STAR --help
Usage: STAR [options]... --genomeDir /path/to/genome/index/ --readFilesIn R1.fq R2.fq
Spliced Transcripts Alignment to a Reference (c) Alexander Dobin, 2009-2020
STAR version=2.7.9a
STAR compilation time,server,dir=2021-05-04T09:43:56-0400 vega:/home/dobin/data/STAR/STARcode/STAR.master/source
For more details see:
<https://github.com/alexdobin/STAR>
<https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf>
### versions
versionGenome 2.7.4a
string: earliest genome index version compatible with this STAR release. Please do not change this value!
### Parameter Files
parametersFiles -
string: name of a user-defined parameters file, "-": none. Can only be defined on the command line.
### System
sysShell -
string: path to the shell binary, preferably bash, e.g. /bin/bash.
- ... the default shell is executed, typically /bin/sh. This was reported to fail on some Ubuntu systems - then you need to specify path to bash.
### Run Parameters
runMode alignReads
string: type of the run.
alignReads ... map reads
genomeGenerate ... generate genome files
inputAlignmentsFromBAM ... input alignments from BAM. Presently only works with --outWigType and --bamRemoveDuplicates.
liftOver ... lift-over of GTF files (--sjdbGTFfile) between genome assemblies using chain file(s) from --genomeChainFiles.
soloCellFiltering </path/to/raw/count/dir/> </path/to/output/prefix> ... STARsolo cell filtering ("calling") without remapping, followed by the path to raw count directory and output (filtered) prefix
runThreadN 1
int: number of threads to run STAR
runDirPerm User_RWX
string: permissions for the directories created at the run-time.
User_RWX ... user-read/write/execute
All_RWX ... all-read/write/execute (same as chmod 777)
runRNGseed 777
int: random number generator seed.
### Genome Parameters
genomeDir ./GenomeDir/
string: path to the directory where genome files are stored (for --runMode alignReads) or will be generated (for --runMode generateGenome)
genomeLoad NoSharedMemory
string: mode of shared memory usage for the genome files. Only used with --runMode alignReads.
LoadAndKeep ... load genome into shared and keep it in memory after run
LoadAndRemove ... load genome into shared but remove it after run
LoadAndExit ... load genome into shared memory and exit, keeping the genome in memory for future runs
Remove ... do not map anything, just remove loaded genome from memory
NoSharedMemory ... do not use shared memory, each job will have its own private copy of the genome
genomeFastaFiles -
string(s): path(s) to the fasta files with the genome sequences, separated by spaces. These files should be plain text FASTA files, they *cannot* be zipped.
Required for the genome generation (--runMode genomeGenerate). Can also be used in the mapping (--runMode alignReads) to add extra (new) sequences to the genome (e.g. spike-ins).
genomeChainFiles -
string: chain files for genomic liftover. Only used with --runMode liftOver .
genomeFileSizes 0
uint(s)>0: genome files exact sizes in bytes. Typically, this should not be defined by the user.
genomeTransformOutput None
string(s) which output to transform back to original genome
SAM ... SAM/BAM alignments
SJ ... splice junctions (SJ.out.tab)
None ... no transformation of the output
### Genome Indexing Parameters - only used with --runMode genomeGenerate
genomeChrBinNbits 18
int: =log2(chrBin), where chrBin is the size of the bins for genome storage: each chromosome will occupy an integer number of bins. For a genome with large number of contigs, it is recommended to scale this parameter as min(18, log2[max(GenomeLength/NumberOfReferences,ReadLength)]).
genomeSAindexNbases 14
int: length (bases) of the SA pre-indexing string. Typically between 10 and 15. Longer strings will use much more memory, but allow faster searches. For small genomes, the parameter --genomeSAindexNbases must be scaled down to min(14, log2(GenomeLength)/2 - 1).
genomeSAsparseD 1
int>0: suffux array sparsity, i.e. distance between indices: use bigger numbers to decrease needed RAM at the cost of mapping speed reduction
genomeSuffixLengthMax -1
int: maximum length of the suffixes, has to be longer than read length. -1 = infinite.
genomeTransformType None
string: type of genome transformation
None ... no transformation
Haploid ... replace reference alleles with alternative alleles from VCF file (e.g. consensus allele)
Diploid ... create two haplotypes for each chromosome listed in VCF file, for genotypes 1|2, assumes perfect phasing (e.g. personal genome)
genomeTransformVCF -
string: path to VCF file for genome transformation
#####UnderDevelopment_begin : not supported - do not use
genomeType Full
string: type of genome to generate
Full ... full (normal) genome
Transcriptome ... genome consists of transcript sequences
SuperTransriptome ... genome consists of superTranscript sequences
#####UnderDevelopment_end
# DEPRECATED: please use --genomeTransformVCF and --genomeTransformType options instead.
#genomeConsensusFile -
# string: VCF file with consensus SNPs (i.e. alternative allele is the major (AF>0.5) allele)
# DEPRECATED
### Splice Junctions Database
sjdbFileChrStartEnd -
string(s): path to the files with genomic coordinates (chr <tab> start <tab> end <tab> strand) for the splice junction introns. Multiple files can be supplied wand will be concatenated.
sjdbGTFfile -
string: path to the GTF file with annotations
sjdbGTFchrPrefix -
string: prefix for chromosome names in a GTF file (e.g. 'chr' for using ENSMEBL annotations with UCSC genomes)
sjdbGTFfeatureExon exon
string: feature type in GTF file to be used as exons for building transcripts
sjdbGTFtagExonParentTranscript transcript_id
string: GTF attribute name for parent transcript ID (default "transcript_id" works for GTF files)
sjdbGTFtagExonParentGene gene_id
string: GTF attribute name for parent gene ID (default "gene_id" works for GTF files)
sjdbGTFtagExonParentGeneName gene_name
string(s): GTF attrbute name for parent gene name
sjdbGTFtagExonParentGeneType gene_type gene_biotype
string(s): GTF attrbute name for parent gene type
sjdbOverhang 100
int>0: length of the donor/acceptor sequence on each side of the junctions, ideally = (mate_length - 1)
sjdbScore 2
int: extra alignment score for alignments that cross database junctions
sjdbInsertSave Basic
string: which files to save when sjdb junctions are inserted on the fly at the mapping step
Basic ... only small junction / transcript files
All ... all files including big Genome, SA and SAindex - this will create a complete genome directory
### Variation parameters
varVCFfile -
string: path to the VCF file that contains variation data. The 10th column should contain the genotype information, e.g. 0/1
### Input Files
inputBAMfile -
string: path to BAM input file, to be used with --runMode inputAlignmentsFromBAM
### Read Parameters
readFilesType Fastx
string: format of input read files
Fastx ... FASTA or FASTQ
SAM SE ... SAM or BAM single-end reads; for BAM use --readFilesCommand samtools view
SAM PE ... SAM or BAM paired-end reads; for BAM use --readFilesCommand samtools view
readFilesSAMattrKeep All
string(s): for --readFilesType SAM SE/PE, which SAM tags to keep in the output BAM, e.g.: --readFilesSAMtagsKeep RG PL
All ... keep all tags
None ... do not keep any tags
readFilesIn Read1 Read2
string(s): paths to files that contain input read1 (and, if needed, read2)
readFilesManifest -
string: path to the "manifest" file with the names of read files. The manifest file should contain 3 tab-separated columns:
paired-end reads: read1_file_name $tab$ read2_file_name $tab$ read_group_line.
single-end reads: read1_file_name $tab$ - $tab$ read_group_line.
Spaces, but not tabs are allowed in file names.
If read_group_line does not start with ID:, it can only contain one ID field, and ID: will be added to it.
If read_group_line starts with ID:, it can contain several fields separated by $tab$, and all fields will be be copied verbatim into SAM @RG header line.
readFilesPrefix -
string: prefix for the read files names, i.e. it will be added in front of the strings in --readFilesIn
readFilesCommand -
string(s): command line to execute for each of the input file. This command should generate FASTA or FASTQ text and send it to stdout
For example: zcat - to uncompress .gz files, bzcat - to uncompress .bz2 files, etc.
readMapNumber -1
int: number of reads to map from the beginning of the file
-1: map all reads
readMatesLengthsIn NotEqual
string: Equal/NotEqual - lengths of names,sequences,qualities for both mates are the same / not the same. NotEqual is safe in all situations.
readNameSeparator /
string(s): character(s) separating the part of the read names that will be trimmed in output (read name after space is always trimmed)
readQualityScoreBase 33
int>=0: number to be subtracted from the ASCII code to get Phred quality score
### Read Clipping
clipAdapterType Hamming
string: adapter clipping type
Hamming ... adapter clipping based on Hamming distance, with the number of mismatches controlled by --clip5pAdapterMMp
CellRanger4 ... 5p and 3p adapter clipping similar to CellRanger4. Utilizes Opal package by Martin ?o?i?: https://github.com/Martinsos/opal
None ... no adapter clipping, all other clip* parameters are disregarded
clip3pNbases 0
int(s): number(s) of bases to clip from 3p of each mate. If one value is given, it will be assumed the same for both mates.
clip3pAdapterSeq -
string(s): adapter sequences to clip from 3p of each mate. If one value is given, it will be assumed the same for both mates.
polyA ... polyA sequence with the length equal to read length
clip3pAdapterMMp 0.1
double(s): max proportion of mismatches for 3p adapter clipping for each mate. If one value is given, it will be assumed the same for both mates.
clip3pAfterAdapterNbases 0
int(s): number of bases to clip from 3p of each mate after the adapter clipping. If one value is given, it will be assumed the same for both mates.
clip5pNbases 0
int(s): number(s) of bases to clip from 5p of each mate. If one value is given, it will be assumed the same for both mates.
#####UnderDevelopment_begin : not supported - do not use
clip5pAdapterSeq -
string(s): adapter sequences to clip from 5p of each mate. If one value is given, it will be assumed the same for both mates.
clip5pAdapterMMp 0.1
double(s): max proportion of mismatches for 5p adapter clipping for each mate. If one value is given, it will be assumed the same for both mates.
clip5pAfterAdapterNbases 0
int(s): number of bases to clip from 5p of each mate after the adapter clipping. If one value is given, it will be assumed the same for both mates.
#####UnderDevelopment_end
### Limits
limitGenomeGenerateRAM 31000000000
int>0: maximum available RAM (bytes) for genome generation
limitIObufferSize 30000000 50000000
int>0: max available buffers size (bytes) for input/output, per thread
limitOutSAMoneReadBytes 100000
int>0: max size of the SAM record (bytes) for one read. Recommended value: >(2*(LengthMate1+LengthMate2+100)*outFilterMultimapNmax
limitOutSJoneRead 1000
int>0: max number of junctions for one read (including all multi-mappers)
limitOutSJcollapsed 1000000
int>0: max number of collapsed junctions
limitBAMsortRAM 0
int>=0: maximum available RAM (bytes) for sorting BAM. If =0, it will be set to the genome index size. 0 value can only be used with --genomeLoad NoSharedMemory option.
limitSjdbInsertNsj 1000000
int>=0: maximum number of junction to be inserted to the genome on the fly at the mapping stage, including those from annotations and those detected in the 1st step of the 2-pass run
limitNreadsSoft -1
int: soft limit on the number of reads
### Output: general
outFileNamePrefix ./
string: output files name prefix (including full or relative path). Can only be defined on the command line.
outTmpDir -
string: path to a directory that will be used as temporary by STAR. All contents of this directory will be removed!
- the temp directory will default to outFileNamePrefix_STARtmp
outTmpKeep None
string: whether to keep the tempporary files after STAR runs is finished
None ... remove all temporary files
All .. keep all files
outStd Log
string: which output will be directed to stdout (standard out)
Log ... log messages
SAM ... alignments in SAM format (which normally are output to Aligned.out.sam file), normal standard output will go into Log.std.out
BAM_Unsorted ... alignments in BAM format, unsorted. Requires --outSAMtype BAM Unsorted
BAM_SortedByCoordinate ... alignments in BAM format, sorted by coordinate. Requires --outSAMtype BAM SortedByCoordinate
BAM_Quant ... alignments to transcriptome in BAM format, unsorted. Requires --quantMode TranscriptomeSAM
outReadsUnmapped None
string: output of unmapped and partially mapped (i.e. mapped only one mate of a paired end read) reads in separate file(s).
None ... no output
Fastx ... output in separate fasta/fastq files, Unmapped.out.mate1/2
outQSconversionAdd 0
int: add this number to the quality score (e.g. to convert from Illumina to Sanger, use -31)
outMultimapperOrder Old_2.4
string: order of multimapping alignments in the output files
Old_2.4 ... quasi-random order used before 2.5.0
Random ... random order of alignments for each multi-mapper. Read mates (pairs) are always adjacent, all alignment for each read stay together. This option will become default in the future releases.
### Output: SAM and BAM
outSAMtype SAM
strings: type of SAM/BAM output
1st word:
BAM ... output BAM without sorting
SAM ... output SAM without sorting
None ... no SAM/BAM output
2nd, 3rd:
Unsorted ... standard unsorted
SortedByCoordinate ... sorted by coordinate. This option will allocate extra memory for sorting which can be specified by --limitBAMsortRAM.
outSAMmode Full
string: mode of SAM output
None ... no SAM output
Full ... full SAM output
NoQS ... full SAM but without quality scores
outSAMstrandField None
string: Cufflinks-like strand field flag
None ... not used
intronMotif ... strand derived from the intron motif. This option changes the output alignments: reads with inconsistent and/or non-canonical introns are filtered out.
outSAMattributes Standard
string: a string of desired SAM attributes, in the order desired for the output SAM. Tags can be listed in any combination/order.
***Presets:
None ... no attributes
Standard ... NH HI AS nM
All ... NH HI AS nM NM MD jM jI MC ch
***Alignment:
NH ... number of loci the reads maps to: =1 for unique mappers, >1 for multimappers. Standard SAM tag.
HI ... multiple alignment index, starts with --outSAMattrIHstart (=1 by default). Standard SAM tag.
AS ... local alignment score, +1/-1 for matches/mismateches, score* penalties for indels and gaps. For PE reads, total score for two mates. Stadnard SAM tag.
nM ... number of mismatches. For PE reads, sum over two mates.
NM ... edit distance to the reference (number of mismatched + inserted + deleted bases) for each mate. Standard SAM tag.
MD ... string encoding mismatched and deleted reference bases (see standard SAM specifications). Standard SAM tag.
jM ... intron motifs for all junctions (i.e. N in CIGAR): 0: non-canonical; 1: GT/AG, 2: CT/AC, 3: GC/AG, 4: CT/GC, 5: AT/AC, 6: GT/AT. If splice junctions database is used, and a junction is annotated, 20 is added to its motif value.
jI ... start and end of introns for all junctions (1-based).
XS ... alignment strand according to --outSAMstrandField.
MC ... mate's CIGAR string. Standard SAM tag.
ch ... marks all segment of all chimeric alingments for --chimOutType WithinBAM output.
cN ... number of bases clipped from the read ends: 5' and 3'
***Variation:
vA ... variant allele
vG ... genomic coordinate of the variant overlapped by the read.
vW ... 1 - alignment passes WASP filtering; 2,3,4,5,6,7 - alignment does not pass WASP filtering. Requires --waspOutputMode SAMtag.
***STARsolo:
CR CY UR UY ... sequences and quality scores of cell barcodes and UMIs for the solo* demultiplexing.
GX GN ... gene ID and gene name.
CB UB ... error-corrected cell barcodes and UMIs for solo* demultiplexing. Requires --outSAMtype BAM SortedByCoordinate.
sM ... assessment of CB and UMI.
sS ... sequence of the entire barcode (CB,UMI,adapter).
sQ ... quality of the entire barcode.
***Unsupported/undocumented:
ha ... haplotype (1/2) when mapping to the diploid genome. Requires genome generated with --genomeTransformType Diploid .
rB ... alignment block read/genomic coordinates.
vR ... read coordinate of the variant.
outSAMattrIHstart 1
int>=0: start value for the IH attribute. 0 may be required by some downstream software, such as Cufflinks or StringTie.
outSAMunmapped None
string(s): output of unmapped reads in the SAM format
1st word:
None ... no output
Within ... output unmapped reads within the main SAM file (i.e. Aligned.out.sam)
2nd word:
KeepPairs ... record unmapped mate for each alignment, and, in case of unsorted output, keep it adjacent to its mapped mate. Only affects multi-mapping reads.
outSAMorder Paired
string: type of sorting for the SAM output
Paired: one mate after the other for all paired alignments
PairedKeepInputOrder: one mate after the other for all paired alignments, the order is kept the same as in the input FASTQ files
outSAMprimaryFlag OneBestScore
string: which alignments are considered primary - all others will be marked with 0x100 bit in the FLAG
OneBestScore ... only one alignment with the best score is primary
AllBestScore ... all alignments with the best score are primary
outSAMreadID Standard
string: read ID record type
Standard ... first word (until space) from the FASTx read ID line, removing /1,/2 from the end
Number ... read number (index) in the FASTx file
outSAMmapqUnique 255
int: 0 to 255: the MAPQ value for unique mappers
outSAMflagOR 0
int: 0 to 65535: sam FLAG will be bitwise OR'd with this value, i.e. FLAG=FLAG | outSAMflagOR. This is applied after all flags have been set by STAR, and after outSAMflagAND. Can be used to set specific bits that are not set otherwise.
outSAMflagAND 65535
int: 0 to 65535: sam FLAG will be bitwise AND'd with this value, i.e. FLAG=FLAG & outSAMflagOR. This is applied after all flags have been set by STAR, but before outSAMflagOR. Can be used to unset specific bits that are not set otherwise.
outSAMattrRGline -
string(s): SAM/BAM read group line. The first word contains the read group identifier and must start with "ID:", e.g. --outSAMattrRGline ID:xxx CN:yy "DS:z z z".
xxx will be added as RG tag to each output alignment. Any spaces in the tag values have to be double quoted.
Comma separated RG lines correspons to different (comma separated) input files in --readFilesIn. Commas have to be surrounded by spaces, e.g.
--outSAMattrRGline ID:xxx , ID:zzz "DS:z z" , ID:yyy DS:yyyy
outSAMheaderHD -
strings: @HD (header) line of the SAM header
outSAMheaderPG -
strings: extra @PG (software) line of the SAM header (in addition to STAR)
outSAMheaderCommentFile -
string: path to the file with @CO (comment) lines of the SAM header
outSAMfilter None
string(s): filter the output into main SAM/BAM files
KeepOnlyAddedReferences ... only keep the reads for which all alignments are to the extra reference sequences added with --genomeFastaFiles at the mapping stage.
KeepAllAddedReferences ... keep all alignments to the extra reference sequences added with --genomeFastaFiles at the mapping stage.
outSAMmultNmax -1
int: max number of multiple alignments for a read that will be output to the SAM/BAM files. Note that if this value is not equal to -1, the top scoring alignment will be output first
-1 ... all alignments (up to --outFilterMultimapNmax) will be output
outSAMtlen 1
int: calculation method for the TLEN field in the SAM/BAM files
1 ... leftmost base of the (+)strand mate to rightmost base of the (-)mate. (+)sign for the (+)strand mate
2 ... leftmost base of any mate to rightmost base of any mate. (+)sign for the mate with the leftmost base. This is different from 1 for overlapping mates with protruding ends
outBAMcompression 1
int: -1 to 10 BAM compression level, -1=default compression (6?), 0=no compression, 10=maximum compression
outBAMsortingThreadN 0
int: >=0: number of threads for BAM sorting. 0 will default to min(6,--runThreadN).
outBAMsortingBinsN 50
int: >0: number of genome bins fo coordinate-sorting
### BAM processing
bamRemoveDuplicatesType -
string: mark duplicates in the BAM file, for now only works with (i) sorted BAM fed with inputBAMfile, and (ii) for paired-end alignments only
- ... no duplicate removal/marking
UniqueIdentical ... mark all multimappers, and duplicate unique mappers. The coordinates, FLAG, CIGAR must be identical
UniqueIdenticalNotMulti ... mark duplicate unique mappers but not multimappers.
bamRemoveDuplicatesMate2basesN 0
int>0: number of bases from the 5' of mate 2 to use in collapsing (e.g. for RAMPAGE)
### Output Wiggle
outWigType None
string(s): type of signal output, e.g. "bedGraph" OR "bedGraph read1_5p". Requires sorted BAM: --outSAMtype BAM SortedByCoordinate .
1st word:
None ... no signal output
bedGraph ... bedGraph format
wiggle ... wiggle format
2nd word:
read1_5p ... signal from only 5' of the 1st read, useful for CAGE/RAMPAGE etc
read2 ... signal from only 2nd read
outWigStrand Stranded
string: strandedness of wiggle/bedGraph output
Stranded ... separate strands, str1 and str2
Unstranded ... collapsed strands
outWigReferencesPrefix -
string: prefix matching reference names to include in the output wiggle file, e.g. "chr", default "-" - include all references
outWigNorm RPM
string: type of normalization for the signal
RPM ... reads per million of mapped reads
None ... no normalization, "raw" counts
### Output Filtering
outFilterType Normal
string: type of filtering
Normal ... standard filtering using only current alignment
BySJout ... keep only those reads that contain junctions that passed filtering into SJ.out.tab
outFilterMultimapScoreRange 1
int: the score range below the maximum score for multimapping alignments
outFilterMultimapNmax 10
int: maximum number of loci the read is allowed to map to. Alignments (all of them) will be output only if the read maps to no more loci than this value.
Otherwise no alignments will be output, and the read will be counted as "mapped to too many loci" in the Log.final.out .
outFilterMismatchNmax 10
int: alignment will be output only if it has no more mismatches than this value.
outFilterMismatchNoverLmax 0.3
real: alignment will be output only if its ratio of mismatches to *mapped* length is less than or equal to this value.
outFilterMismatchNoverReadLmax 1.0
real: alignment will be output only if its ratio of mismatches to *read* length is less than or equal to this value.
outFilterScoreMin 0
int: alignment will be output only if its score is higher than or equal to this value.
outFilterScoreMinOverLread 0.66
real: same as outFilterScoreMin, but normalized to read length (sum of mates' lengths for paired-end reads)
outFilterMatchNmin 0
int: alignment will be output only if the number of matched bases is higher than or equal to this value.
outFilterMatchNminOverLread 0.66
real: sam as outFilterMatchNmin, but normalized to the read length (sum of mates' lengths for paired-end reads).
outFilterIntronMotifs None
string: filter alignment using their motifs
None ... no filtering
RemoveNoncanonical ... filter out alignments that contain non-canonical junctions
RemoveNoncanonicalUnannotated ... filter out alignments that contain non-canonical unannotated junctions when using annotated splice junctions database. The annotated non-canonical junctions will be kept.
outFilterIntronStrands RemoveInconsistentStrands
string: filter alignments
RemoveInconsistentStrands ... remove alignments that have junctions with inconsistent strands
None ... no filtering
### Output splice junctions (SJ.out.tab)
outSJtype Standard
string: type of splice junction output
Standard ... standard SJ.out.tab output
None ... no splice junction output
### Output Filtering: Splice Junctions
outSJfilterReads All
string: which reads to consider for collapsed splice junctions output
All ... all reads, unique- and multi-mappers
Unique ... uniquely mapping reads only
outSJfilterOverhangMin 30 12 12 12
4 integers: minimum overhang length for splice junctions on both sides for: (1) non-canonical motifs, (2) GT/AG and CT/AC motif, (3) GC/AG and CT/GC motif, (4) AT/AC and GT/AT motif. -1 means no output for that motif
does not apply to annotated junctions
outSJfilterCountUniqueMin 3 1 1 1
4 integers: minimum uniquely mapping read count per junction for: (1) non-canonical motifs, (2) GT/AG and CT/AC motif, (3) GC/AG and CT/GC motif, (4) AT/AC and GT/AT motif. -1 means no output for that motif
Junctions are output if one of outSJfilterCountUniqueMin OR outSJfilterCountTotalMin conditions are satisfied
does not apply to annotated junctions
outSJfilterCountTotalMin 3 1 1 1
4 integers: minimum total (multi-mapping+unique) read count per junction for: (1) non-canonical motifs, (2) GT/AG and CT/AC motif, (3) GC/AG and CT/GC motif, (4) AT/AC and GT/AT motif. -1 means no output for that motif
Junctions are output if one of outSJfilterCountUniqueMin OR outSJfilterCountTotalMin conditions are satisfied
does not apply to annotated junctions
outSJfilterDistToOtherSJmin 10 0 5 10
4 integers>=0: minimum allowed distance to other junctions' donor/acceptor
does not apply to annotated junctions
outSJfilterIntronMaxVsReadN 50000 100000 200000
N integers>=0: maximum gap allowed for junctions supported by 1,2,3,,,N reads
i.e. by default junctions supported by 1 read can have gaps <=50000b, by 2 reads: <=100000b, by 3 reads: <=200000. by >=4 reads any gap <=alignIntronMax
does not apply to annotated junctions
### Scoring
scoreGap 0
int: splice junction penalty (independent on intron motif)
scoreGapNoncan -8
int: non-canonical junction penalty (in addition to scoreGap)
scoreGapGCAG -4
GC/AG and CT/GC junction penalty (in addition to scoreGap)
scoreGapATAC -8
AT/AC and GT/AT junction penalty (in addition to scoreGap)
scoreGenomicLengthLog2scale -0.25
extra score logarithmically scaled with genomic length of the alignment: scoreGenomicLengthLog2scale*log2(genomicLength)
scoreDelOpen -2
deletion open penalty
scoreDelBase -2
deletion extension penalty per base (in addition to scoreDelOpen)
scoreInsOpen -2
insertion open penalty
scoreInsBase -2
insertion extension penalty per base (in addition to scoreInsOpen)
scoreStitchSJshift 1
maximum score reduction while searching for SJ boundaries in the stitching step
### Alignments and Seeding
seedSearchStartLmax 50
int>0: defines the search start point through the read - the read is split into pieces no longer than this value
seedSearchStartLmaxOverLread 1.0
real: seedSearchStartLmax normalized to read length (sum of mates' lengths for paired-end reads)
seedSearchLmax 0
int>=0: defines the maximum length of the seeds, if =0 seed length is not limited
seedMultimapNmax 10000
int>0: only pieces that map fewer than this value are utilized in the stitching procedure
seedPerReadNmax 1000
int>0: max number of seeds per read
seedPerWindowNmax 50
int>0: max number of seeds per window
seedNoneLociPerWindow 10
int>0: max number of one seed loci per window
seedSplitMin 12
int>0: min length of the seed sequences split by Ns or mate gap
seedMapMin 5
int>0: min length of seeds to be mapped
alignIntronMin 21
minimum intron size: genomic gap is considered intron if its length>=alignIntronMin, otherwise it is considered Deletion
alignIntronMax 0
maximum intron size, if 0, max intron size will be determined by (2^winBinNbits)*winAnchorDistNbins
alignMatesGapMax 0
maximum gap between two mates, if 0, max intron gap will be determined by (2^winBinNbits)*winAnchorDistNbins
alignSJoverhangMin 5
int>0: minimum overhang (i.e. block size) for spliced alignments
alignSJstitchMismatchNmax 0 -1 0 0
4*int>=0: maximum number of mismatches for stitching of the splice junctions (-1: no limit).
(1) non-canonical motifs, (2) GT/AG and CT/AC motif, (3) GC/AG and CT/GC motif, (4) AT/AC and GT/AT motif.
alignSJDBoverhangMin 3
int>0: minimum overhang (i.e. block size) for annotated (sjdb) spliced alignments
alignSplicedMateMapLmin 0
int>0: minimum mapped length for a read mate that is spliced
alignSplicedMateMapLminOverLmate 0.66
real>0: alignSplicedMateMapLmin normalized to mate length
alignWindowsPerReadNmax 10000
int>0: max number of windows per read
alignTranscriptsPerWindowNmax 100
int>0: max number of transcripts per window
alignTranscriptsPerReadNmax 10000
int>0: max number of different alignments per read to consider
alignEndsType Local
string: type of read ends alignment
Local ... standard local alignment with soft-clipping allowed
EndToEnd ... force end-to-end read alignment, do not soft-clip
Extend5pOfRead1 ... fully extend only the 5p of the read1, all other ends: local alignment
Extend5pOfReads12 ... fully extend only the 5p of the both read1 and read2, all other ends: local alignment
alignEndsProtrude 0 ConcordantPair
int, string: allow protrusion of alignment ends, i.e. start (end) of the +strand mate downstream of the start (end) of the -strand mate
1st word: int: maximum number of protrusion bases allowed
2nd word: string:
ConcordantPair ... report alignments with non-zero protrusion as concordant pairs
DiscordantPair ... report alignments with non-zero protrusion as discordant pairs
alignSoftClipAtReferenceEnds Yes
string: allow the soft-clipping of the alignments past the end of the chromosomes
Yes ... allow
No ... prohibit, useful for compatibility with Cufflinks
alignInsertionFlush None
string: how to flush ambiguous insertion positions
None ... insertions are not flushed
Right ... insertions are flushed to the right
### Paired-End reads
peOverlapNbasesMin 0
int>=0: minimum number of overlap bases to trigger mates merging and realignment
peOverlapMMp 0.01
real, >=0 & <1: maximum proportion of mismatched bases in the overlap area
### Windows, Anchors, Binning
winAnchorMultimapNmax 50
int>0: max number of loci anchors are allowed to map to
winBinNbits 16
int>0: =log2(winBin), where winBin is the size of the bin for the windows/clustering, each window will occupy an integer number of bins.
winAnchorDistNbins 9
int>0: max number of bins between two anchors that allows aggregation of anchors into one window
winFlankNbins 4
int>0: log2(winFlank), where win Flank is the size of the left and right flanking regions for each window
winReadCoverageRelativeMin 0.5
real>=0: minimum relative coverage of the read sequence by the seeds in a window, for STARlong algorithm only.
winReadCoverageBasesMin 0
int>0: minimum number of bases covered by the seeds in a window , for STARlong algorithm only.
### Chimeric Alignments
chimOutType Junctions
string(s): type of chimeric output
Junctions ... Chimeric.out.junction
SeparateSAMold ... output old SAM into separate Chimeric.out.sam file
WithinBAM ... output into main aligned BAM files (Aligned.*.bam)
WithinBAM HardClip ... (default) hard-clipping in the CIGAR for supplemental chimeric alignments (default if no 2nd word is present)
WithinBAM SoftClip ... soft-clipping in the CIGAR for supplemental chimeric alignments
chimSegmentMin 0
int>=0: minimum length of chimeric segment length, if ==0, no chimeric output
chimScoreMin 0
int>=0: minimum total (summed) score of the chimeric segments
chimScoreDropMax 20
int>=0: max drop (difference) of chimeric score (the sum of scores of all chimeric segments) from the read length
chimScoreSeparation 10
int>=0: minimum difference (separation) between the best chimeric score and the next one
chimScoreJunctionNonGTAG -1
int: penalty for a non-GT/AG chimeric junction
chimJunctionOverhangMin 20
int>=0: minimum overhang for a chimeric junction
chimSegmentReadGapMax 0
int>=0: maximum gap in the read sequence between chimeric segments
chimFilter banGenomicN
string(s): different filters for chimeric alignments
None ... no filtering
banGenomicN ... Ns are not allowed in the genome sequence around the chimeric junction
chimMainSegmentMultNmax 10
int>=1: maximum number of multi-alignments for the main chimeric segment. =1 will prohibit multimapping main segments.
chimMultimapNmax 0
int>=0: maximum number of chimeric multi-alignments
0 ... use the old scheme for chimeric detection which only considered unique alignments
chimMultimapScoreRange 1
int>=0: the score range for multi-mapping chimeras below the best chimeric score. Only works with --chimMultimapNmax > 1
chimNonchimScoreDropMin 20
int>=0: 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
chimOutJunctionFormat 0
int: formatting type for the Chimeric.out.junction file
0 ... no comment lines/headers
1 ... comment lines at the end of the file: command line and Nreads: total, unique/multi-mapping
### Quantification of Annotations
quantMode -
string(s): types of quantification requested
- ... none
TranscriptomeSAM ... output SAM/BAM alignments to transcriptome into a separate file
GeneCounts ... count reads per gene
quantTranscriptomeBAMcompression 1 1
int: -2 to 10 transcriptome BAM compression level
-2 ... no BAM output
-1 ... default compression (6?)
0 ... no compression
10 ... maximum compression
quantTranscriptomeBan IndelSoftclipSingleend
string: prohibit various alignment type
IndelSoftclipSingleend ... prohibit indels, soft clipping and single-end alignments - compatible with RSEM
Singleend ... prohibit single-end alignments
### 2-pass Mapping
twopassMode None
string: 2-pass mapping mode.
None ... 1-pass mapping
Basic ... basic 2-pass mapping, with all 1st pass junctions inserted into the genome indices on the fly
twopass1readsN -1
int: number of reads to process for the 1st step. Use very large number (or default -1) to map all reads in the first step.
### WASP parameters
waspOutputMode None
string: WASP allele-specific output type. This is re-implementation of the original WASP mappability filtering by Bryce van de Geijn, Graham McVicker, Yoav Gilad & Jonathan K Pritchard. Please cite the original WASP paper: Nature Methods 12, 1061–1063 (2015), https://www.nature.com/articles/nmeth.3582 .
SAMtag ... add WASP tags to the alignments that pass WASP filtering
### STARsolo (single cell RNA-seq) parameters
soloType None
string(s): type of single-cell RNA-seq
CB_UMI_Simple ... (a.k.a. Droplet) one UMI and one Cell Barcode of fixed length in read2, e.g. Drop-seq and 10X Chromium.
CB_UMI_Complex ... one UMI of fixed length, but multiple Cell Barcodes of varying length, as well as adapters sequences are allowed in read2 only, e.g. inDrop.
CB_samTagOut ... output Cell Barcode as CR and/or CB SAm tag. No UMI counting. --readFilesIn cDNA_read1 [cDNA_read2 if paired-end] CellBarcode_read . Requires --outSAMtype BAM Unsorted [and/or SortedByCoordinate]
SmartSeq ... Smart-seq: each cell in a separate FASTQ (paired- or single-end), barcodes are corresponding read-groups, no UMI sequences, alignments deduplicated according to alignment start and end (after extending soft-clipped bases)
soloCBwhitelist -
string(s): file(s) with whitelist(s) of cell barcodes. Only --soloType CB_UMI_Complex allows more than one whitelist file.
None ... no whitelist: all cell barcodes are allowed
soloCBstart 1
int>0: cell barcode start base
soloCBlen 16
int>0: cell barcode length
soloUMIstart 17
int>0: UMI start base
soloUMIlen 10
int>0: UMI length
soloBarcodeReadLength 1
int: length of the barcode read
1 ... equal to sum of soloCBlen+soloUMIlen
0 ... not defined, do not check
soloBarcodeMate 0
int: identifies which read mate contains the barcode (CB+UMI) sequence
0 ... barcode sequence is on separate read, which should always be the last file in the --readFilesIn listed
1 ... barcode sequence is a part of mate 1
2 ... barcode sequence is a part of mate 2
soloCBposition -
strings(s) position of Cell Barcode(s) on the barcode read.
Presently only works with --soloType CB_UMI_Complex, and barcodes are assumed to be on Read2.
Format for each barcode: startAnchor_startPosition_endAnchor_endPosition
start(end)Anchor defines the Anchor Base for the CB: 0: read start; 1: read end; 2: adapter start; 3: adapter end
start(end)Position is the 0-based position with of the CB start(end) with respect to the Anchor Base
String for different barcodes are separated by space.
Example: inDrop (Zilionis et al, Nat. Protocols, 2017):
--soloCBposition 0_0_2_-1 3_1_3_8
soloUMIposition -
string position of the UMI on the barcode read, same as soloCBposition
Example: inDrop (Zilionis et al, Nat. Protocols, 2017):
--soloCBposition 3_9_3_14
soloAdapterSequence -
string: adapter sequence to anchor barcodes.
soloAdapterMismatchesNmax 1
int>0: maximum number of mismatches allowed in adapter sequence.
soloCBmatchWLtype 1MM_multi
string: matching the Cell Barcodes to the WhiteList
Exact ... only exact matches allowed
1MM ... only one match in whitelist with 1 mismatched base allowed. Allowed CBs have to have at least one read with exact match.
1MM_multi ... multiple matches in whitelist with 1 mismatched base allowed, posterior probability calculation is used choose one of the matches.
Allowed CBs have to have at least one read with exact match. This option matches best with CellRanger 2.2.0
1MM_multi_pseudocounts ... same as 1MM_Multi, but pseudocounts of 1 are added to all whitelist barcodes.
1MM_multi_Nbase_pseudocounts ... same as 1MM_multi_pseudocounts, multimatching to WL is allowed for CBs with N-bases. This option matches best with CellRanger >= 3.0.0
soloInputSAMattrBarcodeSeq -
string(s): when inputting reads from a SAM file (--readsFileType SAM SE/PE), these SAM attributes mark the barcode sequence (in proper order).
For instance, for 10X CellRanger or STARsolo BAMs, use --soloInputSAMattrBarcodeSeq CR UR .
This parameter is required when running STARsolo with input from SAM.
soloInputSAMattrBarcodeQual -
string(s): when inputting reads from a SAM file (--readsFileType SAM SE/PE), these SAM attributes mark the barcode qualities (in proper order).
For instance, for 10X CellRanger or STARsolo BAMs, use --soloInputSAMattrBarcodeQual CY UY .
If this parameter is '-' (default), the quality 'H' will be assigned to all bases.
soloStrand Forward
string: strandedness of the solo libraries:
Unstranded ... no strand information
Forward ... read strand same as the original RNA molecule
Reverse ... read strand opposite to the original RNA molecule
soloFeatures Gene
string(s): genomic features for which the UMI counts per Cell Barcode are collected
Gene ... genes: reads match the gene transcript
SJ ... splice junctions: reported in SJ.out.tab
GeneFull ... full genes: count all reads overlapping genes' exons and introns
#####UnderDevelopment_begin : not supported - do not use
Transcript3p ... quantification of transcript for 3' protocols
#####UnderDevelopment_end
soloMultiMappers Unique
string(s): counting method for reads mapping to multiple genes
Unique ... count only reads that map to unique genes
Uniform ... uniformly distribute multi-genic UMIs to all genes
Rescue ... distribute UMIs proportionally to unique+uniform counts (~ first iteartion of EM)
PropUnique ... distribute UMIs proportionally to unique mappers, if present, and uniformly if not.
soloUMIdedup 1MM_All
string(s): type of UMI deduplication (collapsing) algorithm
1MM_All ... all UMIs with 1 mismatch distance to each other are collapsed (i.e. counted once).
1MM_Directional_UMItools ... follows the "directional" method from the UMI-tools by Smith, Heger and Sudbery (Genome Research 2017).
1MM_Directional ... same as 1MM_Directional_UMItools, but with more stringent criteria for duplicate UMIs
Exact ... only exactly matching UMIs are collapsed.
NoDedup ... no deduplication of UMIs, count all reads.
1MM_CR ... CellRanger2-4 algorithm for 1MM UMI collapsing.
soloUMIfiltering -
string(s) type of UMI filtering (for reads uniquely mapping to genes)
- ... basic filtering: remove UMIs with N and homopolymers (similar to CellRanger 2.2.0).
MultiGeneUMI ... basic + remove lower-count UMIs that map to more than one gene.
MultiGeneUMI_All ... basic + remove all UMIs that map to more than one gene.
MultiGeneUMI_CR ... basic + remove lower-count UMIs that map to more than one gene, matching CellRanger > 3.0.0 .
Only works with --soloUMIdedup 1MM_CR
soloOutFileNames Solo.out/ features.tsv barcodes.tsv matrix.mtx
string(s) file names for STARsolo output:
file_name_prefix gene_names barcode_sequences cell_feature_count_matrix
soloCellFilter CellRanger2.2 3000 0.99 10
string(s): cell filtering type and parameters
None ... do not output filtered cells
TopCells ... only report top cells by UMI count, followed by the exact number of cells
CellRanger2.2 ... simple filtering of CellRanger 2.2.
Can be followed by numbers: number of expected cells, robust maximum percentile for UMI count, maximum to minimum ratio for UMI count
The harcoded values are from CellRanger: nExpectedCells=3000; maxPercentile=0.99; maxMinRatio=10
EmptyDrops_CR ... EmptyDrops filtering in CellRanger flavor. Please cite the original EmptyDrops paper: A.T.L Lun et al, Genome Biology, 20, 63 (2019): https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1662-y
Can be followed by 10 numeric parameters: nExpectedCells maxPercentile maxMinRatio indMin indMax umiMin umiMinFracMedian candMaxN FDR simN
The harcoded values are from CellRanger: 3000 0.99 10 45000 90000 500 0.01 20000 0.01 10000
soloOutFormatFeaturesGeneField3 "Gene Expression"
string(s): field 3 in the Gene features.tsv file. If "-", then no 3rd field is output.
#####UnderDevelopment_begin : not supported - do not use
soloClusterCBfile -
string: file containing the cluster information for cell barcodes, two columns: CB cluster_index. Only used with --soloFeatures Transcript3p
#####UnderDevelopment_end
