Abstract
Quantification of gene expression and characterization of gene transcript structures are central problems in molecular biology. RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (anchor-Seq) are important methods, but can be cumbersome and difficult for beginners to learn. To teach interested students and scientists how to analyze RNA-Seq and anchor-Seq data, we present a start-to-finish tutorial for analyzing RNA-Seq and anchor-Seq data: SeqAcademy ( source code: https://github.com/NCBI-Hackathons/seqacademy, webpage: http://www.seqacademy.org/). This user-friendly pipeline, fully written in markdown language, emphasizes the use of publicly available RNA-Seq and anchor-Seq data and strings together popular tools that bridge that gap between raw sequencing reads and biological insight. We demonstrate practical and conceptual considerations for various RNA-Seq and anchor-Seq analysis steps with a biological use case - a previously published yeast experiment. This work complements existing sophisticated RNA-Seq and anchor-Seq pipelines designed for advanced users by gently introducing the critical components of RNA-Seq and anchor-Seq analysis to the novice bioinformatician. In conclusion, this well-documented pipeline will introduce state-of-the-art RNA-Seq and anchor-Seq analysis tools to beginning bioinformaticians and help facilitate the analysis of the burgeoning amounts of public RNA-Seq and anchor-Seq data.
Highlights
RNA sequencing (RNA-Seq) is a rapidly expanding technique used to answer broad questions in the life sciences, ranging from mitochondrial function (Mercer et al, 2011) to the pathogenesis of breast cancer (Li et al, 2017)
The expanding importance of RNA-Seq and ChIP-Seq data is reflected by its explosive growth in terabytes in the primary public repository for storing this data - the Sequence Read Archive (SRA) (Wheeler et al, 2008)
We developed a proof-of-concept, well-documented “tutorial pipeline” over the course of a three-day NCBIsponsored hackathon intended to teach RNA-seq and ChIP-seq analysis to beginners. This tutorial pipeline, “SeqAcademy,” incorporates state-of-the-art RNA-Seq and ChIP-seq analysis tools into a simple, easy to use workflow tutorial and we demonstrate its use with publicly available data
Summary
RNA sequencing (RNA-Seq) is a rapidly expanding technique used to answer broad questions in the life sciences, ranging from mitochondrial function (Mercer et al, 2011) to the pathogenesis of breast cancer (Li et al, 2017). Chromatin immunoprecipitation sequencing (ChIP-Seq) is a genome-wide technique for profiling histone modifications, protein interactions, and transcription factor binding sites (Barski et al, 2007) Using this technique to analyze protein interactions involves very large data sets for computational analysis. The expanding importance of RNA-Seq and ChIP-Seq data is reflected by its explosive growth in terabytes in the primary public repository for storing this data - the Sequence Read Archive (SRA) (Wheeler et al, 2008). This incredible increase in the amount of public data has not been met with an equal increase in the number of scientists who can skillfully and thoughtfully analyze this important resource. Given the fundamental role that RNA-Seq and ChIP-Seq data, among other next-generation sequencing data types, are likely to play in the coming decades, there is a critical need to teach RNA-Seq and ChIP-Seq analysis to life scientists with diverse interests and backgrounds
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