RESUMO
New sequencing technologies pose significant challenges in terms of data complexity and magnitude. It is essential that efficient software is developed with performance that scales with this growth in sequence information. Here we present a comprehensive and integrated set of tools for the analysis of data from large scale sequencing experiments. It supports adapter detection and removal, demultiplexing of barcodes, paired-end data, a range of read architectures and the efficient removal of sequence redundancy. Sequences can be trimmed and filtered based on length, quality and complexity. Quality control plots track sequence length, composition and summary statistics with respect to genomic annotation. Several use cases have been integrated into a single streamlined pipeline, including both mRNA and small RNA sequencing experiments. This pipeline interfaces with existing tools for genomic mapping and differential expression analysis.
Assuntos
Ensaios de Triagem em Larga Escala , Controle de Qualidade , Análise de Sequência de RNA/métodos , Software , Algoritmos , Processamento Eletrônico de Dados , HumanosRESUMO
BACKGROUND: miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). METHODS: We screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation. RESULTS AND CONCLUSION: We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency.
Assuntos
Reprogramação Celular/genética , Proteínas F-Box/genética , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , MicroRNAs/genética , MicroRNAs/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Sequência de Bases , Biologia Computacional , Proteína 7 com Repetições F-Box-WD , Fibroblastos/citologia , Regulação da Expressão Gênica , CamundongosRESUMO
Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3' UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.