RNA Sequencing

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

zygote blastocyst
muscle cells
kidney cells

zygote blastocyst
muscle cells
kidney cells
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
gene SLC25A3
2
Testes
log10(reads)
0
2
0 Liver
2
0 Skeletal Muscle
Heart
2
0
3B AK074759
3B BC011574
3A AK092689
log2(intensity)
10
0 Testes
Liver
10
Skeletal Muscle
10
Heart
10
Wang*, Sandberg* et al. 2008 Nature

2
0
log10(reads)
2
0
2
0
2
0 %' %&("
3B %&
3A
7654
$
10
#
log2(probe intensity)
0
10 "
0 ! !(%" !(%!
10
)*+, -,./+, -,.0/10,23
0
10
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-N-
glycosylase
(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
Normalize for different transcripts lengths and
ACGCG...
different sequence depths in different samples.
TCGAG...
AGGTA...
RPKM (Reads per kilobase and million mappable reads):
CCGTG...
Given 10 million mappable reads:
CTGCG...
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 1 263
Cdx2 124 6256 1 1
Sox17 11 5 9814 99
Sox3 151 1234 6 796
Shh 0 0 0 1
Ihh 4 12 107 17
Dhh 10 212 575 80

Relative expressions on microarrays
Sandberg*, Neilsen* et al. Science 2008

Lowly expressed genes (r=0.099)
Improvement over microarrays? High expressed genes (r=0.177)
!"#$%&'($)*+,,
$"!!!
$!!!!
#"!!!
#!!!!
"!!!
!
! "!!! #!!!! #"!!! $!!!! $"!!!
RNA-Seq Expression Rank
Wang et al. 2009 Nat Rev Gen Guttman et al. Nat Biotech 2010

Lowly expressed genes (r=0.099)
Improvement over microarrays? High expressed genes (r=0.177)
!"#$%&'($)*+,,
$"!!!
$!!!!
#"!!!
#!!!!
"!!!
!
! "!!! #!!!! #"!!! $!!!! $"!!!
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

More sensitive due to low background
More quantitative and larger dynamic range Cost (higher depths for RNA
isoforms still more expensive)
Improved power to detect RNA isoforms
generated by alternative splicing, promoters Limited number of packages and
and polyadenylation
tools for non-bioinformaticians
De novo identification of transcripts and
junctions

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
0.05 RPKM
expressed at 1 RPKM: 1 RPKM
1 x 1.5 x 20 = 30 reads

Brain Brain
Figure 4. Ramsköld et al
Muscle Muscle
Avoiding 3‘UTRs help expression estimates

a Liver b Liver
2
10
0 500 r=0.80 1500
1000 2000 0 500 1000 1500 2000 2500 3000
Portion of genes (cumulative)

Length inn=465
Solexa (expression relative to POLR2A)

nucleotides Length in nucleotides
101

Position on chromosome 19
TandemUTR genes (brain)
TandemUTR genes (UHR) 7933000 7931000
Other 0
genes (brain) 1.0
10
Portion of genes (cumulative)
Other genes (UHR)
Fraction of reads

10-1

0.5

10-2

0.0
non-unique/ without 3'UTR)
Expression (full transcript

10-3
polyA sites
# reads
10-4 60 140 brain

-3 -2 165 34 UHR
10-4 value10
Solexa / TaqMan 10
value 10-1 10 101 Fisher's
0
102 exact test: p=10-27
TaqMan (expression relative to POLR2A)
c
Genes with one

annotated isoform TandemUTR genes

Correlation Correlation
Brain UHR p-value UHR p-value
Full transcript 0.78 0.79 0.70
Without 3'UTR 0.81 0.81 0.0007 0.76 0.0003
Without 5'UTR 0.77 0.79 0.04 0.70 0.3
Coding region 0.81 0.81 0.001 0.76 0.00002
Internal exons 0.81 0.81 0.9 0.75 0.2
Constitutive exons
0.79 0.78 0.6 0.70 0.02
Truncated at first 0.79 0.80 0.03 0.74 0.002

predicted poly(A) site
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
Suppleme
a b
1.0
Read density in introns
300 Gene expression (exons)
0.8
250
Portion of mRNA pool

200 0.6
Genes per sample
150
0.4
100
0.2
50
! 0.0
-4 -3 -2 -1 0 1 2 3 4
10 10 10 10 10 10 10 10 10 1 10 10
Reads per kilobase and million mappable reads Number of most
The density of reads in exons and introns of Ensembl genes with one Human cerebellum samples
annotated isoform, for 10 human tissue samples. The density is an average Ramskold purple and Comp
et al. PLoS black, Biol
and 2009
other tis
across
Wednesday, March all introns or exons of a gene.
16, 2011 This illustrates the reproducib
Suppleme
a b
1.0
300 Gene expression (exons)
0.8
250 80% 92%

200 0.6
Genes per sample
150
0.4
100
0.2
50
! 0.0
-4 -3 -2 -1 0 1 2 3 4
10 10 10 10 10 10 10 10 10 1 10 10
and 2009
other tis
across
Suppleme
a b
1.0
300 Gene expression (exons) 11-13,000 genes
per tissue
0.8
250 80% 92%
absolute expression
levels

200 0.6
Genes per sample
150
0.4
100
0.2
50
! 0.0
-4 -3 -2 -1 0 1 2 3 4
10 10 10 10 10 10 10 10 10 1 10 10
and 2009
other tis
across
A shared set of genes in tissue transcriptomes
KLQJ
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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
Ramsköld et al. PLoS Comp Biol 2009

%-()*#%$$"$*)
Inside-outside rule of ubiquitous
transcription
#
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:%9"%!#%8&C%#'N'#7OGM7,'!;'!B
./. ./2 ./1 ./0 ./3 4/.

5)*#('6!7!6!8",'9"'(6"&
Ramskold D., et al. PLoS Comp Biol 2009
Tissue transcriptome compositions:
The contribution of highly expressed genes
Human tissues Brain
Mouse tissues
Lymph node Colon
Muscle Fat Brain
Heart Liver Liver
Testes Breast Muscle
100% 100%
Human cell lines
HME
mfc7
80% 80% bt474
mb435
t47d
P ortion of mR NA pool
P ortion of mR NA pool
Mouse liver Human muscle 60%

60% Mouse muscle Heart
Human liver
Colon
Lymph node
40% Fat 40%
Breast
Testes
Mouse brain
20% Human brain
20%
0% 0%
1 10 100 1000 10000 1 10
Number of most expressed genes Num

Read coverage of untranslated regions
a d e
Brain Muscle Liver 5'UTR
5'UTR
3'UTR
Extracellular
0.0 0.2 0.4 0.6 0.8 1.0
Read coverage relative to coding region
b All genes Plasma membrane
5'UTR CDS 3'UTR Relative number of 0 20

Brain tissue-specific genes
Muscle
f
Liver
xpression level (RPKM)

0 500 1000 1500 2000
Length in nucleotides
c Tissue-specific
5'UTR CDS 3'UTR

Brain
a d Testese
Liver
5'UTR Skeletal Muscl
Heart
3'UTR AK074759
Extracellular BC011574
AK092689
0.0 0.2 0.4 0.6 0.8 1.0
b All genes Plasma membrane

Brain tissue-specific genes
Muscle
f
Liver

0 500 1000 1500 2000
Length in nucleotides
c Tissue-specific
5'UTR CDS 3'UTR

Brain
a d Testese
Liver
5'UTR Skeletal Muscl
a d Heart
3'UTR Brain
AK074759
Muscle
5'UTR Extracellular BC011574
AK092689
0.0 0.2 0.4 0.6 0.8 1.0
3'UTR
Extracellula
b All genes 0.2 0.0 0.4 0.6 0.8 Plasma
1.0 membrane
Expression weighted UTR length
Read coverage relative toestimates
coding region
b All genes Brain Plasma membr
tissue-specific genes
5'UTR CDS 3'UTR Relative numbe
Muscle
Brain tissue-specific
f g
Liver
Muscle

0 500 1000 1500 2000
Length in nucleotides Liver
c Tissue-specific
0 500 1000 1500 2000
5'UTR CDSLength in nucleotides 3'UTR
c Tissue-specific 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

MXE1 MXE2
Extens. Core
Mutually Exclusive Exons

MXE1 MXE2 5‘ Exon SE 3‘ Exon

Extens. Core
Skipped Exons

pA pA
MXE1 MXE2 5‘ Exon SE 3‘ Exon

Extens. Core
Skipped Exons
Alternative Polyadenylation

Alternative Splicing as a Switch and as a Tuner

Switching on the Fas receptor
5 6 7
Cascino et al. 1995

5 7 Soluble Inhibition of apoptosis

5 6 7
Cascino et al. 1995


5 6 7
5 6 7 Membrane-bound Apoptosis
Cascino et al. 1995


5 6 7
Cascino et al. 1995


5 6 7
Cascino et al. 1995
Tuning the inner ear:

splicing of calcium-activated potassium channels in hair cells
2 2v 3
Ramanathan et al. 1999


5 6 7
Cascino et al. 1995

2 2v 3 Low frequencies
2 2v 3


5 6 7
Cascino et al. 1995

2 2v 3 Low frequencies
2 2v 3
2 3 High frequencies

Genome-wide detection of mRNA isoforms
• Expressed Sequence Tags

• Traditional 3’UTR 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
Extent of Alternative Splicing

ORJUHDGV

Ability to detect alternative isoforms will depend on read

"
coverage
#
E Multi-exon genes F
Isoform 1
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samples)

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0.2
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Isoform 1
Figure S2. Assessment of frequency of alternativ
a, Fraction of alternatively spliced genes binned by r
Isoform 2
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
Sandberg* et high cove
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

from Expression
G to Regulation, “RNA-maps”
6 (

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Binding of NOVA splicing factor

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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 Ramsköld Dept of Biology, MIT, USA
Helena Storvall Chris Burge
Ersen Kavak, PhD Eric Wang
Mats Ensterö, PhD
Gösta Winberg, PhD
Liudmila Matskova, PhD
Cancer Center, MIT, USA
Phillip A. Sharp
Joel Neilson
Sick Children Hospital,

Dept of Cell and Mol Biol LICR, Stockholm Toronto, Canada
Urban Lendahl Jonas Muhr Janet Rossant
Johan Ericson Fredrik Lanner

Questions?

RNA Sequencing

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RNA Sequencing

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RNA-Sequencing

Wednesday, March 16, 2011

hair cells hippocampal neuron

Wednesday, March 16, 2011

hair cells hippocampal neuron

Wednesday, March 16, 2011

hair cells hippocampal neuron

Wednesday, March 16, 2011

hair cells hippocampal neuron

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

Eric H. Davidson, Caltech

Barbara Wold, Caltech

Davidson et al. 2002, Science

Wednesday, March 16, 2011

Isolate polyA+ RNA

Wang et al. 2009 Nat Rev Gen

Wednesday, March 16, 2011

Isolate polyA+ RNA

Wang et al. 2009 Nat Rev Gen

Wednesday, March 16, 2011

Isolate polyA+ RNA

Wang et al. 2009 Nat Rev Gen

Wednesday, March 16, 2011

Wang*, Sandberg* et al. 2008 Nature

Wednesday, March 16, 2011

Many protocols exists:

in-house data using Illumina short RNA kit

Wednesday, March 16, 2011

Wednesday, March 16, 2011

Wednesday, March 16, 2011

The number of fragments are proportional to the

Wednesday, March 16, 2011

The number of fragments are proportional to the

Wednesday, March 16, 2011

The number of fragments are proportional to the

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

Wang*, Sandberg* et al. 2008 Nature

<10M reads Mortazavi et al. 2008 Nat Methods

Wednesday, March 16, 2011

Read counts for selected genes

Wednesday, March 16, 2011

Sandberg*, Neilsen* et al. Science 2008

Wednesday, March 16, 2011

RNA-Seq Expression Rank

Wednesday, March 16, 2011

RNA-Seq Expression Rank

RNA-Seq data is richer than microarray data

Wednesday, March 16, 2011

background RPKM ~ 0.05 RPKM

expressed at 0.3 RPKM:

Wednesday, March 16, 2011

Avoiding 3‘UTRs help expression estimates

Portion of genes (cumulative)

Solexa (expression relative to POLR2A)

 Other genes (UHR) 

 10-4 60 140 brain

annotated isoform TandemUTR genes

Ramskold et al. PLoS Comp Biol 2009

Wednesday, March 16, 2011

Oshlack and Wakefield Biology Direct 2009

Wednesday, March 16, 2011

How many genes are expressed in a tissue?

How many genes are house-keeping (or expressed in

Wang, Sandberg et al. 2008 Nature

Wang, Sandberg et al. 2008 Nature

Sandberg, Neilsen et al. Science 2008

Other genes (UHR)

10-4 60 140 brain