Name | Modified | Size | Downloads / Week |
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DHC-MEGE.sh | 2013-02-05 | 13.8 kB | |
README.txt | 2013-01-31 | 5.2 kB | |
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README for DHC-MEGE 31-1-2013 DHC-MEGE is a program for the identification of enriched motifs in genes found to be differentially expressed in a microarray or RNA-seq experiment. What sets this program apart from others is that it utilises a DNase hypersensitivity connectivity map which allows for the identification of cis elements which may be hundreds of kilobases away, but could be acting as enhancers/silencers/etc. From the gene lists, DHC-MEGE uses HOMER motif finding and then outputs the occurance of that motif in other DH regions across the genome and outputs this as a GMT file. The GMT file then can be used in GSEA analysis. Program Inputs You need a list of up genes, down genes, a DNase hypersensitivity connectivity map (BED8 format) and genome sequence (fasta format). These need to specified with a config file. See the section on the config file below. Running the program Simply run the program as you would any Unix shell script, make the script executable with chmod then execute, specifying the config file as the first argument. Here is an example: chmod +x DHC-MEGE.sh ./DHC-MEGE.sh /path/to/config.cfg Program Outputs The program will create a results directory with a unique timestamp and deposit all results there. The main output is the GMT file, which is a list of gene lists which details the occurance of sequence motifs across the hypersensitive regions of the genome. Homer generates reports for each of the identified motifs and even makes sequence logos if the software is correctly configured. How can I use the output file? GMT files can be used in gene set enrichment analysis (GSEA) of the array/mRNA-seq experiment. Check out the Broad Institute website for more info on GSEA. http://www.broadinstitute.org/gsea/msigdb/index.jsp Need more help/information? We have our article accepted in Bioinformation: “Motif analysis in DNAse hypersensitivity regions uncovers distal cis-elements associated with gene expression” where you can see an example of DHC-MEGE in action. The configuration file Here is what an example config file looks like: ##################################################################################### ##CONFIG FILE FOR DHC-MEGE PIPELINE ##ENTER THE CORRELATION THRESHOLD CORRTHRESH=0.8 ##ENTER THE DESIRED SIZE OF GENESETS GENESETSIZE=1000 ##ENTER SIMILARITY THRESHOLD SIMTHRESHOLD=10 ##NUMBER CPUS NR_CPUS=4 #ENTER THE LIST OF UPREGULATED GENES UPGENELIST=/path/to/UPgenes.txt #ENTER THE LIST OF DOWNREGULATED GENES DOWNGENELIST=/path/to/DOWNgenes.txt #PROVIDE AN IDENTIFICATION FOR THE EXPERIMENT RUNNAME=ExperimentName #PROVIDE THE PATH TO THE Genome GENOME=/path/to/genome/sequence.fa #PROVIDE THE PATH TO THE DHC-MAP #HUMAN DHC MAP CAN BE OBTAINED FROM ENCODE DHCMAP=/path/to/DHC-MAP.bed ##################################################################################### Notes on the parameters in the confguration file CORRTHRESH is the correlation coefficient between the proximal and distal hypersensitivity regions. The more correlated they are, the more influential the distal element is for gene expression. The minimum CORRTHRESH that ENCODE use as significant is 0.7. In our paper, we use 0.8. GENESETSIZE is the maximum size of gene sets which will be output. This is an important parameter because is we don't limit the gene set size, then we may have uniquitous sequence motifs gene sets containing nearly all genes which is not informative when it comes to GSEA. We recommend using a limit of 1000. This limit ensures that only the top 1000 sequence matches SIMTHRESHOLD is the similarity threshold for inclusion of genes into the GMT. It is log odds value computed by Homer and is used to sort identified motif instances. We have used a threshold of 10. NR_CPUS is the number of parallel threads to run. The program may use a couple more or less at any point in time depending on the available resources. We will be working to refine this behaviour in future versions. UPGENELIST/DOWNGENELIST these files need to contain a list of genes, on on each new line. Please ensure that the gene names used EXACTLY match those in the DHC map otherwise you could be missing a large number of motifs! RUNNAME if you like, you can give your experiment a name for identification of the results directory. GENOME during the motif searching process, Homer will sample bed regions from the genes selected and needs to extract the actual sequence from the fasta file. DHCMAP the path to the DHC map. The ENCODE DHC map for human is available from the EBI website. It has an 8 field bed format. Please see the ENCODE DNase hypersensitivity analysis paper supplementary data (PMID: 22955617) for the link. ##################################################################################### # This software is free to distribute and use under the GPL license. # If you use this software in your academic work please cite our article in # bioinformation - Ziemann et al, 2012. # Please report bugs and give suggestions for future improvement. # mark.ziemann@gmail.com #####################################################################################