RNA-seq Analysis

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RNA

– Transcribed form of the DNA

– Active state of the DNA

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RNA sequencing

– RNA quantification at
single base resolution
– Cost efficient analysis of
the whole transcriptome in
a high-throughput manner

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Where does data come from?

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Challenges of RNA sequencing

Presence of
Different origin for
incompletely Sequencing biases
the sample RNA and
processed RNAs or (e.g. PCR library
the reference
transcriptional preparation)
genome
background noise

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Benefits of RNA sequencing

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2 main research applications for RNA-Seq
– Transcript discovery

– Which RNA molecules are in my sample?

– Novel isoforms and alternative splicing, Non-coding RNAs, Single nucleotide

variations, Fusion genes

– RNA quantification

– What is the concentration of RNAs?

– Absolute gene expression (within sample), Differential expression (between

biological samples)

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How to analyze
RNA seq data
for RNA
quantification?

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Overview of the Data Processing

• No available standardized workflow

• Multiple possible best practices for every dataset

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Data Pre-processing

– Adapter clipping to trim the sequencing adapters

– Quality trimming to remove wrongly called and low quality bases

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Annotation of
RNA-Seq reads

– Simple mapping on a
reference genome?
– More challenging

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Annotation of RNA-Seq reads

– 3 main strategies for annotations

– Transcriptome mapping

– Genome mapping

– De novo transcriptome assembly and annotation

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Transcriptome
mapping

– Need reliable gene models

– No detection of novel genes

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Genome mapping

Splice-aware read alignment

Detection of novel genes and isoforms

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Transcriptome and Genome mapping

– Needed

– Reference genome/transcriptome in FASTA

– Annotations of known genes, ... in GTF

– Where to find?

– Joint projects to produce and maintain annotations on selected organisms:

EMBL-EBI, UCSC, RefSeq, Ensembl, ...

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De novo transcriptome assembly

– No need for a reference genome ...

1. Assembly into transcripts

2. Map reads back

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Quantification
– What is the expression level of the genomic features?

– Counting the number of reads per features: Easy!!

– Challenges

– How to handle multi-mapped reads (i.e. reads with multiple alignments)?

– How to distinguish between different isoforms?

– At gene level?

– At transcript level?

– At exon level?

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Differential
Expression Analysis

– Account for variability of

expression across biological
replicates with the help of
counts

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Differential Expression Analysis: Normalization

– Make the expression levels comparable across

– By Features: genes, isoforms
– By Samples
– Methods
– FPKM/RPKM (Cufflinks/Cuffdiff)
– TMM (edgeR)
– DESeq2 (DESeq2)

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Visualization
– Integrative Genomics Viewer (IGV) or Trackster
– Visualization of the aligned BAM files

– Sashimi plots
– Quantitative visualization of read coverage along exons and splice junctions

– CummeRbund
– Visualization package for Cufflinks high-throughput sequencing data

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 Using Galaxy
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Galaxy

– Galaxy is a web-based data analysis platform.

– It is easy to use, and completely free.

– Galaxy offers over eight thousand analysis tools.

– Galaxy is widely used. It currently has over ten thousand publications.

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Why use Galaxy?

– It’s easy!

– No installation, all you need is a browser.

– No complex commands, just point and click!

– Makes your research reproducible

– Galaxy keeps track of all analysis details

– Cross-domain: bioinformatics, chemistry, ecology, climate science, ..

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 How to use
Galaxy?

– Find a Galaxy server

– UseGalaxy.*:
– Many other smaller, often
domain-specific Galaxies
available
– List of all public Galaxies
(135+): galaxyproject.org/use

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Main Galaxy servers
– There are three main Galaxy servers: Galaxy Main, Galaxy Europe, and Galaxy
Australia.

– These three Galaxies have the biggest teams behind them and offer the most
tools and resources.

– You can register an account for free on any of these servers.

– In addition, there are many smaller Galaxy servers to choose from.

– Many of these are domain specific. For example, Galaxy Proteomics focuses on
proteomics tools and workflows.

– A lot of universities and other institutions have local private servers.

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The Galaxy Interface

– Three main panels

– Left: Available Tools

– Middle: View your data and run tools

– Right: Full record of your analysis history

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Uploading data

– Upload from your computer

– Import files from URL

– Import from public data stores

– UCSC, NCBI, ENA, many more..

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Finding a tool

– After your data is uploaded, you are ready to run tools.

– You can find tools by exploring the tool list on the left.

– If you already know the name of the tool you want to use, you can enter this in
the search box at the top of the tool panel.

– If you have found a tool you like, you can add it to your favorites by clicking the
star at the top of the tool.

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Running a tool

– When you click on a tool, it will show up in the middle panel.

– Here you can select your input files and set the parameters for the tool.

– Then you can hit the Execute button to start the tool.

– Older versions of a tool are usually kept available to ensure reproducibility.

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Analysis Results

– Tool outputs are added to the history

– Different dataset states
– waiting , running , success, failed
– Expand for more options
– galaxy-save Download dataset
– galaxy-info Information about tool run
– galaxy-refresh Reload tool with the same parameters
– galaxy-barchart Visualize dataset
– Red dataset?
– galaxy-bug Click Bug icon
– view error message
– submit error report ]

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Analysis Results
– The results of the analysis will be added to your history.
– You will see these output files go through various states.
– When a dataset is grey, it means it is waiting to run.
– When it turns orange, it means the tool is running.
– When the tool is finished, the outputs will turn green if the tool ran successfully.
– You can then click on the history item to get more information and options.
– For example, you can download the file to your computer.
– Or you can reload the tool with the same parameters.
– There is also an option to visualize your data.
– If there was a problem with the tool, it will turn red.
– You can click on the bug icon to view the error message or submit a error report
to the Galaxy administrators.
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