Workflow Type: Galaxy

ATACseq Workflow

This workflow is highly concordant with the corresponding training material. You can have more information about ATAC-seq analysis in the slides and the tutorial.

Inputs dataset

  • The workflow needs a single input which is a list of dataset pairs of fastqsanger.

Inputs values

  • reference_genome: this field will be adapted to the genomes available for bowtie2 and the genomes available for bedtools slopbed (dbkeys table)
  • effective_genome_size: this is used by macs2 and may be entered manually (indications are provided for heavily used genomes)

Processing

  • The workflow will remove nextera adapters and low quality bases and filter out any read smaller than 15bp.
  • The filtered reads are mapped with bowtie2 allowing dovetail and fragment length up to 1kb.
  • The BAM is filtered to keep only MAPQ30, concordant pairs and pairs outside of the mitochondria.
  • The PCR duplicates are removed with Picard.
  • The BAM is converted to BED to enable macs2 to take both pairs into account.
  • The peaks are called with macs2 which at the same time generates a coverage file.
  • The coverage file is converted to bigwig
  • The amount of reads 500bp from summits and the total number of reads are computed if further normalization is wanted.
  • Other QC are performed:
    • A histogram with fragment length is computed.
    • The evaluation of percentage of reads to chrM or MT is computed.
  • A multiQC is run to have an overview of the QC.

Warning

  • The coverage output is not normalized.
  • The reference_genome parameter value is used to select references in bowtie2 and bedtools slopbed. Only references that are present in bowtie2 and bedtools slopbed are selectable. If your favorite reference genome is not available ask your administrator to make sure that each bowtie2 reference has a corresponding len file for use in bedtools slopbed.

Inputs

ID Name Description Type
PE fastq input PE fastq input Should be a paired collection with ATAC-seq fastqs
  • File[]
effective_genome_size effective_genome_size Used by macs2:\nH. sapiens: 2700000000, M. musculus: 1870000000, D. melanogaster: 120000000, C. elegans: 90000000
  • int
reference_genome reference_genome reference_genome
  • string

Steps

ID Name Description
3 Cutadapt (remove adapter + bad quality bases) toolshed.g2.bx.psu.edu/repos/lparsons/cutadapt/cutadapt/4.0+galaxy0
4 Bowtie2 map on reference toolshed.g2.bx.psu.edu/repos/devteam/bowtie2/bowtie2/2.4.5+galaxy1
5 filter MAPQ30 concordant pairs and not mitochondrial pairs toolshed.g2.bx.psu.edu/repos/devteam/bamtools_filter/bamFilter/2.5.1+galaxy0
6 Get number of reads per chromosome toolshed.g2.bx.psu.edu/repos/devteam/samtools_idxstats/samtools_idxstats/2.0.4
7 remove PCR duplicates toolshed.g2.bx.psu.edu/repos/devteam/picard/picard_MarkDuplicates/2.18.2.3
8 reads in chrM/MT for multiQC toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/1.1.2
9 convert BAM to BED to improve peak calling toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_bamtobed/2.30.0+galaxy2
10 Compute fragment length histogram toolshed.g2.bx.psu.edu/repos/iuc/pe_histogram/pe_histogram/1.0.1
11 number of reads toolshed.g2.bx.psu.edu/repos/iuc/samtools_view/samtools_view/1.15.1+galaxy0
12 Call Peak with MACS2 toolshed.g2.bx.psu.edu/repos/iuc/macs2/macs2_callpeak/2.2.7.1+galaxy0
13 get summits +/-500kb toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_slopbed/2.30.0+galaxy1
14 summary of MACS2 toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_grep_tool/1.1.1
15 Bigwig from MACS2 wig_to_bigWig
16 Merge summits +/-500kb toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_mergebed/2.30.0
17 Compute coverage on summits +/-500kb toolshed.g2.bx.psu.edu/repos/iuc/bedtools/bedtools_coveragebed/2.30.0
18 number of reads in peaks toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/1.1.2
19 Combine number of reads in peaks with total number of reads cat1
20 reads in peaks multiQC toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/1.1.2
21 MultiQC toolshed.g2.bx.psu.edu/repos/iuc/multiqc/multiqc/1.11+galaxy0

Outputs

ID Name Description Type
mapping stats mapping stats n/a
  • File
MarkDuplicates metrics MarkDuplicates metrics n/a
  • File
BAM filtered rmDup BAM filtered rmDup n/a
  • File
histogram of fragment length histogram of fragment length n/a
  • File
MACS2 narrowPeak MACS2 narrowPeak n/a
  • File
MACS2 report MACS2 report n/a
  • File
Coverage from MACS2 (bigwig) Coverage from MACS2 (bigwig) n/a
  • File
1kb around summits 1kb around summits n/a
  • File
Nb of reads in summits +-500bp Nb of reads in summits +-500bp n/a
  • File
MultiQC on input dataset(s): Stats MultiQC on input dataset(s): Stats n/a
  • File
MultiQC webpage MultiQC webpage n/a
  • File

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