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April 21, 2020

Genome assembly and annotation of the Trichoplusia ni Tni-FNL insect cell line enabled by long-read technologies.

Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL.By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL.Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly.This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.

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April 21, 2020

De novo assembly of the goldfish (Carassius auratus) genome and the evolution of genes after whole-genome duplication.

For over a thousand years, the common goldfish (Carassius auratus) was raised throughout Asia for food and as an ornamental pet. As a very close relative of the common carp (Cyprinus carpio), goldfish share the recent genome duplication that occurred approximately 14 million years ago in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies provides an exciting opportunity to link genotype to phenotype and to understand the dynamics of genome evolution and speciation. We generated a high-quality draft sequence and gene annotations of a “Wakin” goldfish using 71X PacBio long reads. The two subgenomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged substantially across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression levels across all tissues, while loss of conserved noncoding elements determined expression variance between different tissues. This assembly provides an important resource for comparative genomics and understanding the causes of goldfish variants.

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April 21, 2020

The comparative genomics and complex population history of Papio baboons.

Recent studies suggest that closely related species can accumulate substantial genetic and phenotypic differences despite ongoing gene flow, thus challenging traditional ideas regarding the genetics of speciation. Baboons (genus Papio) are Old World monkeys consisting of six readily distinguishable species. Baboon species hybridize in the wild, and prior data imply a complex history of differentiation and introgression. We produced a reference genome assembly for the olive baboon (Papio anubis) and whole-genome sequence data for all six extant species. We document multiple episodes of admixture and introgression during the radiation of Papio baboons, thus demonstrating their value as a model of complex evolutionary divergence, hybridization, and reticulation. These results help inform our understanding of similar cases, including modern humans, Neanderthals, Denisovans, and other ancient hominins.

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April 21, 2020

Shared and unique microbes between Small hive beetles (Aethina tumida) and their honey bee hosts.

The small hive beetle (SHB) is an opportunistic parasite that feeds on bee larvae, honey, and pollen. While SHBs can also feed on fruit and other plant products, like its plant-feeding relatives, SHBs prefer to feed on hive resources and only reproduce inside bee colonies. As parasites, SHBs are inevitably exposed to bee-associated microbes, either directly from the bees or from the hive environment. These microbes have unknown impacts on beetles, nor is it known how extensively beetles transfer microbes among their bee hosts. To identify sets of beetle microbes and the transmission of microbes from bees to beetles, a metagenomic analysis was performed. We identified sets of herbivore-associated bacteria, as well as typical bee symbiotic bacteria for pollen digestion, in SHB larvae and adults. Deformed wing virus was highly abundant in beetles, which colonize SHBs as suggested by a controlled feeding trial. Our data suggest SHBs are vectors for pathogen transmission among bees and between colonies. The dispersal of host pathogens by social parasites via floral resources and the hive environment increases the threats of these parasites to honey bees. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas, Mycoplasma and by sequences assigned to the order Clostridiales. Applying Sanger sequencing on the full-length bacterial 16S rRNA gene from the pool of 46 isolates obtained in this study showed a clear assignment of the PacBio full-length bacterial 16S rRNA gene sequences down to the genus level. One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions. Comparisons of sequence assignments between full-length and in silico derived variable 16S rRNA gene regions showed different microbial profiles with variable effects between phylogenetic groups and taxonomic ranks. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

SMRT sequencing analysis reveals the full-length transcripts and alternative splicing patterns in Ananas comosus var. bracteatus.

Ananas comosus var. bracteatus is an herbaceous perennial monocot cultivated as an ornamental plant for its chimeric leaves. Because of its genomic complexity, and because no genomic information is available in the public GenBank database, the complete structure of the mRNA transcript is unclear and there are limited molecular mechanism studies for Ananas comosus var. bracteatus.Three size fractionated full-length cDNA libraries (1-2 kb, 2-3 kb, and 3-6 kb) were constructed and subsequently sequenced in five single-molecule real-time (SMRT) cells (2 cells, 2 cells, and 1 cell, respectively).In total, 19,838 transcripts were identified for alternative splicing (AS) analysis. Among them, 19,185 (96.7%) transcripts were functionally annotated. A total of 9,921 genes were identified by mapping the non-redundant isoforms to the reference genome. A total of 10,649 AS events were identified, the majority of which were intron retention events. The alternatively spliced genes had functions in the basic metabolism processes of the plant such as carbon metabolism, amino acid biosynthesis, and glycolysis. Fourteen genes related to chlorophyll biosynthesis were identified as having AS events. The distribution of the splicing sites and the percentage of conventional and non-canonical AS sites of the genes categorized in pathways related to the albino leaf phenotype (ko00860, ko00195, ko00196, and ko00710) varied greatly. The present results showed that there were 8,316 genes carrying at least one poly (A) site, which generated 21,873 poly (A) sites. These findings indicated that the quality of the gene structure and functional information of the obtained genome was greatly improved, which may facilitate further genetic study of Ananas comosus var. bracteatus.

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April 21, 2020

Biodiversity seen through the perspective of insects: 10 simple rules on methodological choices and experimental design for genomic studies.

Massively parallel DNA sequencing opens up opportunities for bridging multiple temporal and spatial dimensions in biodiversity research, thanks to its efficiency to recover millions of nucleotide polymorphisms. Here, we identify the current status, discuss the main challenges, and look into future perspectives on biodiversity genomics focusing on insects, which arguably constitute the most diverse and ecologically important group among all animals. We suggest 10 simple rules that provide a succinct step-by-step guide and best-practices to anyone interested in biodiversity research through the study of insect genomics. To this end, we review relevant literature on biodiversity and evolutionary research in the field of entomology. Our compilation is targeted at researchers and students who may not yet be specialists in entomology or molecular biology. We foresee that the genomic revolution and its application to the study of non-model insect lineages will represent a major leap to our understanding of insect diversity.

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April 21, 2020

Genes of the pig, Sus scrofa, reconstructed with EvidentialGene.

The pig is a well-studied model animal of biomedical and agricultural importance. Genes of this species, Sus scrofa, are known from experiments and predictions, and collected at the NCBI reference sequence database section. Gene reconstruction from transcribed gene evidence of RNA-seq now can accurately and completely reproduce the biological gene sets of animals and plants. Such a gene set for the pig is reported here, including human orthologs missing from current NCBI and Ensembl reference pig gene sets, additional alternate transcripts, and other improvements. Methodology for accurate and complete gene set reconstruction from RNA is used: the automated SRA2Genes pipeline of EvidentialGene project.

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April 21, 2020

Rapid antigen diversification through mitotic recombination in the human malaria parasite Plasmodium falciparum.

Malaria parasites possess the remarkable ability to maintain chronic infections that fail to elicit a protective immune response, characteristics that have stymied vaccine development and cause people living in endemic regions to remain at risk of malaria despite previous exposure to the disease. These traits stem from the tremendous antigenic diversity displayed by parasites circulating in the field. For Plasmodium falciparum, the most virulent of the human malaria parasites, this diversity is exemplified by the variant gene family called var, which encodes the major surface antigen displayed on infected red blood cells (RBCs). This gene family exhibits virtually limitless diversity when var gene repertoires from different parasite isolates are compared. Previous studies indicated that this remarkable genome plasticity results from extensive ectopic recombination between var genes during mitotic replication; however, the molecular mechanisms that direct this process to antigen-encoding loci while the rest of the genome remains relatively stable were not determined. Using targeted DNA double-strand breaks (DSBs) and long-read whole-genome sequencing, we show that a single break within an antigen-encoding region of the genome can result in a cascade of recombination events leading to the generation of multiple chimeric var genes, a process that can greatly accelerate the generation of diversity within this family. We also found that recombinations did not occur randomly, but rather high-probability, specific recombination products were observed repeatedly. These results provide a molecular basis for previously described structured rearrangements that drive diversification of this highly polymorphic gene family.

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April 21, 2020

Deep repeat resolution-the assembly of the Drosophila Histone Complex.

Though the advent of long-read sequencing technologies has led to a leap in contiguity of de novo genome assemblies, current reference genomes of higher organisms still do not provide unbroken sequences of complete chromosomes. Despite reads in excess of 30 000 base pairs, there are still repetitive structures that cannot be resolved by current state-of-the-art assemblers. The most challenging of these structures are tandemly arrayed repeats, which occur in the genomes of all eukaryotes. Untangling tandem repeat clusters is exceptionally difficult, since the rare differences between repeat copies are obscured by the high error rate of long reads. Solving this problem would constitute a major step towards computing fully assembled genomes. Here, we demonstrate by example of the Drosophila Histone Complex that via machine learning algorithms, it is possible to exploit the underlying distinguishing patterns of single nucleotide variants of repeats from very noisy data to resolve a large and highly conserved repeat cluster. The ideas explored in this paper are a first step towards the automated assembly of complex repeat structures and promise to be applicable to a wide range of eukaryotic genomes. © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.

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April 21, 2020

Infection mechanisms and putative effector repertoire of the mosquito pathogenic oomycete Pythium guiyangense uncovered by genomic analysis.

Pythium guiyangense, an oomycete from a genus of mostly plant pathogens, is an effective biological control agent that has wide potential to manage diverse mosquitoes. However, its mosquito-killing mechanisms are almost unknown. In this study, we observed that P. guiyangense could utilize cuticle penetration and ingestion of mycelia into the digestive system to infect mosquito larvae. To explore pathogenic mechanisms, a high-quality genome sequence with 239 contigs and an N50 contig length of 1,009 kb was generated. The genome assembly is approximately 110 Mb, which is almost twice the size of other sequenced Pythium genomes. Further genome analysis suggests that P. guiyangense may arise from a hybridization of two related but distinct parental species. Phylogenetic analysis demonstrated that P. guiyangense likely evolved from common ancestors shared with plant pathogens. Comparative genome analysis coupled with transcriptome sequencing data suggested that P. guiyangense may employ multiple virulence mechanisms to infect mosquitoes, including secreted proteases and kazal-type protease inhibitors. It also shares intracellular Crinkler (CRN) effectors used by plant pathogenic oomycetes to facilitate the colonization of plant hosts. Our experimental evidence demonstrates that CRN effectors of P. guiyangense can be toxic to insect cells. The infection mechanisms and putative virulence effectors of P. guiyangense uncovered by this study provide the basis to develop improved mosquito control strategies. These data also provide useful knowledge on host adaptation and evolution of the entomopathogenic lifestyle within the oomycete lineage. A deeper understanding of the biology of P. guiyangense effectors might also be useful for management of other important agricultural pests.

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April 21, 2020

Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus.

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April 21, 2020

Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches.

Fungi are ubiquitous on earth and are essential for the maintenance of the global ecological equilibrium. Despite providing benefits to living organisms, they can also target specific hosts and inflict damage. These fungal pathogens are known to affect, for example, plants and mam- mals and thus reduce crop production necessary to sustain food supply and cause mortality in humans and animals. Designing defenses against these fungi is essential for the control of food resources and human health. As far as fungal pathogens are concerned, the principal option has been the use of antifungal agents, also called fungicides when they are used in the environment.

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April 21, 2020

Integrating Hi-C links with assembly graphs for chromosome-scale assembly.

Long-read sequencing and novel long-range assays have revolutionized de novo genome assembly by automating the reconstruction of reference-quality genomes. In particular, Hi-C sequencing is becoming an economical method for generating chromosome-scale scaffolds. Despite its increasing popularity, there are limited open-source tools available. Errors, particularly inversions and fusions across chromosomes, remain higher than alternate scaffolding technologies. We present a novel open-source Hi-C scaffolder that does not require an a priori estimate of chromosome number and minimizes errors by scaffolding with the assistance of an assembly graph. We demonstrate higher accuracy than the state-of-the-art methods across a variety of Hi-C library preparations and input assembly sizes. The Python and C++ code for our method is openly available at https://github.com/machinegun/SALSA.

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