Transcript Computational approaches to the analysis of mRNA

I519 Introduction to Bioinformatics, Fall, 2012
From ChIP-chip to ChIP-Seq: the
study of mammalian transcription
factor binding sites and epigenetics
From Chip-Chip to Chip-Seq
 ChIP-chip (ChIP on tiled microarrays)
 ChIP-sequencing (ChIP-seq) combines
chromatin immunoprecipitation (ChIP) and
massively parallel sequencing to identify
mammalian DNA sequences bound by
transcription factors in vivo.
Chromatin immunoprecipitation (ChIP)
between the side chains of two lysines
between lysine & cytosine
Formaldehyde (CH2O) is a very reactive dipolar
compound (the carbon atom is the nucleophilic
center). Amino and imino groups of proteins (e.g.,
the side chains of lysine and arginine) and of nucleic
acids (e.g., cytosine) react with formaldehyde,
leading to the formation of a Schiff base (reaction I)
Chip-Seq workflow
Solexa sequencing technology
provided short read length
sequences of approx 30 base
pairs that were ideal for
characterizing ChIP-derived
fragments.
Nature Methods - 4, 613 - 614 (2007)
Advantages of ChIP-Seq
 Single base-pair resolution of direct sequencing
 ChIP-seq data are likely to have less noise or
artifacts
 potential binding regions need not be specified
prior to experiment
 lower cost, minimal hands-on processing and a
requirement for fewer replicate experiments as
well as less input material.
 Epigenetics meets next-generation sequencing.
Epigenetics. 2008 Nov;3(6):318-21
Next generation sequencing (NGS)
techniques
454 Sequencing
Illumina/Solexa
ABI SOLiD
Sequencing
Chemistry
Pyrosequencing
Polymerase-based
sequence-bysynthesis
Ligation-based
sequencing
Amplification
approach
Emulsion PCR
Bridge amplification
Emulsion PCR
Paired end (PED)
separation
3 kb
200-500 bp
3 kb
Mb per run
100 Mb
1300 Mb
3000 Mb
Time per PED run
<0.5 day
4 days
5 days
Read length
(update)
250-400 bp
35, 75 and 100 bp
35 and 50 bp
Cost per run
$ 8,438 USD
$ 8,950 USD
$ 17,447 USD
Cost per Mb
$ 84.39 USD
$ 5.97 USD
$ 5.81 USD
Tools for extracting transcription factor
targets from ChIP-Seq data
 CisGenome uses a conditional binomial model to identify
enriched regions when a control data set is provided (Nat.
Biotechnol. 26:1293–1300, 2008)
 MACS (Model-based Analysis of ChIP-Seq) uses the control
dataset to model the tag distribution across the genome
using the Poisson distribution lBG (Genome Biol, 9:R137,
2009)
 PeakSeq enables systematic scoring of ChIP-seq
experiments relative to controls (Biotechnol, 27:66–75, 2009)
 QuEST (Quantitative Enrichment of Sequence Tags) Nat.
Methods, 5:829–834, 2008
 GLITR (GLobal Identifier of Target Regions) identifies
enriched regions in target data by calculating a fold-change
based on random samples of control (input chromatin) data
Why peak detection is difficult
PeakSeq: Biotechnol,
27:66–75, 2009
The signal for a given transcription
factor is the 'convolution' of various
effects: the density of mappable
bases in a region, the underlying
chromatin structure and the actual
signal from transcription factor
binding.
Some fraction of the peaks in the
ChIP-seq signal map for a
transcription factor might be due to
the nature of the open chromatin
structure instead of the presence of
transcription factor binding--one
must compare the signal against
one from a control.
PeakSeq scoring procedure
Biotechnol, 27:66–75, 2009
High-Resolution Profiling of Histone
methylations in the human genome
 Ref: Cell, 129(4):823-837, 2007
 Generated high-resolution maps for the genome-wide
distribution of 20 histone lysine and arginine methylations and
others across the human genome using the Solexa 1G
sequencing technology (The cells were digested with MNase to
generate mainly mononucleosomes with minor fraction of
dinucleosomes for histone modification mapping)
 Typical patterns of histone methylations exhibited at promoters,
insulators, enhancers, and transcribed regions are identified.
– The monomethylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 are
all linked to gene activation
– trimethylations of H3K27, H3K9, and H3K79 are linked to repression.
– H2A.Z (a Histone variant) associates with functional regulatory elements,
and CTCF marks boundaries of histone methylation domains.
– …
BS-seq for epigenetic profiling
 BS-seq (bisulphite sequencing) combines
bisulphite treatment of genomic DNA with ultrahigh-throughput sequencing
 Cytosine DNA methylation is important in
regulating gene expression and in silencing
transposons and other repetitive sequences
Bisulphite sequencing
References
 Genome-wide profiles of STAT1 DNA
association using chromatin immunoprecipitation
and massively parallel sequencing. Nature
Methods - 4, 651 - 657 (2007)