2021/10/21, 24 インストール追記
FLEXBARはMultiplexで読んだシーケンスのdemultiplexやアダプタートリミングに使われるツール。柔軟な条件でランできる。よく使われているらしく、現在Flexbar3まで発表されている。解析時間は短く、100Mのリードなら数秒〜10秒程度の時間でアダプターをトリミングできる。ペアリードの順番は破壊しないので安心して使える。
マニュアル
インストール
Binaryをダウンロードして（リンクのmac版）、環境変数にパスを通す。
export DYLD_LIBRARY_PATH=/path/FlexbarDir:$DYLD_LIBRARY_PATH #カレントがダウンロードしたFlexbarDir/なら後半は ='pwd':$DYLD_LIBRARY_PATH
#conda (20211021追記)
mamba install -c bioconda -y flexbar
> flexbar --help
$ flexbar -h
flexbar - flexible barcode and adapter removal
==============================================
SYNOPSIS
flexbar -r reads [-b barcodes] [-a adapters] [options]
DESCRIPTION
The program Flexbar preprocesses high-throughput sequencing data
efficiently. It demultiplexes barcoded runs and
removes adapter sequences. Several adapter removal presets for
Illumina libraries are included. Flexbar computes
exact overlap alignments using SIMD and multicore parallelism.
Moreover, trimming and filtering features are
provided, e.g. trimming of homopolymers at read ends. Flexbar
increases read mapping rates and improves genome as
well as transcriptome assemblies. Unique molecular identifiers can
be extracted in a flexible way. The software
supports data in fasta and fastq format from multiple sequencing
platforms. Refer to the manual on
github.com/seqan/flexbar/wiki or contact Johannes Roehr on
github.com/jtroehr for support with this application.
OPTIONS
-h, --help
Display the help message.
-hh, --full-help
Display the help message with advanced options.
-v, --versions
Print Flexbar and SeqAn version numbers.
-c, --cite
Show program references for citation.
Basic options:
-n, --threads INTEGER
Number of threads to employ. Default: 1.
-t, --target OUTPUT_PREFIX
Prefix for output file names or paths. Default: flexbarOut.
-r, --reads INPUT_FILE
Fasta/q file or stdin (-) with reads that may contain barcodes.
-p, --reads2 INPUT_FILE
Second input file of paired reads, gz and bz2 files supported.
Barcode detection:
-b, --barcodes INPUT_FILE
Fasta file with barcodes for demultiplexing, may contain N.
-br, --barcode-reads INPUT_FILE
Fasta/q file containing separate barcode reads for detection.
-bo, --barcode-min-overlap INTEGER
Minimum overlap of barcode and read. Default: barcode length.
-be, --barcode-error-rate DOUBLE
Error rate threshold for mismatches and gaps. Default: 0.0.
-bt, --barcode-trim-end STRING
Type of detection, see section trim-end modes. Default: LTAIL.
Adapter removal:
-a, --adapters INPUT_FILE
Fasta file with adapters for removal that may contain N.
-a2, --adapters2 INPUT_FILE
File with extra adapters for second read set in paired mode.
-aa, --adapter-preset STRING
One of TruSeq, SmallRNA, Methyl, Ribo, Nextera, and NexteraMP.
-ao, --adapter-min-overlap INTEGER
Minimum overlap for removal without pair overlap. Default: 3.
-ae, --adapter-error-rate DOUBLE
Error rate threshold for mismatches and gaps. Default: 0.1.
-at, --adapter-trim-end STRING
Type of removal, see section trim-end modes. Default: RIGHT.
-ap, --adapter-pair-overlap STRING
Overlap detection of paired reads. One of ON, SHORT, and ONLY.
Filtering and trimming:
-u, --max-uncalled INTEGER
Allowed uncalled bases N for each read. Default: 0.
-x, --pre-trim-left INTEGER
Trim given number of bases on 5' read end before detection.
-y, --pre-trim-right INTEGER
Trim specified number of bases on 3' end prior to detection.
-m, --min-read-length INTEGER
Minimum read length to remain after removal. Default: 18.
Quality-based trimming:
-q, --qtrim STRING
Quality-based trimming mode. One of TAIL, WIN, and BWA.
-qf, --qtrim-format STRING
Quality format. One of sanger, solexa, i1.3, i1.5, and i1.8.
-qt, --qtrim-threshold INTEGER
Minimum quality as threshold for trimming. Default: 20.
Trimming of homopolymers:
-hr, --htrim-right STRING
Trim certain homopolymers on right read end after removal.
-hi, --htrim-min-length INTEGER
Minimum length of homopolymers at read ends. Default: 3.
-he, --htrim-error-rate DOUBLE
Error rate threshold for mismatches. Default: 0.1.
Output selection:
-f, --fasta-output
Prefer non-quality format fasta for output.
-z, --zip-output STRING
Direct compression of output files. One of GZ and BZ2.
-1, --stdout-reads
Write reads to stdout, tagged and interleaved if needed.
Logging and tagging:
-l, --align-log STRING
Print chosen read alignments. One of ALL, MOD, and TAB.
-o, --stdout-log
Write statistics to stdout instead of target log file.
-g, --removal-tags
Tag reads that are subject to adapter or barcode removal.
TRIM-END MODES
ANY: longer side of read remains after removal of overlap
LEFT: right side remains after removal, align <= read end
RIGHT: left part remains after removal, align >= read start
LTAIL: consider first n bases of reads in alignment
RTAIL: use only last n bases, see tail-length options
EXAMPLES
flexbar -r reads.fq -t target -q TAIL -qf i1.8
flexbar -r reads.fq -b barcodes.fa -bt LTAIL
flexbar -r reads.fq -a adapters.fa -ao 3 -ae 0.1
flexbar -r r1.fq -p r2.fq -a a1.fa -a2 a2.fa -ap ON
flexbar -r r1.fq -p r2.fq -aa TruSeq -ap ON
VERSION
Last update: May 2019
flexbar version: 3.5.0
SeqAn version: 2.4.0
Available on github.com/seqan/flexbar
ラン
アダプターを除く。
flexbar -r input.fastq -a adaptor.fa -be LTAIL -t target -n 4
- -t Prefix for output file names or paths. Default: flexbarOut.
- -r Fasta/q file or stdin (-) with reads that may contain barcodes.
- -p Second input file of paired reads, gz and bz2 files supported.
- -a Fasta file with adapters for removal that may contain N.
- -n Number of threads to employ. Default: 1.
- -be Type of detection, see section trim-end modes. Default: LTAIL.
TRIM-END MODES
- ANY: longer side of read remains after removal of overlap
- LEFT: right side remains after removal, align <= read end
- RIGHT: left part remains after removal, align >= read start
- LTAIL: consider first n bases of reads in alignment
- RTAIL: use only last n bases, see tail-length options
引用
Flexbar 3.0 - SIMD and multicore parallelization.
Roehr JT, Dieterich C, Reinert K.
Bioinformatics. 2017 Sep 15;33(18):2941-2942.
FLEXBAR-Flexible Barcode and Adapter Processing for Next-Generation Sequencing Platforms.
Dodt M, Roehr JT, Ahmed R, Dieterich C.
Biology (Basel). 2012 Dec 14;1(3):895-905.
