ATAC-Seq 101 for Biologists

July 10, 2024Learning Series
Caitlin Winkler, PhD
Caitlin Winkler, PhDSenior Solutions Scientist

ATAC-Seq 101 for Biologists

Are you hearing about ATAC-seq but not sure where to get started? Maybe you're already running ATAC-seq experiments but want to better understand the assay and downstream analysis steps. This post will help biologists and bioinformaticists understand the foundations for designing an ATAC-Seq experiment and analyzing your data to maximize novel insight gained.

Background: A work-horse technique

Since its development in 2013, Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) has provided a reliable method the study of chromatin accessibility. By providing a comprehensive view of the open chromatin regions in the genome, ATAC-seq experiments allowing researchers to identify regulatory elements and understand gene regulation mechanisms. This technique is faster and requires fewer cells compared to prior methods like DNase-seq and FAIRE-seq.

In a Nutshell - ATAC-Seq Method

  • Measures the accessibility of chromatin by detecting open regions in the genome
  • Provides insights into regulatory elements, transcription factor binding sites, and nucleosome positioning
  • Sequences are used to identify regions of active chromatin and infer regulatory networks
  • Enables a deeper understanding of gene regulation and cellular states

Applications of ATAC-seq

ATAC-Seq is widely used for various applications, including:

  • Chromatin Accessibility Profiling: Mapping open chromatin regions across the genome
  • Transcription Factor Footprinting: Identifying binding sites of transcription factors
  • Differential Accessibility Analysis: Comparing chromatin accessibility between different conditions or treatments
  • Epigenetic Studies: Understanding changes in chromatin structure during development and disease
  • Single cell ATAC-seq: Profiling chromatin accessibility at the single-cell level (requires a modified protocol)

What Do I Need for ATAC-Seq?

Reagents and Materials

  • Transposase (Tn5) Kit: For fragmenting and tagging open chromatin regions
  • Library Preparation Kit: For converting fragmented DNA into a sequencing-compatible library
  • Sequencing Platform: Illumina, PacBio, or Oxford Nanopore for sequencing the libraries
  • Computational Resources: For data analysis and storage.

Designing Your ATAC-Seq Experiment

A well-designed ATAC-Seq experiment includes:

Biological Replicates: Ensuring statistical power and reproducibility. Controls: Including untreated or baseline samples to compare against experimental conditions. Cell Quality: High-quality cells with minimal damage are crucial for accurate results. Library Complexity: Adequate sequencing depth to capture low-abundance accessible regions. Analyzing Your ATAC-Seq Data Pipeline to Generate Peaks After you've prepared your samples and sent them off for sequencing, you'll receive FASTQ files. These large, raw sequencing files need to be processed through a multi-step ATAC-Seq pipeline to ultimately generate peak calls.

Common steps in an ATAC-Seq pipeline (and some of the software used to perform them) include:

Quality Control (QC): FASTQC and MultiQC. Adapter Trimming: TrimGalore! Alignment to Reference Genome: Bowtie2 or BWA. Peak Calling: MACS2 or F-Seq. Normalization: To control for sequencing depth and technical variability. Differential Accessibility Analysis: DESeq2, edgeR, or DiffBind. Running the above tools yourself will require computational infrastructure and coding expertise. Expect the ATAC-Seq pipeline to run in about 1-3 hours per sample, depending on your compute resources and parallelization approach.

Want to get to insights faster? With Pluto, you can run an end-to-end ATAC-Seq pipeline in your browser, with no infrastructure or coding required. Learn more with a live, 15-minute demo.

Analyses and Visualization There are a wide variety of analyses and visualizations you can use to investigate your ATAC-Seq data. Here are a few ideas to get you started:

Differential Accessibility Analysis: Identify changes in chromatin accessibility between conditions. Motif Analysis: Identify enriched motifs within accessible regions. Volcano Plots: Visualize differential accessibility results. Heatmaps: Display accessibility levels of differentially accessible regions. Dimensionality Reduction (PCA, UMAP, t-SNE): Visualize how samples cluster based on chromatin accessibility. Create all of these plots (and more!) in Pluto. Contact us to get started running bioinformatics analyses to create the interactive plots shown in this blog, all from your browser.

ATAC-Seq Data Analysis Analyzing ATAC-Seq data in your browser, without the need to manage pipelines, can be a big win for your team. Allowing collaboration and agile discovery live in a meeting or presentation will transform how you iterate on your scientific research.

Ready to analyze some ATAC-Seq data? Thanks for reading this brief overview of ATAC-Seq experiments! To learn more about how your team can collaboratively analyze ATAC-Seq data in your browser with Pluto, visit our website or our ATAC-Seq page to get started today.

References & additional resources

  • Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature Methods, 10(12), 1213-1218 (2013).
  • Corces, M. R., Trevino, A. E., Hamilton, E. G., et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nature Methods, 14(10), 959-962 (2017).
  • Schep, A. N., Wu, B., Buenrostro, J. D., & Greenleaf, W. J. chromVAR: inferring transcription-factor-associated accessibility from single-cell epigenomic data. Nature Methods, 14(10), 975-978 (2017).