Assay for Transposase‐Accessible Chromatin Using Sequencing (ATAC‐seq) Data Analysis

Kristy L.S. Miskimen1, E. Ricky Chan2, Jonathan L. Haines2

1 Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio, 2 Institute of Computational Biology, Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 20.4
DOI:  10.1002/cphg.32
Online Posting Date:  January, 2017
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Abstract

The study of epigenetic properties of the human genome, including structural modifications of DNA and chromatin, has increased tremendously as mounting evidence has demonstrated how much epigenetics affects human gene expression. Buenrostro et al. have developed a rapid method, requiring low numbers of living cells as input, for examining chromatin accessibility across the epigenome, known as the assay for transposase‐accessible chromatin using sequencing (ATAC‐seq). The overall goal of this unit is to provide a thorough ATAC‐seq data analysis plan, as well as describe how primary human blood samples can be processed for use in ATAC‐seq. In addition, a number of quality control parameters are discussed to ensure the integrity and confidence in the ATAC‐seq data. © 2017 by John Wiley & Sons, Inc.

Keywords: ATAC‐seq; chromatin accessibility; epigenetics; peak‐calling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: ATAC‐seq Library Preparation Using Primary Blood Samples
  • Basic Protocol 2: ATAC‐seq
  • Basic Protocol 3: ATAC‐seq Data Analysis Tools and Procedure
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: ATAC‐seq Library Preparation Using Primary Blood Samples

  Materials
  • Human blood obtained in EDTA tubes (BD, cat. no. 367862 or 367899) or cell preparation tubes (CPT) with sodium citrate (BD, cat. no. 362760 or 362761)
  • Erythrocyte lysis buffer (Buffer EL, Qiagen, cat. no. 79217), if using EDTA tubes
  • Phosphate‐buffered saline (PBS, HyClone, cat. no. SH30256FS)
  • Trypan blue, 0.4% solution in PBS (MP Biomedicals, cat. no. 091691049)
  • Benchtop centrifuge
  • Sterile 15‐ml conical tubes
  • Refrigerated swinging bucket centrifuge (with holder for 16 × 125–mm or 13 × 100–mm tubes)
  • Vortex
  • Aspirator
  • Hemacytometer
  • Inverted microscope

Basic Protocol 2: ATAC‐seq

  Materials
  • Nextera kit (Illumina, cat. no. FC‐121‐1030)
    • TD (2× reaction buffer)
    • TDE1 (Nextera Tn5 transposase)
  • Nuclease‐free H 2O (available from various suppliers)
  • Nuclei pellet (see protocol 1)
  • MinElute PCR Purification Kit (Qiagen)
  • 25 μM PCR primer 1 (custom‐synthesized by Integrated DNA Technologies, IDT); sequences provided in Buenrostro et al. ( )
  • 25 μM barcoded PCR primer 2 (custom‐synthesized by Integrated DNA Technologies, IDT); sequences provided in Buenrostro et al. ( )
  • NEBNext High‐Fidelity 2× PCR Master Mix (New England Biolabs, cat. no. M0541)
  • 25 μM custom Nextera PCR primer 1
  • 25 μM custom Nextera PCR primer 2
  • 100× SYBR Green I (Invitrogen, cat. no. S‐7563)
  • 5% TBE polyacrylamide gel (optional)
  • 100‐bp DNA ladder (New England Biolabs; optional)
  • Bioanalyzer High‐Sensitivity DNA Analysis kit (Agilent; optional)
  • 37°C water bath
  • 0.2‐ml PCR tubes
  • PCR thermal cycler
  • qPCR instrument (Applied Biosystems StepOnePlus Real‐Time PCR System, cat. no. 4376600)
  • Typhoon TRIO Variable Mode Imager (Amersham Biosciences; optional)
  • Bioanalyzer (Agilent; optional)

Basic Protocol 3: ATAC‐seq Data Analysis Tools and Procedure

  Materials
  • Access to a Linux/Unix environment and familiarity with the operating system is required for running the analysis pipeline. Most of the pipeline will be run on the command line. For the installation of the required packages, consult the developers’ sites for support and troubleshooting. The analysis will typically require ∼16 GB of RAM and access to multiple CPU cores with a minimum of four processors. Make sure the locations of the programs are listed in the path. Additionally, for visualization, a web browser with the latest update is often required.
The following packages (or comparable packages) are required:
  • FastQC (read QC): http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
  • Picard Tools (sequence file tools): http://broadinstitute.github.io/picard/
  • Samtools (alignment file tools): http://www.htslib.org/
  • Cutadapt (quality and adapter trimmer): https://pypi.python.org/pypi/cutadapt
  • Trim Galore! (Cutadapt and FastQC wrapper): http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
  • bowtie2 (short read aligner): http://bowtie‐bio.sourceforge.net/bowtie2/index.shtml
  • MACS2 (peak detection): https://pypi.python.org/pypi/MACS2
Files
  • User‐generated ATAC‐seq sequencing files in fastq format; if fastq files are not zipped, zip them (fastq.gz), as most data analysis packages accept the zipped format, which will require approximately one‐third the original data storage space
  • Human genome in FASTA format (http://www.gencodegenes.org/releases/current.html): the genome build that is chosen should be consistent throughout the experimental process—use of the latest reference build (currently GRCh38/hg38) is recommended
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Figures

Videos

Literature Cited

Literature Cited
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