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11/4/15 1 Jonas Ungerback Benjamin Wilkins Yoonha Choi Mandy Wong Adriana Contreras 2.11.15 Computacional and ComparaDve Genomics Compara’ve analysis of different RNA-Seq pipelines • Experimental design (Biology) • EvaluaDon of RNA-seq results by different approaches • StaDsDcs and PCA • Relevance: differenDal Expression, GO • Conclusions Overview

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Page 1: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

11/4/15

1

JonasUngerbackBenjaminWilkinsYoonhaChoiMandyWong

AdrianaContreras

2.11.15

ComputacionalandComparaDveGenomics

Compara'veanalysisofdifferentRNA-Seqpipelines

•  Experimentaldesign(Biology)•  EvaluaDonofRNA-seqresultsbydifferentapproaches

•  StaDsDcsandPCA•  Relevance:differenDalExpression,GO•  Conclusions

Overview

Page 2: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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2

Phase 1: Precommitment

Rapid proliferation Multipotent

Phase 2: T-identity Slowed proliferation TCR rearrangement

THYMUS

Flt3 Kit, CD44 IL2Ra (CD25)

IL7Ra

DP

CD3e pre-TCR

Notch Notch

b-selection Commitment

Phase 3: Post b-selection Rapid proliferation

Loss of Notch dependence

CD8 CD4

DN4 DN1/ETP

Pre-TCR signal

b,d,g-TCR rearrangements a-TCR rearrangements

Quick overview of early T-cell development

CD45

FSC

c-Kit

CD25

CD44

CD25

CD11b

CD25

GFP

FSC

EV GFP PU1-WT-HA PU1-ENG-HA PU1-ETS-HA

%Live

%CD45+

%GFP+

%GFP+

%GFP+

Quick overview of early T-cell development

Page 3: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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3

Experimental layout -- file generation:

1. Isolate hematopoietic progenitors from fetal liver (FLP) or use a DN3-like cell line (P2C2). 2. Expand on OP9DL1 feeder cells (FLPs only).

3. Enriched for CD25+ cells and infect with HA-tagged version of PU.1-WT, PU.1-ENG (DNA-binding domain linked to an obligate repressor) and PU.1-ETS (DNA-binding domain only).

OP9DL1 culture

5-7d

2d

OP9DL1 culture

4. Expand on OP9DL1 feeder cells.

5. Fluorescence activated cell sorting (FACS).

6. Chromatin Immunoprecipitation /ChIP) and RNA-isolation.

7. ChIP -or RNA-sequencing.

8. Data analysis

Page 4: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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4

Samples QC

10Samples4(oldrun)5Experimental

5Controls

6(newrun)

Comparison between different pipelines

Page 5: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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5

Comparison between different pipelines

TopHatCufflinksTrimmed

TopHatcufflinksnontrimmed

TopHatHT-seqnontrimmed

TopHatHT-seqtrimmed

Page 6: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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6

BowDeExpressnontrimmed BowDeExpressTrimmed

PCA

Page 7: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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7

PCA

GO

0" 2" 4" 6" 8" 10" 12" 14" 16" 18" 20"

ReDnolandDrugmetabolism

MusclecontracDon

HCM

cardiomyopathy

Metabolismcytoch

Arachidonicacidm

ViralmyocardiDs

Steroidhor

Linoleicacid

CoagulaDon

PPARsign

Androgensm

Page 8: Compara’ve analysis of different RNA-Seq pipelines · RNA-seq presentation.pptx Author: wrplab Created Date: 11/4/2015 2:38:20 PM

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8

Conclusions

Thank you!

BillLisaAaronAllTAsAlllecturers

….andofcourseallthecourseparDcipantforafunDme!!!


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