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Another tool for quality trimming ... this is probably the best known.


Trimmomatic is quite unique in the way it is called.

It is a java program, so this requires the jar file (naturally) and then:

  1. whether single- or pair-end reads (SE|PE)
  2. number of threads with hyphen, i.e. -threads N
  3. whether quality scoring is phred33 or phred64 (although it will try to auto-detect)
  4. next come the two input pair-ended file with no hyphens, i.e just the plain filenames.
  5. next come the ouput files and four names are required here, because two files of these will be single-end (unpaired reads), i.e. their corresponding read in the pair did not reach the quality standard and so was deleted.

Adapter file specification

You can use an environmental variable for the adapter fasta file, because they have been set by the module and will point to the right place. Here is a list, the most commonly used one is $TS3PE.

  • $TS3SE is TruSeq3-SE.fa
  • $TS2SE is TruSeq2-SE.fa
  • $TS2PE is TruSeq2-PE.fa
  • $NXPE is NexteraPE-PE.fa
  • $TS3PE is TruSeq3-PE.fa
  • $TS3PE2 is TruSeq3-PE-2.fa

Example use-cases

java -jar $PATHTRIMMOJAR/trimmomatic-0.32.jar PE -threads 6 -phred33 $DATASETPATH/Pair1.fastq.gz $DATASETPATH/Pair1.fastq.gz forward_paired.fq forward_unpaired.fq reverse_paired.fq reverse_unpaired.fq ILLUMINACLIP:$TS3PE:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:50