Run Trim Galore! on the Cluster¶
- Trim Galore! is a wrapper script to automate quality and adapter trimming as well as quality control, with some added functionality to remove biased methylation positions for RRBS sequence files (for directional, non-directional (or paired-end) sequencing).
- This guide will cover how to run Trim Galore! on the Cluster.
- Here is a link to Trim Galore!'s Homepage.
- Running Trim Galore! is as simple as loading the required modules are running
trim_galore file.fqreplacing file.fq with your FASTQ file.
Walkthrough: Run Trim Galore! on the Cluster¶
- This walkthrough will have Trim Galore! remove base calls with a Phred score of 20 or lower (assuming Sanger encoding) and remove sequences that became shorter than 20 bp from a FASTQ file.
- SP1.fq can be downloaded here which is sourced from this webpage.
- PBS Script can be found here.
- You can transfer the files to your account on the cluster to follow along. The file transfer guide may be helpful.
Part 1: The PBS Script¶
#PBS -N trimgaloreTest #PBS -A [Account] #PBS -l nodes=1:ppn=2 #PBS -l pmem=2gb #PBS -l walltime=3:00 #PBS -q inferno #PBS -j oe #PBS -o trimgaloreTest.out cd $PBS_O_WORKDIR module load python/2.7 module load fastqc/0.10.1 module load trim_galore/0.3.7 trim_galore SP1.fq
#PBSdirectives are standard, requesting just 3 minutes of walltime and 1 node with 2 cores. More on
#PBSdirectives can be found in the PBS guide
$PBS_O_WORKDIRis a variable that represents the directory you submit the PBS script from. Make sure the files you want to use are in the same directory you put the PBS script.
- Output Files will also show up in this dir as well
module load trim_galore/0.3.7loads the 0.3.7 version of Trim Galore!. To see what versions of a software are available, run
module avail [Software], and load the one you want. The other modules are dependencies that must be loaded before Trim Galore! is loaded.
trim_galore SP1.fqgets Trim Galore! to remove base calls with a Phred score of 20 or lower (assuming Sanger encoding) and remove sequences that became shorter than 20 bp.
Part 2: Submit Job and Check Status¶
- Make sure you're in the dir that contains the
PBSScript as well as the
- Submit as normal, with
qsub <pbs script name>. In this case
- Check job status with
qstat -t 22182721, replacing the number with the job id returned after running qsub
- You can delete the job with
qdel 22182721, again replacing the number with the jobid returned after running qsub
Part 3: Collecting Results¶
- In the directory where you submitted the
PBSscript, you should see a
trimgaloreTest.outfile which contains the results of the job,
SP1.fq_trimming_report.txtwhich contains a report of the trimming performed on the FASTQ file, and
SP1_trimmed.fqwhich contains the trimmed version of
cator open the file in a text editor to take a look.
trimgaloreTest.outshould look like SP1.fq_trimming_report.txt except with the PBS Prologue at the top and PBS Epilogue at the bottom.
SP1.fq_trimming_report.txtshould look like this:
SUMMARISING RUN PARAMETERS ========================== Input filename: SP1.fq Trimming mode: single-end Trim Galore version: 0.3.7 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length before a sequence gets removed: 20 bp This is cutadapt 1.8.1 with Python 2.7.9 Command line parameters: -f fastq -e 0.1 -q 20 -O 1 -a AGATCGGAAGAGC SP1.fq Trimming 1 adapter with at most 10.0% errors in single-end mode ... Finished in 0.01 s (40 us/read; 1.50 M reads/minute). === Summary === Total reads processed: 250 Reads with adapters: 102 (40.8%) Reads written (passing filters): 250 (100.0%) Total basepairs processed: 7,750 bp Quality-trimmed: 549 bp (7.1%) Total written (filtered): 6,625 bp (85.5%) === Adapter 1 === Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 102 times. No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1 Bases preceding removed adapters: A: 19.6% C: 58.8% G: 5.9% T: 15.7% none/other: 0.0% Overview of removed sequences length count expect max.err error counts 1 45 62.5 0 45 2 14 15.6 0 14 3 4 3.9 0 4 4 2 1.0 0 2 5 1 0.2 0 1 6 5 0.1 0 5 8 3 0.0 0 3 9 2 0.0 0 2 11 4 0.0 1 2 2 12 3 0.0 1 2 1 13 2 0.0 1 2 14 4 0.0 1 4 15 5 0.0 1 1 4 16 1 0.0 1 1 17 2 0.0 1 2 18 1 0.0 1 0 1 24 3 0.0 1 0 3 29 1 0.0 1 1 RUN STATISTICS FOR INPUT FILE: SP1.fq ============================================= 250 sequences processed in total Sequences removed because they became shorter than the length cutoff of 20 bp: 39 (15.6%)