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BIOINFORMATICS LAB
Episode VIII – ChIP-Seq
Analysis
Andrew N. Holding, PhDFederico M. Giorgi, PhD
Chiara Cabrelle, TA
Department of Pharmacy and Biotechnology
First Cycle Degree in Genomics
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• Ferrè and/or Giorgi
• 27 June, 10:00 AULA 1 SC FARMACEUTICHE Via Belmeloro 6
• 18 July, 10:00 AULA B Via Irnerio 42
• Giorgi Exam
– One tool picked from the list (1+ slides presentation)
– Oral Questions about the Lab topics
• Ferrè Exam
– Ask Ferrè
Exam Sessions - Bioinformatics
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Andrew Holdinghttp://andrewholding.com/
Proteomics Genomics Cancer Game Theory
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Science in Cambridge!
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Science in Cambridge!Downing College
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Science in Cambridge!Gryffindor House
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Science in Cambridge!
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Science in Cambridge! Holding's Office (Former Giorgi's Office)
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UPDATE R
BA
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TRANSCRIPTION FACTORS
BA
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TRANSCRIPTION FACTORS
BA
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CHIP-SEQ
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CHIP-SEQ
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ESTROGEN RECEPTOR & BREAST CANCER
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ESTROGEN RECEPTOR & BREAST CANCER
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INSTALL DIFFBIND
if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("DiffBind")
library(DiffBind)
First we must install DiffBind.
You may previously have come across EdgeR and DESeq2 as methods of analysing RNA-seq
data.
The methods we are going to apply to the analysis of ChIP-seq data are based on these two
packages. Hopefully many of the ideas we use will there for be familiar.
Load Rstudio and run the following commands.
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LOAD THE SAMPLE SHEET
DiffBind requires a sample sheet.
- A sample sheet provides the meta data on each of the samples.
- Metadata includes the tissue type, if the cells were resistant or not, and replicate numbers
- The sample sheet also provides a list of where the files that include where the reads and the peak
files (generated by MACS2) are stored .
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LOAD THE SAMPLE SHEET
DiffBind requires a sample sheet.
Helpfully there is example data included in the DiffBind package from a real experiment.
The following code lets us see what files are included in the DiffBind package.
pathDiffBind<-system.file("extra", package="DiffBind")
list.files(pathDiffBind)
Output:
The file we want is tamoxifen.csv
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LOAD THE SAMPLE SHEET
We can load the Sample Sheet using read.csv(). This function imports the CSV file into R.
pathDiffBind<-system.file("extra", package="DiffBind")
sampleSheet<-read.csv(paste0(pathDiffBind,"/","tamoxifen.csv"))
View(sampleSheet)
Then view with:
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TABLES
pathDiffBind<-system.file("extra", package="DiffBind")
sampleSheet<-read.csv(paste0(pathDiffBind,"/","tamoxifen.csv"))
View(sampleSheet[sampleSheet$Tissue=='MCF7',])
We can adapt this code if we only want to look at the samples labelled MCF7 in the Tissue column.
Exercise: How would we view only the resistant samples?
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CREATING THE DIFFBIND OBJECT
Now we know we can load the sample sheet, we can create the DiffBind Object.
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CREATING THE DIFFBIND OBJECT
setwd(system.file("extra", package="DiffBind"))
pathDiffBind<-system.file("extra", package="DiffBind")
dbaObject<-dba(sampleSheet=paste0(pathDiffBind,"/","tamoxifen.csv"))
dbaObject
Now we know we can load the sample sheet, we can create the DiffBind Object.
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CLUSTERING THE SAMPLES
setwd(system.file("extra", package="DiffBind"))
pathDiffBind<-system.file("extra", package="DiffBind")
dbaObject<-dba(sampleSheet=paste0(pathDiffBind,"/","tamoxifen.csv"))
plot(dbaObject)
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CLUSTERING THE SAMPLES
dba.plotVenn(dbaObject,mask=dbaObject$masks$T47D)
## If you need to reload data
data(tamoxifen_peaks)
dbaObject<-tamoxifen
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CLUSTERING THE SAMPLES
dba.plotVenn(dbaObject,mask=dbaObject$masks$T47D)
## If you need to reload data
data(tamoxifen_peaks)
dbaObject<-tamoxifen
Exercise: How would we view the peak overlap of the resistant samples?
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PEAK OVERLAP
overlap<-dba.overlap(dbaObject,mode=DBA_OLAP_RATE)
plot(overlap)
## If you need to reload data
data(tamoxifen_peaks)
dbaObject<-tamoxifen
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READ COUNTS
dba.count(dbaObject, minOverlap=3)
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READ COUNTS
dba.count(dbaObject, minOverlap=3)
data(tamoxifen_counts)
dbaObject<-tamoxifen
plot(dbaObject)
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READ COUNTS
data(tamoxifen_counts)
dbaObject<-tamoxifen
plot(dbaObject)
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PRINCPAL COMPONENT ANALYSIS
dba.plotPCA(dbaObject)
Exercise: How would we view the 2nd and 3rd principal component,
and colour by condition?
data(tamoxifen_counts) #If you need to reload data
dbaObject<-tamoxifen
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PRINCPAL COMPONENT ANALYSIS
dba.plotPCA(dbaObject,components = 2:3,attributes=DBA_CONDITION)
data(tamoxifen_counts) #If you need to reload data
dbaObject<-tamoxifen
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DIFFBIND ANALYSIS
dbaObject<-dba.contrast(dbaObject, categories = DBA_CONDITION)
dbaObject<-dba.analyze(dbaObject)
dba.plotMA(dbaObject)
data(tamoxifen_counts) #If you need to reload data
dbaObject<-tamoxifen
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Alternative AnalysisExercise: Up to this point we have focused on the difference in ER binding between
Tamoxifen resistant and sensitive cells.
What if we want to look at the difference between two different types of breast cancer?
MCF7 cells have a heterozygous loss of Progesterone receptor, while T47D have a gain. How
does this change ER binding in a patient?
Start from count data.
data(tamoxifen_counts)
dbaObject<-tamoxifen
count contrast analyze plot
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MCF7 vs T47D
Load count data
dba.contrast
dba.analyze
dba.plotMA
dbaObject
minMembers=2
categories=DBA_TISSUE
dbaObject
dbaObject
contrast=?
data(tamoxifen_counts)
dbaObject<-tamoxifen
Suggested Parameters
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Alternative Analysis
#Load count data
data(tamoxifen_counts)
dbaObject<-tamoxifen
#Establish contrast
dbaObject_tissue<-dba.contrast(dbaObject, categories = DBA_TISSUE, minMembers = 2)
#Analyse
dbaObject_tissue<-dba.analyze(dbaObject_tissue)
#Gives BT474 vs MCF7, we need to choose correct contrast
dba.plotMA(dbaObject_tissue)
#list contrasts and let them find interesting ones.
dba.contrast(dbaObject_tissue,categories = DBA_TISSUE,minMembers = 2)
#plot correct contrast
dba.plotMA(dbaObject_tissue,contrast = 4)
Answer
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Alternative to MA plot
dbaObject<-dba.contrast(dbaObject, categories = DBA_CONDITION)
dbaObject<-dba.analyze(dbaObject)
dba.plotMA(dbaObject)
data(tamoxifen_analysis) #If you need to reload data
dbaObject<-tamoxifen
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Alternative to MA plot
dbaObject<-dba.contrast(dbaObject, categories = DBA_CONDITION)
dbaObject<-dba.analyze(dbaObject)
dba.plotVolcano
data(tamoxifen_analysis) #If you need to reload data
dbaObject<-tamoxifen
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Alternative to MA plot
dbaObject<-dba.contrast(dbaObject, categories = DBA_CONDITION)
dbaObject<-dba.analyze(dbaObject)
dba.plotVolcano
data(tamoxifen_analysis) #If you need to reload data
dbaObject<-tamoxifen
There is something
missing here! Can
you guess what?
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REPORT
View(as.data.frame(dba.report(dbaObject,contrast = 1)))
data(tamoxifen_analysis) #If you need to reload data
dbaObject<-tamoxifen
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REPORTif (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
if (!requireNamespace("ChIPpeakAnno", quietly = TRUE))
BiocManager::install("ChIPpeakAnno")
library(ChIPpeakAnno)
### Reload data
data(tamoxifen_analysis)
dbaObject<-tamoxifen
### Export report
dfReport<-dba.report(dbaObject,contrast = 1)
### Annotate
data(TSS.human.GRCh37)
dfReport <- annotatePeakInBatch(dfReport, AnnotationData=TSS.human.GRCh37)
View(as.data.frame(dfReport))
You should now have a feature column with Ensembl IDs with for each peak
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REPORTif (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
if (!requireNamespace("org.Hs.eg.db", quietly = TRUE))
BiocManager::install("org.Hs.eg.db")
if (!requireNamespace("ChIPpeakAnno", quietly = TRUE))
BiocManager::install("ChIPpeakAnno")
library(ChIPpeakAnno)
### Reload data
data(tamoxifen_analysis)
dbaObject<-tamoxifen
### Export report and annotate
dfReport<-dba.report(dbaObject,contrast = 1)
data(TSS.human.GRCh37)
dfReport <- annotatePeakInBatch(dfReport, AnnotationData=TSS.human.GRCh37)
### Add gene symbols
library(org.Hs.eg.db)
dfReport<- addGeneIDs(annotatedPeak=dfReport,
orgAnn="org.Hs.eg.db",
IDs2Add="symbol")
View(as.data.frame(dfReport))
You should now have a symbol column with the gene symbol
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Exercise!Visualize your peaks on the Human Genome
# Generate a BED file
output<-as.data.frame(dba.report(dbaObject,contrast = 1))
bed<-output[,1:3]
outfile<-paste0(file.path(Sys.getenv("USERPROFILE"),"Desktop"),"/example.bed")
write.table(bed,quote=FALSE,row=FALSE,col=FALSE,file=outfile,sep="\t")
Open the BED file: it contains ChIP-Seq peak coordinates
-> Go to UCSC Genome Browser
Click on Genomes, Human, hg19
My Data, Custom Tracks, Choos File, Select your BED file, submit
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Exercise!
Click on GO!
See where the peaks are (ER binding sites)
You already know which genes it binds (on chromosome 18)
Check those genes on the Genome Browser!
View(as.data.frame(dfReport))
Last column is what you need (symbol)
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Exercise!
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Further Readings
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Further Readings