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These scripts allow to allow the user to take ATAC-sequences through to shifted.bam.
There are 5 scripts that need to be run in succession -- each prior step must complete before the next.
Eventually we will separate out this part from the ATAC-seq repository -- but for now we shall leave it here because of the dependencies that remain to the directory of scripts
[ login to helix ]
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[ mkdir /data/Banchereau-Lab/ATAC-seq/<atac_seq_analysis_name> ]
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[ cd /data/Banchereau-Lab/ATAC-seq/<atac_seq_analysis_name> ]
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[ git clone the ATAC-seq repository ]
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[ ./make_atac_seq_shifted_bam_1_fastqc.sh \</data/Banchereau-Lab/GT-delivery/ATAC-seq/\<atac_seq_analysis_name\>/ \>
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[ ./make_atac_seq_shifted_bam_2_trimmomatic.sh \</data/Banchereau-Lab/GT-delivery/ATAC-seq/\<atac_seq_analysis_name\>/ \>
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[ ./make_atac_seq_shifted_bam_3_bwa.sh ]
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[ ./make_atac_seq_shifted_bam_4_shift_sam.sh ]
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[ ./make_atac_seq_shifted_bam_5_bamtobed.sh ]
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[ ./make_atac_seq_shifted_bam_6_cleanup.sh ]
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[ done! - send an email to the analyst who is to take it from here ]
#Begin - Make a directory in Banchereau-Lab ATAC-seq directory and one in Banchereau-Lab GT-delivery/ATAC-seq
Once it is known that the files are ready from Genome Technologies (GT) These files are copied to /data/Banchereau-Lab/GT-delivery/ATAC-seq maintaining the project information encoded in the directory name
Once copied, the user goes to the /data/Banchereau-Lab/ATAC-seq directory makes an appropriately named subdirectory -- and begins the process this may be facilitated by aligning all files in the project at once once they are all delivered for example, and it may be over the entire course of several months -- a single run may be performed and the job can be job arrayed
To facilitate this -- symbolic links are made to a representatively named directory structure and these may then be referenced in this first call
Pick a name that is appropriate for the study for which this sequencing was done.
Make a directory with that name /data/Banchereau-Lab/ATAC-seq/<atac_seq_analysis_name>/working
#Change directory to this newly named directory in the ATAC-seq directory
Make sure you are in this newly named directory before you clone this repository.
cd /data/Banchereau-Lab/ATAC-seq/<atac_seq_analysis_name>/
#Get the scripts.
git clone this repository
#Call the first script - run fastqc
make_atac_seq_shifted_bam_1_fastqc.sh </data/Banchereau-Lab/GT-delivery/ATAC-seq/<atac_seq_analysis_name>/ >
Purpose: calls the fastqc to create quality control files for each of the fastq's
What else happens -- a working directory is created
#Call the second script - trim files
make_atac_seq_shifted_bam_2_trimmomatic.sh
Call: make_atac_seq_shifted_bam_1_trimmomatic.sh </data/Banchereau-Lab/GT-delivery/ATAC-seq/<atac_seq_analysis_name>/ >
Purpose: fastqc has been run now we run trimmomatic to identified the trimmend and untrimmed fastq files
Assumptions:
1. code has been checked out - fastqc has been run - working directory created
2. we are in the same directory as the scripts.
#Call the third script - align to genome
make_atac_seq_shifted_bam_3_bwa.sh
Call: make_atac_seq_shifted_bam_3_bwa.sh
Purpose: bwa is the alignment of the trimmed fastq files to the genome
Assumptions:
1. code has been checked out - fastqc has been run - working directory created
2. we are in the same directory as the scripts.
3. trimmomatic has been run
#Call the fourth script - shift the sam file
make_atac_seq_shifted_bam_4_shift_sam.sh
Call: make_atac_seq_shifted_bam_4_shift_sam.sh
Purpose: This routine calls ATAC_BAM_shifter_gappedAlign.pl
From Asli Uyar, PhD
"The bam file needs to be adjusted because Tn5 has a 9bp binding site, and it binds in the middle.
Functionally that means that the DNA had to be accessible at least 4.5bp on either site of the insertion.
To my understanding the 4 for positive and 5 for negative was chosen at random, that could be wrong
for the negative strand the start position is the 5'-most, which doesn't actually change, it's the
3'-position that changes, and to modify that you only need to change the read sequence and size.
If the read is on the positive strand (as determined by the sam flag)
1. it will add 4 to the start,
2. and subtract 5 from the partner start
if the read is on the negative strand,
1. 5 will subtracted from it's start
2. and 4 added to its mate start.
The length and read type will be adjusted in both cases and the read and quality string trimmed appropriately,
Example of how a BAM file will be altered
Original: HWI-ST374:226:C24HPACXX:4:2214:15928:96004 99 chrI 427 255 101M = 479 153 HWI-ST374:226:C24HPACXX:4:2214:15928:96004 147 chrI 479 255 101M = 427 -153
Altered: HWI-ST374:226:C24HPACXX:4:2214:15928:96004 99 chrI 431 255 97M = 474 144 HWI-ST374:226:C24HPACXX:4:2214:15928:96004 147 chrI 479 255 96M = 431 -144
Call: make_atac_seq_shifted_bam_4_shift_sam.sh
Assumptions:
1. code has been checked out - fastqc has been run - working directory created
2. we are in the same directory as the scripts.
3. trimmomatic has been run
4. bwa has been run
#Call the fifth script - convert to shifted_sorted.bam
make_atac_seq_shifted_bam_5_bamtobed.sh
Call: make_atac_seq_shifted_bam_5_bamtobed.sh
Purpose: This routine sorts the shifted sam file and converts it to a bed file.
Assumptions:
1. code has been checked out - fastqc has been run - working directory created
2. we are in the same directory as the scripts.
3. trimmomatic has been run
4. bwa has been run
5. shifted_bam has been called to make the sam file correct for ATAC_seq
#Call the sixth and final script -- this cleans up no longer needed files
make_atac_seq_shifted_bam_6_cleanup.sh
Call: make_atac_seq_shifted_bam_6_cleanup.sh
Purpose: This routine removes all the trim and trimU fastq files (intermediate files that may be reproduced) It also removes all sam files, and the unsorted bam file, also all .o and .e files for readibility.
Assumptions:
1. code has been checked out - fastqc has been run - working directory created
2. we are in the same directory as the scripts.
3. trimmomatic has been run
4. bwa has been run
5. shifted_bam has been called to make the sam file correct for ATAC_seq
6. the shifted_bam script and bedtobam has been called so all that is left to do is cleanup!
#What happens next?
Now the real work begins -- peak calling, analysis, etc -- all the downstream work that is necessary to come to biologically meaningful conclusions.