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Open AccessJournal ArticleDOI

A survey of best practices for RNA-seq data analysis

TLDR
All of the major steps in RNA-seq data analysis are reviewed, including experimental design, quality control, read alignment, quantification of gene and transcript levels, visualization, differential gene expression, alternative splicing, functional analysis, gene fusion detection and eQTL mapping.
Abstract
RNA-sequencing (RNA-seq) has a wide variety of applications, but no single analysis pipeline can be used in all cases. We review all of the major steps in RNA-seq data analysis, including experimental design, quality control, read alignment, quantification of gene and transcript levels, visualization, differential gene expression, alternative splicing, functional analysis, gene fusion detection and eQTL mapping. We highlight the challenges associated with each step. We discuss the analysis of small RNAs and the integration of RNA-seq with other functional genomics techniques. Finally, we discuss the outlook for novel technologies that are changing the state of the art in transcriptomics.

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Journal ArticleDOI

Integrating single-cell transcriptomic data across different conditions, technologies, and species.

TL;DR: An analytical strategy for integrating scRNA-seq data sets based on common sources of variation is introduced, enabling the identification of shared populations across data sets and downstream comparative analysis.

Integrative analysis of 111 reference human epigenomes

TL;DR: In this article, the authors describe the integrative analysis of 111 reference human epigenomes generated as part of the NIH Roadmap Epigenomics Consortium, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression.
Journal ArticleDOI

Multi-omics approaches to disease.

TL;DR: This review provides an overview of omics technologies and methods for their integration across multiple omics layers and offers the opportunity to understand the flow of information that underlies disease.
Journal ArticleDOI

Differential analysis of RNA-seq incorporating quantification uncertainty.

TL;DR: A method for the differential analysis of gene expression data that utilizes bootstrapping in conjunction with response error linear modeling to decouple biological variance from inferential variance is described.
Journal ArticleDOI

Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap

TL;DR: This protocol describes pathway enrichment analysis of gene lists from RNA-seq and other genomics experiments using g:Profiler, GSEA, Cytoscape and EnrichmentMap software, and describes innovative visualization techniques.
References
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Journal ArticleDOI

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

TL;DR: This work presents DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates, which enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression.
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Fast and accurate short read alignment with Burrows–Wheeler transform

TL;DR: Burrows-Wheeler Alignment tool (BWA) is implemented, a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps.
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Trimmomatic: a flexible trimmer for Illumina sequence data

TL;DR: Timmomatic is developed as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data and is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested.
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Fast gapped-read alignment with Bowtie 2

TL;DR: Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
Journal ArticleDOI

Gene Ontology: tool for the unification of biology

TL;DR: The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing.
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