Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.
- MeSH
- alternativní sestřih MeSH
- analýza jednotlivých buněk metody MeSH
- antigeny CD45 * genetika metabolismus MeSH
- leukocyty metabolismus imunologie MeSH
- lidé MeSH
- myši MeSH
- protein - isoformy genetika MeSH
- sekvenční analýza RNA metody MeSH
- software * MeSH
- stanovení celkové genové exprese metody MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.
- MeSH
- genom virový MeSH
- lidé MeSH
- počátek transkripce * MeSH
- promotorové oblasti (genetika) MeSH
- RNA virová genetika MeSH
- sekvenční analýza RNA * metody MeSH
- stanovení celkové genové exprese MeSH
- transkriptom * MeSH
- virus opičích neštovic genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.
- MeSH
- DNA genetika MeSH
- genetická predispozice k nemoci * MeSH
- lidé MeSH
- nádory genetika diagnóza MeSH
- reprodukovatelnost výsledků MeSH
- RNA genetika MeSH
- sekvenční analýza DNA metody MeSH
- sekvenční analýza RNA metody MeSH
- vysoce účinné nukleotidové sekvenování * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We aimed to explore the development and cell communication of osteoblasts and osteoclasts with aneuploidy variation in giant cell tumour of bone (GCTB). We predicted the diploid and aneuploid cells in tissue samples using the CopyKAT package. The Monocle2 package was used to analyse differentiation trajectories of aneuploid cells. We used the CellChat package to observe the signalling pathways and ligand-receptor pairs for the two interaction types, "Cell-Cell Contact" and "Secreted Signalling", respectively. A total of 9,117 cells were obtained including eight cell types. Most aneuploid cells were osteoblasts. As the cell differentiation trajectory matured, we found that aneuploid osteoblasts first increased the inflammatory response activity and then enhanced the ability to activate T cells, whereas osteoclasts gradually enhanced the cellular energy metabolism, cell adhesion, cell proliferation and immune response; the activated biological functions were gradually weakened. The analysis by CellChat indicated that CTLA4 or TIGIT might act as important immune checkpoint genes to attenuate the inhibitory effect of aneuploid osteoclasts on NK/T cells, thereby enhancing the activity of NK/T cells. Our study found that both osteoblasts and osteoclasts might be involved in the development of GCTB, which may provide a new direction for the treatment of GCTB.
- MeSH
- analýza jednotlivých buněk * MeSH
- aneuploidie * MeSH
- buněčná diferenciace genetika MeSH
- lidé MeSH
- mezibuněčná komunikace * genetika MeSH
- nádory kostí genetika patologie metabolismus MeSH
- obrovskobuněčný nádor kosti * genetika patologie MeSH
- osteoblasty * metabolismus MeSH
- osteoklasty * metabolismus patologie MeSH
- sekvenční analýza RNA metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen), as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq), or unique molecular identifiers (QIAseq). There was no single protocol outperforming others across all metrics. Limited overlap of measured miRNA profiles was documented between methods largely owing to protocol-specific biases. Methods designed to minimize bias largely differ in their performance, and contributing factors were identified. Usage of unique molecular identifiers has rather negligible effect and, if designed incorrectly, can even introduce spurious results. Together, these results identify strengths and weaknesses of all current methods and provide guidelines for applications of small RNA-Seq in biomarker research.
The role of alternative promoter usage in tissue-specific gene expression has been well established; however, its role in complex diseases is poorly understood. We performed cap analysis of gene expression (CAGE) sequencing from the left ventricle of a rat model of hypertension, the spontaneously hypertensive rat (SHR), and a normotensive strain, Brown Norway to understand the role of alternative promoter usage in complex disease. We identified 26,560 CAGE-defined transcription start sites in the rat left ventricle, including 1,970 novel cardiac transcription start sites. We identified 28 genes with alternative promoter usage between SHR and Brown Norway, which could lead to protein isoforms differing at the amino terminus between two strains and 475 promoter switching events altering the length of the 5' UTR. We found that the shift in Insr promoter usage was significantly associated with insulin levels and blood pressure within a panel of HXB/BXH recombinant inbred rat strains, suggesting that hyperinsulinemia due to insulin resistance might lead to hypertension in SHR. Our study provides a preliminary evidence of alternative promoter usage in complex diseases.
- MeSH
- genetická transkripce genetika MeSH
- hypertenze * genetika metabolismus MeSH
- krysa rodu rattus MeSH
- potkani inbrední SHR MeSH
- promotorové oblasti (genetika) genetika MeSH
- sekvenční analýza RNA metody MeSH
- stanovení celkové genové exprese metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- alternativní sestřih * MeSH
- DNA vazebné proteiny genetika MeSH
- fúzní onkogenní proteiny genetika MeSH
- lidé MeSH
- lipoblastom genetika patologie MeSH
- sekvenční analýza RNA metody MeSH
- transkripční faktor Zeb2 genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
Extracellular RNAs present in biofluids have emerged as potential biomarkers for disease. Where most studies focus on blood-derived fluids, other biofluids may be more informative. We present an atlas of messenger, circular, and small RNA transcriptomes of a comprehensive collection of 20 human biofluids. By means of synthetic spike-in controls, we compare RNA content across biofluids, revealing a 10,000-fold difference in concentration. The circular RNA fraction is increased in most biofluids compared to tissues. Each biofluid transcriptome is enriched for RNA molecules derived from specific tissues and cell types. Our atlas enables an informed selection of the most relevant biofluid to monitor particular diseases. To verify the biomarker potential in these biofluids, four validation cohorts representing a broad spectrum of diseases were profiled, revealing numerous differential RNAs between case and control subjects. Spike-normalized data are publicly available in the R2 web portal for further exploration.
- MeSH
- biologické markery * MeSH
- kohortové studie MeSH
- lidé MeSH
- RNA genetika metabolismus MeSH
- sekvenční analýza RNA metody MeSH
- stanovení celkové genové exprese metody MeSH
- tělesné tekutiny metabolismus MeSH
- transkriptom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Dědičné neuropatie, včetně nejčastější formy Charcot-Marie-Tooth (CMT), jsou skupinou onemocnění, pro které je charakteristické distální postižení končetin - svalová slabost a deformity nohou i rukou, svalové atrofie. V naší laboratoři je díky dlouholeté diagnostice této skupiny chorob pro celou ČR shromážděn velký soubor pacientů. Máme vybráno několik velkých rodin (minimálně 4), u kterých příčina onemocnění doposud není známa, a to i po provedení vyšetření nejčastějších genových poruch spojených s CMT, genového mapovaní na SNP čipech, nejnovější metody masivně paralelního sekvenování vyšetřením panelů všech genů, které jsou v současnosti spojovány s CMT a celoexomového sekvenování. Cílem projektu je provedení dalších moderních analýz v těchto rodinách, a tím objasnění příčiny dědičné neuropatie. Využijeme zcela nové možnosti a metody a to celogenomové sekvenování a transkriptomové sekvenování. V první fázi se zaměříme na objasnění příčiny u čtyř velkých rodin, ve druhé fázi budou do projektu zahrnuty i další rodiny (celkem až 10 rodin).; Hereditary neuropathies (IPN) are a group of inherited peripheral neuropathies, which are characterized by distal muscle weakness, foot deformities and muscle atrophy. We have gathered a few families in whom the cause of the disease remained unknown – even after using a variety of methods like linkage analysis, targeted massively parallel sequencing of a gene panel and whole exome sequencing (WES). The aim of the project is to implement advanced analyses, clarify the causes of CMT disease in this group of patients with previously unexplained cause of hereditary neuropathy. We will use new possibilities and methods such as whole genome sequencing and RNA sequencing. Firstly, we will focus on four larger families, with clinically well documented inherited peripheral neuropathy and multiple affected members. Secondly, other families will also be included in the project (up to ten families).
- MeSH
- hereditární motorické a senzitivní neuropatie diagnóza genetika MeSH
- lidé MeSH
- sekvenční analýza RNA metody MeSH
- sekvenování celého genomu metody MeSH
- sekvenování transkriptomu MeSH
- vzácné nemoci diagnóza genetika MeSH
- Check Tag
- lidé MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- genetika, lékařská genetika
- neurologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
