RESUMEN
Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas de Diagnóstico Molecular/métodos , Análisis de Secuencia de ADN/métodos , Exoma , Humanos , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.
Asunto(s)
Exorribonucleasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Strongylocentrotus purpuratus/embriología , Animales , Secuencia de Bases , Diferenciación Celular , Separación Celular , Citometría de Flujo , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Strongylocentrotus purpuratus/enzimología , Factores de Tiempo , TranscriptomaRESUMEN
Brown adipose tissue (BAT) plays a pivotal role in promoting energy expenditure by the virtue of uncoupling protein-1 (UCP-1) that differentiates BAT from its energy storing white adipose tissue (WAT) counterpart. The clinical implication of "classical" BAT (originates from Myf5 positive myoblastic lineage) or the "beige" fat (originates through trans-differentiation of WAT) activation in improving metabolic parameters is now becoming apparent. However, the inducers and endogenous molecular determinants that govern the lineage commitment and differentiation of classical BAT remain obscure. We report here that in the absence of any forced gene expression, stimulation with bone morphogenetic protein 6 (BMP6) induces brown fat differentiation from skeletal muscle precursor cells of murine and human origins. Through a comprehensive transcriptional profiling approach, we have discovered that two days of BMP6 stimulation in C2C12 myoblast cells is sufficient to induce genes characteristic of brown preadipocytes. This developmental switch is modulated in part by newly identified regulators, Optineurin (Optn) and Cyclooxygenase-2 (Cox2). Furthermore, pathway analyses using the Causal Reasoning Engine (CRE) identified additional potential causal drivers of this BMP6 induced commitment switch. Subsequent analyses to decipher key pathway that facilitates terminal differentiation of these BMP6 primed cells identified a key role for Insulin Like Growth Factor-1 Receptor (IGF-1R). Collectively these data highlight a therapeutically innovative role for BMP6 by providing a means to enhance the amount of myogenic lineage derived brown fat.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Proteína Morfogenética Ósea 6/metabolismo , Mioblastos/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Proteína Morfogenética Ósea 6/genética , Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/metabolismo , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Línea Celular , Análisis por Conglomerados , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Ácidos Grasos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Proteínas de Transporte de Membrana , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción , Fenotipo , Receptor IGF Tipo 1 , Transducción de Señal , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Proteína Desacopladora 1RESUMEN
The analysis of the differential expression of genes has been the key goal of many molecular biology methods for decades and will remain with us for decades to come. It constitutes a fundamental resource at our disposal for determining the relationship between products of transcription, biology and disease. The completed genome sequencing of many common species allowed microarrays and RNA sequencing (RNAseq) to become major tools in Systems Biology. However, we estimate that at least half of all experiments ignore transcripts that change less than some subjectively chosen threshold, typically around 2-3 fold. Here we show that a majority of the informative RNAs and differentially expressed transcripts can exhibit fold changes less than 2. We use highly quantitative single-molecule sequencing of total cellular RNA derived from a time course of inflammatory response, a process critical to a large number of diseases. Furthermore, we show that enrichment of biologically-relevant functions occurs even at very low fold changes in RNA levels. In addition, we show that most of the common statistical methods can reliably detect transcripts with low fold change when as few as 3 biological replicates are sequenced using single-molecule based RNAseq. In conclusion, given the prevalence of expression profiling in current research, the loss of data in half of all expression studies results in a significant, yet needless drain on the discovery process.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/genética , Biología de Sistemas , Secuencia de Bases , Humanos , Inflamación/genética , Inflamación/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/genéticaRESUMEN
We present a comprehensive map of over 1 million polyadenylation sites and quantify their usage in major cancers and tumor cell lines using direct RNA sequencing. We built the Expression and Polyadenylation Database to enable the visualization of the polyadenylation maps in various cancers and to facilitate the discovery of novel genes and gene isoforms that are potentially important to tumorigenesis. Analyses of polyadenylation sites indicate that a large fraction (â¼30%) of mRNAs contain alternative polyadenylation sites in their 3' untranslated regions, independent of the cell type. The shortest 3' untranslated region isoforms are preferentially upregulated in cancer tissues, genome-wide. Candidate targets of alternative polyadenylation-mediated upregulation of short isoforms include POLR2K, and signaling cascades of cell-cell and cell-extracellular matrix contact, particularly involving regulators of Rho GTPases. Polyadenylation maps also helped to improve 3' untranslated region annotations and identify candidate regulatory marks such as sequence motifs, H3K36Me3 and Pabpc1 that are isoform dependent and occur in a position-specific manner. In summary, these results highlight the need to go beyond monitoring only the cumulative transcript levels for a gene, to separately analysing the expression of its RNA isoforms.
Asunto(s)
Regiones no Traducidas 3' , Neoplasias/genética , Poliadenilación , Línea Celular Tumoral , Genoma Humano , Humanos , Neoplasias/metabolismo , Isoformas de ARN/química , Isoformas de ARN/metabolismo , ARN no Traducido/metabolismo , Secuencias Reguladoras de Ácido Ribonucleico , Análisis de Secuencia de ARNRESUMEN
Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Metástasis de la Neoplasia/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/genética , Animales , Supervivencia Celular , Inhibición de Contacto , Modelos Animales de Enfermedad , Genes Relacionados con las Neoplasias/genética , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Análisis de Secuencia de ARN , Proteínas Wnt/genética , Proteína wnt2/genética , Proteína wnt2/metabolismoRESUMEN
It has recently been shown that RNA 3'-end formation plays a more widespread role in controlling gene expression than previously thought. To examine the impact of regulated 3'-end formation genome-wide, we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and describe the effects of 3'-end formation on genome organization. We reveal extreme heterogeneity in RNA 3' ends, discover previously unrecognized noncoding RNAs and propose widespread reannotation of the genome. We explain the origin of most poly(A)(+) antisense RNAs and identify cis elements that control 3'-end formation in different registers. These findings are essential to understanding what the genome actually encodes, how it is organized and how regulated 3'-end formation affects these processes.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Secuencia de Bases , Mapeo Cromosómico , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Poliadenilación , Procesamiento de Término de ARN 3' , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/química , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , ARN no Traducido/química , ARN no Traducido/genética , ARN no Traducido/metabolismo , Análisis de Secuencia de ARNRESUMEN
The single-stranded genome of adeno-associated viral (AAV) vectors is one of the key factors leading to slow-rising but long-term transgene expression kinetics. Previous molecular studies have established what is now considered a textbook molecular model of AAV genomes with two copies of inverted tandem repeats at either end. In this study, we profiled hundreds of thousands of individual molecules of AAV vector DNA directly isolated from capsids, using single-molecule sequencing (SMS), which avoids any intermediary steps such as plasmid cloning. The sequence profile at 3' ends of both the regular and oversized vector did show the presence of an inverted terminal repeat (ITR), which provided direct confirmation that AAV vector packaging initiates from its 3' end. Furthermore, the vector 5'-terminus profile showed inconsistent termination for oversized vectors. Such incomplete vectors would not be expected to undergo canonical synthesis of the second strand of their genomic DNA and thus could function only via annealing of complementary strands of DNA. Furthermore, low levels of contaminating plasmid DNA were also detected. SMS may become a valuable tool during the development phase of vectors that are candidates for clinical use and for facilitating/accelerating studies on vector biology.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Genoma Viral , Secuencias Repetidas Terminales/genética , Secuencia de Bases , ADN Viral/genética , ADN Viral/metabolismo , Dependovirus/metabolismo , Vectores Genéticos/aislamiento & purificación , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Plásmidos/genética , Plásmidos/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Transfección , Ensamble de VirusRESUMEN
Genetic testing for disease risk is an increasingly important component of medical care. However, testing can be expensive, which can lead to patients and physicians having limited access to the genetic information needed for medical decisions. To simplify DNA sample preparation and lower costs, we have developed a system in which any gene can be captured and sequenced directly from human genomic DNA without amplification, using no proteins or enzymes prior to sequencing. Extracted whole-genome DNA is acoustically sheared and loaded in a flow cell channel for single-molecule sequencing. Gene isolation, amplification, or ligation is not necessary. Accurate and low-cost detection of DNA sequence variants is demonstrated for the BRCA1 gene. Disease-causing mutations as well as common variants from well-characterized samples are identified. Single-molecule sequencing generates very reproducible coverage patterns, and these can be used to detect any size insertion or deletion directly, unlike PCR-based methods, which require additional assays. Because no gene isolation or amplification is required for sequencing, the exceptionally low costs of sample preparation and analysis could make genetic tests more accessible to those who wish to know their own disease susceptibility. Additionally, this approach has applications for sequencing integration sites for gene therapy vectors, transposons, retroviruses, and other mobile DNA elements in a more facile manner than possible with other methods.
Asunto(s)
Análisis Mutacional de ADN/métodos , Genes BRCA1 , Mutación , Secuencia de Bases , Línea Celular Tumoral , Exones , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Alineación de Secuencia , Eliminación de SecuenciaRESUMEN
The importance of short (<200 nt) RNAs in cell biogenesis has been well documented. These short RNAs include crucial classes of molecules such as transfer RNAs, small nuclear RNA, microRNAs, and many others (reviewed in Storz et al., Annu Rev Biochem 74:199-217, 2005; Ghildiyal and Zamore, Nat Rev Genet 10:94-108, 2009). Furthermore, the realm of functional RNAs that fall within this size range is growing to include less well-characterized RNAs such as short RNAs found at the promoters and 3' termini of genes (Affymetrix ENCODE Transcriptome Project et al., Nature 457:1028-1032, 2009; Davis and Ares, Proc Natl Acad Sci USA 103:3262-3267, 2006; Kapranov et al., Science 316:1484-1488, 2007; Taft et al., Nat Genet 41:572-578, 2009; Kapranov et al., Nature 466:642-646, 2010), short RNAs involved in paramutation (Rassoulzadegan et al., Nature 441:469-474, 2006), and others (reviewed in Kawaji and Hayashizaki, PLoS Genet 4:e22, 2008). Discovery and accurate quantification of these RNA molecules, less than 200 bases in size, is thus an important and also challenging aspect of understanding the full repertoire of cellular and extracellular RNAs. Here, we describe the strategies and procedures we developed to profile short RNA species using single-molecule sequencing (SMS) and the advantages SMS offers.
Asunto(s)
Perfilación de la Expresión Génica/métodos , ARN Pequeño no Traducido/análisis , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , Biología Computacional/instrumentación , Biología Computacional/métodos , ADN Complementario , Perfilación de la Expresión Génica/instrumentación , Humanos , ARN/aislamiento & purificación , ARN Pequeño no Traducido/aislamiento & purificación , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/instrumentaciónRESUMEN
Methods for in-depth genome-wide characterization of transcriptomes and quantification of transcript levels using various microarray and next-generation sequencing technologies have emerged as valuable tools for understanding cellular physiology and human disease biology and have begun to be utilized in various clinical diagnostic applications. Current methods, however, typically require RNA to be converted to complementary DNA prior to measurements. This step has been shown to introduce many biases and artifacts. In order to best characterize the 'true' transcriptome, the single-molecule direct RNA sequencing (DRS) technology was developed. This review focuses on the underlying principles behind the DRS, sample preparation steps, and the current and novel avenues of research and applications DRS offers.
Asunto(s)
Análisis de Secuencia de ARN/métodos , ADN Complementario/biosíntesis , ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Genoma Humano , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Dispositivos Ópticos , Análisis de Secuencia de ARN/instrumentaciónRESUMEN
5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.
Asunto(s)
Citosina/análogos & derivados , Células Madre Embrionarias/metabolismo , Genoma/genética , Análisis de Secuencia de ADN/métodos , 5-Metilcitosina/análogos & derivados , Animales , Biotinilación , Línea Celular , Citosina/análisis , Citosina/aislamiento & purificación , Citosina/metabolismo , Metilación de ADN , Exones/genética , Regulación del Desarrollo de la Expresión Génica/genética , Glucosa/metabolismo , Ratones , Ácido Peryódico/metabolismo , Regiones Promotoras Genéticas/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética/genéticaRESUMEN
RNA Seq provides unparalleled levels of information about the transcriptome including precise expression levels over a wide dynamic range. It is essential to understand how technical variation impacts the quality and interpretability of results, how potential errors could be introduced by the protocol, how the source of RNA affects transcript detection, and how all of these variations can impact the conclusions drawn. Multiple human RNA samples were used to assess RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting. Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells. PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome. Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage. Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols. Detection of short RNAs was also hampered by RNA fragmentation. Thus, this work will help researchers choose from among a range of options when analyzing gene expression, each with its own advantages and disadvantages.
Asunto(s)
Análisis de Secuencia de ARN/métodos , Encéfalo/metabolismo , Línea Celular Tumoral , ADN Complementario , Humanos , Hígado/metabolismoRESUMEN
The second wave of next generation sequencing technologies, referred to as single-molecule sequencing (SMS), carries the promise of profiling samples directly without employing polymerase chain reaction steps used by amplification-based sequencing (AS) methods. To examine the merits of both technologies, we examine mRNA sequencing results from single-molecule and amplification-based sequencing in a set of human cancer cell lines and tissues. We observe a characteristic coverage bias towards high abundance transcripts in amplification-based sequencing. A larger fraction of AS reads cover highly expressed genes, such as those associated with translational processes and housekeeping genes, resulting in relatively lower coverage of genes at low and mid-level abundance. In contrast, the coverage of high abundance transcripts plateaus off using SMS. Consequently, SMS is able to sequence lower- abundance transcripts more thoroughly, including some that are undetected by AS methods; however, these include many more mapping artifacts. A better understanding of the technical and analytical factors introducing platform specific biases in high throughput transcriptome sequencing applications will be critical in cross platform meta-analytic studies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Sesgo , Fusión Génica , Genes Relacionados con las Neoplasias/genética , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
With the advent of high-throughput sequencing technologies, multiple bacterial genomes can be sequenced in days. While the ultimate goal of de novo assembly of bacterial genomes is progressing, changes in the genomic sequence of closely related bacterial strains and isolates are now easily monitored by comparison of their sequences to those of a reference genome. Such studies can be applied to the fields of bacterial evolution, epidemiology, and diagnostics. We present a protocol for single-molecule sequencing of bacterial DNA whose end result is the identification of single nucleotide variants, and various size insertions and deletions relative to a reference genome. The protocol is characterized by the simplicity of sample preparation and the lack of amplification-related sequencing bias.
Asunto(s)
Genoma Bacteriano/genética , Análisis de Secuencia de ADN/métodos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Poli A/metabolismo , Poliadenilación , UltrasonidoRESUMEN
Methods for in-depth characterization of transcriptomes and quantification of transcript levels have emerged as valuable tools for understanding cellular physiology and human disease biology, and have begun to be utilized in various clinical diagnostic applications. Today, current methods utilized by the scientific community typically require RNA to be converted to cDNA prior to comprehensive measurements. However, this cDNA conversion process has been shown to introduce many biases and artifacts that interfere with the proper characterization and quantitation of transcripts. We have developed a direct RNA sequencing (DRS) approach, in which, unlike other technologies, RNA is sequenced directly without prior conversion to cDNA. The benefits of DRS include the ability to use minute quantities (e.g. on the order of several femtomoles) of RNA with minimal sample preparation, the ability to analyze short RNAs which pose unique challenges for analysis using cDNA-based approaches, and the ability to perform these analyses in a low-cost and high-throughput manner. Here, we describe the strategies and procedures we employ to prepare various RNA species for analysis with DRS.
Asunto(s)
Perfilación de la Expresión Génica/métodos , ARN/genética , Análisis de Secuencia de ARN/métodos , Animales , Secuencia de Bases , Humanos , Ratones , Hibridación de Ácido Nucleico , Poliadenilación , ARN/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
The recent transition in gene expression analysis technology to ultra high-throughput cDNA sequencing provides a means for higher quantitation sensitivity across a wider dynamic range than previously possible. Sensitivity of detection is mostly a function of the sheer number of sequence reads generated. Typically, RNA is converted to cDNA using random hexamers and the cDNA is subsequently sequenced (RNA-Seq). With this approach, higher read numbers are generated for long transcripts as compared to short ones. This length bias necessitates the generation of very high read numbers to achieve sensitive quantitation of short, low-expressed genes. To eliminate this length bias, we have developed an ultra high-throughput sequencing approach where only a single read is generated for each transcript molecule (single-molecule sequencing Digital Gene Expression (smsDGE)). So, for example, equivalent quantitation accuracy of the yeast transcriptome can be achieved by smsDGE using only 25% of the reads that would be required using RNA-Seq. For sample preparation, RNA is first reverse-transcribed into single-stranded cDNA using oligo-dT as a primer. A poly-A tail is then added to the 3' ends of cDNA to facilitate the hybridization of the sample to the Helicos(®) single-molecule sequencing Flow-Cell to which a poly dT oligo serves as the substrate for subsequent sequencing by synthesis. No PCR, sample-size selection, or ligation steps are required, thus avoiding possible biases that may be introduced by such manipulations. Each tailed cDNA sample is injected into one of 50 flow-cell channels and sequenced on the Helicos(®) Genetic Analysis System. Thus, 50 samples are sequenced simultaneously generating 10-20 million sequence reads on average for each sample channel. The sequence reads can then be aligned to the reference of choice such as the transcriptome, for quantitation of known transcripts, or the genome for novel transcript discovery. This chapter provides a summary of the methods required for smsDGE.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Análisis de Secuencia de ADN/métodos , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/metabolismo , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/metabolismo , Desoxirribonucleasas/metabolismo , Hibridación de Ácido Nucleico , Poli A/metabolismo , Poliadenilación , ARN/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN/métodosRESUMEN
Single-molecule sequencing enables DNA or RNA to be sequenced directly from biological samples, making it well-suited for diagnostic and clinical applications. Here we review the properties and applications of this rapidly evolving and promising technology.
Asunto(s)
ADN/análisis , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nanotecnología/métodos , ARN/análisis , Análisis de Secuencia de ADN/métodos , Animales , ADN/genética , Cartilla de ADN/química , Cartilla de ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Genómica/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Nanotecnología/instrumentación , Puntos Cuánticos , ARN/genética , Análisis de Secuencia de ADN/instrumentaciónRESUMEN
In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.
Asunto(s)
ARN/análisis , Empalme Alternativo , Animales , Humanos , ARN/genética , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.