BACKGROUND: Limited accessibility to intestinal epithelial tissue in wild animals and humans makes it challenging to study patterns of intestinal gene regulation, and hence to monitor physiological status and health in field conditions. To explore solutions to this limitation, we have used a noninvasive approach via fecal RNA-seq, for the quantification of gene expression markers in gastrointestinal cells of free-range primates and a forager human population. Thus, a combination of poly(A) mRNA enrichment and rRNA depletion methods was used in tandem with RNA-seq to quantify and compare gastrointestinal gene expression patterns in fecal samples of wild Gorilla gorilla gorilla (n = 9) and BaAka hunter-gatherers (n = 10) from The Dzanga Sangha Protected Areas, Central African Republic. RESULTS: Although only a small fraction (< 4.9%) of intestinal mRNA signals was recovered, the data was sufficient to detect significant functional differences between gorillas and humans, at the gene and pathway levels. These intestinal gene expression differences were specifically associated with metabolic and immune functions. Additionally, non-host RNA-seq reads were used to gain preliminary insights on the subjects' dietary habits, intestinal microbiomes, and infection prevalence, via identification of fungi, nematode, arthropod and plant RNA. CONCLUSIONS: Overall, the results suggest that fecal RNA-seq, targeting gastrointestinal epithelial cells can be used to evaluate primate intestinal physiology and gut gene regulation, in samples obtained in challenging conditions in situ. The approach used herein may be useful to obtain information on primate intestinal health, while revealing preliminary insights into foraging ecology, microbiome, and diet.

• Klíčová slova
• Gene expression, Nonhuman primate, Noninvasive method, RNA-seq,
• MeSH
• feces * MeSH
• gastrointestinální trakt metabolismus   MeSH
• Gorilla gorilla genetika   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• poly A genetika   MeSH
• sekvenování transkriptomu * MeSH
• stanovení celkové genové exprese * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• poly A MeSH

The immune system is a complex and sophisticated biological system, spanning multiple levels of complexity, from the molecular level to that of tissue. Our current understanding of its function and complexity, of the heterogeneity of leukocytes, is a result of decades of concentrated efforts to delineate cellular markers using conventional methods of antibody screening and antigen identification. In mammalian models, this led to in-depth understanding of individual leukocyte subsets, their phenotypes, and their roles in health and disease. The field was further propelled forward by the development of single-cell (sc) RNA-seq technologies, offering an even broader and more integrated view of how cells work together to generate a particular response. Consequently, the adoption of scRNA-seq revealed the unexpected plasticity and heterogeneity of leukocyte populations and shifted several long-standing paradigms of immunology. This review article highlights the unprecedented opportunities offered by scRNA-seq technology to unveil the individual contributions of leukocyte subsets and their crosstalk in generating the overall immune responses in bony fishes. Single-cell transcriptomics allow identifying unseen relationships, and formulating novel hypotheses tailored for teleost species, without the need to rely on the limited number of fish-specific antibodies and pre-selected markers. Several recent studies on single-cell transcriptomes of fish have already identified previously unnoticed expression signatures and provided astonishing insights into the diversity of teleost leukocytes and the evolution of vertebrate immunity. Without a doubt, scRNA-seq in tandem with bioinformatics tools and state-of-the-art methods, will facilitate studying the teleost immune system by not only defining key markers, but also teaching us about lymphoid tissue organization, development/differentiation, cell-cell interactions, antigen receptor repertoires, states of health and disease, all across time and space in fishes. These advances will invite more researchers to develop the tools necessary to explore the immunology of fishes, which remain non-conventional animal models from which we have much to learn.

• Klíčová slova
• RNA-seq, antibody, flow cytometry, leukocytes, lymphocytes, phenotype, single-cell transcriptome, teleost bony fish,
• MeSH
• analýza jednotlivých buněk * metody   MeSH
• imunita MeSH
• leukocyty imunologie   metabolismus   MeSH
• ryby genetika   imunologie   MeSH
• sekvenování transkriptomu * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Significant innovations in next-generation sequencing techniques and bioinformatics tools have impacted our appreciation and understanding of RNA. Practical RNA sequencing (RNA-Seq) applications have evolved in conjunction with sequence technology and bioinformatic tools advances. In most projects, bulk RNA-Seq data is used to measure gene expression patterns, isoform expression, alternative splicing and single-nucleotide polymorphisms. However, RNA-Seq holds far more hidden biological information including details of copy number alteration, microbial contamination, transposable elements, cell type (deconvolution) and the presence of neoantigens. Recent novel and advanced bioinformatic algorithms developed the capacity to retrieve this information from bulk RNA-Seq data, thus broadening its scope. The focus of this review is to comprehend the emerging bulk RNA-Seq-based analyses, emphasizing less familiar and underused applications. In doing so, we highlight the power of bulk RNA-Seq in providing biological insights.

• Klíčová slova
• RNA-Seq applications, RNA-Seq genomics, bioinformatics advancements, next-generation sequencing, transcriptomicsomics,
• MeSH
• algoritmy MeSH
• alternativní sestřih MeSH
• jednonukleotidový polymorfismus MeSH
• sekvenční analýza RNA metody   MeSH
• výpočetní biologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Transcriptome sequencing (RNA-seq) is widely used to detect gene rearrangements and quantitate gene expression in acute lymphoblastic leukemia (ALL), but its utility and accuracy in identifying copy number variations (CNVs) has not been well described. CNV information inferred from RNA-seq can be highly informative to guide disease classification and risk stratification in ALL due to the high incidence of aneuploid subtypes within this disease. Here we describe RNAseqCNV, a method to detect large scale CNVs from RNA-seq data. We used models based on normalized gene expression and minor allele frequency to classify arm level CNVs with high accuracy in ALL (99.1% overall and 98.3% for non-diploid chromosome arms, respectively), and the models were further validated with excellent performance in acute myeloid leukemia (accuracy 99.8% overall and 99.4% for non-diploid chromosome arms). RNAseqCNV outperforms alternative RNA-seq based algorithms in calling CNVs in the ALL dataset, especially in samples with a high proportion of CNVs. The CNV calls were highly concordant with DNA-based CNV results and more reliable than conventional cytogenetic-based karyotypes. RNAseqCNV provides a method to robustly identify copy number alterations in the absence of DNA-based analyses, further enhancing the utility of RNA-seq to classify ALL subtype.

• MeSH
• algoritmy MeSH
• karyotypizace MeSH
• lidé MeSH
• sekvenování transkriptomu MeSH
• variabilita počtu kopií segmentů DNA * genetika   MeSH
• vysoce účinné nukleotidové sekvenování * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

• Klíčová slova
• NudC, RNA capping, RNA polymerase, RNA-seq, Rai1, nicotinamide adenine dinucleotide, non-canonical initiating nucleotide, transcription, transcription initiation, transcription start site,
• MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• endoribonukleasy metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu genetika   MeSH
• genetická transkripce genetika   MeSH
• NAD metabolismus   MeSH
• nukleotidy genetika   MeSH
• počátek transkripce fyziologie   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• RNA čepičky genetika   MeSH
• transkriptom genetika   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• endoribonukleasy MeSH
• mRNA decapping enzymes MeSH Prohlížeč
• NAD MeSH
• nukleotidy MeSH
• RNA čepičky MeSH

BACKGROUND: Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data. RESULTS: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation. CONCLUSIONS: We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.

• Klíčová slova
• ABE fermentation, Clostridium beijerinckii NRRL B-598, RNA-Seq transcriptome,
• MeSH
• bakteriofágy genetika   MeSH
• butanoly metabolismus   MeSH
• Clostridium beijerinckii genetika   metabolismus   virologie   MeSH
• DNA virů genetika   MeSH
• fermentace MeSH
• genetická transkripce MeSH
• kinetika MeSH
• pseudogeny genetika   MeSH
• sekvenční analýza RNA * MeSH
• stanovení celkové genové exprese * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• butanoly MeSH
• DNA virů MeSH

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized transcripts is still limited to a few models. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four vertebrate models, two amphibians (Xenopus laevis, Ambystoma mexicanum) and two fishes (Acipenser ruthenus, Danio rerio) using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important transcripts such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. It was also observed that there was a poor correlation between the vegetally localized transcripts but a relatively good correlation between the animally localized transcripts. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.

• Klíčová slova
• Amphibians, Egg, Fishes, RNA localization, TOMO-Seq, evo devo,
• MeSH
• biologická evoluce MeSH
• dánio pruhované MeSH
• oocyty * metabolismus   MeSH
• RNA * genetika   metabolismus   MeSH
• Xenopus laevis genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA * MeSH

We review current studies of plant mitochondrial transcriptomes performed by RNA-seq, highlighting methodological challenges unique to plant mitochondria. We propose ways to improve read mapping accuracy and sensitivity such as modifying a reference genome at RNA editing sites, using splicing- and ambiguity-competent aligners, and masking chloroplast- or nucleus-derived sequences. We also outline modified RNA-seq methods permitting more accurate detection and quantification of partially edited sites and the identification of transcription start sites on a genome-wide scale. The application of RNA-seq goes beyond genome-wide determination of transcript levels and RNA maturation events, and emerges as an elegant resource for the comprehensive identification of editing, splicing, and transcription start sites. Thus, improved RNA-seq methods customized for plant mitochondria hold tremendous potential for advancing our understanding of plant mitochondrial evolution and cyto-nuclear interactions in a broad array of plant species.

• MeSH
• editace RNA genetika   MeSH
• genom mitochondriální genetika   MeSH
• mitochondrie genetika   MeSH
• rostliny genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• transkriptom genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology.

• Klíčová slova
• Alternative splicing, Bone cells, Non-coding RNA, RNAseq, Topological domains,
• MeSH
• alternativní sestřih MeSH
• buněčná diferenciace genetika   MeSH
• kultivované buňky MeSH
• lebka fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• osteoblasty fyziologie   MeSH
• osteogeneze genetika   MeSH
• RNA analýza   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA MeSH

BACKGROUND: The international, multicenter registry LOGGIC Core BioClinical Data Bank aims to enhance the understanding of tumor biology in pediatric low-grade glioma (pLGG) and provide clinical and molecular data to support treatment decisions and interventional trial participation. Hence, the question arises whether implementation of RNA sequencing (RNA-Seq) using fresh frozen (FrFr) tumor tissue in addition to gene panel and DNA methylation analysis improves diagnostic accuracy and provides additional clinical benefit. METHODS: Analysis of patients aged 0 to 21 years, enrolled in Germany between April 2019 and February 2021, and for whom FrFr tissue was available. Central reference histopathology, immunohistochemistry, 850k DNA methylation analysis, gene panel sequencing, and RNA-Seq were performed. RESULTS: FrFr tissue was available in 178/379 enrolled cases. RNA-Seq was performed on 125 of these samples. We confirmed KIAA1549::BRAF-fusion (n = 71), BRAF V600E-mutation (n = 12), and alterations in FGFR1 (n = 14) as the most frequent alterations, among other common molecular drivers (n = 12). N = 16 cases (13%) presented rare gene fusions (eg, TPM3::NTRK1, EWSR1::VGLL1, SH3PXD2A::HTRA1, PDGFB::LRP1, GOPC::ROS1). In n = 27 cases (22%), RNA-Seq detected a driver alteration not otherwise identified (22/27 actionable). The rate of driver alteration detection was hereby increased from 75% to 97%. Furthermore, FGFR1 internal tandem duplications (n = 6) were only detected by RNA-Seq using current bioinformatics pipelines, leading to a change in analysis protocols. CONCLUSIONS: The addition of RNA-Seq to current diagnostic methods improves diagnostic accuracy, making precision oncology treatments (MEKi/RAFi/ERKi/NTRKi/FGFRi/ROSi) more accessible. We propose to include RNA-Seq as part of routine diagnostics for all pLGG patients, especially when no common pLGG alteration was identified.

• Klíčová slova
• RNA sequencing, actionable drivers, molecular profiling, pLGG, rare gene fusions,
• MeSH
• dítě MeSH
• DNA vazebné proteiny genetika   MeSH
• gliom * patologie   MeSH
• individualizovaná medicína MeSH
• lidé MeSH
• molekulární patologie MeSH
• protoonkogenní proteiny B-Raf * genetika   MeSH
• protoonkogenní proteiny genetika   MeSH
• sekvenování transkriptomu MeSH
• transkripční faktory genetika   MeSH
• tyrosinkinasy MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• protoonkogenní proteiny B-Raf * MeSH
• protoonkogenní proteiny MeSH
• transkripční faktory MeSH
• tyrosinkinasy MeSH
• VGLL1 protein, human MeSH Prohlížeč