FastQC——測序資料質量分析

下載安裝與配置

cd ~
wget http://www.bioinformatics.babraham.ac.uk/projects/fastqc/fastqc_v0.11.3.zip
unzip fastqc_v0.11.3.zip
cd FastQC/
sudo gedit /etc/profile
新增檔案末尾並儲存： export PATH=/home/WANGLAILAI_ubuntu/FastQC:$PATH
sudo source /etc/profile
fastqc -h

FastQC - A high throughput sequence QC analysis tool 
SYNOPSIS

fastqc seqfile1 seqfile2 .. seqfileN

fastqc [-o output dir] [--(no)extract] [-f fastq|bam|sam] 
       [-c contaminant file] seqfile1 .. seqfileN

DESCRIPTION

FastQC reads a set of sequence files and produces from each one a quality
control report consisting of a number of different modules, each one of 
which will help to identify a different potential type of problem in your
data.

If no files to process are specified on the command line then the program
will start as an interactive graphical application.  If files are provided
on the command line then the program will run with no user interaction
required.  In this mode it is suitable for inclusion into a standardised
analysis pipeline.

The options for the program as as follows:

-h --help       Print this help file and exit

-v --version    Print the version of the program and exit

-o --outdir     Create all output files in the specified output directory.
                Please note that this directory must exist as the program
                will not create it.  If this option is not set then the 
                output file for each sequence file is created in the same
                directory as the sequence file which was processed.
                
--casava        Files come from raw casava output. Files in the same sample
                group (differing only by the group number) will be analysed
                as a set rather than individually. Sequences with the filter
                flag set in the header will be excluded from the analysis.
                Files must have the same names given to them by casava
                (including being gzipped and ending with .gz) otherwise they
                won't be grouped together correctly.
                
--nano          Files come from naopore sequences and are in fast5 format. In
                this mode you can pass in directories to process and the program
                will take in all fast5 files within those directories and produce
                a single output file from the sequences found in all files.                    
                
--nofilter      If running with --casava then don't remove read flagged by
                casava as poor quality when performing the QC analysis.
               
--extract       If set then the zipped output file will be uncompressed in
                the same directory after it has been created.  By default
                this option will be set if fastqc is run in non-interactive
                mode.
                
-j --java       Provides the full path to the java binary you want to use to
                launch fastqc. If not supplied then java is assumed to be in
                your path.
               
--noextract     Do not uncompress the output file after creating it.  You
                should set this option if you do not wish to uncompress
                the output when running in non-interactive mode.
                
--nogroup       Disable grouping of bases for reads >50bp. All reports will
                show data for every base in the read.  WARNING: Using this
                option will cause fastqc to crash and burn if you use it on
                really long reads, and your plots may end up a ridiculous size.
                You have been warned!
                
-f --format     Bypasses the normal sequence file format detection and
                forces the program to use the specified format.  Valid
                formats are bam,sam,bam_mapped,sam_mapped and fastq
                
-t --threads    Specifies the number of files which can be processed
                simultaneously.  Each thread will be allocated 250MB of
                memory so you shouldn't run more threads than your
                available memory will cope with, and not more than
                6 threads on a 32 bit machine
              
-c              Specifies a non-default file which contains the list of
--contaminants  contaminants to screen overrepresented sequences against.
                The file must contain sets of named contaminants in the
                form name[tab]sequence.  Lines prefixed with a hash will
                be ignored.

-a              Specifies a non-default file which contains the list of
--adapters      adapter sequences which will be explicity searched against
                the library. The file must contain sets of named adapters
                in the form name[tab]sequence.  Lines prefixed with a hash
                will be ignored.
                
-l              Specifies a non-default file which contains a set of criteria
--limits        which will be used to determine the warn/error limits for the
                various modules.  This file can also be used to selectively 
                remove some modules from the output all together.  The format
                needs to mirror the default limits.txt file found in the
                Configuration folder.
                
-k --kmers       Specifies the length of Kmer to look for in the Kmer content
                module. Specified Kmer length must be between 2 and 10. Default
                length is 7 if not specified.
                
 -q --quiet       Supress all progress messages on stdout and only report errors.

-d --dir         Selects a directory to be used for temporary files written when
                generating report images. Defaults to system temp directory if
                not specified.
                
BUGS

Any bugs in fastqc should be reported either to simon.andrews@babraham.ac.uk
or in www.bioinformatics.babraham.ac.uk/bugzilla/

例項執行：

我們需要測序檔案FASTQ，為了方便，直接使用 MATLAB 生物資訊學工具箱中的資料，windows下：

C:\Program Files\MATLAB\R2017a\toolbox\bioinfodata\SRR005164_1_50.fastq

在/FastQC下新建目錄/mytest並且將SRR005164_1_50.fastq拷貝到其中

fastqc -t 2 ./mytest/SRR005164_1_50.fastq -o ./mytest
cd /mytest
unzip SRR005164_1_50_fastqc.zip
tree

輸出：

.
├── SRR005164_1_50.fastq
├── SRR005164_1_50_fastqc
│   ├── fastqc_data.txt（資料結果檔案，後來被視覺化成 Images 下的圖片）
│   ├── fastqc.fo（額外說明檔案）
│   ├── fastqc_report.html （HTML報告檔案）
│   ├── Icons
│   │   ├── error.png
│   │   ├── fastqc_icon.png
│   │   ├── tick.png
│   │   └── warning.png
│   ├── Images
│   │   ├── adapter_content.png
│   │   ├── duplication_levels.png
│   │   ├── kmer_profiles.png
│   │   ├── per_base_n_content.png
│   │   ├── per_base_quality.png
│   │   ├── per_base_sequence_content.png
│   │   ├── per_sequence_gc_content.png
│   │   ├── per_sequence_quality.png
│   │   └── sequence_length_distribution.png
│   └── summary.txt （總結檔案）
├── SRR005164_1_50_fastqc.html
└── SRR005164_1_50_fastqc.zip

開啟SRR005164_1_50_fastqc.html報告檔案，有

1. Basic Statistics（基礎統計資訊)
2. Per base sequence quality（平均鹼基質量）
3. Per sequence quality scores （平均每條序列質量）
4. Per base sequence content （每條序列平均鹼基組成）
5. Per sequence GC content （每個序列GC含量）
6. Per base N content （每個位點N含量）
7. Sequence Length Distribution （序列長度分佈）
8. Sequence Duplication Levels （序列重複水平）
9. Overrepresented sequences （過度呈現的序列）
10. Adapter Content （接頭分佈）
11. Kmer Content（K值分佈）