# Trimming FASTQ files with Trimmomatic

### Trimmomatic

[Trimmomatic](https://http://www.usadellab.org/cms/?page=trimmomatic) is a flexible read trimming tool for Illumina NGS data.

#### Generic Trimmomatic command:

> *java -jar trimmomatic-0.39.jar PE inputforward.fq.gz inputreverse.fq.gz outputforwardpaired.fq.gz outputforwardunpaired.fq.gz outputreversepaired.fq.gz outputreverseunpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:keepBothReads LEADING:3 TRAILING:3 MINLEN:36*

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### Created a bash script for running trimmomatic on multiple files at the same time:

This scripts looks for files with "*_R1.fastq.gz" in their name, and applies trimmomaic on them, and their paired "*_R2.fastq.gz" files in the same directory

#!/bin/bash
# arg1: number of threads
# to run: 
# chmod +x trim.sh
# <path>/trim.sh <number of threads>
# Example: ./trim.sh 40

for f in *_R1.fastq.gz # for each sample

do
    n=${f%%_R1.fastq.gz} # strip part of file name
    trimmomatic PE -threads $1 ${n}_R1.fastq.gz  ${n}_R2.fastq.gz \
    ${n}_R1_trimmed.fastq.gz ${n}_R1_unpaired.fastq.gz ${n}_R2_trimmed.fastq.gz \
    ${n}_R2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 \
    LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

done

#### Transfered BASH script from local computer to cloud instance

scp -C /Users/cm/JPL_Google_Drive/scripts/trim.sh cmicro@149.165.171.66:/home/cmicro/scripts

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## On the cloud instance:

#### Create a directory for Trimming

mkdir trim
cd trim

#### Create symbolic links to FASTQ files
(Assuming all fastq files are in the fastqs_backup directory)

ln -s /home/cmicro/fastqs_backup/*.fastq.gz .

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### Create a Conda environment & install Trimmomatic

conda create -y -n trim trimmomatic

Copy TruSeq adapters to local directory so Trimmomatic finds them easily.

cp /opt/miniconda3/pkgs/trimmomatic-*/share/trimmomatic-*/adapters/TruSeq3-PE.fa .

#### Activate Trimmomatic Conda environment

conda activate trim

#### Make BASH script executable & run script (with)

chmod +x trim.sh

#### Running BASH script for Trimmomatic using 40 threads

/home/cmicro/scripts/trim.sh 40

# Move trimmed FASTQ files to specific directory

mkdir trimmed_fastqs
find . -type f -name "*trimmed*" -exec mv '{}' trimmed_fastqs/ \;

Command line output is below:
> TrimmomaticPE: Started with arguments:
-threads 42 sample1_R1.fastq.gz sample1_R2.fastq.gz sample1_R1_trimmed.fastq.gz sample1_R1_unpaired.fastq.gz sample1_R2_trimmed.fastq.gz sample1_R2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Quality encoding detected as phred33
Input Read Pairs: 5342751 Both Surviving: 4283096 (80.17%) Forward Only Surviving: 1020644 (19.10%) Reverse Only Surviving: 15211 (0.28%) Dropped: 23800 (0.45%)
TrimmomaticPE: Completed successfully
TrimmomaticPE: Started with arguments:
-threads 42 sample2_R1.fastq.gz sample2_R2.fastq.gz sample2_R1_trimmed.fastq.gz sample2_R1_unpaired.fastq.gz sample2_R2_trimmed.fastq.gz sample2_R2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Quality encoding detected as phred33
Input Read Pairs: 5528625 Both Surviving: 4327888 (78.28%) Forward Only Surviving: 1156093 (20.91%) Reverse Only Surviving: 17389 (0.31%) Dropped: 27255 (0.49%)
TrimmomaticPE: Completed successfully
TrimmomaticPE: Started with arguments:
-threads 42 sample3_R1.fastq.gz sample3_R2.fastq.gz sample3_R1_trimmed.fastq.gz sample3_R1_unpaired.fastq.gz sample3_R2_trimmed.fastq.gz sample3_R2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Quality encoding detected as phred33
Input Read Pairs: 5342751 Both Surviving: 4283096 (80.17%) Forward Only Surviving: 1020644 (19.10%) Reverse Only Surviving: 15211 (0.28%) Dropped: 23800 (0.45%)
TrimmomaticPE: Completed successfully
TrimmomaticPE: Started with arguments:
-threads 42 sample4_R1.fastq.gz sample4_R2.fastq.gz sample4_R1_trimmed.fastq.gz sample4_R1_unpaired.fastq.gz sample4_R2_trimmed.fastq.gz sample4_R2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Quality encoding detected as phred33
Input Read Pairs: 6718423 Both Surviving: 5270348 (78.45%) Forward Only Surviving: 1386864 (20.64%) Reverse Only Surviving: 22301 (0.33%) Dropped: 38910 (0.58%)
TrimmomaticPE: Completed successfully