ChIP-seq Data Analysis
Dr. G. Ramesh Kumar, PhD.,
AU-KBC Research Centre,
MIT, Anna University,
Chromepet,Chennai-44.
Methods for studying epigenetic
modifications
•DNA methylation– Bisulfite Sequencing (BS)
TTCGCCGACTAA TTCGCCGAuTAA
•Histone modification
• Chromatin ImmunoPrecipitation (ChIP)
© 2013 American Society of Plant Biologists
HAT & HDAC
• Control over the coiling and uncoiling of DNA around histone is
necessary to carry out gene expression and study the
functionality of DNA sequence.
• This can be carried out by a process in which Histone Acetyl
Transferases (HAT) acetylates the lysine residues thus producing
active chromatin.
• As another process HDAC which deacetylates lysine residues in
producing silenced chromatin.
• The dual process of HAT and HDAC plays a vital role in modulation
of chromatin topology and regulation of gene transcription.
Chromatin immunoPrecipitation (ChIP)
• It is a type of immunoprecipitation experimental
technique used to investigate the interaction between
proteins and DNA in the cell.
• It aims to determine whether specific proteins are
associated with specific genomic regions, such as
transcription factors on promoters or other DNA
binding sites.
• ChIP also aims to determine the specific location in the
genome that various histone modifications are
associated with, indicating the target of the histone
modifiers.
ChIP-seq big picture
• Combine high-throughput sequencing with Chromatin
Immuno-precipitation to identify specific protein-DNA
interactions genome-wide, including those of:
– Transcription factors
– Histones (various types and modifications)
– RNA Polymerase (survey of transcription)
– DNA polymerase (investigate DNA replication)
– DNA repair enzymes
• … or fragments of DNA that are modified (e.g.
methylated)
ChIP-Chip vs ChIP-Seq
• Chromatin ImmunoPrecipitation +
microarray or high throughput sequencing
• Detect genome-wide in vivo location of TF
and other DNA-binding proteins
– Find all the DNA sequences bound by TF (TFBS)
• Can learn the regulatory mechanism of a
transcription factor or DNA-binding protein
much better and faster
ChIP-Chip vs ChIP-Seq
“ChIP-Seq is the best thing that happened to ChIP
tech.
It is 100x better than ChIP-Chip since it escapes most
of the problems of microarray probe hybridization.
Plus it is cheaper, and genome wide.
But ChIP-Seq is only the tip of the iceberg - there are
many inventive ways to use a sequencer.”
Quote from intro to Homer software at: [Link]
ChIP-seq technology
Development of novel high-throughput sequencing methods
- Revolutionized genomic studies
- Enabled large scale sequence analysis through
the generation of millions of short sequencing reads
Chip-seq technology
Instead of hybridizing the ChIP DNA to a microarray (ChIP-
chip), each sample is processed directly into a DNA library for
sequencing and subsequent bioinformatics analysis.
ChIP-Seq has improved sensitivity and reduced background
over ChIP-chip.
2-4 times more TFBS are determined, compared with ChiP-chip
Chromatin Immunoprecipitation
Modified histone
(ChiP)
(e.g. H3K4me)
Cross-link DNA to
histone
© 2013 American Society of Plant Biologists
Chromatin Immunoprecipitation
Modified histone
(ChiP)
(e.g. H3K4me)
Cross-link DNA to
histone
Shear DNA to smaller
pieces; add specific
antibody to modified
histone; affinity purify
antibody and bound
histone/DNA complex. `
© 2013 American Society of Plant Biologists
Chromatin Immunoprecipitation
Modified histone
(ChiP)
(e.g. H3K4me)
Cross-link DNA to
histone
Shear DNA to smaller
pieces; add specific
Remove crosslink, antibody to modified
purify DNA, examine by histone; affinity purify
PCR, sequencing or antibody and bound
microarray histone/DNA complex.
© 2013 American Society of Plant Biologists
ChIP experiment
In Nutshell
•Protein cross-linked to DNA in vivo
by treating cells with formaldehyde
•Shear chromatin (sonication)
•IP with specific antibody
•Reverse cross-links, purify DNA
•PCR amplification*
•Identify sequences
•Genome-wide association map
*-unless using a single molecule sequencer
History: From ChIP-chip to ChIP-seq
ChIP-chip (c.2000)
• Resolution (30-100bp)
• Coverage limited by
sequences on the array
• Cross-hybridization between
probes and non-specific targets
creates background noise
Types of Analysis
1. Binding site identification and discovery of
binding sequence motifs (Non-histone ChIP)
2. Epigenomic gene regulation and chromatin
structure (Histone ChIP)
ChIP-seq overview
DNA + bound protein Fragment DNA Immunoprecipitate
Sequence Prepare
Release DNA
Map sequence sequencing
tags to genome library
& identify
peaks
Adapted from slide set by: Stuart M. Brown, Ph.D.,
Center for Health Informatics & Bioinformatics, NYU School of Medicine
ChIP-seq Workflow
Confirm ChIP
Prepare library
Submit for
Sequencing
Get Raw sequence data & do QC
Map sequence reads to genome
Identify ChIP peaks over input background
Downstream bioinformatic analyses
Platforms
Chip-seq can be done using the
Illumina,
SOLiD,
Ion Torrent or
Ion Proton platforms.
General Steps
• Alignment
• Peak Detection
• Motif Detection
• GO
• Pathway Analysis
Peak Detection
• Detect enriched regions in ChIP sample as
compared to the control.
• Detect peaks of transcription factor regulatory
sites using PICS/MACS.
• Identify genes regulated by TF binding sites.
• Detect histone modification sites using the
enriched region detection method.
• (PICS)Model-based Analysis of ChIP-Seq
• (MACS) PICS: probabilistic inference for ChIP-
seq.
Peak Detection tools
• [Link]
[Link]
The ENCODE Project
Dozens of labs did ChIP-seq, under rigorous quality
guidelines, for over 100 transcription factors and histone
modifications, plus related assays for DNA methylation,
chromatin accessibility etc.
Major paper (many others provide additional details):
Encode Project Consortium (over 100 authors) An integrated encyclopedia of DNA
elements in the human genome. Nature. 2012 Sep 6;489(7414):57-74. doi:
10.1038/nature11247.
Some ways to access this data:
[Link]/encode (Nature’s summary & links to all related papers)
[Link] (a way to explore the data in a wiki format)
UCSC genome browser
sra (short read archive, repository for raw data
[Link]
ENCODE/dataMatrix/
[Link]
ml
PFMS-Peak-Finder MetaServer
• A metaServer application designed for analyzing
ChIP-seq data.
• [Link]
• It integrates several peak-finders within a single
interface.
• A ChIP-seq data file can be analyzed using one or
more of the peak-finders (up to 7 among MACS
v1.3.7, CisGenome v2.0, Findpeaks v3.1.9.2,
HPeak v1.1, E-range v.2.1, SeqSite v1.0 and
SISSRs v1.4) and also produces consensus peaks.
MACS
Model-based Analysis of ChIP-Seq (MACS) on short reads
sequencers such as Genome Analyzer (Illumina / Solexa).
MACS empirically models the length of the sequenced ChIP
fragments, which tends to be shorter than sonication or
library construction size estimates, and uses it to improve
the spatial resolution of predicted binding sites.
MACS also uses a dynamic Poisson distribution to
effectively capture local biases in the genome sequence,
allowing for more sensitive and robust prediction.
MACS compares favorably to existing ChIP-Seq peak-finding
algorithms, is publicly available open source, and can be
used for ChIP-Seq with or without control.
GeneProf
• A web-based, graphical software suite that
allows users to analyse data produced using
high-throughput sequencing platforms (RNA-
seq and ChIP-seq).
• [Link]
ChIP-seq Applications
• Protein-DNA interactions
• Chromatin States
• Transcriptional regulation
SUMMARY
• Chromatin immunoprecipitation (ChIP) followed by
sequencing is a powerful method to determine the locations
of DNA binding sites for a protein (transcription factor) of
interest.
• Determining protein-DNA interactions involved in gene
regulation is essential to fully understand biological processes
and diseases.
• For Chip-seq, protein-binding DNA regions that have been
enriched using antibodies or other affinity reagents are
sequenced.
• Applications of Chip-seq include studies of regulation of gene
expression and of chromatin states in epigenetic studies.
