featurecounts

Read counting

Genomic features

Description

featureCounts is a read summarization program for counting reads generated from either RNA or genomic DNA sequencing experiments by implementing highly efficient chromosome hashing and feature blocking techniques. It works with either single or paired-end reads and provides a wide range of options appropriate for different sequencing applications.

Inputs

Name	Type & Properties	Description
--annotation -a	file required	Name of an annotation file. GTF/GFF format by default. See '--format' option for more format information.
--input -i	file required multiple	A list of SAM or BAM format files separated by semi-colon (;). They can be either name or location sorted. Location-sorted paired-end reads are automatically sorted by read names.

Name

Type & Properties

--annotation

-a

file

required

--input

-i

file

required

multiple

Outputs

Name	Type & Properties	Description
--counts -o	file required output	Name of output file including read counts in tab delimited format.
--summary	file output	Summary statistics of counting results in tab delimited format.
--junctions	file output	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).

Name

Type & Properties

--counts

-o

file

required

output

--summary

file

output

--junctions

file

output

Annotation

Name	Type & Properties	Description
--format -F	string	Specify format of the provided annotation file. Acceptable formats include 'GTF' (or compatible GFF format) and 'SAF'. 'GTF' by default.
--feature_type -t	string multiple	Specify feature type(s) in a GTF annotation. If multiple types are provided, they should be separated by ';' with no space in between. 'exon' by default. Rows in the annotation with a matched feature will be extracted and used for read mapping.
--attribute_type -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.
--extra_attributes	string multiple	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 semicolon (;).
--chrom_alias -A	file	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.

Name

Type & Properties

--format

-F

string

--feature_type

-t

string

multiple

--attribute_type

-g

string

--extra_attributes

string

multiple

--chrom_alias

-A

file

Level of summarization

Name	Type & Properties	Description
--feature_level -f	boolean_true	Perform read counting at feature level (eg. counting reads for exons rather than genes).

Name

Type & Properties

--feature_level

-f

boolean_true

Overlap between reads and features

Name	Type & Properties	Description
--overlapping -O	boolean_true	Assign reads to all their overlapping meta-features (or features if '--feature_level' is specified).
--min_overlap	integer	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.
--frac_overlap	double	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 '--min_overlap' option need to be satisfied for read assignment.
--frac_overlap_feature	double	Minimum fraction of overlapping bases in a feature that is required for read assignment. Value should be within range [0,1]. 0 by default.
--largest_overlap	boolean_true	Assign reads to a meta-feature/feature that has the largest number of overlapping bases.
--non_overlap	integer	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.
--non_overlap_feature	integer	Maximum number of non-overlapping bases in a feature that is allowed in read assignment. No limit is set by default.
--read_extension5	integer	Reads are extended upstream by <int> bases from their 5' end.
--read_extension3	integer	Reads are extended upstream by <int> bases from their 3' end.
--read2pos	integer	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.

Name

Type & Properties

--overlapping

-O

boolean_true

--min_overlap

integer

--frac_overlap

double

--frac_overlap_feature

double

--largest_overlap

boolean_true

--non_overlap

integer

--non_overlap_feature

integer

--read_extension5

integer

--read_extension3

integer

--read2pos

integer

Multi-mapping reads

Name	Type & Properties	Description
--multi_mapping -M	boolean_true	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.

Name

Type & Properties

--multi_mapping

-M

boolean_true

Fractional counting

Name	Type & Properties	Description
--fraction	boolean_true	Assign fractional counts to features. This option must be used together with '--multi_mapping' or '--overlapping' or both. When '--multi_mapping' 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 '--overlapping' 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 '--multi_mapping' and '--overlapping' are specified, each alignment will carry a fractional count of 1/(x*y).

Name

Type & Properties

--fraction

boolean_true

Read filtering

Name	Type & Properties	Description
--min_map_quality -Q	integer	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.
--split_only	boolean_true	Count split alignments only (ie. alignments with CIGAR string containing 'N'). An example of split alignments is exon-spanning reads in RNA-seq data.
--non_split_only	boolean_true	If specified, only non-split alignments (CIGAR strings do not contain letter 'N') will be counted. All the other alignments will be ignored.
--primary	boolean_true	Count primary alignments only. Primary alignments are identified using bit 0x100 in SAM/BAM FLAG field.
--ignore_dup	boolean_true	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.

Name

Type & Properties

--min_map_quality

-Q

integer

--split_only

boolean_true

--non_split_only

boolean_true

--primary

boolean_true

--ignore_dup

boolean_true

Strandedness

Name	Type & Properties	Description
--strand -s	integer	Perform strand-specific read counting. A single integer value (applied to all input files) 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).

Name

Type & Properties

--strand

-s

integer

Exon-exon junctions

Name	Type & Properties	Description
--ref_fasta -G	file	Provide the name of a FASTA-format file that contains the reference sequences used in read mapping that produced the provided SAM/BAM files.

Name

Type & Properties

--ref_fasta

-G

file

Parameters specific to paired end reads

Name	Type & Properties	Description
--paired -p	boolean_true	Specify that input data contain paired-end reads. To perform fragment counting (ie. counting read pairs), the '--countReadPairs' parameter should also be specified in addition to this parameter.
--count_read_pairs	boolean_true	Count read pairs (fragments) instead of reads. This option is only applicable for paired-end reads.
--both_aligned -B	boolean_true	Count read pairs (fragments) instead of reads. This option is only applicable for paired-end reads.
--check_pe_dist -P	boolean_true	Check validity of paired-end distance when counting read pairs. Use '--min_length' and '--max_length' to set thresholds.
--min_length -d	integer	Minimum fragment/template length, 50 by default.
--max_length -D	integer	Maximum fragment/template length, 600 by default.
--same_strand -C	boolean_true	Do not count read pairs that have their two ends mapping to different chromosomes or mapping to same chromosome but on different strands.
--donotsort	boolean_true	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.

Name

Type & Properties

--paired

-p

boolean_true

--count_read_pairs

boolean_true

--both_aligned

-B

boolean_true

--check_pe_dist

-P

boolean_true

--min_length

-d

integer

--max_length

-D

integer

--same_strand

-C

boolean_true

--donotsort

boolean_true

Read groups

Name	Type & Properties	Description
--by_read_group	boolean_true	Assign reads by read group. "RG" tag is required to be present in the input BAM/SAM files.

Name

Type & Properties

--by_read_group

boolean_true

Long reads

Name	Type & Properties	Description
--long_reads	boolean_true	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.

Name

Type & Properties

--long_reads

boolean_true

Assignment results for each read

Name	Type & Properties	Description
--detailed_results	file output	Directory to save the detailed assignment results. Use `--detailed_results_format` to determine the format of the detailed results.
--detailed_results_format -R	string	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 documentaiton for more info about these formats.

Name

Type & Properties

--detailed_results

file

output

--detailed_results_format

-R

string

Miscellaneous

Name	Type & Properties	Description
--max_M_op	integer	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	boolean_true	Output verbose information for debugging, such as un-matched chromosome/contig names.

Name

Type & Properties

--max_M_op

integer

--verbose

boolean_true

Run this component

Run the following command to execute this component with Nextflow:

cat > params.yaml <<'EOM'  
counts: "$id.$key.counts.tsv"  
summary: "$id.$key.summary.tsv"  
junctions: "$id.$key.junctions.txt"  
detailed_results: "$id.$key.detailed_results.detailed_results"  
id: "run"  
publish_dir: "output/"  
EOM

nextflow run https://packages.viash-hub.com/vsh/biobox.git \  
  -revision v0.2.0 \  
  -main-script target/nextflow/featurecounts/main.nf \  
  -params-file params.yaml

featurecounts

Inputs

Outputs

Annotation

Level of summarization

Overlap between reads and features

Multi-mapping reads

Fractional counting

Read filtering

Strandedness

Exon-exon junctions

Parameters specific to paired end reads

Read groups

Long reads

Assignment results for each read

Miscellaneous

Run this component

Relationships

Used by

Uses