SYNOPSIS

gsnap [,OPTIONS/...] ,<FASTA file>, or/ cat <FASTA file> | gmap [OPTIONS...]

OPTIONS

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-sarray=,INT/

Whether to use a suffix array, which will give increased speed. Allowed values: 0 (no), 1 (yes, plus GSNAP/GMAP algorithm, default), or 2 (yes, and use only suffix array algorithm). Note that suffix arrays will bias against SNP alleles in SNP-tolerant alignment.

-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 1000)

--barcode-length=,INT/

Amount of barcode to remove from start of read (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)

--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

--gunzip

Uncompress gzipped input files

--bunzip2

Uncompress bzip2-compressed input files

Computation options

Note: GSNAP has an ultrafast algorithm for calculating mismatches up to and including

((readlength+2)/kmer - 2) ("ultrafast mismatches"). The program will run fastest if max-mismatches (plus suboptimal-levels) is within that value. Also, indels, especially end indels, take longer to compute, although the algorithm is still designed to be fast.

-B, --batch=,INT/

Batch mode (default = 2)

         Mode     Offsets       Positions       Genome          Suffix array
           0      see note      mmap            mmap            mmap
           1      see note      mmap & preload  mmap            mmap
           2      see note      mmap & preload  mmap & preload  mmap & preload
           3      see note      allocate        mmap & preload  mmap & preload
 (default) 4      see note      allocate        allocate        mmap & preload
           5      see note      allocate        allocate        allocate

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

If mmap not available and allocate not chosen, then will use fileio (very slow)

Note about offsets: Expansion of offsets can be controlled

independently by the --expand-offsets flag. However, offsets are accessed relatively fast in this version of GSNAP.

--use-shared-memory=,INT/

If 1 (default), then allocated memory is shared among all processes on this node. If 0, 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.

--expand-offsets=,INT/

Whether to expand the genomic offsets index Values: 0 (no, default), or 1 (yes). Expansion gives faster alignment, but requires more memory

-m, --max-mismatches=,FLOAT/

Maximum number of mismatches allowed (if not specified, then defaults to the ultrafast level of ((readlength+index_interval-1)/kmer - 2)) (By default, the genome index interval is 3, but this can be changed by providing a different value for -q to gmap_build when processing 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) For RNA-Seq, you may need to increase this value slightly to align reads extending past the ends of an exon.

--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 0.0.

--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 (default))

--maxsearch=,INT/

Maximum number of alignments to find (default 1000). Must be larger than --npaths, which is the number to report. Keeping this number large will allow for random selection among multiple alignments. Reducing this number can speed up the program.

-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=,INT/

Maximum number of middle insertions allowed (default is readlength - indel-endlength)

-z, --max-middle-deletions=,INT/ Maximum number of middle deletions allowed (default 30)

-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 6)

-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-mismatch-score=,INT/

Score to use for mismatches when trimming at ends (default is -3; to turn off trimming, specify 0). Warning: turning trimming off will give false positive mismatches at the ends of reads

--trim-indel-score=,INT/

Score to use for indels when trimming at ends (default is -2; to turn off trimming, specify 0). Warning: turning trimming off will give false positive indels at the ends of reads

--end-detail=,STRING/

Amount of alignment detail at ends of read: high (default), medium, or low Note: medium detail could increase speed by 20% or so, but will miss some splices at the ends of reads

-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.

Options for GMAP alignment within GSNAP

--gmap-mode=,STRING/

Cases to use GMAP for complex alignments containing multiple splices or indels Allowed values: none, all, pairsearch, indel_knownsplice, terminal, improve

(or multiple values, separated by commas).

Default: all, i.e., pairsearch,indel_knownsplice,terminal,improve

--trigger-score-for-gmap=,INT/

Try GMAP pairsearch on nearby genomic regions if best score (the total of both ends if paired-end) exceeds this value (default 5)

--gmap-min-match-length=,INT/

Keep GMAP hit only if it has this many consecutive matches (default 20)

--gmap-allowance=,INT/

Extra mismatch/indel score allowed for GMAP alignments (default 3)

--max-gmap-pairsearch=,INT/

Perform GMAP pairsearch on nearby genomic regions up to this many many candidate ends (default 50). Requires pairsearch in --gmap-mode

--max-gmap-terminal=,INT/

Perform GMAP terminal on nearby genomic regions up to this many candidate ends (default 50). Requires terminal in --gmap-mode

--max-gmap-improvement=,INT/

Perform GMAP improvement on nearby genomic regions up to this many candidate ends (default 5). Requires improve in --gmap-mode

--microexon-spliceprob=,FLOAT/

Allow microexons only if one of the splice site probabilities is greater than this value (default 0.95)

Splicing options for DNA-Seq

--find-dna-chimeras=,INT/

Look for distant splicing in DNA-Seq data (0=no (default), 1=yes) Automatically inactivated for RNA-Seq data if -N or -s are specified)

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 50000)

-e, --local-splice-penalty=,INT/

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

-E, --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

-K, --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)

-l, --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 1000). 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 200). 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

-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, the worker threads are halted 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-use-0M

Insert 0M in CIGAR between adjacent insertions and deletions Required by Picard, but can cause errors in other tools

--sam-multiple-primaries

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

--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

--action-if-cigar-error

Action to take if there is a disagreement between CIGAR length and sequence length Allowed values: ignore, warning, noprint (default), abort

--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