<style> #title { height: 100% !important; display: flex !important; flex-direction: column !important; justify-content: center !important; } </style> <section id="title" data-background="/images/presentations/bg.svg.png" data-transition-speed="slow"> # Open chromatin and ATAC-seq Nathan Sheffield <div class="bullet"> <img src="/images/external/uva_dgs_logo.svg" height="85"> <img src="/images/logo/logo_databio_long.svg" height="65"> </div> <span style="font-size:0.6em"><a href="http://www.databio.org/slides">www.databio.org/slides</a></span> </section> --- <!-- .slide: data-background="/images/presentations/bg.svg.png" data-transition-speed="slow" --> ### Outline <style> .previewblock { float: left; width: 20px; height: 45px; margin: 0; border: none; white-space: nowrap; box-sizing: border-box; } .questionblock { float: left; width: 100%; margin: 5px 0; border: 1px solid rgba(255, 255, 255, .2); } </style> <div class="previewblock" style="width:20%">Regulatory DNA</div> <div class="previewblock" style="width:60%"></div> <div class="previewblock" style="width:20%">Basic analysis</div> <div class="previewblock" style="width:20%">|</div> <div class="previewblock" style="width:60%"></div> <div class="previewblock" style="width:20%">|</div> <br clear="all"> <div class="previewblock" style="width:20%; background:#883388">20%</div> <div class="previewblock" style="width:60%; background:#333388">60%</div> <div class="previewblock" style="width:20%; background:#338888">20%</div> <div class="previewblock" style="width:20%"></div> <div class="previewblock" style="width:60%">|</div> <div class="previewblock" style="width:20%"></div> <br clear="all"> <div class="previewblock" style="width:20%"></div> <div class="previewblock" style="width:60%">Chromatin accessibility protocols</div> <div class="previewblock" style="width:20%"></div> <div class="questionblock" style="background:#222; color:#eee; font-size: 0.6em; margin-top: 35px">◁ Questions ▷</div> --- ### What is regulatory DNA? <img src="/_modules/open-chromatin/genes1flowchart.svg" width="400" style="background:white"><br clear="all"> Regulatory DNA is a decision-maker --- ### Challenges to studying regulatory DNA - Variation: age, cell-type, environment, disease - Amount: 1-2% protein coding vs 8-20%? regulatory - Target: what gene does it affect? - Function: is it a promoter, silencer, insulator, enhancer? - Rigidity: genetic code vs TF motifs --- <div class="col2">Genetic code <img src="/_modules/open-chromatin/genetic-code.jpg" width="400"> </div> <div class="col2">Transcription factor motif <img src="/_modules/open-chromatin/motif.svg" width="400"><br clear="all"> </div> We can computationally identify genes and even predict function. Regulatory DNA is more difficult. --- ### Chromatin accessibility <div class="well"> Chromatin accessibility is the degree to which nuclear macromolecules are able to physically contact chromatinized DNA... </div> <div class="well fragment"> [It] is determined by the occupancy and topological organization of nucleosomes as well as other chromatin-binding factors that occlude access to DNA. </div> <a href="https://doi.org/10.1038/s41576-018-0089-8">Klemm et al. 2019</a> --- ### How can we identify regulatory DNA? <img src="/_modules/open-chromatin/Chromatin_Structures.png" style="background:white" width="1000"><br clear="all"> https://en.wikipedia.org/wiki/Chromatin --- ### How can we identify regulatory DNA? <img src="/_modules/open-chromatin/beads-on-a-string.jpg" width="600"><br clear="all"> Alberts 2002 --- ### How can we identify regulatory DNA? - [ChIP](https://en.wikipedia.org/wiki/ChIP_sequencing): Chromatin immunoprecipitation - [DNase](https://en.wikipedia.org/wiki/DNase-Seq): classic 'gold standard' to identify open chromatin - [ATAC](https://en.wikipedia.org/wiki/ATAC-seq): Assay for transposase-accessible chromatin - [FAIRE](https://en.wikipedia.org/wiki/FAIRE-Seq): Formaldehyde-assisted isolation of regulatory elements [Trends](https://trends.google.com/trends/explore?date=all&geo=US&q=%2Fm%2F013cd5r3,%2Fm%2F0glsck6,%2Fm%2F0glnrtn) --- ### ChIP-seq <img src="/_modules/open-chromatin/park2009_fig1.png" width="650"> --- ### DNase-seq: Biology <img src="/_modules/open-chromatin/dnase.svg" width="650"> --- <span class="bullet"><img src="/_modules/open-chromatin/sword.svg" width="75" class="bullet">ATAC-seq: Experiment (Buenrostro et al. 2013)</span> <br><img src="/_modules/open-chromatin/buenrostro2013_fig1.jpg" width="600"><br clear="all"> --- <span class="bullet">Transposase Tn5 protein (Reznikoff 2008)</span> <br><img src="/_modules/open-chromatin/reznikoff2008_fig4.jpg" width="475"><br clear="all"> --- <span class="bullet">Chromatin and transcription factors (Thurman et al. 2012)</span><br> <img src="/_modules/open-chromatin/thurman2012_fig2.jpg" width="495"><br clear="all"> --- ### Chromatin accessibility biology summary - Open chromatin usually coincides with active regulatory DNA - ... but exact annotation or binding is not provided - The advantage and disadvantage of ChIP seq is in its target. It also requires antibodies and provides more diffuse signal - ATAC is pronounced 'attack' {.fragment} --- ### Basic data analysis steps - 1. Trim adapters (fastq -> fastq) - 2. Align reads (fastq -> bam) - 3. Shift reads (bam -> bam) - 4. Call peaks (bam -> bed) --- <span class="bullet"><img src="/_modules/open-chromatin/sword.svg" width="75" class="bullet">ATAC-seq: 9 base duplication (Reznikoff 2008)</span> <br><img src="/_modules/open-chromatin/reznikoff2008_fig2.jpg" width="600"><br clear="all"> --- ### Tn5 molecular biology <img src="/_modules/open-chromatin/tn5_shift.svg" width="725" style="background:white"><br clear="all"> --- <style> #acknowledgements { height: 100% !important; display: flex !important; flex-direction: column !important; justify-content: center !important; } </style> <section id="acknowledgements" data-background="/images/presentations/bg.svg.png"> # Thank You <br clear="all"/> <span class="small bullet"><img src="/images/external/github_bug_black.svg" height="20" class="bullet"><a href="http://github.com/nsheff">nsheff</a></span> · <span class="small bullet"><img src="/images/icons/web.svg" height="25" class="bullet"><a href="http://databio.org">databio.org</a></span> · <span class="small bullet"><img src="/images/icons/letter.svg" height="25" class="bullet"><a href="mailto:nsheffield@virginia.edu">nsheffield@virginia.edu</a></span> <div class="bullet" style="background-color:rgb(45,45,45,.65); border-radius: 25px; opacity:0.9"> <img src="/images/external/uva_dgs_logo.svg" height="65"> <img src="/images/logo/logo_databio_long.svg" height="45"> </div> </section>