Trimmomatic performs a variety of useful trimming tasks for illumina paired-end and single ended data.The selection of trimming steps and their associated parameters are supplied on the command line. Trimmomatic was developed at the Usadel lab in Aachen, Germany.
The current trimming steps are:
It works with FASTQ (using phred + 33 or phred + 64 quality scores, depending on the Illumina pipeline used), either uncompressed or gzipp'ed FASTQ. Use of gzip format is determined based on the .gz extension.
For single-ended data, one input and one output file are specified, plus the processing steps. For paired-end data, two input files are specified, and 4 output files, 2 for the 'paired' output where both reads survived the processing, and 2 for corresponding 'unpaired' output where a read survived, but the partner read did not.
Use the modules commands to set up trimmomatic, as in the example below. The module sets environment variables called 'TRIMMOJAR' and 'TRIMMOMATIC_JAR' which point to the location of the trimmomatic java file. The variable 'TRIMMOMATIC_JARPATH' points to the directory in which the trimmomatic jar file is located.
Fasta files of adapter sequences are included with trimmomatic and can be found at
/usr/local/apps/trimmomatic/<version>/adapters
Allocate an interactive session and run the program. Sample session:
[user@biowulf]$ sinteractive --mem 10g salloc.exe: Pending job allocation 46116226 salloc.exe: job 46116226 queued and waiting for resources salloc.exe: job 46116226 has been allocated resources salloc.exe: Granted job allocation 46116226 salloc.exe: Waiting for resource configuration salloc.exe: Nodes cn3144 are ready for job [user@cn3144 ~]$ module load trimmomatic [user@cn3144 ~]$ java -jar $TRIMMOJAR PE -phred33 \ /fdb/app_testdata/fastq/H_sapiens/hg100_1m_pe1.fq.gz /fdb/app_testdata/fastq/H_sapiens/hg100_1m_pe2.fq.gz \ output_forward_paired.fq.gz output_forward_unpaired.fq.gz \ output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz \ ILLUMINACLIP:/usr/local/apps/trimmomatic/0.39/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 TrimmomaticPE: Started with arguments: -phred33 /fdb/app_testdata/fastq/H_sapiens/hg100_1m_pe1.fq.gz /fdb/app_testdata/fastq/H_sapiens/hg100_1m_pe2.fq.gz out1 out2 out3 out4 ILLUMINACLIP:/usr/local/apps/trimmomatic/0.39/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 Multiple cores found: Using 2 threads Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT' ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences Input Read Pairs: 1000000 Both Surviving: 964918 (96.49%) Forward Only Surviving: 25517 (2.55%) Reverse Only Surviving: 7780 (0.78%) Dropped: 1785 (0.18%) TrimmomaticPE: Completed successfully [user@cn3144 ~]$ exit salloc.exe: Relinquishing job allocation 46116226 [user@biowulf ~]$
Create a batch input file (e.g. trimmomatic.sh). For example:
#!/bin/bash ml trimmomatic || exit 1 java -Djava.io.tmpdir=. -jar $TRIMMOJAR PE -phred33 -threads $SLURM_CPUS_PER_TASK \ SRR292678_1.fastq.gz SRR292678_2.fastq.gz \ output_forward_paired.fq.gz output_forward_unpaired.fq.gz \ output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz \ ILLUMINACLIP:/usr/local/apps/trimmomatic/0.39/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 \ SLIDINGWINDOW:4:15 MINLEN:36
Submit this job using the Slurm sbatch command.
sbatch -c 2 --mem=6g trimmomatic.sh
Create a swarmfile (e.g. trimmomatic.swarm). For example:
java -Djava.io.tmpdir=. -jar $TRIMMOJAR PE -phred33 -threads $SLURM_CPUS_PER_TASK \ /fdb/app_testdata/fastq/H_sapiens/hg100_1m_pe1.fq.gz /fdb/app_testdata/fastq/H_sapiens/hg100_1m_pe2.fq.gz \ output_forward_paired.fq.gz output_forward_unpaired.fq.gz \ output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz \ ILLUMINACLIP:/usr/local/apps/trimmomatic/0.39/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 \ SLIDINGWINDOW:4:15 MINLEN:36 [...etc....]
Submit this job using the swarm command.
swarm -f trimmomatic.swarm -g 6 -t 8 --module trimmomaticwhere
-g # | Number of Gigabytes of memory required for each process (1 line in the swarm command file) |
-t # | Number of threads/CPUs required for each process (1 line in the swarm command file). |
--module trimmomatic | Loads the trimmomatic module for each subjob in the swarm |