Curare and GenExVis: a versatile toolkit for analyzing and visualizing RNA-Seq data.

Even though high-throughput transcriptome sequencing is routinely performed in many laboratories, computational analysis of such data remains a cumbersome process often executed manually, hence error-prone and lacking reproducibility. For corresponding data processing, we introduce Curare, an easy-to-use yet versatile workflow builder for analyzing high-throughput RNA-Seq data focusing on differential gene expression experiments. Data analysis with Curare is customizable and subdivided into preprocessing, quality control, mapping, and downstream analysis stages, providing multiple options for each step while ensuring the reproducibility of the workflow. For a fast and straightforward exploration and visualization of differential gene expression results, we provide the gene expression visualizer software GenExVis. GenExVis can create various charts and tables from simple gene expression tables and DESeq2 results without the requirement to upload data or install software packages. In combination, Curare and GenExVis provide a comprehensive software environment that supports the entire data analysis process, from the initial handling of raw RNA-Seq data to the final DGE analyses and result visualizations, thereby significantly easing data processing and subsequent interpretation.

Bioactivity Profiling of In Silico Predicted Linear Toxins from the Ants Myrmica rubra and Myrmica ruginodis.

The venoms of ants (Formicidae) are a promising source of novel bioactive molecules with potential for clinical and agricultural applications. However, despite the rich diversity of ant species, only a fraction of this vast resource has been thoroughly examined in bioprospecting programs. Previous studies focusing on the venom of Central European ants (subfamily Myrmicinae) identified a number of short linear decapeptides and nonapeptides resembling antimicrobial peptides (AMPs). Here, we describe the in silico approach and bioactivity profiling of 10 novel AMP-like peptides from the fellow Central European myrmicine ants Myrmica rubra and Myrmica ruginodis. Using the sequences of known ant venom peptides as queries, we screened the venom gland transcriptomes of both species. We found transcripts of nine novel decapeptides and one novel nonapeptide. The corresponding peptides were synthesized for bioactivity profiling in a broad panel of assays consisting of tests for cytotoxicity as well as antiviral, insecticidal, and antimicrobial activity. U-MYRTX-Mrug5a showed moderately potent antimicrobial effects against several bacteria, including clinically relevant pathogens such as Listeria monocytogenes and Staphylococcus epidermidis, but high concentrations showed negligible cytotoxicity. U-MYRTX-Mrug5a is, therefore, a probable lead for the development of novel peptide-based antibiotics.

Venomics of the Central European Myrmicine Ants Myrmica rubra and Myrmica ruginodis.

Animal venoms are a rich source of novel biomolecules with potential applications in medicine and agriculture. Ants are one of the most species-rich lineages of venomous animals. However, only a fraction of their biodiversity has been studied so far. Here, we investigated the venom components of two myrmicine (subfamily Myrmicinae) ants: Myrmica rubra and Myrmica ruginodis. We applied a venomics workflow based on proteotranscriptomics and found that the venoms of both species are composed of several protein classes, including venom serine proteases, cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 (CAP) superfamily proteins, Kunitz-type serine protease inhibitors and venom acid phosphatases. Several of these protein classes are known venom allergens, and for the first time we detected phospholipase A1 in the venom of M. ruginodis. We also identified two novel epidermal growth factor (EGF) family toxins in the M. ruginodis venom proteome and an array of additional EGF-like toxins in the venom gland transcriptomes of both species. These are similar to known toxins from the related myrmicine ant, Manica rubida, and the myrmecine (subfamily Myrmeciinae) Australian red bulldog ant Myrmecia gullosa, and are possibly deployed as weapons in defensive scenarios or to subdue prey. Our work suggests that M.rubra and M. ruginodis venoms contain many enzymes and other high-molecular-weight proteins that cause cell damage. Nevertheless, the presence of EGF-like toxins suggests that myrmicine ants have also recruited smaller peptide components into their venom arsenal. Although little is known about the bioactivity and function of EGF-like toxins, their presence in myrmicine and myrmecine ants suggests they play a key role in the venom systems of the superfamily Formicoidea. Our work adds to the emerging picture of ant venoms as a source of novel bioactive molecules and highlights the need to incorporate such taxa in future venom bioprospecting programs.

Next-Generation Sequencing Analysis of the Tineola bisselliella Larval Gut Transcriptome Reveals Candidate Enzymes for Keratin Digestion.

The clothes moth Tineola bisselliella is one of a few insects that can digest keratin, leading to the destruction of clothing, textiles and artwork. The mechanism of keratin digestion is not yet fully understood, partly reflecting the lack of publicly available genomic and transcriptomic data. Here we present a high-quality gut transcriptome of T. bisselliella generated from larvae reared on keratin-rich and keratin-free diets. The overall transcriptome consists of 428,221 contigs that were functionally annotated and screened for candidate enzymes involved in keratin utilization. As a mechanism for keratin digestion, we identified cysteine synthases, cystathionine ß-synthases and cystathionine γ-lyases. These enzymes release hydrogen sulfite, which may reduce the disulfide bonds in keratin. The dataset also included 27 differentially expressed contigs with trypsin domains, among which 20 were associated with keratin feeding. Finally, we identified seven collagenases that were upregulated on the keratin-rich diet. In addition to this enzymatic repertoire potentially involved in breaking down keratin, our analysis of poly(A)-enriched and poly(A)-depleted transcripts suggested that T. bisselliella larvae possess an unstable intestinal microbiome that may nevertheless contribute to keratin digestion.

Genomic analysis of novel Yarrowia-like yeast symbionts associated with the carrion-feeding burying beetle Nicrophorus vespilloides.

BACKGROUND: Mutualistic interactions with microbes can help insects adapt to extreme environments and unusual diets. An intriguing example is the burying beetle Nicrophorus vespilloides, which feeds and reproduces on small vertebrate carcasses. Its fungal microbiome is dominated by yeasts that potentially facilitate carcass utilization by producing digestive enzymes, eliminating cadaver-associated toxic volatiles (that would otherwise attract competitors), and releasing antimicrobials to sanitize the microenvironment. Some of these yeasts are closely related to the biotechnologically important species Yarrowia lipolytica. RESULTS: To investigate the roles of these Yarrowia-like yeast (YLY) strains in more detail, we selected five strains from two different phylogenetic clades for third-generation sequencing and genome analysis. The first clade, represented by strain B02, has a 20-Mb genome containing ~ 6400 predicted protein-coding genes. The second clade, represented by strain C11, has a 25-Mb genome containing ~ 6300 predicted protein-coding genes, and extensive intraspecific variability within the ITS-D1/D2 rDNA region commonly used for species assignments. Phenotypic microarray analysis revealed that both YLY strains were able to utilize a diverse range of carbon and nitrogen sources (including microbial metabolites associated with putrefaction), and can grow in environments with extreme pH and salt concentrations. CONCLUSIONS: The genomic characterization of five yeast strains isolated from N. vespilloides resulted in the identification of strains potentially representing new YLY species. Given their abundance in the beetle hindgut, and dominant growth on beetle-prepared carcasses, the analysis of these strains has revealed the genetic basis of a potential symbiotic relationship between yeasts and burying beetles that facilitates carcass digestion and preservation.

Cottonseed Press Cake as a Potential Diet for Industrially Farmed Black Soldier Fly Larvae Triggers Adaptations of Their Bacterial and Fungal Gut Microbiota.

Black soldier fly larvae (Hermetia illucens, Diptera: Stratiomyidae) are used for the bioconversion of organic side products into valuable compounds such as proteins, lipids and chitin. However, the economic competitiveness of farmed insects compared to conventional protein production systems in agriculture and aquaculture depends on the availability of large quantities of inexpensive insect feed. Cottonseed press cake (CPC) is a side-stream of cotton production that is rich in proteins and lipids but unsuitable as feed for several farmed animals, except ruminants, due to the presence of the anti-nutritional sesquiterpenoid gossypol. Here, we tested CPC as a feed for black soldier fly larvae and studied the impact of this diet on the gut microbiome. Larvae reared on CPC developed normally and even showed a shorter life-cycle, but were smaller at the end of larval development than control larvae reared on chicken feed. The adaptability of the larvae to different diets is mediated by their versatile gut microbiome, which facilitates digestion and detoxification. We therefore used amplicon sequencing to analyze the bacterial and fungal communities associated with larvae reared on each diet, revealing differences between the larval guts and frass (residual feed substrate) as well as differences between the two diet groups. For example, Actinomycetaceae and Aspergillaceae were significantly enriched in guts of the CPC diet group and may help to metabolize compounds such as gossypol. Potentially probiotic yeasts and beneficial Enterobacteriaceae, which presumably belong to the core microbiota, were detected in high relative abundance in the gut and frass, indicating a functional role of these microbes, especially the protection against pathogens. We conclude that CPC may be suitable as an inexpensive and environmentally sustainable feed for the industrial rearing of black soldier flies.

Expansion and re-classification of the extracytoplasmic function (ECF) σ factor family.

Extracytoplasmic function σ factors (ECFs) represent one of the major bacterial signal transduction mechanisms in terms of abundance, diversity and importance, particularly in mediating stress responses. Here, we performed a comprehensive phylogenetic analysis of this protein family by scrutinizing all proteins in the NCBI database. As a result, we identified an average of ∼10 ECFs per bacterial genome and 157 phylogenetic ECF groups that feature a conserved genetic neighborhood and a similar regulation mechanism. Our analysis expands previous classification efforts ∼50-fold, enriches many original ECF groups with previously unclassified proteins and identifies 22 entirely new ECF groups. The ECF groups are hierarchically related to each other and are further composed of subgroups with closely related sequences. This two-tiered classification allows for the accurate prediction of common promoter motifs and the inference of putative regulatory mechanisms across subgroups composing an ECF group. This comprehensive, high-resolution description of the phylogenetic distribution of the ECF family, together with the massive expansion of classified ECF sequences and an openly accessible data repository called 'ECF Hub' (https://www.computational.bio.uni-giessen.de/ecfhub), will serve as a powerful hypothesis-generator to guide future research in the field.

Larvae of the Clothing Moth Tineola bisselliella Maintain Gut Bacteria that Secrete Enzyme Cocktails to Facilitate the Digestion of Keratin.

The evolutionary success of insects is promoted by their association with beneficial microbes that enable the utilization of unusual diets. The synanthropic clothing moth Tineola bisselliella provides an intriguing example of this phenomenon. The caterpillars of this species have adapted to feed on keratin-rich diets such as feathers and wool, which cannot be digested by most other animals and are resistant to common digestive enzymes. Inspired by the hypothesis that this ability may be conferred by symbiotic microbes, we utilized a simple assay to detect keratinase activity and a method to screen gut bacteria for candidate enzymes, which were isolated from feather-fed larvae. The isolation of DNA from keratin-degrading bacterial strains followed by de novo genome sequencing resulted in the identification of a novel bacterial strain related to Bacillus sp. FDAARGOS_235. Genome annotation identified 20 genes with keratinase domains. Proteomic analysis of the culture supernatant from this gut bacterium grown in non-nutrient buffer supplemented with feathers revealed several candidate enzymes potentially responsible for keratin degradation, including a thiol-disulfide oxidoreductase and multiple proteases. Our results suggest that the unusual diet of T. bisselliella larvae promotes their association with keratinolytic microorganisms and that the ability of larvae to feed on keratin can at least partially be attributed to bacteria that produce a cocktail of keratin-degrading enzymes.

A reference genome of the Chinese hamster based on a hybrid assembly strategy.

Accurate and complete genome sequences are essential in biotechnology to facilitate genome-based cell engineering efforts. The current genome assemblies for Cricetulus griseus, the Chinese hamster, are fragmented and replete with gap sequences and misassemblies, consistent with most short-read-based assemblies. Here, we completely resequenced C. griseus using single molecule real time sequencing and merged this with Illumina-based assemblies. This generated a more contiguous and complete genome assembly than either technology alone, reducing the number of scaffolds by >28-fold, with 90% of the sequence in the 122 longest scaffolds. Most genes are now found in single scaffolds, including up- and downstream regulatory elements, enabling improved study of noncoding regions. With >95% of the gap sequence filled, important Chinese hamster ovary cell mutations have been detected in draft assembly gaps. This new assembly will be an invaluable resource for continued basic and pharmaceutical research.

Integrative analysis of DNA methylation and gene expression in butyrate-treated CHO cells.

The cellular mechanisms responsible for the versatile properties of CHO cells as the major production cell line for biopharmaceutical molecules are not entirely understood yet, although several 'omics' data facilitate the understanding of CHO cells and their reactions to environmental conditions. However, genome-wide studies of epigenetic processes such as DNA methylation are still limited. To prove the applicability and usefulness of integrating DNA methylation and gene expression data in a biotechnological context, we exemplarily analyzed the time course of cellular reactions upon butyrate addition in antibody-producing CHO cells by whole-genome bisulfite sequencing and CHO-specific cDNA microarrays. Gene expression and DNA methylation analyses showed that pathways known to be affected by butyrate, including cell cycle and apoptosis, as well as pathways potentially involved in butyrate-induced hyperproductivity such as central energy metabolism and protein biosynthesis were affected. Differentially methylated regions were furthermore found to contain binding-site motifs of specific transcription factors and were hypothesized to represent regulatory regions closely connected to the cellular response to butyrate. Generally, our experiment underlines the benefit of integrating DNA methylation and gene expression data, as it provided potential novel candidate genes for rational cell line development and allowed for new insights into the butyrate effect on CHO cells.

The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms.

The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C. jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C. utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid.

Complete Genome Sequence and Annotation of Corynebacterium singulare DSM 44357, Isolated from a Human Semen Specimen.

Corynebacterium singulare DSM 44357 is a urease-positive microorganism isolated from human semen. The complete genome sequence of C. singulare DSM 44357 comprises 2,830,519 bp with a mean G+C content of 60.12% and 2,581 protein-coding genes. The deduced antibiotic resistance pattern of this strain includes macrolides, lincosamides, aminoglycosides, chloramphenicol, and tetracyline.

The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells.

Chinese hamster ovary (CHO) cells represent the most commonly used production cell line for therapeutic proteins. By recent genome and transcriptome sequencing a basis was created for future investigations of genotype-phenotype relationships and for improvement of CHO cell productivity and product quality. In this context information is missing about DNA cytosine methylation as a crucial epigenetic modification and an important element in mammalian genome regulation and development. Here, we present the first DNA methylation map of a CHO cell line in single-base resolution that was generated by whole genome bisulfite sequencing combined with gene expression analysis by CHO microarrays. We show CHO DP-12 cells to exhibit global hypomethylation compared to a majority of mammalian methylomes and hypermethylation of CpG-dense regions at gene promoters called CpG islands. We also observed partially methylated domains that cover 62% of the CHO DP-12 cell genome and contain functional clusters of genes. Gene expression analysis showed these clusters to be either highly or weakly expressed with regard to CHO-specific characteristics and hence proves DNA methylation in CHO cells to be an important link between genomics and transcriptomics.

Complete Genome Sequence of Corynebacterium ureicelerivorans DSM 45051, a Lipophilic and Urea-Splitting Isolate from the Blood Culture of a Septicemia Patient.

Corynebacterium ureicelerivorans is an opportunistic pathogen with a lipophilic lifestyle and an exceptionally high urease activity. The genome sequence of the type strain revealed that lipophilism is caused by the lack of a fatty acid synthase gene. The ureABCEFGD genes are similar to the urease gene region of Corynebacterium glucuronolyticum.

Discovery of transcription start sites in the Chinese hamster genome by next-generation RNA sequencing.

Chinese hamster ovary (CHO) cell lines are one of the major production tools for monoclonal antibodies, recombinant proteins, and therapeutics. Although many efforts have significantly improved the availability of sequence information for CHO cells in the last years, forthcoming draft genomes still lack the information depth known from the mouse or human genomes. Many genes annotated for CHO cells and the Chinese hamster reference genome still are in silico predictions, only insufficiently verified by biological experiments. The correct annotation of transcription start sites (TSSs) is of special interest for CHO cells, as these directly define the location of the eukaryotic core promoter. Our study aims to elucidate these largely unexplored regions, trying to shed light on promoter landscapes in the Chinese hamster genome. Based on a 5' enriched dual library RNA sequencing approach 6547 TSSs were identified, of which over 90% were assigned to known genes. These TSSs were used to perform extensive promoter studies using a novel, modular bioinformatics pipeline, incorporating analyses of important regulatory elements of the eukaryotic core promoter on per-gene level and on genomic scale.

Complete Genome Sequence of Corynebacterium falsenii DSM 44353 To Study the Evolution of Corynebacterium Cluster 3 Species.

Corynebacterium falsenii is a member of the natural microflora of wild and domesticated birds and is rarely detected in human clinical specimens. The chromosomal sequence of the type strain C. falsenii DSM 44353 comprises 2,677,607 bp and provides detailed insights into the evolution of Corynebacterium species assigned to the highly diverse cluster 3.

Transcriptome analyses of CHO cells with the next-generation microarray CHO41K: development and validation by analysing the influence of the growth stimulating substance IGF-1 substitute LongR(3.).

The increasing importance of Chinese hamster ovary (CHO) cells for the production of pharmaceutical proteins has awakened the demand to understand the cellular metabolism of these cells. However, satisfactory gene expression studies have yet been impractical due to insufficient coverage of sequences. In this work, previously determined sequence information of CHO cells and newly derived data from 454 and Illumina sequencing was used to establish the CHO41K microarray which contains 41,304 probes. Self-hybridisation was performed for replica determination and samples were run in triplicates to increase statistical power. For determination of technical variance, confidence intervals at an M-value of ±0.6 for 95% and at ±2.3 for 99% of the probes were calculated. Intra-microarray and slide to slide variance was not detectable. In a first application, this microarray enabled an in-depth look inside the cellular transcriptome of CHO cells cultured in the presence or absence of the growth supporting substance "insulin like growth factor 1" (IGF-1) analogue LongR(3). Its effect on the cells ranged from enhanced growth to delay of cell death as well as cytoskeletal installation. Suggesting that under supplementation, a minimised cellular effort in installation of a large cytoskeleton occurs, possibly in favour of promoting faster cell division.

Construction of a public CHO cell line transcript database using versatile bioinformatics analysis pipelines.

Chinese hamster ovary (CHO) cell lines represent the most commonly used mammalian expression system for the production of therapeutic proteins. In this context, detailed knowledge of the CHO cell transcriptome might help to improve biotechnological processes conducted by specific cell lines. Nevertheless, very few assembled cDNA sequences of CHO cells were publicly released until recently, which puts a severe limitation on biotechnological research. Two extended annotation systems and web-based tools, one for browsing eukaryotic genomes (GenDBE) and one for viewing eukaryotic transcriptomes (SAMS), were established as the first step towards a publicly usable CHO cell genome/transcriptome analysis platform. This is complemented by the development of a new strategy to assemble the ca. 100 million reads, sequenced from a broad range of diverse transcripts, to a high quality CHO cell transcript set. The cDNA libraries were constructed from different CHO cell lines grown under various culture conditions and sequenced using Roche/454 and Illumina sequencing technologies in addition to sequencing reads from a previous study. Two pipelines to extend and improve the CHO cell line transcripts were established. First, de novo assemblies were carried out with the Trinity and Oases assemblers, using varying k-mer sizes. The resulting contigs were screened for potential CDS using ESTScan. Redundant contigs were filtered out using cd-hit-est. The remaining CDS contigs were re-assembled with CAP3. Second, a reference-based assembly with the TopHat/Cufflinks pipeline was performed, using the recently published draft genome sequence of CHO-K1 as reference. Additionally, the de novo contigs were mapped to the reference genome using GMAP and merged with the Cufflinks assembly using the cuffmerge software. With this approach 28,874 transcripts located on 16,492 gene loci could be assembled. Combining the results of both approaches, 65,561 transcripts were identified for CHO cell lines, which could be clustered by sequence identity into 17,598 gene clusters.

Whole-Genome Sequence of the Clinical Strain Corynebacterium argentoratense DSM 44202, Isolated from a Human Throat Specimen.

Corynebacterium argentoratense is part of the human skin microbiota and is occasionally detected in the upper respiratory tract of patients suffering from tonsillitis. The complete DNA sequence of the type strain DSM 44202 comprises 2,031,902 bp, yielding the smallest genome sequenced thus far for a corynebacterium associated with humans.