RNA-Seq Data Analysis talk given by Andreas Wilm (iGap & GIS) during the joint Illumina and Genome Institute of Singapore workshop on 24th May 2018

1. Getting Started With RNA-Seq Data Analysis
Genome Institute of Singapore
Andreas Wilm, PhD :: Team Lead, Bioinformatics Core
24th May 2018

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Sequencing, Data Management & Genome Analytics Services
http://www.igap.io | igap@gis.a-star.edu.sg

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This is not an RNASeq tutorial
Disclaimer

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• Bulk vs. Single Cell
• Transcript discovery
• Differential gene expression
• Allele-specific expression
• Detection of RNA editing
• Viral detection
• Gene fusion detection
• Alternative splicing
• De novo transcript assembly
• …
“The analysis goals of RNA-Seq experiments are diverse. Each of
these analysis goals has distinct requirements and challenges”
Source: Griffith et al. (2015)

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Outstanding Training Resource
Griffith et al. (PLoS Comp Biol, 2015)
Informatics for RNA Sequencing: A Web Resource for
Analysis on the Cloud

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Companion Web Resource: www.rnaseq.wiki

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Short Read Mapping
• Naïve mapping to genome won’t
work: exon-exon junctions
• Use splice-aware mappers (e.g.
STAR). Require annotation
• Recent (fast) alternative: pseudo-
aligners (e.g. Kallisto), which
map to transcriptome
Source: Wikipedia

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File Formats Plain Reads: FastQ
(usually >= 20M per sample)
Aligned: BAM Courtesy: Jonathan Göke

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Common Analysis Flowchart
• Example: Tuxedo suite
• Large number of tools
• Number of representative tools
listed in Griffith et al. (2015): >100
• Multiple versions available
Source: Griffith et al. 2015

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Tools Evolve
Source: Twitter
• Keep upgrading !
• Try different programs

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Installation: Experts Only?
• Often requires Unix/Linux knowledge
• Install package dependencies first
• Not being root/admin can complicate things
• How to install different version of the same program?
Source: XKCD

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Package Managers
Homebrew-Bio
Bioinformatics formulae
for Linuxbrew and Homebrew
More than 3000 bioinformatics
packages ready to use with conda
• Precompiled packages
• Installation through single command
• Multiple versions available
• Support for isolated environments

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RNACocktail Protocol Dependencies
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Sahraeianet al. 2017

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Containers
Singularity
• Containers package software, dependencies etc.
• No installation needed at all
• Think: entire operating system in a file
• Extreme Portability

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Example: New Tuxedo Protocol (Pertea et al., 2016)
Source: https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual
• Replaces TopHat, Cuffdiff etc.
• How to scale to many samples?
• What if a step fails?
• How to make useable for others?

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Workflow Managers
• Automation
• Scalability
• Robustness
• Reentrancy
• Reproducibility
• Sharing
Di Tommaso et al. (NBT, 2017) Köster & Rahmann (Bioinformatics, 2012)
snakemake

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Putting it all Together
• Portable
• Validated
• Well documented
• Community maintained
Courtesy: Paolo Di Tommaso
Presented at Bio-IT World 2018

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RNASeq Pipelines, Written in Nextflow and Using Containers
• NF-Core RNASeq: expression analysis and extensive QC
• Tuxedo: Transcript-level expression analysis and DE
• CalliNGS-NF: Variant Calling with GATK, incl. ASE
• …

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Demo
• Example: GATK best practices for
variant calling for RNAseq
• Plus SNVs post-processing and
quantification for allele specific
expression
• Note: multiple samples, no software
installation, fully orchestrated etc.
Source: https://github.com/CRG-CNAG/CalliNGS-NF

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Honorable Mentions
Cloud based analytics portals:
And others…
Software for Single Cell Gene Expression:
Cell Ranger Pipelines and Loupe Cell Browser

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We can do the Heavy Lifting for you:
From Off-the-shelf to “Artisan” Analysis
http://www.igap.io | igap@gis.a-star.edu.sg

22. Thank you