genomic instability and allelic mismatches, which may further drive the swift diversification of the Sp185/333 gene family that may improve the immune fitness of the species.

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