1107 system 1 account iewed the asian big cats doc
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tzerland from 7-11 July 2014. This report is specifically with regard to the Standing Committee's deliberations under Agenda item 38 concerning Asian big cats. The writer has read and reviewed the Asian big cats documents posted on the CITES website, under Agenda item 38, for the present meeting and also the Environmental Investigation Agency's document titled 'EIA Briefing Document on Asian big cats for the 65th Meeting of thTRANSCRIPT
EpigenomicsThe many garments of the genome sequence
Winterschool Brisbane, 2014 !
Dr Fabian Buske Garvan Institute of Medical Research
Sequencing has revolutionised life sciences
Epigenetics!ChIP-Seq,
WGBS, HiC,
DNaseHS, Repli-seq,
…
Transcriptomics!RNA-seq, CAGE-seq
Capture-seq, …
Genomics!WGS,
ExonCapture …
Epigenetics
the study of heritable changes that occur without a change in the DNA sequence
Epigenetics
http://www.youtube.com/watch?v=Tj_6DcUTRnM
Outline
• DNA methylation
- Whole Genome Bisulphite Sequencing
• Histone modification
- Chromatin Immunoprecipitation Sequencing
• DNA looping
- Chromosome Conformation Capture (HiC)
DNA methylation• Addition of a methyl group to the 5-carbon of
cytosine in DNA (5mC)
• In mammals, almost exclusively occurs at CpG dinucleotides in a strand symmetrical manner
- Strand symmetry allows for stable inheritance through cell divisions via DNMT1 maintenance
- ~28M CpG sites in the human genome
- Majority are methylated
- Except the 3.9M in/adjacent to CpG islands
Why study DNA methylation?
• Has demonstrated roles in!- Cellular programming
- dynamic during development/differentiation - Genomic imprinting/X-inactivation
!• 5mC presence is anti-correlated with “activity” of a DNA sequence!
- Promoters, gene bodies, distal regulatory elements, insulators - MBPs bind 5mC to repress the surrounding chromatin
!• Is stable and relatively easily assayable!
- Covalent modification of the DNA
DNA methylation & cancer• Aberrant promoter methylation in cancer is associated with tumour
suppressor gene silencing!- Occurs at enhancers/insulators as well !
!!!!!!!!!
• Alterations in other diseases are relatively poorly studied
How do we study DNA methylation?
• Bisulfite treatment deaminates unmethylated cytosines to uracil!!
- Uracil is converted to thymine via PCR!- 5mC is unaffected, therefore remains as
cytosine after PCR!!
‣ Methylation is then assayable as a SNP
Shear DNA
Methylated DNA
C GTCT
C GTUT
C GTTT
PCR
Whole genome bisulphite sequencing
Benefits!• Assays all mappable CpG sites (~27M)!• Get a “free” genome sequence at the same time!!Caveats!• Quantitation ability is proportional to depth of sequencing (count Cs vs Ts)!
- To detect a 10% change in 5mC at a single site, requires lots of coverage!- Pooling possible as adjacent CpG sites are correlated!
• Expensive, low throughput, µgs of DNA needed!• Analysis is not straightforward, few methods are available!!Library preparation is basically the same of WGS but with a bisulfite step and different polymerase (Uracil tolerant proofreader)
Data analysis of methylated regions
• Mapping against an in-‐silico bisulfite-‐treated genome (Bismark) • Discovery of ac>ve regulatory regions de novo (MethylSeekR -‐ HMM)
!!!!!
• Differen>ally Methylated Regions between pa>ent cohorts/treatments/condi>ons (bioconductor bsseq)
Histones
the nucleosome is composed of two copies of each of the four core histones (ie, H2A, H2B, H3, and H4), which are wrapped around by 146 bp of DNA
The N-terminal tails of histone polypeptides can be modified by more than 100 different post-translational modifications including methylation, acetylation, phosphorylation, and ubiquitination
Why study Histone modifications?
important epigenetic mechanism in transcriptional regulation through modification of the chromatin structure or through chromatin condensation
interplay between histone modifications and DNA methylation define developmental potential of a cell
chromatin profiling is especially well suited to the characterisation of non-coding portions of the genome in a tissue-specific manner
How do we study Histones?• Chromatin Immunoprecipitation
with subsequent sequencing (ChIP-Seq)!!
- crosslinking of proteins to DNA!- enrichment with specific antibody!- sequencing!!
‣ Analysis of histone mark deposition via read density
DNA-protein complex
DNA extraction
Sample fragmentation
Crosslink proteins and DNA
Immunoprecipitate
ChIP-Seq
Benefits!
• Captures genome-wide tissue-specific protein-DNA interactions !
• Relatively cheap compared to WGBS, HiC!
!Caveats!
• Highly dependent on an available antibody and its specificity !
• ~20-60M reads depending on the fraction of the genome anticipated to be
bound!
• Controls (input) need to be sequenced deeper that actual IP library!
!
ChIP-Seq data analysis
Mapping to the sequence space
Transcribed in cancer cells
Transcribed in normal cells
DNA looping
• The DNA fiber is a flexible polymer
• DNA looping enables genomic regions that are distant in sequence space to come in close physical proximity and thus relay signals (e.g. enhancers and promoters)
1D 3Dvs
Sequencing based 3D assaysCardinality
Resolution
3C 4C
5C
Chia-Pet
HiC
High (bp)
Low (mb)
One-to-one All-to-All
HiC
Chia-Pet
5Csweet spot
Capture-C
Capture-c
$
$$$
quadratic nature of “all versus all” data
3C/HiC protocol
• HiC: Before ligation, the restriction ends are filled in with biotin-labeled nucleotides.
DNACrosslink proteins and DNA Sample fragmentation Ligation PCR amplify ligated junctions
via restriction enzymes
HiC data processing
http://www.bioinformatics.babraham.ac.uk/projects/hicup/
Take Home Messages• Epigenetics: the study of heritable changes that
occur without a change in the DNA sequence
• Variety of assays available for the interrogation of the epigenetic state genome-wide
• Lots of public data available (ENCODE, Epigenome Roadmap, GEO)
• Understand the biological question and the wet-lab protocol… choose your tools accordingly!
• Check out Illumina’s poster http://bit.ly/1kxGdzz