June 1, 2021  |  

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark discoveries in biochemistry and genetics. We show that de novo assembly of a new strain yields complete assemblies of entire chromosomes, and additionally contains entire centromeric sequences. Base-modification analyses reveal candidate sites of increased interpulse duration (IPD) ratio, that may signify regions of 5mC, 5hmC, or 6mA base modifications. Iso-seq method provides full-length transcript evidence for comprehensive gene annotation, as well as context to the base-modifications in the newly assembled genome. Projects that integrate multiple genome-wide assays could become common practice for identifying genomic elements and understanding their function in new strains and organisms.

April 21, 2020  |  

Genomic and transcriptomic insights into the survival of the subaerial cyanobacterium Nostoc flagelliforme in arid and exposed habitats.

The cyanobacterium Nostoc flagelliforme is an extremophile that thrives under extraordinary desiccation and ultraviolet (UV) radiation conditions. To investigate its survival strategies, we performed whole-genome sequencing of N. flagelliforme CCNUN1 and transcriptional profiling of its field populations upon rehydration in BG11 medium. The genome of N. flagelliforme is 10.23 Mb in size and contains 10 825 predicted protein-encoding genes, making it one of the largest complete genomes of cyanobacteria reported to date. Comparative genomics analysis among 20 cyanobacterial strains revealed that genes related to DNA replication, recombination and repair had disproportionately high contributions to the genome expansion. The ability of N. flagelliforme to thrive under extreme abiotic stresses is supported by the acquisition of genes involved in the protection of photosynthetic apparatus, the formation of monounsaturated fatty acids, responses to UV radiation, and a peculiar role of ornithine metabolism. Transcriptome analysis revealed a distinct acclimation strategy to rehydration, including the strong constitutive expression of genes encoding photosystem I assembly factors and the involvement of post-transcriptional control mechanisms of photosynthetic resuscitation. Our results provide insights into the adaptive mechanisms of subaerial cyanobacteria in their harsh habitats and have important implications to understand the evolutionary transition of cyanobacteria from aquatic environments to terrestrial ecosystems. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

April 21, 2020  |  

Genomic variation and strain-specific functional adaptation in the human gut microbiome during early life.

The human gut microbiome matures towards the adult composition during the first years of life and is implicated in early immune development. Here, we investigate the effects of microbial genomic diversity on gut microbiome development using integrated early childhood data sets collected in the DIABIMMUNE study in Finland, Estonia and Russian Karelia. We show that gut microbial diversity is associated with household location and linear growth of children. Single nucleotide polymorphism- and metagenomic assembly-based strain tracking revealed large and highly dynamic microbial pangenomes, especially in the genus Bacteroides, in which we identified evidence of variability deriving from Bacteroides-targeting bacteriophages. Our analyses revealed functional consequences of strain diversity; only 10% of Finnish infants harboured Bifidobacterium longum subsp. infantis, a subspecies specialized in human milk metabolism, whereas Russian infants commonly maintained a probiotic Bifidobacterium bifidum strain in infancy. Groups of bacteria contributing to diverse, characterized metabolic pathways converged to highly subject-specific configurations over the first two years of life. This longitudinal study extends the current view of early gut microbial community assembly based on strain-level genomic variation.

April 21, 2020  |  

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

April 21, 2020  |  

Whole-genome sequencing reveals recent and frequent genetic recombination between clonal lineages of Cryphonectria parasitica in western Europe.

Changes in the mode of reproduction are frequently observed in invasive fungal populations. The ascomycete Cryphonectria parasitica, which causes Chestnut Blight, was introduced to Europe from North America and Asia in the 20th century. Previous genotyping studies based on ten microsatellite markers have identified several clonal lineages which have spread throughout western Europe, suggesting that asexuality was the main reproductive mode of this species during colonization, although occasional sexual reproduction is not excluded. Based on the whole-genome sequences alignment of 46 C. parasitica isolates from France, North America and Asia, genealogy and population structure analyses mostly confirmed these lineages as clonal. However, one of these clonal lineages showed a signal of strong recombination, suggesting different strategies of reproduction in western Europe. Signatures of several recent recombination events within all the French clonal lineages studied here were also identified, indicating that gene flow is regular between these lineages. In addition, haplotype identification of seven French clonal lineages revealed that emergences of new clonal lineages during colonization were the result of hybridization between the main expanding clonal lineages and minor haplotypes non-sequenced in the present study. This whole-genome sequencing study underlines the importance of recombination events in the invasive success of these clonal populations, and suggests that sexual reproduction may be more frequent within and between the western European clonal lineages of C. parasitica than previously assumed using few genetic markers.Copyright © 2019 Elsevier Inc. All rights reserved.

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