Project description:Long-read RNA sequencing (RNA-seq) holds great potential for characterizing transcriptome variation and full-length transcript isoforms, but the relatively high error rate of current long-read sequencing platforms poses a major challenge. We present ESPRESSO, a computational tool for robust discovery and quantification of transcript isoforms from error-prone long reads. ESPRESSO jointly considers alignments of all long reads aligned to a gene and uses error profiles of individual reads to improve the identification of splice junctions and the discovery of their corresponding transcript isoforms. On both a synthetic spike-in RNA sample and human RNA samples, ESPRESSO outperforms multiple contemporary tools in not only transcript isoform discovery but also transcript isoform quantification. In total, we generated and analyzed ~1.1 billion nanopore RNA-seq reads covering 30 human tissue samples and three human cell lines. ESPRESSO and its companion dataset provide a useful resource for studying the RNA repertoire of eukaryotic transcriptomes.
Project description:Long-read RNA sequencing (RNA-seq) holds great potential for characterizing transcriptome variation and full-length transcript isoforms, but the relatively high error rate of current long-read sequencing platforms poses a major challenge. We present ESPRESSO, a computational tool for robust discovery and quantification of transcript isoforms from error-prone long reads. ESPRESSO jointly considers alignments of all long reads aligned to a gene and uses error profiles of individual reads to improve the identification of splice junctions and the discovery of their corresponding transcript isoforms. On both a synthetic spike-in RNA sample and human RNA samples, ESPRESSO outperforms multiple contemporary tools in not only transcript isoform discovery but also transcript isoform quantification. In total, we generated and analyzed ~1.1 billion nanopore RNA-seq reads covering 30 human tissue samples and three human cell lines. ESPRESSO and its companion dataset provide a useful resource for studying the RNA repertoire of eukaryotic transcriptomes.
Project description:Antimicrobial-induced DNA damage, and subsequent repair via upregulation of DNA repair factors, including error-prone translesion polymerases, can lead to the increased accumulation of mutations in the microbial genome, and ultimately increased risk of acquired mutations associated with antimicrobial resistance. While this phenotype is well described in bacterial species, it is less thoroughly investigated amongst microbial fungi. Here, we monitor DNA damage induced by antifungal agents in the fungal pathogen Candida albicans, and find that commonly used antifungal drugs are able to induce DNA damage, leading to the upregulation of transcripts encoding predicted error-prone polymerases and related factors. We focus on REV1, encoding a putative error-prone polymerase, and find that while deleting this gene in C. albicans leads to increased sensitivity to DNA damage, it also unexpectedly renders cells more likely to incur mutations and evolve resistance to antifungal agents. We further find that deletion of REV1 leads to a significant depletion in the uncharacterized protein Shm1, which itself plays a role in fungal mutagenesis. Together, this work lends new insight into previously uncharacterized factors with important roles in the DNA damage response, mutagenesis, and the evolution of antifungal drug resistance.
Project description:3' based single-cell RNA sequencing (scRNA-seq) has enabled the study of gene expression at the individual cell level and the development of few methods to quantify transcriptomic variability at the single-cell level. Yet, most of these methods have low sensitivity and specificity. Here, we present SCALPEL, a new tool to quantify and characterize transcript isoforms at the single-cell level using standard 3' based single-cell transcriptomics data. SCALPEL predictions have higher sensitivity than that of other tools and can be validated experimentally. We have used SCALPEL to study the changes in isoform usage during mouse spermatogenesis and in the differentiation of induced pluripotent stem cells (iPSCs) to neural progenitors (NPCs). These analyses confirmed known changes in the 3' UTR length during cell differentiation and allowed the identification of new cell populations and cell type specific microRNA signatures controlling isoform expression in individual cells. Together, our results highlight the importance of isoform quantification to gain insight on the gene regulatory mechanisms at the single-cell level.
Project description:Medical applications of human iPS cells(hiPSC) are evolving, but detailed elucidation of the mechanisms of reprogramming remains to be elucidated. Reprogramming is accompanied by an increase in the expression of related genes that maintain pluripotency, such as OCT3 /4 and NANOG. Also, through reprogramming, many CNVs and point mutation arise in genomes, which constitute a major barrier to the use of hiPSC for regenerative medicine. On the other hand, we recently found that DNA repair-related genes expression are altered through reprogramming, elevated expression of genes that accurately convey genomic information, such as homologous recombination (HR) and mismatch repair(MMR), and decreased expression of error-prone translesion Synthesis polymerase(TLS). Here, we confirmed this expression change in another cell-line, and further found that this expression change was maintained by overlapping passages as well as OCT3 /4 and NANOG. This suggests that changes in the expression of DNA repair-related genes associated with reprogramming and their maintenance may be new indicators of the quality control of cells exhibiting pluripotency.
Project description:Age-related neurodegenerative diseases (NDDs) and neuronal dysfunction are associated with the aggregation and propagation of specific pathogenic protein species (e.g. Aβ, α-synuclein, tau). However, whether the disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation with its frequent outcome of random protein misfolding is sufficient to recapitulate many early features of NDDs, including proteostasis dysregulation, perturbed Ca2+ signalling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in abnormal behavioral changes reminiscent of early Alzheimer's disease (AD), such as learning and memory deficits, maladaptive responses to novel stimuli, spontaneous epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation. Ectopic hippocampal NPY expression and cerebral glucose hypometabolism (18F-fluorodeoxyglucose PET) were further signs of neuronal hyperexcitability. Collectively, our findings strongly suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs providing an alternative framework for understanding the initiation of Alzheimer’s disease.