Day 5 Session 29: Questions and follow-up…. James C. Fleet, PhD Distinguished Professor Department of Nutrition Science Pete Pascuzzi, PhD Assistant Professor Purdue Libraries

Day 5 Session 30: DNA-seq Applications James C. Fleet, PhD Distinguished Professor Department of Nutrition Science Pete Pascuzzi, PhD Assistant Professor Purdue Libraries

An Overview of Chromatin Immuno-Precipitation (ChIP)

A Generalized Scheme for Evaluating Chromatin Biology by NGS Meyer and Liu et al., 2014, Nat Rev Genet 15:709 (focuses on bias in these approaches)

ChIP-seq Analysis Workflow Well Designed Study Data Filters Interpret Experiment Normalized Vetted Data Network building High Quality DNA Statistical Analysis Additional QC analysis Library Preparation Pathway and Geneset Analysis Differentially Expressed Peak/Gene List Sequencing and QC analysis Process Data Mapping Peak Calling Motif Discovery Raw Reads

Study Design: ChIP-seq 37oC No IFNg Ascorbate b glycerol PO4 IDG-SW3 cells n = 2/group Permissive Culture (33oC, IFNg) 3 35 GEO ID = GSE54782 +/- 100 nM 1,25(OH)2 D 3 h prior Histone ChIP-seq: 3 and 35 days (n = 1) H3K4me1, me2, me3 H3K9ac H3K9me3 H3K36me3 H4K5ac H4K20me1 TF ChIP-seq: VDR, RXR (n = 1) St. John et al., 2014, Mol Endocrinol 28:1150 Fleet 2016

Common Modifications in ENCODE What Do the Histone Marks Predict? Common Modifications in ENCODE Histone Mark Function H3K4Me3 Active promoter H3K9ac Promoters + enhancers H3K27ac Active enhancer H3K4me1 Active + Poised enhancers Lawrence et al. 2016, Trends in Genetics 32:42

Day 5 Session 31: Processing, Visualization, QC of DNA-seq Data James C. Fleet, PhD Distinguished Professor Department of Nutrition Science Pete Pascuzzi, PhD Assistant Professor Purdue Libraries

Tools for Critical Steps in ChIP-seq Sequencing Mapping TF: 20 x 106 reads Pol2 or histone marks: 100 x 106 reads Bowtie or MAC Peak Calling Differential Binding Analysis MACS2, PeakRanger DBChIP Peak Annotation BEDtools, ChIPPeakAnno Motif Discovery Cistrome, Lasagna 2.0 Bailey et al., 2013, PLOS Comput Biol 9: e1003326

Converting Bam to Bed or bigWig Files Samtools bedtools http://www.htslib.org/ Galaxy http://bedtools.readthedocs.io/en/latest/# https://usegalaxy.org/

This is the GitHub repository for Bedtools

Friday BREAK #1

Day 5 Session 32: Special Lecture Machine Learning Jennifer Neville, PhD Associate Professor Departments of Computer Science and Statistics Purdue University

Day 5 Session 33-34: Integrating Multiple Types of NGS Data James C. Fleet, PhD Distinguished Professor Department of Nutrition Science Pete Pascuzzi, PhD Assistant Professor Purdue Libraries

IGV to Visualize Multi-protein Regulation of Genes bigwig files used ENCODE MCF-7 Data from Human Genome (hg19)

New Methods to Study Higher Order Structure Sajan and Hawkins (2012) Ann Rev Genomics Human Genet 13:59

ChiaPET Data is Starting to Appear in ENCODE ENCODE Data from Human Genome (hg19)

Searching for Transcription Factor Binding Sites VDR ChIP-seq in RWPE1 cells Vehicle Vit D ~500 bp Published ChIP-seq From the UCSC Browser

Searching for Transcription Factor Binding Sites in DNA OMIC TOOLS https://omictools.com/motif-scanning-category VDR element PWM Proximal 500 bp in hCYP24A1 Gene Gene Regulation.com 52,790,626 52,790,586

Friday BREAK #2

Day 5 Session 35: Week 2 Preview Wanqing Liu, PhD Assistant Professor Department of Medicinal Chemistry and Molecular Pharmacology Min Zhang, PhD Associate Professor Department of Statistics