L. Collado-Torres' ENAR 2015 presentation about using `derfinder` to identify differentially expressed regions in the DLPFC across the lifespan of normal individuals. Described in Jaffe et al, Nature Neuroscience, 2014

1. Dissecting human brain development
at high resolution using RNA-seq
Leonardo Collado-Torres
@fellgernon
#ENAR2015

2. motivating problem: identify and validate
regions of the genome that change
expression during brain development

3. Fetal
Infant
Child
Teen
Adult
50+
6 / group, N = 36
Jaffe et al, Nat. Neuroscience, 2014
LIBD data
• Gender balanced
• Similar other covariates
like RNA Integrity Number
(RIN)

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

5. coverage
vector
2 6 0 11 6
Genome
(DNA)
Read coverage
Adapted from @jtleek

6. Jaffe et al, Nat. Neuroscience, 2014

7. derfinder: input data
n
samples
→
~348
million
nt
11.24%
coverage
Rows with at least 1 sample with coverage > 5
Adapted from @jtleek

8. derfinder: statistical model (every base)
• Null model
• Alternative Model
• F-statistic
i: base-pair
j: sample

9. derfinder: annotation-agnostic
Collado-Torres et al, bioRxiv, 2015
BrainSpan data

10. DERs vs null regions by permutation

11. Compare DERs vs annotation
Collado-Torres et al, bioRxiv, 2015
BrainSpan data

12. Identifying DERs
Fetal
Infant
Child
Teen
Adult
50+
6 / group, N = 36
Discovery data Null:
Alt:
Models
Cutoff
Details
• Rank DERs by area
• 1000 permutations
• Control FWER (≤
5%) by max area
per permutation
Results
63,135 DERs
20.509
Corresponds to p-value 10-08
Jaffe et al, Nat. Neuroscience, 2014

13. Jaffe et al, Nat. Neuroscience, 2014

14. Replicating DERs
Fetal
Infant
Child
Teen
Adult
50+
6 / group, N = 36
Replication data Null:
Alt:
Models
Cutoff
Details
Per sample and per DER calculate
average expression
Results
50,650 DERs replicated
Single F-statistic per DER
p-value < 0.05
Jaffe et al, Nat. Neuroscience, 2014

15. Widespread differential expression of novel
transcriptional activity
Jaffe et al, Nat. Neuroscience, 2014

16. DERs validate: Cytosolic vs total mRNA
fractions
Jaffe et al, Nat. Neuroscience, 2014

17. CBC:
28
MD:
24
STR:
28
AMY:
31
HIP:
32
DFC:
34
Total N samples: 487
BrainSpan data
Coverage
Data
from
BrainSpan:
hOp://download.alleninsRtute.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

18. Age-associated DERs lack regional specificity
in the human brain
BrainSpan data
Jaffe et al, Nat. Neuroscience, 2014

19. Age-associated DERs are conserved in the
developing mouse cortex
Jaffe et al, Nat. Neuroscience, 2014
Fetal:
E17
Adult:
C57BL/6
Data
from
Dillman
2013
n= 4 n = 3

20. ProporRon
of
Cells
Expression changes across development may
represent a changing neuronal phenotype
Jaffe et al, Nat. Neuroscience, 2014
Estimation method: Houseman et al, BMC Bioinformatics, 2012

21. Collado-Torres et al, in prep
regionReport

22. LIBD Human DLPFC Development
• UCSC “Track Hub”
Jaffe et al, Nat. Neuroscience, 2014

23. motivating problem: identify and validate
regions of the genome that change
expression during brain development
1. derfinder permits discovery of novel
expressed regions
2. we identified & validated gene
expression changes the developing brain
3. we have developed tools for
reproducible/shareable reporting

24. Acknowledgements
Leek Group
Jeffrey Leek
Alyssa Frazee
Hopkins
Sarven Sabunciyan
Ben Langmead
LIBD
Andrew Jaffe
Jooheon Shin
Nikolay Ivanov
Amy Deep
Ran Tao
Yankai Jia
Thomas Hyde
Joel Kleinman
Daniel Weinberger
Harvard
Rafael Irizarry
Michael Love
Funding
NIH
LIBD
CONACyT México

25. References + software + code
• Collado-Torres L, et al. derfinder: Software for annotation-agnostic RNA-seq
differential expression analysis. bioRxiv 015370 (2015). doi:10.1101/015370
– http://bioconductor.org/packages/release/bioc/html/derfinder.html
– http://lcolladotor.github.io/derSoftware/
• Jaffe AE, Shin J, Collado-Torres L, Leek JT, et al. Developmental regulation of
human cortex transcription and its clinical relevance at single base resolution.
Nat. Neurosci. (2014) doi:10.1038/nn.3898.
– https://github.com/lcolladotor/libd_n36
– https://github.com/lcolladotor/enrichedRanges
• http://www.bioconductor.org/packages/release/bioc/html/regionReport.html
• http://lcolladotor.github.io/regionReportSupp/