Systematic comparison of experimental assays and analytical pipelines for identification of active enhancers genome-wide

Mounting evidence supports the idea that transcriptional patterns serve as more specific identifiers of active enhancers than histone marks; however, the optimal strategy to identify active enhancers both experimentally and computationally has not been determined. In this study, we compared 13 genome-wide RNA sequencing assays in K562 cells and showed that the nuclear run-on followed by cap-selection assay (namely, GRO/PRO-cap) has significant advantages in eRNA detection and active enhancer identification. We also introduced a new analytical tool, Peak Identifier for Nascent-Transcript Sequencing (PINTS), to identify active promoters and enhancers genome-wide and pinpoint the precise location of the 5′ transcription start sites (TSSs) within these regulatory elements. Finally, we compiled a comprehensive enhancer candidate compendium based on the detected eRNA TSSs available in 120 cell and tissue types. To facilitate the exploration and prioritization of these enhancer candidates, we also built a user-friendly web server (https://pints.yulab.org) for the compendium with various additional genomic and epigenomic annotations. With the knowledge of the best available assays and pipelines, this large-scale annotation of candidate enhancers will pave the road for selection and characterization of their functions in a time-, labor-, and cost-effective manner in future.

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Use of a counterselectable transposon to create markerless knockouts from a 18,432-clone ordered M. bovis BCG mutant resource

10.1101/817007 ◽

2019 ◽

Author(s):

Katlyn Borgers ◽

Kristof Vandewalle ◽

Annelies Van Hecke ◽

Gitte Michielsen ◽

Evelyn Plets ◽

...

Keyword(s):

Vaccine Strain ◽

Vaccine Development ◽

Cost Effective ◽

Essential Genes ◽

Selection Marker ◽

Marker Genes ◽

Gene Effects ◽

Genome Wide ◽

Effective Manner ◽

Slow Growing

AbstractMutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach; despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian Pooling-Coordinate Sequencing allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which is undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described, which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the M. tb complex (18,432 clones, mutating 83% of the non-essential M. tb homologues), from which clean knockouts can be generated.ImportanceWhile speeding up research for many fields of biology (e.g. yeast, plant, and C. elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner, and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of M. bovis BCG Danish targets 83% of all non-essential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g. drug resistance research, vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the M. tb complex. The stretch to a full collection of mutants in all non-essential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

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Enterprise Crowdsourcing Platform to Improve HR Workflow

Management of the personnel and intellectual resources in Russia ◽

10.12737/2305-7807-2020-5-8 ◽

2020 ◽

Vol 9 (5) ◽

pp. 5-8 ◽

Cited By ~ 1

Author(s):

G. Zuev

Keyword(s):

Large Scale ◽

Best Practice ◽

Cost Effective ◽

Hr Management ◽

Practical Project ◽

Effective Manner ◽

Wide Range ◽

Hr Managers ◽

Short Time ◽

Small Parts

Crowdsourcing technologies may solve a wide range of business issues: improve efficiency of HR management, increase customer loyalty and maximize economic efficiency of whole enterprise. The recent years best practice has shown how crowdsourcing is gaining particular relevance of human resource management, allowing HR managers to resolve organization relevant problems in quick and cost-effective manner. Important advantage of crowdsourcing сomes from his main ability: decomposition of tasks into small parts and the ability to perform it’s remotely, via Internet. Thanks to this, not only large corporations, but also small and medium-sized businesses can execute a large-scale projects in a short time. This article discusses the main approaches and principles of practical project management via crowdsourcing platforms, using as the example “Beorg Smart Vision” solution.

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Benchmarking non-targeted metabolomics using yeast derived libraries

10.1101/2020.10.06.319160 ◽

2020 ◽

Author(s):

Evelyn Rampler ◽

Gerrit Hermann ◽

Gerlinde Grabmann ◽

Yasin El Abiead ◽

Harald Schoeny ◽

...

Keyword(s):

Large Scale ◽

High Resolution Mass Spectrometry ◽

Chemical Space ◽

Large Fraction ◽

Cost Effective ◽

Regulatory Elements ◽

Instrumental Performance ◽

Compound Identification ◽

Targeted Metabolomics ◽

Targeted Analysis

AbstractNon-targeted analysis by high-resolution mass spectrometry (HRMS) is the essential discovery tool in metabolomics. Up to date, standardization and validation remain a challenge. Community wide accepted, cost-effective benchmark materials are lacking. In this work, we propose yeast (Pichia pastoris) extracts, derived from fully controlled fermentations for this purpose. We established an open-source metabolite library of > 200 metabolites, reproducibly recovered in ethanolic extracts by orthogonal LCHRMS methods, different fermentations (over three years) and different laboratories. More specifically, compound identification was based on accurate mass, matching retention times, and MS/MS spectra as compared to authentic standards and internal databases. The library includes metabolites from the classes of 1) organic acids and derivatives (2) nucleosides, nucleotides and analogues, (3) lipids and lipid-like molecules, (4) organic oxygen compounds, (5) organoheterocyclic compounds, (6) organic nitrogen compounds and (7) benzoids at expected concentrations ranges of sub-nM to µM. As yeast is a eukaryotic organism, key regulatory elements are highly conserved between yeast and all annotated metabolites were also reported in the Human metabolome data base (HMDB). A large fraction of metabolites was found to be stable for several years when stored at −80°C. Thus, the yeast benchmark material enabled not only to test for the chemical space and coverage upon method implementation and developments, but enabled in-house routines for instrumental performance tests. Finally, the benchmark material opens new avenues for batch to batch corrections in large scale non-targeted metabolomics studies.

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Integration of methylation QTL and enhancer–target gene maps with schizophrenia GWAS summary results identifies novel genes

Bioinformatics ◽

10.1093/bioinformatics/btz161 ◽

2019 ◽

Vol 35 (19) ◽

pp. 3576-3583 ◽

Cited By ~ 7

Author(s):

Chong Wu ◽

Wei Pan

Keyword(s):

Quantitative Trait ◽

Large Scale ◽

Target Gene ◽

Association Studies ◽

Regulatory Elements ◽

Supplementary Information ◽

Genome Wide Association Studies ◽

Novel Genes ◽

Gene Pairs ◽

Genome Wide

Abstract Motivation Most trait-associated genetic variants identified in genome-wide association studies (GWASs) are located in non-coding regions of the genome and thought to act through their regulatory roles. Results To account for enriched association signals in DNA regulatory elements, we propose a novel and general gene-based association testing strategy that integrates enhancer-target gene pairs and methylation quantitative trait locus data with GWAS summary results; it aims to both boost statistical power for new discoveries and enhance mechanistic interpretability of any new discovery. By reanalyzing two large-scale schizophrenia GWAS summary datasets, we demonstrate that the proposed method could identify some significant and novel genes (containing no genome-wide significant SNPs nearby) that would have been missed by other competing approaches, including the standard and some integrative gene-based association methods, such as one incorporating enhancer-target gene pairs and one integrating expression quantitative trait loci. Availability and implementation Software: wuchong.org/egmethyl.html Supplementary information Supplementary data are available at Bioinformatics online.

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Cis-regulatory elements and human evolution

10.1101/005652 ◽

2014 ◽

Author(s):

Adam Siepel ◽

Leonardo Arbiza

Keyword(s):

Transcriptional Regulation ◽

Human Evolution ◽

Large Scale ◽

Regulatory Elements ◽

Data Sets ◽

Regulatory Evolution ◽

Genome Wide ◽

A Genome ◽

Wide Scale ◽

Human Polymorphism

Modification of gene regulation has long been considered an important force in human evolution, particularly through changes to cis-regulatory elements (CREs) that function in transcriptional regulation. For decades, however, the study of cis-regulatory evolution was severely limited by the available data. New data sets describing the locations of CREs and genetic variation within and between species have now made it possible to study CRE evolution much more directly on a genome-wide scale. Here, we review recent research on the evolution of CREs in humans based on large-scale genomic data sets. We consider inferences based on primate divergence,human polymorphism, and combinations of divergence and polymorphism. We then consider "new frontiers" in this field stemming from recent research on transcriptional regulation.

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Computational annotation of miRNA transcription start sites

Briefings in Bioinformatics ◽

10.1093/bib/bbz178 ◽

2020 ◽

Author(s):

Saidi Wang ◽

Amlan Talukder ◽

Mingyu Cha ◽

Xiaoman Li ◽

Haiyan Hu

Keyword(s):

Computational Methods ◽

Large Scale ◽

Transcription Start ◽

Protein Coding ◽

Functional Roles ◽

Transcription Start Sites ◽

Small Noncoding Rnas ◽

Mirna Genes ◽

Genome Wide ◽

Computational Annotation

Abstract Motivation MicroRNAs (miRNAs) are small noncoding RNAs that play important roles in gene regulation and phenotype development. The identification of miRNA transcription start sites (TSSs) is critical to understand the functional roles of miRNA genes and their transcriptional regulation. Unlike protein-coding genes, miRNA TSSs are not directly detectable from conventional RNA-Seq experiments due to miRNA-specific process of biogenesis. In the past decade, large-scale genome-wide TSS-Seq and transcription activation marker profiling data have become available, based on which, many computational methods have been developed. These methods have greatly advanced genome-wide miRNA TSS annotation. Results In this study, we summarized recent computational methods and their results on miRNA TSS annotation. We collected and performed a comparative analysis of miRNA TSS annotations from 14 representative studies. We further compiled a robust set of miRNA TSSs (RSmirT) that are supported by multiple studies. Integrative genomic and epigenomic data analysis on RSmirT revealed the genomic and epigenomic features of miRNA TSSs as well as their relations to protein-coding and long non-coding genes. Contact [email protected], [email protected]

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EnhancerAtlas 2.0: an updated resource with enhancer annotation in 586 tissue/cell types across nine species

Nucleic Acids Research ◽

10.1093/nar/gkz980 ◽

2019 ◽

Cited By ~ 8

Author(s):

Tianshun Gao ◽

Jiang Qian

Keyword(s):

Large Scale ◽

Target Gene ◽

Target Genes ◽

Cell Types ◽

Regulatory Elements ◽

Tissue Cell ◽

Normal Tissues ◽

Genome Wide ◽

Wide Range ◽

User Friendly

Abstract Enhancers are distal cis-regulatory elements that activate the transcription of their target genes. They regulate a wide range of important biological functions and processes, including embryogenesis, development, and homeostasis. As more and more large-scale technologies were developed for enhancer identification, a comprehensive database is highly desirable for enhancer annotation based on various genome-wide profiling datasets across different species. Here, we present an updated database EnhancerAtlas 2.0 (http://www.enhanceratlas.org/indexv2.php), covering 586 tissue/cell types that include a large number of normal tissues, cancer cell lines, and cells at different development stages across nine species. Overall, the database contains 13 494 603 enhancers, which were obtained from 16 055 datasets using 12 high-throughput experiment methods (e.g. H3K4me1/H3K27ac, DNase-seq/ATAC-seq, P300, POLR2A, CAGE, ChIA-PET, GRO-seq, STARR-seq and MPRA). The updated version is a huge expansion of the first version, which only contains the enhancers in human cells. In addition, we predicted enhancer–target gene relationships in human, mouse and fly. Finally, the users can search enhancers and enhancer–target gene relationships through five user-friendly, interactive modules. We believe the new annotation of enhancers in EnhancerAtlas 2.0 will facilitate users to perform useful functional analysis of enhancers in various genomes.

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Economic Analysis as a Basis for Large-Scale Nitrogen Control Decisions: Reducing Nitrogen Loads to the Gulf of Mexico

The Scientific World JOURNAL ◽

10.1100/tsw.2001.270 ◽

2001 ◽

Vol 1 ◽

pp. 968-975 ◽

Cited By ~ 2

Author(s):

Otto C. Doering ◽

Marc Ribaudo ◽

Fransisco Diaz-Hermelo ◽

Ralph Heimlich ◽

Fred Hitzhusen ◽

...

Keyword(s):

Economic Analysis ◽

Large Scale ◽

Management Practices ◽

Scientific Information ◽

Cost Effective ◽

Economic Consequences ◽

N Losses ◽

Policy Makers ◽

Nitrogen Loads ◽

Effective Manner

Economic analysis can be a guide to determining the level of actions taken to reduce nitrogen (N) losses and reduce environmental risk in a cost-effective manner while also allowing consideration of relative costs of controls to various groups. The biophysical science of N control, especially from nonpoint sources such as agriculture, is not certain. Widespread precise data do not exist for a river basin (or often even for a watershed) that couples management practices and other actions to reduce nonpoint N losses with specific delivery from the basin. The causal relationships are clouded by other factors influencing N flows, such as weather, temperature, and soil characteristics. Even when the science is certain, economic analysis has its own sets of uncertainties and simplifying economic assumptions. The economic analysis of the National Hypoxia Assessment provides an example of economic analysis based on less than complete scientific information that can still provide guidance to policy makers about the economic consequences of alternative approaches. One critical value to policy makers comes from bounding the economic magnitude of the consequences of alternative actions. Another value is the identification of impacts outside the sphere of initial concerns. Such analysis can successfully assess relative impacts of different degrees of control of N losses within the basin as well as outside the basin. It can demonstrate the extent to which costs of control of any one action increase with the intensity of application of control.

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Epigenetics of Ancient DNA

Acta Naturae ◽

10.32607/20758251-2016-8-3-72-76 ◽

2016 ◽

Vol 8 (3) ◽

pp. 72-76 ◽

Cited By ~ 1

Author(s):

S. V. Zhenilo ◽

A. S. Sokolov ◽

E. B. Prokhortchou

Keyword(s):

Ancient Dna ◽

Methylation Status ◽

Regulation Of Gene Expression ◽

Genetic Selection ◽

Regulatory Elements ◽

Transcription Start Sites ◽

Genome Wide ◽

Evolutionary Changes ◽

Hoxd Cluster ◽

Different Populations

Initially, the study of DNA isolated from ancient specimens had been based on the analysis of the primary nucleotide sequence. This approach has allowed researchers to study the evolutionary changes that occur in different populations and determine the influence of the environment on genetic selection. However, the improvement of methodological approaches to genome-wide analysis has opened up new possibilities in the search for the epigenetic mechanisms involved in the regulation of gene expression. It was discovered recently that the methylation status of the regulatory elements of the HOXD cluster and MEIS1 gene changed during human evolution. Epigenetic changes in these genes played a key role in the evolution of the limbs of modern humans. Recent works have demonstrated that it is possible to determine the transcriptional activity of genes in ancient DNA samples by combining information on DNA methylation and the DNAaseI hypersensitive sequences located at the transcription start sites of genes. In the nearest future, if a preserved fossils brain is found, it will be possible to identify the evolutionary changes in the higher nervous system associated with epigenetic differences.

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funMotifs: Tissue-specific transcription factor motifs

10.1101/683722 ◽

2019 ◽

Cited By ~ 1

Author(s):

Husen M. Umer ◽

Karolina Smolinska-Garbulowska ◽

Nour-al-dain Marzouka ◽

Zeeshan Khaliq ◽

Claes Wadelius ◽

...

Keyword(s):

Large Scale ◽

Tissue Specificity ◽

Functional Characterization ◽

Regulatory Elements ◽

Web Based ◽

Tissue Specific ◽

Functional Variants ◽

Genome Wide ◽

Reporter Assays

ABSTRACTTranscription factors (TF) regulate gene expression by binding to specific sequences known as motifs. A bottleneck in our knowledge of gene regulation is the lack of functional characterization of TF motifs, which is mainly due to the large number of predicted TF motifs, and tissue specificity of TF binding. We built a framework to identify tissue-specific functional motifs (funMotifs) across the genome based on thousands of annotation tracks obtained from large-scale genomics projects including ENCODE, RoadMap Epigenomics and FANTOM. The annotations were weighted using a logistic regression model trained on regulatory elements obtained from massively parallel reporter assays. Overall, genome-wide predicted motifs of 519 TFs were characterized across fifteen tissue types. funMotifs summarizes the weighted annotations into a functional activity score for each of the predicted motifs. funMotifs enabled us to measure tissue specificity of different TFs and to identify candidate functional variants in TF motifs from the 1000 genomes project, the GTEx project, the GWAS catalogue, and in 2,515 cancer samples from the Pan-cancer analysis of whole genome sequences (PCAWG) cohort. To enable researchers annotate genomic variants or regions of interest, we have implemented a command-line pipeline and a web-based interface that can publicly be accessed on: http://bioinf.icm.uu.se/funmotifs.

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