RNA-seq analysis
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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
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.
- 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
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.
Melanoma phenotype plasticity underlies tumour dissemination and resistance to therapy, yet its regulation is incompletely understood. In vivo switching between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype is directed by the tumour microenvironment. We found that treatment of partially dedifferentiated, invasive A375M2 cells with two structurally unrelated p38 MAPK inhibitors, SB2021920 and BIRB796, induces a phenotype switch in 3D collagen, as documented by increased expression of melanocyte differentiation markers and a loss of invasive phenotype markers. The phenotype is accompanied by morphological change corresponding to amoeboid-mesenchymal transition. We performed RNA sequencing with an Illumina HiSeq platform to fully characterise transcriptome changes underlying the switch. Gene expression results obtained with RNA-seq were validated by comparing them with RT-qPCR. Transcriptomic data generated in the study will extend the present understanding of phenotype plasticity in melanoma and its contribution to invasion and metastasis.
- MeSH
- buněčná diferenciace účinky léků genetika MeSH
- fenotyp MeSH
- genová ontologie MeSH
- imidazoly farmakologie MeSH
- inhibitory proteinkinas farmakologie MeSH
- kolagen metabolismus MeSH
- lidé MeSH
- melanom genetika patologie MeSH
- mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové mikroprostředí účinky léků genetika MeSH
- naftaleny farmakologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proliferace buněk účinky léků genetika MeSH
- pyrazoly farmakologie MeSH
- pyridiny farmakologie MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- sekvenování transkriptomu metody MeSH
- stanovení celkové genové exprese metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.
- MeSH
- analýza jednotlivých buněk * metody MeSH
- B-lymfocyty metabolismus cytologie MeSH
- buněčný cyklus * genetika MeSH
- ChiP sekvenování metody MeSH
- chromatin * metabolismus chemie genetika MeSH
- fibroblasty metabolismus cytologie MeSH
- lidé MeSH
- sekvenování transkriptomu metody MeSH
- telomery * genetika MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Aim: Human induced pluripotent stem cells (iPSCs) are inefficiently derived from somatic cells by overexpression of defined transcription factors. Overexpression of H2A histone variant macroH2A1.1, but not macroH2A1.2, leads to increased iPSC reprogramming by unclear mechanisms. Materials & methods: Cleavage under targets and tagmentation (CUT&Tag) allows robust epigenomic profiling of a low cell number. We performed an integrative CUT&Tag-RNA-Seq analysis of macroH2A1-dependent orchestration of iPSCs reprogramming using human endothelial cells. Results: We demonstrate wider genome occupancy, predicted transcription factors binding, and gene expression regulated by macroH2A1.1 during reprogramming, compared to macroH2A1.2. MacroH2A1.1, previously associated with neurodegenerative pathologies, specifically activated ectoderm/neural processes. Conclusion: CUT&Tag and RNA-Seq data integration is a powerful tool to investigate the epigenetic mechanisms occurring during cell reprogramming.
- MeSH
- endoteliální buňky metabolismus MeSH
- histony * metabolismus MeSH
- indukované pluripotentní kmenové buňky * metabolismus MeSH
- lidé MeSH
- přeprogramování buněk genetika MeSH
- sekvenování transkriptomu MeSH
- transkripční faktory genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- 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
Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U-containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by alkylation repair homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble U-containing tRNAs. In addition, both our approaches revealed ALKBH8 binding to several other types of noncoding RNAs, in particular C/D box snoRNAs.
Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.
