Updated 2019-07-15

Run Trim Galore! on the Cluster

Overview

  • 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.

Summary

  • Running Trim Galore! is as simple as loading the required modules are running trim_galore file.fq replacing 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 -l nodes=1:ppn=2
#PBS -l pmem=2gb
#PBS -l walltime=3:00
#PBS -q force-6
#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

  • The #PBS directives are standard, requesting just 3 minutes of walltime and 1 node with 2 cores. More on #PBS directives can be found in the PBS guide
  • $PBS_O_WORKDIR is 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/0.3.7 loads 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.fq gets 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 PBS Script as well as the SP1.fq FASTQ file.
  • Submit as normal, with qsub <pbs script name>. In this case qsub trim_galore.pbs
  • 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 PBS script, you should see a trimgaloreTest.out file which contains the results of the job, SP1.fq_trimming_report.txt which contains a report of the trimming performed on the FASTQ file, and SP1_trimmed.fq which contains the trimmed version of SP1.fq. Use cat or open the file in a text editor to take a look.
  • trimgaloreTest.out should 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.txt should 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%) 
  • SP1_trimmed.fq should look like this.
  • After the result files are produced, you can move the files off the cluster, refer to the file transfer guide for help.
  • Congratulations! You successfully ran Trim Galore! on the cluster.