Given a profile generated from the multiple sequence alignment, or, retrieved from motif library such as PROSITE or Pfam, you can align a protein sequence with the profile. The procedure is similar to the one to search against the motif library database, however, you should provide a name of the file containing profile matrix instead of the. Motif runtime libraries and executables: OpenMandriva Main Release i686 Official: motif-2.3.8-2-omv4000.i686.rpm: Motif runtime libraries and executables: OpenMandriva Main Release x8664 Official: motif-2.3.8-2-omv4002.x8664.rpm: Motif runtime libraries and executables. If you installed oagtcltk.darwin-x86.tar.gz you will have mplmotif in your path, which is our Motif plotting program. Motif doesn't work well with the newer versions of XQuartz. First try to run mplmotif to see if it brings up an empty plotting window.
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Finding Enriched Motifs in Genomic Regions (findMotifsGenome.pl)
HOMER was initially developed to automate the process of finding enriched motifs in ChIP-Seq peaks. More generally, HOMER analyzes genomic positions, not limited to only ChIP-Seq peaks, for enriched motifs. The main idea is that all the user really needs is a file containing genomic coordinates (i.e. a HOMER peak file or BED file), and HOMER will generally take care of the rest. To analyze a peak file for motifs, run the following command:i.e. findMotifsGenome.pl ERpeaks.txt hg18 ER_MotifOutput/ -size 200-mask
A variety of output files will be placed in the , including html pages showing the results. The '-mask' is optional and tells the program to use the repeat-masked sequence. (The old shorthand hg18r will also work). The -size parameter is now mandatory when running findMotifsGenome.pl to avoid confusion - plus it's always a good idea to know exactly what size the regions you are analyzing are.
The findMotifsGenome.pl program is a wrapper that helps set up the data for analysis using the HOMER motif discovery algorithm. By default this will perform de novo motif discovery as well as check the enrichment of known motifs. If you have not done so already, please look over this page describing how HOMER analyzes sequences for enriched motifs.
An important prerequisite for analyzing genomic motifs is that the appropriate genome must by configured for use with HOMER. In version v3.1, HOMER now handles custom/arbitrary genomes. Instead of intalling/configuring a genome, you can specify the path to a file or directory containing the genomic sequence in FASTA format. The genome can be in a single FASTA file, or you specify a directory where where each chromosome can be in a separate file (named chrXXX.fa or chrXXX.fa.masked). In either case, the FASTA headers must contain the chromosome names followed by white space, i.e. '>chr blahblahblah', not '>chr1-blahblahblah', or prefereably only '>chr1'. (also note that homer will create a 'preparsed/' directory where the genome is, so make sure you have write permissions in the genomic directory.
Selecting the size of the region for motif finding (-size # or -size given, default: 200)
This is one of the most important parameters and also a source of confusion for many. If you wish to find motifs using your peaks using their exact sizes, use the option '-size given'). However, for Transcription Factor peaks, most of the motifs are found +/- 50-75 bp from the peak center, making it better to use a fixed size rather than depend on your peak size.
Acceptable Input files
findMotifsGenome.pl accepts HOMER peak files or BED files:HOMER peak files should have at minimum 5 columns (separated by TABs, additional columns will be ignored):
- Column1: Unique Peak ID
- Column2: chromosome
- Column3: starting position
- Column4: ending position
- Column5: Strand (+/- or 0/1, where 0='+', 1='-')
- Column1: chromosome
- Column2: starting position
- Column3: ending position
- Column4: Unique Peak ID
- Column5: not used
- Column6: Strand (+/- or 0/1, where 0='+', 1='-')
Mac Users: If using a EXCEL to prepare input files, make sure to save files as a 'Text (Windows)' if running MacOS - saving as 'Tab delimited text' in Mac produces problems for the software. Otherwise, you can run the script 'changeNewLine.pl ' to convert the Mac-formatted text file to a Windows/Dos/Unix formatted text file.
If errors occur, it is likely that the file is not in the correct format, or the first column is not actually populated with unique identifiers.
Custom Background Regions
How findMotifsGenome.pl works
There are a series of steps that the program goes through to find quality motifs:1. Verify peak/BED file
2. Extract sequences from the genome corresponding to the regions in the input file, filtering sequences that are >70% 'N'
3. Calculate GC/CpG content of peak sequences.
4. Preparse the genomic sequences of the selected size to serve as background sequences.
5. Randomly select background regions for motif discovery.
If custom background regions are provided ('-bg '), HOMER will automatically ensure that these regions do NOT overlap with the target regions (using mergePeaks). Customregionswill still be normalized for GC-content.
6. Autonormalization of sequence bias.
7. Check enrichment of known motifs
8. de novo motif finding
findMotifsGenome.pl Output
A full description of motif finding output and the output can be found here.Several files are produced in the output directory:
homerMotifs.all.motifs : Simply the concatenated file composed of all the homerMotifs.motifs<#> files.
motifFindingParameters.txt : command used to execute findMotifsGenome.pl
knownResults.txt : text file containing statistics about known motif enrichment (open in EXCEL).
seq.autonorm.tsv : autonormalization statistics for lower-order oligo normalization.
homerResults.html : formatted output of de novo motif finding.
homerResults/ directory: contains files for the homerResults.html webpage, including motif<#>.motif files for use in finding specific instance of each motif.
knownResults.html : formatted output of known motif finding.
knownResults/ directory: contains files for the knownResults.html webpage, including known<#>.motif files for use in finding specific instance of each motif.
Interpreting motif finding results
In general, when analyzing ChIP-Seq / ChIP-Chip peaks you should expect to see strong enrichment for a motif resembling the site recognized by the DNA binding domain of the factor you are studying. Enrichment p-values reported by HOMER should be very very significant (i.e. << 1e-50). If this is not the case, there is a strong possibility that the experiment may have failed in one way or another. For example, the peaks could be of low quality because the factor is not expressed very high.
Practical Tips for Motif finding
Important motif finding parameters
Masked vs. Unmasked Genome ('-mask' or hg18 vs. hg18r) Region Size ('-size <#>', '
Motif length ('-len <#>' or '-len <#>,<#>,..', default 8,10,12)
Mismatches allowed in global optimization phase ('-mis <#>', default: 2)
Number of CPUs to use ('-p <#>', default 1)
Number of motifs to find ('-S <#>', default 25)
Normalize CpG% content instead of GC% content ('-cpg')
Region level autonormalization ('-nlen <#>', default 3, '-nlen 0' to disable)
Motif level autonormalization (-olen <#>, default 0 i.e. disabled)
User defined background regions ('-bg ')
Hypergeometric enrichment scoring ('-h')
Find enrichment of individual oligos ('-oligo').
Force findMotifsGenome.pl to re-preparse genome for the given region size ('-preparse').
Only search for motifs on + strand ('-norevopp')
Search for RNA motifs ('-rna')
Mask motifs ('-mask ')
Optimize motifs ('-opt ')
Dump FASTA files ('-dumpFasta')
Finding Instance of Specific Motifs
By default, HOMER does not return the locations of each motif found in the motif discovery process. To recover the motif locations, you must first select the motifs you're interested in by getting the 'motif file' output by HOMER. You can combine multiple motifs in single file if you like to form a 'motif library'. To identify motif locations, you have two options:1. Run findMotifsGenome.pl with the '-find ' option. This will output a tab-delimited text file with each line containing an instance of the motif in the target peaks. The output is sent to stdout.
For example: findMotifsGenome.pl ERalpha.peaks hg18 MotifOutputDirectory/ -find motif1.motif > outputfile.txt
The output file will contain the columns:
- Peak/Region ID
- Offset from the center of the region
- Sequence of the site
- Name of the Motif
- Strand
- Motif Score (log odds score of the motif matrix, higher scores are better matches)
For example: annotatePeaks.pl ERalpha.peaks hg18 -m motif1.motif > outputfile.txt
The output file will contain columns:
- Peak/Region ID
- Chromosome
- Start
- End
- Strand of Peaks
19. CpG%
20. GC%
21. Motif Instances
..
Motif Instances have the following format:
i.e -29(TAAATCAACA,+,0.00)
You can also find histogram of motif density this way by adding '-hist <#>' to the command. For example:
Graphing the output with EXCEL:
Command-line options for findMotifsGenome.pl
Program will find de novo and known motifs in regions in the genomeUsage: findMotifsGenome.pl [additional options]
Example: findMotifsGenome.pl peaks.txt mm8r peakAnalysis -size 200 -len 8
Possible Genomes:
..
Custom: provide the path to genome FASTA files (directory or single file)
Heads up: will create the directory 'preparsed/' in same location.
Basic options:
-bg (genomic positions to be used as background, default=automatic)
removes background positions overlapping with target positions
-chopify (chop up large background regions to the avg size of target regions)
-len <#>[,<#>,<#>..] (motif length, default=8,10,12) [NOTE: values greater 12 may cause the program
to run out of memory - in these cases decrease the number of sequences analyzed (-N),
or try analyzing shorter sequence regions (i.e. -size 100)]
-size <#> (fragment size to use for motif finding, default=200)
-size <#,#> (i.e. -size -100,50 will get sequences from -100 to +50 relative from center)
-size given (uses the exact regions you give it)
-S <#> (Number of motifs to optimize, default: 25)
-mis <#> (global optimization: searches for strings with # mismatches, default: 2)
-norevopp (don't search reverse strand for motifs)
-nomotif (don't search for de novo motif enrichment)
-rna (output RNA motif logos and compare to RNA motif database, automatically sets -norevopp)
Scanning sequence for motifs
-find (This will cause the program to only scan for motifs)
Known Motif Options/Visualization
-bits (scale sequence logos by information content, default: doesn't scale)
-nocheck (don't search for de novo vs. known motif similarity)
-mcheck (known motifs to check against de novo motifs,
default: /bioinformatics/homer/data/knownTFs/all.motifs
-float (allow adjustment of the degeneracy threshold for known motifs to improve p-value[dangerous])
-noknown (don't search for known motif enrichment, default: -known)
-mknown (known motifs to check for enrichment,
default: /bioinformatics/homer/data/knownTFs/known.motifs
Sequence normalization options:
-gc (use GC% for sequence content normalization, now the default)
-cpg (use CpG% instead of GC% for sequence content normalization)
-noweight (no CG correction)
Advanced options:
-h (use hypergeometric for p-values, binomial is default)
-N <#> (Number of sequences to use for motif finding, default=max(50k, 2x input)
-noforce (will attempt to reuse sequence files etc. that are already in output directory)
-local <#> (use local background, # of equal size regions around peaks to use i.e. 2)
-redundant <#> (Remove redundant sequences matching greater than # percent, i.e. -redundant 0.5)
-mask [motif file 2].. (motifs to mask before motif finding)
-opt [motif file 2].. (motifs to optimize or change length of)
-refine (motif to optimize)
-rand (randomize target and background sequences labels)
-ref (use file for target and background - first argument is list of peak ids for targets)
-oligo (perform analysis of individual oligo enrichment)
-dumpFasta (Dump fasta files for target and background sequences for use with other programs)
-preparse (force new background files to be created)
-keepFiles (keep temporary files)
homer2 specific options:
-homer2 (use homer2 instead of original homer, default)
-nlen <#> (length of lower-order oligos to normalize in background, default: -nlen 3)
-nmax <#> (Max normalization iterations, default: 160)
-olen <#> (lower-order oligo normalization for oligo table, use if -nlen isn't working well)
-p <#> (Number of processors to use, default: 1)
-e <#> (Maximum expected motif instance per bp in random sequence, default: 0.01)
-cache <#> (size in MB for statistics cache, default: 500)
-quickMask (skip full masking after finding motifs, similar to original homer)
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Original homer specific options:
-homer1 (to force the use of the original homer)
-depth [low|med|high|allnight] (time spent on local optimization default: med)
Use the Common Desktop Environment Motif and X11R5 libraries to develop a Common Desktop Environment Motif-compliant application for the X Window System. The Common Desktop Environment Motif libraries are the Motif 2.1 libraries with bug fixes and enhancements.
Motif Library (libXm)
Google chat application for mac. The Common Desktop Environment provides all the Motif 2.1 header files.
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Motif UIL library (libUil)
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The Motif User Interface Language (UIL) is a specification language for describing the initial state of a Motif application's user interface. The Common Desktop Environment version is essentially unchanged from the Motif version.
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Include the UilDef.h header file (found in the uil directory) to access UIL.
Motif Resource Manager Library (libMrm)
The Motif resource manager (MRM) is responsible for creating widgets based on definitions contained in User Interface Definition (UID) files created by the UIL compiler. MRM interprets the output of the UIL compiler and generates the appropriate argument lists for widget creation functions. Valhallaroom free download mac. Use libMrm to access the Motif resource manager. The Common Desktop Environment version is essentially unchanged from the Motif version.
Include the Mrm/MrmPublic.h header files to access libMrm in your application.