scispace - formally typeset
Search or ask a question
Journal ArticleDOI

RNA-Seq: a revolutionary tool for transcriptomics

01 Jan 2009-Nature Reviews Genetics (Nature Publishing Group)-Vol. 10, Iss: 1, pp 57-63
TL;DR: The RNA-Seq approach to transcriptome profiling that uses deep-sequencing technologies provides a far more precise measurement of levels of transcripts and their isoforms than other methods.
Abstract: RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq also provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes.

Content maybe subject to copyright    Report

Citations
More filters
Journal ArticleDOI
TL;DR: This Opinion article focuses on the use of genome editing to target S genes for the development of transgene-free and durable disease-resistant crop varieties.

178 citations

Journal ArticleDOI
TL;DR: An update of MS-based PTM analyses is provided, with a focus on current enrichment strategies coupled with revolutionary advances in high performance MS, and examples of the discovery of recently described biological roles of PTMs are discussed.
Abstract: Recent advances in mass spectrometry (MS)-based proteomics allow the identification and quantitation of thousands of posttranslational modification (PTM) sites in a single experiment. This follows from the development of more effective class enrichment strategies, new high performance instrumentation and bioinformatic algorithms with rigorous scoring strategies. More widespread use of these combined capabilities have led to a vast expansion in our knowledge of the complexity of biological processes mediated by PTMs. The classes most actively pursued include phosphorylation, ubiquitination, O-GlcNAcylation, methylation, and acetylation. Very recently succinylation, SUMOylation, and citrullination have emerged. Among the some 260 000 PTM sites that have been identified in the human proteome thus far, only a few have been assigned to key regulatory and/or other biological roles. Here, we provide an update of MS-based PTM analyses, with a focus on current enrichment strategies coupled with revolutionary advances in high performance MS. Furthermore, we discuss examples of the discovery of recently described biological roles of PTMs and address the challenges of defining site-specific functions.

178 citations

Journal ArticleDOI
TL;DR: The findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription, and summarizes the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription.

177 citations

Journal ArticleDOI
TL;DR: A change in focus toward screening functional genomic elements as opposed to genes sensu stricto will be required if the authors are to capitalize fully on recent technical and conceptual advances and identify new types of disease‐associated mutation within noncoding regions remote from the genes whose function they disrupt.
Abstract: The number of reported germline mutations in human nuclear genes, either underlying or associated with inherited disease, has now exceeded 100,000 in more than 3,700 different genes. The availability of these data has both revolutionized the study of the morbid anatomy of the human genome and facilitated “personalized genomics.” With ∼300 new “inherited disease genes” (and ∼10,000 new mutations) being identified annually, it is pertinent to ask how many “inherited disease genes” there are in the human genome, how many mutations reside within them, and where such lesions are likely to be located? To address these questions, it is necessary not only to reconsider how we define human genes but also to explore notions of gene “essentiality” and “dispensability.” Answers to these questions are now emerging from recent novel insights into genome structure and function and through complete genome sequence information derived from multiple individual human genomes. However, a change in focus toward screening functional genomic elements as opposed to genes sensu stricto will be required if we are to capitalize fully on recent technical and conceptual advances and identify new types of disease-associated mutation within noncoding regions remote from the genes whose function they disrupt.

176 citations


Cites background from "RNA-Seq: a revolutionary tool for t..."

  • ..., 2010; Prabhu and Pe’er, 2009], but also of the successful application of direct RNA sequencing of the human transcriptome [Ozsolak et al., 2009; Wang et al., 2009b] and whole-genome sequencing [Lupski et al....

    [...]

  • ...…functional elements including intron splice enhancers and silencers, cis-acting RNA elements that regulate alternative splicing [Tress et al., 2007; Wang et al., 2009a], and potentially also trans-splicing elements [Akiva et al., 2006; Gingeras 2009; Shao et al., 2006], as well as other regulatory…...

    [...]

Journal ArticleDOI
TL;DR: ReCount is an online resource of RNA-seq gene count tables and auxilliary data built from raw RNA sequencing data from 18 different published studies comprising 475 samples and over 8 billion reads, allowing statistical and computational scientists to consider alternative parameter values.
Abstract: RNA sequencing is a flexible and powerful new approach for measuring gene, exon, or isoform expression. To maximize the utility of RNA sequencing data, new statistical methods are needed for clustering, differential expression, and other analyses. A major barrier to the development of new statistical methods is the lack of RNA sequencing datasets that can be easily obtained and analyzed in common statistical software packages such as R. To speed up the development process, we have created a resource of analysis-ready RNA-sequencing datasets. ReCount is an online resource of RNA-seq gene count tables and auxilliary data. Tables were built from raw RNA sequencing data from 18 different published studies comprising 475 samples and over 8 billion reads. Using the Myrna package, reads were aligned, overlapped with gene models and tabulated into gene-by-sample count tables that are ready for statistical analysis. Count tables and phenotype data were combined into Bioconductor ExpressionSet objects for ease of analysis. ReCount also contains the Myrna manifest files and R source code used to process the samples, allowing statistical and computational scientists to consider alternative parameter values. By combining datasets from many studies and providing data that has already been processed from. fastq format into ready-to-use. RData and. txt files, ReCount facilitates analysis and methods development for RNA-seq count data. We anticipate that ReCount will also be useful for investigators who wish to consider cross-study comparisons and alternative normalization strategies for RNA-seq.

176 citations


Cites methods from "RNA-Seq: a revolutionary tool for t..."

  • ...RNA-seq, or short-read sequencing of mRNA, has emerged as a powerful and flexible tool for studying gene expression [1]....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors.
Abstract: We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41–52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3′ untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 × 10 5 distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices. The mRNA population specifies a cell’s identity and helps to govern its present and future activities. This has made transcriptome analysis a general phenotyping method, with expression microarrays of many kinds in routine use. Here we explore the possibility that transcriptome analysis, transcript discovery and transcript refinement can be done effectively in large and complex mammalian genomes by ultra-high-throughput sequencing. Expression microarrays are currently the most widely used methodology for transcriptome analysis, although some limitations persist. These include hybridization and cross-hybridization artifacts 1–3 , dye-based detection issues and design constraints that preclude or seriously limit the detection of RNA splice patterns and previously unmapped genes. These issues have made it difficult for standard array designs to provide full sequence comprehensiveness (coverage of all possible genes, including unknown ones, in large genomes) or transcriptome comprehensiveness (reliable detection of all RNAs of all prevalence classes, including the least abundant ones that are physiologically relevant). Other

12,293 citations

PatentDOI
04 Oct 2000-Science
TL;DR: Serial analysis of gene expression (SAGE) should provide a broadly applicable means for the quantitative cataloging and comparison of expressed genes in a variety of normal, developmental, and disease states.
Abstract: PROBLEM TO BE SOLVED: To provide a method for preparing a short nucleotide sequence (tag) which is useful to identify a cDNA oligonucleotide and is derived from a restricted position in a mRNA or a cDNA. SOLUTION: This is the method of preparing a tag for identifying the cDNA oligonucleotide. The above method comprises preparing the cDNA oligonucleotide bearing 5' and 3' terminals, collecting cDNA fragments by cutting the cDNA oligonucleotide with a restriction enzyme at the first restriction endonuclease site, separating a cDNA oligonucleotide bearing 5' or 3' terminal and connecting an oligonucleotide linker to the isolated cDNA fragment bearing the cDNA oligonucleotide 5' or 3' terminal. Here, the oligonucleotide linker contains the recognition site of the second restriction endonuclease enzyme and the isolated cDNA fragment is cut with the second restriction endonuclease enzyme which cuts the cDNA fragment in a section separated from the recognition site to obtain the tag for identifying the cDNA oligonucleotide.

4,437 citations

Journal ArticleDOI
TL;DR: This work describes the software MAQ, software that can build assemblies by mapping shotgun short reads to a reference genome, using quality scores to derive genotype calls of the consensus sequence of a diploid genome, e.g., from a human sample.
Abstract: New sequencing technologies promise a new era in the use of DNA sequence. However, some of these technologies produce very short reads, typically of a few tens of base pairs, and to use these reads effectively requires new algorithms and software. In particular, there is a major issue in efficiently aligning short reads to a reference genome and handling ambiguity or lack of accuracy in this alignment. Here we introduce the concept of mapping quality, a measure of the confidence that a read actually comes from the position it is aligned to by the mapping algorithm. We describe the software MAQ that can build assemblies by mapping shotgun short reads to a reference genome, using quality scores to derive genotype calls of the consensus sequence of a diploid genome, e.g., from a human sample. MAQ makes full use of mate-pair information and estimates the error probability of each read alignment. Error probabilities are also derived for the final genotype calls, using a Bayesian statistical model that incorporates the mapping qualities, error probabilities from the raw sequence quality scores, sampling of the two haplotypes, and an empirical model for correlated errors at a site. Both read mapping and genotype calling are evaluated on simulated data and real data. MAQ is accurate, efficient, versatile, and user-friendly. It is freely available at http://maq.sourceforge.net.

2,927 citations

Journal ArticleDOI
TL;DR: It is found that the Illumina sequencing data are highly replicable, with relatively little technical variation, and thus, for many purposes, it may suffice to sequence each mRNA sample only once (i.e., using one lane).
Abstract: Ultra-high-throughput sequencing is emerging as an attractive alternative to microarrays for genotyping, analysis of methylation patterns, and identification of transcription factor binding sites. Here, we describe an application of the Illumina sequencing (formerly Solexa sequencing) platform to study mRNA expression levels. Our goals were to estimate technical variance associated with Illumina sequencing in this context and to compare its ability to identify differentially expressed genes with existing array technologies. To do so, we estimated gene expression differences between liver and kidney RNA samples using multiple sequencing replicates, and compared the sequencing data to results obtained from Affymetrix arrays using the same RNA samples. We find that the Illumina sequencing data are highly replicable, with relatively little technical variation, and thus, for many purposes, it may suffice to sequence each mRNA sample only once (i.e., using one lane). The information in a single lane of Illumina sequencing data appears comparable to that in a single array in enabling identification of differentially expressed genes, while allowing for additional analyses such as detection of low-expressed genes, alternative splice variants, and novel transcripts. Based on our observations, we propose an empirical protocol and a statistical framework for the analysis of gene expression using ultra-high-throughput sequencing technology.

2,834 citations

Journal ArticleDOI
TL;DR: The program SOAP is designed to handle the huge amounts of short reads generated by parallel sequencing using the new generation Illumina-Solexa sequencing technology, which supports multi-threaded parallel computing and has a batch module for multiple query sets.
Abstract: Summary: We have developed a program SOAP for efficient gapped and ungapped alignment of short oligonucleotides onto reference sequences. The program is designed to handle the huge amounts of short reads generated by parallel sequencing using the new generation Illumina-Solexa sequencing technology. SOAP is compatible with numerous applications, including single-read or pair-end resequencing, small RNA discovery and mRNA tag sequence mapping. SOAP is a command-driven program, which supports multi-threaded parallel computing, and has a batch module for multiple query sets. Availability: http://soap.genomics.org.cn Contact: soap@genomics.org.cn

2,729 citations


"RNA-Seq: a revolutionary tool for t..." refers methods in this paper

  • ...There are several programs for mapping reads to the genome, including ELAND, SOA...

    [...]