Blat Suite Program Specifications and User Guide

Blat produces two major classes of alignments:

• at the DNA level between two sequences that are of 95% or greater identity, but which may include large inserts.
• at the protein or translated DNA level between sequences that are of 80% or greater identity and may also include large inserts.

The output of blat is flexible. By default, it is a simple tab-delimited file that describes the alignment, but which does not include the sequence of the alignment itself. Alternatively, blat can produce output compatible with BLAST or WU-BLAST, as well as several other formats.

The main programs in the blat suite are:

• gfServer – a server that maintains an index of the genome in memory and uses the index to quickly find regions with high levels of sequence similarity to a query sequence.
• gfClient – a program that queries gfServer over the network and does a detailed alignment of the query sequence with regions found by gfServer.
• blat – client and server combined into a single program, first building the index, then using the index, and then exiting.
• webBlat – a web-based version of gfClient that presents the alignments in an interactive fashion.

Building an index of the genome typically takes 10 or 15 minutes. For interactive applications, it is typical to use gfServer to build a whole-genome index. gfClient or webBlat can then align a single query within a few seconds. When aligning a large number of sequences in a batch mode, it may be more efficient to use blat, particularly if it is run on a cluster of computers. Each blat run is typically done against a single chromosome, but with a large number of query sequences.

Other programs in the blat suite are:

• pslSort – combines and sorts the output of multiple blat runs. The blat default output format is psl.
• pslReps – selects the best alignments for a particular query sequence, using a "near best in genome" approach.
• pslPretty – converts alignments from psl format, which is a tab-delimited format that does not include the bases themselves, to a more readable alignment format.
• faToTwoBit – converts fasta format sequence files to the dense, randomly accessible .2bit format that gfClient can use.
• twoBitToFa – converts from .2bit format back to fasta format.
• faToNib – converts from fasta to the somewhat less dense, randomly accessible .nib format that predated .2bit format. Note that each .nib file can contain only a single sequence.
• nibFrag – converts portions of a .nib file back to fasta format.

Usage details for each of the above programs are described in the sections below. For all programs except webBlat, the usage options may also be viewed by typing the name of the program (without arguments) on the UNIX command line.

The gfServer program requires approximately 1 byte of memory for every 3 bases in the genome it is indexing in DNA mode, and 1.5 bytes for each unmasked base in translated mode. The blat program requires approximately 2 bytes of memory for each base in the genome in DNA mode, and 3 bytes of memory for each base in translated mode. The other programs use relatively little memory.

You may also be interested in the following programs that are not part of the blat suite:

• in-silico PCR – quickly finds the sequence between two primers. This includes webPCR, an interface similar to webBlat.
• Genome Browser – displays annotations as a series of tracks on top of the genome.

All of the above software and source code are freely available from the University of California Santa Cruz for non-profit academic use. A license is required for commercial use. For blat licensing, contact Kent Informatics. All other licensing is handled by the UCSC Genome Browser project.

blat

blat – Standalone blat 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, 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 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 is used.

Options:

-t=type                Database type, one of:
                          dna - (default) DNA sequence
                          prot - protein sequence
                          dnax - DNA sequence translated in six frames to protein

-q=type                Query type, 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

-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. Relevant only for minMatch > 1.

-noHead                Suppresses .psl header (so it's just a tab-separated file).

-makeOoc=N.ooc         Makes overused tile file. Target must 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 needed only with makeOoc. 
                       Also affected by stepSize: when stepSize is halved, repMatch is doubled to 
                       compensate.

-mask=type             Masks 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 - masks out lower-cased sequence
                             upper - masks out upper-cased sequence
                             out - masks according to database.out RepeatMasker .out file
                             file.out - masks database according to RepeatMasker file.out

-qMask=type            Masks 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. Relevant only for masking using RepeatMasker .out files.

-dots=N                Outputs a dot every N sequences to show program's progress.

-trimT                 Trims leading poly-T.

-noTrimA               Don't trim trailing poly-A.

-trimHardA             Removes poly-A tail from qSize as well as alignments in psl output.

-fastMap               Run for fast DNA/DNA remapping, not allowing introns and requiring high %ID.

-out=type              Controls output file format, 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, looks 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. 

Blat settings for common usage scenarios:

Mapping ESTs to the genome within the same species: -ooc=11.ooc

Mapping full length mRNAs to the genome in the same species: -ooc=11.ooc -fine -q=rna

Mapping ESTs to the genome across species: -q=dnax -t=dnax

Mapping mRNA to the genome across species: -q=rnax -t=dnax

Mapping proteins to the genome: -q=prot -t=dnax

Mapping DNA to DNA in the same species: -ooc=11.ooc -fastMap

Mapping DNA from one species to another species: -q=dnax -t=dnax
Note that the query side of the alignment should be split into chunks of 25 KB or less for best performance.

gfServer

gfServer – Make a server to quickly find where DNA occurs in genome.

Usage:

To set up a server:
   gfServer start host port file(s) where files are in .nib or .2bit format

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 1 probe fa file against .nib format genome (not starting server):
   gfServer direct probe.fa file(s).nib

To test pcr without starting server:
   gfServer pcrDirect fPrimer rPrimer file(s).nib

To figure out usage level:
   gfServer status host port

To get input file list:
   gfServer files host port

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 triggers 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: when using this option, it 
                       is best to run on RepeatMasked data.

-log=logFile           Keep a log file that records server requests.

-seqLog                Include sequences in log file (not logged with -syslog).

-syslog                Log to syslog.

-logFacility=facility  Log to the specified syslog facility. Default is local0.

-mask                  Use masking from .nib file.

-repMatch=N            Number of occurrences of a tile (n-mer) that triggers repeat-masking of the 
                       tile. Default is 1024.

-maxDnaHits=N          Maximum number of hits sent from the server for a DNA query. Default is 100.

-maxTransHits=N        Maximum number of hits sent from the server for a translated query. 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.

-canStop               If set, a quit message will actually take down the server. 

gfClient

gfClient – 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 used for the gfServer.

seqDir is the path of the .nib or .2bit 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 the output file.

Options:

-t=type          Database type, one of:
                    dna - (Default) DNA sequence
                    prot - protein sequence
                    dnax - DNA sequence translated in six frames to protein

-q=type          Query type, 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 -d=prot -q=prot

-dots=N          Outputs a dot every N query sequences.

-nohead          Suppresses psl header.

-minScore=N      Sets minimum score. This is twice the matches minus the mismatches and minus a 
		    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, 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. 

faToTwoBit

faToTwoBit – Convert DNA from fasta to .2bit format.

Usage:

faToTwoBit in.fa [in2.fa in3.fa ...] out.2bit

Options:

-noMask          Ignore lower-case masking in fa file.

-stripVersion    Strip off version number after "." for GenBank accessions.

-ignoreDups      Convert only first sequence if there are duplicate sequence names. Use 
                 "twoBitDup" to find duplicate sequences.

twoBitToFa

twoBitToFa – Convert all or part of .2bit file to fasta format.

Usage:

twoBitToFa input.2bit output.fa

Options:

-seq=name               Restrict this to just one sequence.

-start=X                Start at given position in sequence (zero-based).

-end=X                  End at given position in sequence (non-inclusive).

-seqList=file           File containing list of the desired sequence names in the format 
                        seqSpec[:start-end], e.g. chr1 or chr1:0-189, where coordinates are 
                        half-open zero-based, i.e. [start,end).

-noMask                 Convert sequence to all upper-case.

-bpt=index.bpt          Use bpt index instead of the built-in one.

-bed=input.bed          Grab sequences specified by input.bed. Will exclude introns.

-bedPos                 With -bed, to use chrom:start-end as the fasta ID in output.fa.

-udcDir=/dir/to/cache   Location of cache for remote bigBeds and bigwigs.

Sequence and range may also be specified as part of the input file name using the syntax:

/path/input.2bit:name
   or
/path/input.2bit:name
   or
/path/input.2bit:name:start-end 

faToNib

faToNib – Convert from .fa to .nib format.

Usage:

faToNib [options] in.fa out.nib

Options:

-softMask   Create nib that soft-masks lower-case sequence.

-hardMask   Create nib that hard-masks lower-case sequence.

nibFrag

nibFrag – Extract part of a .nib file as .fa, with all bases and gaps lower-case by default.

Usage:

nibFrag [options] file.nib start end strand out.fa

Where:

strand is + (plus) or m (minus)

Options:

-masked         Use lower-case characters for bases meant to be masked out.

-hardMasked     Use upper-case characters for bases that are not masked out, and Ns for masked-out bases.

-upper          Use upper-case characters for all bases.

-name=name      Use given name after '>' in output sequence.

-dbHeader=db    Add full database info to the header, with or without -name option.

-tbaHeader=db   Format header for compatibility with tba, takes database name as argument.

pslPretty

pslPretty – Convert PSL to human-readable output

Usage:

pslPretty in.psl target.lst query.lst pretty.out

Options:

-axt              Save in axt format.

-dot=N            Output a dot every N records.

-long             Don't abbreviate long inserts.

-check=fileName   Output alignment checks to filename.

To improve performance, a .psl file containing multiple targets should be sorted by target. The target and query lists can be .fasta, .2bit, or .nib files, or a list of these files, one per line.

pslSort

pslSort – Merge and sort psCluster .psl output files.

Usage:

To sort all of the .psl files in the "inDirs" directories in two stages: first into temporary files in tempDir, and then into outFile: 
    pslSort dirs[1|2] outFile tempDir inDir(s)

The device on tempDir must have sufficient space, typically 15-20 gigabytes if processing a whole genome.

To sort a genome-to-genome alignment, reflecting the alignments across the diagonal: 
    pslSort g2g[1|2] outFile tempDir inDir(s)

Appending [1|2] to the "dirs" or "g2g" option limits the program to only the 
first or second pass of the sort.

Options:

-nohead     Suppress the psl header.

-verbose=N  Set verbosity level, higher for more output. Default is 1.

Note: pslSort will run out of memory on extremely large files. It may be preferable to use this UNIX command in these situations:

sort -k 10 *.psl > sorted.psl

In this case, remove the header lines from the .psl input file using the "-noHead" option to blat before using the UNIX sort command.

pslReps

pslReps – Analyze repeats and generate genome-wide best alignments from a sorted set of local alignments.

Usage:

pslReps in.psl out.psl out.psr

Where:

in.psl is an alignment file generated by psLayout and sorted by pslSort

out.psl is the best alignment output

out.psr contains repeat info

Options:

-nohead            Suppress psl header.

-ignoreSize        Reduce weighing in favor of larger alignments.

-noIntrons         Don't penalize for not having introns when calculating size factor.

-singleHit         Takes single best hit, not splitting into parts.

-minCover=0.N      Minimum coverage to output. Default is 0.

-ignoreNs          Ignore Ns when calculating minCover.

-minAli=0.N        Minimum alignment ratio. Default is 0.93.

-nearTop=0.N       Amount of deviation from top allowed to be taken. Default is 0.01.

-minNearTopSize=N  Minimum size of alignment that is near top for alignment to be kept. Default 30.

-coverQSizes=file  Tab separate file with effective query sizes. When used with -minCover, this 
                   allows polyAs to be excluded from the coverage calculation.

Setting up WebBlat

Installing webBlat

Installing a web-based blat server involves four major steps:

1. Create the sequence databases.
2. Run the gfServer program to create in-memory indexes of the databases.
3. Edit the webBlat.cfg file to set the machine and port that the gfServer(s) are running on, and to optionally customize the webBlat appearance for users.
4. Copy the webBlat executable and webBlat.cfg to a directory where the web server can execute webBlat as a cgi.

Creating sequence databases

Create databases with the program faToTwoBit. Typically, a separate database is created for each genome being indexed. Each database can contain up to four billion bases of sequence in an unlimited number of records. The databases for webPcr and webBlat are identical.

The input to faToTwoBit is one or more fasta format files, each of which can contain multiple records. If the sequence contains repeat sequences, as is the case with vertebrates and many plants, the repeat sequences can be represented in lower-case and the other sequence in upper-case. The gfServer program can then be configured to ignore the repeat sequences. The output of faToTwoBit is a file that is designed for fast random access and efficient storage. The output file stores four bases per byte, and uses a small amount of additional space to store the case of the DNA and to keep track of blocks of Ns in the input. Other ambiguity codes in the input sequence, such as Y and U, will be converted to N.

Here is an example of how a typical installation might create a mouse and a human genome database:

cd /data/genomes
mkdir twoBit
faToTwoBit human/hg19/*.fa  twoBit/hg19.2bit
faToTwoBit mouse/mm10/*.fa  twoBit/mm10.2bit 

It is not necessary to put all of the databases in the same directory, but it can simplify bookkeeping. The databases can use the older blat format, .nib, instead of the .2bit format. The .nib format packs only two bases per byte, and handles only one record per .nib file.

Creating in-memory indexes with gfServer

The gfServer program creates an in-memory index of a nucleotide sequence database that can be used for translated or untranslated searches. Translated indexes enable protein-based blat queries and use approximately two bytes per unmasked base in the database. Untranslated indexes are used for nucleotide-based blat queries and the in-silico PCR utility. A normal blat index uses approximately one-quarter of a byte per base. For blat on smaller (primer-sized) queries or for in-silico PCR, it is recommended that you use a more thorough index that requires one-half of a byte per base. The gfServer program is memory intensive but typically does not require a lot of CPU power. With sufficient memory, multiple gfServers can be run on the same machine.

Here is an example of a typical installation:

ssh bigRamMachine
cd /data/genomes/twoBit
gfServer start bigRamMachine 17779 hg19.2bit &
gfServer -trans -mask start bigRamMachine 17778 hg19.2bit & 

The "trans" flag creates a translated index. It requires approximately 15 minutes to build an untranslated index and 45 minutes to build a translated index.

To build an untranslated index to be shared with in-silico PCR:

gfServer -stepSize=5 bigRamMachine 17779 hg19.2bit & 

This index will be slightly more sensitive with blat, particularly for small query sequences.

Editing the webBlat.cfg file

The webBlat.cfg file tells the webBlat program where to look for gfServers and sequence. The basic format of the .cfg file is line-oriented. The first word of each line is a command, and lines that are blank or start with "#" are ignored. The webBlat.cfg and webPcr.cfg files are similar. The webBlat.cfg commands are:

• gfServer – defines the host and port on which the (untranslated) gfServer is running, the associated sequence directory, and the name of the database to display on the web page.
• gfServerTrans – defines the location of a translated server.
• background – (optional) defines the background image (if any) to display on the web page.
• company – (optional) defines the company name to display on the web page.
• tempDir – specifies where to put temporary files. This path is relative to the directory where the web server executes CGI scripts. It is recommended that an automated mechanism, such as a cron job, be put in place to periodically remove files that haven't been recently accessed.

The background and company commands are optional. The webBlat.cfg file must have at least one valid gfServer or gfServerTrans line, and a tempDir line.

Here is an example of a webBlat.cfg file that you might find for a typical webBlat installation:

company Awesome Research Amalgamated
background /images/dnaPaper.jpg
gfServerTrans bigRamMachine 17778 /data/genomes/twoBit/hg19.2bit Human Genome
gfServer bigRamMachine 17779 /data/genomes/twoBit/hg19.2bit Human Genome
gfServerTrans mouseServer 17780 /data/genomes/twoBit/mm10.2bit Mouse Genome
gfServer mouseServer 17781 /data/genomes/twoBit/mm10.2bit Mouse Genome
tempDir ../trash 

Locating webBlat

The details of this step vary highly among web servers. Unless you are administering your own computer, you will likely have to ask your local system administrators for help with this part of the webBlat installation. Here is an example of a typical installation of Apache:

ssh webServer
cd kent/webBlat
cp webBlat webBlat.cfg /usr/local/apache/cgi-bin/
mkdir /usr/local/apache/trash
chmod 777 /usr/local/apache/trash 

In the above example, the executable and configuration file reside in the directory kent/webBlat/. The program will create some files in the trash directory. It is advisable to periodically remove old files from this directory.

Here is an example of an installation on Mac OS X:

cp webBlat webBlat.cfg /Library/WebServer/CGI-Executables/
mkdir /Library/WebServer/trash
chmod 777 /Library/WebServer/trash