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July 19, 2019

A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples.

VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues.PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module.Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus.This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.

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July 19, 2019

A mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species.

The genome of Fusarium oxysporum (Fo) consists of a set of eleven ‘core’ chromosomes, shared by most strains and responsible for housekeeping, and one or several accessory chromosomes. We sequenced a strain of Fo f.sp. radicis-cucumerinum (Forc) using PacBio SMRT sequencing. All but one of the core chromosomes were assembled into single contigs, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. A central part of this chromosome contains all identified candidate effector genes, including homologs of SIX6, SIX9, SIX11 and SIX 13. We show that SIX6 contributes to virulence of Forc. Through horizontal chromosome transfer (HCT) to a non-pathogenic strain, we also show that the accessory chromosome containing the SIX gene homologs is indeed a pathogenicity chromosome for cucurbit infection. Conversely, complete loss of virulence was observed in Forc016 strains that lost this chromosome. We conclude that also a non-wilt-inducing Fo pathogen relies on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Three out of nine HCT strains investigated have undergone large-scale chromosome alterations, reflecting the remarkable plasticity of Fo genomes.

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July 19, 2019

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

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July 19, 2019

Increased risk of low birth weight in women with placental malaria associated with P. falciparum VAR2CSA clade.

Pregnancy associated malaria (PAM) causes adverse pregnancy and birth outcomes owing to Plasmodium falciparum accumulation in the placenta. Placental accumulation is mediated by P. falciparum protein VAR2CSA, a leading PAM-specific vaccine target. The extent of its antigen diversity and impact on clinical outcomes remain poorly understood. Through amplicon deep-sequencing placental malaria samples from women in Malawi and Benin, we assessed sequence diversity of VAR2CSA’s ID1-DBL2x region, containing putative vaccine targets and estimated associations of specific clades with adverse birth outcomes. Overall, var2csa diversity was high and haplotypes subdivided into five clades, the largest two defined by homology to parasites strains, 3D7 or FCR3. Across both cohorts, compared to women infected with only FCR3-like variants, women infected with only 3D7-like variants delivered infants with lower birthweight (difference: -267.99?g; 95% Confidence Interval [CI]: -466.43?g,-69.55?g) and higher odds of low birthweight (<2500?g) (Odds Ratio [OR] 5.41; 95% CI:0.99,29.52) and small-for-gestational-age (OR: 3.65; 95% CI: 1.01,13.38). In two distinct malaria-endemic African settings, parasites harboring 3D7-like variants of VAR2CSA were associated with worse birth outcomes, supporting differential effects of infection with specific parasite strains. The immense diversity coupled with differential clinical effects of this diversity suggest that an effective VAR2CSA-based vaccine may require multivalent activity.

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July 19, 2019

PacBio sequencing reveals transposable element as a key contributor to genomic plasticity and virulence variation in Magnaporthe oryzae.

The sustainable cultivation of rice, which serves as staple food crop for more than half of the world’s population, is under serious threat due to the huge yield losses inflicted by rice blast disease caused by the globally destructive fungus Magnaporthe oryzae (Pyricularia oryzae) (Dean et al., 2012, Nalley et al., 2016, Deng et al., 2017). This filamentous ascomycete fungus is also capable of causing blast infection on other economically important cereal crops, including wheat, millet, and barley, making it the world’s most important plant pathogenic fungus (Zhong et al., 2016). The advent of whole-genome sequencing technology and the subsequent deployment of next-generation sequencing (NGS) strategies have successfully generated genome assemblies for over 50 isolates of M. oryzae, which have played an instrumental role in enhancing our understanding of how rice blast fungus undertakes host adaptation, host specificity, and host range expansion to overcome host resistance (Dean et al., 2005, Xue et al., 2012, Wu et al., 2015, Zhang et al., 2016). However, research findings obtained from comparative genomic studies conducted using the NGS-assembled genome do not present an in-depth account of the genomic features that contribute to the prevailing genomic variations among M. oryzae species, because NGS assemblies are highly fragmented and lack most of the lineage-specific (LS) regions, which are more plastic than the core genome and enriched with repeats and effector proteins (Raffaele and Kamoun, 2012, Faino et al., 2016).

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July 19, 2019

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications.The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

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July 19, 2019

Parkinson’s disease associated with pure ATXN10 repeat

Large, non-coding pentanucleotide repeat expansions of ATTCT in intron 9 of the ATXN10 gene typically cause progressive spinocerebellar ataxia with or without seizures and present neuropathologically with Purkinje cell loss resulting in symmetrical cerebellar atrophy. These ATXN10 repeat expansions can be interrupted by sequence motifs which have been attributed to seizures and are likely to act as genetic modifiers. We identified a Mexican kindred with multiple affected family members with ATXN10 expansions. Four affected family members showed clinical features of spinocerebellar ataxia type 10 (SCA10). However, one affected individual presented with early-onset levodopa-responsive parkinsonism, and one family member carried a large repeat ATXN10 expansion, but was clinically unaffected. To characterize the ATXN10 repeat, we used a novel technology of single-molecule real-time (SMRT) sequencing and CRISPR/Cas9-based capture. We sequenced the entire span of ~5.3–7.0kb repeat expansions. The Parkinson’s patient carried an ATXN10 expansion with no repeat interruption motifs as well as an unaffected sister. In the siblings with typical SCA10, we found a repeat pattern of ATTCC repeat motifs that have not been associated with seizures previously. Our data suggest that the absence of repeat interruptions is likely a genetic modifier for the clinical presentation of L-Dopa responsive parkinsonism, whereas repeat interruption motifs contribute clinically to epilepsy. Repeat interruptions are important genetic modifiers of the clinical phenotype in SCA10. Advanced sequencing techniques now allow to better characterize the underlying genetic architecture for determining accurate phenotype–genotype correlations.

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July 19, 2019

Genome sequencing of strain Cellulosimicrobium sp. TH-20 with ginseng biotransformation ability.

Biotransformation for increasing the pharmaceutical effect of ginsenosides is getting more and more attractions. Strain Cellulosimicrobium sp. TH-20 isolated from ginseng soil samples was identified to produce enzymes contributing to its excellent biotransformation activity against ginsenosides, the main active components of ginseng. Based on phylogenetic tree and homology analysis, the strain can be designated as Cellulosimicrobium sp. Genome sequencing was performed using the Illumina Miseq to explore the functional genes involved in ginsenoside transformation. The draft genome of Cellulosimicrobium sp. TH-20 encoded 3450 open reading frames, 51 tRNA, and 9 rRNA. All ORFs were annotated using NCBI BLAST with non-redundant proteins, Gene Ontology, Cluster of Orthologous Gene, and Kyoto Encyclopedia of Genes and Genomes databases. A total of 11 genes were selected based on the functional annotation analysis. These genes are relevant to ginsenoside biotransformation, including 6 for beta-glucosidase, 1 for alpha-N-arabinofuranosidase, 1 for alpha-1,6-glucosidase, 1 for endo-1,4-beta-xylanase, 1 for alpha-L-arabinofuranosidase, and 1 for beta-galactosidase. These glycosidases were predicted to catalyze the hydrolysis of sugar moieties attached to the aglycon of ginsenosides and led to the transformation of PPD-type and PPT-type ginsenosides.

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July 19, 2019

Omics approaches to study gene regulatory networks for development in echinoderms.

Gene regulatory networks (GRNs) describe the interactions for a developmental process at a given time and space. Historically, perturbation experiments represent one of the key methods for analyzing and reconstructing a GRN, and the GRN governing early development in the sea urchin embryo stands as one of the more deeply dissected so far. As technology progresses, so do the methods used to address different biological questions. Next-generation sequencing (NGS) has become a standard experimental technique for genome and transcriptome sequencing and studies of protein-DNA interactions and DNA accessibility. While several efforts have been made toward the integration of different omics approaches for the study of the regulatory genome in many animals, in a few cases, these are applied with the purpose of reconstructing and experimentally testing developmental GRNs. Here, we review emerging approaches integrating multiple NGS technologies for the prediction and validation of gene interactions within echinoderm GRNs. These approaches can be applied to both ‘model’ and ‘non-model’ organisms. Although a number of issues still need to be addressed, advances in NGS applications, such as assay for transposase-accessible chromatin sequencing, combined with the availability of embryos belonging to different species, all separated by various evolutionary distances and accessible to experimental regulatory biology, place echinoderms in an unprecedented position for the reconstruction and evolutionary comparison of developmental GRNs. We conclude that sequencing technologies and integrated omics approaches allow the examination of GRNs on a genome-wide scale only if biological perturbation and cis-regulatory analyses are experimentally accessible, as in the case of echinoderm embryos.© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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July 19, 2019

Characterisation of MHC class I genes in the koala.

Koala (Phascolarctos cinereus) populations are on the decline across the majority of Australia’s mainland. Two major diseases threatening the long-term survival of affected koala populations are caused by obligate intracellular pathogens: Chlamydia and koala retrovirus (KoRV). To improve our understanding of the koala immune system, we characterised their major histocompatibility complex (MHC) class I genes, which are centrally involved in presenting foreign peptides derived from intracellular pathogens to cytotoxic T cells. A total of 11 class I genes were identified in the koala genome. Three genes, Phci-UA, UB and UC, showed relatively high genetic variability and were expressed in all 12 examined tissues, whereas the other eight genes had tissue-specific expression and limited polymorphism. Evidence of diversifying selection was detected in Phci-UA and UC, while gene conversion may have played a role in creating new alleles at Phci-UB. We propose that Phci-UA, UB and UC are likely classical MHC genes of koalas, and further research is needed to understand their role in koala chlamydial and KoRV infections.

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July 19, 2019

A novel approach using long-read sequencing and ddPCR to investigate gonadal mosaicism and estimate recurrence risk in two families with developmental disorders.

De novo mutations contribute significantly to severe early-onset genetic disorders. Even if the mutation is apparently de novo, there is a recurrence risk due to parental germ line mosaicism, depending on in which gonadal generation the mutation occurred.We demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction.In the first family, a TCOF1 variant c.3156C>T was identified in the proband with Treacher Collins syndrome. The variant affects splicing and was determined to be of paternal origin. It was present in <1% of the paternal germ cells, suggesting a very low recurrence risk. In the second family, the couple had undergone several unsuccessful pregnancies where a de novo mutation PTPN11 c.923A>C causing Noonan syndrome was identified. The variant was present in 40% of the paternal germ cells suggesting a high recurrence risk.Our findings highlight a successful strategy to identify the parental origin of mutations and to investigate the recurrence risk in couples that have undergone assisted reproduction with an unknown donor or in couples with gonadal mosaicism that will undergo preimplantation genetic diagnosis.© 2017 The Authors Prenatal Diagnosis published by John Wiley & Sons Ltd.

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July 19, 2019

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction.

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tapirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

ALF: a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysis.

The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.

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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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