title: “Quantitative-ChIPseq-Workshop” author: “Rory Stark” date: “November 25, 2020” output: html_document —

Instructor

Rory Stark, Principal Scientist Computational Biology, University of Cambridge, Cancer Research UK Cambridge Institute

Workshop Description

This workshop will demonstrate the steps involved in performing a quantitative analysis of ChIP-seq data in Bioconductor (specifically differential binding analysis), with some discussion of related assays such as ATAC-seq. Particular attention will be paid to processing of aligned reads, including blacklisting, greylisting, filtering for quality and duplication, and the particular challenges presented when normalizing these data. While the workshop follows the DiffBind package vignette, the use of a number of other Bioconductor packages is discussed, including csaw, ChIPQC, edgeR, DESeq2, and GreyListChIP.

Pre-requisites

Attendees should be familiar with the following:

  • Basic knowledge of R syntax
  • Familiarity with core Bioconductor classes, such as GRanges.
  • Familiarity with ChIP-seq or a related assay, including aligning sequencing reads.
  • Familiarity with generalized linear models (GLMs), especially as realized in the edgeR and/or DESeq2 packages.

Helpful background reading:

  • Read through the DiffBind package vignette
  • Read through the csaw package vignette

There is a book chapter which offers a detailed outline of the design and analysis of ChIP-seq experiments:

  • Stark, Rory, and James Hadfield. “Characterization of DNA-protein interactions: design and analysis of ChIP-seq experiments.” Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing. Springer, Cham, 2016. 223-260.

Email the instructor if you would like a copy.

Workshop participation

Students will participate by following along an R markdown document, and asking questions throughout the workshop.

The conference organizers have arranged for attendees to have access to a RStudio session running inside a Docker instance that contains everything you need to follow the workshop.

NOTE: The workshop Docker instance does not have much compute power. As a result, it is not possible to run all the code in the time allowed. Two operations are affected: building greylists, and counting reads. In these cases, the workshop includes pre-computed data objects that can be loaded to avoid the time-consuming code. This is noted inline.

Workshop participation – local Docker image

If you do not have access to the RStudio instance provided by the conference organizers, or you would like to run the Docker image locally and you have a Docker client installed on your machine, you run this workshop locally via:

docker pull crukcibioinformatics/quantitative_chip_workshop:latest
docker images
docker run -e PASSWORD=DiffBind -p 8787:8787 <imageid> 

Then open a browser and go to the URL localhost:8787. Log into RStudio with username:rstudio and password:DiffBind.

Workshop participation – local R installation

The workshop uses a Docker container with Bioconductor Release version 3.12. If you would like to install Bioconductor on your computer, install R and the required Bioconductor packages, including this workshop package.

How to follow the workshop

First load the workshop package:

library(QuantitativeChIPseqWorkshop)

There are a number of ways to follow the workshop:

Follow the workshop in the pre-built HTML in RStudio

You can open the fully built workshop (with all code and results) in the “Help” pane in RStudio as follows:

vignette("Quantitative-ChIPseq-Workshop","QuantitativeChIPseqWorkshop")

Follow the workshop in the pre-built HTML in a separate Browser window or tab

If you are using the Docker image, You can open the fully built workshop (with all code and results) in a browser tab as follows:

rstudioapi::viewer(paste(system.file("doc",package="QuantitativeChIPseqWorkshop"),
                         "Quantitative-ChIPseq-Workshop.html", sep="/"))

It may be helpful to move the tab into its own window so you can see the vignette and the RStudio window at the same time.

Follow the workshop directly in the workshop mark-down file

You can open the workshop “source” .Rmd file in the RStudio “Source” pane, and follow along, executing each code chunk in sequence, to build it as you go.

file.edit("vignettes/Quantitative-ChIPseq-Workshop.Rmd")

R / Bioconductor packages used

  • DiffBind
  • csaw
  • ChIPQC
  • DESeq2
  • edgeR
  • GreyListChIP

Time outline

The workshop duration is 90 min. Approximate timing of activities:

Activity Time
Introduction 5m
QC of DNA enrichment assays 5m
Peaks vs. Windows 5m
DiffBind Sample sheet 5m
Blacklists and Greylists 10m
Counting aligned reads 10m
Examining and normalizing the binding matrix 10m
Modeling and Testing 5m
Examining analysis results 10m
Multi-factor Design 5m
Normalization discussion 15m
Conclusions 5m

Workshop goals and objectives

Participants will learn a complete set of steps for conducting a differential binding analysis of data from a ChIP-seq or related DNA enrichment experiment within Bioconductor.

Learning goals

  • Gain an understanding of the processing flow for performing quantitative analysis of data from ChIP-seq and related DNA enrichment assays
  • Be able to filter and count aligned reads to prepare them for analysis
  • Appreciate the challenges inherent in normalizing these data
  • Complete a differential binding analysis

Learning objectives

  • Load experimental data into DiffBind
  • Filter aligned reads using blacklists, greylists, and mapping quality
  • Derive a consensus peak set and compute a matrix of read counts
  • Normalize read counts appropriately
  • Establish a design to model the data and one or more contrasts to test hypotheses based on the model
  • Report, plot, and export results for further analysis