ChIP-seq
13 likes12,068 views
ChIP-seq is a technique to identify where proteins bind to DNA in the genome. It involves cross-linking proteins to DNA in cells, fragmenting the DNA, immunoprecipitating the protein-DNA complexes using an antibody for the protein of interest, and then sequencing the retrieved DNA. This allows mapping of the genomic binding sites for the protein. The document discusses experimental design considerations for ChIP-seq, such as antibody choice and controls. It also reviews data analysis steps including read mapping, peak calling to identify enriched regions, and downstream analyses like motif finding. Higher resolution techniques like ChIP-exo are also introduced that can identify protein binding sites at base pair level.
ChIP-seq
- 2. Sebastian Schmeier 2 DNA sequencing RNA sequencing Biological sample Gene regulation, chromatin structure Genome variation Gene expression Genome assembly Transcriptome assembly, Splice variant detection Metabarcoding Common analyses overview
- 3. Sebastian Schmeier Gene regulation, chromatin structure • Overview • How do we analyse it? • ChIP • Experimental design considerations • Data analyses • Downstream analyses • Methods for higher base-pair resolution 3
- 4. Sebastian Schmeier RNA Pol II promoter 4http://www.nature.com/nrg/journal/v13/n4/full/nrg3163.html
- 5. Sebastian Schmeier Transcription factors 5 Homeodomain Zinc-finger
- 6. Sebastian Schmeier Transcription factors cooperate 6 Molecular Biology of the Cell, Bruce Alberts, 5th ed., Page 447 Directly Indirectly http://www.nature.com/nrg/journal/v13/n9/full/nrg3207.html
- 7. Sebastian Schmeier Location location location 7http://www.nature.com/nrg/journal/v13/n9/full/nrg3207.html
- 9. Sebastian Schmeier Histone modifications • Histone modifications specify if DNA is compacted 9https://en.wikipedia.org/wiki/Histone#/media/File:Histone_modifications.png
- 10. Sebastian Schmeier Where are the proteins on the DNA? 10 H3K4me3: Marks the location of promoters H3K36me3: Indicates the bodies of transcribed genes and non-coding RNA H3K4me1: Identifies functional enhancer elements outside of promoters http://www.nature.com/nrg/journal/v11/n7/full/nrg2795.html
- 11. Sebastian Schmeier Where are the proteins on the DNA? 11http://www.nature.com/nrg/journal/v11/n7/full/nrg2795.html
- 12. Sebastian Schmeier •A way to detect what DNA a certain protein is binding to •You cross-link the sample, and fragment the DNA into pieces •Immunoprecipitate using an antibody to your protein of interest •Reverse the cross-links, and isolate the DNA •PCR to see if your DNA is there •You can also sequence the DNA and map to a reference genome (ChIP-seq) or hybridise it to a microarray (ChIP-chip) 12http://www.nature.com/nature/journal/v461/n7261/full/nature08451.html Chromatin immunoprecipitation (ChIP)
- 13. Sebastian Schmeier chip vs. seq 13https://www.ebi.ac.uk/training/online/sites/ebi.ac.uk.training.online/files/user/1317/documents/chipseq_loos.pdf Chromatin immunoprecipitation (ChIP)
- 14. Sebastian Schmeier Lab procedures 14https://www.ebi.ac.uk/training/online/sites/ebi.ac.uk.training.online/files/user/1317/documents/chipseq_loos.pdf Chromatin immunoprecipitation (ChIP)
- 15. Sebastian Schmeier Experimental Design 15 • Antibody quality • A sensitive and specific antibody will give a high level of enrichment • Limited efficiency of antibody is the main reason for a failed ChIP-seq experiments • Check your antibody ahead if possible, e.g.Western blotting to check the reactivity of the antibody with unmodified and non-histone proteins. Chromatin immunoprecipitation (ChIP)
- 16. Sebastian Schmeier Experimental Design 16 • Control experiments • Open chromatin regions are fragmented more easily than closed regions. • Uneven distribution of sequence tags across the genome • Some fraction of the peaks in the ChIP-seq signal map for aTF might be due to the nature of the chromatin structure in regions of open chromatin • A ChIP-seq peak should be compared with the same region in a matched control Chromatin immunoprecipitation (ChIP) http://www.nature.com/nbt/journal/v27/n1/full/nbt.1518.html
- 17. Sebastian Schmeier Experimental Design 17 • Control type • Input DNA • Mock IP - DNA obtained from IP without antibody • Very little material can be pulled down leading to inconsistent results of multiple mock IPs. • Nonspecific IP - using an antibody against a protein that is not known to be involved in DNA binding • There is no consensus on which is the most appropriate • Sequencing a control can be avoided when looking at: • time points • differential binding pattern between conditions Chromatin immunoprecipitation (ChIP)
- 18. Sebastian Schmeier 18 • Sequencing depth • More prominent peaks are identified with fewer reads, whereas weaker peaks require greater depth • Number of putative target regions continues to increase significantly as a function of sequencing depth Chromatin immunoprecipitation (ChIP) http://www.nature.com/nbt/journal/v27/n1/full/nbt.1518.html Experimental Design
- 19. Sebastian Schmeier Experimental Design 19 • Multiplexing • Number of reads per run continue to increase • The ability to sequence multiple samples (e.g. DNA from several ChIP experiments) at the same time becomes important, especially for small genomes • Different barcode adaptors are ligated to different samples • After sequencing reads are separated according to barcodes • Paired-end sequencing • Reads are sequenced from both ends • Increase “mappability” - especially in repetitive regions • Costs more as single end reads • For ChIP-seq, usually not worth the extra cost, unless you have a specific interest in repeat regions Chromatin immunoprecipitation (ChIP)
- 20. Sebastian Schmeier Data analysis overview 20 Chromatin immunoprecipitation (ChIP) http://www.nature.com/nrg/journal/v11/n7/full/nrg2795.html
- 21. Sebastian Schmeier 21 • Very short reads • Use dynamic trimming to get rid of bad read ends • e.g. FastQC / SolexaQA++ Chromatin immunoprecipitation (ChIP) http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
- 22. Sebastian Schmeier Mapping reads 22 • Not all of the genome is ‘available’ for mapping • Align your reads to the unmasked genome • For ChIP-seq, usually short reads are used (36bp) • Limited gain in using longer reads Chromatin immunoprecipitation (ChIP) http://www.nature.com/nbt/journal/v27/n1/full/nbt.1518.html
- 23. Sebastian Schmeier Mapping challenges 23 • Enormous amount of reads to align • Done against large genome - needs pre-indexing structure and large memory • Has to be fast and memory efficient • Shorter read length • Mismatches • Repetitive regions • Multi-mapping reads Chromatin immunoprecipitation (ChIP)
- 24. Sebastian Schmeier Peak calling • Basic - regions are scored by the number of tags in a window of a given size. • Each region is assessed by enrichment over control and minimum tag density. • Advanced - take advantage of the directionality of the reads. Challenges • Adjust for sequence alignability - regions that contain repetitive elements have different expected tag count • Different ChIP-seq applications produce different type of peaks. Most current tools have been designed to detect sharp peaks (TF binding, histone modifications at regulatory elements) 24 Chromatin immunoprecipitation (ChIP) http://www.nature.com/nrg/journal/v10/n10/abs/nrg2641.html
- 25. Sebastian Schmeier Data analysis overview 25 Chromatin immunoprecipitation (ChIP) http://www.nature.com/nrg/journal/v11/n7/full/nrg2795.html
- 26. Sebastian Schmeier • Find some function based on the genes that are close to your peak regions 26 http://bejerano.stanford.edu/great/public/html/ GREAT
- 28. Sebastian Schmeier Find those motifs • MEME suite 28http://meme-suite.org/
- 29. Sebastian Schmeier Find those motifs • MEME suite • MEME-ChIP 29http://meme-suite.org/
- 30. Sebastian Schmeier Find those motifs • MEME suite • MEME-ChIP 30http://meme-suite.org/
- 31. Sebastian Schmeier Base-pair resolution digital epigenome profiling • The resolution of most epigenomic techniques, e.g. traditional ChIP-seq, is not high enough. • It has been limited by the methods used to prepare chromatin. • Traditional ChIP-seq gives you peak regions 31http://www.nature.com/nrg/journal/v10/n10/abs/nrg2641.html
- 32. Sebastian Schmeier Base-pair resolution digital epigenome profiling But, base-pair resolution is essential because… • single-base shifts in nucleosome positioning can alter chromatin structure • the precise DNA sequence bound by aTF is important to deduce binding site motifs • it allows the identification of single binding sites within a cluster of closely spaced sites 32
- 33. Sebastian Schmeier ChIP-exo • Crosslinked cells are sonicated to fragment and solubilise chromatin. • ChIP is carried out with an antibody directed against the protein of interest. • Immunoprecipitated DNA is digested with exonucleases to remove DNA that is not protected by the protein • Resected DNA is then purified and sequenced • Less straightforward than standard ChIP– seq 33http://www.nature.com/nrg/journal/v15/n12/full/nrg3798.html
- 34. Sebastian Schmeier High-resolution X-ChIP • Crosslinked cells are lysed and chromatin is digested with micrococcal nuclease (MNase). • Chromatin is then sonicated to improve solubility. • The use of both MNase and sonication results in near-complete solubilisation of some chromatin-bound proteins, making it especially useful for large complexes that resist solubilisation • An antibody directed against the protein of interest is then used to immunoprecipitate DNA, which is then purified and sequenced 34http://www.nature.com/nrg/journal/v15/n12/full/nrg3798.html
- 35. Sebastian Schmeier ORGANIC • In the occupied regions of genomes from affinity- purified naturally isolated chromatin (ORGANIC) method, soluble chromatin extracted from MNase treatment of nuclei is used as an input to ChIP • The input sample provides a genome-wide footprinting of factors • ChIP pulldown provides a factor-specific map in a single experiment using a simple library preparation protocol • Highly-specific and identifies more binding sites with consensus motifs than previous X-ChIP studies 35http://www.nature.com/nrg/journal/v15/n12/full/nrg3798.html
- 36. Sebastian Schmeier X-ChIP vs. native ChIP • Native ChIP is performed without crosslinking. • It is usually applied to nucleosomes • The assumption being that the wrapping of DNA around histones precludes rearrangement during chromatin preparation and immunoprecipitation • It is often assumed that native ChIP is unsuitable for profiling non-histone proteins owing to potential rearrangement. • Can be addressed by ORGANIC 36 http://www.nature.com/nrg/journal/v15/n12/full/nrg3798.html • Solubility of proteins of interest can also be an issue with native ChIP • As harsh detergents and sonication are not used, recovery might be lower than for X-ChIP, especially for large complexes, in which case high-resolution X-ChIP is preferred
- 37. References Lenhard B et al., Metazoan promoters: emerging characteristics and insights into transcriptional regulation, Nature Reviews Genetics 2012 Zentner GE, Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014 Hawkins RD1, Hon GC, Ren B. Next-generation genomics: an integrative approach. Nat Rev Genet. 2010 Rozowsky J1 et al. PeakSeq enables systematic scoring of ChIP-seq experiments relative to controls. Nat Biotechnol. 2009 Sebastian Schmeier s.schmeier@gmail.com http://sschmeier.github.io/bioinf-workshop/