Program options for hisat2: HISAT2 version 2.2.0 by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo) Usage: hisat2 [options]* -x {-1 -2 | -U } [-S ] Index filename prefix (minus trailing .X.ht2). Files with #1 mates, paired with files in . Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). Files with #2 mates, paired with files in . Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). Files with unpaired reads. Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2). File for SAM output (default: stdout) , , can be comma-separated lists (no whitespace) and can be specified many times. E.g. '-U file1.fq,file2.fq -U file3.fq'. Options (defaults in parentheses): Input: -q query input files are FASTQ .fq/.fastq (default) --qseq query input files are in Illumina's qseq format -f query input files are (multi-)FASTA .fa/.mfa -r query input files are raw one-sequence-per-line -c , , are sequences themselves, not files -s/--skip skip the first reads/pairs in the input (none) -u/--upto stop after first reads/pairs (no limit) -5/--trim5 trim bases from 5'/left end of reads (0) -3/--trim3 trim bases from 3'/right end of reads (0) --phred33 qualities are Phred+33 (default) --phred64 qualities are Phred+64 --int-quals qualities encoded as space-delimited integers Presets: Same as: --fast --no-repeat-index --sensitive --bowtie2-dp 1 -k 30 --score-min L,0,-0.5 --very-sensitive --bowtie2-dp 2 -k 50 --score-min L,0,-1 Alignment: --bowtie2-dp use Bowtie2's dynamic programming alignment algorithm (0) - 0: no dynamic programming, 1: conditional dynamic programming, and 2: unconditional dynamic programming (slowest) --n-ceil func for max # non-A/C/G/Ts permitted in aln (L,0,0.15) --ignore-quals treat all quality values as 30 on Phred scale (off) --nofw do not align forward (original) version of read (off) --norc do not align reverse-complement version of read (off) --no-repeat-index do not use repeat index Spliced Alignment: --pen-cansplice penalty for a canonical splice site (0) --pen-noncansplice penalty for a non-canonical splice site (12) --pen-canintronlen penalty for long introns (G,-8,1) with canonical splice sites --pen-noncanintronlen penalty for long introns (G,-8,1) with noncanonical splice sites --min-intronlen minimum intron length (20) --max-intronlen maximum intron length (500000) --known-splicesite-infile provide a list of known splice sites --novel-splicesite-outfile report a list of splice sites --novel-splicesite-infile provide a list of novel splice sites --no-temp-splicesite disable the use of splice sites found --no-spliced-alignment disable spliced alignment --rna-strandness specify strand-specific information (unstranded) --tmo reports only those alignments within known transcriptome --dta reports alignments tailored for transcript assemblers --dta-cufflinks reports alignments tailored specifically for cufflinks --avoid-pseudogene tries to avoid aligning reads to pseudogenes (experimental option) --no-templatelen-adjustment disables template length adjustment for RNA-seq reads Scoring: --mp , max and min penalties for mismatch; lower qual = lower penalty <6,2> --sp , max and min penalties for soft-clipping; lower qual = lower penalty <2,1> --no-softclip no soft-clipping --np penalty for non-A/C/G/Ts in read/ref (1) --rdg , read gap open, extend penalties (5,3) --rfg , reference gap open, extend penalties (5,3) --score-min min acceptable alignment score w/r/t read length (L,0.0,-0.2) Reporting: -k It searches for at most distinct, primary alignments for each read. Primary alignments mean alignments whose alignment score is equal to or higher than any other alignments. The search terminates when it cannot find more distinct valid alignments, or when it finds , whichever happens first. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM ‘secondary’ bit (which equals 256) set in its FLAGS field. For reads that have more than distinct, valid alignments, hisat2 does not guarantee that the alignments reported are the best possible in terms of alignment score. Default: 5 (linear index) or 10 (graph index). Note: HISAT2 is not designed with large values for -k in mind, and when aligning reads to long, repetitive genomes, large -k could make alignment much slower. --max-seeds HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that will be extended. For DNA-read alignment (--no-spliced-alignment), HISAT2 extends up to these many seeds and skips the rest of the seeds. For RNA-read alignment, HISAT2 skips extending seeds and reports no alignments if the number of seeds is larger than the number specified with the option, to be compatible with previous versions of HISAT2. Large values for --max-seeds may improve alignment sensitivity, but HISAT2 is not designed with large values for --max-seeds in mind, and when aligning reads to long, repetitive genomes, large --max-seeds could make alignment much slower. The default value is the maximum of 5 and the value that comes with -k times 2. -a/--all HISAT2 reports all alignments it can find. Using the option is equivalent to using both --max-seeds and -k with the maximum value that a 64-bit signed integer can represent (9,223,372,036,854,775,807). --repeat report alignments to repeat sequences directly Paired-end: -I/--minins minimum fragment length (0), only valid with --no-spliced-alignment -X/--maxins maximum fragment length (500), only valid with --no-spliced-alignment --fr/--rf/--ff -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr) --no-mixed suppress unpaired alignments for paired reads --no-discordant suppress discordant alignments for paired reads Output: -t/--time print wall-clock time taken by search phases --un write unpaired reads that didn't align to --al write unpaired reads that aligned at least once to --un-conc write pairs that didn't align concordantly to --al-conc write pairs that aligned concordantly at least once to (Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g. --un-gz , to gzip compress output, or add '-bz2' to bzip2 compress output.) --summary-file print alignment summary to this file. --new-summary print alignment summary in a new style, which is more machine-friendly. --quiet print nothing to stderr except serious errors --met-file send metrics to file at (off) --met-stderr send metrics to stderr (off) --met report internal counters & metrics every secs (1) --no-head suppress header lines, i.e. lines starting with @ --no-sq suppress @SQ header lines --rg-id set read group id, reflected in @RG line and RG:Z: opt field --rg add ("lab:value") to @RG line of SAM header. Note: @RG line only printed when --rg-id is set. --omit-sec-seq put '*' in SEQ and QUAL fields for secondary alignments. Performance: -o/--offrate override offrate of index; must be >= index's offrate -p/--threads number of alignment threads to launch (1) --reorder force SAM output order to match order of input reads --mm use memory-mapped I/O for index; many 'hisat2's can share Other: --qc-filter filter out reads that are bad according to QSEQ filter --seed seed for random number generator (0) --non-deterministic seed rand. gen. arbitrarily instead of using read attributes --remove-chrname remove 'chr' from reference names in alignment --add-chrname add 'chr' to reference names in alignment --version print version information and quit -h/--help print this usage message ---------------------------------------------- Program options for samtools_idxstats: idxstats: unrecognized option '--help' Usage: samtools idxstats [options] --input-fmt-option OPT[=VAL] Specify a single input file format option in the form of OPTION or OPTION=VALUE -@, --threads INT Number of additional threads to use [0] --verbosity INT Set level of verbosity ---------------------------------------------- Program options for hisat2_build: HISAT2 version 2.2.0 by Daehwan Kim (infphilo@gmail.com, http://www.ccb.jhu.edu/people/infphilo) Usage: hisat2-build [options]* reference_in comma-separated list of files with ref sequences hisat2_index_base write ht2 data to files with this dir/basename Options: -c reference sequences given on cmd line (as ) --large-index force generated index to be 'large', even if ref has fewer than 4 billion nucleotides -a/--noauto disable automatic -p/--bmax/--dcv memory-fitting -p number of threads --bmax max bucket sz for blockwise suffix-array builder --bmaxdivn max bucket sz as divisor of ref len (default: 4) --dcv diff-cover period for blockwise (default: 1024) --nodc disable diff-cover (algorithm becomes quadratic) -r/--noref don't build .3/.4.ht2 (packed reference) portion -3/--justref just build .3/.4.ht2 (packed reference) portion -o/--offrate SA is sampled every 2^offRate BWT chars (default: 5) -t/--ftabchars # of chars consumed in initial lookup (default: 10) --localoffrate SA (local) is sampled every 2^offRate BWT chars (default: 3) --localftabchars # of chars consumed in initial lookup in a local index (default: 6) --snp SNP file name --haplotype haplotype file name --ss Splice site file name --exon Exon file name --repeat-ref Repeat reference file name --repeat-info Repeat information file name --repeat-snp Repeat snp file name --repeat-haplotype Repeat haplotype file name --seed seed for random number generator -q/--quiet disable verbose output (for debugging) -h/--help print detailed description of tool and its options --usage print this usage message --version print version information and quit ---------------------------------------------- Program options for samtools_sort: sort: unrecognized option '--help' 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 samtools_view: samtools view: unrecognised option '--help' Usage: samtools view [options] || [region ...] Options: -b output BAM -C output CRAM (requires -T) -1 use fast BAM compression (implies -b) -u uncompressed BAM output (implies -b) -h include header in SAM output -H print SAM header only (no alignments) -c print only the count of matching records -o FILE output file name [stdout] -U FILE output reads not selected by filters to FILE [null] -t FILE FILE listing reference names and lengths (see long help) [null] -X include customized index file -L FILE only include reads overlapping this BED FILE [null] -r STR only include reads in read group STR [null] -R FILE only include reads with read group listed in FILE [null] -d STR:STR only include reads with tag STR and associated value STR [null] -D STR:FILE only include reads with tag STR and associated values listed in FILE [null] -q INT only include reads with mapping quality >= INT [0] -l STR only include reads in library STR [null] -m INT only include reads with number of CIGAR operations consuming query sequence >= INT [0] -f INT only include reads with all of the FLAGs in INT present [0] -F INT only include reads with none of the FLAGS in INT present [0] -G INT only EXCLUDE reads with all of the FLAGs in INT present [0] -s FLOAT subsample reads (given INT.FRAC option value, 0.FRAC is the fraction of templates/read pairs to keep; INT part sets seed) -M use the multi-region iterator (increases the speed, removes duplicates and outputs the reads as they are ordered in the file) -x STR read tag to strip (repeatable) [null] -B collapse the backward CIGAR operation -? print long help, including note about region specification -S ignored (input format is auto-detected) --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 -T, --reference FILE Reference sequence FASTA FILE [null] -@, --threads INT Number of additional threads to use [0] --write-index Automatically index the output files [off] --verbosity INT Set level of verbosity ---------------------------------------------- Program options for samtools_index: index: invalid option -- '-' 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] ----------------------------------------------