scholarly journals Hybrid genome assembly and annotation of Danionella translucida, a transparent fish with the smallest known vertebrate brain

2019 ◽  
Author(s):  
Mykola Kadobianskyi ◽  
Lisanne Schulze ◽  
Markus Schuelke ◽  
Benjamin Judkewitz
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Large Fraction ◽  
Model Organism ◽  
Close Relative ◽  
Vertebrate Brain ◽  
Hybrid Genome ◽  
Long Read ◽  
Optical Turbidity ◽  
Low Coverage

Studying the activity of distributed neuronal circuits at a cellular resolution in vertebrates is very challenging due to the size and optical turbidity of their brains. We recently presented Danionella translucida, a close relative of zebrafish, as a model organism suited for studying large-scale neural network interactions in adult individuals. Danionella remains transparent throughout its life, has the smallest known vertebrate brain and possesses a rich repertoire of complex behaviours. Here we sequenced, assembled and annotated the Danionella translucida genome employing a hybrid Illumina/Nanopore read library as well as RNA-seq of embryonic, larval and adult mRNA. We achieved high assembly continuity using low-coverage long-read data and annotated a large fraction of the transcriptome. This dataset will pave the way for molecular research and targeted genetic manipulation of the smallest known vertebrate brain.

scholarly journals Growth Dynamics and Survival of Liberibacter crescens BT-1, an Important Model Organism for the Citrus Huanglongbing Pathogen “Candidatus Liberibacter asiaticus”

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Marta Sena-Vélez ◽  
Sean D. Holland ◽  
Manu Aggarwal ◽  
Nick G. Cogan ◽  
Mukesh Jain ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Genetic Manipulation ◽  
Model Organism ◽  
Growth Dynamics ◽  
Culture Conditions ◽  
Citrus Greening ◽  
Close Relative ◽  
Logarithmic Phase ◽  
Content Type ◽  
Liberibacter Asiaticus

ABSTRACT Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen “Candidatus Liberibacter asiaticus,” associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than “Ca. Liberibacter asiaticus” and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10-day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria. IMPORTANCE Liberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by “Candidatus Liberibacter asiaticus” and carried by the Asian citrus psyllid. L. crescens is the only close relative of “Ca. Liberibacter asiaticus” that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens.

scholarly journals Increasing the efficiency of long-read sequencing for hybrid assembly with k-mer-based multiplexing

2019 ◽  
Author(s):  
Alexander Dilthey ◽  
Sebastian A. Meyer ◽  
Achim J. Kaasch
Keyword(s):  
Large Scale ◽  
Bacterial Genome ◽  
Labor Cost ◽  
Hybrid Assembly ◽  
Molecular Barcoding ◽  
Hybrid Genome ◽  
Illumina Data ◽  
Sequencing Cost ◽  
Long Read ◽  
Labor Requirements

AbstractHybrid genome assembly has emerged as an important technique in bacterial genomics, but cost and labor requirements limit large-scale application. We present Ultraplexing, a method to improve per-sample sequencing cost and hands-on-time of Nanopore sequencing for hybrid assembly by at least 50%, compared to molecular barcoding while maintaining high assembly quality (Quality Value; QV ≥ 42). Ultraplexing requires the availability of Illumina data and uses inter-sample genetic variability to assign reads to isolates, which obviates the need for molecular barcoding. Thus, Ultraplexing can enable significant sequencing and labor cost reductions in large-scale bacterial genome projects.

scholarly journals LazyB: fast and cheap genome assembly

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Thomas Gatter ◽  
Sarah von Löhneysen ◽  
Jörg Fallmann ◽  
Polina Drozdova ◽  
Tom Hartmann ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Hybrid Methods ◽  
Computational Effort ◽  
Sequencing Data ◽  
Long Reads ◽  
Hybrid Genome ◽  
Long Read ◽  
Low Coverage ◽  
Genome Assembler ◽  
Large Genomes

Abstract Background Advances in genome sequencing over the last years have lead to a fundamental paradigm shift in the field. With steadily decreasing sequencing costs, genome projects are no longer limited by the cost of raw sequencing data, but rather by computational problems associated with genome assembly. There is an urgent demand for more efficient and and more accurate methods is particular with regard to the highly complex and often very large genomes of animals and plants. Most recently, “hybrid” methods that integrate short and long read data have been devised to address this need. Results is such a hybrid genome assembler. It has been designed specificially with an emphasis on utilizing low-coverage short and long reads. starts from a bipartite overlap graph between long reads and restrictively filtered short-read unitigs. This graph is translated into a long-read overlap graph G. Instead of the more conventional approach of removing tips, bubbles, and other local features, stepwisely extracts subgraphs whose global properties approach a disjoint union of paths. First, a consistently oriented subgraph is extracted, which in a second step is reduced to a directed acyclic graph. In the next step, properties of proper interval graphs are used to extract contigs as maximum weight paths. These path are translated into genomic sequences only in the final step. A prototype implementation of , entirely written in python, not only yields significantly more accurate assemblies of the yeast and fruit fly genomes compared to state-of-the-art pipelines but also requires much less computational effort. Conclusions is new low-cost genome assembler that copes well with large genomes and low coverage. It is based on a novel approach for reducing the overlap graph to a collection of paths, thus opening new avenues for future improvements. Availability The prototype is available at https://github.com/TGatter/LazyB.

scholarly journals Large-scale CRISPRi and transcriptomics of Staphylococcus epidermidis identify genetic factors implicated in commensal-pathogen lifestyle versatility

2021 ◽  
Author(s):  
Michelle Spoto ◽  
Elizabeth Fleming ◽  
Yvette Ondouah Nzutchi ◽  
Changhui Guan ◽  
Julia Oh
Keyword(s):  
Human Skin ◽  
Large Scale ◽  
Genetic Factors ◽  
Genetic Manipulation ◽  
Transcriptional Profiling ◽  
Acid Stress ◽  
Model Organism ◽  
Essential Genes ◽  
Skin Microbiome ◽  
Cell Wall Modification

AbstractStaphylococcus (S.) epidermidis is a ubiquitous human commensal skin bacterium that is also one of the most prevalent nosocomial pathogens. The genetic factors underlying this remarkable lifestyle plasticity are incompletely understood, much due to the difficulties of genetic manipulation, precluding high-throughput functional profiling of this species. To probe S. epidermdis’ versatility to survive across a diversity of skin sites and infection niches, we developed a large-scale CRISPR interference (CRISPRi) screen complemented by transcriptional profiling (RNA-seq) across 24 diverse environmental conditions and piloted a droplet-based CRISPRi approach to enhance throughput and sensitivity. We identified putative essential genes, importantly, revealing amino acid metabolism as crucial to survival across diverse environments, demonstrated the importance of trace metal uptake for survival under multiple stress conditions, and identified condition-specific essential genes for each of our 24 different environments. We identified pathways significantly enriched and repressed across our range of stress and nutrient limited conditions, demonstrating the considerable plasticity of S. epidermidis in responding to environmental stressors. In particular, we postulate a mechanism by which nitrogen metabolism is linked to lifestyle versatility in response to hyperosmotic challenges, such as those encountered on human skin. Finally, by leveraging both transcriptomic and CRISPRi data, we performed a comprehensive analysis of S. epidermidis survival under acid stress and hypothesize a role for cell wall modification as a vital component of the survival response in acidic conditions. Taken together, this study represents one of the first large-scale CRISPRi studies in a non-model organism and the first to integrate transcriptomics data across multiple conditions to provide new biological insights into a keystone member of the human skin microbiome. Our results additionally provide a valuable benchmarking analysis for CRISPRi screens and are rich resource for other staphylococcal researchers.

scholarly journals In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1422
Author(s):  
Ousama Al Shanaa ◽  
Andrey Rumyantsev ◽  
Elena Sambuk ◽  
Marina Padkina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Potential Model ◽  
Model Organism ◽  
Rna Aptamers ◽  
Early Stages ◽  
Near Future

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.

scholarly journals FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yanping Long ◽  
Zhijian Liu ◽  
Jinbu Jia ◽  
Weipeng Mo ◽  
Liang Fang ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Walls ◽  
Large Scale ◽  
Full Length ◽  
Cell Level ◽  
Root Cells ◽  
Rna Profiling ◽  
Different Types ◽  
Long Read ◽  
Single Nucleus

AbstractThe broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.

scholarly journals Recombining Your Way Out of Trouble: The Genetic Architecture of Hybrid Fitness under Environmental Stress

2019 ◽  
Vol 37 (1) ◽  
pp. 167-182 ◽  
Author(s):  
Zebin Zhang ◽  
Devin P Bendixsen ◽  
Thijs Janzen ◽  
Arne W Nolte ◽  
Duncan Greig ◽  
...  
Keyword(s):  
Environmental Stress ◽  
Evolutionary Potential ◽  
Genetic Trait ◽  
Hybrid Fitness ◽  
Mixed Effect ◽  
Mixed Effect Models ◽  
Hybrid Genome ◽  
Chromosome Associations ◽  
Low Coverage ◽  
Negative Epistasis

Abstract Hybridization between species can either promote or impede adaptation. But we know very little about the genetic basis of hybrid fitness, especially in nondomesticated organisms, and when populations are facing environmental stress. We made genetically variable F2 hybrid populations from two divergent Saccharomyces yeast species. We exposed populations to ten toxins and sequenced the most resilient hybrids on low coverage using ddRADseq to investigate four aspects of their genomes: 1) hybridity, 2) interspecific heterozygosity, 3) epistasis (positive or negative associations between nonhomologous chromosomes), and 4) ploidy. We used linear mixed-effect models and simulations to measure to which extent hybrid genome composition was contingent on the environment. Genomes grown in different environments varied in every aspect of hybridness measured, revealing strong genotype–environment interactions. We also found selection against heterozygosity or directional selection for one of the parental alleles, with larger fitness of genomes carrying more homozygous allelic combinations in an otherwise hybrid genomic background. In addition, individual chromosomes and chromosomal interactions showed significant species biases and pervasive aneuploidies. Against our expectations, we observed multiple beneficial, opposite-species chromosome associations, confirmed by epistasis- and selection-free computer simulations, which is surprising given the large divergence of parental genomes (∼15%). Together, these results suggest that successful, stress-resilient hybrid genomes can be assembled from the best features of both parents without paying high costs of negative epistasis. This illustrates the importance of measuring genetic trait architecture in an environmental context when determining the evolutionary potential of genetically diverse hybrid populations.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals Transposition of Tn5367 in Mycobacterium marinum, Using a Conditionally Recombinant Mycobacteriophage

2003 ◽  
Vol 185 (5) ◽  
pp. 1745-1748 ◽  
Author(s):  
Jan Rybniker ◽  
Martina Wolke ◽  
Christiane Haefs ◽  
Georg Plum
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Skin Infection ◽  
Model Organism ◽  
Transposon Mutagenesis ◽  
Mycobacterium Marinum ◽  
Close Relative ◽  
Mutant Library ◽  
Human Pathogen ◽  
Valid Model ◽  
Highly Efficient

ABSTRACT Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis. As with M. tuberculosis, M. marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. Conditions permissive for phage replication in M. tuberculosis facilitated highly efficient transposon delivery in M. marinum. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M. marinum.

scholarly journals Meeting Highlights: Genome Sequencing and Biology 2001

2001 ◽  
Vol 2 (4) ◽  
pp. 243-251
Author(s):  
Jo Wixon
Keyword(s):  
Comparative Genomics ◽  
Functional Genomics ◽  
Genome Sequencing ◽  
Large Scale ◽  
Model Organism

We bring you a report from the CSHL Genome Sequencing and Biology Meeting, which has a long and prestigious history. This year there were sessions on large-scale sequencing and analysis, polymorphisms (covering discovery and technologies and mapping and analysis), comparative genomics of mammalian and model organism genomes, functional genomics and bioinformatics.

Sign in / Sign up

Export Citation Format

Share Document