RNA Sequencing PDF

RNA-Sequencing
Rickard Sandberg
Assistant Professor
Ludwig Institute for Cancer Research
Department of Cell and Molecular Biology
Karolinska Institutet
Wednesday, March 16, 2011
Cell/Tissue differences are reflected in

gene expression patterns
Mammals: 100s of cell types, tissues, organs, systems
muscle cells
kidney cells
hair cells
hippocampal neuron

muscle cells
kidney cells
hair cells
hippocampal neuron

zygote
blastocyst
muscle cells
kidney cells
hair cells
hippocampal neuron

zygote
blastocyst
muscle cells
kidney cells
hair cells
hippocampal neuron
All the information needed to encode an organisms is captured in the

genome of the zygote together with the proteins that act on the genome
Gene Regulatory Network:

with new technologies
Eric H. Davidson, Caltech
Barbara Wold, Caltech
Davidson et al. 2002, Science

mRNA-seq protocol
Isolate polyA+ RNA
Wang et al. 2009 Nat Rev Gen

mRNA-seq protocol
Isolate polyA+ RNA

mRNA-seq protocol
Isolate polyA+ RNA
polyA+ RNAs
rRNA- RNAs
short RNAs (e.g. miRNAs)
Ribosome footprint
sequencing
! GRO-Seq (Global Run On
sequencing)
! CLIP-Seq (RNA-protein
interactions)
!
!
!
!
! non-RNA applications:
ChIP-Seq, DNAse
hypersensitive sites,...
mRNA-SEQ read distributions
log10(reads)
gene SLC25A3
Testes
Liver
Skeletal Muscle
Heart
2
0
2
0
2
0
2
0
AK074759
BC011574
AK092689
3B
3B
log2(intensity)
3A
10
Testes
Liver
Skeletal Muscle
Heart
0
10
0
10
0
10
0
3A
log2(probe intensity)
10
0
10
0
10
$
#
"
!
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)*+, -,./+, -,.0/10,23
0
10
0
3B
%' %&("
%&
7654
log10(reads)
Wang*, Sandberg* et al. 2008 Nature

2
0
2
0
2
0
2
0
Wang*, Sandberg* et al. 2008 Nature Mortazavi et al. 2008 Nat Methods
Strand-specific RNA-Seq
Many protocols exists:
RNA ligation preserves strand information (Illumina short RNA kit)
Incorporation of dUTP in second strand synthesis, uracil-Nglycosylase
(Parkhomchuk et al. NAR 2009)
Digital RNA counting methods
in-house data using Illumina short RNA kit

Strand-specific RNA-Seq protocols
How gene expression levels are estimated

gene A (2 kb transcript)
gene B (600 bp transcript)

The number of fragments are proportional to the

Fragmentation abundance and length of the transcript.


Sequencing
ACGCG...
TCGAG...
AGGTA...
CCGTG...
CTGCG...


Sequencing
ACGCG...
TCGAG...
AGGTA...
CCGTG...
CTGCG...
Normalize for different transcripts lengths and

different sequence depths in different samples.
RPKM (Reads per kilobase and million mappable reads):
Given 10 million mappable reads:
RPKM, Gene A: 500 reads x 1000/2000 x 106/107
500 / (2 x 10) = 25 RPKM
RPKM roughly corresponds to transcripts per cell (Mortazavi et al. 2008)

(assuming a standard cell with ~ 300.000 transcripts)
mRNA-SEQ is highly quantitative
Biol. Replicates
MAQC samples
UHR (cell line mix)
Brain
Spiked-in RNAs
<10M reads
Mortazavi et al. 2008 Nat Methods
~20M reads
RNA sequencing of blastocyst-derived

cell lines
Read counts for selected genes
ES
TS
XEN
EpiSC
Nanog
6525
20
263
Cdx2
124
6256
Sox17
11
9814
99
Sox3
151
1234
796
Shh
Ihh
12
107
17
Dhh
10
212
575
80
Relative expressions on microarrays
Sandberg*, Neilsen* et al. Science 2008

Lowly expressed genes (r=0.099)

High expressed genes (r=0.177)
!"#$%&'($)*+,,
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Improvement over microarrays?
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RNA-Seq Expression Rank
Guttman et al. Nat Biotech 2010
Lowly expressed genes (r=0.099)

High expressed genes (r=0.177)
!"#$%&'($)*+,,
"!!!
#!!!!
#"!!!
$!!!!
$"!!!
Improvement over microarrays?
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#!!!!
#"!!!
$!!!!
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RNA-Seq Expression Rank
Guttman et al. Nat Biotech 2010
RNA-Seq data is richer than microarray data

More sensitive due to low background
More quantitative and larger dynamic range
Improved power to detect RNA isoforms
generated by alternative splicing, promoters
and polyadenylation
De novo identification of transcripts and
junctions
Cost (higher depths for RNA

isoforms still more expensive)
Limited number of packages and
tools for non-bioinformaticians
Significance of expression level

background RPKM ~ 0.05 RPKM
detection level of 0.3 RPKM
an average 1 500 nt transcript
20 M uniquely mapping reads
background model:
0.05 x 1.5 x 20 = 1.5 reads
expressed at 0.3 RPKM:
0.3 x 1.5 x 20 = 9 reads
binomial test for 9 reads out of 20 M mapping
to transcript given a background probability
of 1.5 / 20x109
gives a p-value of 2.8e-5
expressed at 1 RPKM:
1 x 1.5 x 20 = 30 reads
0.05 RPKM
1 RPKM
Brain
Brain
Figure 4. Ramskld et al
Muscle
Muscle
Avoiding 3UTRs help expression estimates

Portion of genes (cumulative)

2000
500
1000
1500
2000
Length in nucleotides
2500
Liver
3000

Position on chromosome 19
TandemUTR genes (brain)

TandemUTR genes (UHR)
0
Other
genes
(brain)
10
Other genes (UHR)
7933000
1.0
Fraction of reads
Solexa (expression relative to POLR2A)
10
r=0.80 1500
500
1000
Length inn=465
nucleotides
101
Liver
Portion of genes (cumulative)
10-1
10-2
0.5

0.0
10-3

7931000

non-unique/ without 3'UTR)
Expression (full transcript
polyA sites
# reads
brain
UHR
0
60
140
10-4
165
34

-3

-2
10-4 value10
10-1
10
101
102 exact test: p=10-27
Fisher's
Solexa
/ TaqMan 10
value
TaqMan (expression relative to POLR2A)

Genes with one

annotated isoform

TandemUTR genes
Correlation
Correlation
Brain UHR p-value UHR p-value
Full transcript

Without 3'UTR

Without 5'UTR
Coding region
Internal exons
Constitutive exons
Truncated at first
predicted poly(A) site

0.78
0.79
0.70
0.81
0.81
0.0007
0.76 0.0003
0.77
0.79
0.04
0.70 0.3
0.81
0.81
0.001
0.76 0.00002
0.81
0.81
0.9
0.75 0.2
0.79
0.78
0.6
0.70 0.02
0.79
0.80
0.03
0.74 0.002
Ramskold et al. PLoS Comp Biol 2009

Differential expression
Transcript length effects

differential expression tests
Oshlack and Wakefield Biology Direct 2009

General Feature of Tissue Transcriptomes
How many genes are expressed in a tissue?

How many genes are house-keeping (or expressed in
all human/mammalian cells)?
Are genes regulated by all-or-none expression, graded
expression or by different isoforms?
General Feature of Tissue Transcriptomes
How many genes are expressed in a tissue?
Number of expressed genes? 8-20,000?
How many genes are house-keeping (or expressed in

all human/mammalian cells)?
1,000-2,000 or 10,000?
Are genes regulated by all-or-none expression, graded

expression or by different isoforms?
would require single-cell measurements.
e.g. 30-50% of genes with alternative
splicing
More on background/level of detection

a
b
1.0
Read density in introns

Gene expression (exons)
300
0.8
Portion of mRNA pool
250
Genes per sample
Suppleme
200
150
100
0.6
0.4
0.2
50
10
-4
10
-3
-2
-1
10
10
10
10
10
Reads per kilobase and million mappable reads
10
The density of reads in exons and introns of Ensembl genes with one
annotated isoform, for 10 human tissue samples. The density is an average
across
all introns or exons of a gene.
Wednesday, March
16, 2011
10
0.0
1
10
10
Number of most
Human cerebellum samples

and Comp
black, Biol
and 2009
other tis
Ramskold purple
et al. PLoS
This illustrates the reproducib

a
b
1.0

300
80%
0.8
92%
250
Genes per sample
Suppleme
200
150
100
0.6
0.4
0.2
50
10
-4
10
-3
-2
-1
10
10
10
10
10
10
across
Wednesday, March
16, 2011
10
0.0
1
10
10
Number of most

and Comp
black, Biol
and 2009
other tis
Ramskold purple
et al. PLoS

a
b
1.0

300
11-13,000 genes
per tissue
80%
250
0.8
92%
absolute expression
levels
Genes per sample
Suppleme
200
150
100
0.6
0.4
0.2
50
10
-4
10
-3
-2
-1
10
10
10
10
10
10
across
Wednesday, March
16, 2011
10
0.0
1
10
10
Number of most

and Comp
black, Biol
and 2009
other tis
Ramskold purple
et al. PLoS
A shared set of genes in tissue transcriptomes
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The number of shared genes are sensitive to the expression level used for detection,
but 5-10 times higher than microarray and SAGE based estimates
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Ramskld et al. PLoS Comp Biol 2009
Inside-outside rule of ubiquitous

transcription
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Tissue transcriptome compositions:

The contribution of highly expressed genes
Human tissues
Lymph node
Muscle
Heart
Testes
Brain
Colon
Fat
Liver
Breast
Mouse tissues
Brain
Liver
Muscle
80%
80%
Colon
Lymph node
40%
Fat
Breast
Testes
Mouse brain
Human brain
20%
0%
Human muscle
Heart
Mouse liver
Mouse muscle
Human liver
60%
10
100
1000
Number of most expressed genes
P ortion of mR NA pool
100%
P ortion of mR NA pool
100%
Human cell lines

HME
mfc7
bt474
mb435
t47d
60%
40%
20%
10000
0%
10
Num

Read coverage of untranslated regions

a
d
Brain
5'UTR
3'UTR
0.0
b
0.2
0.4
0.6
0.8
Read coverage relative to coding region
1.0
Liver 5'UTR
Muscle
Extracellular
Plasma membrane
All genes
5'UTR
CDS
3'UTR
Brain
Relative number of
tissue-specific genes
Muscle
0 20
0
c
500
1000
1500
2000
Tissue-specific
5'UTR
CDS
3'UTR
Brain
xpression level (RPKM)
Liver


a
Testese
Liver
5'UTR
Liver
Skeletal Muscl
Heart
d
Brain
5'UTR
3'UTR
0.0
b
0.2
0.4
0.6
0.8
1.0
Muscle
Extracellular
AK074759
BC011574
AK092689
Plasma membrane
All genes
5'UTR
CDS
3'UTR
Brain
Relative number of
Muscle
0 20
0
c
500
1000
1500
2000
Tissue-specific
5'UTR
CDS
3'UTR
Brain
Liver


a
d
Brain
5'UTR
3'UTR
5'UTR
0.0
0.2
0.4
0.6
0.8
3'UTR
1.0
Testese
Liver
5'UTR
Liver
Skeletal Muscl
Heart
Muscle
d
Brain
Extracellular
AK074759
BC011574
AK092689
Muscle
Extracellula
Plasma
1.0 membrane
0.2
0.4
0.6
0.8
Expression weighted
UTR length
Read coverage
relative toestimates
coding region
5'UTR
CDS
3'UTR
Relative number of
0 20
Plasma membr
b All genes
Brain
All genes
0.0
5'UTR
CDS
3'UTR
Muscle
Liver
0
c
500
Tissue-specific
0
5'UTR
c
1000
1500
500
Tissue-specific
Muscle
2000
1000
1500
CDSLength in nucleotides 3'UTR
Brain
Relative numbe
f g
tissue-specific
Liver
2000
Brain
RNA-Sequencing:
Transcriptome Reconstruction
Nature Biotechnology, April 2010

RNA-Sequencing:
Transcriptome Reconstruction

Transcript reconstruction
Transcript reconstruction

Increased depth improves reconstruction

Discovery of cell-type specific

alternative isoforms

Our default view of gene expression?
mRNA isoform regulation
Alternative Promoters
Extens.
Core
Alternative Splice Sites
Extens.
Core
Mutually Exclusive Exons
MXE1
MXE2
Extens.
Core
Skipped Exons
MXE1
MXE2
5 Exon
SE
3 Exon
pA
Extens.
Core
Skipped Exons
Alternative Polyadenylation
MXE1
MXE2
5 Exon
SE
3 Exon
pA
Alternative Splicing as a Switch and as a Tuner

Switching on the Fas receptor
Cascino et al. 1995

5
5
Soluble
Inhibition of apoptosis
Cascino et al. 1995

5
5
7
5
Soluble
7 Membrane-bound
Apoptosis
Cascino et al. 1995

5
5
7
5
Soluble
7 Membrane-bound
Apoptosis
Cascino et al. 1995

5
5
7
5
Soluble
7 Membrane-bound
Apoptosis
Cascino et al. 1995
Tuning the inner ear:

splicing of calcium-activated potassium channels in hair cells
2v
Ramanathan et al. 1999

5
5
7
5
Soluble
7 Membrane-bound
Apoptosis
Cascino et al. 1995

2 2v 3
2
2v
Low frequencies

5
5
7
5
Soluble
7 Membrane-bound
Apoptosis
Cascino et al. 1995

2 2v 3
2
2v
Low frequencies
3
2
High frequencies
Genome-wide detection of mRNA isoforms
Expressed Sequence Tags

Traditional 3UTR focused microarrays (e.g. Affymetrix)
Exon and Tiling Arrays
Deep Sequencing using Illumina/Solexa, SOLiD, (454)
Mismapping rates for splice

junctions
discovery of novel junctions
D
ORJUHDGV
Extent of Alternative Splicing

#
Ability to detect alternative isoforms will depend on read
coverage
"
Multi-exon genes
Isoform 2

Isoform 1
Isoform 2

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(9 tissue samples)
0.8
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(random subsets of 9 out
of 14 tissue and cell line
samples)
0.6
0.4
0.2
0.0
2
3
4
No. of reads (log10)
Figure S2. Assessment of frequency of alternativ

a, Fraction of alternatively spliced genes binned by r
of 250 genes per bin. The upper asymptote of the sig
observed data was used to infer the true fraction of
using bin size of 250).Wang*,
To assess
whether
high cove
Sandberg* et
al. 2008 Nature
fraction of alternative splicing, a subsampling appr
Identify alternatively spliced transcripts
Katz et al. Nature Methods 2010

Using splice-junction alone is noisy

Power of paired-end reads

Tissue-regulated mRNA isoforms

Tissue-regulated mRNA isoforms

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Conclusions
RNA-seq enables genome-wide transcriptome quantification with

more accurate and absolute expression estimates
Low background enables quantification of lowly expressed
transcripts (~1 copy per cell)
Investigate alternative promoters, splicing and polyadenylation,
non-coding RNAs
Current Team and Collaborators

Sandberg Lab
Daniel Ramskld
Helena Storvall
Ersen Kavak, PhD
Mats Enster, PhD
Gsta Winberg, PhD
Liudmila Matskova, PhD
Dept of Biology, MIT, USA

Chris Burge
Eric Wang
Cancer Center, MIT, USA

Phillip A. Sharp
Joel Neilson
Dept of Cell and Mol Biol

Urban Lendahl
Johan Ericson
LICR, Stockholm
Jonas Muhr
Sick Children Hospital,

Toronto, Canada
Janet Rossant
Fredrik Lanner
Questions?

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RNA-Sequencing

Wednesday, March 16, 2011

Cell/Tissue differences are reflected in

Wednesday, March 16, 2011

Cell/Tissue differences are reflected in

Wednesday, March 16, 2011

Cell/Tissue differences are reflected in

Wednesday, March 16, 2011

Cell/Tissue differences are reflected in

All the information needed to encode an organisms is captured in the

Gene Regulatory Network:

Eric H. Davidson, Caltech

Barbara Wold, Caltech

Davidson et al. 2002, Science

Wang et al. 2009 Nat Rev Gen

Wang et al. 2009 Nat Rev Gen

mRNA-SEQ read distributions

Wednesday, March 16, 2011

Wang*, Sandberg* et al. 2008 Nature

in-house data using Illumina short RNA kit

Strand-specific RNA-Seq protocols

Wednesday, March 16, 2011

How gene expression levels are estimated

Wednesday, March 16, 2011

How gene expression levels are estimated

The number of fragments are proportional to the

Wednesday, March 16, 2011

How gene expression levels are estimated

The number of fragments are proportional to the

Wednesday, March 16, 2011

How gene expression levels are estimated

The number of fragments are proportional to the

Normalize for different transcripts lengths and

RPKM roughly corresponds to transcripts per cell (Mortazavi et al. 2008)

mRNA-SEQ is highly quantitative

Wang*, Sandberg* et al. 2008 Nature

Mortazavi et al. 2008 Nat Methods

RNA sequencing of blastocyst-derived

Wednesday, March 16, 2011

Relative expressions on microarrays

Sandberg*, Neilsen* et al. Science 2008

Lowly expressed genes (r=0.099)

Improvement over microarrays?

RNA-Seq Expression Rank

Wang et al. 2009 Nat Rev Gen

Wednesday, March 16, 2011

Guttman et al. Nat Biotech 2010

Lowly expressed genes (r=0.099)

Improvement over microarrays?

RNA-Seq Expression Rank

Wang et al. 2009 Nat Rev Gen

Guttman et al. Nat Biotech 2010

RNA-Seq data is richer than microarray data

Cost (higher depths for RNA

Significance of expression level

Wednesday, March 16, 2011

Avoiding 3UTRs help expression estimates

Portion of genes (cumulative)

TandemUTR genes (brain)

Solexa (expression relative to POLR2A)

Portion of genes (cumulative)

   

Genes with one

Wang, Sandberg et al. 2008 Nature

Wang, Sandberg et al. 2008 Nature

Sandberg, Neilsen et al. Science 2008

0.76 0.0003

0.70 0.3

0.76 0.00002

0.75 0.2

0.70 0.02

0.74 0.002