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Dissecting human brain development
at high resolution using RNA-seq
Leonardo Collado-Torres
@fellgernon
#ENAR2015
motivating problem: identify and validate
regions of the genome that change
expression during brain development
Fetal	
   Infant	
  
Child	
   Teen	
  
Adult	
   50+	
  
6 / group, N = 36
Jaffe et al, Nat. Neuroscience, 2014
LIBD data...
RNA-seq
reads
Genome
(DNA)
RNA transcripts
(many possible
variants)
Measuring gene expression: RNA-seq
Adapted from @jtleek
coverage
vector
2 6 0 11 6
Genome
(DNA)
Read coverage
Adapted from @jtleek
Jaffe et al, Nat. Neuroscience, 2014
derfinder: input data
n	
  samples	
  →	
  
~348	
  million	
  nt	
  
11.24%	
  	
  
coverage	
  
Rows with at least 1 sam...
derfinder: statistical model (every base)
•  Null model
•  Alternative Model
•  F-statistic
i: base-pair
j: sample
derfinder: annotation-agnostic
Collado-Torres et al, bioRxiv, 2015
BrainSpan data
DERs vs null regions by permutation
Compare DERs vs annotation
Collado-Torres et al, bioRxiv, 2015
BrainSpan data
Identifying DERs
Fetal	
   Infant	
  
Child	
   Teen	
  
Adult	
   50+	
  
6 / group, N = 36
Discovery data Null:
Alt:
Mod...
Jaffe et al, Nat. Neuroscience, 2014
Replicating DERs
Fetal	
   Infant	
  
Child	
   Teen	
  
Adult	
   50+	
  
6 / group, N = 36
Replication data Null:
Alt:
M...
Widespread differential expression of novel
transcriptional activity
Jaffe et al, Nat. Neuroscience, 2014
DERs validate: Cytosolic vs total mRNA
fractions
Jaffe et al, Nat. Neuroscience, 2014
CBC:	
  28	
  
MD:	
  24	
  STR:	
  28	
  AMY:	
  31	
  HIP:	
  32	
  
DFC:	
  34	
  
Total N samples: 487
BrainSpan data
...
Age-associated DERs lack regional specificity
in the human brain
BrainSpan data
Jaffe et al, Nat. Neuroscience, 2014
Age-associated DERs are conserved in the
developing mouse cortex
Jaffe et al, Nat. Neuroscience, 2014
Fetal:	
  E17	
  	
 ...
ProporRon	
  of	
  Cells	
  
Expression changes across development may
represent a changing neuronal phenotype
Jaffe et al...
Collado-Torres et al, in prep
regionReport
LIBD Human DLPFC Development
•  UCSC “Track Hub”
Jaffe et al, Nat. Neuroscience, 2014
motivating problem: identify and validate
regions of the genome that change
expression during brain development
1. derfind...
Acknowledgements
Leek Group
Jeffrey Leek
Alyssa Frazee
Hopkins
Sarven Sabunciyan
Ben Langmead
LIBD
Andrew Jaffe
Jooheon Sh...
References + software + code
•  Collado-Torres L, et al. derfinder: Software for annotation-agnostic RNA-seq
differential ...
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Dissecting human brain development at high resolution using RNA-seq

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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

Published in: Science
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Dissecting human brain development at high resolution using RNA-seq

  1. 1. Dissecting human brain development at high resolution using RNA-seq Leonardo Collado-Torres @fellgernon #ENAR2015
  2. 2. motivating problem: identify and validate regions of the genome that change expression during brain development
  3. 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. 4. RNA-seq reads Genome (DNA) RNA transcripts (many possible variants) Measuring gene expression: RNA-seq Adapted from @jtleek
  5. 5. coverage vector 2 6 0 11 6 Genome (DNA) Read coverage Adapted from @jtleek
  6. 6. Jaffe et al, Nat. Neuroscience, 2014
  7. 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. 8. derfinder: statistical model (every base) •  Null model •  Alternative Model •  F-statistic i: base-pair j: sample
  9. 9. derfinder: annotation-agnostic Collado-Torres et al, bioRxiv, 2015 BrainSpan data
  10. 10. DERs vs null regions by permutation
  11. 11. Compare DERs vs annotation Collado-Torres et al, bioRxiv, 2015 BrainSpan data
  12. 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. 13. Jaffe et al, Nat. Neuroscience, 2014
  14. 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. 15. Widespread differential expression of novel transcriptional activity Jaffe et al, Nat. Neuroscience, 2014
  16. 16. DERs validate: Cytosolic vs total mRNA fractions Jaffe et al, Nat. Neuroscience, 2014
  17. 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. 18. Age-associated DERs lack regional specificity in the human brain BrainSpan data Jaffe et al, Nat. Neuroscience, 2014
  19. 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. 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. 21. Collado-Torres et al, in prep regionReport
  22. 22. LIBD Human DLPFC Development •  UCSC “Track Hub” Jaffe et al, Nat. Neuroscience, 2014
  23. 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. 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. 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/

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