sysem 3 2801 google d for solid color areas in a displayed or r
2007 sysem 4 google vking and the use of innovative techno
-
Upload
nga.nguyen -
Category
Education
-
view
10 -
download
0
description
Transcript of 2007 sysem 4 google vking and the use of innovative techno
![Page 1: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/1.jpg)
EpigenomicsThe many garments of the genome sequence
Winterschool Brisbane, 2014 !
Dr Fabian Buske Garvan Institute of Medical Research
![Page 2: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/2.jpg)
Sequencing has revolutionised life sciences
Epigenetics!ChIP-Seq,
WGBS, HiC,
DNaseHS, Repli-seq,
…
Transcriptomics!RNA-seq, CAGE-seq
Capture-seq, …
Genomics!WGS,
ExonCapture …
![Page 3: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/3.jpg)
Epigenetics
the study of heritable changes that occur without a change in the DNA sequence
![Page 4: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/4.jpg)
Epigenetics
http://www.youtube.com/watch?v=Tj_6DcUTRnM
![Page 5: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/5.jpg)
Outline
• DNA methylation
- Whole Genome Bisulphite Sequencing
• Histone modification
- Chromatin Immunoprecipitation Sequencing
• DNA looping
- Chromosome Conformation Capture (HiC)
![Page 6: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/6.jpg)
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
![Page 7: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/7.jpg)
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
![Page 8: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/8.jpg)
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
![Page 9: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/9.jpg)
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
![Page 10: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/10.jpg)
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)
![Page 11: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/11.jpg)
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)
![Page 12: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/12.jpg)
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
![Page 13: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/13.jpg)
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
![Page 14: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/14.jpg)
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
![Page 15: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/15.jpg)
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!
!
![Page 16: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/16.jpg)
ChIP-Seq data analysis
![Page 17: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/17.jpg)
Mapping to the sequence space
Transcribed in cancer cells
Transcribed in normal cells
![Page 18: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/18.jpg)
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)
![Page 19: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/19.jpg)
1D 3Dvs
![Page 20: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/20.jpg)
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
![Page 21: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/21.jpg)
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
![Page 22: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/22.jpg)
HiC data processing
http://www.bioinformatics.babraham.ac.uk/projects/hicup/
![Page 23: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/23.jpg)
![Page 24: 2007 sysem 4 google vking and the use of innovative techno](https://reader034.fdocuments.net/reader034/viewer/2022042520/5f1506ba8d9bab1f446037e7/html5/thumbnails/24.jpg)
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