# STAR 2.7.10a # STAR - Spliced Transcripts Alignment to a Reference ## Abstract **Motivation:** Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. **Results:** To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. [Link to manual](../docs/STAR/STARmanual.pdf) ## STAR 2.7.10a --- 2022/01/14 ::: New features, behavior changes and bug fixes **New options and features:** * Implemented --soloCellReadStats Standard option to output read statistics for each cell barcode. * Allow to define --clip5pAdapterSeq with --clipAdapterType CellRanger4 option. * Implemented --soloCBmatchWLtype ED2 to allow mismatches and one insertion+deletion (edit distance <=2) for --soloType CB_UMI_Complex. * Implemented Solo BAM tags gx gn: output ';'-separated gene IDs and names for both unique- and multi-gene reads. Note that GX/GN tags are used to output gene ID/name for unique-gene reads. * Implemented --soloFeatures GeneFull_ExonOverIntron GeneFull_Ex50pAS options which prioritize exonic over intronic overlaps for pre-mRNA counting. * Added script extras/scripts/soloCountMatrixFromBAM.awk to re-create Solo count matrix from the BAM output. **Changes in behavior:** * Changed --soloType CB_samTagOut behavior: if barcode cennot be matched to the passlist, CB:Z:- will be recorded (previously CB tag was absent for such reads). * Changed Solo summary statistics outputs in Barcodes.stats and Features.stats files. * Changed Solo BAM tags GX GN behavior: for missing values, "-" is output instead of omitting the tag. * Changed Solo BAM tags output for multiple --soloFeatures: now the first feature on the list is used for GX,GN,XB,UB tags. * Changed Solo SJ behavior: it no longer depends on the whether the alignment is concordant to a Gene. * Fixed a bug that resulted in slightly different solo counts if --soloFeatures Gene and GeneFull were used together with --soloCBmatchWLtype 1MM_multi_pseudocounts option. **Bug fixes** * PR #1425: Assign supplementary alignment to correct mate when mates fully overlap. Many thanks to Sebastian @suhrig for resolving this problem in the chimeric detection. * Fixed a bug introduced in 2.7.9a for --quantMode TranscriptomeSAM output that resulted in both mapped and unmapped output for some reads. Many thanks to Diane Trout (@Caltech) for helping to track this bug. * Issue #1223: fixed the N_unmapped value reported in ReadsPerGene.out.tab. The single-end (i.e. partially mapped alignment are not excluded from N_unmapped. * Issues #535, #1350: fixed a long-standing problem that resulted in a seg-fault whem mapping to the rabbit genome. * Issue #1316: fixed the seg-fault which occurred if --soloType CB_samTagOut and --soloCBwhitelist None are used together. * Issue #1177: throw an error in case the BAM file does not contain NH and AS tags for duplication removal jobs (--runMode inputAlignmentsFromBAM --bamRemoveDuplicatesType UniqueIdenticalNotMulti). * Issue #1262: fixed the bug that prevented EM matrix output when only EM option is specified in --soloMultiMappers. * Issue #1230: fixed the bug that caused seg-faults for --runMode soloCellFiltering runs. ## Location and version ```console $ which STAR /local/cluster/bin/STAR $ STAR --version 2.7.10a ``` ## help message ```console $ 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=2.7.10a STAR compilation time,server,dir=2022-01-14T18:50:00-05:00 :/home/dobin/data/STAR/STARcode/STAR.master/source For more details see: ### 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 options. liftOver ... lift-over of GTF files (--sjdbGTFfile) between genome assemblies using chain file(s) from --genomeChainFiles. soloCellFiltering ... 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 ofthe 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 themapping (--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 genomeChrSetMitochondrial chrM M MT string(s) names of the mitochondrial chromosomes. Presently only used for STARsolo statisics 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 start end 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 toit. 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 Šošić: 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 samefor 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 willbe 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, separated by space. clip5pAdapterMMp 0.1 double(s): max proportion of mismatches for 5p adapter clipping for each mate, separated by space clip5pAfterAdapterNbases 0 int(s): number of bases to clip from 5p of each mate after the adapter clipping, separated by space. #####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 valuecan 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 anycombination/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 junctionis 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 for unique-gene reads. gx gn ... gene IDs and gene names for unique- and multi-gene reads. 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 appliedafter 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 theread 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 thestart (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. Specify >0 valueto switch on the "merginf of overlapping mates" algorithm. 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 chimericalignments (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 filteringby 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 ... multiple Cell Barcodes of varying length, one UMI of fixed length and one adapter sequence of fixed length are allowed in read2 only (e.g. inDrop, ddSeq). 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 onRead2. 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. Only one adapter sequence is allowed. 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 baseallowed. 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 addedto 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 EditDist_2 ... allow up to edit distance of 3 fpr each of the barcodes. May include one deletion + one insertion. Only works with --soloType CB_UMI_Complex. Matches to multiple passlist barcdoes are not allowed. Similar to ParseBio Split-seq pipeline. soloInputSAMattrBarcodeSeq - string(s): when inputting reads from a SAM file (--readsFileType SAM SE/PE), these SAM attributesmark the barcode sequence (in proper order). For instance, for 10X CellRanger or STARsolo BAMs, use --soloInputSAMattrBarcodeSeq CRUR . 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 attributesmark 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 gene (pre-mRNA): count all reads overlapping genes' exons andintrons GeneFull_ExonOverIntron ... full gene (pre-mRNA): count all reads overlapping genes' exons and introns: prioritize 100% overlap with exons GeneFull_Ex50pAS ... full gene (pre-RNA): count all reads overlapping genes' exons and introns: prioritize >50% overlap with exons. Do not count reads with 100% exonic overlap in the antisense direction. #####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 iteration of EM) PropUnique ... distribute UMIs proportionally to unique mappers, if present, and uniformly if not. EM ... multi-gene UMIs are distributed using Expectation Maximization algorithm 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 bySmith, 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. soloCellReadStats None string: Output reads statistics for each CB Standard ... standard 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 ``` software ref: research ref: