Early-stage idiopathic Parkinson's disease is associated with reduced circular RNA expression.

Neurodegeneration in Parkinson's disease (PD) precedes diagnosis by years. Early neurodegeneration may be reflected in RNA levels and measurable as a biomarker. Here, we present the largest quantification of whole blood linear and circular RNAs (circRNA) in early-stage idiopathic PD, using RNA sequencing data from two cohorts (PPMI = 259 PD, 161 Controls; ICICLE-PD = 48 PD, 48 Controls). We identified a replicable increase in TMEM252 and LMNB1 gene expression in PD. We identified novel differences in the expression of circRNAs from ESYT2, BMS1P1 and CCDC9, and replicated trends of previously reported circRNAs. Overall, using circRNA as a diagnostic biomarker in PD did not show any clear improvement over linear RNA, minimising its potential clinical utility. More interestingly, we observed a general reduction in circRNA expression in both PD cohorts, accompanied by an increase in RNASEL expression. This imbalance implicates the activation of an innate antiviral immune response and suggests a previously unknown aspect of circRNA regulation in PD.

Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision.

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation.

LINE retrotransposons characterize mammalian tissue-specific and evolutionarily dynamic regulatory regions.

BACKGROUND: To investigate the mechanisms driving regulatory evolution across tissues, we experimentally mapped promoters, enhancers, and gene expression in the liver, brain, muscle, and testis from ten diverse mammals. RESULTS: The regulatory landscape around genes included both tissue-shared and tissue-specific regulatory regions, where tissue-specific promoters and enhancers evolved most rapidly. Genomic regions switching between promoters and enhancers were more common across species, and less common across tissues within a single species. Long Interspersed Nuclear Elements (LINEs) played recurrent evolutionary roles: LINE L1s were associated with tissue-specific regulatory regions, whereas more ancient LINE L2s were associated with tissue-shared regulatory regions and with those switching between promoter and enhancer signatures across species. CONCLUSIONS: Our analyses of the tissue-specificity and evolutionary stability among promoters and enhancers reveal how specific LINE families have helped shape the dynamic mammalian regulome.

Cell type-specific novel long non-coding RNA and circular RNA in the BLUEPRINT hematopoietic transcriptomes atlas.

Transcriptional profiling of hematopoietic cell subpopulations has helped to characterize the developmental stages of the hematopoietic system and the molecular bases of malignant and non-malignant blood diseases. Previously, only the genes targeted by expression microarrays could be profiled genome-wide. High-throughput RNA sequencing, however, encompasses a broader repertoire of RNA molecules, without restriction to previously annotated genes. We analyzed the BLUEPRINT consortium RNA-sequencing data for mature hematopoietic cell types. The data comprised 90 total RNA-sequencing samples, each composed of one of 27 cell types, and 32 small RNA-sequencing samples, each composed of one of 11 cell types. We estimated gene and isoform expression levels for each cell type using existing annotations from Ensembl. We then used guided transcriptome assembly to discover unannotated transcripts. We identified hundreds of novel non-coding RNA genes and showed that the majority have cell type-dependent expression. We also characterized the expression of circular RNA and found that these are also cell type-specific. These analyses refine the active transcriptional landscape of mature hematopoietic cells, highlight abundant genes and transcriptional isoforms for each blood cell type, and provide a valuable resource for researchers of hematologic development and diseases. Finally, we made the data accessible via a web-based interface: https://blueprint.haem.cam.ac.uk/bloodatlas/.

GENCODE 2021.

The GENCODE project annotates human and mouse genes and transcripts supported by experimental data with high accuracy, providing a foundational resource that supports genome biology and clinical genomics. GENCODE annotation processes make use of primary data and bioinformatic tools and analysis generated both within the consortium and externally to support the creation of transcript structures and the determination of their function. Here, we present improvements to our annotation infrastructure, bioinformatics tools, and analysis, and the advances they support in the annotation of the human and mouse genomes including: the completion of first pass manual annotation for the mouse reference genome; targeted improvements to the annotation of genes associated with SARS-CoV-2 infection; collaborative projects to achieve convergence across reference annotation databases for the annotation of human and mouse protein-coding genes; and the first GENCODE manually supervised automated annotation of lncRNAs. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.

Ensembl 2021.

The Ensembl project (https://www.ensembl.org) annotates genomes and disseminates genomic data for vertebrate species. We create detailed and comprehensive annotation of gene structures, regulatory elements and variants, and enable comparative genomics by inferring the evolutionary history of genes and genomes. Our integrated genomic data are made available in a variety of ways, including genome browsers, search interfaces, specialist tools such as the Ensembl Variant Effect Predictor, download files and programmatic interfaces. Here, we present recent Ensembl developments including two new website portals. Ensembl Rapid Release (http://rapid.ensembl.org) is designed to provide core tools and services for genomes as soon as possible and has been deployed to support large biodiversity sequencing projects. Our SARS-CoV-2 genome browser (https://covid-19.ensembl.org) integrates our own annotation with publicly available genomic data from numerous sources to facilitate the use of genomics in the international scientific response to the COVID-19 pandemic. We also report on other updates to our annotation resources, tools and services. All Ensembl data and software are freely available without restriction.

An improved pig reference genome sequence to enable pig genetics and genomics research.

BACKGROUND: The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. RESULTS: We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. CONCLUSIONS: These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.

Sequencing-based microsatellite instability testing using as few as six markers for high-throughput clinical diagnostics.

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer-predisposition, and can be used to predict response to immunotherapy. Here, we present a single-molecule molecular inversion probe and sequencing-based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward-forward stepwise selection was used to identify a 6-marker subset of equal accuracy to the 24-marker panel. Assessment of assay detection limits showed that the 24-marker panel is marginally more robust to sample variables than the 6-marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Ensembl 2020.

The Ensembl (https://www.ensembl.org) is a system for generating and distributing genome annotation such as genes, variation, regulation and comparative genomics across the vertebrate subphylum and key model organisms. The Ensembl annotation pipeline is capable of integrating experimental and reference data from multiple providers into a single integrated resource. Here, we present 94 newly annotated and re-annotated genomes, bringing the total number of genomes offered by Ensembl to 227. This represents the single largest expansion of the resource since its inception. We also detail our continued efforts to improve human annotation, developments in our epigenome analysis and display, a new tool for imputing causal genes from genome-wide association studies and visualisation of variation within a 3D protein model. Finally, we present information on our new website. Both software and data are made available without restriction via our website, online tools platform and programmatic interfaces (available under an Apache 2.0 license) and data updates made available four times a year.

An integrated transcriptional analysis of the developing human retina.

The scarcity of embryonic/foetal material as a resource for direct study means that there is still limited understanding of human retina development. Here, we present an integrated transcriptome analysis combined with immunohistochemistry in human eye and retinal samples from 4 to 19 post-conception weeks. This analysis reveals three developmental windows with specific gene expression patterns that informed the sequential emergence of retinal cell types and enabled identification of stage-specific cellular and biological processes, and transcriptional regulators. Each stage is characterised by a specific set of alternatively spliced transcripts that code for proteins involved in the formation of the photoreceptor connecting cilium, pre-mRNA splicing and epigenetic modifiers. Importantly, our data show that the transition from foetal to adult retina is characterised by a large increase in the percentage of mutually exclusive exons that code for proteins involved in photoreceptor maintenance. The circular RNA population is also defined and shown to increase during retinal development. Collectively, these data increase our understanding of human retinal development and the pre-mRNA splicing process, and help to identify new candidate disease genes.

Ensembl 2019.

The Ensembl project (https://www.ensembl.org) makes key genomic data sets available to the entire scientific community without restrictions. Ensembl seeks to be a fundamental resource driving scientific progress by creating, maintaining and updating reference genome annotation and comparative genomics resources. This year we describe our new and expanded gene, variant and comparative annotation capabilities, which led to a 50% increase in the number of vertebrate genomes we support. We have also doubled the number of available human variants and added regulatory regions for many mouse cell types and developmental stages. Our data sets and tools are available via the Ensembl website as well as a through a RESTful webservice, Perl application programming interface and as data files for download.

GENCODE reference annotation for the human and mouse genomes.

The accurate identification and description of the genes in the human and mouse genomes is a fundamental requirement for high quality analysis of data informing both genome biology and clinical genomics. Over the last 15 years, the GENCODE consortium has been producing reference quality gene annotations to provide this foundational resource. The GENCODE consortium includes both experimental and computational biology groups who work together to improve and extend the GENCODE gene annotation. Specifically, we generate primary data, create bioinformatics tools and provide analysis to support the work of expert manual gene annotators and automated gene annotation pipelines. In addition, manual and computational annotation workflows use any and all publicly available data and analysis, along with the research literature to identify and characterise gene loci to the highest standard. GENCODE gene annotations are accessible via the Ensembl and UCSC Genome Browsers, the Ensembl FTP site, Ensembl Biomart, Ensembl Perl and REST APIs as well as https://www.gencodegenes.org.

Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular.

BACKGROUND: Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. RESULTS: A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. CONCLUSIONS: Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species.

Ensembl 2018.

The Ensembl project has been aggregating, processing, integrating and redistributing genomic datasets since the initial releases of the draft human genome, with the aim of accelerating genomics research through rapid open distribution of public data. Large amounts of raw data are thus transformed into knowledge, which is made available via a multitude of channels, in particular our browser (http://www.ensembl.org). Over time, we have expanded in multiple directions. First, our resources describe multiple fields of genomics, in particular gene annotation, comparative genomics, genetics and epigenomics. Second, we cover a growing number of genome assemblies; Ensembl Release 90 contains exactly 100. Third, our databases feed simultaneously into an array of services designed around different use cases, ranging from quick browsing to genome-wide bioinformatic analysis. We present here the latest developments of the Ensembl project, with a focus on managing an increasing number of assemblies, supporting efforts in genome interpretation and improving our browser.

PTESFinder: a computational method to identify post-transcriptional exon shuffling (PTES) events.

BACKGROUND: Transcripts, which have been subject to Post-transcriptional exon shuffling (PTES), have an exon order inconsistent with the underlying genomic sequence. These have been identified in a wide variety of tissues and cell types from many eukaryotes, and are now known to be mostly circular, cytoplasmic, and non-coding. Although there is no uniformly ascribed function, several have been shown to be involved in gene regulation. Accurate identification of these transcripts can, however, be difficult due to artefacts from a wide variety of sources. RESULTS: Here, we present a computational method, PTESFinder, to identify these transcripts from high throughput RNAseq data. Uniquely, it systematically excludes potential artefacts emanating from pseudogenes, segmental duplications, and template switching, and outputs both PTES and canonical exon junction counts to facilitate comparative analyses. In comparison with four existing methods, PTESFinder achieves highest specificity and comparable sensitivity at a variety of read depths. PTESFinder also identifies between 13 % and 41.6 % more structures, compared to publicly available methods recently used to identify human circular RNAs. CONCLUSIONS: With high sensitivity and specificity, user-adjustable filters that target known sources of false positives, and tailored output to facilitate comparison of transcript levels, PTESFinder will facilitate the discovery and analysis of these poorly understood transcripts.

Circular RNA enrichment in platelets is a signature of transcriptome degradation.

In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17- to 188-fold relative to nucleated tissues and 14- to 26-fold relative to samples digested with RNAse R to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched on average 12.7 times in platelets relative to nucleated tissues and identify 3162 genes significantly enriched for circRNAs, including some where all RNAseq reads appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in cultured megakaryocytes, and demonstrate that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost >90% of their progenitor mRNAs and that translation in platelets occurs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts previously classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artifacts.