Basic Applied Bioinformatics

CHAPTER 42Basics of RNA‐Seq Data Analysis

GVPPSR Kumar, AP Sahoo and A Kumar

Animal Biotechnology Division, IVRI, UP, India

42.1 INTRODUCTION

The complete set of transcripts (transcribed RNA products) in a cell is called the transcriptome (in terms of both type and quantity). Transcriptome analysis helps in understanding the pattern of gene expression to address basic biological questions, and unravels biological pathways and molecular mechanisms that regulate cell fate, development and disease progression. The transcriptome is analyzed through RNA‐sequencing (RNA‐seq) or microarray experiments. RNA‐sequencing involves sequencing of the entire transcriptome, using next‐generation sequencing (NGS) platforms.

The data generated through various platforms go into secondary analysis, which mainly involves alignment (if it is reference‐based) or assembly (if it is de novo). In this book, RNA‐seq data generated are analyzed through distinct pipelines to identify differentially expressed genes under different experimental conditions. The basics of RNA‐sequencing data analysis will be discussed in this chapter.

42.2 AIM OF AN RNA‐SEQ EXPERIMENT

1. To quantify RNA abundance.
2. Annotating the transcription start site, 5′ and 3′ ends and splicing patterns of genes.
3. To quantify the changing expression levels of each transcript during development, and under different conditions.
4. To identify variants on the transcripts.

With proper depth/coverage, NGS addresses the limitations of a microarray ...