Lecture 5 : ChIP-seq
Modern Genomic Technologies  (LF:DSMGT01 )
Vojta Bystry
vojtech.bystry@ceitec.muni.cz

NGS data analysis
2
2
Raw data
.fastq
Genome/Transcriptome Reference Mapping
.bam
Interaction
analysis
CHIP-seq
Expression analysis
RNAseq
Variant analysis
WES
de-multiplexing
Not known
reference
QC
QC
>
Experiment design
Not ”classic” reference
Metagenomics
Reference assembly
Immunogenetic
VDJ-genes
CRISPR
sgRNA
Methylation
Bisulfide-seq
…

ChIP-seq
ChIP Sequencing Service, Sequencing Analysis Services - Nucleome Informatics Private Limited,
Hyderabad | ID: 15911548030
●Very laborious and hard laboratory preparation
●Not a good success rate
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○

Primary analysis + QC
●Alignment – standard DNA (RNA for CLIP)
●QC standard
○Check sequencing quality
○RSeQC – Read Dstribution
○
Graphical user interface, application Description automatically generated

Primary analysis + QC
●IP experiment quality control
○Sample correlation
■Replicates control treatment
○Strand cross-correlation
■Shift of strand mapping
■Shift should correlate with expected fragment size
○
Figure 1 Treemap chart Description automatically generated with medium confidence

Primary analysis + QC
●IP experiment quality control
○Sample correlation
■Replicates control treatment
○Strand cross-correlation
■Shift of strand mapping
■Shift should correlate with expected fragment size
○
Figure 1 Cross-correlation analysis

Primary analysis + QC
●Fingerprint profile
○profile of cumulative read coverages
○how evenly are the reads distributed over the genome
●
○

Deduplication
●Remove all duplicates!
○It is very low probability that the fragment would be cut at exactly same position
○Usually experiments without UMIs

Peak calling
●Read extension


Peak calling
●Statistical assessment of peaks against background
●Background
○Control sample – recommended
○Model background from overall coverage of the sample
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●
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●Peak calling annotation
●Differential peak calling
Graphical user interface, application Description automatically generated

Post peak calling QC
●FRIP score = fraction of reads in peaks
○High number is good
○However can be low in specific experiments and still the experiment be OK
●Average peak profile
Chart, bar chart Description automatically generated Graphical user interface Description
automatically generated Chart, line chart Description automatically generated

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Peak calling results
Table Description automatically generated

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Peak calling results
Table Description automatically generated
●peaks.annotated.bed
●control.bigWig
●bigWig
Graphical user interface, application, Excel Description automatically generated

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www.ceitec.eu
CEITEC
@CEITEC_Brno
Vojta Bystry
vojtech.bystry@ceitec.muni.cz
Thank you for your attention!

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