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生物信息学/使用fastp进行数据质量控制

维基教科书,自由的教学读本

fastp的特性

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  • 对数据自动进行全方位质控,生成人性化的报告
  • 过滤功能(低质量,太短,太多N……);
  • 对每一个序列的头部或尾部,计算滑动窗内的质量均值,并将均值较低的子序列进行切除(类似Trimmomatic的做法,但是快非常多);
  • 全局剪裁 (在头/尾部,不影响去重),对于Illumina下机数据往往最后一到两个cycle需要这样处理;
  • 去除接头污染。厉害的是,你不用输入接头序列,因为算法会自动识别接头序列并进行剪裁;
  • 对于双端测序(PE)的数据,软件会自动查找每一对read的重叠区域,并对该重叠区域中不匹配的碱基对进行校正;
  • 去除尾部的polyG。对于Illumina NextSeq/NovaSeq的测序数据,因为是两色法发光,polyG是常有的事,所以该特性对该两类测序平台默认打开;
  • 对于PE数据中的overlap区间中不一致的碱基对,依据质量值进行校正;
  • 可以对带分子标签(UMI)的数据进行预处理,不管UMI在插入片段还是在index上,都可以轻松处理; -可以将输出进行分拆,而且支持两种模式,分别是指定分拆的个数,或者分拆后每个文件的行数;

fastp完美支持gzip的输入和输出,同时支持SE和PE数据,而且不但支持像Illumina平台的short read数据,也在一定程度上支持了PacBio/Nanopore的long reads数据。

fastp软件会生成HTML格式的报告,而且该报告中没有任何一张静态图片,所有的图表都是使用JavaScript动态绘制,非常具有交互性。想要看一下样板报告的,可以去以下链接:http://opengene.org/fastp/fastp.html

而且软件的开发者还充分考虑到了各种自动化分析的需求,不但生成了人可读的HTML报告,还生成了程序可读性非常强的JSON结果,该JSON报告中的数据包含了HTML报告100%的信息,而且该JSON文件的格式还是特殊定制的,不但程序读得爽,你用任何一款文本编辑器打开,一眼过去也会看得明明白白。想要看一下JSON结果长什么样的,可以去以下链接:http://opengene.org/fastp/fastp.json

fastp的安装

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fastp软件下载网址

Bioconda源安装

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# 不一定最新
conda install -c bioconda fastp

安装二进制命令

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wget http://opengene.org/fastp/fastp
chmod a+x ./fastp

从源码安装(Mac和Linux)

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git clone https://github.com/OpenGene/fastp.git

# build
cd fastp
make

#安装
sudo make install

从源码安装(Windows)

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git clone -b master --depth=1 https://github.com/OpenGene/fastp.git
cd fastp
make

fastp的参数和选项

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usage: fastp -i <in1> -o <out1> [-I <in1> -O <out2>] [options...]
options:
  # I/O options   即输入输出文件设置
  -i, --in1                          read1 input file name (string)
  -o, --out1                         read1 output file name (string [=])
  -I, --in2                          read2 input file name (string [=])
  -O, --out2                         read2 output file name (string [=])
  -6, --phred64                      indicates the input is using phred64 scoring (it'll be converted to phred33, so the output will still be phred33)
  -z, --compression                  compression level for gzip output (1 ~ 9). 1 is fastest, 9 is smallest, default is 2. (int [=2])
    --reads_to_process               specify how many reads/pairs to be processed. Default 0 means process all reads. (int [=0])
  
  # adapter trimming options   过滤序列接头参数设置
  -A, --disable_adapter_trimming     adapter trimming is enabled by default. If this option is specified, adapter trimming is disabled
  -a, --adapter_sequence               the adapter for read1. For SE data, if not specified, the adapter will be auto-detected. For PE data, this is used if R1/R2 are found not overlapped. (string [=auto])
      --adapter_sequence_r2            the adapter for read2 (PE data only). This is used if R1/R2 are found not overlapped. If not specified, it will be the same as <adapter_sequence> (string [=])
    
  # global trimming options   剪除序列起始和末端的低质量碱基数量参数
  -f, --trim_front1                  trimming how many bases in front for read1, default is 0 (int [=0])
  -t, --trim_tail1                   trimming how many bases in tail for read1, default is 0 (int [=0])
  -F, --trim_front2                  trimming how many bases in front for read2. If it's not specified, it will follow read1's settings (int [=0])
  -T, --trim_tail2                   trimming how many bases in tail for read2. If it's not specified, it will follow read1's settings (int [=0])

  # polyG tail trimming, useful for NextSeq/NovaSeq data   polyG剪裁
  -g, --trim_poly_g                  force polyG tail trimming, by default trimming is automatically enabled for Illumina NextSeq/NovaSeq data
      --poly_g_min_len                 the minimum length to detect polyG in the read tail. 10 by default. (int [=10])
  -G, --disable_trim_poly_g          disable polyG tail trimming, by default trimming is automatically enabled for Illumina NextSeq/NovaSeq data

  # polyX tail trimming
  -x, --trim_poly_x                    enable polyX trimming in 3' ends.
      --poly_x_min_len                 the minimum length to detect polyX in the read tail. 10 by default. (int [=10])
  
  # per read cutting by quality options   划窗裁剪
  -5, --cut_by_quality5              enable per read cutting by quality in front (5'), default is disabled (WARNING: this will interfere deduplication for both PE/SE data)
  -3, --cut_by_quality3              enable per read cutting by quality in tail (3'), default is disabled (WARNING: this will interfere deduplication for SE data)
  -W, --cut_window_size              the size of the sliding window for sliding window trimming, default is 4 (int [=4])
  -M, --cut_mean_quality             the bases in the sliding window with mean quality below cutting_quality will be cut, default is Q20 (int [=20])
  
  # quality filtering options   根据碱基质量来过滤序列
  -Q, --disable_quality_filtering    quality filtering is enabled by default. If this option is specified, quality filtering is disabled
  -q, --qualified_quality_phred      the quality value that a base is qualified. Default 15 means phred quality >=Q15 is qualified. (int [=15])
  -u, --unqualified_percent_limit    how many percents of bases are allowed to be unqualified (0~100). Default 40 means 40% (int [=40])
  -n, --n_base_limit                 if one read's number of N base is >n_base_limit, then this read/pair is discarded. Default is 5 (int [=5])
  
  # length filtering options   根据序列长度来过滤序列
  -L, --disable_length_filtering     length filtering is enabled by default. If this option is specified, length filtering is disabled
  -l, --length_required              reads shorter than length_required will be discarded, default is 15. (int [=15])

  # low complexity filtering
  -y, --low_complexity_filter          enable low complexity filter. The complexity is defined as the percentage of base that is different from its next base (base[i] != base[i+1]).
  -Y, --complexity_threshold           the threshold for low complexity filter (0~100). Default is 30, which means 30% complexity is required. (int [=30])

  # filter reads with unwanted indexes (to remove possible contamination)
      --filter_by_index1               specify a file contains a list of barcodes of index1 to be filtered out, one barcode per line (string [=])
      --filter_by_index2               specify a file contains a list of barcodes of index2 to be filtered out, one barcode per line (string [=])
      --filter_by_index_threshold      the allowed difference of index barcode for index filtering, default 0 means completely identical. (int [=0])

  # base correction by overlap analysis options   通过overlap来校正碱基
  -c, --correction                   enable base correction in overlapped regions (only for PE data), default is disabled
  
  # UMI processing
  -U, --umi                          enable unique molecular identifer (UMI) preprocessing
      --umi_loc                      specify the location of UMI, can be (index1/index2/read1/read2/per_index/per_read, default is none (string [=])
      --umi_len                      if the UMI is in read1/read2, its length should be provided (int [=0])
      --umi_prefix                   if specified, an underline will be used to connect prefix and UMI (i.e. prefix=UMI, UMI=AATTCG, final=UMI_AATTCG). No prefix by default (string [=])
      --umi_skip                       if the UMI is in read1/read2, fastp can skip several bases following UMI, default is 0 (int [=0])

  # overrepresented sequence analysis
  -p, --overrepresentation_analysis    enable overrepresented sequence analysis.
  -P, --overrepresentation_sampling    One in (--overrepresentation_sampling) reads will be computed for overrepresentation analysis (1~10000), smaller is slower, default is 20. (int [=20])

  # reporting options
  -j, --json                         the json format report file name (string [=fastp.json])
  -h, --html                         the html format report file name (string [=fastp.html])
  -R, --report_title                 should be quoted with ' or ", default is "fastp report" (string [=fastp report])
  
  # threading options   设置线程数
  -w, --thread                       worker thread number, default is 3 (int [=3])
  
  # output splitting options
  -s, --split                        split output by limiting total split file number with this option (2~999), a sequential number prefix will be added to output name ( 0001.out.fq, 0002.out.fq...), disabled by default (int [=0])
  -S, --split_by_lines               split output by limiting lines of each file with this option(>=1000), a sequential number prefix will be added to output name ( 0001.out.fq, 0002.out.fq...), disabled by default (long [=0])
  -d, --split_prefix_digits          the digits for the sequential number padding (1~10), default is 4, so the filename will be padded as 0001.xxx, 0 to disable padding (int [=4])
  
  # help
  -?, --help                         print this message

虽然参数看起来比较多,但常用的主要包括以下几个部分:

  • 输入输出文件设置
  • 接头处理
  • 全局裁剪(即直接剪掉起始和末端低质量碱基)
  • 滑窗质量剪裁 (与trimmomatic相似)
  • 过滤过短序列
  • 校正碱基(用于双端测序)
  • 质量过滤
1、接头处理
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fastp默认启用了接头处理,但是可以使用-A命令来关掉。fastp可以自动化地查找接头序列并进行剪裁,也就是说你可以不输入任何的接头序列,fastp全自动搞定了!对于SE数据,你还是可以-a参数来输入你的接头,而对于PE数据则完全没有必要,fastp基于PE数据的overlap分析可以更准确地查找接头,去得更干净,而且对于一些接头本身就有碱基不匹配情况处理得更好。fastp对于接头去除会有一个汇总的报告。

2、全局裁剪
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fastp可以对所有read在头部和尾部进行统一剪裁,该功能在去除一些测序质量不好的cycle比较有用,比如151*2的PE测序中,最后一个cycle通常质量是非常低的,需要剪裁掉。使用-f和-t分别指定read1的头部和尾部的剪裁,使用-F和-T分别指定read2的头部和尾部的剪裁。

3、滑窗质量剪裁
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很多时候,一个read的低质量序列都是集中在read的末端,也有少部分是在read的开头。fastp支持像Trimmomatic那样对滑动窗口中的碱基计算平均质量值,然后将不符合的滑窗直接剪裁掉。使用-5参数开启在5’端,也就是read的开头的剪裁,使用-3参数开启在3’端,也就是read的末尾的剪裁。使用-W参数指定滑动窗大小,默认是4,使用-M参数指定要求的平均质量值,默认是20,也就是Q20。

4、过滤过短序列
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默认开启多序列过滤,默认值为15,使用-L(--disable_length_filtering)禁止此默认选项。或使用-l(--length_required)自定义最短序列。

5、校正碱基(用于双端测序)
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fastp支持对PE数据的每一对read进行分析,查找它们的overlap区间,然后对于overlap区间中不一致的碱基,如果发现其中一个质量非常高,而另一个非常低,则可以将非常低质量的碱基改为相应的非常高质量值的碱基值。此选项默认关闭,可使用-c(--correction)开启。

6、质量过滤
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fastp可以对低质量序列,较多N的序列,该功能默认是启用的,但可以使用-Q参数关闭。使用-q参数来指定合格的phred质量值,比如-q 15表示质量值大于等于Q15的即为合格,然后使用-u参数来指定最多可以有多少百分比的质量不合格碱基。比如-q 15 -u 40表示一个read最多只能有40%的碱基的质量值低于Q15,否则会被扔掉。使用-n可以限定一个read中最多能有多少个N。

fastp的使用示例

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#!/bin/bash

for i in 74 75 76 82 83 84 85 86 87 88; do
    {
    fastp -i ~/RNAseq/cleandata/SRR17343${i}_1.fastq.gz -o SRR17343${i}_1.fastq.gz \
        -I ~/RNAseq/cleandata/SRR17343${i}_2.fastq.gz -O SRR17343${i}_2.fastq.gz \
        -Q --thread=5 --length_required=50 --n_base_limit=6 --compression=6
    }&
done
wait