Program options for multiqc: Usage: multiqc [OPTIONS] Main MultiQC run command for use with the click command line, complete with all click function decorators. To make it easy to use MultiQC within notebooks and other locations that don't need click, we simply pass the parsed variables on to a vanilla python function. Options: -f, --force Overwrite any existing reports -d, --dirs Prepend directory to sample names -dd, --dirs-depth INTEGER Prepend [INT] directories to sample names. Negative number to take from start of path. -s, --fullnames Do not clean the sample names (leave as full file name) -i, --title TEXT Report title. Printed as page header, used for filename if not otherwise specified. -b, --comment TEXT Custom comment, will be printed at the top of the report. -n, --filename TEXT Report filename. Use 'stdout' to print to standard out. -o, --outdir TEXT Create report in the specified output directory. -t, --template [default|default_dev|geo|sections|simple] Report template to use. --tag TEXT Use only modules which tagged with this keyword, eg. RNA --view-tags, --view_tags View the available tags and which modules they load -x, --ignore TEXT Ignore analysis files (glob expression) --ignore-samples TEXT Ignore sample names (glob expression) --ignore-symlinks Ignore symlinked directories and files --sample-names PATH File containing alternative sample names -l, --file-list Supply a file containing a list of file paths to be searched, one per row -e, --exclude [module name] Do not use this module. Can specify multiple times. -m, --module [module name] Use only this module. Can specify multiple times. --data-dir Force the parsed data directory to be created. --no-data-dir Prevent the parsed data directory from being created. -k, --data-format [tsv|json|yaml] Output parsed data in a different format. Default: tsv -z, --zip-data-dir Compress the data directory. -p, --export Export plots as static images in addition to the report -fp, --flat Use only flat plots (static images) -ip, --interactive Use only interactive plots (HighCharts Javascript) --lint Use strict linting (validation) to help code development --pdf Creates PDF report with 'simple' template. Requires Pandoc to be installed. --no-megaqc-upload Don't upload generated report to MegaQC, even if MegaQC options are found -c, --config PATH Specific config file to load, after those in MultiQC dir / home dir / working dir. --cl-config, --cl_config TEXT Specify MultiQC config YAML on the command line -v, --verbose Increase output verbosity. -q, --quiet Only show log warnings --no-ansi Disable coloured log output --version Show the version and exit. -h, --help Show this message and exit. ---------------------------------------------- Program options for samtools_sort: samtools sort: failed to read header from "-" sort: invalid option -- 'h' Usage: samtools sort [options...] [in.bam] Options: -l INT Set compression level, from 0 (uncompressed) to 9 (best) -m INT Set maximum memory per thread; suffix K/M/G recognized [768M] -n Sort by read name -t TAG Sort by value of TAG. Uses position as secondary index (or read name if -n is set) -o FILE Write final output to FILE rather than standard output -T PREFIX Write temporary files to PREFIX.nnnn.bam --no-PG do not add a PG line --input-fmt-option OPT[=VAL] Specify a single input file format option in the form of OPTION or OPTION=VALUE -O, --output-fmt FORMAT[,OPT[=VAL]]... Specify output format (SAM, BAM, CRAM) --output-fmt-option OPT[=VAL] Specify a single output file format option in the form of OPTION or OPTION=VALUE --reference FILE Reference sequence FASTA FILE [null] -@, --threads INT Number of additional threads to use [0] --verbosity INT Set level of verbosity ---------------------------------------------- Program options for star: 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=2.7.6a STAR compilation time,server,dir=Sat Sep 19 11:48:47 EDT 2020 vega:/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. liftOver ... lift-over of GTF files (--sjdbGTFfile) between genome assemblies using chain file(s) from --genomeChainFiles. 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. genomeConsensusFile - string: VCF file with consensus SNPs (i.e. alternative allele is the major (AF>0.5) allele) ### 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. #####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 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_end ### 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 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 -: no prefix 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 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. 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. 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. 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. ### Limits limitGenomeGenerateRAM 31000000000 int>0: maximum available RAM (bytes) for genome generation limitIObufferSize 150000000 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, unsorted. 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. ***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 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 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. Similar to CellRanger 2.2.0 1MM_multi_pseudocounts ... same as 1MM_Multi, but pseudocounts of 1 are added to all whitelist barcodes. Similar to CellRanger 3.x.x 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 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 ... follows the "directional" method from the UMI-tools by Smith, Heger and Sudbery (Genome Research 2017). Exact ... only exactly matching UMIs are collapsed NoDedup ... no deduplication of UMIs, count all reads. Allowed for --soloType SmartSeq soloUMIfiltering - string(s) type of UMI filtering - ... basic filtering: remove UMIs with N and homopolymers (similar to CellRanger 2.2.0) MultiGeneUMI ... remove lower-count UMIs that map to more than one gene (introduced in CellRanger 3.x.x) 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 CellRanger2.2 ... simple filtering of CellRanger 2.2, followed by three numbers: number of expected cells, robust maximum percentile for UMI count, maximum to minimum ratio for UMI count TopCells ... only report top cells by UMI count, followed by the exact number of cells None ... do not output filtered cells 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 ---------------------------------------------- Program options for samtools_view: [main_samview] fail to read the header from "-". [main_samview] fail to read the header from "-". ---------------------------------------------- Program options for samtools_index: Usage: samtools index [-bc] [-m INT] [out.index] Options: -b Generate BAI-format index for BAM files [default] -c Generate CSI-format index for BAM files -m INT Set minimum interval size for CSI indices to 2^INT [14] -@ INT Sets the number of threads [none] index: invalid option -- 'h' Usage: samtools index [-bc] [-m INT] [out.index] Options: -b Generate BAI-format index for BAM files [default] -c Generate CSI-format index for BAM files -m INT Set minimum interval size for CSI indices to 2^INT [14] -@ INT Sets the number of threads [none] ----------------------------------------------