Design and Analysis of RNA Sequencing Data
11
Richa Bharti and Dominik G. Grimm
Contents
11.1
Introduction . . . . . . . .. . .. . . .. . .. . . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . . .. . .. . 144
11.2
RNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
11.3
RNA-Seq Library Preparation . . .... ... ... ... .... ... ... .... ... ... ... .... ... ... .... ... ... ... 147
11.4
Choice of Sequencing Platform . . . .. . .. . . .. . . .. . .. . . .. . .. . . .. . . .. . .. . . .. . . .. . .. . . .. . . .. . .. 147
11.5
Quality Check (QC) and Sequence Pre-processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
11.6
RNA-Seq Analysis .... .... .... ..... .... .... .... .... .... .... .... .... .... .... .... ..... .... ... 150
11.6.1 Reference-Based Alignment . . . .. . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . .. . 150
11.6.2 De novo or Reference-Free Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
11.7
Functional Annotation of de novo Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
11.8
Post-alignment/assembly Assessment and Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
11.9
Visualization of Mapped Reads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
11.10 Quantification of Gene Expression . ..... .... ..... ..... .... ..... .... ..... .... ..... .... ..... 161
11.11 Counting Reads Per Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
11.12 Counting Reads Per Transcripts . . . .... .... .... ... .... .... ... .... .... ... .... .... .... ... .... 163
11.13 Counting Reads Per Exons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
11.14 Normalization and Differential Expression (DE) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
11.15 Functional Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
R. Bharti · D. G. Grimm (*)
Technical University of Munich, Campus Straubing for Biotechnology and Sustainability,
Bioinformatics, Straubing, Germany
Weihenstephan-Triesdorf University of Applied Sciences, Straubing, Germany
e-mail: dominik.grimm@hswt.de
# Springer Nature Switzerland AG 2021
M. Kappelmann-Fenzl (ed.), Next Generation Sequencing and Data Analysis, Learning
Materials in Biosciences, https://doi.org/10.1007/978-3-030-62490-3_11
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