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.
Overview of major steps
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).
A configuration file required to execute the pipeline.
bash ASAP.sh [-h] [-v] [-c]
Options
-c CONFIGFILE
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)
-h/-v
Print out the help/current version
About the configuration file:
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 parameters
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
Mapping step parameters
map:
Set to "yes/no". If mapping is skipped (map=no), a BAM file must be provided to proceed.
(see post-mapping steps).
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
Filtering parameters
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
Coverage
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
Read extraction
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)
Fragment length
fragDist:
Set to "yes/no" if fragment length distribution should be computed or not
Peak calling
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)
Output files
ASAP outputs mapping files, coverage files, fragments distribution table/plot and MACS2 peak calling results.
Mapping output
*.mapped.sorted.bam:
Contains mapped reads (bowtie2 raw mapping results)
Filtering/post-processing outputs
*.(un)masked.(un)shifted.bam:
Contains the selected set of reads after filtering. Ideally, accessible peaks are called using this file.
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