FASTX-Toolkit

FASTX-Toolkit介绍

背景介绍

高通量测序数据下机后的原始fastq文件，包含4行，其中一行为质量值，另外一行则为对应序列，高通量的数据处理首先要进行质量控制，这些过程包括去接头、过滤低质量reads、去除低质量的3’和5’端，去除N较多的reads等，针对高通量测序数据的质控软件有很多，在此介绍质控工具：fastx_toolkit

FASTX-Toolkit

FASTX-Toolkit是用于短读FASTA / FASTQ文件预处理的命令行工具的集合。 新一代测序仪通常生成FASTA或FASTQ文件，包含多个短读序列（可能带有质量信息）。 这种FASTA / FASTQ文件的主要处理是使用专门程序将序列映射（也称为比对）到参考基因组或其他数据库。 这种映射程序的示例是：Blat，SHRiMP，LastZ，MAQ以及许多其他程序。 但是，在将序列映射到基因组之前预处理FASTA / FASTQ文件有时会更有效率 - 操作序列以产生更好的映射结果。 FASTX-Toolkit工具执行其中一些预处理任务。

可用工具

• FASTQ-to-FASTA converter
  Convert FASTQ files to FASTA files.
  将FASTQ文件转换为FASTA文件
• FASTQ Information
  Chart Quality Statistics and Nucleotide Distribution
  图表质量统计和核苷酸分布
• FASTQ/A Collapser
  Collapsing identical sequences in a FASTQ/A file into a single sequence (while maintaining reads counts)
  将FASTQ / A文件中的相同序列折叠成单个序列（同时保持读取计数）
• FASTQ/A Trimmer
  Shortening reads in a FASTQ or FASTQ files (removing barcodes or noise)
  缩短FASTQ或FASTQ文件中的读数。
• FASTQ/A Renamer
  Renames the sequence identifiers in FASTQ/A file
  在FASTQ / A文件中重命名序列标识符
• FASTQ/A Clipper
  Removing sequencing adapters / linkers
  删除测序适配器/连接器
• FASTQ/A Reverse-Complement
  Producing the Reverse-complement of each sequence in a FASTQ/FASTA file
  在FASTQ / FASTA文件中生成每个序列的反向补码
• FASTQ/A Barcode splitter
  Splitting a FASTQ/FASTA files containning multiple samples
  拆分包含多个样本的FASTQ / FASTA文件
• FASTA Formatter
  changes the width of sequences line in a FASTA file
  更改FASTA文件中序列行的宽度
• FASTA Nucleotide Changer
  Convets FASTA sequences from/to RNA/DNA
  将FASTA序列从/转换为RNA / DNA
• FASTQ Quality Filter
  Filters sequences based on quality
  根据质量过滤序列
• FASTQ Quality Trimmer
  Trims (cuts) sequences based on quality
  根据质量修剪（剪切）序列
• FASTQ Masker
  Masks nucleotides with ‘N’ (or other character) based on quality
  根据质量，使用’N’（或其他字符）掩蔽核苷酸

下载

下载地址：fastx_toolkit下载链接

wget http://hannonlab.cshl.edu/fastx_toolkit/fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64.tar.bz2
tar xjvf fastx_toolkit_0.0.13_binaries_Linux_2.6_amd64.tar.bz2

使用

注意事项

fastx_toolkit由一系列的命令组成，每个命令提供一个实用的小功能。在使用时需要注意以下几点:

• 不支持压缩格式的输入文件
• 不允许序列中存在N碱基，这样的序列会自动去除
• 可视化命令依赖gunplot软件和perl的GD模块
• 默认情况下认为fastq文件的碱基编码格式为phred64

在安装该软件时尤其时运时如果遇到:make命令报错：“fgets called with bigger size than length of destination buffer”，安装比较新版本，就能解决问题。

如果在运行fastx_quality_stats 过程中出现“fastx_quality_stats: Invalid quality score value (char ‘#’ ord 35 quality value -29) on line 4”，请在参数中加入“-Q 33”

参数及其使用

FASTQ-to-FASTA

usage: fastq_to_fasta [-h] [-r] [-n] [-v] [-z] [-i INFILE] [-o OUTFILE]

version 0.0.6
   [-h]         = This helpful help screen.
   [-r]         = Rename sequence identifiers to numbers.
   [-n]         = keep sequences with unknown (N) nucleotides.
          Default is to discard such sequences.
   [-v]         = Verbose - report number of sequences.
          If [-o] is specified,  report will be printed to STDOUT.
          If [-o] is not specified (and output goes to STDOUT),
          report will be printed to STDERR.
   [-z]         = Compress output with GZIP.
   [-i INFILE]  = FASTA/Q input file. default is STDIN.
   [-o OUTFILE] = FASTA output file. default is STDOUT.

FASTX Statistics

usage: fastx_quality_stats [-h] [-i INFILE] [-o OUTFILE]

version 0.0.6 (C) 2008 by Assaf Gordon (gordon@cshl.edu)
   [-h] = This helpful help screen.
   [-i INFILE]  = FASTA/Q input file. default is STDIN.
                  If FASTA file is given, only nucleotides
          distribution is calculated (there's no quality info).
   [-o OUTFILE] = TEXT output file. default is STDOUT.

The output TEXT file will have the following fields (one row per column):
    column    = column number (1 to 36 for a 36-cycles read solexa file)
    count   = number of bases found in this column.
    min     = Lowest quality score value found in this column.
    max     = Highest quality score value found in this column.
    sum     = Sum of quality score values for this column.
    mean    = Mean quality score value for this column.
    Q1    = 1st quartile quality score.
    med    = Median quality score.
    Q3    = 3rd quartile quality score.
    IQR    = Inter-Quartile range (Q3-Q1).
    lW    = 'Left-Whisker' value (for boxplotting).
    rW    = 'Right-Whisker' value (for boxplotting).
    A_Count    = Count of 'A' nucleotides found in this column.
    C_Count    = Count of 'C' nucleotides found in this column.
    G_Count    = Count of 'G' nucleotides found in this column.
    T_Count    = Count of 'T' nucleotides found in this column.
    N_Count = Count of 'N' nucleotides found in this column.
    max-count = max. number of bases (in all cycles)

FASTQ Quality Chart

Usage: /usr/local/bin/fastq_quality_boxplot_graph.sh [-i INPUT.TXT] [-t TITLE] [-p] [-o OUTPUT]

  [-p]           - Generate PostScript (.PS) file. Default is PNG image.
  [-i INPUT.TXT] - Input file. Should be the output of "solexa_quality_statistics" program.
  [-o OUTPUT]    - Output file name. default is STDOUT.
  [-t TITLE]     - Title (usually the solexa file name) - will be plotted on the graph.

FASTA/Q Nucleotide Distribution

Usage: /usr/local/bin/fastx_nucleotide_distribution_graph.sh [-i INPUT.TXT] [-t TITLE] [-p] [-o OUTPUT]

  [-p]           - Generate PostScript (.PS) file. Default is PNG image.
  [-i INPUT.TXT] - Input file. Should be the output of "fastx_quality_statistics" program.
  [-o OUTPUT]    - Output file name. default is STDOUT.
  [-t TITLE]     - Title - will be plotted on the graph.

FASTA/Q Clipper

usage: fastx_clipper [-h] [-a ADAPTER] [-D] [-l N] [-n] [-d N] [-c] [-C] [-o] [-v] [-z] [-i INFILE] [-o OUTFILE]

version 0.0.6
   [-h]         = This helpful help screen.
   [-a ADAPTER] = ADAPTER string. default is CCTTAAGG (dummy adapter).
   [-l N]       = discard sequences shorter than N nucleotides. default is 5.
   [-d N]       = Keep the adapter and N bases after it.
          (using '-d 0' is the same as not using '-d' at all. which is the default).
   [-c]         = Discard non-clipped sequences (i.e. - keep