Analysis of bulk RNA-seq data

Analysis of Next-Generation Sequencing Data

An introduction to transcriptome or
gene expression

05 June 2023
Phuc Loi Luu, PhD
Loi.lp@pacificinformatics.com.vn

Analysis of bulk RNA-seq data

 Analysis of bulk RNA-seq data
Analysis of Next-Generation Sequencing Data

Friederike Dündar
Applied Bioinformatics Core
Slides at https://bit.ly/2T3sjRg 1

February 18, 2020

1
https://physiology.med.cornell.edu/faculty/skrabanek/lab/angsd/schedule_2020/
Friederike Dündar (ABC, WCM) Analysis of bulk RNA-seq data February 18, 2020 2 / 51
1 Why study RNA?

2 Different types of RNA – different library preps

3 Gene expression : Microarray vs RNA-seq

Why study RNA?

Why study RNA?

 Why study RNA?

DNA is just the blueprint, it is not an effector molecule.

 Why study RNA?

DNA is just the blueprint, it is not an effector molecule

GENOMICS TRANSCRIPTOMICS
DNA sequence of an organism = characterization of gene
genetic basis of phenotypic products
differences identification of specific RNAs
sites of DNA-protein or quantification of RNAs
DNA-RNA interactions RNA-protein interactions
sites of open vs. closed RNA structure
chromatin

In order to understand the functional consequences (capacity) of a DNA

sequence, we need to study its products, i.e. RNA and proteins.
 Different types of RNA – different library preps

Different types of RNA – different library preps

 Different types of RNA – different library preps

DNA and RNA have different properties

DNA RNA
usually double-stranded generally single-stranded, but with
very stable the capacity for complementary base
mutations are heritable pairing ⇒ ability to form myriad
same amount in (almost) all cells different shapes
same sequence in every cell of an usually fairly short-lived (minutes to
organism hours)
easily degraded/damaged without
protection
mutations are not passed on
individual transcript amounts differ
greatly depending on the gene, the
cell type, the developmental status,
the environment etc.
transcript sizes range from 10-20bp
to several kb
 Different types of RNA – different library preps

Different types of RNA

There are numerous different types of functional RNA moleculesin addition to messenger RNA, which
does not carry out a function of its own except transporting the DNA code (genetic
information) into the cytoplasm where it can be translated into proteins.
 Different types of RNA – different library preps

Different types of RNA

Ca. 75% of the human genome can be transcribed( = copied into RNA)
but <3% of the genome is subsequently translatedinto proteins genescan
therefore be
coding(⇒ final product: protein) or
non-coding(⇒ final product: RNA)
Non-coding RNAs cover a wide range of functions including protein assembly
(⇒ ribosomal RNA, transfer RNA) and gene expressionregulation

See Wilkes et al. [2017], Bartoszewski

and Sikorski [2018] and Dai et al.
[2020] for an introduction into the
diverse RNA families and their func-
tions.
 Different types of RNA – different library preps

Different types of RNA (there are more!)

 Different types of RNA – different library preps

Typical applications of RNA-seq

Identification of transcripts – which portions of the genome are

expressed?
► identification of splice variants
► transcriptome assembly
► detection of gene fusion events
Quantification of transcripts
► comparison of different cell types/conditions/diseases and their effect on
individual mRNA quantities
► allele-specific expression

Illumina technology is best suited for the quantification of

known transcripts; its short reads are not a good match for the
identification of novel transcripts in very complex transcriptomes
such as the ones found in mammals.
 Different types of RNA – different library preps

Sequencing prep protocol depends on the RNA properties

It is not a one-size-fits-all situation!

abundanceand stability
► rRNA: 90-95% (!)
► tRNA: 3-5%
► mRNA: 2%
► all other non-coding RNAs: well
below 1%
cellular location
► most are in the cytoplasm
size
► miRNAs: 18-23bp
► mRNA: several 100 to 1000 bp
specific sequences/modifications
► poly(A) tails of mRNA
► 2D structure
► antisense transcripts
 Different types of RNA – different library preps

Focus today: messenger RNA

mRNA amounts are used as a proxy for the amounts of their corresponding
proteins within a given tissue.
 Different types of RNA – different library preps

Focus today: messenger RNA

How to profile gene expression?
How to profile gene expression?

1. Quantitative Reverse Transcription and

Polymerase Chain Reaction (qRT/PCR)
2. Northern Blotting
3. Fluorescence In situ Hybridization (FISH)
4. Microarrays
5. RNA-seq
10.1007/978-1-4939-7834-2_2
Microarray vs RNA-seq

https://www.otogenetics.com/rna-sequencing-vs-microarray/
10.1007/978-1-4939-7834-2_2
10.1007/978-1-4939-7834-2_2
10.1007/978-1-4939-7834-2_2
10.1007/978-1-4939-7834-2_2
10.1007/978-1-4939-7834-2_2
Microarray vs RNA-seq
How to choose gene expression assay?
10.1007/978-1-4939-7834-2_2
10.1007/978-1-4939-7834-2_2
 Different types of RNA – different library preps

Focus today: messenger RNA

Bulk RNA-seq of mRNA

expression quantification of (mostly) mRNA transcripts

extracted from populations of cells
and tested for gene-specific differences between distinct
conditions
 Different types of RNA – different library preps

Typical questions addressed with bulk RNA-seq

Does a certain treatment induce gene expression changes? And if it

does, which genes are most strongly affected?
How does the gene expression profile of a cancer cell differ from a
healthy cell?
Which genes are turned on/off during the course of embryonic
development?
Which genes differ in mice that have been engineered to lack a certain
gene? E.g., which genes – in addition to the one that’s been
“knocked-out” – may be depleted or overcompensating for the loss?
Which genes are activated in response to an environmental stimulus,
e.g. heat shock or alcohol poisoining?
How does the gene expression profile change in the same tissue in an
aging individual?
...
 Different types of RNA – different library preps

General steps of RNA-seq preparation

1 RNA extraction2 (cell lysis, RNA purification)

2 enrichment of the RNA of interest
3 fragmentation (ca. 200 bp)
4 cDNA synthesis
5 library prep to obtain cDNA with adapters for
sequencing

2
Most standard extraction methods will lose RNA <100 bp!
 Different types of RNA – different library preps

QC of RNA extraction

Avoid degraded RNA! Optimum: RNA Integraty Score (RIN) of 10.

 Different types of RNA – different library preps

General steps of RNA-seq preparation

1 RNA extraction (cell lysis, RNA purification)

2 enrichment of the RNA of interest
► mRNA: poly(A) enrichment vs. ribosomal-depletion
► small RNAs: size-based enrichment
3
fragmentation (ca. 200 bp)
4 cDNA synthesis
5 library prep to obtain cDNA with adapters for
sequencing
 Different types of RNA – different library preps

Every step has consequences – example: mRNA

enrichment strategies
 Different types of RNA – different library preps

The most common library preparation methods

(A) classical
unstranded
mRNA library
prep
 Different types of RNA – different library preps

The most common library preparation methods

(A) classical
unstranded
mRNA library
prep
(B) stranded
mRNA
(dUTP-based)
(see Levin et al.
[2010] and Zhao
et al. [2015] for
details)
 Different types of RNA – different library preps

The most common library preparation methods

Unstranded vs. stranded
 Different types of RNA – different library preps

The most common library preparation methods

(A) classical
unstranded mRNA
library prep
(B) stranded
mRNA
(dUTP-based) (see
Levin et al. [2010]
and Zhao et al.
[2015] for details)
(C) small RNAs
(miRNA, piRNA,
tRNA, ... <100 bp)
using 2 adapters –
not optimal for
differential
expression
analyses!
 Different types of RNA – different library preps

Every step has consequences

Do not mix different strategies for samples that are to be

compared to each other!
► extraction, enrichment, library prep

There are many papers comparing different aspects of different RNA-seq

approaches, e.g.
Library preparation methods for next-generation sequencing: Tone
down the bias [van Dijk et al., 2014]
Systematic comparison of small RNA library preparation protocols for
next-generation sequencing [Dard-Dascot et al., 2018]
A comprehensive assessment of RNA-seq protocols for degraded and
low-quantity samples. [Schuierer et al., 2017]
many more – PubMed is your friend!

Make an informed decision!