================================================================ ======== blat ==================================== ================================================================ ### kent source version 443 ### blat - Standalone BLAT v. 37x1 fast sequence search command line tool usage: blat database query [-ooc=11.ooc] output.psl where: database and query are each either a .fa, .nib or .2bit file, or a list of these files with one file name per line. -ooc=11.ooc tells the program to load over-occurring 11-mers from an external file. This will increase the speed by a factor of 40 in many cases, but is not required. output.psl is the name of the output file. Subranges of .nib and .2bit files may be specified using the syntax: /path/file.nib:seqid:start-end or /path/file.2bit:seqid:start-end or /path/file.nib:start-end With the second form, a sequence id of file:start-end will be used. options: -t=type Database type. Type is one of: dna - DNA sequence prot - protein sequence dnax - DNA sequence translated in six frames to protein The default is dna. -q=type Query type. Type is one of: dna - DNA sequence rna - RNA sequence prot - protein sequence dnax - DNA sequence translated in six frames to protein rnax - DNA sequence translated in three frames to protein The default is dna. -prot Synonymous with -t=prot -q=prot. -ooc=N.ooc Use overused tile file N.ooc. N should correspond to the tileSize. -tileSize=N Sets the size of match that triggers an alignment. Usually between 8 and 12. Default is 11 for DNA and 5 for protein. -stepSize=N Spacing between tiles. Default is tileSize. -oneOff=N If set to 1, this allows one mismatch in tile and still triggers an alignment. Default is 0. -minMatch=N Sets the number of tile matches. Usually set from 2 to 4. Default is 2 for nucleotide, 1 for protein. -minScore=N Sets minimum score. This is the matches minus the mismatches minus some sort of gap penalty. Default is 30. -minIdentity=N Sets minimum sequence identity (in percent). Default is 90 for nucleotide searches, 25 for protein or translated protein searches. -maxGap=N Sets the size of maximum gap between tiles in a clump. Usually set from 0 to 3. Default is 2. Only relevent for minMatch > 1. -noHead Suppresses .psl header (so it's just a tab-separated file). -makeOoc=N.ooc Make overused tile file. Target needs to be complete genome. -repMatch=N Sets the number of repetitions of a tile allowed before it is marked as overused. Typically this is 256 for tileSize 12, 1024 for tile size 11, 4096 for tile size 10. Default is 1024. Typically comes into play only with makeOoc. Also affected by stepSize: when stepSize is halved, repMatch is doubled to compensate. -noSimpRepMask Suppresses simple repeat masking. -mask=type Mask out repeats. Alignments won't be started in masked region but may extend through it in nucleotide searches. Masked areas are ignored entirely in protein or translated searches. Types are: lower - mask out lower-cased sequence upper - mask out upper-cased sequence out - mask according to database.out RepeatMasker .out file file.out - mask database according to RepeatMasker file.out -qMask=type Mask out repeats in query sequence. Similar to -mask above, but for query rather than target sequence. -repeats=type Type is same as mask types above. Repeat bases will not be masked in any way, but matches in repeat areas will be reported separately from matches in other areas in the psl output. -minRepDivergence=NN Minimum percent divergence of repeats to allow them to be unmasked. Default is 15. Only relevant for masking using RepeatMasker .out files. -dots=N Output dot every N sequences to show program's progress. -trimT Trim leading poly-T. -noTrimA Don't trim trailing poly-A. -trimHardA Remove poly-A tail from qSize as well as alignments in psl output. -fastMap Run for fast DNA/DNA remapping - not allowing introns, requiring high %ID. Query sizes must not exceed 5000. -out=type Controls output file format. Type is one of: psl - Default. Tab-separated format, no sequence pslx - Tab-separated format with sequence axt - blastz-associated axt format maf - multiz-associated maf format sim4 - similar to sim4 format wublast - similar to wublast format blast - similar to NCBI blast format blast8- NCBI blast tabular format blast9 - NCBI blast tabular format with comments -fine For high-quality mRNAs, look harder for small initial and terminal exons. Not recommended for ESTs. -maxIntron=N Sets maximum intron size. Default is 750000. -extendThroughN Allows extension of alignment through large blocks of Ns. ================================================================ ======== gfClient ==================================== ================================================================ ### kent source version 443 ### gfClient v. 37x1 - A client for the genomic finding program that produces a .psl file usage: gfClient host port seqDir in.fa out.psl where host is the name of the machine running the gfServer port is the same port that you started the gfServer with seqDir is the path of the .2bit or .nib files relative to the current dir (note these are needed by the client as well as the server) in.fa is a fasta format file. May contain multiple records out.psl is where to put the output options: -t=type Database type. Type is one of: dna - DNA sequence prot - protein sequence dnax - DNA sequence translated in six frames to protein The default is dna. -q=type Query type. Type is one of: dna - DNA sequence rna - RNA sequence prot - protein sequence dnax - DNA sequence translated in six frames to protein rnax - DNA sequence translated in three frames to protein -prot Synonymous with -t=prot -q=prot. -dots=N Output a dot every N query sequences. -nohead Suppresses 5-line psl header. -minScore=N Sets minimum score. This is twice the matches minus the mismatches minus some sort of gap penalty. Default is 30. -minIdentity=N Sets minimum sequence identity (in percent). Default is 90 for nucleotide searches, 25 for protein or translated protein searches. -out=type Controls output file format. Type is one of: psl - Default. Tab-separated format without actual sequence pslx - Tab-separated format with sequence axt - blastz-associated axt format maf - multiz-associated maf format sim4 - similar to sim4 format wublast - similar to wublast format blast - similar to NCBI blast format blast8- NCBI blast tabular format blast9 - NCBI blast tabular format with comments -maxIntron=N Sets maximum intron size. Default is 750000. -genome=name When using a dynamic gfServer, The genome name is used to find the data files relative to the dynamic gfServer root, named in the form $genome.2bit, $genome.untrans.gfidx, and $genome.trans.gfidx -genomeDataDir=path When using a dynamic gfServer, this is the dynamic gfServer root directory that contained the genome data files. Defaults to being the root directory. ================================================================ ======== gfPcr ==================================== ================================================================ ### kent source version 443 ### gfPcr - In silico PCR version 37x1 using gfServer index. usage: gfPcr host port seqDir fPrimer rPrimer output or gfPcr host port seqDir batch output Where: host is the name of the machine running the gfServer port is the gfServer port (usually 17779) seqDir is where the nib or 2bit files for the genome database are fPrimer is the forward strand primer rPrimer is the reverse strand primer output is the output file. Use 'stdout' for output to standard output batch is a space or tab delimited file with the following fields on each line name/fPrimer/rPrimer/maxProductSize options: -maxSize=N - Maximum size of PCR product (default 4000) -minPerfect=N - Minimum size of perfect match at 3' end of primer (default 15) -minGood=N - Minimum size where there must be 2 matches for each mismatch (default 18) -out=XXX - Output format. Either fa - fasta with position, primers in header (default) bed - tab delimited format. Fields: chrom/start/end/name/score/strand psl - blat format. -name=XXX - Name to use in bed output. -genome=name When using a dynamic gfServer, The genome name is used to find the data files relative to the dynamic gfServer root, named in the form $genome.2bit, and $genome.untrans.gfidx. -genomeDataDir=path When using a dynamic gfServer, this is the dynamic gfServer root directory that contained the genome data files. Defaults to being the root directory. ================================================================ ======== gfServer ==================================== ================================================================ ### kent source version 443 ### gfServer v 37x1 - Make a server to quickly find where DNA occurs in genome To set up a server: gfServer start host port file(s) where the files are .2bit or .nib format files specified relative to the current directory To remove a server: gfServer stop host port To query a server with DNA sequence: gfServer query host port probe.fa To query a server with protein sequence: gfServer protQuery host port probe.fa To query a server with translated DNA sequence: gfServer transQuery host port probe.fa To query server with PCR primers: gfServer pcr host port fPrimer rPrimer maxDistance To process one probe fa file against a .2bit format genome (not starting server): gfServer direct probe.fa file(s).2bit To test PCR without starting server: gfServer pcrDirect fPrimer rPrimer file(s).2bit To figure out if server is alive, on static instances get usage statics as well: gfServer status host port For dynamic gfServer instances, specify -genome and optionally the -genomeDataDir to get information on an untranslated genome index. Include -trans to get about information about a translated genome index To get input file list: gfServer files host port To generate a precomputed index: gfServer index gfidx file(s) where the files are .2bit or .nib format files. Separate indexes are be created for untranslated and translated queries. These can be used with a persistent server as with 'start -indexFile or a dynamic server. They must follow the naming convention for for dynamic servers. To run a dynamic server (usually called by xinetd): gfServer dynserver rootdir Data files for genomes are found relative to the root directory. Queries are made using the prefix of the file path relative to the root directory. The files $genome.2bit, $genome.untrans.gfidx, and $genome.trans.gfidx are required. Typically the structure will be in the form: $rootdir/$genomeDataDir/$genome.2bit $rootdir/$genomeDataDir/$genome.untrans.gfidx $rootdir/$genomeDataDir/$genome.trans.gfidx in this case, one would call gfClient with -genome=$genome -genomeDataDir=$genomeDataDir Often $genomeDataDir will be the same name as $genome, however it can be a multi-level path. For instance: GCA/902/686/455/GCA_902686455.1_mSciVul1.1/ The translated or untranslated index maybe omitted if there is no need to handle that type of request. The -perSeqMax functionality can be implemented by creating a file $rootdir/$genomeDataDir/$genome.perseqmax options: -tileSize=N Size of n-mers to index. Default is 11 for nucleotides, 4 for proteins (or translated nucleotides). -stepSize=N Spacing between tiles. Default is tileSize. -minMatch=N Number of n-mer matches that trigger detailed alignment. Default is 2 for nucleotides, 3 for proteins. -maxGap=N Number of insertions or deletions allowed between n-mers. Default is 2 for nucleotides, 0 for proteins. -trans Translate database to protein in 6 frames. Note: it is best to run this on RepeatMasked data in this case. -log=logFile Keep a log file that records server requests. -seqLog Include sequences in log file (not logged with -syslog). -ipLog Include user's IP in log file (not logged with -syslog). -debugLog Include debugging info in log file. -syslog Log to syslog. -logFacility=facility Log to the specified syslog facility - default local0. -mask Use masking from .2bit file. -repMatch=N Number of occurrences of a tile (n-mer) that triggers repeat masking the tile. Default is 1024. -noSimpRepMask Suppresses simple repeat masking. -maxDnaHits=N Maximum number of hits for a DNA query that are sent from the server. Default is 100. -maxTransHits=N Maximum number of hits for a translated query that are sent from the server. Default is 200. -maxNtSize=N Maximum size of untranslated DNA query sequence. Default is 40000. -maxAaSize=N Maximum size of protein or translated DNA queries. Default is 8000. -perSeqMax=file File contains one seq filename (possibly with ':seq' suffix) per line. -maxDnaHits will be applied to each filename[:seq] separately: each may have at most maxDnaHits/2 hits. The filename MUST not include the directory. Useful for assemblies with many alternate/patch sequences. -canStop If set, a quit message will actually take down the server. -indexFile Index file create by `gfServer index'. Saving index can speed up gfServer startup by two orders of magnitude. The parameters must exactly match the parameters when the file is written or bad things will happen. -timeout=N Timeout in seconds. Default is 90. ================================================================ ======== isPcr ==================================== ================================================================ ### kent source version 443 ### isPcr - Standalone v 37x1 In-Situ PCR Program usage: isPcr database query output where database is a fasta, nib, or twoBit file or a text file containing a list of these files, query is a text file file containing three columns: name, forward primer, and reverse primer, and output is where the results go. The names 'stdin' and 'stdout' can be used as file names to make using the program in pipes easier. options: -ooc=N.ooc Use overused tile file N.ooc. N should correspond to the tileSize -tileSize=N the size of match that triggers an alignment. Default is 11 . -stepSize=N spacing between tiles. Default is 5. -maxSize=N - Maximum size of PCR product (default 4000) -minSize=N - Minimum size of PCR product (default 0) -minPerfect=N - Minimum size of perfect match at 3' end of primer (default 15) -minGood=N - Minimum size where there must be 2 matches for each mismatch (default 15) -mask=type Mask out repeats. Alignments won't be started in masked region but may extend through it in nucleotide searches. Masked areas are ignored entirely in protein or translated searches. Types are lower - mask out lower cased sequence upper - mask out upper cased sequence out - mask according to database.out RepeatMasker .out file file.out - mask database according to RepeatMasker file.out -makeOoc=N.ooc Make overused tile file. Database needs to be complete genome. -repMatch=N sets the number of repetitions of a tile allowed before it is marked as overused. Typically this is 256 for tileSize 12, 1024 for tile size 11, 4096 for tile size 10. Default is 1024. Only comes into play with makeOoc -noSimpRepMask Suppresses simple repeat masking. -flipReverse Reverse complement reverse (second) primer before using -out=XXX - Output format. Either fa - fasta with position, primers in header (default) bed - tab delimited format. Fields: chrom/start/end/name/score/strand psl - blat format.