RESUMEN
Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.
Asunto(s)
Genoma , Urocordados , Animales , Urocordados/genética , Urocordados/clasificación , Evolución Molecular , Femenino , Filogenia , Masculino , SinteníaRESUMEN
In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.
RESUMEN
The development of complex methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where optima are sought in a multidimensional parameter space through step-by-step optimizations. The difficulty of miniaturizing reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates 648 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterization of biochemical systems.
Asunto(s)
Fenómenos Bioquímicos , Transcripción Reversa , Animales , Automatización de Laboratorios , Células HeLa , Humanos , Ratones , Miniaturización , Reacción en Cadena de la Polimerasa , Análisis de la Célula IndividualRESUMEN
BACKGROUND: The larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. RESULTS: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is contained within five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups in OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. CONCLUSIONS: We show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica's highly polymorphic genome. This genome assembly opens up the possibility of cross-genome comparison between O. dioica populations, as well as of studies of chromosomal evolution in this lineage.
Asunto(s)
Secuenciación de Nanoporos , Nanoporos , Urocordados , Animales , Genoma , Masculino , Telómero/genética , Urocordados/genéticaRESUMEN
Maturation of human-induced pluripotent stem cells (hiPSCs)-derived hepatocytes-like cells (HLCs) toward a complete hepatocyte phenotype remains a challenge as primitiveness patterns are still commonly observed. In this study, we propose a modified differentiation protocol for those cells which includes a prematuration in Petri dishes and a maturation in microfluidic biochip. For the first time, a large range of biomolecular families has been extracted from the same sample to combine transcriptomic, proteomic, and metabolomic analysis. After integration, these datasets revealed specific molecular patterns and highlighted the hepatic regeneration profile in biochips. Overall, biochips exhibited processes of cell proliferation and inflammation (via TGFB1) coupled with anti-fibrotic signaling (via angiotensin 1-7, ATR-2, and MASR). Moreover, cultures in this condition displayed physiological lipid-carbohydrate homeostasis (notably via PPAR, cholesterol metabolism, and bile synthesis) coupled with cell respiration through advanced oxidative phosphorylation (through the overexpression of proteins from the third and fourth complex). The results presented provide an original overview of the complex mechanisms involved in liver regeneration using an advanced in vitro organ-on-chip technology.
Asunto(s)
Diferenciación Celular , Genómica , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Regeneración Hepática , Hígado/metabolismo , Proteómica , HumanosRESUMEN
The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.
Asunto(s)
Diferenciación Celular/genética , Hepatocitos/citología , Células Madre Pluripotentes Inducidas/citología , Regeneración Hepática , Hígado/crecimiento & desarrollo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2C8/genética , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Evaluación Preclínica de Medicamentos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Factor Nuclear 1 de Respiración/genética , Oncostatina M/farmacología , Factor de Transcripción Sp3/genética , Transcriptoma/efectos de los fármacosRESUMEN
The larvacean Oikopleura dioica is a planktonic chordate and is a tunicate that belongs to the closest relatives to vertebrates. Its simple and transparent body, invariant embryonic cell lineages, and short life cycle of 5 days make it a promising model organism for the study of developmental biology. The genome browser OikoBase was established in 2013 using Norwegian O. dioica. However, genome information for other populations is not available, even though many researchers have studied local populations. In the present study, we sequenced using Illumina and PacBio RSII technologies the genome of O. dioica from a southwestern Japanese population that was cultured in our laboratory for 3 years. The genome of Japanese O. dioica was assembled into 576 scaffold sequences with a total length and N50 length of 56.6 and 1.5 Mb, respectively. A total of 18,743 gene models (transcript models) were predicted in the genome assembly, named OSKA2016. In addition, 19,277 non-redundant transcripts were assembled using RNA-seq data. The OSKA2016 has global sequence similarity of only 86.5% when compared with the OikoBase, highlighting the sequence difference between the two far distant O. dioica populations on the globe. The genome assembly, transcript assembly, and transcript models were incorporated into ANISEED (https://www.aniseed.cnrs.fr/) for genome browsing and BLAST searches. Mapping of reads obtained from male- or female-specific genome libraries yielded male-specific scaffolds in the OSKA2016 and revealed that over 2.6 Mb of sequence were included in the male-specific Y-region. The genome and transcriptome resources from two distinct populations will be useful datasets for developmental biology, evolutionary biology, and molecular ecology using this model organism.
Asunto(s)
Bases de Datos Genéticas , Modelos Genéticos , Urocordados/genética , Animales , Japón , TranscriptomaRESUMEN
A core promoter is a stretch of DNA surrounding the transcription start site (TSS) that integrates regulatory inputs and recruits general transcription factors to initiate transcription. The nature and causative relationship of the DNA sequence and chromatin signals that govern the selection of most TSSs by RNA polymerase II remain unresolved. Maternal to zygotic transition represents the most marked change of the transcriptome repertoire in the vertebrate life cycle. Early embryonic development in zebrafish is characterized by a series of transcriptionally silent cell cycles regulated by inherited maternal gene products: zygotic genome activation commences at the tenth cell cycle, marking the mid-blastula transition. This transition provides a unique opportunity to study the rules of TSS selection and the hierarchy of events linking transcription initiation with key chromatin modifications. We analysed TSS usage during zebrafish early embryonic development at high resolution using cap analysis of gene expression, and determined the positions of H3K4me3-marked promoter-associated nucleosomes. Here we show that the transition from the maternal to zygotic transcriptome is characterized by a switch between two fundamentally different modes of defining transcription initiation, which drive the dynamic change of TSS usage and promoter shape. A maternal-specific TSS selection, which requires an A/T-rich (W-box) motif, is replaced with a zygotic TSS selection grammar characterized by broader patterns of dinucleotide enrichments, precisely aligned with the first downstream (+1) nucleosome. The developmental dynamics of the H3K4me3-marked nucleosomes reveal their DNA-sequence-associated positioning at promoters before zygotic transcription and subsequent transcription-independent adjustment to the final position downstream of the zygotic TSS. The two TSS-defining grammars coexist, often physically overlapping, in core promoters of constitutively expressed genes to enable their expression in the two regulatory environments. The dissection of overlapping core promoter determinants represents a framework for future studies of promoter structure and function across different regulatory contexts.
Asunto(s)
Regiones Promotoras Genéticas/genética , Sitio de Iniciación de la Transcripción , Pez Cebra/genética , Animales , Secuencia de Bases , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/metabolismo , Metilación , Madres , Nucleosomas/genética , Iniciación de la Transcripción Genética , Transcriptoma/genética , Pez Cebra/embriología , Cigoto/metabolismoRESUMEN
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
Asunto(s)
Atlas como Asunto , Anotación de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Transcriptoma/genética , Animales , Línea Celular , Células Cultivadas , Análisis por Conglomerados , Secuencia Conservada/genética , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Genes Esenciales/genética , Genoma/genética , Humanos , Ratones , Sistemas de Lectura Abierta/genética , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética/genéticaRESUMEN
Published single-cell datasets are rich resources for investigators who want to address questions not originally asked by the creators of the datasets. The single-cell datasets might be obtained by different protocols and diverse analysis strategies. The main challenge in utilizing such single-cell data is how we can make the various large-scale datasets to be comparable and reusable in a different context. To challenge this issue, we developed the single-cell centric database 'SCPortalen' (http://single-cell.clst.riken.jp/). The current version of the database covers human and mouse single-cell transcriptomics datasets that are publicly available from the INSDC sites. The original metadata was manually curated and single-cell samples were annotated with standard ontology terms. Following that, common quality assessment procedures were conducted to check the quality of the raw sequence. Furthermore, primary data processing of the raw data followed by advanced analyses and interpretation have been performed from scratch using our pipeline. In addition to the transcriptomics data, SCPortalen provides access to single-cell image files whenever available. The target users of SCPortalen are all researchers interested in specific cell types or population heterogeneity. Through the web interface of SCPortalen users are easily able to search, explore and download the single-cell datasets of their interests.
Asunto(s)
Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Ratones/genética , Análisis de la Célula Individual , Transcriptoma , Animales , Exactitud de los Datos , Curaduría de Datos , Expresión Génica , Ontología de Genes , Humanos , Anotación de Secuencia Molecular , Interfaz Usuario-Computador , Flujo de TrabajoRESUMEN
In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for complete and partial on-chip differentiation were tested. Unlike full on-chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin-1 or albumin, while no stabilin-1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 106 cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha ( ESRRA), hepatic nuclear factor 1 ( HNF1A), hepatic nuclear factor 4 ( HNF4A), transcription factor 4 ( TCF4), and CCAAT enhancer binding protein alpha ( CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the É-fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.
Asunto(s)
Diferenciación Celular , Hepatocitos , Células Madre Pluripotentes Inducidas , Hígado , Técnicas Analíticas Microfluídicas , Nicho de Células Madre , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/citología , Hígado/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Underlying the complexity of the mammalian brain is its network of neuronal connections, but also the molecular networks of signaling pathways, protein interactions, and regulated gene expression within each individual neuron. The diversity and complexity of the spatially intermingled neurons pose a serious challenge to the identification and quantification of single neuron components. To address this challenge, we present a novel approach for the study of the ribosome-associated transcriptome-the translatome-from selected subcellular domains of specific neurons, and apply it to the Purkinje cells (PCs) in the rat cerebellum. We combined microdissection, translating ribosome affinity purification (TRAP) in nontransgenic animals, and quantitative nanoCAGE sequencing to obtain a snapshot of RNAs bound to cytoplasmic or rough endoplasmic reticulum (rER)-associated ribosomes in the PC and its dendrites. This allowed us to discover novel markers of PCs, to determine structural aspects of genes, to find hitherto uncharacterized transcripts, and to quantify biophysically relevant genes of membrane proteins controlling ion homeostasis and neuronal electrical activities.
Asunto(s)
Perfilación de la Expresión Génica , Células de Purkinje/metabolismo , Animales , Sitios de Unión , Mapeo Cromosómico , Análisis por Conglomerados , Citoplasma/metabolismo , Dendritas/metabolismo , Retículo Endoplásmico Rugoso/metabolismo , Familia de Multigenes , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN no Traducido/genética , ARN no Traducido/metabolismo , Ratas , Ribosomas/fisiología , TranscriptomaRESUMEN
Many eukaryotic genes possess multiple alternative promoters with distinct expression specificities. Therefore, comprehensively annotating promoters and deciphering their individual regulatory dynamics is critical for gene expression profiling applications and for our understanding of regulatory complexity. We introduce RAMPAGE, a novel promoter activity profiling approach that combines extremely specific 5'-complete cDNA sequencing with an integrated data analysis workflow, to address the limitations of current techniques. RAMPAGE features a streamlined protocol for fast and easy generation of highly multiplexed sequencing libraries, offers very high transcription start site specificity, generates accurate and reproducible promoter expression measurements, and yields extensive transcript connectivity information through paired-end cDNA sequencing. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive data set that represents the first available developmental time-course of promoter usage. We found that >40% of developmentally expressed genes have at least two promoters and that alternative promoters generally implement distinct regulatory programs. Transposable elements, long proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, contribute at least 1300 promoters shaping the developmental transcriptome of D. melanogaster. Hundreds of these promoters drive the expression of annotated genes, and transposons often impart their own expression specificity upon the genes they regulate. These observations provide support for the theory that transposons may drive regulatory innovation through the distribution of stereotyped cis-regulatory modules throughout their host genomes.
Asunto(s)
Elementos Transponibles de ADN , Regulación del Desarrollo de la Expresión Génica , Regiones Promotoras Genéticas , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Biblioteca de Genes , Genes del Desarrollo , Genes de Insecto , Estadios del Ciclo de Vida/genética , Análisis de Secuencia de ADN/métodos , Sitio de Iniciación de la Transcripción , Transcripción Genética , TranscriptomaRESUMEN
We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica , Papillomavirus Humano 16/genética , Biología Molecular/métodos , ARN Viral/biosíntesis , ARN Viral/aislamiento & purificación , Femenino , Papillomavirus Humano 16/aislamiento & purificación , Humanos , ARN sin Sentido/biosíntesis , ARN sin Sentido/genética , ARN sin Sentido/aislamiento & purificación , ARN no Traducido/biosíntesis , ARN no Traducido/genética , ARN no Traducido/aislamiento & purificación , ARN Viral/genética , Neoplasias del Cuello Uterino/virologíaRESUMEN
Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.
Asunto(s)
Regiones Promotoras Genéticas , Receptores Odorantes/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Orden Génico , Sitios Genéticos , Factores de Transcripción MEF2 , Ratones , Ratones Transgénicos , Factores Reguladores Miogénicos/metabolismo , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Tissues contain complex populations of cells. Like countries, which are comprised of mixed populations of people, tissues are not homogeneous. Gene expression studies that analyze entire populations of cells from tissues as a mixture are blind to this diversity. Thus, critical information is lost when studying samples rich in specialized but diverse cells such as tumors, iPS colonies, or brain tissue. High throughput methods are needed to address, model and understand the constitutive and stochastic differences between individual cells. Here, we describe microfluidics technologies that utilize a combination of molecular biology and miniaturized labs on chips to study gene expression at the single cell level. We discuss how the characterization of the transcriptome of each cell in a sample will open a new field in gene expression analysis, population transcriptomics, that will change the academic and biomedical analysis of complex samples by defining them as quantified populations of single cells.
Asunto(s)
Células Eucariotas/clasificación , Microfluídica/métodos , ARN Mensajero/análisis , Análisis de la Célula Individual/métodos , Transcriptoma , Células Eucariotas/citología , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Dispositivos Laboratorio en un Chip , Microfluídica/instrumentación , ARN Mensajero/genética , Análisis de la Célula Individual/instrumentaciónRESUMEN
Template switching (TS) has been an inherent mechanism of reverse transcriptase, which has been exploited in several transcriptome analysis methods, such as CAGE, RNA-Seq and short RNA sequencing. TS is an attractive option, given the simplicity of the protocol, which does not require an adaptor mediated step and thus minimizes sample loss. As such, it has been used in several studies that deal with limited amounts of RNA, such as in single cell studies. Additionally, TS has also been used to introduce DNA barcodes or indexes into different samples, cells or molecules. This labeling allows one to pool several samples into one sequencing flow cell, increasing the data throughput of sequencing and takes advantage of the increasing throughput of current sequences. Here, we report TS artifacts that form owing to a process called strand invasion. Due to the way in which barcodes/indexes are introduced by TS, strand invasion becomes more problematic by introducing unsystematic biases. We describe a strategy that eliminates these artifacts in silico and propose an experimental solution that suppresses biases from TS.
Asunto(s)
Artefactos , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Animales , Humanos , Ratones , ARN/sangre , ARN/química , Ratas , Sensibilidad y Especificidad , Moldes GenéticosRESUMEN
Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.
Asunto(s)
Evolución Molecular , Regiones Promotoras Genéticas , Regiones no Traducidas 3' , Animales , Secuencia de Bases , ADN , Genoma , Proteoma , TATA BoxRESUMEN
BACKGROUND: The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson's disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown. RESULTS: By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains. CONCLUSIONS: Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Regulación de la Expresión Génica , Mesencéfalo/citología , Mesencéfalo/metabolismo , Odorantes , Receptores Odorantes/genética , Animales , Línea Celular , Análisis por Conglomerados , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Especificidad de Órganos/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Receptores Odorantes/metabolismo , Proteínas Recombinantes , Sustancia Negra/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Analyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting. With current high-speed sequencers, it is necessary to flank the cDNAs with specific adapters. Adding template-switching oligonucleotides to reverse transcription reactions is the most commonly used approach when working with very small quantities of RNA even from single cells. RESULTS: Here we compared the performance of DNA-RNA, DNA-LNA and DNA oligonucleotides in template-switching during nanoCAGE library preparation. Test libraries from rat muscle and HeLa cell RNA were prepared in technical triplicates and sequenced for comparison of the gene coverage and distribution of the reads within transcripts. The DNA-RNA oligonucleotide showed the highest specificity for capped 5' ends of mRNA, whereas the DNA-LNA provided similar gene coverage with more reads falling within exons. CONCLUSIONS: While confirming the cap-specific preference of DNA-RNA oligonucleotides in template-switching reactions, our data indicate that DNA-LNA hybrid oligonucleotides could potentially find other applications in random RNA sequencing.