Abstract
RNA-seq analysis is becoming a standard method for global gene expression profiling. However, open and standard pipelines to perform RNA-seq analysis by non-experts remain challenging due to the large size of the raw data files and the hardware requirements for running the alignment step. Here we introduce a reproducible open source RNA-seq pipeline delivered as an IPython notebook and a Docker image. The pipeline uses state-of-the-art tools and can run on various platforms with minimal configuration overhead. The pipeline enables the extraction of knowledge from typical RNA-seq studies by generating interactive principal component analysis (PCA) and hierarchical clustering (HC) plots, performing enrichment analyses against over 90 gene set libraries, and obtaining lists of small molecules that are predicted to either mimic or reverse the observed changes in mRNA expression. We apply the pipeline to a recently published RNA-seq dataset collected from human neuronal progenitors infected with the Zika virus (ZIKV). In addition to confirming the presence of cell cycle genes among the genes that are downregulated by ZIKV, our analysis uncovers significant overlap with upregulated genes that when knocked out in mice induce defects in brain morphology. This result potentially points to the molecular processes associated with the microcephaly phenotype observed in newborns from pregnant mothers infected with the virus. In addition, our analysis predicts small molecules that can either mimic or reverse the expression changes induced by ZIKV. The IPython notebook and Docker image are freely available at: http://nbviewer.jupyter.org/github/maayanlab/Zika-RNAseq-Pipeline/blob/master/Zika.ipynband https://hub.docker.com/r/maayanlab/zika/.
Highlights
The increase in awareness about the irreproducibility of scientific research requires the development of methods that make experimental and computational protocols repeatable and transparent[1]
Here we present an interactive IPython notebook that serves as a tutorial for performing a standard RNA-seq pipeline
The following step is to identify the differentially expressed genes (DEG) between the two conditions. This is achieved with a unique method we developed called the Characteristic Direction (CD)[9]
Summary
The increase in awareness about the irreproducibility of scientific research requires the development of methods that make experimental and computational protocols repeatable and transparent[1]. The advent of interactive notebooks for data analysis pipelines significantly enhances the recording and sharing of data, source code, and figures[2]. In a subset of recent publications, an interactive notebook was published alongside customary manuscripts[3]. Here we present an interactive IPython notebook (http://nbviewer.jupyter.org/github/maayanlab/ZikaRNAseq-Pipeline/blob/master/Zika.ipynb) that serves as a tutorial for performing a standard RNA-seq pipeline. We applied the pipeline to RNA-seq data from a recent publication where human induced pluripotent stem cells were differentiated to neuronal progenitors and infected with Zika virus (ZIKV)[4]. The aim of the study was to begin to understand the molecular mechanisms that induce the observed devastating phenotype of newborn-microcephaly from pregnant mothers infected with the virus
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