Fastq adapter removal and QC

Note

Required OS:OS x or Linux. Windows users, please contact Saranga Wijeratne
Software:Trimmomatic
Purpose:This document provides instructions about how to remove adapters and filter low quality bases from a Fastq file
More:Read more about Read trimming adapter removing here:
Author:This document is created by Saranga Wijeratne

Load the software

Note

If you are runing this on MCBL mcic-ender-svr following command will load the software module to your environment.

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$ module load Trimmomatic/3.2.2

then you can get the help how to run Trimmomatic,

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$ java -jar $TRIMHOME/trimmomatic-0.33.jar

Files needed

Input Files:

In put files should be in fastq format/compressed fastq( .fq, .fastq, .fq.gz, .fastq.gz). Read Introduction e.g :C8EC8ANXX_s2_1_illumina12index_1_SL143785.fastq, C8EC8ANXX_s2_1_illumina12index_1_SL143785.fastq.gz, s_1_1_sequence.txt.gz lane1_forward.fq.gz
Adapter File:

Currently, following Adapter sequence files are hosted in MCBL server.

  • TruSeq2-PE.fa
  • TruSeq2-SE.fa
  • TruSeq3-PE.fa
  • TruSeq3-SE.fa
  • NexteraPE-PE.fa

Warning

If you want to make your own adapter sequence file, please read the The Adapter Fasta section and Making cutome clipping files here before you make your Adapter sequence file.

Code examples

Single-end fastq files

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$java -jar $TRIMHOME/trimmomatic-0.33.jar SE -threads 12 s_1_1_sequence.txt.gz lane1_forward.fq.gz ILLUMINACLIP:$TRIMHOME/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Paired End Fastq Files

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$java -jar $TRIMHOME/trimmomatic-0.33.jar PE -threads 12 C8EC8ANXX_s2_1_illumina12index_1_SL143785.fastq.gz C8EC8ANXX_s2_2_illumina12index_1_SL143785.fastq.gz C8EC8ANXX_s2_1_Trimmed_1P.fastq.gz C8EC8ANXX_s2_1_Trimmed_1U.fastq.gz C8EC8ANXX_s2_2_Trimmed_1P.fastq.gz C8EC8ANXX_s2_2_Trimmed_1U.fastq.gz ILLUMINACLIP:$TRIMHOME/adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Multiple fastq files

Tip

Assumption has been made that your data in “Raw_Data” folder

Input Files:C6EF7ANXX_s3_1_illumina12index_10_SL100996.fastq.gz C6EF7ANXX_s3_1_illumina12index_19_SL100997.fastq.gz C6EF7ANXX_s3_1_illumina12index_22_SL100998.fastq.gz C6EF7ANXX_s3_1_illumina12index_25_SL100999.fastq.gz C6EF7ANXX_s3_1_illumina12index_27_SL101000.fastq.gz C6EF7ANXX_s3_1_illumina12index_3_SL100994.fastq.gz C6EF7ANXX_s3_1_illumina12index_5_SL100995.fastq.gz C6EF7ANXX_s3_2_illumina12index_10_SL100996.fastq.gz C6EF7ANXX_s3_2_illumina12index_19_SL100997.fastq.gz C6EF7ANXX_s3_2_illumina12index_22_SL100998.fastq.gz C6EF7ANXX_s3_2_illumina12index_25_SL100999.fastq.gz C6EF7ANXX_s3_2_illumina12index_27_SL101000.fastq.gz C6EF7ANXX_s3_2_illumina12index_3_SL100994.fastq.gz C6EF7ANXX_s3_2_illumina12index_5_SL100995.fastq.gz

These are paired-end fastq files. e.g C6EF7ANXX_s3_1_illumina12index_10_SL100996.fastq.gz and C6EF7ANXX_s3_2_illumina12index_10_SL100996.fastq.gz belongs to single sample.

Adapter File:

$TRIMHOME/adapters/TruSeq3-PE.fa (Make sure you change this accordingly)
Output Files:

Each paired-end read (e.g C6EF7ANXX_s3_1_illumina12index_10_SL100996.fastq.gz and C6EF7ANXX_s3_2_illumina12index_10_SL100996.fastq.gz) will give 4 outputs:

  • Q_trimmed_6EF7ANXX_s3_1_illumina12index_10_SL100996_1P.fastq.gz - for paired forwad reads
  • Q_trimmed_6EF7ANXX_s3_1_illumina12index_10_SL100996_1U.fastq.gz - for unpaired forward reads
  • Q_trimmed_6EF7ANXX_s3_2_illumina12index_10_SL100996_1P.fastq.gz - for paired reverse reads
  • Q_trimmed_6EF7ANXX_s3_2_illumina12index_10_SL100996_1U.fastq.gz - for unpaired reverse reads
 
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$cd Raw_Data #make sure you change the folder name accordingly
$mkdir Trimmed_Data # Output will be saved here
$files_1=(*_s3_1_*.fastq.gz);files_2=(*_s3_2_*.fastq.gz);sorted_files_1=($(printf "%s\n" "${files_1[@]}" | sort -u));sorted_files_2=($(printf "%s\n" "${files_2[@]}" | sort -u));for ((i=0; i<${#sorted_files_1[@]}; i+=1));do java -jar $TRIMHOME/trimmomatic-0.33.jar PE -threads 12  -trimlog Trimmed_Data/log-j3.stat -phred33   ${sorted_files_1[i]} ${sorted_files_2[i]} Trimmed_Data/Q_trimmed_${sorted_files_1[i]%%.*}_1P.fastq.gz Trimmed_Data/Q_trimmed_${sorted_files_1[i]%%.*}-U.fastq.gz Trimmed_Data/Q_trimmed_${sorted_files_2[i]%%.*}_1P.fastq.gz Trimmed_Data/Q_trimmed_${sorted_files_1[i]%%.*}-U.fastq.gz ILLUMINACLIP:$TRIMHOME/adapters/TruSeq3-PE:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:40  &>Trimmed_Data/stat.txt; done