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NGS関連のインフォマティクス情報についてまとめています。

QC、エラー修復、トリミング、レポート作成を自動実行する AfterQC

 

AfterQCはfastqのフィルタリング、トリミング、エラー修復、およびクオリティチェックを全て自動で行なってくれるツールである。エラー修復はオーバーラップするペアードエンドリードのクオリティを比較して実行される。2017年に論文が発表された。

 

 インストール

Github

git clone https://github.com/OpenGene/AfterQC.git

$ after.py -h

 

Usage: Automatic Filtering, Trimming, Error Removing and Quality Control for Illumina fastq data 

 

Simplest usage:

cd to the folder containing your fastq data, run <python after.py>

 

Options:

  --version             show program's version number and exit

  -h, --help            show this help message and exit

  -1 READ1_FILE, --read1_file=READ1_FILE

                        file name of read1, required. If input_dir is

                        specified, then this arg is ignored.

  -2 READ2_FILE, --read2_file=READ2_FILE

                        file name of read2, if paired. If input_dir is

                        specified, then this arg is ignored.

  -7 INDEX1_FILE, --index1_file=INDEX1_FILE

                        file name of 7' index. If input_dir is specified, then

                        this arg is ignored.

  -5 INDEX2_FILE, --index2_file=INDEX2_FILE

                        file name of 5' index. If input_dir is specified, then

                        this arg is ignored.

  -d INPUT_DIR, --input_dir=INPUT_DIR

                        the input dir to process automatically. If read1_file

                        are input_dir are not specified, then current dir (.)

                        is specified to input_dir

  -g GOOD_OUTPUT_FOLDER, --good_output_folder=GOOD_OUTPUT_FOLDER

                        the folder to store good reads, by default it is named

                        'good', in the current directory

  -b BAD_OUTPUT_FOLDER, --bad_output_folder=BAD_OUTPUT_FOLDER

                        the folder to store bad reads, by default it is named

                        'bad', in the same folder as good_output_folder

  -r REPORT_OUTPUT_FOLDER, --report_output_folder=REPORT_OUTPUT_FOLDER

                        the folder to store QC reports, by default it is named

                        'QC', in the same folder as good_output_folder

  --read1_flag=READ1_FLAG

                        specify the name flag of read1, default is R1, which

                        means a file with name *R1* is read1 file

  --read2_flag=READ2_FLAG

                        specify the name flag of read2, default is R2, which

                        means a file with name *R2* is read2 file

  --index1_flag=INDEX1_FLAG

                        specify the name flag of index1, default is I1, which

                        means a file with name *I1* is index2 file

  --index2_flag=INDEX2_FLAG

                        specify the name flag of index2, default is I2, which

                        means a file with name *I2* is index2 file

  -f TRIM_FRONT, --trim_front=TRIM_FRONT

                        number of bases to be trimmed in the head of read. -1

                        means auto detect

  -t TRIM_TAIL, --trim_tail=TRIM_TAIL

                        number of bases to be trimmed in the tail of read. -1

                        means auto detect

  --trim_pair_same=TRIM_PAIR_SAME

                        use same trimming configuration for read1 and read2 to

                        keep their sequence length identical, default is true

  -q QUALIFIED_QUALITY_PHRED, --qualified_quality_phred=QUALIFIED_QUALITY_PHRED

                        the quality value that a base is qualifyed. Default 15

                        means phred base quality >=Q15 is qualified.

  -u UNQUALIFIED_BASE_LIMIT, --unqualified_base_limit=UNQUALIFIED_BASE_LIMIT

                        if exists more than unqualified_base_limit bases that

                        quality is lower than qualified quality, then this

                        read/pair is bad. Default is 60

  -p POLY_SIZE_LIMIT, --poly_size_limit=POLY_SIZE_LIMIT

                        if exists one polyX(polyG means GGGGGGGGG...), and its

                        length is >= poly_size_limit, then this read/pair is

                        bad. Default is 35

  -a ALLOW_MISMATCH_IN_POLY, --allow_mismatch_in_poly=ALLOW_MISMATCH_IN_POLY

                        the count of allowed mismatches when detection polyX.

                        Default 2 means allow 2 mismatches for polyX detection

  -n N_BASE_LIMIT, --n_base_limit=N_BASE_LIMIT

                        if exists more than maxn bases have N, then this

                        read/pair is bad. Default is 5

  -s SEQ_LEN_REQ, --seq_len_req=SEQ_LEN_REQ

                        if the trimmed read is shorter than seq_len_req, then

                        this read/pair is bad. Default is 35

  --debubble            specify whether apply debubble algorithm to remove the

                        reads in the bubbles. Default is False

  --debubble_dir=DEBUBBLE_DIR

                        specify the folder to store output of debubble

                        algorithm, default is debubble

  --draw=DRAW           specify whether draw the pictures or not, when use

                        debubble or QC. Default is on

  --barcode=BARCODE     specify whether deal with barcode sequencing files,

                        default is on, which means all files with barcode_flag

                        in filename will be treated as barcode sequencing

                        files

  --barcode_length=BARCODE_LENGTH

                        specify the designed length of barcode

  --barcode_flag=BARCODE_FLAG

                        specify the name flag of a barcoded file, default is

                        barcode, which means a file with name *barcode* is a

                        barcoded file

  --barcode_verify=BARCODE_VERIFY

                        specify the verify sequence of a barcode which is

                        adjunct to the barcode

  --store_overlap=STORE_OVERLAP

                        specify whether store only overlapped bases of the

                        good reads

  --overlap_output_folder=OVERLAP_OUTPUT_FOLDER

                        the folder to store only overlapped bases of the good

                        reads

  --qc_only             if qconly is true, only QC result will be output, this

                        can be much fast

  --qc_sample=QC_SAMPLE

                        sample up to qc_sample reads when do QC, 0 means

                        sample all reads. Default is 200,000

  --qc_kmer=QC_KMER     specify the kmer length for KMER statistics for QC,

                        default is 8

  --no_correction       disable base correction for mismatched base pairs in

                        overlapped areas

  --mask_mismatch       set the qual num to 0 for mismatched base pairs in

                        overlapped areas to mask them out

  --no_overlap          disable overlap analysis (usually much faster with

                        this option)

——

 

ラン

シングルエンド

python after.py -1 R1.fq

ペアードエンド

python after.py -1 R1.fq -2 R2.fq

fastqのディレクトリを指定してラン(fastqを全て解析)。 

python after.py -d input/

 

ラン中にlogがほとんど出ないので不安になるが、200MBx2のfastqで10分くらいかかるのでいじらず待つ。解析が終わると、ランしたパスに3つのディレクトリができる。

f:id:kazumaxneo:20170917002342j:plain

上では2組のペアードエンドfastqフォルダを解析している。badにはトリミングをパスしなかったリードが収納されており、goodにトリミングされて最低限の長さを満たしたリードが収納されている。ペアリードの順番が崩れないように、goodにはペアリード両方についてPASSしたリードだけ出力されている。

 

 Q>30でトリミングし、25-bp以下になったリードは破棄する。

python after.py -1 R1.fq -2 R2.fq -q 30 -s 25

  フィルタリングオプション

  • -f <int> number of bases to be trimmed in the head of read. -1 means auto detect.
  • -t <int> number of bases to be trimmed in the tail of read. -1 means auto detect.
  • -q <int> the quality value that a base is qualifyed. Default 20 means base quality >=Q20 is qualified.
  • -u <int> if exists more than unqualified_base_limit bases that quality is lower than qualified quality, then this read/pair is bad. Default 0 means do not filter reads by low quality base count.
  • -p <int> POLY_SIZE_LIMIT if exists one polyX(polyG means GGGGGGGGG...), and its length is >= poly_size_limit, then this read/pair is bad. Default is 35.
  • -a <int> the count of allowed mismatches when evaluating poly_X. Default 5 means disallow any mismatches.
  • -n <int> if exists more than maxn bases have N, then this read/pair is bad. Default is 5.
  • -s <int> if the trimmed read is shorter than seq_len_req, then this read/pair is bad. Default is 35.

バブル除去オプション(一般のシーケンスには非推奨)

  • --debubble enable debubble algorithm to remove the reads in the bubbles. Default is False

バーコードオプション

  • --barcode specify whether deal with barcode sequencing files, default is on

クオリティチェックオプション

  • --qc_only enable this option, only QC result will be output, this can be much faster
  • --qc_kmer <int> specify the kmer length for KMER statistics for QC, default is 8

 

QC/には分析結果がhtmlで出力される。html5で描画されており、編集可能である。

f:id:kazumaxneo:20170917004054j:plain

f:id:kazumaxneo:20170917003650j:plain

f:id:kazumaxneo:20170917003652j:plain

f:id:kazumaxneo:20170917003656j:plain

f:id:kazumaxneo:20170917003659j:plain

f:id:kazumaxneo:20170917003703j:plain

f:id:kazumaxneo:20170917003706j:plain

f:id:kazumaxneo:20170917003709j:plain

f:id:kazumaxneo:20170917003713j:plain

f:id:kazumaxneo:20170917003717j:plain

f:id:kazumaxneo:20170917003723j:plain

f:id:kazumaxneo:20170917003729j:plain

f:id:kazumaxneo:20170917003726j:plain

f:id:kazumaxneo:20170917003732j:plain

 

 

AfterQCの 動作はfastqcとBBtools、scytheなどのツールを合わせたようなものになっている。動作はやや遅いため、後継のfastpの論文も発表されている。

   

引用

AfterQC: automatic filtering, trimming, error removing and quality control for fastq data.

Shifu Chen, Tanxiao Huang, Yanqing Zhou, Yue Han, Mingyan Xu and Jia Gu.

BMC Bioinformatics 2017 18(Suppl 3):80