Component: ExpressionQuantifier

ExpressionQuantifier

Quantifies expression from a bam file from RNA-Seq. Currently supports eXpress, Cufflinks, BitSeq, and RSEM. Tool specific instructions:

Cufflinks: align with tophat or if using STAR include --outSAMstrandField intronMotif parameter when aligning. Requires genome reference. Example parameters for cufflinks: "--no-effective-length-correction --max-bundle-length 10000000 --multi-read-correct --upper-quartile-norm -q".

eXpress: align with bowtie or if using STAR include --quantMode TranscriptomeSAM --quantTranscriptomeBan Singleend. Requires transcriptome reference that can be prepared using gffread -w transcripts.fa -g genome.fa annotation.gtf (gffread comes with cufflinks). Example parameters for eXpress: "--rf-stranded".

RSEM: align with bowtie or if using STAR include --quantMode TranscriptomeSAM --quantTranscriptomeBan IndelSoftclipSingleend. This component only supports the rsem-calculate-expression command; so you need to prepare the reference on your own (using RSEM-prepare-reference). For reference here provide the directory where you created the reference, and the string that you use to name the reference in this command needs to be given as the "extra" parameter in this component. Example parameters for RSEM: "-p 8 --paired-end --bam --estimate-rspd --append-names"; example extra: "rsemRef".

BitSeq: align with bowtie or if using STAR use either parameters for eXpress or RSEM (both seem to work and I don't know which ones are better). BitSeq runs in two steps, first it runs parse-alignment command and then estimateExpression. In "parameters" you can define the parameters you want for the parseAlignment command, and in "extra" you can define the parameters used for estimating expression. Example parameters for BitSeq: "--uniform"; example extra: "--outType rpkm".

Version	2.0
Bundle	sequencing
Categories	Analysis
Authors	Alejandra Cervera (alejandra.cervera@helsinki.fi)
Issue tracker	View/Report issues
Requires	installer (bash)
Source files	component.xml quant.sh
Usage	Example with default values ExpressionQuantifier( //alignment = AlignedReadSet, //reference = FASTA, //referenceFolder = BinaryFolder, //annotation = GTF, //mask = GTF, //array = AlignedReadSet, extra = "", parameters = "", tool = "" )

Version

2.0

Bundle

sequencing

Categories

Analysis

Authors

Alejandra Cervera (alejandra.cervera@helsinki.fi)

Issue tracker

View/Report issues

Requires

installer (bash)

Source files

component.xml quant.sh

Usage

Example with default values

Inputs

Name	Type	Mandatory	Description
alignment	AlignedReadSet	Optional	Aligned reads to transcriptome or genome.
reference	FASTA	Optional	Target sequences, either transcripts or genome. For Cufflinks a genome fasta is expected; for eXpress and BitSeq a transcriptome fasta.
referenceFolder	BinaryFolder	Optional	Reference folder, only for RSEM. Include in the extra parameter the name of the reference inside the folder without extension.
annotation	GTF	Optional	Only for Cufflinks, it sets -g/--GTF-guide parameter.
mask	GTF	Optional	Only for Cufflinks, gtf with transcripts to be excluded from the analysis. If this file is provided the -M paramter will be added automatically, do not include in paramters below.
array	Array<AlignedReadSet>	Optional	Alignments supplied as an array will be comma separated in the input for eXpress.

Name

Type

Mandatory

Description

alignment

AlignedReadSet

Optional

Aligned reads to transcriptome or genome.

reference

FASTA

Optional

Target sequences, either transcripts or genome. For Cufflinks a genome fasta is expected; for eXpress and BitSeq a transcriptome fasta.

referenceFolder

BinaryFolder

Optional

Reference folder, only for RSEM. Include in the extra parameter the name of the reference inside the folder without extension.

annotation

GTF

Optional

Only for Cufflinks, it sets -g/--GTF-guide parameter.

mask

GTF

Optional

Only for Cufflinks, gtf with transcripts to be excluded from the analysis. If this file is provided the -M paramter will be added automatically, do not include in paramters below.

array

Array<AlignedReadSet>

Optional

Alignments supplied as an array will be comma separated in the input for eXpress.

Outputs

Name	Type	Description
folder	BinaryFolder	All files produced by the tool.
genes	CSV	Gene level expression table, if available (eXpress does not produce one).
isoforms	CSV	Transcript level expression table. Gene level expression values are not produced by express and from cufflinks they are available in the output array.

Name

Type

Description

folder

BinaryFolder

All files produced by the tool.

genes

CSV

Gene level expression table, if available (eXpress does not produce one).

isoforms

CSV

Transcript level expression table. Gene level expression values are not produced by express and from cufflinks they are available in the output array.

Parameters

Name	Type	Default	Description
extra	string	""	When using RSEM define here the name given to the reference (using rsem-prepare-reference); for BitSeq define here any parameters wanted in the estimateExpression step.
parameters	string	""	Define parameters to be used with either tool, do not include any that requiere an input file defined above.
tool	string	""	The quantifier to be used, either cufflinks, express or rsem.

Name

Type

Default

Description

extra

string

When using RSEM define here the name given to the reference (using rsem-prepare-reference); for BitSeq define here any parameters wanted in the estimateExpression step.

parameters

string

Define parameters to be used with either tool, do not include any that requiere an input file defined above.

tool

string

The quantifier to be used, either cufflinks, express or rsem.

Test cases

Test case	Parameters▼	IN alignment	IN reference	IN referenceFolder	IN annotation	IN mask	IN array	OUT folder	OUT genes	OUT isoforms
case1_express	properties	alignment	reference	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	tool=express
case2_cufflinks	properties	alignment	reference	(missing)	annotation	(missing)	(missing)	(missing)	(missing)	(missing)
	tool=cufflinks,
case3_rsem	properties	alignment	(missing)	referenceFolder	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	tool=rsem, parameters=-p 8 --bam --estimate-rspd --append-names --paired-end, extra=RSEMref
case4_bitseq	properties	alignment	reference	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	tool=bitseq, parameters=--uniform, extra=--outType rpkm

Test case

Parameters▼

IN
alignment

IN
reference

IN
referenceFolder

IN
annotation

IN
mask

IN
array

OUT
folder

OUT
genes

OUT
isoforms

case1_express