Introductory Analysis and Validation of CUT&RUN Sequencing Data

Summary

This protocol guides bioinformatics beginners through an introductory CUT&RUN analysis pipeline that enables users to complete an initial analysis and validation of CUT&RUN sequencing data. Completing the analysis steps described here, combined with downstream peak annotation, will allow users to draw mechanistic insights into chromatin regulation.

Abstract

The CUT&RUN technique facilitates detection of protein-DNA interactions across the genome. Typical applications of CUT&RUN include profiling changes in histone tail modifications or mapping transcription factor chromatin occupancy. Widespread adoption of CUT&RUN is driven, in part, by technical advantages over conventional ChIP-seq that include lower cell input requirements, lower sequencing depth requirements, and increased sensitivity with reduced background signal due to a lack of cross-linking agents that otherwise mask antibody epitopes. Widespread adoption of CUT&RUN has also been achieved through the generous sharing of reagents by the Henikoff lab and the development of commercial kits to accelerate adoption for beginners. As technical adoption of CUT&RUN increases, CUT&RUN sequencing analysis and validation become critical bottlenecks that must be surmounted to enable complete adoption by predominantly wet lab teams. CUT&RUN analysis typically begins with quality control checks on raw sequencing reads to assess sequencing depth, read quality, and potential biases. Reads are then aligned to a reference genome sequence assembly, and several bioinformatics tools are subsequently employed to annotate genomic regions of protein enrichment, confirm data interpretability, and draw biological conclusions. Although multiple in silico analysis pipelines have been developed to support CUT&RUN data analysis, their complex multi-module structure and usage of multiple programming languages render the platforms difficult for bioinformatics beginners who may lack familiarity with multiple programming languages but wish to understand the CUT&RUN analysis procedure and customize their analysis pipelines. Here, we provide a single-language step-by-step CUT&RUN analysis pipeline protocol designed for users with any level of bioinformatics experience. This protocol includes completing critical quality checks to validate that the sequencing data is suitable for biological interpretation. We expect that following the introductory protocol provided in this article combined with downstream peak annotation will allow users to draw biological insights from their own CUT&RUN datasets.

Introduction

The ability to measure interactions between proteins and genomic DNA is fundamental to understanding the biology of chromatin regulation. Effective assays that measure chromatin occupancy for a given protein provide at least two key pieces of information: i) genomic localization and ii) protein abundance at a given genomic region. Tracking the recruitment and localization changes of a protein of interest in chromatin can reveal direct target loci of the protein and reveal mechanistic roles of that protein in chromatin-based biological processes such as regulation of transcription, DNA repair, or DNA replication. The techniques available today to profile protein-DNA in....

Protocol

NOTE: Information for CUT&RUN fastq files in GSE126612 are available in Table 1. Information related to the software applications used in this study are listed in the Table of Materials.

1. Downloading Easy-Shells_CUTnRUN pipeline from its Github page

1. Open terminal from operating system.
   NOTE: If user is not sure how to open terminal in macOS and Windows, review this webpage (https://discovery.cs.illinois.edu/guides/Syst.......

Discussion

The ability to map protein occupancy on chromatin is fundamental to conducting mechanistic studies in the field of chromatin biology. As laboratories adopt new wet lab techniques to profile chromatin, the ability to analyze sequencing data from those wet lab experiments becomes a common bottleneck for wet lab scientists. Therefore, we describe an introductory step-by-step protocol to enable bioinformatics beginners to overcome the analysis bottleneck, and initiate analysis and quality control checks of their own CUT&.......

Materials

Name	Company	Catalog Number	Comments
bedGraphToBigWig	ENCODE	https://hgdownload.soe.ucsc.edu/admin/exe/	Software to compress and convert readcounts bedGraph to bigWig
bedtools-2.31.1	The Quinlan Lab @ the U. of Utah	https://bedtools.readthedocs.io/en/latest/index.html	Software to process bam/bed/bedGraph files
bowtie2 2.5.4	Johns Hopkins University	https://bowtie-bio.sourceforge.net/bowtie2/index.shtml	Software to build bowtie index and perform alignment
CollectInsertSizeMetrics (Picard)	Broad institute	https://github.com/broadinstitute/picard	Software to perform insert size distribution analysis
Cutadapt	NBIS	https://cutadapt.readthedocs.io/en/stable/index.html	Software to perform adapter trimming
Deeptoolsv3.5.1	Max Planck Institute	https://deeptools.readthedocs.io/en/develop/index.html	Software to perform Pearson coefficient correlation analysis, Principal component analysis, and Heatmap/average plot analysis
FastQC Version 0.12.0	Babraham Bioinformatics	https://github.com/s-andrews/FastQC	Software to check quality of fastq file
Intervenev0.6.1	Computational Biology & Gene regulation - Mathelier group	https://intervene.readthedocs.io/en/latest/index.html	Software to perform venn diagram analysis using peak files
MACSv2.2.9.1	Chan Zuckerberg initiative	https://github.com/macs3-project/MACS/tree/macs_v2	Software to call peaks
MACSv3.0.2	Chan Zuckerberg initiative	https://github.com/macs3-project/MACS/tree/master	Software to call peaks
Samtools-1.21	Wellcome Sanger Institute	https://github.com/samtools/samtools	Software to process sam/bam files
SEACRv1.3	Howard Hughes Medial institute	https://github.com/FredHutch/SEACR	Software to call peaks
SRA Toolkit Release 3.1.1	NCBI	https://github.com/ncbi/sra-tools	Software to download SRR from GEO
Trim_Galore v0.6.10	Babraham Bioinformatics	https://github.com/FelixKrueger/TrimGalore	Software to perform quality and atapter trimming