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STAR_2.7.10a.md

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STAR Aligner v2.7.10a

STAR is a parameter-rich RNAseq aligner. This tool is based on this release: https://github.com/alexdobin/STAR/tree/2.7.10a. Depending on the parameters used, run time is typically around 5-10 hours, with spot instance cost of $2.50 - $8.00

Alignment tool

tools/star_2.7.10a_align.cwl

Required references:

Required inputs:

  • readFilesIn1: Fastq reads file, either uncompressed or gzipped
  • readFilesIn2: Required IF data is paired end. Same format as readFilesIn1
  • outSAMattrRGline: Suggested setting, with TABS SEPARATING THE TAGS, format is: ID:sample_name LB:aliquot_id PL:platform SM:BSID. For example ID:
    7316-242 LB:750189 PL:ILLUMINA SM:BS_W72364MN
  • outFileNamePrefix: output files name prefix (including full or relative path). Can only be defined on the command line. Tool will add '.' after prefix to easily delineate between file name and suffix

Optional inputs

Try not to panic, but there are many options. We do set many defaults to make life easier, and should yield reasonable results. Below is a table of params and values we set by default, as well as suggested changes based on downstream fusion callers, which should have little effect on expression calculations, but potentially valuable increases in sensitivity for fusion calling.

Param/Description WF Default (arriba recommended) GTEx/Broad STAR-Fusion Recommended
runThreadN 16 16 16
twopassMode Basic Basic Basic
alignSJoverhangMin 8 8 8
outFilterMismatchNoverLmax 0.1 0.1 0.1
outFilterType BySJout BySJout BySJout
outFilterScoreMinOverLread 0.33 0.33 0.33
outFilterMatchNminOverLread 0.33 0.33 0.33
outReadsUnmapped None None None
limitSjdbInsertNsj 1200000 1200000 1200000
outSAMstrandField intronMotif intronMotif intronMotif
outFilterIntronMotifs None None None
alignSoftClipAtReferenceEnds Yes Yes Yes
quantMode TranscriptomeSAM GeneCounts TranscriptomeSAM GeneCounts TranscriptomeSAM GeneCounts
outSAMtype BAM Unsorted BAM Unsorted BAM Unsorted
outSAMunmapped Within Within Within
genomeLoad NoSharedMemory NoSharedMemory NoSharedMemory
chimMainSegmentMultNmax 1 1 1
outSAMattributes NH HI AS nM NM ch NH HI AS nM NM ch NH HI AS nM NM ch
alignInsertionFlush None None Right
alignIntronMax 1000000 1000000 100000
alignMatesGapMax 1000000 1000000 100000
alignSJDBoverhangMin 1 1 10
outFilterMismatchNmax 999 999 999
alignSJstitchMismatchNmax 5 -1 5 5 0 -1 0 0 5 -1 5 5
alignSplicedMateMapLmin 0 0 30
alignSplicedMateMapLminOverLmate 0.5 0.66 0
chimJunctionOverhangMin 10 15 8
chimMultimapNmax 50 0 20
chimMultimapScoreRange 1 1 3
chimNonchimScoreDropMin 20 20 10
chimOutJunctionFormat 1 1 1
chimOutType Junctions WithinBAM SoftClip Junctions SeparateSAMold WithinBAM SoftClip Junctions WithinBAM SoftClip
chimScoreDropMax 30 20 20
chimScoreJunctionNonGTAG -1 -1 -4
chimScoreSeparation 1 10 10
chimSegmentMin 10 15 12
chimSegmentReadGapMax 3 0 0
outFilterMultimapNmax 50 20 20
peOverlapMMp 0.01 0.01 0
peOverlapNbasesMin 10 0 12

Outputs:

  • log_progress_out: Simple progress output. Can use to gauge speed and run time
  • log_out: Contains a summary of all params used and reference files
  • log_final_out: Overall summary of read mapping statistics
  • genomic_bam_out: UNSORTED read mapping to genomic coordinates
  • junctions_out: high confidence collapsed splice junctions in tab-delimited form
  • transcriptome_bam_out: Read mapping to transcriptome
  • chimeric_sam_out: Deprecated output. Incompatible with certain options, has chimeric read alignments
  • chimeric_junctions: Chimeric junctions output file. May be used for downstream tools for fusion analysis
  • gene_counts: STAR-generated read counts by gene

Reference generation tool

tools/star_2.7.10a_genome_generate.cwl
This tool is used to create the necessary genome indices for STAR. You should only need to do this once for each gene model, fasta reference, and possibly read length of input files (STAR author says values of 100 should work for most cases)

Required references:

  • genomeDir: Output dirname. Recommend STAR_{version}_GENCODE{version num}
  • genome_fa: Fasta file to index. Recommend from GENCODE, PRI assembly. Must unzip first if compressed
  • gtf: Matched GTF file to index. Recommend from GENCODE, PRI assembly

Optional inputs:

  • runThreadN: Number of threads to use. Default 16
  • sjdbOverhang: Splice junction database overhang. Ideal value is read len minus 1, but default 100 ok for most cases

Outputs:

  • star_ref: A tar gzipped package with all required files and indices to run STAR