Long-read technologies are overcoming early limitations in accuracy and throughput, broadening their application domains in genomics. Dedicated analysis tools that take into account the ...characteristics of long-read data are thus required, but the fast pace of development of such tools can be overwhelming. To assist in the design and analysis of long-read sequencing projects, we review the current landscape of available tools and present an online interactive database, long-read-tools.org, to facilitate their browsing. We further focus on the principles of error correction, base modification detection, and long-read transcriptomics analysis and highlight the challenges that remain.
Activated caspases are a hallmark of apoptosis induced by the intrinsic pathway, but they are dispensable for cell death and the apoptotic clearance of cells in vivo. This has led to the suggestion ...that caspases are activated not just to kill but to prevent dying cells from triggering a host immune response. Here, we show that the caspase cascade suppresses type I interferon (IFN) production by cells undergoing Bak/Bax-mediated apoptosis. Bak and Bax trigger the release of mitochondrial DNA. This is recognized by the cGAS/STING-dependent DNA sensing pathway, which initiates IFN production. Activated caspases attenuate this response. Pharmacological caspase inhibition or genetic deletion of caspase-9, Apaf-1, or caspase-3/7 causes dying cells to secrete IFN-β. In vivo, this precipitates an elevation in IFN-β levels and consequent hematopoietic stem cell dysfunction, which is corrected by loss of Bak and Bax. Thus, the apoptotic caspase cascade functions to render mitochondrial apoptosis immunologically silent.
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•Activation of Bak and Bax, essential mediators of apoptosis, triggers mtDNA release•mtDNA stimulates the cGAS/STING pathway to induce IFN-β production•Activation of the apoptotic caspase cascade blocks the cGAS/STING pathway•Inhibition or genetic deletion of caspases causes apoptotic cells to secrete IFN-β
In the absence of a functional caspases, dying cells behave as if virally infected, activating the cGAS/STING pathway to produce type I IFN, showing that the apoptotic caspase cascade functions to render apoptosis immunologically silent.
limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental ...designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described.
Despite accumulating evidence for a mammary differentiation hierarchy, the basal compartment comprising stem cells remains poorly characterized. Through gene expression profiling of Lgr5
basal ...epithelial cells, we identify a new marker, Tetraspanin8 (Tspan8). Fractionation based on Tspan8 and Lgr5 expression uncovered three distinct mammary stem cell (MaSC) subsets in the adult mammary gland. These exist in a largely quiescent state but differ in their reconstituting ability, spatial localization, and their molecular and epigenetic signatures. Interestingly, the deeply quiescent MaSC subset (Lgr5
Tspan8
) resides within the proximal region throughout life, and has a transcriptome strikingly similar to that of claudin-low tumours. Lgr5
Tspan8
cells appear to originate from the embryonic mammary primordia before switching to a quiescent state postnatally but can be activated by ovarian hormones. Our findings reveal an unexpected degree of complexity within the adult MaSC compartment and identify a dormant subset poised for activation in response to physiological stimuli.
graphics for RNA-sequencing and microarray gene expression analyses may contain upwards of tens of thousands of points. Details about certain genes or samples of interest are easily obscured in such ...dense summary displays. Incorporating interactivity into summary plots would enable additional information to be displayed on demand and facilitate intuitive data exploration.
The open-source Glimma package creates interactive graphics for exploring gene expression analysis with a few simple R commands. It extends popular plots found in the limma package, such as multi-dimensional scaling plots and mean-difference plots, to allow individual data points to be queried and additional annotation information to be displayed upon hovering or selecting particular points. It also offers links between plots so that more information can be revealed on demand. Glimma is widely applicable, supporting data analyses from a number of well-established Bioconductor workflows ( limma , edgeR and DESeq2 ) and uses D3/JavaScript to produce HTML pages with interactive displays that enable more effective data exploration by end-users. Results from Glimma can be easily shared between bioinformaticians and biologists, enhancing reporting capabilities while maintaining reproducibility.
The Glimma R package is available from http://bioconductor.org/packages/Glimma/ .
su.s@wehi.edu.au , law@wehi.edu.au or mritchie@wehi.edu.au.
Innate lymphoid cells (ILCs) are enriched at mucosal surfaces, where they provide immune surveillance. All ILC subsets develop from a common progenitor that gives rise to pre-committed progenitors ...for each of the ILC lineages. Currently, the temporal control of gene expression that guides the emergence of these progenitors is poorly understood. We used global transcriptional mapping to analyze gene expression in different ILC progenitors. We identified PD-1 to be specifically expressed in PLZF+ ILCp and revealed that the timing and order of expression of the transcription factors NFIL3, ID2, and TCF-1 was critical. Importantly, induction of ILC lineage commitment required only transient expression of NFIL3 prior to ID2 and TCF-1 expression. These findings highlight the importance of the temporal program that permits commitment of progenitors to the ILC lineage, and they expand our understanding of the core transcriptional program by identifying potential regulators of ILC development.
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•ILCp transcriptomics define the blueprint for hierarchical ILC development•PD-1 identifies the PLZF-expressing ILC precursor in the bone marrow•Transient NFIL3 expression prior to ID2 expression is required for ILC development•ID2 and TCF-1 are required to extinguish stem cell and B and T cell gene programs
Seillet et al. define the hierarchical blueprint for ILC development using global transcriptomic analyses of ILC progenitors. This revealed that PD-1 is a key marker of ILCp and uncovered a regulatory circuit governed by NFIL3 in regulating ID2 and TCF-1 essential for ILC differentiation.
The prosurvival protein BCL-2 is frequently overexpressed in estrogen receptor (ER)-positive breast cancer. We have generated ER-positive primary breast tumor xenografts that recapitulate the primary ...tumors and demonstrate that the BH3 mimetic ABT-737 markedly improves tumor response to the antiestrogen tamoxifen. Despite abundant BCL-XL expression, similar efficacy was observed with the BCL-2 selective inhibitor ABT-199, revealing that BCL-2 is a crucial target. Unexpectedly, BH3 mimetics were found to counteract the side effect of tamoxifen-induced endometrial hyperplasia. Moreover, BH3 mimetics synergized with phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitors in eliciting apoptosis. Importantly, these two classes of inhibitor further enhanced tumor response in combination therapy with tamoxifen. Collectively, our findings provide a rationale for the clinical evaluation of BH3 mimetics in therapy for breast cancer.
•BH3 mimetics improve response of breast tumor xenografts to endocrine therapy•The efficacy of the BCL-2-selective inhibitor ABT-199 reveals BCL-2 as a key target•Synergy with PI3K/mTOR inhibitors reveals additional combinatorial therapy options•BCL-2 inhibitors counteract tamoxifen-induced endometrial hyperplasia
Single-cell RNA sequencing (scRNA-seq) technology allows researchers to profile the transcriptomes of thousands of cells simultaneously. Protocols that incorporate both designed and random barcodes ...have greatly increased the throughput of scRNA-seq, but give rise to a more complex data structure. There is a need for new tools that can handle the various barcoding strategies used by different protocols and exploit this information for quality assessment at the sample-level and provide effective visualization of these results in preparation for higher-level analyses. To this end, we developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols that include CEL-seq, MARS-seq, Chromium 10X, Drop-seq and Smart-seq. scPipe produces a count matrix that is essential for downstream analysis along with an HTML report that summarises data quality. These results can be used as input for downstream analyses including normalization, visualization and statistical testing. scPipe performs this processing in a few simple R commands, promoting reproducible analysis of single-cell data that is compatible with the emerging suite of open-source scRNA-seq analysis tools available in R/Bioconductor and beyond. The scPipe R package is available for download from https://www.bioconductor.org/packages/scPipe.
Abstract
Systematic variation in the methylation of cytosines at CpG sites plays a critical role in early development of humans and other mammals. Of particular interest are regions of differential ...methylation between parental alleles, as these often dictate monoallelic gene expression, resulting in parent of origin specific control of the embryonic transcriptome and subsequent development, in a phenomenon known as genomic imprinting. Using long-read nanopore sequencing we show that, with an average genomic coverage of ∼10, it is possible to determine both the level of methylation of CpG sites and the haplotype from which each read arises. The long-read property is exploited to characterize, using novel methods, both methylation and haplotype for reads that have reduced basecalling precision compared to Sanger sequencing. We validate the analysis both through comparison of nanopore-derived methylation patterns with those from Reduced Representation Bisulfite Sequencing data and through comparison with previously reported data. Our analysis successfully identifies known imprinting control regions (ICRs) as well as some novel differentially methylated regions which, due to their proximity to hitherto unknown monoallelically expressed genes, may represent new ICRs.
The KRAS oncoprotein, a critical driver in 33% of lung adenocarcinoma (LUAD), has remained an elusive clinical target due to its perceived undruggable nature. The identification of dependencies borne ...through common co-occurring mutations are sought to more effectively target KRAS-mutant lung cancer. Approximately 20% of KRAS-mutant LUAD carry loss-of-function mutations in KEAP1, a negative regulator of the antioxidant response transcription factor NFE2L2/NRF2. We demonstrate that Keap1-deficient Kras
lung tumors arise from a bronchiolar cell-of-origin, lacking pro-tumorigenic macrophages observed in tumors originating from alveolar cells. Keap1 loss activates the pentose phosphate pathway, inhibition of which, using 6-AN, abrogated tumor growth. These studies highlight alternative therapeutic approaches to specifically target this unique subset of KRAS-mutant LUAD cancers.
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