2019 6/19 インストール追記
2019 6/19 追記
2019 8/14 help追加
2019 8/15 run log追加
2020 11/1 コマンド追加
RNA reqのリードカウントツールを紹介する。
featureCounts
ダウンロード
sourceforgeリンク
https://sourceforge.net/projects/subread/files/subread-1.5.2/
インストール
ソースコードをダウンロードして解凍し、/srcに移動。macでは以下のようにしてビルドする。
cd subread-1.4.6-source/src
make -f Makefile.MacOS
#bioconda (link)
conda install -c bioconda subread> featureCounts
$ featureCounts
Version 1.6.4
Usage: featureCounts [options] -a <annotation_file> -o <output_file> input_file1 [input_file2] ...
## Mandatory arguments:
-a <string> Name of an annotation file. GTF/GFF format by default. See
-F option for more format information. Inbuilt annotations
(SAF format) is available in 'annotation' directory of the
package. Gzipped file is also accepted.
-o <string> Name of output file including read counts. A separate file
including summary statistics of counting results is also
included in the output ('<string>.summary'). Both files
are in tab delimited format.
input_file1 [input_file2] ... A list of SAM or BAM format files. They can be
either name or location sorted. If no files provided,
<stdin> input is expected. Location-sorted paired-end reads
are automatically sorted by read names.
## Optional arguments:
# Annotation
-F <string> Specify format of the provided annotation file. Acceptable
formats include 'GTF' (or compatible GFF format) and
'SAF'. 'GTF' by default. For SAF format, please refer to
Users Guide.
-t <string> Specify feature type in GTF annotation. 'exon' by
default. Features used for read counting will be
extracted from annotation using the provided value.
-g <string> Specify attribute type in GTF annotation. 'gene_id' by
default. Meta-features used for read counting will be
extracted from annotation using the provided value.
--extraAttributes Extract extra attribute types from the provided GTF
annotation and include them in the counting output. These
attribute types will not be used to group features. If
more than one attribute type is provided they should be
separated by comma.
-A <string> Provide a chromosome name alias file to match chr names in
annotation with those in the reads. This should be a two-
column comma-delimited text file. Its first column should
include chr names in the annotation and its second column
should include chr names in the reads. Chr names are case
sensitive. No column header should be included in the
file.
# Level of summarization
-f Perform read counting at feature level (eg. counting
reads for exons rather than genes).
# Overlap between reads and features
-O Assign reads to all their overlapping meta-features (or
features if -f is specified).
--minOverlap <int> Minimum number of overlapping bases in a read that is
required for read assignment. 1 by default. Number of
overlapping bases is counted from both reads if paired
end. If a negative value is provided, then a gap of up
to specified size will be allowed between read and the
feature that the read is assigned to.
--fracOverlap <float> Minimum fraction of overlapping bases in a read that is
required for read assignment. Value should be within range
[0,1]. 0 by default. Number of overlapping bases is
counted from both reads if paired end. Both this option
and '--minOverlap' option need to be satisfied for read
assignment.
--fracOverlapFeature <float> Minimum fraction of overlapping bases in a
feature that is required for read assignment. Value
should be within range [0,1]. 0 by default.
--largestOverlap Assign reads to a meta-feature/feature that has the
largest number of overlapping bases.
--nonOverlap <int> Maximum number of non-overlapping bases in a read (or a
read pair) that is allowed when being assigned to a
feature. No limit is set by default.
--nonOverlapFeature <int> Maximum number of non-overlapping bases in a feature
that is allowed in read assignment. No limit is set by
default.
--readExtension5 <int> Reads are extended upstream by <int> bases from their
5' end.
--readExtension3 <int> Reads are extended upstream by <int> bases from their
3' end.
--read2pos <5:3> Reduce reads to their 5' most base or 3' most base. Read
counting is then performed based on the single base the
read is reduced to.
# Multi-mapping reads
-M Multi-mapping reads will also be counted. For a multi-
mapping read, all its reported alignments will be
counted. The 'NH' tag in BAM/SAM input is used to detect
multi-mapping reads.
# Fractional counting
--fraction Assign fractional counts to features. This option must
be used together with '-M' or '-O' or both. When '-M' is
specified, each reported alignment from a multi-mapping
read (identified via 'NH' tag) will carry a fractional
count of 1/x, instead of 1 (one), where x is the total
number of alignments reported for the same read. When '-O'
is specified, each overlapping feature will receive a
fractional count of 1/y, where y is the total number of
features overlapping with the read. When both '-M' and
'-O' are specified, each alignment will carry a fractional
count of 1/(x*y).
# Read filtering
-Q <int> The minimum mapping quality score a read must satisfy in
order to be counted. For paired-end reads, at least one
end should satisfy this criteria. 0 by default.
--splitOnly Count split alignments only (ie. alignments with CIGAR
string containing 'N'). An example of split alignments is
exon-spanning reads in RNA-seq data.
--nonSplitOnly If specified, only non-split alignments (CIGAR strings do
not contain letter 'N') will be counted. All the other
alignments will be ignored.
--primary Count primary alignments only. Primary alignments are
identified using bit 0x100 in SAM/BAM FLAG field.
--ignoreDup Ignore duplicate reads in read counting. Duplicate reads
are identified using bit Ox400 in BAM/SAM FLAG field. The
whole read pair is ignored if one of the reads is a
duplicate read for paired end data.
# Strandness
-s <int or string> Perform strand-specific read counting. A single integer
value (applied to all input files) or a string of comma-
separated values (applied to each corresponding input
file) should be provided. Possible values include:
0 (unstranded), 1 (stranded) and 2 (reversely stranded).
Default value is 0 (ie. unstranded read counting carried
out for all input files).
# Exon-exon junctions
-J Count number of reads supporting each exon-exon junction.
Junctions were identified from those exon-spanning reads
in the input (containing 'N' in CIGAR string). Counting
results are saved to a file named '<output_file>.jcounts'
-G <string> Provide the name of a FASTA-format file that contains the
reference sequences used in read mapping that produced the
provided SAM/BAM files. This optional argument can be used
with '-J' option to improve read counting for junctions.
# Parameters specific to paired end reads
-p If specified, fragments (or templates) will be counted
instead of reads. This option is only applicable for
paired-end reads.
-B Only count read pairs that have both ends aligned.
-P Check validity of paired-end distance when counting read
pairs. Use -d and -D to set thresholds.
-d <int> Minimum fragment/template length, 50 by default.
-D <int> Maximum fragment/template length, 600 by default.
-C Do not count read pairs that have their two ends mapping
to different chromosomes or mapping to same chromosome
but on different strands.
--donotsort Do not sort reads in BAM/SAM input. Note that reads from
the same pair are required to be located next to each
other in the input.
# Number of CPU threads
-T <int> Number of the threads. 1 by default.
# Read groups
--byReadGroup Assign reads by read group. "RG" tag is required to be
present in the input BAM/SAM files.
# Long reads
-L Count long reads such as Nanopore and PacBio reads. Long
read counting can only run in one thread and only reads
(not read-pairs) can be counted. There is no limitation on
the number of 'M' operations allowed in a CIGAR string in
long read counting.
# Assignment results for each read
-R <format> Output detailed assignment results for each read or read-
pair. Results are saved to a file that is in one of the
following formats: CORE, SAM and BAM. See Users Guide for
more info about these formats.
--Rpath <string> Specify a directory to save the detailed assignment
results. If unspecified, the directory where counting
results are saved is used.
# Miscellaneous
--tmpDir <string> Directory under which intermediate files are saved (later
removed). By default, intermediate files will be saved to
the directory specified in '-o' argument.
--maxMOp <int> Maximum number of 'M' operations allowed in a CIGAR
string. 10 by default. Both 'X' and '=' are treated as 'M'
and adjacent 'M' operations are merged in the CIGAR
string.
--verbose Output verbose information for debugging, such as un-
matched chromosome/contig names.
-v Output version of the program.
マニュアル
featureCounts - quick guide
WEHI Bioinformatics - featureCounts
実行方法
inputはsam/bamファイルと、リファレンスのカウントするfeatureの場所を記したgtfファイルか、よりシンプルなSAFというフォーマットである。タブ区切りの5フィールドの形式で、公式サイトに例がある。
GeneID Chr Start End Strand 497097 chr1 3204563 3207049 - 497097 chr1 3411783 3411982 - 497097 chr1 3660633 3661579 -
シングルエンドのデータをカウント。例えば3つのbamからカウントデータを取得する。
featureCounts -T 8 -t exon -g gene_id -a annotation.gtf -o counts.txt input1.bam input2.bam input3.bam
- -T Number of the threads. 1 by default.
- -t Specify the feature type. Only rows which have the matched matched feature type in the provided GTF annotation file will be included for read counting. `exon' by default.
- -g Specify the attribute type used to group features (eg. exons) into meta-features (eg. genes), when GTF annotation is provided. `gene_id' by default.
- -a Give the name of the annotation file. The program assumes that the provided annotation file is in GTF format. Use -F option to specify other annotation formats.
- -o Give the name of the output file.
6サンプルカウントした結果のキャプチャが以下となる。
先頭部分数十行を載せている。出力はタブ区切りのテキストファイルで、1行目はコメント行（#）である。2行目以下は以下のような並びになっている。
‘Geneid’, ‘Chr’, ‘Start’, ‘End’, ‘Strand’,‘Length’,‘sample1_count’,‘sample2_count’ ...
２列目に1;1;1;1;1;1とあるのは、exsonが6あることを意味している。lengthは１つの遺伝子のオーバーラップをのぞいたexonの合計サイズ (bp) である。右端にあるのがカウントの列である。サンプル数分だけできる。
strand specificにカウント。
featureCounts -s 1 -T 8 -t exon -g gene_id -a annotation.gtf -o counts.txt <input.bam>
- -s Indicate if strand-specific read counting should be performed. It has three possible values: 0 (unstranded), 1 (stranded) and 2 (reversely stranded). 0 by default.
"-s 1"はfeatrureと同じ向きにアライメントされたリードだけカウント。"-s 2"はfeatureの反対向きにアライメントされたリードだけカウント。
ペアリードのフラグメントをカウント。
featureCounts -p -T 8 -t exon -g gene_id -a annotation.gtf -o counts.txt <input_PE.bam>
- -p If specified, fragments (or templates) will be counted instead of reads. This option is only applicable for paired-end reads. The two reads from the same fragment must be adjacent to each other in the provided SAM/BAM file.
複数ファイルを同時にカウントして出力する。
featureCounts -T 8 -t exon -g gene_id -a annotation.gtf -o counts.txt library1.bam library2.bam library3.bam
bam/samをスペース区切りで記載する。
ペアエンドの両方がマッピングされたものだけカウント。
featureCounts -p -B -t exon -g gene_id -a annotation.gtf -o counts.txt <input_PE.bam>
- -B If specified, only fragments that have both ends successfully aligned will be considered for summarization. This option is only applicable for paired-end reads.
ペアエンドの両方がマッピングされたものだけカウント。unstranded。featureは CDS、rRNA、tmRNA、tRNAをカウント（フィーチャーは実際にannotation.gtfを開いて確認すること*1）。
featureCounts -p -B -s 0 -T 8 -t CDS,rRNA,tmRNA,tRNA -g transcript_id -a annotation.gtf -o counts.txt <input_PE.bam>
紹介したオプション以外に、インサートサイズに下限、上限を設けるオプション（-P, -d, -D）や、異なるクロモソームにアライメントされたpaired-endを排除するオプション（-C）、マッピングクオリティの閾値（-Q）、複数箇所にマッピングされたリードのカウント（-M）、など多様なオプションがあります。詳細は公式quickマニュアルを確認してください。
実行時は標準出力にlogも表示される。
========== _____ _ _ ____ _____ ______ _____
===== / ____| | | | _ \| __ \| ____| /\ | __ \
===== | (___ | | | | |_) | |__) | |__ / \ | | | |
==== \___ \| | | | _ <| _ /| __| / /\ \ | | | |
==== ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
========== |_____/ \____/|____/|_| \_\______/_/ \_\_____/
v1.6.4
//========================== featureCounts setting ===========================\\
|| ||
|| Input files : 6 BAM files ||
|| S SRX2391505.bam ||
|| S SRX2391506.bam ||
|| S SRX2391507.bam ||
|| S SRX2391512.bam ||
|| S SRX2391513.bam ||
|| S SRX2391514.bam ||
|| ||
|| Output file : counts.txt ||
|| Summary : counts.txt.summary ||
|| Annotation : Escherichia_coli_k_12.ASM80076v1.44.gtf (GTF) ||
|| Dir for temp files : ./ ||
|| ||
|| Threads : 8 ||
|| Level : meta-feature level ||
|| Paired-end : no ||
|| Multimapping reads : not counted ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
\\============================================================================//
//================================= Running ==================================\\
|| ||
|| Load annotation file Escherichia_coli_k_12.ASM80076v1.44.gtf ... ||
|| Features : 4258 ||
|| Meta-features : 4257 ||
|| Chromosomes/contigs : 1 ||
|| ||
|| Process BAM file SRX2391505.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 11635683 ||
|| Successfully assigned alignments : 8362707 (71.9%) ||
|| Running time : 0.03 minutes ||
|| ||
|| Process BAM file SRX2391506.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 9923480 ||
|| Successfully assigned alignments : 7888505 (79.5%) ||
|| Running time : 0.02 minutes ||
|| ||
|| Process BAM file SRX2391507.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 15194419 ||
|| Successfully assigned alignments : 12625859 (83.1%) ||
|| Running time : 0.04 minutes ||
|| ||
|| Process BAM file SRX2391512.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 14376088 ||
|| Successfully assigned alignments : 11823380 (82.2%) ||
|| Running time : 0.03 minutes ||
|| ||
|| Process BAM file SRX2391513.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 13734901 ||
|| Successfully assigned alignments : 11896845 (86.6%) ||
|| Running time : 0.03 minutes ||
|| ||
|| Process BAM file SRX2391514.bam... ||
|| Single-end reads are included. ||
|| Assign alignments to features... ||
|| Total alignments : 11465553 ||
|| Successfully assigned alignments : 9222513 (80.4%) ||
|| Running time : 0.03 minutes ||
|| ||
|| ||
|| Summary of counting results can be found in file "counts.txt.summary" ||
|| ||
\\============================================================================//
引用
featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.
Liao Y, Smyth GK and Shi W (2014).
Bioinformatics. 2014 Apr 1;30(7):923-30.
関連
オプションについてはbioinformaticsさんのページが詳しいです。
*1
featureCountsラン開始時にfeature数が画面に表示されるので、合っているか確認すること。
