Publications by authors named "Thomas Gingeras"

98 Publications

Ground tissue circuitry regulates organ complexity in maize and .

Science 2021 12 2;374(6572):1247-1252. Epub 2021 Dec 2.

Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY 10003, USA.

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http://dx.doi.org/10.1126/science.abj2327DOI Listing
December 2021

Selective time-dependent changes in activity and cell-specific gene expression in human postmortem brain.

Sci Rep 2021 03 23;11(1):6078. Epub 2021 Mar 23.

University of Illinois at Chicago, Chicago, IL, 60612, USA.

As a means to understand human neuropsychiatric disorders from human brain samples, we compared the transcription patterns and histological features of postmortem brain to fresh human neocortex isolated immediately following surgical removal. Compared to a number of neuropsychiatric disease-associated postmortem transcriptomes, the fresh human brain transcriptome had an entirely unique transcriptional pattern. To understand this difference, we measured genome-wide transcription as a function of time after fresh tissue removal to mimic the postmortem interval. Within a few hours, a selective reduction in the number of neuronal activity-dependent transcripts occurred with relative preservation of housekeeping genes commonly used as a reference for RNA normalization. Gene clustering indicated a rapid reduction in neuronal gene expression with a reciprocal time-dependent increase in astroglial and microglial gene expression that continued to increase for at least 24 h after tissue resection. Predicted transcriptional changes were confirmed histologically on the same tissue demonstrating that while neurons were degenerating, glial cells underwent an outgrowth of their processes. The rapid loss of neuronal genes and reciprocal expression of glial genes highlights highly dynamic transcriptional and cellular changes that occur during the postmortem interval. Understanding these time-dependent changes in gene expression in post mortem brain samples is critical for the interpretation of research studies on human brain disorders.
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http://dx.doi.org/10.1038/s41598-021-85801-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988150PMC
March 2021

Single-cell RNA sequencing of developing maize ears facilitates functional analysis and trait candidate gene discovery.

Dev Cell 2021 02 4;56(4):557-568.e6. Epub 2021 Jan 4.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. Electronic address:

Crop productivity depends on activity of meristems that produce optimized plant architectures, including that of the maize ear. A comprehensive understanding of development requires insight into the full diversity of cell types and developmental domains and the gene networks required to specify them. Until now, these were identified primarily by morphology and insights from classical genetics, which are limited by genetic redundancy and pleiotropy. Here, we investigated the transcriptional profiles of 12,525 single cells from developing maize ears. The resulting developmental atlas provides a single-cell RNA sequencing (scRNA-seq) map of an inflorescence. We validated our results by mRNA in situ hybridization and by fluorescence-activated cell sorting (FACS) RNA-seq, and we show how these data may facilitate genetic studies by predicting genetic redundancy, integrating transcriptional networks, and identifying candidate genes associated with crop yield traits.
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http://dx.doi.org/10.1016/j.devcel.2020.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904613PMC
February 2021

A limited set of transcriptional programs define major cell types.

Genome Res 2020 07 29;30(7):1047-1059. Epub 2020 Jul 29.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, E-08003 Barcelona, Catalonia, Spain.

We have produced RNA sequencing data for 53 primary cells from different locations in the human body. The clustering of these primary cells reveals that most cells in the human body share a few broad transcriptional programs, which define five major cell types: epithelial, endothelial, mesenchymal, neural, and blood cells. These act as basic components of many tissues and organs. Based on gene expression, these cell types redefine the basic histological types by which tissues have been traditionally classified. We identified genes whose expression is specific to these cell types, and from these genes, we estimated the contribution of the major cell types to the composition of human tissues. We found this cellular composition to be a characteristic signature of tissues and to reflect tissue morphological heterogeneity and histology. We identified changes in cellular composition in different tissues associated with age and sex, and found that departures from the normal cellular composition correlate with histological phenotypes associated with disease.
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http://dx.doi.org/10.1101/gr.263186.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397875PMC
July 2020

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

Nature 2020 07 29;583(7818):699-710. Epub 2020 Jul 29.

Department of Biological Science, Florida State University, Tallahassee, FL, USA.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE and Roadmap Epigenomics data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.
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http://dx.doi.org/10.1038/s41586-020-2493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410828PMC
July 2020

Perspectives on ENCODE.

Nature 2020 07 29;583(7818):693-698. Epub 2020 Jul 29.

HudsonAlpha Institute for Biotechnology, Huntsville, AL, USA.

The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community.
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http://dx.doi.org/10.1038/s41586-020-2449-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410827PMC
July 2020

Processing by RNase 1 forms tRNA halves and distinct Y RNA fragments in the extracellular environment.

Nucleic Acids Res 2020 08;48(14):8035-8049

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

Extracellular RNAs participate in intercellular communication, and are being studied as promising minimally invasive diagnostic markers. Several studies in recent years showed that tRNA halves and distinct Y RNA fragments are abundant in the extracellular space, including in biofluids. While their regulatory and diagnostic potential has gained a substantial amount of attention, the biogenesis of these extracellular RNA fragments remains largely unexplored. Here, we demonstrate that these fragments are produced by RNase 1, a highly active secreted nuclease. We use RNA sequencing to investigate the effect of a null mutation of RNase 1 on the levels of tRNA halves and Y RNA fragments in the extracellular environment of cultured human cells. We complement and extend our RNA sequencing results with northern blots, showing that tRNAs and Y RNAs in the non-vesicular extracellular compartment are released from cells as full-length precursors and are subsequently cleaved to distinct fragments. In support of these results, formation of tRNA halves is recapitulated by recombinant human RNase 1 in our in vitro assay. These findings assign a novel function for RNase 1, and position it as a strong candidate for generation of tRNA halves and Y RNA fragments in biofluids.
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http://dx.doi.org/10.1093/nar/gkaa526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430647PMC
August 2020

Management, Analyses, and Distribution of the MaizeCODE Data on the Cloud.

Front Plant Sci 2020 31;11:289. Epub 2020 Mar 31.

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, United States.

MaizeCODE is a project aimed at identifying and analyzing functional elements in the maize genome. In its initial phase, MaizeCODE assayed up to five tissues from four maize strains (B73, NC350, W22, TIL11) by RNA-Seq, Chip-Seq, RAMPAGE, and small RNA sequencing. To facilitate reproducible science and provide both human and machine access to the MaizeCODE data, we enhanced SciApps, a cloud-based portal, for analysis and distribution of both raw data and analysis results. Based on the SciApps workflow platform, we generated new components to support the complete cycle of MaizeCODE data management. These include publicly accessible scientific workflows for the reproducible and shareable analysis of various functional data, a RESTful API for batch processing and distribution of data and metadata, a searchable data page that lists each MaizeCODE experiment as a reproducible workflow, and integrated JBrowse genome browser tracks linked with workflows and metadata. The SciApps portal is a flexible platform that allows the integration of new analysis tools, workflows, and genomic data from multiple projects. Through metadata and a ready-to-compute cloud-based platform, the portal experience improves access to the MaizeCODE data and facilitates its analysis.
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http://dx.doi.org/10.3389/fpls.2020.00289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136414PMC
March 2020

Dynamics of microRNA expression during mouse prenatal development.

Genome Res 2019 11 23;29(11):1900-1909. Epub 2019 Oct 23.

Department of Developmental and Cell Biology, University of California Irvine, Irvine, California 92697, USA.

MicroRNAs (miRNAs) play a critical role as posttranscriptional regulators of gene expression. The ENCODE Project profiled the expression of miRNAs in an extensive set of organs during a time-course of mouse embryonic development and captured the expression dynamics of 785 miRNAs. We found distinct organ-specific and developmental stage-specific miRNA expression clusters, with an overall pattern of increasing organ-specific expression as embryonic development proceeds. Comparative analysis of conserved miRNAs in mouse and human revealed stronger clustering of expression patterns by organ type rather than by species. An analysis of messenger RNA expression clusters compared with miRNA expression clusters identifies the potential role of specific miRNA expression clusters in suppressing the expression of mRNAs specific to other developmental programs in the organ in which these miRNAs are expressed during embryonic development. Our results provide the most comprehensive time-course of miRNA expression as part of an integrated ENCODE reference data set for mouse embryonic development.
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http://dx.doi.org/10.1101/gr.248997.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836743PMC
November 2019

Genome-wide analysis of polymerase III-transcribed elements suggests cell-type-specific enhancer function.

Genome Res 2019 09 14;29(9):1402-1414. Epub 2019 Aug 14.

Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

elements are one of the most successful families of transposons in the human genome. A portion of elements is transcribed by RNA Pol III, whereas the remaining ones are part of Pol II transcripts. Because elements are highly repetitive, it has been difficult to identify the Pol III-transcribed elements and quantify their expression levels. In this study, we generated high-resolution, long-genomic-span RAMPAGE data in 155 biosamples all with matching RNA-seq data and built an atlas of 17,249 Pol III-transcribed elements. We further performed an integrative analysis on the ChIP-seq data of 10 histone marks and hundreds of transcription factors, whole-genome bisulfite sequencing data, ChIA-PET data, and functional data in several biosamples, and our results revealed that although the human-specific elements are transcriptionally repressed, the older, expressed elements may be exapted by the human host to function as cell-type-specific enhancers for their nearby protein-coding genes.
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http://dx.doi.org/10.1101/gr.249789.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6724667PMC
September 2019

The long noncoding RNA regulates inflammatory gene expression.

EMBO J 2019 04 27;38(8). Epub 2019 Mar 27.

Department of Pediatrics, University of California San Diego School of Medicine, La Jolla, CA, USA

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in (locally) or in (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of -acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B We focused on elucidating the function of one lncRNA, named or (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the promoter. In turn, -APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing transcription and subsequent Ca signaling and inflammatory gene expression. Finally, genetic variants affecting expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of -acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.
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http://dx.doi.org/10.15252/embj.2018100041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463213PMC
April 2019

The fractured landscape of RNA-seq alignment: the default in our STARs.

Nucleic Acids Res 2018 06;46(10):5125-5138

Stanley Institute for Cognitive Genomics, Cold Spring Harbor Laboratory, Woodbury, NY 11797, USA.

Many tools are available for RNA-seq alignment and expression quantification, with comparative value being hard to establish. Benchmarking assessments often highlight methods' good performance, but are focused on either model data or fail to explain variation in performance. This leaves us to ask, what is the most meaningful way to assess different alignment choices? And importantly, where is there room for progress? In this work, we explore the answers to these two questions by performing an exhaustive assessment of the STAR aligner. We assess STAR's performance across a range of alignment parameters using common metrics, and then on biologically focused tasks. We find technical metrics such as fraction mapping or expression profile correlation to be uninformative, capturing properties unlikely to have any role in biological discovery. Surprisingly, we find that changes in alignment parameters within a wide range have little impact on both technical and biological performance. Yet, when performance finally does break, it happens in difficult regions, such as X-Y paralogs and MHC genes. We believe improved reporting by developers will help establish where results are likely to be robust or fragile, providing a better baseline to establish where methodological progress can still occur.
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http://dx.doi.org/10.1093/nar/gky325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007662PMC
June 2018

Advances, challenges, and opportunities in extracellular RNA biology: insights from the NIH exRNA Strategic Workshop.

JCI Insight 2018 04 5;3(7). Epub 2018 Apr 5.

Cancer Immunology, Hematology, and Etiology Branch, Division of Cancer Biology, National Cancer Institute, Bethesda, Maryland, USA.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.
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http://dx.doi.org/10.1172/jci.insight.98942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928855PMC
April 2018

Conserved noncoding transcription and core promoter regulatory code in early development.

Elife 2017 12 20;6. Epub 2017 Dec 20.

Watson School of Biological Sciences, Cold Spring Harbor Laboratory, New York, United States.

Multicellular development is driven by regulatory programs that orchestrate the transcription of protein-coding and noncoding genes. To decipher this genomic regulatory code, and to investigate the developmental relevance of noncoding transcription, we compared genome-wide promoter activity throughout embryogenesis in 5 species. Core promoters, generally not thought to play a significant regulatory role, in fact impart restrictions on the developmental timing of gene expression on a global scale. We propose a hierarchical regulatory model in which core promoters define broad windows of opportunity for expression, by defining a range of transcription factors from which they can receive regulatory inputs. This two-tiered mechanism globally orchestrates developmental gene expression, including extremely widespread noncoding transcription. The sequence and expression specificity of noncoding RNA promoters are evolutionarily conserved, implying biological relevance. Overall, this work introduces a hierarchical model for developmental gene regulation, and reveals a major role for noncoding transcription in animal development.
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http://dx.doi.org/10.7554/eLife.29005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754203PMC
December 2017

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

Nat Genet 2017 Dec 6;49(12):1731-1740. Epub 2017 Nov 6.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain.

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.
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http://dx.doi.org/10.1038/ng.3988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709232PMC
December 2017

FANTOM5 CAGE profiles of human and mouse samples.

Sci Data 2017 08 29;4:170112. Epub 2017 Aug 29.

Scottish Centre for Regenerative Medicine, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, UK.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.
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http://dx.doi.org/10.1038/sdata.2017.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574368PMC
August 2017

Comparative transcriptomics in human and mouse.

Nat Rev Genet 2017 07 8;18(7):425-440. Epub 2017 May 8.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain.

Cross-species comparisons of genomes, transcriptomes and gene regulation are now feasible at unprecedented resolution and throughput, enabling the comparison of human and mouse biology at the molecular level. Insights have been gained into the degree of conservation between human and mouse at the level of not only gene expression but also epigenetics and inter-individual variation. However, a number of limitations exist, including incomplete transcriptome characterization and difficulties in identifying orthologous phenotypes and cell types, which are beginning to be addressed by emerging technologies. Ultimately, these comparisons will help to identify the conditions under which the mouse is a suitable model of human physiology and disease, and optimize the use of animal models.
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http://dx.doi.org/10.1038/nrg.2017.19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413734PMC
July 2017

Gene-specific patterns of expression variation across organs and species.

Genome Biol 2016 07 8;17(1):151. Epub 2016 Jul 8.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona, 08003, Spain.

Background: A comparison of transcriptional profiles derived from different tissues in a given species or among different species assumes that commonalities reflect evolutionarily conserved programs and that differences reflect species or tissue responses to environmental conditions or developmental program staging. Apparently conflicting results have been published regarding whether organ-specific transcriptional patterns dominate over species-specific patterns, or vice versa, making it unclear to what extent the biology of a given organism can be extrapolated to another. These studies have in common that they treat the transcriptomes monolithically, implicitly ignoring that each gene is likely to have a specific pattern of transcriptional variation across organs and species.

Results: We use linear models to quantify this pattern. We find a continuum in the spectrum of expression variation: the expression of some genes varies considerably across species and little across organs, and simply reflects evolutionary distance. At the other extreme are genes whose expression varies considerably across organs and little across species; these genes are much more likely to be associated with diseases than are genes whose expression varies predominantly across species.

Conclusions: Whether transcriptomes, when considered globally, cluster preferentially according to one component or the other may not be a property of the transcriptomes, but rather a consequence of the dominant behavior of a subset of genes. Therefore, the values of the components of the variance of expression for each gene could become a useful resource when planning, interpreting, and extrapolating experimental data from mouse to humans.
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http://dx.doi.org/10.1186/s13059-016-1008-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937605PMC
July 2016

Optimizing RNA-Seq Mapping with STAR.

Methods Mol Biol 2016 ;1415:245-62

Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, NY, 11746, USA.

Recent advances in high-throughput sequencing technology made it possible to probe the cell transcriptomes by generating hundreds of millions of short reads which represent the fragments of the transcribed RNA molecules. The first and the most crucial task in the RNA-seq data analysis is mapping of the reads to the reference genome. STAR (Spliced Transcripts Alignment to a Reference) is an RNA-seq mapper that performs highly accurate spliced sequence alignment at an ultrafast speed. STAR alignment algorithm can be controlled by many user-defined parameters. Here, we describe the most important STAR options and parameters, as well as best practices for achieving the maximum mapping accuracy and speed.
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http://dx.doi.org/10.1007/978-1-4939-3572-7_13DOI Listing
December 2017

Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA.

RNA 2015 Nov 21;21(11):1966-79. Epub 2015 Sep 21.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

Extracellular vesicles (EVs) have been proposed as a means to promote intercellular communication. We show that when human primary cells are exposed to cancer cell EVs, rapid cell death of the primary cells is observed, while cancer cells treated with primary or cancer cell EVs do not display this response. The active agents that trigger cell death are 29- to 31-nucleotide (nt) or 22- to 23-nt processed fragments of an 83-nt primary transcript of the human RNY5 gene that are highly likely to be formed within the EVs. Primary cells treated with either cancer cell EVs, deproteinized total RNA from either primary or cancer cell EVs, or synthetic versions of 31- and 23-nt fragments trigger rapid cell death in a dose-dependent manner. The transfer of processed RNY5 fragments through EVs may reflect a novel strategy used by cancer cells toward the establishment of a favorable microenvironment for their proliferation and invasion.
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http://dx.doi.org/10.1261/rna.053629.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4604435PMC
November 2015

Mapping RNA-seq Reads with STAR.

Curr Protoc Bioinformatics 2015 Sep 3;51:11.14.1-11.14.19. Epub 2015 Sep 3.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Mapping of large sets of high-throughput sequencing reads to a reference genome is one of the foundational steps in RNA-seq data analysis. The STAR software package performs this task with high levels of accuracy and speed. In addition to detecting annotated and novel splice junctions, STAR is capable of discovering more complex RNA sequence arrangements, such as chimeric and circular RNA. STAR can align spliced sequences of any length with moderate error rates, providing scalability for emerging sequencing technologies. STAR generates output files that can be used for many downstream analyses such as transcript/gene expression quantification, differential gene expression, novel isoform reconstruction, and signal visualization. In this unit, we describe computational protocols that produce various output files, use different RNA-seq datatypes, and utilize different mapping strategies. STAR is open source software that can be run on Unix, Linux, or Mac OS X systems.
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http://dx.doi.org/10.1002/0471250953.bi1114s51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631051PMC
September 2015

Enhanced transcriptome maps from multiple mouse tissues reveal evolutionary constraint in gene expression.

Nat Commun 2015 Jan 13;6:5903. Epub 2015 Jan 13.

Functional Genomics Group, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

Mice have been a long-standing model for human biology and disease. Here we characterize, by RNA sequencing, the transcriptional profiles of a large and heterogeneous collection of mouse tissues, augmenting the mouse transcriptome with thousands of novel transcript candidates. Comparison with transcriptome profiles in human cell lines reveals substantial conservation of transcriptional programmes, and uncovers a distinct class of genes with levels of expression that have been constrained early in vertebrate evolution. This core set of genes captures a substantial fraction of the transcriptional output of mammalian cells, and participates in basic functional and structural housekeeping processes common to all cell types. Perturbation of these constrained genes is associated with significant phenotypes including embryonic lethality and cancer. Evolutionary constraint in gene expression levels is not reflected in the conservation of the genomic sequences, but is associated with conserved epigenetic marking, as well as with characteristic post-transcriptional regulatory programme, in which sub-cellular localization and alternative splicing play comparatively large roles.
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http://dx.doi.org/10.1038/ncomms6903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308717PMC
January 2015

Comparison of the transcriptional landscapes between human and mouse tissues.

Proc Natl Acad Sci U S A 2014 Dec 20;111(48):17224-9. Epub 2014 Nov 20.

Department of Genetics, Stanford University, Stanford, CA 94305;

Although the similarities between humans and mice are typically highlighted, morphologically and genetically, there are many differences. To better understand these two species on a molecular level, we performed a comparison of the expression profiles of 15 tissues by deep RNA sequencing and examined the similarities and differences in the transcriptome for both protein-coding and -noncoding transcripts. Although commonalities are evident in the expression of tissue-specific genes between the two species, the expression for many sets of genes was found to be more similar in different tissues within the same species than between species. These findings were further corroborated by associated epigenetic histone mark analyses. We also find that many noncoding transcripts are expressed at a low level and are not detectable at appreciable levels across individuals. Moreover, the majority lack obvious sequence homologs between species, even when we restrict our attention to those which are most highly reproducible across biological replicates. Overall, our results indicate that there is considerable RNA expression diversity between humans and mice, well beyond what was described previously, likely reflecting the fundamental physiological differences between these two organisms.
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http://dx.doi.org/10.1073/pnas.1413624111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4260565PMC
December 2014

A comparative encyclopedia of DNA elements in the mouse genome.

Nature 2014 Nov;515(7527):355-64

Bioinformatics and Genomics, Centre for Genomic Regulation (CRG) and UPF, Doctor Aiguader, 88, 08003 Barcelona, Catalonia, Spain.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.
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http://dx.doi.org/10.1038/nature13992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4266106PMC
November 2014

Comparative analysis of the transcriptome across distant species.

Nature 2014 Aug;512(7515):445-8

Functional Genomics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.
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http://dx.doi.org/10.1038/nature13424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155737PMC
August 2014

Considerations when investigating lncRNA function in vivo.

Elife 2014 Aug 14;3:e03058. Epub 2014 Aug 14.

Chris P Ponting is in the MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom; Wellcome Trust Sanger Institute, Cambridge, United Kingdom

Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.
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http://dx.doi.org/10.7554/eLife.03058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132285PMC
August 2014

A genome-wide survey of sexually dimorphic expression of Drosophila miRNAs identifies the steroid hormone-induced miRNA let-7 as a regulator of sexual identity.

Genetics 2014 Oct 31;198(2):647-68. Epub 2014 Jul 31.

Max Planck Research Group of Gene Expression and Signaling, Max Planck Institute for Biophysical Chemistry, Göttingen 37077, Germany

MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.
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http://dx.doi.org/10.1534/genetics.114.169268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4196619PMC
October 2014

Multiplicity of 5' cap structures present on short RNAs.

PLoS One 2014 31;9(7):e102895. Epub 2014 Jul 31.

RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Japan; RIKEN Omics Science Center, Yokohama, Kanagawa, Japan.

Most RNA molecules are co- or post-transcriptionally modified to alter their chemical and functional properties to assist in their ultimate biological function. Among these modifications, the addition of 5' cap structure has been found to regulate turnover and localization. Here we report a study of the cap structure of human short (<200 nt) RNAs (sRNAs), using sequencing of cDNA libraries prepared by enzymatic pretreatment of the sRNAs with cap sensitive-specificity, thin layer chromatographic (TLC) analyses of isolated cap structures and mass spectrometric analyses for validation of TLC analyses. Processed versions of snoRNAs and tRNAs sequences of less than 50 nt were observed in capped sRNA libraries, indicating additional processing and recapping of these annotated sRNAs biotypes. We report for the first time 2,7 dimethylguanosine in human sRNAs cap structures and surprisingly we find multiple type 0 cap structures (mGpppC, 7mGpppG, GpppG, GpppA, and 7mGpppA) in RNA length fractions shorter than 50 nt. Finally, we find the presence of additional uncharacterized cap structures that wait determination by the creation of needed reference compounds to be used in TLC analyses. These studies suggest the existence of novel biochemical pathways leading to the processing of primary and sRNAs and the modifications of their RNA 5' ends with a spectrum of chemical modifications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102895PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117478PMC
October 2015

Defining functional DNA elements in the human genome.

Proc Natl Acad Sci U S A 2014 Apr 21;111(17):6131-8. Epub 2014 Apr 21.

Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02139.

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease.
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http://dx.doi.org/10.1073/pnas.1318948111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035993PMC
April 2014
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