A pipeline to trim the reads and remove ribosomal RNA from RNA-Seq data
This pipeline trims the adaptor from the ends of reads, and move plant Ribosomal reads from RNAseq reads. (This tool also works for data from animal species, when corresponding rRNA sequences is provide.) The process is based on two tools, Trimmomatic and STAR for trimming and mapping respectively.
First of all please make sure how to run these two tools on your system.
On Sapelo/UGA, it is like this:
Trimmomatic
module load ava/jdk1.8.0_20
/usr/local/apps/trimmomatic/0.33/trimmomatic-0.33.jar
STAR
modle load java/jdk1.7.0_67
/usr/local/apps/star/latest/bin/STAR
Dependency:
Trimmomatic http://www.usadellab.org/cms/?page=trimmomatic
STAR https://github.com/alexdobin/STAR
copy NGSclean directory to your system, for example
/lustre1/lxue/NGSclean
prepare rRNA reference for STAR
NGSclean=/lustre1/lxue/NGSclean
cd $NGSclean
mkdir rRNA_ref
module load java/jdk1.7.0_67
/usr/local/apps/star/2.4.2a/bin/STAR \
--runThreadN 2 \
--runMode genomeGenerate \
--genomeDir rRNA_ref \
--genomeChrBinNbits 5 \
--genomeFastaFiles rRNA_only_NR.fas
Keep '--genomeDir rRNA_ref' as it is change 'rRNA_only_NR.fas' if a new reference file is necessary.
cd /lustre1/lxue/RNAseq/01clean
NGSclean=/lustre1/lxue/NGSclean
python $NGSclean/generate_design_file.py -f /lustre1/lxue/RNAseq/00reads -d RNAseq_design.txt -p
Check the design file. Revise the sample names if necessary, it will be used as a prefix for fastq files
more RNAseq_design.txt
cd /lustre1/lxue/RNAseq/01clean
settings
NGSclean=/lustre1/lxue/NGSclean
trimmoFull=/usr/local/apps/trimmomatic/0.33/trimmomatic-0.33.jar
starFull=/usr/local/apps/star/latest/bin/STAR
adaptor=/usr/local/apps/trimmomatic/latest/adapters/TruSeq3-PE.fa
trimmo_module=java/jdk1.8.0_20
star_module=java/jdk1.7.0_67
generate shell files
-s how to treat the singleton reads:merge, keep , discard
-q queue to run the jobs: queue(defualt), inter(run interactive jobs)
python $NGSclean/trim_and_clean.py -d RNAseq_design.txt -t 8 -s merge \
--run_trimmomatic $trimmoFull --load_trimmo_module $trimmo_module --adaptor $adaptor \
--run_star $starFull --load_star_module $star_module
python $NGSclean/trim_and_clean.py -d RNAseq_design.txt -t 8 -s keep \
--run_trimmomatic $trimmoFull --load_trimmo_module $trimmo_module --adaptor $adaptor \
--run_star $starFull --load_star_module $star_module
python $NGSclean/trim_and_clean.py -d RNAseq_design.txt -t 8 -s discard -q inter \
--run_trimmomatic $trimmoFull --load_trimmo_module $trimmo_module --adaptor $adaptor \
--run_star $starFull --load_star_module $star_module
And then run the master shell script to submit the jobs or run them interactively.
For example:
chmod 750 RNAseq_design.sh
./RNAseq_design.sh
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