RESUMO
Long-range and highly accurate de novo assembly from short-read data is one of the most pressing challenges in genomics. Recently, it has been shown that read pairs generated by proximity ligation of DNA in chromatin of living tissue can address this problem, dramatically increasing the scaffold contiguity of assemblies. Here, we describe a simpler approach ("Chicago") based on in vitro reconstituted chromatin. We generated two Chicago data sets with human DNA and developed a statistical model and a new software pipeline ("HiRise") that can identify poor quality joins and produce accurate, long-range sequence scaffolds. We used these to construct a highly accurate de novo assembly and scaffolding of a human genome with scaffold N50 of 20 Mbp. We also demonstrated the utility of Chicago for improving existing assemblies by reassembling and scaffolding the genome of the American alligator. With a single library and one lane of Illumina HiSeq sequencing, we increased the scaffold N50 of the American alligator from 508 kbp to 10 Mbp.
Assuntos
Cromossomos , Biologia Computacional/métodos , Ligação Genética , Biblioteca Genômica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Linhagem Celular , Humanos , Reprodutibilidade dos TestesRESUMO
SUMMARY: Most genes in human, mouse and rat produce more than one transcript isoform. The Affymetrix Exon Array is a tool for studying the many processes that regulate RNA production, with separate probesets measuring RNA levels at known and putative exons. For insights on how exons levels vary between normal tissues, we constructed the Affy Exon Tissues track from tissue data published by Affymetrix. This track reports exon probeset intensities as log ratios relative to median values across the dataset and renders them as colored heat maps, to yield quick visual identification of exons with intensities that vary between normal tissues. AVAILABILITY: Affy Exon Tissues track is freely available under the UCSC Genome Browser (http://genome.ucsc.edu/) for human (hg18), mouse (mm8 and mm9), and rat (rn4).
Assuntos
Biologia Computacional/métodos , Éxons , Animais , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA/metabolismo , Ratos , SoftwareRESUMO
Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.
Assuntos
Processamento Alternativo/genética , Éxons/genética , Íntrons/genética , Proteínas Adaptadoras de Transdução de Sinal/classificação , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sequência de Bases , Encéfalo/metabolismo , Sequência Conservada , Evolução Molecular , Humanos , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Músculos/metabolismo , Neuropeptídeos/classificação , Neuropeptídeos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Isoformas de Proteínas/genética , Alinhamento de SequênciaRESUMO
Genetic and biochemical studies of Schizosaccharomyces pombe and Saccharomyces cerevisiae have identified gene products that play essential functions in both pre-mRNA splicing and cell cycle control. Among these are the conserved, Myb-related CDC5 (also known as Cef1p in S. cerevisiae) proteins. The mechanism by which loss of CDC5/Cef1p function causes both splicing and cell cycle defects has been unclear. Here we provide evidence that cell cycle arrest in a new temperature-sensitive CEF1 mutant, cef1-13, is an indirect consequence of defects in pre-mRNA splicing. Although cef1-13 cells harbor global defects in pre-mRNA splicing discovered through intron microarray analysis, inefficient splicing of the alpha-tubulin-encoding TUB1 mRNA was considered as a potential cause of the cef1-13 cell cycle arrest because cef1-13 cells arrest uniformly at G(2)/M with many hallmarks of a defective microtubule cytoskeleton. Consistent with this possibility, cef1-13 cells possess reduced levels of total TUB1 mRNA and alpha-tubulin protein. Removing the intron from TUB1 in cef1-13 cells boosts TUB1 mRNA and alpha-tubulin expression to near wild-type levels and restores microtubule stability in the cef1-13 mutant. As a result, cef1-13 tub1Deltai cells progress through mitosis and their cell cycle arrest phenotype is alleviated. Removing the TUB1 intron from two other splicing mutants that arrest at G(2)/M, prp17Delta and prp22-1 strains, permits nuclear division, but suppression of the cell cycle block is less efficient. Our data raise the possibility that although cell cycle arrest phenotypes in prp mutants can be explained by defects in pre-mRNA splicing, the transcript(s) whose inefficient splicing contributes to cell cycle arrest is likely to be prp mutant dependent.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA , Genes Fúngicos , RNA Helicases , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Tubulina (Proteína)/genética , Sequência de Bases , Ciclo Celular/genética , RNA Helicases DEAD-box , DNA Fúngico/genética , Proteínas Fúngicas/genética , Íntrons , Microtúbulos/metabolismo , Mitose/genética , Mutação , Fenótipo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , TemperaturaRESUMO
The University of California Santa Cruz (UCSC) Table Browser (http://genome.ucsc.edu/cgi-bin/hgText) provides text-based access to a large collection of genome assemblies and annotation data stored in the Genome Browser Database. A flexible alternative to the graphical-based Genome Browser, this tool offers an enhanced level of query support that includes restrictions based on field values, free-form SQL queries and combined queries on multiple tables. Output can be filtered to restrict the fields and lines returned, and may be organized into one of several formats, including a simple tab- delimited file that can be loaded into a spreadsheet or database as well as advanced formats that may be uploaded into the Genome Browser as custom annotation tracks. The Table Browser User's Guide located on the UCSC website provides instructions and detailed examples for constructing queries and configuring output.
Assuntos
Bases de Dados Genéticas , Genoma , Armazenamento e Recuperação da Informação , Software , Animais , Biologia Computacional , Genômica , Humanos , Internet , Interface Usuário-ComputadorRESUMO
Splicing and alternative splicing are major processes in the interpretation and expression of genetic information for metazoan organisms. The study of splicing is moving from focused attention on the regulatory mechanisms of a selected set of paradigmatic alternative splicing events to questions of global integration of splicing regulation with genome and cell function. For this reason, parallel methods for detecting and measuring alternative splicing are necessary. We have adapted the splicing-sensitive oligonucleotide microarrays used to estimate splicing efficiency in yeast to the study of alternative splicing in vertebrate cells and tissues. We use gene models incorporating knowledge about splicing to design oligonucleotides specific for discriminating alternatively spliced mRNAs from each other. Here we present the main strategies for design, application, and analysis of spotted oligonucleotide arrays for detection and measurement of alternative splicing. We demonstrate these strategies using a two-intron yeast gene that has been altered to produce different amounts of alternatively spliced RNAs, as well as by profiling alternative splicing in NCI 60 cancer cell lines.
Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sequência de Aminoácidos , Dinamina II/metabolismo , Genoma Humano , Humanos , Processamento de Imagem Assistida por Computador/métodos , Modelos Biológicos , Dados de Sequência Molecular , Sítios de Splice de RNA , Sensibilidade e Especificidade , Homologia de Sequência do Ácido NucleicoRESUMO
Introns interrupt almost every eukaryotic protein-coding gene, yet how the splicing apparatus interprets the genome during messenger RNA (mRNA) synthesis is poorly understood. We designed microarrays to distinguish spliced from unspliced RNA for each intron-containing yeast gene and measured genomewide effects on splicing caused by loss of 18 different mRNA processing factors. After accommodating changes in transcription and decay by using gene-specific indexes, functional relationships between mRNA processing factors can be identified through their common effects on spliced and unspliced RNA. Groups of genes with different dependencies on mRNA processing factors are also apparent. Quantitative polymerase chain reactions confirm the array-based finding that Prp17p and Prp18p are not dispensable for removal of introns with short branchpoint-to-3' splice site distances.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Genoma Fúngico , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Íntrons , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleoproteína Nuclear Pequena U5 , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.