Function: Star2Pass

Star2Pass

Runs STAR aligner in two pass mode for an array of samples together. The two pass mode means that the samples are aligned to the reference genome provided and STAR will create a list of identified splice junctions in each sample. In the in-build STAR two pass mode, those splice junctions are used to improve the reference genome and the samples are realigned to this enhanced genome. However, in this function we pool together the splice junctions of all the samples to make one single enhanced genome to which all the samples are aligned to. If you do not want to pool together the splice junctions of different samples you can run the two pass mode by using the STAR component or Align function and adding the two pass mode option (according to STAR manual) as a parameter. You can control which splice junctions are included by using the lowBound and highBound parameters. For example, lowBound="UniqueMapping=5" discards all splice junctons which do not have at least 5 uniquely mapped reads overlapping the junction. Columns available in the splice junctions file are "Chromosome,Start,End,Strand,IntronMotif,Annotated,UniqueMapping,MultiMapping,MaxOverhang". Only splice junctions from canonical chromosomes (1-22,X,Y) are kept. It is recommended to supply your own initial genome because if you need to rerun any parameters related to genome generation are used both for the first and second pass genome. lowBound and highBound parameters work exactly as defined in the CSVFilter component on each individual splice junctions file.

Version	1.0
Bundle	sequencing
Categories	Alignment
Authors	Alejandra Cervera (alejandra.cervera@helsinki.fi)
Issue tracker	View/Report issues
Requires	STAR
Source files	component.xml function.scala
Usage	Example with default values Star2Pass( reference = BinaryFile, reads = BinaryFile, //mates = BinaryFile, //genome = BinaryFolder, //annotation = BinaryFile, //parameterFile = TextFile, //custom = TextFile, alignParameters = "", execute = "changed", genomeLoad = "LoadAndRemove", genomeParameters = "", highBound = "", lowBound = "", mainAlignmentType = "", memory = 10000, readFilesCommand = "", threads = 1, useEncodeParams = true )

Version

1.0

Bundle

sequencing

Categories

Alignment

Authors

Alejandra Cervera (alejandra.cervera@helsinki.fi)

Issue tracker

View/Report issues

Requires

STAR

Source files

component.xml function.scala

Usage

Example with default values

Inputs

Name	Type	Mandatory	Description
reference	BinaryFile	Mandatory	Reference genome.
reads	Array<BinaryFile>	Mandatory	FASTA or FASTQ file containing reads for the alignment.
mates	Array<BinaryFile>	Optional	FASTA or FASTQ file containing mates. Required for paired end data.
genome	BinaryFolder	Optional	A STAR genome for first pass.
annotation	BinaryFile	Optional	Genome annotation. A GTF file will work by default. For a GFF3 file add to genomeParameters: "--sjdbGTFtagExonParentTranscript Parent"
parameterFile	TextFile	Optional	This file overrides default STAR parameters, but will itself be overridden by the command line. Use parametersDefault from STAR source as template. It is needed unless you have specified every single parameter (even default ones as a parameter string).
custom	TextFile	Optional	if you want to add custom parameters, aka sample specific such as readgroups, provide them here as they should be added to the STAR call (only for second pass). It must have two columns: Key and Custom. The keys should match the input keys.

Name

Type

Mandatory

Description

reference

BinaryFile

Mandatory

Reference genome.

reads

Array<BinaryFile>

Mandatory

FASTA or FASTQ file containing reads for the alignment.

mates

Array<BinaryFile>

Optional

FASTA or FASTQ file containing mates. Required for paired end data.

genome

BinaryFolder

Optional

A STAR genome for first pass.

annotation

BinaryFile

Optional

Genome annotation. A GTF file will work by default. For a GFF3 file add to genomeParameters: "--sjdbGTFtagExonParentTranscript Parent"

parameterFile

TextFile

Optional

This file overrides default STAR parameters, but will itself be overridden by the command line. Use parametersDefault from STAR source as template. It is needed unless you have specified every single parameter (even default ones as a parameter string).

custom

TextFile

Optional

if you want to add custom parameters, aka sample specific such as readgroups, provide them here as they should be added to the STAR call (only for second pass). It must have two columns: Key and Custom. The keys should match the input keys.

Outputs

Name	Type	Description
folder	Array<BinaryFolder>	All files created by STAR in the output folder.
alignments	Array<BAM>	(Sorted) alignment. A coordinate sorted file will be indexed, i.e. there is a .bai file.
spliceJunctions	CSV	Splice junctions. This CSV file is created by adding a header to STAR output. ("Chromosome\tStart\tEnd\tStrand\tIntronMotif\tAnnotated\tUniqueMapping\tMultiMapping\tMaxOverhang"): Column 1: chromosome Column 2: first base of the intron (1-based) Column 3: last base of the intron (1-based) Column 4: strand Column 5: intron motif: 0: non-canonical; 1: GT/AG, 2: CT/AC, 3: GC/AG, 4: CT/GC, 5: AT/AC, 6: GT/AT Column 6: 0: unannotated, 1: annotated (only if splice junctions database is used) Column 7: number of uniquely mapping reads crossing the junction Column 8: number of multi-mapping reads crossing the junction Column 9: maximum spliced alignment overhang Of these, the following awk expression is relevant for 2-pass STAR: "if($5>0){print $1,$2,$3,strChar[$4]}}" In other words, chromosome and intron boundaries known, and intron motif is classified. Rest of the columns are useful for filtering interesting junctions in 2-pass STAR.

Name

Type

Description

folder

Array<BinaryFolder>

All files created by STAR in the output folder.

alignments

Array<BAM>

(Sorted) alignment. A coordinate sorted file will be indexed, i.e. there is a .bai file.

spliceJunctions

CSV

Splice junctions. This CSV file is created by adding a header to STAR output. ("Chromosome\tStart\tEnd\tStrand\tIntronMotif\tAnnotated\tUniqueMapping\tMultiMapping\tMaxOverhang"):

Column 1: chromosome
Column 2: first base of the intron (1-based)
Column 3: last base of the intron (1-based)
Column 4: strand
Column 5: intron motif: 0: non-canonical; 1: GT/AG, 2: CT/AC, 3: GC/AG, 4: CT/GC, 5: AT/AC, 6: GT/AT
Column 6: 0: unannotated, 1: annotated (only if splice junctions database is used)
Column 7: number of uniquely mapping reads crossing the junction
Column 8: number of multi-mapping reads crossing the junction
Column 9: maximum spliced alignment overhang

Of these, the following awk expression is relevant for 2-pass STAR: "if($5>0){print $1,$2,$3,strChar[$4]}}" In other words, chromosome and intron boundaries known, and intron motif is classified. Rest of the columns are useful for filtering interesting junctions in 2-pass STAR.

Parameters

Name	Type	Default	Description
alignParameters	string	""	Parameters passed on to STAR on the second alignment step.
execute	string	"changed"	Change it to "once" if you do not want to re-execute the first pass and making genome steps if you change any parameter (such as threads)
genomeLoad	string	"LoadAndRemove"	LoadAndRemove works for parallel STAR instances and if everything goes fine, should free memory after the last STAR exits. LoadAndKeep, LoadAndRemove, Remove, LoadAndExit and NoSharedMemory are the options.
genomeParameters	string	""	Parameters passed on to STAR in any of the two possible generating genome steps.
highBound	string	""	Same as lowBound but for max values (instead of min).
lowBound	string	""	For subsetting the splice junctions files used for generating the second pass genome. Define column name and threshold, ex: UniqueMapping=5.
mainAlignmentType	string	""	Depending on thparameters more than one alignment may be produced (ex. sortedByCoord or toTranscriptome). The alignment not selected will still be available in the folder output. The string defined here will define which alignment will be linked to the alignment output of this component.
memory	int	10000	Memory passed to STAR call.
readFilesCommand	string	""	Used when input reads are compressed, ex. zcat or acat
threads	int	1	Number of threads passed to STAR.
useEncodeParams	boolean	true	set the parameters used for the Encode project specified in the manual (max and min intron size and max number of multiple alignments)

Name

Type

Default

Description

alignParameters

string

Parameters passed on to STAR on the second alignment step.

execute

string

"changed"

Change it to "once" if you do not want to re-execute the first pass and making genome steps if you change any parameter (such as threads)

genomeLoad

string

"LoadAndRemove"

LoadAndRemove works for parallel STAR instances and if everything goes fine, should free memory after the last STAR exits. LoadAndKeep, LoadAndRemove, Remove, LoadAndExit and NoSharedMemory are the options.

genomeParameters

string

Parameters passed on to STAR in any of the two possible generating genome steps.

highBound

string

Same as lowBound but for max values (instead of min).

lowBound

string

For subsetting the splice junctions files used for generating the second pass genome. Define column name and threshold, ex: UniqueMapping=5.

mainAlignmentType

string

Depending on thparameters more than one alignment may be produced (ex. sortedByCoord or toTranscriptome). The alignment not selected will still be available in the folder output. The string defined here will define which alignment will be linked to the alignment output of this component.

memory

int

10000

Memory passed to STAR call.

readFilesCommand

string

Used when input reads are compressed, ex. zcat or acat

threads

int

Number of threads passed to STAR.

useEncodeParams

boolean

true

set the parameters used for the Encode project specified in the manual (max and min intron size and max number of multiple alignments)

Test cases

Test case	Parameters▼	IN reference	IN reads	IN mates	IN genome	IN annotation	IN parameterFile	IN custom	OUT folder	OUT alignments	OUT spliceJunctions
case1	properties	reference	reads	mates	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)	(missing)
	genomeLoad=NoSharedMemory, readFilesCommand=zcat, threads=10, genomeParameters=--genomeSAindexNbases=5
case2	properties	reference	reads	mates	(missing)	(missing)	(missing)	custom	(missing)	(missing)	(missing)
	genomeLoad=NoSharedMemory, readFilesCommand=zcat, threads=10

Test case

Parameters▼

IN
reference

IN
reads

IN
mates

IN
genome

IN
annotation

IN
parameterFile

IN
custom

OUT
folder

OUT
alignments

OUT
spliceJunctions