Background Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. Results We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30–40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. Conclusions This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

Abstract The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

Fungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by diverse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome shot-gun sequencing with a portable DNA sequencing device as a method for the detection of fungal pathogens from wheat (Triticum aestivum) in a standard molecular biology laboratory. The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused by Puccinia striiformis f. sp. tritici), Septoria tritici blotch (caused by Zymoseptoria tritici), and yellow leaf spot (caused by Pyrenophora tritici repentis). We also identified the bacterial genus Pseudomonas co-present with Puccinia and Zymoseptoria but not Pyrenophora infections. One limitation of the method is the over-representation of redundant wheat genome sequences from samples. This could be addressed by long-range amplicon-based sequencing approaches in future studies, which specifically target nonhost organisms. Our work outlines a new approach for detection of a broad range of plant pathogens and associated microbes using a portable sequencer in a standard laboratory, providing the basis for future development of an on-site disease monitoring system. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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Different cupric oxide (CuO) structures have attracted intensive interest because of their promising applications in various fields. In this study, three kinds of CuO structures, namely, CuO microdisks, CuO nanoblocks, and CuO microspheres, are synthesized by solution-based synthetic methods. The morphologies and crystal structures of these CuO structures are characterized by field-emission scanning electron microscope and X-ray diffractometer, respectively. They are used as thermal conductive fillers to prepare silicone-based thermal greases, giving rise to great enhancement in thermal conductivity. Compared with pure silicone base, the thermal conductivities of thermal greases with CuO microdisks, CuO nanoblocks, and CuO microspheres are 0.283, 0256, and 0.239 W/mK, respectively, at filler loading of 9 vol.%, which increases 139%, 116%, and 99%, respectively. These thermal greases present a slight descendent tendency in thermal conductivity at elevated temperatures. These experimental data are compared with Nan's model prediction, indicating that the shape factor has a great influence on thermal conductivity improvement of thermal greases with different CuO structures. Meanwhile, due to large aspect ratio of CuO microdisks, they can form thermal networks more effectively than the other two structures, resulting in higher thermal conductivity enhancement.

Background Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. Results We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5′ uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5′ adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. Conclusions We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Background European brown hares (Lepus europaeus) and European rabbits (Oryctolagus cuniculus) are invasive pest species in Australia, with rabbits having a substantially larger environmental impact than hares. As their spatial distribution in Australia partially overlaps, we conducted a comparative microbiome study to determine how the composition of gastrointestinal microbiota varies between these species, since this may indicate species differences in diet, physiology, and other internal and external factors. Methods We analysed the faecal microbiome of nine wild hares and twelve wild rabbits from a sympatric periurban reserve in Canberra, Australia, using a 16S rRNA amplicon-based sequencing approach. Additionally, we compared the concordance between results from Illumina and Nanopore sequencing platforms. Results We identified significantly more variation in faecal microbiome composition between individual rabbits compared to hares, despite both species occupying a similar habitat. The faecal microbiome in both species was dominated by the phyla Firmicutes and Bacteroidetes, typical of many vertebrates. Many phyla, including Actinobacteria, Proteobacteria and Patescibacteria, were shared between rabbits and hares. In contrast, bacteria from phylum Verrucomicrobia were present only in rabbits, while phyla Lentisphaerae and Synergistetes were represented only in hares. We did not identify phylum Spirochaetes in Australian hares; this phylum was previously shown to be present at high relative abundance in European hare faecal samples. These differences in the composition of faecal microbiota may be indicative of less discriminate foraging behaviour in rabbits, which in turn may enable them to adapt quicker to new environments, and may reflect the severe environmental impacts that this species has in Australia.

Background: Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false positive signals. Results: We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome (~0.7% heterozygosity) and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. Conclusions: This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.