Molecular Profiling of RNA Tumors Using High-Throughput RNA Sequencing: From Raw Data to Systems Level Analyses

Willian A da Silveira; E Starr Hazard; Dongjun Chung; Gary Hardiman

doi:10.1007/978-1-4939-9004-7_13

Molecular Profiling of RNA Tumors Using High-Throughput RNA Sequencing: From Raw Data to Systems Level Analyses

Methods Mol Biol. 2019:1908:185-204. doi: 10.1007/978-1-4939-9004-7_13.

Authors

Willian A da Silveira^{1

2}, E Starr Hazard^{1

3}, Dongjun Chung⁴, Gary Hardiman^{5

6

7

8}

Affiliations

¹ MUSC Bioinformatics, Center for Genomics Medicine, Medical University of South Carolina (MUSC), Charleston, SC, USA.
² Institute for Global Food Security, Queens University Belfast, Belfast, UK.
³ Library Science and Informatics, Medical University of South Carolina (MUSC), Charleston, SC, USA.
⁴ Department of Public Health Sciences, Medical University of South Carolina (MUSC), Charleston, SC, USA.
⁵ MUSC Bioinformatics, Center for Genomics Medicine, Medical University of South Carolina (MUSC), Charleston, SC, USA. G.Hardiman@qub.ac.uk.
⁶ Department of Public Health Sciences, Medical University of South Carolina (MUSC), Charleston, SC, USA. G.Hardiman@qub.ac.uk.
⁷ Department of Medicine, Medical University of South Carolina (MUSC), Charleston, SC, USA. G.Hardiman@qub.ac.uk.
⁸ School of Biological Sciences & Institute for Global Food Security, Queens University Belfast, Stranmillis Road, Belfast, BT9 5AG, UK. G.Hardiman@qub.ac.uk.

PMID: 30649729
DOI: 10.1007/978-1-4939-9004-7_13

Abstract

RNAseq is a powerful technique enabling global profiles of transcriptomes in healthy and diseased states. In this chapter we review pipelines to analyze the data generated by sequencing RNA, from raw data to a system level analysis. We first give an overview of workflow to generate mapped reads from FASTQ files, including quality control of FASTQ, filtering and trimming of reads, and alignment of reads to a genome. Then, we compare and contrast three popular options to determine differentially expressed (DE) transcripts (The Tuxedo Pipeline, DESeq2, and Limma/voom). Finally, we examine four tool sets to extrapolate biological meaning from the list of DE genes (Genecards, The Human Protein Atlas, GSEA, and ToppGene). We emphasize the need to ask a concise scientific question and to clearly under stand the strengths and limitations of the methods.

Keywords: DESeq 2; FASTQ; Gene Set Enrichment Analysis/GSEA; HTSeq; High-throughput sequencing (HTS); Limma/Voom; RNAsequencing (RNAseq); TOPPGENE; Tuxedo pipeline.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Algorithms
Computational Biology / methods*
Gene Expression Profiling / methods*
High-Throughput Nucleotide Sequencing / methods*
Humans
Molecular Sequence Annotation / methods
Quality Control
Sequence Analysis, RNA / methods
Software*
Workflow