defense2016

Note that the podium blocks the
view!

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Annotation-agnostic differential
expression and binding analyses
Leonardo Collado-Torres
@fellgernon

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Mo6va6ng problem: iden6fy and validate
regions of the genome that change expression
during brain development

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Research theme
- annota6on-agnos6c
- reproducible analyses
- easily accessible data
- sta6s6cal tools for the genomics community

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RNA-seq
reads
Genome
(DNA)
RNA transcripts
(many possible
variants)
Measuring gene expression: RNA-seq
Adapted from @jtleek

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Genome
(DNA)
Mapped reads
Common analysis pipelines:
• Feature coun6ng (gene or exon level)
• Transcript assembly

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Challenges in counting
hFp://www-huber.embl.de/users/anders/HTSeq/doc/count.html

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Annotation variation
Frazee et al, Biostatistics, 2014

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DER finder approach
•  Find con6guous base pairs with
Diﬀeren6al Expression signal à DE
Regions or DERs
•  Find nearest annotated feature

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coverage
vector
2 6 0 11 6
Genome
(DNA)
Read coverage

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Collado-Torres et al, bioRxiv, 2016
GTEx data
0 4 8 16 32 64
Liver Heart Testis
Coverage
STK19, 0 bp from tss: covers
−
+
Genes
31978500 31979500 31980500 31981500
+
Tx
chr6

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0 1 2
Neo.F Neo.A notNeo.F notNeo.A CBC.F CBC.A
Mean coverage
IL17RD, 72831 bp from tss: overlaps 3'
−
+
Genes
57124000 57126000 57128000 57130000
−
Tx
chr3
Coverage dip
Detect artifacts

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Input data
n samples →
~348 million nt
11.24%
coverage
Rows with at least 1 sample with coverage > 5

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•  Null model
•  Alterna6ve Model
Finding DERs by expressed regions
• Find regions of the genome with expression data
• Compute coverage matrix
• Apply sta6s6cal tests
i: expressed region
j: sample

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Project size is increasing
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2009
(11)
2010
(46)
2011
(121)
2012
(235)
2013
(408)
2014
(625)
2015
(548)
2016
(18)
7
8
9
10
11
12
Project size in base−pairs over the years
log10 base−pairs per project
hFps://jhubiosta6s6cs.shinyapps.io/recount/

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• Data: 3 6ssues (liver, tes6s, heart), 8 samples each
• Align with
• Iden6fy expressed regions with derﬁnder
– Adjust coverage (40 million)
– Find expressed regions (cutoﬀ 5)
– Discard ERs < 9 bp
GTEx: DERs via expressed regions

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Presence of intronic ERs
Can strictly intronic ERs diﬀeren6ate 6ssues?

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PCs differentiate tissues

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Differential intronic ERs | exonic ERs

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Differential intronic ERs | exonic ERs
0 8 32 64 128
Liver Heart Testis
Coverage
RBX1, 395 bp from tss: inside
−
+
Genes
40951000 40951500 40952000 40952500
+
Tx
chr22

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Simulation setup
3 replicates:
2 groups, each with 5 samples
~2 million paired-end reads for chr17
1/6 high, 1/6 low in group 2 vs group 1
Annota6on:
complete
missing 20% of transcripts (8.28% exons)
Reference set:
3868 exons that overlap only 1 transcript

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Simulation results
•  Similar power to methods that have complete
annota6on
•  Methods with incorrect annota6on lose a lot of power
•  Higher empirical FDR/FPR

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Identifying brain development DERs
Fetal Infant
Child Teen
Adult 50+
6 / group, N = 36
Discovery data
Null:
Alt:
Models
Initial results
Jaffe et al, Nat. Neuroscience, 2015
50,650 DERs replicated
63,135 DERs
Final results

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Age-associated DERs lack regional specificity
in the human brain
BrainSpan data

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Propor6on of Cells
Expression changes across development may
represent a changing neuronal phenotype
Estimation method: Houseman et al, BMC Bioinformatics, 2012

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Collado-Torres et al, F1000Research, 2015
regionReport
chr start end strand p-value
chr1 1000 2000 + 0.9
chr2 5000 8000 - 0.001
chr3 2468 2668 + 0.051
. . . . .
. . . . .
. . . . .
chrX 6000 6300 + 0.009
chrX 6500 6800 - 0.5
Genomic workflow:
identify regions
renderReport
(A) default (B) custom
(C) derfinderReport
(D) DESeq2Report (E) edgeReport
Create HTML/PDF
report

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regionReport

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Interactive HTML reports
(A)
(B) Clickable buttons:
show/hide code

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Interactive HTML reports
(A)
(B)

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Chromatin immunoprecipitation seq.
http://assets.illumina.com/content/dam/illumina-marketing/images/techniques/large/web-graphic-chipseq-workflow-large.jpg

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Common ChIP-seq analysis pipeline
Sample 1
Sample 2
...
Sample N
1
call peaks
call peaks
call peaks
U
11
U
12
U
1N
Number of
unique peaks
Merge unique
peaks
Total peaks:
U
merged
≤ U
1
+ U
2
Determine differentially
bound peaks
between groups 1 & 2
Total tests: U
merged
1
Total: U
1
Sample 1
Sample 2
...
Sample N
2
call peaks
call peaks
call peaks
U
21
U
22
U
2N
Number of
unique peaks
2
Total: U
2
Group 1
Group 2
(A)
(A)
(B)
(C)

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Common ChIP-seq analysis pipeline
10400 10600 10800 11000 11200 11400
0 8 16 32 64 128 256
(−1,0] (0,1] (1,10] (10,20] (20,30] (30,100]
Coverage
MROH1 , 84229 bp from tss: inside
−
+
Genes
145287800 145288000 145288200 145288400 145288600 145288800
−
+
Tx
chr8
0 8 16 32 64 128 256
(−1,0] (0,1] (1,10] (10,20] (20,30] (30,100]
Coverage
MROH1 , 84229 bp from tss: inside
−
+
Genes
145287800 145288000 145288200 145288400 145288600 145288800
−
+
Tx
chr8
Merged peak
Non-overlapping
peaks
(A) (B)
Shuhla et al, PLoS Gene6cs 2013

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derfinder applied to ChIP-seq data
• Find regions of the genome with binding data
• Smooth binding signal
• Compute coverage matrix
• Apply sta6s6cal tests

View Slide

View Slide

Brain ChIP-seq data
EpiMap project

View Slide

Regions with binding signal
DBRs overlap with Ensembl v75 features
exon intergenic
intron
0
6170
5963
0
711
8269
923
8720
DBRs overlap with Ensembl v75 features
exon intergenic
intron
0
155897
57577
0
8426
36591
2833
7125
H3K4me3 H3K27ac

View Slide

Variation mostly explained
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0 20 40 60 80 100
Percentage variance explained
Brain
region
C
ell type
Age
at death
H
em
isphere
PM
I
pH
Sex
H
eight
BM
I
C
hrom
atin
am
ount
M
apped
reads
Individual
Flow
cell batch
Library
batch
R
esidual variation
All Regions
H3K4me3 data: all regions with binding signal

View Slide

Differentially bound regions
0 16 64 128 256 512
A:N−:− A:N+:− A:N−:+ A:N+:+ D:N−:− D:N+:− D:N−:+ D:N+:+
Coverage
PRB2, 0 bp from tss: overlaps 5'
−
+
Genes
11652500 11653000 11653500 11654000 11654500
−
Tx
chr12

View Slide

Differentially bound regions
H3K4me3 DBRs by main covariates
Brain region Cell type
Age at death
0
1
14604
209
0
0
48
0
H3K27ac DBRs by main covariates
Brain region Cell type
Age at death
0
2
178869
1235
0
0
156
0
H3K4me3 H3K27ac

View Slide

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0 20 40 60 80 100
Percentage variance explained
Brain
region
C
ell type
Age
at death
H
em
isphere
PM
I
pH
Sex
H
eight
BM
I
C
hrom
atin
am
ount
M
apped
reads
Individual
Flow
cell batch
Library
batch
R
esidual variation
All Regions
Variation in differentially bound regions
H3K4me3 data: only diﬀeren6ally bound regions
Differential binding by brain region, cell type or age at death

View Slide

•  Resource with data from 2,040 projects
•  Aligned with
•  Total RNA-seq samples:
49,657 + 9,662 = 59,319

View Slide

recount: via the web

View Slide

View Slide

Mo6va6ng problem: iden6fy and validate
regions of the genome that change expression
during brain development
1. derfinder permits discovery of novel expressed
& differen6ally bound regions
2.  we iden6fied & validated gene expression
changes in the developing brain
3.  we have developed tools for reproducible
shareable repor6ng
4.  these tools can be easily be used to process
2,040 projects via recount

View Slide

References + software + code
•  Collado-Torres, et al. bioRxiv (2016) doi:10.1101/015370
–  http://bioconductor.org/packages/derfinder
–  http://leekgroup.github.io/derSupplement/
•  Collado-Torres, et al. F1000Research (2015) doi:10.12688/f1000research.6379.1
-  http://www.bioconductor.org/packages/regionReport
-  http://leekgroup.github.io/regionReportSupp/
•  Collado-Torres and Nellore, et al. in prep
–  https://github.com/leekgroup/recount
–  https://jhubiostatistics.shinyapps.io/recount
•  Nellore, Collado-Torres, et al. bioRxiv (2015) doi:10.1101/019067
-  rail.bio
• Nellore, …, Collado-Torres, et al. bioRxiv (2016) doi:10.1101/038224
- intropolis.rail.bio
•  Jaffe, Shin, Collado-Torres, et al. Nat. Neurosci. (2015) doi:10.1038/nn.3898
–  https://github.com/lcolladotor/libd_n36
–  https://github.com/leekgroup/enrichedRanges

View Slide

Acknowledgements
Committee members
Jeffrey Leek
Daniele Fallin
Kasper Hansen
Alexis Battle
Andrew Jaffe
Hongkai Ji
Fernando Pineda
Collaborators
Alyssa Frazee
Abhinav Nellore
Michael Love
Ben Langmead
Rafael Irizarry
Funding
NIH
LIBD
JHU-Biostats
CONACyT México

View Slide

View Slide

Single-base F-statistics
•  Null model
•  Alterna6ve Model
•  F-sta6s6c
i: base-pair
j: sample

View Slide

Single-base F-statistics
BrainSpan data

View Slide

Compare DERs vs annotation
BrainSpan data

View Slide


View Slide

Widespread differential expression of novel
transcriptional activity

View Slide

DERs validate: Cytosolic vs total mRNA
fractions

View Slide

Age-associated DERs are conserved in the
developing mouse cortex
Mouse cerebral cortex, comparing E17 (N=4) to
adult (N=3) C57BL/6 mice Data from Dillman 2013

View Slide

CBC: 28
MD: 24
STR: 28
AMY: 31
HIP: 32
DFC: 34
Total N samples: 487
BrainSpan data
Coverage Data from BrainSpan:
hFp://download.allenins6tute.org/brainspan/MRF_BigWig_Gencode_v10/
VFC: 30 MFC: 32 OFC: 30 M1C: 25
S1C: 26 IPC: 33 A1C: 30 STC: 35 ITC: 33
V1C: 33

View Slide

defense2016

defense2016

Leonardo Collado-Torres

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