Resources for the DNA block
Foundational work
These studies below laid the methodological groundwork for high throughput chromatin characterization by DNA sequencing. Some of the authors are here at CU Anschutz!
Data sets from these studies would be excellent choices as starting points for your final projects.
DNase-seq
Hesselberth JR, Chen X, Zhang Z, Sabo PJ, Sandstrom R, Reynolds AP, Thurman RE, Neph S, Kuehn MS, Noble WS, Fields S, Stamatoyannopoulos JA. Global mapping of protein-DNA interactions in vivo by digital genomic footprinting. Nat Methods. 2009 19305407; PMCID: PMC2668528. [Link]
ATAC-seq
Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013 PMID: 24097267; PMCID: PMC3959825. [Link]
MNase-seq
Ramachandran S, Ahmad K, Henikoff S. Transcription and Remodeling Produce Asymmetrically Unwrapped Nucleosomal Intermediates. Mol Cell. 2017 PMID: 29225036; PMCID: PMC6421108. [Link]
CUT&RUN / CUT&TAG
Skene PJ, Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife. 2017 PMID: 28079019; PMCID: PMC5310842. [Link]
Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019 PMID: 31036827; PMCID: PMC6488672. [Link]
Software we’ll use in class
Read over the GViz vignette to understand how we’ll use it to vissualize genome-scale data on a reference sequence.
Read over the valr vignette to understand how we’ll do BEDtools-like (see below) analysis within RStudio.
Look over the ComplexHeatmap and EnrichedHeatmap documentation, especially XXX. These tools will help us make “meta-plots”: figures that plot genomic signals relative to features.
Other important tools
These are other tools I’ll mention in class. We’re not going to use them directly, but they are important tools in upstream data processing and analysis.
Alignment software
Bowtie2 and BWA are popular choices for short read alignment. They are fast, free, and well-maintained.
Peak calling
MACS is the gold-standard in peak calling. It models read coverage as a Poisson process, straightforward identification of regions of higher than expected coverage (i.e., peaks) to be identified using a single parmaeter (lambda) that captures the mean and variance of read coverage.
Interval analysis
- BEDtools is the “Swiss Army knife” of genome interval analysis. It provides a host of command-line tools that can be linked together for powerful genome signal manipulation.
Experimental rigor in chromatin analysis
These studies identified pervasive artifacts in genomewide chromatin analysis and provide recommendations and solutions to the issues.
Teytelman L, Thurtle DM, Rine J, van Oudenaarden A. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18602-7. doi: 10.1073/pnas.1316064110. Epub 2013 Oct 30. PMID: 24173036; PMCID: PMC3831989.
Shah RN, Grzybowski AT, Cornett EM, Johnstone AL, Dickson BM, Boone BA, Cheek MA, Cowles MW, Maryanski D, Meiners MJ, Tiedemann RL, Vaughan RM, Arora N, Sun ZW, Rothbart SB, Keogh MC, Ruthenburg AJ. Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies. Mol Cell. 2018 Oct 4;72(1):162-177.e7. doi: 10.1016/j.molcel.2018.08.015. Epub 2018 Sep 20. PMID: 30244833; PMCID: PMC6173622.