Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3' end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3' processing are deployed across human tissues, with nearly half of multi-transcript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

The ENCODE Uniform Analysis Pipelines.

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses.

The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models.

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × âˆ¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

New developments on the Encyclopedia of DNA Elements (ENCODE) data portal.

The Encyclopedia of DNA Elements (ENCODE) is an ongoing collaborative research project aimed at identifying all the functional elements in the human and mouse genomes. Data generated by the ENCODE consortium are freely accessible at the ENCODE portal (https://www.encodeproject.org/), which is developed and maintained by the ENCODE Data Coordinating Center (DCC). Since the initial portal release in 2013, the ENCODE DCC has updated the portal to make ENCODE data more findable, accessible, interoperable and reusable. Here, we report on recent updates, including new ENCODE data and assays, ENCODE uniform data processing pipelines, new visualization tools, a dataset cart feature, unrestricted public access to ENCODE data on the cloud (Amazon Web Services open data registry, https://registry.opendata.aws/encode-project/) and more comprehensive tutorials and documentation.

The ENCODE Portal as an Epigenomics Resource.

The Encyclopedia of DNA Elements (ENCODE) web portal hosts genomic data generated by the ENCODE Consortium, Genomics of Gene Regulation, The NIH Roadmap Epigenomics Consortium, and the modENCODE and modERN projects. The goal of the ENCODE project is to build a comprehensive map of the functional elements of the human and mouse genomes. Currently, the portal database stores over 500 TB of raw and processed data from over 15,000 experiments spanning assays that measure gene expression, DNA accessibility, DNA and RNA binding, DNA methylation, and 3D chromatin structure across numerous cell lines, tissue types, and differentiation states with selected genetic and molecular perturbations. The ENCODE portal provides unrestricted access to the aforementioned data and relevant metadata as a service to the scientific community. The metadata model captures the details of the experiments, raw and processed data files, and processing pipelines in human and machine-readable form and enables the user to search for specific data either using a web browser or programmatically via REST API. Furthermore, ENCODE data can be freely visualized or downloaded for additional analyses. © 2019 The Authors. Basic Protocol: Query the portal Support Protocol 1: Batch downloading Support Protocol 2: Using the cart to download files Support Protocol 3: Visualize data Alternate Protocol: Query building and programmatic access.

Recompleting the Caenorhabditis elegans genome.

Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted ≥53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology.

Prevention of data duplication for high throughput sequencing repositories.

Database URL: https://www.encodeproject.org/.

The Encyclopedia of DNA elements (ENCODE): data portal update.

The Encyclopedia of DNA Elements (ENCODE) Data Coordinating Center has developed the ENCODE Portal database and website as the source for the data and metadata generated by the ENCODE Consortium. Two principles have motivated the design. First, experimental protocols, analytical procedures and the data themselves should be made publicly accessible through a coherent, web-based search and download interface. Second, the same interface should serve carefully curated metadata that record the provenance of the data and justify its interpretation in biological terms. Since its initial release in 2013 and in response to recommendations from consortium members and the wider community of scientists who use the Portal to access ENCODE data, the Portal has been regularly updated to better reflect these design principles. Here we report on these updates, including results from new experiments, uniformly-processed data from other projects, new visualization tools and more comprehensive metadata to describe experiments and analyses. Additionally, the Portal is now home to meta(data) from related projects including Genomics of Gene Regulation, Roadmap Epigenome Project, Model organism ENCODE (modENCODE) and modERN. The Portal now makes available over 13000 datasets and their accompanying metadata and can be accessed at: https://www.encodeproject.org/.

Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens.

Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples.

SnoVault and encodeD: A novel object-based storage system and applications to ENCODE metadata.

The Encyclopedia of DNA elements (ENCODE) project is an ongoing collaborative effort to create a comprehensive catalog of functional elements initiated shortly after the completion of the Human Genome Project. The current database exceeds 6500 experiments across more than 450 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the H. sapiens and M. musculus genomes. All ENCODE experimental data, metadata, and associated computational analyses are submitted to the ENCODE Data Coordination Center (DCC) for validation, tracking, storage, unified processing, and distribution to community resources and the scientific community. As the volume of data increases, the identification and organization of experimental details becomes increasingly intricate and demands careful curation. The ENCODE DCC has created a general purpose software system, known as SnoVault, that supports metadata and file submission, a database used for metadata storage, web pages for displaying the metadata and a robust API for querying the metadata. The software is fully open-source, code and installation instructions can be found at: http://github.com/ENCODE-DCC/snovault/ (for the generic database) and http://github.com/ENCODE-DCC/encoded/ to store genomic data in the manner of ENCODE. The core database engine, SnoVault (which is completely independent of ENCODE, genomic data, or bioinformatic data) has been released as a separate Python package.

A streamlined tethered chromosome conformation capture protocol.

BACKGROUND: Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application. RESULTS: Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations. CONCLUSION: Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation.

Principles of metadata organization at the ENCODE data coordination center.

The Encyclopedia of DNA Elements (ENCODE) Data Coordinating Center (DCC) is responsible for organizing, describing and providing access to the diverse data generated by the ENCODE project. The description of these data, known as metadata, includes the biological sample used as input, the protocols and assays performed on these samples, the data files generated from the results and the computational methods used to analyze the data. Here, we outline the principles and philosophy used to define the ENCODE metadata in order to create a metadata standard that can be applied to diverse assays and multiple genomic projects. In addition, we present how the data are validated and used by the ENCODE DCC in creating the ENCODE Portal (https://www.encodeproject.org/). Database URL: www.encodeproject.org.

ENCODE data at the ENCODE portal.

The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments.

Gamete-type dependent crossover interference levels in a defined region of Caenorhabditis elegans chromosome V.

In certain organisms, numbers of crossover events for any single chromosome are limited ("crossover interference") so that double crossover events are obtained at much lower frequencies than would be expected from the simple product of independent single-crossover events. We present a number of observations during which we examined interference over a large region of Caenorhabditis elegans chromosome V. Examining this region for multiple crossover events in heteroallelic configurations with limited dimorphism, we observed high levels of crossover interference in oocytes with only partial interference in spermatocytes.

On topological indices for small RNA graphs.

The secondary structure of RNAs can be represented by graphs at various resolutions. While it was shown that RNA secondary structures can be represented by coarse grain tree-graphs and meaningful topological indices can be used to distinguish between various structures, small RNAs are needed to be represented by full graphs. No meaningful topological index has yet been suggested for the analysis of such type of RNA graphs. Recalling that the second eigenvalue of the Laplacian matrix can be used to track topological changes in the case of coarse grain tree-graphs, it is plausible to assume that a topological index such as the Wiener index that represents all Laplacian eigenvalues may provide a similar guide for full graphs. However, by its original definition, the Wiener index was defined for acyclic graphs. Nevertheless, similarly to cyclic chemical graphs, small RNA graphs can be analyzed using elementary cuts, which enables the calculation of topological indices for small RNAs in an intuitive way. We show how to calculate a structural descriptor that is suitable for cyclic graphs, the Szeged index, for small RNA graphs by elementary cuts. We discuss potential uses of such a procedure that considers all eigenvalues of the associated Laplacian matrices to quantify the topology of small RNA graphs.

The RNAmute web server for the mutational analysis of RNA secondary structures.

RNA mutational analysis at the secondary-structure level can be useful to a wide-range of biological applications. It can be used to predict an optimal site for performing a nucleotide mutation at the single molecular level, as well as to analyze basic phenomena at the systems level. For the former, as more sequence modification experiments are performed that include site-directed mutagenesis to find and explore functional motifs in RNAs, a pre-processing step that helps guide in planning the experiment becomes vital. For the latter, mutations are generally accepted as a central mechanism by which evolution occurs, and mutational analysis relating to structure should gain a better understanding of system functionality and evolution. In the past several years, the program RNAmute that is structure based and relies on RNA secondary-structure prediction has been developed for assisting in RNA mutational analysis. It has been extended from single-point mutations to treat multiple-point mutations efficiently by initially calculating all suboptimal solutions, after which only the mutations that stabilize the suboptimal solutions and destabilize the optimal one are considered as candidates for being deleterious. The RNAmute web server for mutational analysis is available at http://www.cs.bgu.ac.il/~xrnamute/XRNAmute.

FineStr: a web server for single-base-resolution nucleosome positioning.

SUMMARY: The DNA in eukaryotic cells is packed into the chromatin that is composed of nucleosomes. Positioning of the nucleosome core particles on the sequence is a problem of great interest because of the role nucleosomes play in different cellular processes including gene regulation. Using the sequence structure of 10.4 base DNA repeat presented in our previous works and nucleosome core DNA sequences database, we have derived the complete nucleosome DNA bendability matrix of Caenorhabditis elegans. We have developed a web server named FineStr that allows users to upload genomic sequences in FASTA format and to perform a single-base-resolution nucleosome mapping on them. AVAILABILITY: FineStr server is freely available for use on the web at http:/www.cs.bgu.ac.il/ approximately nucleom. The site contains a help file with explanation regarding the exact usage. CONTACT: gabdank@cs.bgu.ac.il.

Energy minimization methods applied to riboswitches: a perspective and challenges.

Energy minimization methods for RNA secondary structure prediction have been used extensively for studying a variety of biological systems. Here, we demonstrate their applicability in riboswitch studies, exemplified in both the expression platform and aptamer domains. In the expression platform domain, energy minimization methods can be used to predict in silico a unique point mutation positioned in the non-conserved region of the TPP riboswitch that will transform it from a termination to an anti-termination state, thus backing the prediction experimentally. Furthermore, a successive prediction can be made for a compensatory mutation that is positioned over half the sequence length of the riboswitch from the original mutation and that completely overturns the anti-termination effect of the original mutation. This approach can be used to computationally predict rational modifications in riboswitches for both research and practical applications. In the aptamer domain, energy minimization methods can be used when attempting to detect a novel purine riboswitch in eukaryotes based on the consensus sequence and structure of the bacterial guanine binding aptamer. In the process, some interesting candidates are identified, and although they are attractive enough to be tested experimentally, they are not detectable by sequence based methods alone. These brief examples represent the important lessons to be learned as to the strengths and limitations of energy minimization methods. In light of our growing knowledge in the energy minimization field, future challenges can be advanced for the rational design of known riboswitches and the detection of novel riboswitches. Unlike analyses of specific cases, it is stressed that all the results described here are predictive in scope with direct applicability and an attempt to validate the predictions experimentally.