STAR

STAR (Spliced Transcripts Alignment to a Reference) is an RNA-seq mapper that performs highly accurate spliced sequence alignment at an ultrafast speed. STAR alignment algorithm can be controlled by many user-defined parameters. Mammal genomes require at least 16GB of RAM, ideally 32GB. The outputs include both short reads aligned to reference genome and transcriptome. In addition, chimeric alignments may be used to produce a separate output file with supporting alignments for putative gene fusion events.

Overview

Dependencies

• star 2.7.10b
• picard 2.19.2

Usage

Cromwell

java -jar cromwell.jar run star.wdl --inputs inputs.json

Inputs

Required workflow parameters:

Parameter	Value	Description
inputGroups	Array[InputGroup]	Array of fastq files to align with STAR and the merged filename
outputFileNamePrefix	String	Prefix for filename
reference	String	Reference id, hg19 or hg38

Optional task parameters:

Parameter	Value	Default	Description
runStar.genomeIndexDir	String	"$HG38_STAR_INDEX100_ROOT/"	Path to STAR index
runStar.starSuffix	String	"Aligned.sortedByCoord.out"	Suffix for sorted file
runStar.transcriptomeSuffix	String	"Aligned.toTranscriptome.out"	Suffix for transcriptome-aligned file
runStar.chimericjunctionSuffix	String	"Chimeric.out"	Suffix for chimeric junction file
runStar.genereadSuffix	String	"ReadsPerGene.out"	ReadsPerGene file suffix
runStar.addParam	String?	None	Additional STAR parameters
runStar.modules	String	"hg38-star-index100/2.7.10b"	modules for running STAR
runStar.chimOutType	String	"WithinBAM SoftClip Junctions"	Indicate where chimeric reads are to be written
runStar.outFilterMultimapNmax	Int	50	max number of multiple alignments allowed for a read: if exceeded, the read is considered unmapped
runStar.chimScoreDropMax	Int	30	max drop (difference) of chimeric score (the sum of scores of allchimeric segments) from the read length
runStar.uniqMAPQ	Int	255	Score for unique mappers
runStar.saSparsed	Int	2	saSparsed parameter for STAR
runStar.multiMax	Int	-1	multiMax parameter for STAR
runStar.chimSegmin	Int	10	minimum length of chimeric segment length
runStar.chimJunOvMin	Int	10	minimum overhang for a chimeric junction
runStar.alignSJDBOvMin	Int	10	minimum overhang for annotated spliced alignments
runStar.alignMatGapMax	Int	100000	maximum gap between two mates
runStar.alignIntMax	Int	100000	maximum intron size
runStar.chimMulmapScoRan	Int	3	the score range for multi-mapping chimeras below the best chimeric score
runStar.chimScoJunNonGTAG	Int	-1	penalty for a non-GTAG chimeric junction
runStar.chimScoreSeparation	Int	1	minimum difference (separation) between the best chimeric score and the next one
runStar.chimMulmapNmax	Int	50	maximum number of chimeric multi-alignments
runStar.chimNonchimScoDMin	Int	10	to trigger chimeric detection, the drop in the best non-chimeric alignment score with respect to the read length has to be greater than this value
runStar.chimOutJunForm	Int?	None	flag to add metadata to chimeric junction output for functionality with starFusion - 1 for metadata, 0 for no metadata
runStar.peOvNbasesMin	Int	10	minimum number of overlap bases to trigger mates merging and realignment
runStar.chimSegmentReadGapMax	Int	3	maximum gap in the read sequence between chimeric segments
runStar.peOvMMp	Float	0.1	maximum proportion of mismatched bases in the overlap area
runStar.threads	Int	6	Requested CPU threads
runStar.jobMemory	Int	64	Memory allocated for this job
runStar.timeout	Int	72	hours before task timeout
indexBam.jobMemory	Int	12	Memory allocated indexing job
indexBam.modules	String	"picard/2.19.2"	modules for running indexing job
indexBam.timeout	Int	48	hours before task timeout

Outputs

Output	Type	Description
starBam	File	Output bam aligned to genome
starIndex	File	Output index file for bam aligned to genome
transcriptomeBam	File	Output bam aligned to transcriptome
starChimeric	File	Output chimeric junctions file
geneReadFile	File	Output raw read counts per transcript

Commands

This section lists command(s) run by the STAR workflow.

run STAR aligner

STAR --twopassMode Basic
--genomeDir ~{genomeIndexDir}
--readFilesIn ~{sep="," read1s} ~{sep="," read2s}
--readFilesCommand zcat
--outFilterIntronMotifs RemoveNoncanonical
--outFileNamePrefix ~{outputFileNamePrefix}.
--outSAMmultNmax ~{multiMax}
--outSAMattrRGline ~{sep=" , " readGroups}
--outSAMstrandField intronMotif
--outSAMmapqUnique ~{uniqMAPQ}
--outSAMunmapped Within KeepPairs
--genomeSAsparseD ~{saSparsed}
--outSAMtype BAM SortedByCoordinate
--quantMode TranscriptomeSAM GeneCounts
--chimSegmentMin ~{chimSegmin}
--chimJunctionOverhangMin ~{chimJunOvMin}
--alignSJDBoverhangMin ~{alignSJDBOvMin}
--alignMatesGapMax ~{alignMatGapMax}
--alignIntronMax ~{alignIntMax}
--alignSJstitchMismatchNmax 5 -1 5 5
--chimMultimapScoreRange ~{chimMulmapScoRan}
--chimScoreJunctionNonGTAG ~{chimScoJunNonGTAG}
--chimMultimapNmax ~{chimMulmapNmax}
--chimNonchimScoreDropMin ~{chimNonchimScoDMin}
~{"--chimOutJunctionFormat " + chimOutJunForm}
--peOverlapNbasesMin ~{peOvNbasesMin}
--peOverlapMMp ~{peOvMMp}
--outFilterMultimapNmax ~{outFilterMultimapNmax}
--runThreadN ~{threads} --chimOutType ~{chimOutType}
--chimScoreDropMax ~{chimScoreDropMax}
--chimScoreSeparation ~{chimScoreSeparation}
--chimSegmentReadGapMax ~{chimSegmentReadGapMax} ~{addParam}

Process Chimeric junctions file for downstream use by STAR-Fusion

awk 'NR<2{print $0;next}{print $0| "sort -V"}' {outputFileNamePrefix}.{chimericjunctionSuffix}.junction \

tmp && mv tmp {outputFileNamePrefix}.{chimericjunctionSuffix}.junction

Index Bam file for random access

java -Xmx~{jobMemory-6}G -jar $PICARD_ROOT/picard.jar BuildBamIndex
VALIDATION_STRINGENCY=LENIENT
OUTPUT="{basename(inputBam, '.bam')}.bai"
INPUT={inputBam}

Support

For support, please file an issue on the Github project or send an email to gsi@oicr.on.ca .

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