fastp软件介绍

1、软件介绍

  在18年之前，fq质控用FASTQC等软件，去接头序列用cutadapt软件，数据过滤用Trimmomatic等软件。
  后来海普洛斯的CTO开发了一款新软件fastp，整合了上述3个功能，实现了又快又好又个性化。

2、重要参数解析

2.1 全部参数

fastp: an ultra-fast all-in-one FASTQ preprocessor
version 0.19.5
usage: fastp [options] ... 
options:
  -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                        indicate 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 4. (int [=4])
      --stdin                          input from STDIN. If the STDIN is interleaved paired-end FASTQ, please also add --interleaved_in.
      --stdout                         stream passing-filters reads to STDOUT. This option will result in interleaved FASTQ output for paired-end input. Disabled by defaut.
      --interleaved_in                 indicate that  is an interleaved FASTQ which contains both read1 and read2. Disabled by defaut.
      --reads_to_process               specify how many reads/pairs to be processed. Default 0 means process all reads. (int [=0])
      --dont_overwrite                 don't overwrite existing files. Overwritting is allowed by default.
  -V, --verbose                        output verbose log information (i.e. when every 1M reads are processed).
  -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 [=auto])
      --detect_adapter_for_pe          by default, the auto-detection for adapter is for SE data input only, turn on this option to enable it for PE data.
  -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])
  -b, --max_len1                       if read1 is longer than max_len1, then trim read1 at its tail to make it as long as max_len1. Default 0 means no limitation (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])
  -B, --max_len2                       if read2 is longer than max_len2, then trim read2 at its tail to make it as long as max_len2. Default 0 means no limitation. If it's not specified, it will follow read1's settings (int [=0])
  -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
  -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])
  -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])
  -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])
  -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])
      --length_limit                   reads longer than length_limit will be discarded, default 0 means no limitation. (int [=0])
  -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_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])
  -c, --correction                     enable base correction in overlapped regions (only for PE data), default is disabled
      --overlap_len_require            the minimum length of the overlapped region for overlap analysis based adapter trimming and correction. 30 by default. (int [=30])
      --overlap_diff_limit             the maximum difference of the overlapped region for overlap analysis based adapter trimming and correction. 5 by default. (int [=5])
  -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])
  -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])
  -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])
  -w, --thread                         worker thread number, default is 2 (int [=2])
  -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                           print this message

  简书上有人以功能划分，重新归纳了参数如下，更加方便查看：

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  (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

2.2 使用示例

（1）最简单的使用示例

fastp -i in.fq -o out.fq # SE测序数据
fastp -i in.R1.fq -o out.R1.fq -I in.R2.fq -O out.R2.fq # PE测序书
fastp -i in.R1.fq.gz -I in.R2.fq.gz -o out.R1.fq.gz -O out.R2.fq.gz # 输入压缩文件，输出也为压缩文件

（2）结合常用参数的使用示例

fastp 
-i *_R1_raw.fastq.gz  # reads1 fastq
-I *_R2_raw.fastq.gz  # reads2 fastq
-o *_R1_trim.fastq.gz # reads1 处理结果
-O *_R2_trim.fastq.gz # reads2 处理结果
--adapter_sequence=AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC  # reads1 接头序列
--adapter_sequence_r2=AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT  # reads2 接头序列 
--thread=4 # 设置线程数
--length_required=55 # 过滤过短序列,自定义55以下为短序列
--compression=4 # 压缩比例，1最快, 9最慢
--trim_poly_g  # 开启polyG剪裁, 适用于Illumina NextSeq和NovaSeq系列数据
--cut_by_quality3 # 开启在3’端，也就是read末尾的剪裁
--correction # 通过overlap来校正碱基
--umi # 添加了umi技术的测序数据
--umi_loc per_read # 指定UMI所在的位置, per_read指在每个插入序列中
--umi_len 5 # UMI所占碱基长度
--umi_skip 3 # 去除UMI后,再去除3bp
-j *.trim.fastp.json # 适合程序读的JSON格式质控结果
-h *.trim.fastp.html # 适合人看的网页格式质控结果

2.3 重要参数详解

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  本部分内容来自参考文件：质控软件fastp常用参数说明_fastp参数_青灯照颦微的博客-CSDN博客；欢迎各位去读原文。

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（1）UMI去除

  分子标签(UMI)，来自于相同的分子的标记，用于去重，错误校正。常用在ctDNA测序，illumina测序的UMI位于两个不同位置：index和read开头。

--umi 启用UMI处理参数；

--umi_loc   指定UMI的位置，可设置下面几种：

"index1": 第一个index作为UMI, 对双端数据，则作用于R1/R2；
"index2": 第二个index作为UMI, 对双端数据，则作用于R1/R2；
"read1": read1的头部作为UMI,  对双端数据，则作用于R1/R2；
"read2": read2的头部作为UMI,  对双端数据，则作用于R1/R2；
"pre_index", "index1_index2": 
"pre_read": read1的头部定义'umi1', read2的头部定义'umi2', 'umi1_umi2'作为UMI, 作用于R1/R2

--umi_len   UMI的长度，当指定UMI的位置为read1, read2,per_read时，应指定UMI长度；

--umi_prefix   UMI设置前缀，例： UMI=AATTCCGG，prefix=ATC，即设置–umi_prefix=ATC，则被加在read_name行的UMI序列将会是ATC_AATTCCGG ；

--umi_skip    UMI去除并加到read_name后，再去除(跳过)的碱基数；例：–umi_skip=4 表示去除UMI后再去除4bp。

  fastp是将UMI提取后加在对应read的name行，如果UMI在read中，那么UMI会从read中移除，如果UMI在index中，会被保留。

（2）质量过滤

-q, --qualified_quality_phred   设置碱基质量值不小于多少时，该碱基为合格碱基，默认碱基质量值是15，即默认碱基质量>=15是合格碱基，<15为不合格碱基；

-u --unqualified_percent_limit   设置允许不合格碱基的占比为多少时，去掉这条read，默认是40，即默认不合格碱基占比>40%时，去掉该read；

-Q, --disable_quality_filtering   设置该参数则禁用默认质量过滤参数(-q, -u)。

（3）长度过滤

-l, --length_required   设置read的最小长度，默认是15，即长度<15的read被去掉；

--length_limit   设置read的最大长度, 默认为0是没有最大长度限制；

（4）低复杂度过滤

-Y, --complexity_threshold   设置read的复杂度过滤阈值，默认为30，即当read复杂度<30时，去掉该read。复杂度：

- 复杂度的定义为 一个碱基与其下一个相邻碱基不同的碱基个数占比；
- 例：一条长为51bp的read，有3个碱基与其下一个碱基不同
   seq = 'AAAATTTTTTTTTTTTTTTTTTTTTGGGGGGGGGGGGGGGGGGGGGGCCCC'
   其复杂度为：complexity = 3/(51-1) = 6%

-y, --low_complexity_filter   设置该参数则禁用默认复杂度过滤参数(-Y)

（5）adapter过滤

-A, --disable_adapter_trimming   设置该参数则禁用默认adapter过滤参数；

-a, --adapter_sequence   指定引物序列(对应SE数据的引物序列 或 对应PE数据的R1的引物序列)。对单端(SE)数据，可通过自动检测前~1Mreads的尾巴，去识别adapter，若设置该参数，则表示禁用自动识别adapter；

--adapter_sequence_r2   指定R2引物序列(对PE数据的R2)。对双端(PE)数据，是通过两条reads的overlap去adapter（由于该方法比较稳定，通常不必设置引物序列）。如果为找到overlap，用使用这些序列去adapter(是否设置都先通过overlap去adapter?)；

--detect_adapter_for_pe   默认对双端数据则默认不使用自动检测adapter(SE可自动检测)，设置该参数，表示对双端数据也启用自动检测方法；

--adapter_fasta   接头序列文件(fasta格式)，注意该fasta文件中的fasta序列长度至少6bp，否则会被跳过。

​   注：fastp首先去除自动化检测到的接头序列，或者使用–adapter_sequence |–adapter_sequence_r2指定的接头序列，然后去除由–adapter_fasta设置的接头序列。去除的接头序列分布可以在html/json文件中查看。

（6）通过质量值过滤每条read

  下面参数是通过滑动窗的平均质量值切除reads。

-W, --cut_window_size   设置滑动窗口大小；

-M, --cut_mean_quality   设置滑动窗口的平均质量值阈值，低于这个阈值则被切除；

可对两端分别进行切除：

对5'端的参数，与Trimmomatic中的LEADING参数方法相似：
 -5, --cut_front 是去除5'端低质量碱基，具体是指滑动窗从5'向末尾3’滑动，如果窗口内的碱基平均质量值低于阈值，则切除这些碱基，然后窗口继续滑动，直到达到阈值则不再去除；
     --cut_front_window_size 是设置从5'端开始的滑动窗的大小，即每个滑动窗包含几个碱基；
     --cut_front_mean_quality 设置从5'端开始的滑动窗平均质量值阈值，低于该阈值则切除这些碱基；
对3'端开始切除的参数与5'端类似，也与Trimmomatic中的TRAILING参数的方法类似：
  -3, --cut_tail 是去除3'端低质量碱基，具体是指滑动窗从3'向起始5’滑动，如果窗口内的碱基平均质量值低于阈值，则切除这些碱基，然后窗口继续滑动，直到达到阈值则不再去除；
      --cut_tail_window_size 是设置从3'端开始的滑动窗的大小；
      --cut_tail_mean_quality 设置从3'端开始的滑动窗平均质量值阈值，低于该阈值则切除这些碱基；

还有切除序列的其他参数：

-r, --cut_right   是切除右侧序列，-3与-r参数的差别是，前者是先进行碱基去除，达到阈值则不再去除碱基，然后继续滑动窗口；后者是前者进行后，继续滑动滑动窗，直到发现窗口内碱基的平均质量值低于阈值，则切除该窗口及右侧所有碱基。也就是使用该参数，就没必要设置–cut_tail参数 。

（7）ployG/ployX

  对Illumina的NextSeq/NovaSeq测序数据，常会用ployG发生(是因为这两个平台使用两个荧光信号，而没有信号时表示G)。fastp能够检测到ployG并去除（默认是NextSeq/NovaSeq平台，通过测序仪ID和fastq识别)

-g, --trim_poly_g   启用去除尾巴ployG；

--poly_g_min_len   设置去除尾巴’G’的最小长度，默认为10即尾巴ployG长度>10时，会被去除；

-G, --disable_trim_poly_g   禁用去除尾巴ployG；

-x, --polyX    启用去除polyX(polyA, polyT, polyC, polyG)，若同时设置–trim_poly_g和–ployX，则先进行ployG尾巴去重，再进行ployX(这样设置有助于ployA尾巴在G尾巴之前时，去重ployA尾巴[常见于mRNA-Seq])。

（8）PE数据的碱基校正(correction)

  fastp通过overlap进行分析，如果找到合适的overlap，当overlap区域的两个错配碱基中，一个碱基质量值较高，一个碱基质量值极低，该软件会将错配的两个碱基进行校正（？将低质量碱基校正为与高质量碱基互补的碱基）。对应的碱基质量值也校正为相同的值。

-c, --correction 对碱基校正，默认不启用该参数；使用该参数是基于检测overlap，overlap的可调参数有：

   --overlap_len_require overlap的长度要求，默认是30，即默认overlap区域的长度不低于30bp；否则认为无overlap；

   --overlap_diff_limit overlap中最大错配数，默认是5，即默认overlap时最多有5个错配；否则认为无overlap；

   --overlap_diff_percent_limit overlap中最大错配数在重叠区的占比，默认是20，即默认最大错配数的碱基占比不高于20%；否则认为无overlap。

（9）整体切除 【global trimming】

  整体切除一般是考虑到，illumina测序最后1个cycle或最后n个cycle测序质量较低，使用-t 1, --trim_tai1l=1参数将所有reads的末尾1bp去除；

-f, --trim_front1   对R1起始几bp进行去除，例如：-f 1或–trim_front1=1表示去除R1起始位置1bp碱基；

-t, --trim_tail1   对R1末尾几bp进行去除，例如：-t 2或–trim_tail1=2表示去除R1末尾位置1bp碱基；

-b, --max_len1   设置R1最大长度阈值，即R1的长度大于阈值，则在尾巴开始切除read直到与阈值相等，默认不切除。注意最大长度在最后一步处理；

-F, --trim_front2    与R1相似；不设置默认则与R1指定的参数相同；

-T, --trim_tail2   与R1相似；不设置默认则与R1指定的参数相同；

-B, --max_len2   设置R2最大长度，同-b参数。[注意最大长度在最后一步处理]

过滤reads顺序

## 过滤reads顺序：
1. 对UMI进行处理("--umi")
2. 整体切除的起始位置切除("-f", "-F") 
   # 比如UMI在5‘端却不知道序列时,可trim_front1 10 trim_front2 10来强制去除插入序列
3. 整体切除的尾巴位置切除("-t", "-T")
4. 5'端质量值切除("-cut_front")
5. 滑动窗切除("--cut_right")
6. 3'端质量值切除("--cut_tail")
7. ployG切除("--trim_ploy_g", 默认作用于'NovaSeq/NextSeq'的数据)
8. 根据overlap分析去adapter(PE数据)
9. 根据adapter序列去apapter("--adapter_sequence", "--adapter_sequence_r2", 对PE数据则跳过该步骤)
10. 去除polyX("--trim_poly_x")
11. 去除最大长度("--max_len")

（10）输出文件切分

  可通过设置分割成几个文件或者设置每个文件的行数 ，两者不可同时设置。

-s, --split   指定最多分割成几个文件；

-S, --split_by_lines   指定分割后的每个文件最多几行；

-d, --split_prefix_digits   设置输出文件的前缀数字位数，例如：–split_prefix_digits=4 --split=3 --out1=out.fq ， 则输出文件为0001.out.fq, 0002.out.fq, 0002.out.fq

（11）过表达序列分析 【overrepresented sequence analysis】

-p,--overrepresentation_analysis   启用该分析，默认仅统计序列长度为10bp, 20bp, 40bp, 100bp或 cycle -2 ；

-P, --overrepresentation_sampling   指定用于统计的reads数比例，默认20，即默认1/20的reads用于序列统计。例：设置-P 100 表示将1/100的reads用序列统计，设置-P 1 表示将所有reads用于统计(运行会很慢，默认20是平衡了速度和精确度)

  不仅有过表达序统计结果，还有循环中(cycles)的分布情况，并用图展示检测到的过表达序列，以便找到最多的序列。

3、软件质控结果文件部分说明

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  本部分结果，主要来自于博客生信学习笔记：fastp质控处理生成的report结果解读_fastp结果解读_twocanis的博客-CSDN博客，欢迎去读原文。

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3.1 Summary(整体结果)

General
  版本号、序列循环数、质控之前的平均长度、质控之后的平均长度、插入片段的峰值
Before filtering
  数据质控之前的（反应测序质量）：总的reads长度、总碱基长度、Q20合格率、Q30合格率、GC含量
After filtering
  质控之后的：内容同上
Filtering result
  reads的通过率、低质量的reads、含太多N值的reads

3.2 Adapter

3.3 Insert size estimation

  配对末端重叠分析，不同长度的Insert在reads中占的比例，相当于是DNA被打断后的长度分布。当插入片段大小<30或> 270，或包含太多错误，则不能被read读取，比如我这里就有28%的不可读reads）

3.4 Before filtering

  质控之前的数据质量、碱基含量以及kmer分析等，可直接在网页上用鼠标拖动放大缩小以及查看具体数据细节，或进行图片保存等操作

（1）reads质量
  在不同位置上的碱基质量分布，一般来讲质量应 >30 且波动较小为不错的数据

（2）碱基质量
  read各个位置上碱基比例分布，这个是为了分析碱基的分离程度。
  何为碱基分离？已知AT配对，CG配对，假如测序过程是比较随机的话（随机意味着好），那么在每个位置上A和T比例应该差不多，C和G的比例也应该差不多。
  如下图所示，两者之间即使有偏差也不应该太大，最好平均在1%以内，如果过高，除非有合理的原因，比如某些特定的捕获测序所致，否则都需要注意是不是测序过程有什么偏差。

（3）KMER计数

  fastp对5个碱基长度的所有组合的出现次数进行了统计，然后把它放在了一张表格中，表格的每一个元素为深背景白字，背景越深，则表示重复次数越多。这样，一眼望去，就可以发现有哪些异常的信息。鼠标可停留在某一具体组合上看出现次数和平均占比。

4、参考文件

1、GitHub - fastp: An ultra-fast all-in-one FASTQ preprocessor

2、fastp参数说明

3、fastp: 一款超快速全功能的FASTQ文件自动化质控+过滤+校正+预处理软件 - 知乎

4、质控软件fastp常用参数说明_fastp参数_青灯照颦微的博客-CSDN博客
5、生信学习笔记：fastp质控处理生成的report结果解读_fastp结果解读_twocanis的博客-CSDN博客