fastpは既に5000回以上引用されている(PubMedより)人気のシークエンシングデータの前処理ツールだが、最近のアップグレード(*1)でいくつか新機能が追加された。新機能を簡単に確認しておく。
インストール
iMetaの論文ではv0.23.2が最新のバージョンのfastpとして、v0.20.0の古いバージョンのfastpと比較されている。
https://github.com/OpenGene/fastp
#CentOS/Ubuntu
wget http://opengene.org/fastp/fastp.0.23.4
mv fastp.0.23.4 fastp
chmod a+x ./fastp
#conda(versionに注意)
mamba install -c bioconda fastp -y
> fastp
$ fastp
fastp: an ultra-fast all-in-one FASTQ preprocessor
version 0.23.4
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])
--umi_delim delimiter to use between the read name and the UMI, default is : (string [=:])
-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
Citation:
Shifu Chen. 2023. Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta 2: e107
新機能(以前紹介していない機能も含む)
1,オーバーラップしたペアエンドリード領域のエラーコレクション
PEデータのオーバーラップ解析を行い、適切なオーバーラップが見つかった場合、ペアエンドリードのオーバーラップ領域のエラーを修正できる。具体的には、一方の塩基が高品質で、もう一方の塩基が超低品質であった場合、品質がより低い方の塩基を品質がより高い方の塩基に修正する。この機能はデフォルトでは有効になっていない。有効にするには -c または --correctionフラグを立てる。オーバーラップ検出のパラメータは、--overlap_len_require (デフォルト30)、 --overlap_diff_limit (デフォルト5)、 --overlap_diff_percent_limit (デフォルト20%)を調整できる。エラーコレクションの対象にするにはこれら3つの条件を同時に満たす必要がある。
fastp -i in.R1.fq.gz -I in.R2.fq.gz -o out.R1.fq.gz -O out.R2.fq.gz \
-c --overlap_len_require 30 --overlap_diff_limit 5 --overlap_diff_percent_limit 20 -h fastp.html
- -c 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])
2、Overrepresented sequence analysis
overrepresented配列解析はデフォルトでは無効で、有効にするには-pまたは--overrepresentation_analysisを指定する。速度とメモリを考慮して、fastpは10bp, 20bp, 40bp, 100bp, (cycles - 2 ) の長さの配列のみをカウントする。
fastp -i in.R1.fq.gz -I in.R2.fq.gz -o out.R1.fq.gz -O out.R2.fq.gz \
-p -P 20 -h fastp.html
- -p enable overrepresented sequence analysis.
- -P One in (--overrepresentation_sampling) reads will be computed for overrepresentation analysis (1~10000), smaller is slower, default is 20. (int [=20])
デフォルトでは、fastpは配列カウントに1/20リードだけ使用される。-Pオプションを指定することで変更できる。例えば、-P 100を指定すると、1/100のリードだけがカウントに使われ、-P 1を指定すると、すべてのリードが使われるが、非常に遅くなる。
分析結果はレポート中のタイル表に提示される。fastp はオーバープレゼンテーショ ン配列のカウント数だけでなく、その配列がサイクル間でどのように分布している かという情報も与えてくれる。
3,ペアエンドのマージ
ペアエンド入力では、-m/--mergeオプションを指定することでマージをサポートする。マージされたリードの保存ファイルは --merged_out <name> を、そうでない場合は --stdout を有効にして STDOUT にストリームする。
#マージされたリードのみ出力
fastp -i in.R1.fq.gz -I in.R2.fq.gz -m --merged_out merged.fq.gz
#マージされなかったリードも出力(ペアは同期)
fastp -i in.R1.fq.gz -I in.R2.fq.gz -m --merged_out merged.fq.gz \
--out1 QT1.fq.gz --out2 QT2.fq.gz
#マージされなかったリードも出力するがクオリティトリミングで同期されなくなったリードは破棄される。これを防ぐには--unpairedを付ける。
fastp -i in.R1.fq.gz -I in.R2.fq.gz -m --merged_out merged.fq.gz \
--out1 QT1.fq.gz --out2 QT2.fq.gz --unpaired1 unpair1.fq.gz --unpaired2 unpair2.fq.gz
- -m 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 [=])
- --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 [=])
4,duplication rate
デフォルトではfastpは重複率を評価する。このモジュールは1Gのメモリを使用し、10%~20%の実行時間を要する。不要な場合は--dont_eval_duplicationを付ける。FASTQデータの重複リードを実際に排除するには、-Dまたは--dedupを指定する。dedupが有効な場合、dup_calc_accuracyレベルはデフォルトで3に設定され、1〜6の任意の値に変更できる。(;fastpはハッシュアルゴリズムを使って同一の配列を見つける。ハッシュが衝突する可能性があるため、全リードの約0.01%が重複排除リードと誤って認識される可能性がある。重複計算の精度は、ハッシュバッファ数を増やすか、バッファサイズを大きくすることで向上する。オプション --dup_calc_accuracy でレベルを指定できる (1 ~ 6)。レベルが高いほどメモリ使用量が多くなり、実行時間も長くなる)
fastp -i in.R1.fq.gz -I in.R2.fq.gz -o out.R1.fq.gz -O out.R2.fq.gz \
-D --dup_calc_accuracy 3
#重複評価を無効にして高速化
fastp -i in.R1.fq.gz -I in.R2.fq.gz -o out.R1.fq.gz -O out.R2.fq.gz \
--dont_eval_duplication
- -D 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.
引用
*1
Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp
Shifu Chen
iMeta, Volume 2, Issue 2 • 1 May 2023
関連
オーバーラップするペアエンドリードのエラー修復にはBBMergeが利用できる