Input options (must include -d)

-D, --dir=directory

Genome directory. Default (as specified by --with-gmapdb to the configure program) is /var/cache/gmap

-d, --db=STRING

Genome database

--use-localdb=INT

Whether to use the local hash tables, which help with finding extensions to the ends of alignments in the presence of splicing or indels (0=no, 1=yes if available (default))

Transcriptome-guided options (optional)

-C, --transcriptdir=directory

Transcriptome directory. Default is the value for --dir above

-c, --transcriptdb=STRING

Transcriptome database

--use-transcriptome-only

Use only the transcriptome index and not the genome index

Computation options

-k, --kmer=INT

kmer size to use in genome database (allowed values: 16 or less) If not specified, the program will find the highest available kmer size in the genome database

--sampling=INT

Sampling to use in genome database. If not specified, the program will find the smallest available sampling value in the genome database within selected k-mer size

-q, --part=INT/INT

Process only the i-th out of every n sequences e.g., 0/100 or 99/100 (useful for distributing jobs to a computer farm).

--input-buffer-size=INT

Size of input buffer (program reads this many sequences at a time for efficiency) (default 10000)

--barcode-length=INT

Amount of barcode to remove from start of every read before alignment (default 0)

--endtrim-length=INT

Amount of trim to remove from the end of every read before alignment (default 0)

--orientation=STRING

Orientation of paired-end reads Allowed values: FR (fwd-rev, or typical Illumina; default), RF (rev-fwd, for circularized inserts), or FF (fwd-fwd, same strand), or 10X (single-cell where read 1 has barcode information; read 2 is rev)

--10x-whitelist=FILE

Whitelist of 10X Genomics GEM bead barcodes, needed to perform correction of cellular barcodes. This file can be obtained at cellranger-x.y.z/lib/python/cellranger/barcodes (for Cell Ranger version >= 4) cellranger-x.y.z/lib/cellranger-cs/x.y.z/lib/python/cellranger/barcodes (<= 3)

--10x-well-position=INT

Position of well information in the accession, when separated by colons If set to 0, then no well information will be printed in the CB field (default: 4)

--fastq-id-start=INT

Starting position of identifier in FASTQ header, space-delimited (>= 1)

--fastq-id-end=INT

Ending position of identifier in FASTQ header, space-delimited (>= 1)

Examples:

@HWUSI-EAS100R:6:73:941:1973#0/1

start=1, end=1 (default) => identifier is HWUSI-EAS100R:6:73:941:1973#0

@SRR001666.1 071112_SLXA-EAS1_s_7:5:1:817:345 length=36

start=1, end=1 => identifier is SRR001666.1 start=2, end=2 => identifier is 071112_SLXA-EAS1_s_7:5:1:817:345 start=1, end=2 => identifier is SRR001666.1 071112_SLXA-EAS1_s_7:5:1:817:345

--force-single-end

When multiple FASTQ files are provided on the command line, GSNAP assumes they are matching paired-end files. This flag treats each file as single-end.

--filter-chastity=STRING

Skips reads marked by the Illumina chastity program. Expecting a string after the accession having a 'Y' after the first colon, like this:

@accession 1:Y:0:CTTGTA

where the 'Y' signifies filtering by chastity. Values: off (default), either, both. For 'either', a 'Y' on either end of a paired-end read will be filtered. For 'both', a 'Y' is required on both ends of a paired-end read (or on the only end of a single-end read).

--allow-pe-name-mismatch

Allows accession names of reads to mismatch in paired-end files

--interleaved

Input is in interleaved format (one read per line, tab-delimited

--gunzip

Uncompress gzipped input files

--bunzip2

Uncompress bzip2-compressed input files

Computation options

-B, --batch=INT

Batch mode (default = 2)

Mode Hash offsets Hash positions Genome Local hash offsets Local hash positions

0

allocate mmap mmap allocate mmap

1

allocate mmap & preload mmap allocate mmap & preload

2

allocate mmap & preload mmap & preload allocate mmap & preload

3

allocate allocate mmap & preload allocate allocate

(default)

4 allocate allocate allocate allocate allocate

Note: For a single sequence, all data structures use mmap

A batch level of 5 means the same as 4, and is kept only for backward compatibility

--use-shared-memory=INT

If 1, then allocated memory is shared among all processes on this node If 0 (default), then each process has private allocated memory

--preload-shared-memory

Load files indicated by --batch mode into shared memory for use by other GMAP/GSNAP processes on this node, and then exit. Ignore any input files.

--unload-shared-memory

Unload files indicated by --batch mode into shared memory, or allow them to be unloaded when existing GMAP/GSNAP processes on this node are finished with them. Ignore any input files.

-m, --max-mismatches=FLOAT

Maximum number of mismatches allowed (if not specified, then GSNAP tries to find the best possible match in the genome) If specified between 0.0 and 1.0, then treated as a fraction of each read length. Otherwise, treated as an integral number of mismatches (including indel and splicing penalties). Default is 0.3

--max-ref-mismatches=FLOAT

If GSNAP is run under SNP-tolerant or masked genome mode, then the --max-mismatches parameter above is for mismatches against reference and SNPs, or against the unmasked genome, respectively. The --max-ref-mismatches parameter is against mismatches against the reference genome or masked genome.

--min-coverage=FLOAT

Minimum coverage required for an alignment. If specified between 0.0 and 1.0, then treated as a fraction of each read length. Otherwise, treated as an integral number of base pairs. Default value is 0.5.

--filter-within-trims=INT

Whether to count mismatches in trimmed part of alignment (1, yes) or mismatches to the ends of the read (0, no), when applying the --max-mismatches and --max-ref-mismatches parameters. Default for RNA-Seq is 1 (yes) so we can allow for reads that align past the ends of an exon. Default for DNA-Seq is 0 (no).

For RNA-Seq, trimmed ends should be ignored, because trimming

is performed at probable splice sites, to allow for reads that align past the ends of an exon.

--query-unk-mismatch=INT

Whether to count unknown (N) characters in the query as a mismatch (0=no (default), 1=yes)

--genome-unk-mismatch=INT

Whether to count unknown (N) characters in the genome as a mismatch (0=no, 1=yes). If --use-mask is specified, default is no, otherwise yes.

-i, --indel-penalty=INT

Penalty for an indel (default 2). Counts against mismatches allowed. To find indels, make indel-penalty less than or equal to max-mismatches. A value < 2 can lead to false positives at read ends

--indel-endlength=INT

Minimum length at end required for indel alignments (default 4)

-y, --max-middle-insertions=FLOAT

Maximum number of middle insertions allowed (default is 0.2) If specified between 0.0 and 1.0, then treated as a fraction of each read length. Otherwise, treated as an integral number of base pairs

-z, --max-middle-deletions=FLOAT

Maximum number of middle deletions allowed (default 0.2) If specified between 0.0 and 1.0, then treated as a fraction of each read length. Otherwise, treated as an integral number of base pairs

-Y, --max-end-insertions=INT

Maximum number of end insertions allowed (default 3)

-Z, --max-end-deletions=INT

Maximum number of end deletions allowed (default 3)

-M, --suboptimal-levels=INT

Report suboptimal hits beyond best hit (default 0) All hits with best score plus suboptimal-levels are reported

-a, --adapter-strip=STRING

Method for removing adapters from reads. Currently allowed values: off, paired. Default is "off". To turn on, specify "paired", which removes adapters from paired-end reads if they appear to be present.

--trim-indel-score=INT

Score to use for indels when trimming at ends. To turn off trimming, specify 0. Default is -2 for both RNA-Seq and DNA-Seq. Warning: Turning trimming off in RNA-Seq can give false positive indels at the ends of reads

-e, --use-mask=STRING

Use genome containing masks (e.g. for non-exons) for scoring preference

-V, --snpsdir=STRING

Directory for SNPs index files (created using snpindex) (default is location of genome index files specified using -D and -d)

-v, --use-snps=STRING

Use database containing known SNPs (in <STRING>.iit, built previously using snpindex) for tolerance to SNPs

--cmetdir=STRING

Directory for methylcytosine index files (created using cmetindex) (default is location of genome index files specified using -D, -V, and -d)

--atoidir=STRING

Directory for A-to-I RNA editing index files (created using atoiindex) (default is location of genome index files specified using -D, -V, and -d)

--mode=STRING

Alignment mode: standard (default), cmet-stranded, cmet-nonstranded, atoi-stranded, atoi-nonstranded, ttoc-stranded, or ttoc-nonstranded. Non-standard modes requires you to have previously run the cmetindex or atoiindex programs (which also cover the ttoc modes) on the genome

-t, --nthreads=INT

Number of worker threads

--max-anchors=INT

Controls number of candidate segments returned by the complete set algorithm Default is 10. Can be increased to higher values to solve alignments with evenly spaced mismatches at close distances. However, higher values will cause GSNAP to run more slowly. A value of 1000, for example, slows down the program by a factor of 10 or so. Therefore, change this value only if absolutely necessary.

Splicing options for DNA-Seq

--find-dna-chimeras=INT

Look for distant splicing involving poor splice sites (0=no, 1=yes) If not specified, then default is to be on unless only known splicing is desired (--use-splicing is specified and --novelsplicing is off)

Splicing options for RNA-Seq

-N, --novelsplicing=INT

Look for novel splicing (0=no (default), 1=yes)

--splicingdir=STRING

Directory for splicing involving known sites or known introns, as specified by the -s or --use-splicing flag (default is directory computed from -D and -d flags). Note: can just give full pathname to the -s flag instead.

-s, --use-splicing=STRING

Look for splicing involving known sites or known introns (in <STRING>.iit), at short or long distances See README instructions for the distinction between known sites and known introns

--ambig-splice-noclip

For ambiguous known splicing at ends of the read, do not clip at the splice site, but extend instead into the intron. This flag makes sense only if you provide the --use-splicing flag, and you are trying to eliminate all soft clipping with --trim-mismatch-score=0

-w, --localsplicedist=INT

Definition of local novel splicing event (default 200000)

--novelend-splicedist=INT

Distance to look for novel splices at the ends of reads (default 80000)

--local-splice-penalty=INT

Penalty for a local splice (default 0). Counts against mismatches allowed

--fusion-sensitivity=INT

Sensitivity for finding fusions

--distant-splice-penalty=INT

Penalty for a distant splice (default 1). A distant splice is one where the intron length exceeds the value of -w, or --localsplicedist, or is an inversion, scramble, or translocation between two different chromosomes Counts against mismatches allowed

--distant-splice-endlength=INT

Minimum length at end required for distant spliced alignments (default 20, min allowed is the value of -k, or kmer size)

--shortend-splice-endlength=INT

Minimum length at end required for short-end spliced alignments (default 2, but unless known splice sites are provided with the -s flag, GSNAP may still need the end length to be the value of -k, or kmer size to find a given splice

--distant-splice-identity=FLOAT

Minimum identity at end required for distant spliced alignments (default 0.95)

--antistranded-penalty=INT

(Not currently implemented, since it leads to poor results) Penalty for antistranded splicing when using stranded RNA-Seq protocols. A positive value, such as 1, expects antisense on the first read and sense on the second read. Default is 0, which treats sense and antisense equally well

--merge-distant-samechr

Report distant splices on the same chromosome as a single splice, if possible. Will produce a single SAM line instead of two SAM lines, which is also done for translocations, inversions, and scramble events

Options for paired-end reads

--pairmax-dna=INT

Max total genomic length for DNA-Seq paired reads, or other reads without splicing (default 2000). Used if -N or -s is not specified. This value is also used for circular chromosomes when splicing in linear chromosomes is allowed

--pairmax-rna=INT

Max total genomic length for RNA-Seq paired reads, or other reads that could have a splice (default 200000). Used if -N or -s is specified. Should probably match the value for -w, --localsplicedist.

--pairexpect=INT

Expected paired-end length, used for calling splices in medial part of paired-end reads (default 500). Was turned off in previous versions, but reinstated.

--pairdev=INT

Allowable deviation from expected paired-end length, used for calling splices in medial part of paired-end reads (default 100). Was turned off in previous versions, but reinstated.

Options for quality scores

--quality-protocol=STRING

Protocol for input quality scores. Allowed values: illumina (ASCII 64-126) (equivalent to -J 64 -j -31) sanger (ASCII 33-126) (equivalent to -J 33 -j 0)

Default is sanger (no quality print shift)

SAM output files should have quality scores in sanger protocol

Or you can customize this behavior with these flags:

-J, --quality-zero-score=INT

FASTQ quality scores are zero at this ASCII value (default is 33 for sanger protocol; for Illumina, select 64)

-j, --quality-print-shift=INT

Shift FASTQ quality scores by this amount in output (default is 0 for sanger protocol; to change Illumina input to Sanger output, select -31)

Output options

-n, --npaths=INT

Maximum number of paths to print (default 100).

-Q, --quiet-if-excessive

If more than maximum number of paths are found, then nothing is printed.

-O, --ordered

Print output in same order as input (relevant only if there is more than one worker thread)

--show-refdiff

For GSNAP output in SNP-tolerant alignment, shows all differences relative to the reference genome as lower case (otherwise, it shows all differences relative to both the reference and alternate genome)

--clip-overlap

For paired-end reads whose alignments overlap, clip the overlapping region.

--merge-overlap

For paired-end reads whose alignments overlap, merge the two ends into a single end (beta implementation)

--print-snps

Print detailed information about SNPs in reads (works only if -v also selected) (not fully implemented yet)

--failsonly

Print only failed alignments, those with no results

--nofails

Exclude printing of failed alignments

--only-concordant

Print only concordant alignments (concordant_uniq, concordant_mult, concordant_circular)

--omit-concordant-uniq

Do not print any concordant_uniq alignments

--omit-concordant-mult

Do not print any concordant_mult alignments

--omit-softclipped

Do not allow any alignments with soft clips

-A, --format=STRING

Another format type, other than default. Currently implemented: sam, m8 (BLAST tabular format)

--split-output=STRING

Basename for multiple-file output, separately for nomapping, halfmapping_uniq, halfmapping_mult, unpaired_uniq, unpaired_mult, paired_uniq, paired_mult, concordant_uniq, and concordant_mult results

-o, --output-file=STRING

File name for a single stream of output results.

--failed-input=STRING

Print completely failed alignments as input FASTA or FASTQ format, to the given file, appending .1 or .2, for paired-end data. If the --split-output flag is also given, this file is generated in addition to the output in the .nomapping file.

--append-output

When --split-output or --failed-input is given, this flag will append output to the existing files. Otherwise, the default is to create new files.

--order-among-best=STRING

Among alignments tied with the best score, order those alignments in this order. Allowed values: genomic, random (default)

--output-buffer-size=INT

Buffer size, in queries, for output thread (default 1000). When the number of results to be printed exceeds this size, worker threads wait until the backlog is cleared

Options for SAM output

--no-sam-headers

Do not print headers beginning with '@'

--add-paired-nomappers

Add nomapper lines as needed to make all paired-end results alternate between first end and second end

--paired-flag-means-concordant=INT

Whether the paired bit in the SAM flags means concordant only (1) or paired plus concordant (0, default)

--sam-headers-batch=INT

Print headers only for this batch, as specified by -q

--sam-hardclip-use-S

Use S instead of H for hardclips

--sam-use-0M

Insert 0M in CIGAR between adjacent insertions, deletions, and introns Picard disallows 0M, other tools may require it

--sam-extended-cigar

Use extended CIGAR format (using X and = symbols instead of M, to indicate matches and mismatches, respectively

--sam-multiple-primaries

Allows multiple alignments to be marked as primary if they have equally good mapping scores

--sam-sparse-secondaries

For secondary alignments (in multiple mappings), uses '*' for SEQ and QUAL fields, to give smaller file sizes. However, the output will give warnings in Picard to give warnings and may not work with downstream tools

--force-xs-dir

For RNA-Seq alignments, disallows XS:A:? when the sense direction is unclear, and replaces this value arbitrarily with XS:A:+. May be useful for some programs, such as Cufflinks, that cannot handle XS:A:?. However, if you use this flag, the reported value of XS:A:+ in these cases will not be meaningful.

--md-lowercase-snp

In MD string, when known SNPs are given by the -v flag, prints difference nucleotides as lower-case when they, differ from reference but match a known alternate allele

--extend-soft-clips

Extends alignments through soft clipped regions. CIGAR string and coordinates will be revised, but currently the MD string will reflect the clipped CIGAR

--action-if-cigar-error

Action to take if there is a disagreement between CIGAR length and sequence length Allowed values: ignore, warning (default), noprint, abort Note that the noprint option does not print the CIGAR string at all if there is an error, so it may break a SAM parser

--read-group-id=STRING

Value to put into read-group id (RG-ID) field

--read-group-name=STRING

Value to put into read-group name (RG-SM) field

--read-group-library=STRING

Value to put into read-group library (RG-LB) field

--read-group-platform=STRING

Value to put into read-group library (RG-PL) field

Help options

--check

Check compiler assumptions

--version

Show version

--help

Show this help message

Other tools of GMAP suite are located in /usr/lib/gmap