Dissecting human brain development at high resolution using RNA-seq

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

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Transcript of Dissecting human brain development at high resolution using RNA-seq

Page 1: Dissecting human brain development at high resolution using RNA-seq

Dissecting human brain development at high resolution using RNA-seq

Leonardo Collado-Torres @fellgernon #ENAR2015

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

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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)

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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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coverage vector 2 6 0 11 6

Genome (DNA)

Read coverage

Adapted from @jtleek

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Jaffe et al, Nat. Neuroscience, 2014

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derfinder: input data n  samples  →  

~348  million  nt  11.24%    

coverage  

Rows with at least 1 sample with coverage > 5

Adapted from @jtleek

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derfinder: statistical model (every base)

•  Null model

•  Alternative Model

•  F-statistic

i: base-pair j: sample

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derfinder: annotation-agnostic

Collado-Torres et al, bioRxiv, 2015 BrainSpan data

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DERs vs null regions by permutation

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Compare DERs vs annotation

Collado-Torres et al, bioRxiv, 2015 BrainSpan data

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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

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Jaffe et al, Nat. Neuroscience, 2014

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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

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Widespread differential expression of novel transcriptional activity

Jaffe et al, Nat. Neuroscience, 2014

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DERs validate: Cytosolic vs total mRNA fractions

Jaffe et al, Nat. Neuroscience, 2014

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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  

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Age-associated DERs lack regional specificity in the human brain

BrainSpan data Jaffe et al, Nat. Neuroscience, 2014

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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

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Prop

orRo

n  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

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Collado-Torres et al, in prep

regionReport

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LIBD Human DLPFC Development

•  UCSC “Track Hub”

Jaffe et al, Nat. Neuroscience, 2014

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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

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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

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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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