1  Introduction

Single-molecule footprinting is a technique used to study protein-DNA interactions, chromatin accessibility, and nucleosome positioning at the level of individual DNA molecules. Unlike bulk footprinting methods, which provide an averaged signal across a population of molecules, single-molecule footprinting captures heterogeneity in DNA-protein interactions and chromatin accessibility.

This book covers various tasks for the analysis of single-molecule footprinting data using footprintR:

describes how to read footprinting data from modBam files and other formats into R. It also introduces how the data is represented in memory.

shows how to create informative summaries to judge the quality of an experimental dataset, and how low-quality reads can be filtered out from loaded data or from modBam files.

illustrates the use of plotRegion to visualize read- or summary-level information for a single genomic region, optionally with additional data and annotation tracks.

describes various nucleosome-related analyses, such as how to measure the average distance between nucleosomes, how to place nucleosomes onto individual reads, or how to quantify nucleosome phasing in a regions.

introduces scanForHighScoringRegions, a modular framework that can be used to scan (parts of) the genome for regions of interest, for example differentially modified regions or regions containing phased nucleosomes. If you are looking for a “peak finder” for footprinting data, this is the chapter to read.

describes how to group reads, for example by allele or methylation status using regroupReads, or across multiple genomic regions using getAnchorRegions. The latter is useful to create meta-plots of your data, for example around transcript start sites or transcription factor binding sites.

illustrates the power of combining multiple data modalities (for example genetic variability, accessibility, endogenous CpG methylation and ChIP data) into an integrated analysis: The allele-specific binding of a transcription factor.