用于执行FASTQ数据 的质量控制，支持单端和双端短读数据，作为一体化的 FASTQ预处理器，fastp提供了 诸如质量分析，接头修整，读 取过滤和基本校正等功能。 在处理完所有读取后，这些值 将合并，并且报告者将生成 HTML和JSON格式的报告 。它可以仅仅扫描FASTQ文件一次，就完成比FASTQC+cutadapt+Trimmomatic这三个软件加起来的功能还多很多的功能，而且速度上比仅仅使用Trimmomatic一个软件还要快3倍左右，因为它使用C++开发，处处使用了高效算法，而且完美支持多线程！
Fig.1
Fig.2
安装
使用conda安装conda install -c bioconda fastp=0.23.2。
#看一下是否成功，顺便看一下help文档
fastp
fastp: an ultra-fast all-in-one FASTQ preprocessor
version 0.23.2
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 [=])
--unpaired1 for PE input, if read1 passed QC but read2 not, it will be written to unpaired1\. Default is to discard it. (string [=])
--unpaired2 for PE input, if read2 passed QC but read1 not, it will be written to unpaired2\. If --unpaired2 is same as --unpaired1 (default mode), both unpaired reads will be written to this same file. (string [=])
--overlapped_out for each read pair, output the overlapped region if it has no any mismatched base. (string [=])
--failed_out specify the file to store reads that cannot pass the filters. (string [=])
-m, --merge for paired-end input, merge each pair of reads into a single read if they are overlapped. The merged reads will be written to the file given by --merged_out, the unmerged reads will be written to the files specified by --out1 and --out2\. The merging mode is disabled by default.
--merged_out in the merging mode, specify the file name to store merged output, or specify --stdout to stream the merged output (string [=])
--include_unmerged in the merging mode, write the unmerged or unpaired reads to the file specified by --merge. Disabled by default.
-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 output. Disabled by default.
--interleaved_in indicate that is an interleaved FASTQ which contains both read1 and read2\. Disabled by default.
--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.
--fix_mgi_id the MGI FASTQ ID format is not compatible with many BAM operation tools, enable this option to fix it.
-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 (string [=auto])
--adapter_fasta specify a FASTA file to trim both read1 and read2 (if PE) by all the sequences in this FASTA file (string [=])
--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])
-D, --dedup enable deduplication to drop the duplicated reads/pairs
--dup_calc_accuracy accuracy level to calculate duplication (1~6), higher level uses more memory (1G, 2G, 4G, 8G, 16G, 24G). Default 1 for no-dedup mode, and 3 for dedup mode. (int [=0])
--dont_eval_duplication don't evaluate duplication rate to save time and use less memory.
-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_front move a sliding window from front (5') to tail, drop the bases in the window if its mean quality < threshold, stop otherwise.
-3, --cut_tail move a sliding window from tail (3') to front, drop the bases in the window if its mean quality < threshold, stop otherwise.
-r, --cut_right move a sliding window from front to tail, if meet one window with mean quality < threshold, drop the bases in the window and the right part, and then stop.
-W, --cut_window_size the window size option shared by cut_front, cut_tail or cut_sliding. Range: 1~1000, default: 4 (int [=4])
-M, --cut_mean_quality the mean quality requirement option shared by cut_front, cut_tail or cut_sliding. Range: 1~36 default: 20 (Q20) (int [=20])
--cut_front_window_size the window size option of cut_front, default to cut_window_size if not specified (int [=4])
--cut_front_mean_quality the mean quality requirement option for cut_front, default to cut_mean_quality if not specified (int [=20])
--cut_tail_window_size the window size option of cut_tail, default to cut_window_size if not specified (int [=4])
--cut_tail_mean_quality the mean quality requirement option for cut_tail, default to cut_mean_quality if not specified (int [=20])
--cut_right_window_size the window size option of cut_right, default to cut_window_size if not specified (int [=4])
--cut_right_mean_quality the mean quality requirement option for cut_right, default to cut_mean_quality if not specified (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])
-e, --average_qual if one read's average quality score =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])
--cut_by_quality5 DEPRECATED, use --cut_front instead.
--cut_by_quality3 DEPRECATED, use --cut_tail instead.
--cut_by_quality_aggressive DEPRECATED, use --cut_right instead.
--discard_unmerged DEPRECATED, no effect now, see the introduction for merging.
-?, --help print this message
功能
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。
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，该校正功能默认没有开启使用-c参数可以启用，对于一些对噪声容忍度低的应用，比如液体活检，建议开启。
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。
使用
fastp使用非常简单，如果不清楚具体参数该如何设置，直接定义好输入及输出数据就可以快速实现。
##单端数据
fastp -i R1.fastq.gz -o R1_clean.fastq.gz
##双端数据
fastp -i R1.fastq.gz -o R1_clean.fastq.gz -I R2.fastq.gz -O R2_clean.fastq.gz
本次测试使用之前下载好的原始数据(GSE176393)，我的代码
#批量操作
cat SRR_Acc_List.txt | while read line;
do
fastp -i 0-rawdata/$line\_1.fastq.gz -o 1-fastp/$line\_clean_1.fastq.gz -I 0-rawdata/$line\_2.fastq.gz -O 1-fastp/$line\_clean_2.fastq.gz -j 1-fastp/$line.json -h 1-fastp/$line.html -z 9 -f 0 -t 0 -T 0 -F 0 -l 75 -p --thread=10
done
##这里我添加了-p参数，查看过度表达序列的情况，也可不加，开启会增加运行时间
输出文件如下，两个版本的报告和输出结果文件。
Fig.3
报告详解
报告全部包括这些内容，点击每一个条目可以收缩或展开
Fig.4
Summary
Summary中包含了本次分析的情况概要。
Fig.5
- 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
Adapters
这里列出了read1和read2从1到几十位的adapters的发生次数，以及其他未列出的接头数
Fig.6
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，则不能被读取，比如我这里就有33.870706%的不可读reads）
Fig.7
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
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
Fig.8
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，如上图所示，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。
Fig.9
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。
Fig.10
过表达序列
提供了这些overrepresented sequence的序列个数和占比，还提供了他们在测序cycles中的分布情况，这有利于分析各种问题
Fig.11
参考资料：
生信学习笔记：fastp质控处理生成的report结果解读
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