README.md

MP_preSeq_FastQC

Description

Quality control for NGS reads using FastQC

Install and configure

conda is used to configure the pipeline environment.

# The conda environment can be named here
NID="pipeline_QC"
# Create a new environment
conda create -n $NID
# Activate the environment to install softwares
source activate $NID
conda install fastqc
# Deactivate it after finish installation
source deactivate $NID

Input files

NGS reads either SE or PE in FASTQ format with or without .gz suffix, such as (take the NA12878 as the sample ID for the example):

• SE reads: NA12878.fq, NA12878.fq.gz ; NA12878.fastq, NA12878.fastq.gz
• PE reads: NA12878_1.fq, NA12878_2.fq; NA12878_1.fq.gz, NA12878_2.fq.gz; NA12878_1.fastq, NA12878_2.fastq; NA12878_1.fastq.gz, NA12878_2.fastq.gz

Usage synopsis

Main usage

# Before working, the pipeline environemnt should be activated
source activate $NID
bash fastqc.sh $in $out $cpus
source deactivate $NID

Parameters explanation

1. $in:
   • input folder containing FASTQ files, or
   • input FASTQ files
2. $out: output folder
3. $cpus: threads to use

Output files

Two files will be output for each sample (take the NA12878 as the sample ID for the example):

1. NA12878_fastqc.html: the main output for view
2. NA12878_fastqc.zip: the file can be used for script parser

Key notes

QC

Every parts should be checked in the .html file, especially:

1. Basic Statistics: Encoding, Total sequences, Sequence length
2. Per base sequence quality
3. Per sequence quality scores
4. Sequence Length Distribution
5. Adapter Content

Tricks

FastQC set a thread for each file, so it has no real effect if the threads are set to an integer larger than file number.

FAQ

Others

Encoding in Basic Statistics should be checked especially for old NGS data.

Usage example

All input and output files are stored in test_data/pipeline_QC.

Inputs

• NA12878_chr1_2Mb.fastq.gz: FASTQ Gzip file for SE reads

Run pipeline

source activate $NID
# make sure you are in the right working directory
bash fastqc.sh test_data/pipeline_QCNA12878_chr1_2Mb.fastq.gz test_data/pipeline_QC/output 1
source deactivate $NID

Outputs

• NA12878_chr1_2Mb_fastqc.html: main output in HTML format

• NA12878_chr1_2Mb_fastqc.zip: a zipped folder contain all data you will need

  • summary.txt is a brief summary
  • fastqc_data.txt is the result in text format
  • fastqc_report.html is same to the main HTML output

Reference

• FastQC: A quality control tool for high throughput sequence data.
• Using FastQC to check the quality of high throughput sequence (YouTube)
• FastQC documentation
• FastQC (GitHub)
• 利用fastqc检测原始序列的质量
• 20160410 测序分析——使用 FastQC 做质控
• 用FastQC检查二代测序原始数据的质量

Author

Yi Xianfu (yixfbio AT gmail DOT com)

License

GPL v3 or later