Recent advances in single-cell RNA sequencing technologies have made detection of transcripts in single cells possible. The level of resolution provided by these technologies can be used to study changes in transcript usage across cell populations and help investigate new biology. Here, we introduce RNA-Scoop, an interactive cell cluster and transcriptome visualization tool to analyze transcript usage across cell categories and clusters. The tool allows users to examine differential transcript expression across clusters and investigate how usage of specific transcript expression mechanisms varies across cell groups.
Olaparib is the first Health Canadaapproved agent in metastatic prostate cancer to use a companion diagnostic to identify alterations in BRCA1, BRCA2, or ATM. As olaparib is introduced, clinicians must learn to access and interpret germline and somatic next-generation sequencing (NGS) results, and how to manage affected patients who appear to have distinct clinical features. The traditional model of referring patients to a hereditary cancer clinic (HCC) for germline testing is likely impractical in this disease, as the metastatic prostate cancer patient population would be overwhelming. Alternate approaches to this are clinician-ordered genetic testing (so-called "mainstreaming"), out-of-pocket payment for third-party private company genetic testing, or germline testing done in conjunction with somatic testing, particularly cell free circulating tumor DNA (ctDNA).Germline testing alone is not sufficient for identifying Olaparib-eligible patients, as less than half of BRCA1, BRCA2, or ATM alterations are germline in origin, but it is critically important to identify family members who are carriers so that risk-reduction measures can be undertaken. Somatic testing is not widely available in Canada, but some patients can access it through research protocols or by paying out-of-pocket. Somatic testing can be performed on archival or fresh solid tissue biopsy samples, or through whole blood samples to access plasma-derived circulating tumor DNA (ctDNA). Both testing approaches have relative advantages and disadvantages, but neither may be informative in all patients and, therefore, ideal somatic NGS pathways should provide options for both tissue and ctDNA testing.We advocate that clinicians begin discussions with their provincial lab formularies, HCC, and molecular pathology labs to highlight the importance of germline and somatic testing in this population and identify pathways for patient access. While olaparib has approval for use in BRCA1, BRCA2, and ATM-altered mCRPC, emerging evidence suggests that PARP inhibitors have variable activity in these three genes, with BRCA2 alterations appearing to be the most responsive. Retrospective and prospective series have reported varying outcomes to standard of care therapies, such as ARATs and taxane-based chemotherapy, in metastatic castration-resistant prostate cancer (mCRPC) patients with DNA damage repair (DDR) gene alterations, such as BRCA2. In the absence of high-level evidence showing a lack of benefit, we believe this patient population should still be considered for these treatments.In addition, platinum-based chemotherapy appears to have activity in DDR gene-altered mCRPC and should be considered another option when access to olaparib is not possible.At present, there is no evidence to support an optimal treatment sequence in this patient population, therefore, physician and patient preferences will need to be taken into consideration when selecting therapies. As olaparib and other PARP inhibitors are tested in different disease states and in combination with other therapies, we will likely see a more refined approach to use of these agents and management of this new biomarker-defined patient population.
Context: Multiple studies have reported on the genomic characteristics of metastatic hormone-sensitive prostate cancer (mHSPC). The impact of these findings on prognostication, treatment selection, and clinical trial design remains unclear.
Objective: To summarise genomic alteration prevalences in liquid and/or tissue biopsies, infer their clinical implications, and compare genomic alteration frequencies across different disease states and clinical phenotypes.
Evidence acquisition: The PubMed and Web of Knowledge databases were systematically searched up to January 2021. Quality assessment was performed using the Joanna Briggs Institute Critical Appraisal tools.
Evidence synthesis: In total, 11 studies encompassing 1682 mHSPC patients were included. High-volume disease was associated with more frequent alterations in TP53, DNA damage repair, and Wnt pathways. Tumours from patients with de novo mHSPC were enriched for alterations in TP53 and CDK12 compared with recurrent disease. Alterations in AR, TP53, cell cycle signalling, and MYC were associated with a poorer clinical outcome. A comparative analysis of gene alteration frequencies across disease states revealed a relative increase from localised to castration-resistant tumours, with noteworthy enrichment of CTNNB1 alterations in mHSPC (5%), which warrants further investigation. This study was limited by variability in methodology and definitions used among the eligible studies, including differences in sequencing methods, analytes (being either tissue or liquid), alteration calling thresholds, and target patient populations with a relative under-representation of recurrent metastatic disease.
Conclusions: Several genomic alterations are associated with differential prognosis and clinical phenotypes in mHSPC. We urge that emerging data on these potential predictive biomarkers must be validated in biomarker-driven randomised controlled trials before any clinical implementation. Alignment of the assay methodology and reporting will be critical for ensuring rapid scalability.
Patient summary: We reviewed current data on genomic alterations of metastatic hormone-sensitive prostate cancer, and summarised key genomic subtypes that associate with specific clinical phenotypes and treatment outcomes.
Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wildtype and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in double mutant cells (CIC-knockout + IDH1-mutant). These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. This article is protected by copyright. All rights reserved.
Objectives: Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF.
Methods: A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF.
Results: We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at -80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy.
Conclusions: Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.
The draft genome sequence of Bacidia gigantensis, a lichenized fungus in the order Lecanorales, was sequenced directly from a herbarium specimen collected from the type locality at Sleeping Giant Provincial Park in Ontario, Canada. Using long-read sequencing on the Oxford Nanopore PromethION platform, we assembled a nearly complete genome sequence.
Generating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads.
LongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of Caenorhabditis elegans, Oryza sativa, and three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 1.2-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently improves upon human assemblies in under five hours using less than 23 GB of RAM.
Due to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch.
In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in D. melanogaster and S. cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sub-lethal, stress response pathway that is tailored to an organism's environmental norms. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse, and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species, highlights the physiological significance of this protein conformation and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events.
Fusobacterium nucleatum is a ubiquitous opportunistic pathogen with an emerging role as an oncomicrobe in colorectal cancer and other cancer settings. F. nucleatum can adhere to and invade host cells in a manner that varies across F. nucleatum strains and host cell phenotypes. Here, we performed pairwise cocultures between three F. nucleatum strains and two immortalized primary host cell types (human colonic epithelial [HCE] cells and human carotid artery endothelial [HCAE] cells) followed by transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate transcriptional and epigenetic host cell responses. We observed that F. nucleatum-induced host cell transcriptional modulation involves strong upregulation of genes related to immune migration and inflammatory processes, such as TNF, CXCL8, CXCL1, and CCL20. Furthermore, we identified genes strongly upregulated in a cell line-specific manner. In HCE cells, overexpressed genes included UBD and DUOX2/DUOXA2, associated with p53 degradation-mediated proliferation and intestinal reactive oxygen species (ROS) production, respectively. In HCAE cells, overexpressed genes included EFNA1 and LIF, two genes commonly upregulated in colorectal cancer and associated with poor patient outcomes, and PTGS2 (COX2), a gene associated with the protective effect of aspirin in the colorectal cancer setting. Interestingly, we also observed downregulation of numerous histone modification genes upon F. nucleatum exposure. We used the ChIP-seq data to annotate chromatin states genome wide and found significant chromatin remodeling following F. nucleatum exposure in HCAE cells, with increased frequencies of active enhancer and low-signal/quiescent states. Thus, our results highlight increased inflammation and chemokine gene expression as conserved host cell responses to F. nucleatum exposure and extensive host cell epigenomic changes specific to host cell type. IMPORTANCE Fusobacterium nucleatum is a bacterium normally found in the healthy oral cavity but also has an emerging role in colorectal cancer and other cancer settings. The host-microbe interactions of F. nucleatum and its involvement in tumor initiation, progression, and treatment resistance are not fully understood. We explored host cell changes that occur in response to F. nucleatum. We identified key genes differentially expressed in response to various conditions of F. nucleatum exposure and determined that the conserved host cell response to F. nucleatum was dominated by increased inflammation and chemokine gene expression. Additionally, we found extensive host cell epigenomic changes as a novel aspect of host modulation associated with F. nucleatum exposure. These results extend our understanding of F. nucleatum as an emerging pathogen and highlight the importance of considering strain heterogeneity and host cell phenotypic variation when exploring pathogenic mechanisms of F. nucleatum.
The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.