FastQ trimming and QC

FastQ trimming and QC

Use the raw reads we downloaded from NCBI.

Installation

Install necessary packages

conda activate bioinfo
conda install fastqc trimmomatic seqtk

Have a look at the fastq file:

zcat can read a .gz compressed fastq file without extract it. Have a look at the first lines of the file.

zcat SRR10561173_1.fastq.gz | head

Check the quality of the raw reads:

mkdir ~/fastqc
fastqc -o ~/fastqc -f fastq ~/raw_data/SRR10561173_1.fastq.gz ~/raw_data/SRR10561173_2.fastq.gz

Notice: A good pratice:

• Always put your file in single directories, for example, put raw data into ~/raw_data/, put fastqc result into ~/fastqc.
• Always use absolute paths when running commands, for example, use ~/raw_data/SRR10561173_1.fastq.gz to tell the machine the position of the file.

Open the fastqc result. Understand the meaning of each section.

Trimming

• Use trimmomatic to trim the adapter. Find out the adaptor sequence according to the sequence platform and trimmomatic document, or use the adapter according to the FastQC adapter content section.

Adapter sequence	Sequence platform
TruSeq2	GAII
TruSeq3	HiSeq, MiSeq

The adaptor sequence is usually in the folder ~/miniconda2/envs/[environment name]/share/trimmomatic/adapters/

For example:

mkdir trimmomatic
FA=~/miniconda2/envs/bioinfo/share/trimmomatic/adapters/TruSeq3-PE-2.fa
trimmomatic PE SRR10561173_1.fastq.gz SRR10561173_2.fastq.gz -baseout ./trimmomatic/SRR10561173.fastq.gz ILLUMINACLIP:$FA:2:30:10

• Use seqtk to do the quality trim.

    mkdit seqtk
    cd trimmomatic
    for i in *fastq.gz; do out=${i%%.*}; seqtk trimfq $i > ~/seqtk/${out}_seqtk.fastq; done
  

• Run fastqc again to check the quality of the reads after trimming.