用于執(zhí)行FASTQ數(shù)據(jù) 的質量控制唐全，支持單端和雙端短讀數(shù)據(jù)，作為一體化的 FASTQ預處理器垃帅，fastp提供了 諸如質量分析剪勿，接頭修整酱固，讀 取過濾和基本校正等功能头朱。 在處理完所有讀取后班眯，這些值 將合并署隘，并且報告者將生成 HTML和JSON格式的報告 亚隙。它可以僅僅掃描FASTQ文件一次诊霹，就完成比FASTQC+cutadapt+Trimmomatic這三個軟件加起來的功能還多很多的功能畅哑，而且速度上比僅僅使用Trimmomatic一個軟件還要快3倍左右荠呐，因為它使用C++開發(fā)泥张，處處使用了高效算法媚创，而且完美支持多線程钞钙！
Fig.1
Fig.2
安裝
使用conda安裝conda install -c bioconda fastp=0.23.2瘫怜。
#看一下是否成功鲸湃，順便看一下help文檔
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 <in1> 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 <adapter_sequence> (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 <avg_qual, then this read/pair is discarded. Default 0 means no requirement (int [=0])
-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 to detect overlapped region of PE reads. This will affect overlap analysis based PE merge, adapter trimming and correction. 30 by default. (int [=30])
--overlap_diff_limit the maximum number of mismatched bases to detect overlapped region of PE reads. This will affect overlap analysis based PE merge, adapter trimming and correction. 5 by default. (int [=5])
--overlap_diff_percent_limit the maximum percentage of mismatched bases to detect overlapped region of PE reads. This will affect overlap analysis based PE merge, adapter trimming and correction. Default 20 means 20%. (int [=20])
-U, --umi enable unique molecular identifier (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 3 (int [=3])
-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])
--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
對數(shù)據(jù)自動進行全方位質控听系，生成人性化的報告靠胜。
過濾功能（低質量毕源，太短霎褐，太多N……）冻璃。
對每一個序列的頭部或尾部，計算滑動窗內的質量均值嫁审，并將均值較低的子序列進行切除（類似Trimmomatic的做法赖晶，但是快非常多）捂贿。
全局剪裁 （在頭/尾部厂僧，不影響去重），對于Illumina下機數(shù)據(jù)往往最后一到兩個cycle需要這樣處理德召。
去除接頭污染福荸。厲害的是敬锐，你不用輸入接頭序列台夺，因為算法會自動識別接頭序列并進行剪裁颤介。
對于雙端測序（PE）的數(shù)據(jù)滚朵，軟件會自動查找每一對read的重疊區(qū)域辕近，并對該重疊區(qū)域中不匹配的堿基對進行校正移宅。
去除尾部的polyG吞杭。對于Illumina NextSeq/NovaSeq的測序數(shù)據(jù)，因為是兩色法發(fā)光绢掰，polyG是常有的事滴劲，所以該特性對該兩類測序平臺默認打開。
fastp支持對PE數(shù)據(jù)的每一對read進行分析萧芙，查找它們的overlap區(qū)間双揪，然后對于overlap區(qū)間中不一致的堿基渔期，如果發(fā)現(xiàn)其中一個質量非常高疯趟，而另一個非常低信峻，則可以將非常低質量的堿基改為相應的非常高質量值的堿基值，該校正功能默認沒有開啟使用-c參數(shù)可以啟用矾策，對于一些對噪聲容忍度低的應用贾虽，比如液體活檢蓬豁，建議開啟取募。
可以對帶分子標簽（UMI）的數(shù)據(jù)進行預處理蟆技，不管UMI在插入片段還是在index上玩敏，都可以輕松處理。
可以將輸出進行分拆质礼，而且支持兩種模式旺聚，分別是指定分拆的個數(shù)，或者分拆后每個文件的行數(shù)眶蕉。
使用
fastp使用非常簡單砰粹，如果不清楚具體參數(shù)該如何設置，直接定義好輸入及輸出數(shù)據(jù)就可以快速實現(xiàn)造挽。
##單端數(shù)據(jù)
fastp -i R1.fastq.gz -o R1_clean.fastq.gz
##雙端數(shù)據(jù)
fastp -i R1.fastq.gz -o R1_clean.fastq.gz -I R2.fastq.gz -O R2_clean.fastq.gz
本次測試使用之前下載好的原始數(shù)據(jù)(GSE176393)碱璃，我的代碼
#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
##這里我添加了-p參數(shù)嵌器，查看過度表達序列的情況庇谆，也可不加，開啟會增加運行時間
輸出文件如下，兩個版本的報告和輸出結果文件。
Fig.3
報告詳解
報告全部包括這些內容饶囚，點擊每一個條目可以收縮或展開
Fig.4
Summary
Summary中包含了本次分析的情況概要。
Fig.5
- General
版本號梨与、序列循環(huán)數(shù)瞄崇、質控之前的平均長度、質控之后的平均長度、插入片段的峰值
- Before filtering
數(shù)據(jù)質控之前的（反應測序質量）：總的reads長度大渤、總堿基長度、Q20合格率、Q30合格率、GC含量
- After filtering
質控之后的：內容同上
- Filtering result
reads的通過率瞳抓、低質量的reads横蜒、含太多N值的reads
Adapters
這里列出了read1和read2從1到幾十位的adapters的發(fā)生次數(shù)，以及其他未列出的接頭數(shù)
Fig.6
Insert size estimation
配對末端重疊分析呆馁，不同長度的Insert在reads中占的比例纺腊，相當于是DNA被打斷后的長度分布次氨。當插入片段大小<30或> 270犀呼，或包含太多錯誤囊骤，則不能被讀取，比如我這里就有33.870706%的不可讀reads）
Fig.7
Before filtering
質控之前的數(shù)據(jù)質量枷踏、堿基含量以及kmer分析等下梢，可直接在網(wǎng)頁上用鼠標拖動放大縮小以及查看具體數(shù)據(jù)細節(jié)岗屏，或進行圖片保存等操作
reads質量
在不同位置上的堿基質量分布暇屋，一般來講質量應 >30 且波動較小為不錯的數(shù)據(jù)
Fig.8
堿基質量
read各個位置上堿基比例分布联予，這個是為了分析堿基的分離程度。何為堿基分離？已知AT配對裳朋，CG配對暖眼，假如測序過程是比較隨機的話（隨機意味著好），那么在每個位置上A和T比例應該差不多丛肢，C和G的比例也應該差不多，如上圖所示猪勇，兩者之間即使有偏差也不應該太大掀泳，最好平均在1%以內马僻，如果過高诗力，除非有合理的原因俺夕，比如某些特定的捕獲測序所致件甥，否則都需要注意是不是測序過程有什么偏差禁灼。
Fig.9
KMER計數(shù)
fastp對5個堿基長度的所有組合的出現(xiàn)次數(shù)進行了統(tǒng)計，然后把它放在了一張表格中斋荞，表格的每一個元素為深背景白字俺驶，背景越深，則表示重復次數(shù)越多塘幅。這樣，一眼望去猪叙，就可以發(fā)現(xiàn)有哪些異常的信息。鼠標可停留在某一具體組合上看出現(xiàn)次數(shù)和平均占比带膀。
Fig.10
過表達序列
提供了這些overrepresented sequence的序列個數(shù)和占比志珍，還提供了他們在測序cycles中的分布情況，這有利于分析各種問題
Fig.11
參考資料：
