摘錄自：
http://www.reibang.com/p/6f492058da5b
https://zhuanlan.zhihu.com/p/33601691
github:https://github.com/OpenGene/fastp
類似軟件：Trimmomatic（成熟淡诗，復(fù)雜）照藻，SOAPnuke（華大基因）
fastp的特性：
- 對(duì)數(shù)據(jù)自動(dòng)進(jìn)行全方位質(zhì)控韵洋，生成人性化的報(bào)告
- 過(guò)濾功能（低質(zhì)量，太短诱担，太多N……）;
- 對(duì)每一個(gè)序列的頭部或尾部，計(jì)算滑動(dòng)窗內(nèi)的質(zhì)量均值，并將均值較低的子序列進(jìn)行切除（類似Trimmomatic的做法，但是快非常多）;
- 全局剪裁 （在頭/尾部本股，不影響去重），對(duì)于Illumina下機(jī)數(shù)據(jù)往往最后一到兩個(gè)cycle需要這樣處理;
- 去除接頭污染桐腌。厲害的是，你不用輸入接頭序列苟径，因?yàn)樗惴〞?huì)自動(dòng)識(shí)別接頭序列并進(jìn)行剪裁;
- 對(duì)于雙端測(cè)序（PE）的數(shù)據(jù)案站，軟件會(huì)自動(dòng)查找每一對(duì)read的重疊區(qū)域，并對(duì)該重疊區(qū)域中不匹配的堿基對(duì)進(jìn)行校正;
- 去除尾部的polyG棘街。對(duì)于Illumina NextSeq/NovaSeq的測(cè)序數(shù)據(jù)蟆盐，因?yàn)槭莾缮òl(fā)光，polyG是常有的事遭殉，所以該特性對(duì)該兩類測(cè)序平臺(tái)默認(rèn)打開;
- 對(duì)于PE數(shù)據(jù)中的overlap區(qū)間中不一致的堿基對(duì)石挂，依據(jù)質(zhì)量值進(jìn)行校正;
- 可以對(duì)帶分子標(biāo)簽（UMI）的數(shù)據(jù)進(jìn)行預(yù)處理，不管UMI在插入片段還是在index上险污，都可以輕松處理;
- 可以將輸出進(jìn)行分拆痹愚，而且支持兩種模式，分別是指定分拆的個(gè)數(shù)蛔糯，或者分拆后每個(gè)文件的行數(shù);
以上功能大多都不需要輸入太多的參數(shù)拯腮，一些功能默認(rèn)已經(jīng)開啟，但是可以用參數(shù)關(guān)閉蚁飒。fastp完美支持gzip的輸入和輸出动壤，同時(shí)支持SE和PE數(shù)據(jù)，而且不但支持像Illumina平臺(tái)的short read數(shù)據(jù)淮逻，也在一定程度上支持了PacBio/Nanopore的long reads數(shù)據(jù)琼懊。
fastp軟件會(huì)生成HTML格式的報(bào)告阁簸，而且該報(bào)告中沒有任何一張靜態(tài)圖片，所有的圖表都是使用JavaScript動(dòng)態(tài)繪制哼丈，非常具有交互性启妹。想要看一下樣板報(bào)告的，可以去以下鏈接：http://opengene.org/fastp/fastp.html
而且軟件的開發(fā)者還充分考慮到了各種自動(dòng)化分析的需求削祈，不但生成了人可讀的HTML報(bào)告翅溺，還生成了程序可讀性非常強(qiáng)的JSON結(jié)果，該JSON報(bào)告中的數(shù)據(jù)包含了HTML報(bào)告100%的信息髓抑，而且該JSON文件的格式還是特殊定制的咙崎，不但程序讀得爽，你用任何一款文本編輯器打開吨拍，一眼過(guò)去也會(huì)看得明明白白褪猛。想要看一下JSON結(jié)果長(zhǎng)什么樣的，可以去以下鏈接：http://opengene.org/fastp/fastp.json
下面我們先來(lái)看看fastp的具體參數(shù)：
usage: fastp -i <in1> -o <out1> [-I <in1> -O <out2>] [options...]
options:
# I/O options 即輸入輸出文件設(shè)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 過(guò)濾序列接頭參數(shù)設(shè)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質(zhì)量堿基數(shù)量參數(shù)
-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 劃窗裁剪
-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 根據(jù)堿基質(zhì)量來(lái)過(guò)濾序列
-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 根據(jù)序列長(zhǎng)度來(lái)過(guò)濾序列
-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 通過(guò)overlap來(lái)校正堿基
-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 設(shè)置線程數(shù)
-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
雖然參數(shù)看起來(lái)比較多羹饰，但常用的主要包括以下幾個(gè)部分：
輸入輸出文件設(shè)置
接頭處理
全局裁剪（即直接剪掉起始和末端低質(zhì)量堿基）
滑窗質(zhì)量剪裁 （與trimmomatic相似）
過(guò)濾過(guò)短序列
校正堿基（用于雙端測(cè)序）
質(zhì)量過(guò)濾
功能
接頭處理
fastp默認(rèn)啟用了接頭處理伊滋，但是可以使用-A命令來(lái)關(guān)掉。fastp可以自動(dòng)化地查找接頭序列并進(jìn)行剪裁队秩，也就是說(shuō)你可以不輸入任何的接頭序列笑旺，fastp全自動(dòng)搞定了！對(duì)于SE數(shù)據(jù)馍资，你還是可以-a參數(shù)來(lái)輸入你的接頭筒主，而對(duì)于PE數(shù)據(jù)則完全沒有必要，fastp基于PE數(shù)據(jù)的overlap分析可以更準(zhǔn)確地查找接頭鸟蟹，去得更干凈乌妙，而且對(duì)于一些接頭本身就有堿基不匹配情況處理得更好。fastp對(duì)于接頭去除會(huì)有一個(gè)匯總的報(bào)告建钥。全局裁剪
fastp可以對(duì)所有read在頭部和尾部進(jìn)行統(tǒng)一剪裁藤韵，該功能在去除一些測(cè)序質(zhì)量不好的cycle比較有用，比如151*2的PE測(cè)序中熊经，最后一個(gè)cycle通常質(zhì)量是非常低的泽艘，需要剪裁掉。使用-f和-t分別指定read1的頭部和尾部的剪裁镐依，使用-F和-T分別指定read2的頭部和尾部的剪裁悉盆。滑窗質(zhì)量剪裁
很多時(shí)候，一個(gè)read的低質(zhì)量序列都是集中在read的末端馋吗，也有少部分是在read的開頭焕盟。fastp支持像Trimmomatic那樣對(duì)滑動(dòng)窗口中的堿基計(jì)算平均質(zhì)量值，然后將不符合的滑窗直接剪裁掉。使用-5參數(shù)開啟在5’端脚翘，也就是read的開頭的剪裁灼卢，使用-3參數(shù)開啟在3’端，也就是read的末尾的剪裁来农。使用-W參數(shù)指定滑動(dòng)窗大小鞋真，默認(rèn)是4，使用-M參數(shù)指定要求的平均質(zhì)量值沃于，默認(rèn)是20涩咖，也就是Q20。過(guò)濾過(guò)短序列
默認(rèn)開啟多序列過(guò)濾繁莹，默認(rèn)值為15檩互，使用-L（--disable_length_filtering）禁止此默認(rèn)選項(xiàng)∽裳荩或使用-l（--length_required）自定義最短序列闸昨。校正堿基（用于雙端測(cè)序）
fastp支持對(duì)PE數(shù)據(jù)的每一對(duì)read進(jìn)行分析，查找它們的overlap區(qū)間薄风，然后對(duì)于overlap區(qū)間中不一致的堿基饵较，如果發(fā)現(xiàn)其中一個(gè)質(zhì)量非常高，而另一個(gè)非常低遭赂，則可以將非常低質(zhì)量的堿基改為相應(yīng)的非常高質(zhì)量值的堿基值循诉。此選項(xiàng)默認(rèn)關(guān)閉，可使用-c（--correction）開啟撇他。質(zhì)量過(guò)濾
fastp可以對(duì)低質(zhì)量序列茄猫，較多N的序列，該功能默認(rèn)是啟用的逆粹，但可以使用-Q參數(shù)關(guān)閉。使用-q參數(shù)來(lái)指定合格的phred質(zhì)量值炫惩，比如-q 15表示質(zhì)量值大于等于Q15的即為合格僻弹，然后使用-u參數(shù)來(lái)指定最多可以有多少百分比的質(zhì)量不合格堿基。比如-q 15 -u 40表示一個(gè)read最多只能有40%的堿基的質(zhì)量值低于Q15他嚷，否則會(huì)被扔掉蹋绽。使用-n可以限定一個(gè)read中最多能有多少個(gè)N。
例子
最后筋蓖，附一個(gè)簡(jiǎn)單的例子：
#!/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
fastp結(jié)果解讀
接下來(lái)卸耘，我們?cè)倏匆幌氯绾卫斫鈌astp生成的質(zhì)控報(bào)告。fastp的報(bào)告在單一文件中同時(shí)包含了過(guò)濾前和過(guò)濾后的統(tǒng)計(jì)結(jié)果粘咖，如果是PE數(shù)據(jù)蚣抗，則同時(shí)包含了read1和read2的統(tǒng)計(jì)結(jié)果。之前我們已經(jīng)說(shuō)過(guò)了瓮下，fastp會(huì)生成HTML的報(bào)告和JSON格式的報(bào)告翰铡。HTML報(bào)告的默認(rèn)文件名是fastp.html钝域，但是可以通過(guò)-h參數(shù)修改，JSON報(bào)告的默認(rèn)文件名是fastp.json锭魔，但是可以通過(guò)-j參數(shù)修改例证。而且fastp報(bào)告還有一個(gè)標(biāo)題，默認(rèn)是fastp report迷捧，這個(gè)也可以通過(guò)-R參數(shù)修改為你想要的標(biāo)題织咧。JSON格式的報(bào)告是優(yōu)化過(guò)的，人機(jī)皆可讀漠秋，適合進(jìn)階的用戶使用程序解析笙蒙，而這里我們重點(diǎn)關(guān)注HTML格式的報(bào)告。
-
質(zhì)量分布曲線圖
我們第一關(guān)注的當(dāng)然是質(zhì)量膛堤，所以fastp提供了質(zhì)量分布曲線手趣，即每一個(gè)cycle的平均質(zhì)量值，而且fastp同時(shí)提供了A/T/C/G四種不同堿基的平均質(zhì)量肥荔，以及總的平均質(zhì)量绿渣，如下圖所示：
image
從上圖我們可以看到，一共有5條曲線燕耿，分別是A/T/C/G和mean中符。而且HTML報(bào)告中的每一個(gè)項(xiàng)目和分項(xiàng)目都是可以點(diǎn)擊進(jìn)行隱藏和展開的。
-
堿基含量分布曲線
和質(zhì)量分布曲線類似誉帅，堿基含量分布曲線也是按照每一個(gè)cycle來(lái)的淀散，顯示了每一個(gè)位置的堿基含量。如下圖所示：
image
image
從圖中可以看到蚜锨，fastp同時(shí)顯示了A/T/C/G/N/GC
的每一個(gè)位置的比例和總的比例档插。而且如果你覺得頭部那里比較亂看不清的話，可以用鼠標(biāo)拉一個(gè)框亚再，它就放大了郭膛。 -
KMER統(tǒng)計(jì)表格
fastp對(duì)5個(gè)堿基長(zhǎng)度的所有組合的出現(xiàn)次數(shù)進(jìn)行了統(tǒng)計(jì)，然后把它放在了一張表格中氛悬，表格的每一個(gè)元素為深背景白字则剃，背景越深，則表示重復(fù)次數(shù)越多如捅。這樣棍现，一眼望去，就可以發(fā)現(xiàn)有哪一些異常的信息镜遣。
image
從上面的KMER表格中己肮，我們可以發(fā)現(xiàn)，GGGGG的顏色特別深，從鼠標(biāo)移上去之后顯示的信息中我們可以發(fā)現(xiàn)它的出現(xiàn)次數(shù)是平均次數(shù)的12.8倍朴肺，這是不正常的窖剑，因?yàn)镚GGGG的正常倍數(shù)應(yīng)該在1倍左右。幸好我們有fastp戈稿，它可以過(guò)濾掉這種polyG西土，讓數(shù)值較多地回歸正常。
