ASAP is a flexible bioinformatic pipeline for ATAC-seq data analysis. Starting from raw ATAC-seq sequencing reads, ASAP outputs raw and filtered mapping files, coverage files (reads coverage ; tn5 insertion events coverage), fragment length distribution, read exraction based on fragment length, and peak calling results.
- Mapping
- Post-mapping processing and filtering:
- Filter (or not) reads that fall into user-defined blacklisted regions
- Select reads that do not carry more than minMismatch and filter by minimum mapping quality (MAPQ)
- Mark duplicated pairs
- Select concordant, non-duplicated pairs.
- Shift reads by 4bp as described in Schep et al.,2015: shift by 4bp toward to center of the transposition event.
- Compute read coverage
- Compute insertion events coverage
- Fragment length distribution
- Extract reads pairs based on a fragment length range and compute arcs between fragment extremities (protection visualization)
- Peak calling
- ASAP is:
User-friendly: requires a single configuration file. Thus, only one option is required when running the command line (see Usage of ASAP)
Flexible: provides the possibility to skip a given step(s) and target specific post-processing step(s).
- Bowtie2 version >= 2.2.9
- MACS2
- Samtools version >=1.2
- GenomeCoverageBed version >= v2.20.1
A configuration file required to execute the pipeline.
bash ASAP.sh [-h] [-v] [-c]This is the only REQUIRED parameter for ASAP. The configuration file is a text file that gathers the full set of parameters required to execute the pipeline. (check the example ASAP_configFile_example.conf in distribution)
Print out the help/current version
The configuration file gathers the parameters of each step. Note that, when running the pipeline, only the "turned on" steps will performed. A step is turned on by a yes/no argument.
Here we list the different set of parameters to be filled in the configuration file:
General information option about the run. Must be always filled.
- OUTDIR
Main output directory where results are written. OUTDIR is created if does not exist
- sampleName
Name of the processed sample. No space is allowed: use _ or - to mimic space if needed
- CHRLEN
Chromosome info file (tab-delimited format: <Chr name><chr length>)
- path
Full path to the different dependencies, if not already added to $PATH
- map
Set to "yes/no". If mapping is skipped (map=no), a BAM file must be provided to proceed. (see post-mapping steps).
- FASTQ1
fastq file (R1). File can be gizpped
- FASTQ2
fastq file (R2). File can be gizpped
- bowtieIndex
Prefix of bowtie2 indexes
- mappingParameters
Bowtie2 mapping parameters. Default: --very-sensitive -X 2000 -p 10
It is possible to skip the mapping step (map=no) and perform any of the post-mapping steps. To do so, aligned reads must be provided in a BAM file. If* map=yes*, the "turned on" post-mapping steps will performed on the internal mapping results.
- BAM
aligment file in BAM format
- filter
Set to "yes/no". If map=yes, filtering will be performed on internal mapping results, if map=no, filtering will be performed on the provided alignment file in BAM option.
- maxMis
Maximum number of mismatches allowed per read.
- blacklist
Set to "yes/no" if reads should be filtred based on a list of blacklisted regions. If "blacklist=yes", blacklisted regions must be provided in the next parameter.
- blacklistedRegions
Regions used to filter reads.(tab-delimited format: <Chr name><start><end>)
- shift
Set to "yes"/"no". If shift=yes, reads are shifted by 4bp so that read starts reflect the center of the Tn5 transposition event
- readCoverage
Set to "yes/no" if read coverage should be computed or not
- ieventsCoverage
Set to "yes/no" if Tn5 insertion events coverage should be computed or not
- extractReads
Set to "yes/no" if read pairs should be extracted based on a given range of fragment length
- lowBoundary
Lower boundery of the range: [lowBoundary,upBoundary]. Default=100
- upBoundary
Upper boundery of the range: [lowBoundary,upBoundary]. Default=250
- arcs
set to "yes/no" if extracted fragments should represented as arcs (linked extremities)
- fragDist
Set to "yes/no" if fragment length distribution should be computed or not
- callpeak
Set to "yes/no" if peak calling should be computed or not.
- control
Control bam file. Note that peak calling can be performed without a control, however, one can provide a control such as ATAC-seq on genomic DNA. Leave option empty if no control is used.
- MODE
Peak calling mode: <broad/narrow>. Default=broad
- modelParameters
MACS2 shifting options
- fdr
Cutoff for peak detection. Default=0.01
- gsize
Effective genome size of tair10 (gsize=10e7)
ASAP outputs mapping files, coverage files, fragments distribution table/plot and MACS2 peak calling results.
- *.mapped.sorted.bam
Contains mapped reads (bowtie2 raw mapping results)
- *.(un)masked.(un)shifted.bam
Contains the selected set of reads after filtering. Ideally, accessible peaks are called using this file.
- *.csv
Summary of filtering step is CSV format
- *.(un)masked.(un)shifted.ievent.bam
Contains Tn5 insertion events. Basically, instead of showing reads, only the position corresponding to Tn5 insertion event are shown)
- *.(un)masked.(un)shifted.bw
Genome-wide coverage of ATAC reads
- *.(un)masked.(un)shifted.ievent.bw
Genome-wide coverage of Tn5 insertion events
- *.subReads.f3.frag*.bam
Contains the set of extracted reads based on the given rage of fragment length
- *.subReads.f3.frag*.bw
Genome-wide coverage of the set of extracted reads based on the given rage of fragment length
- *.subReads.f3.frag*.arcs.bed
arcs between fragment extremities. This file is visualized on IGV
- *.TLEN.f3F16.txt
Counts/frequencies of fragments length
- *.TLEN.f3F16.png
Plot of fragment length distribution
Output are stored in an directory: peak_calling_<sampleName>. Check MACS2 output list