Hi. I'm currently using HOMER to see known motif enrichment of the list of DEGs I have. Although I followed all the instruction provided in this page (http://homer.ucsd.edu/homer/microarray/index.html) it never seem to get the result correctly. I've tried using Entrez Gene ID instead of TAIR ID, and installed all packages related to arabidopsis and tair10. Can anyone tell me how to solve this problem?
Selected Options: Input file = (file_url).txt Promoter Set = arabidopsis Output Directory = (output_url) Found mset for "arabidopsis", will check against plants motifs
Progress: Step1 - Convert input file to refseq IDs
Percentage of IDs converted into refseq: 97.9% (894 out of 913)
Progress: Step2 - prepare sequence files
Progress: Step3 - creating foreground/background file
Progress: Step4 - removing redundant promoters
Kept 52998 of 56329
Progress: Step5 - adjusting background sequences for GC/CpG content...
Bin # Targets # Background Background Weight
Normalizing lower order oligos using homer2
Reading input files...
0 total sequences read
Autonormalization: 1-mers (4 total)
A inf% inf% -nan
C inf% inf% -nan
G inf% inf% -nan
T inf% inf% -nan
Autonormalization: 2-mers (16 total)
AA inf% inf% -nan
CA inf% inf% -nan
GA inf% inf% -nan
TA inf% inf% -nan
AC inf% inf% -nan
CC inf% inf% -nan
GC inf% inf% -nan
TC inf% inf% -nan
AG inf% inf% -nan
CG inf% inf% -nan
GG inf% inf% -nan
TG inf% inf% -nan
AT inf% inf% -nan
CT inf% inf% -nan
GT inf% inf% -nan
TT inf% inf% -nan
Autonormalization: 3-mers (64 total)
Normalization weights can be found in file: (output_url)/seq.autonorm.tsv
Converging on autonormalization solution:
...............................................................................
Final normalization: Autonormalization: 1-mers (4 total)
A inf% inf% -nan
C inf% inf% -nan
G inf% inf% -nan
T inf% inf% -nan
Autonormalization: 2-mers (16 total)
AA inf% inf% -nan
CA inf% inf% -nan
GA inf% inf% -nan
TA inf% inf% -nan
AC inf% inf% -nan
CC inf% inf% -nan
GC inf% inf% -nan
TC inf% inf% -nan
AG inf% inf% -nan
CG inf% inf% -nan
GG inf% inf% -nan
TG inf% inf% -nan
AT inf% inf% -nan
CT inf% inf% -nan
GT inf% inf% -nan
TT inf% inf% -nan
Autonormalization: 3-mers (64 total)
Progress: Step6 - Gene Ontology Enrichment Analysis
Progress: Step7 - Known motif enrichment
Reading input files...
0 total sequences read
506 motifs loaded
Cache length = 11180
Using hypergeometric scoring
Checking enrichment of 506 motif(s)
|0% 50% 100%|
=================================================================================
Illegal division by zero at (software_url)/bin/findKnownMotifs.pl line 152.
Progress: Step8 - De novo motif finding (HOMER)
Scanning input files...
!!! Something is wrong... are you sure you chose the right length for motif finding? !!! i.e. also check your sequence file!!!
Scanning input files...
!!! Something is wrong... are you sure you chose the right length for motif finding? !!! i.e. also check your sequence file!!!
-blen automatically set to 2
Scanning input files...
!!! Something is wrong... are you sure you chose the right length for motif finding? !!! i.e. also check your sequence file!!! Use of uninitialized value in numeric gt (>) at (software_url)/bin/compareMotifs.pl line 1389. !!! Filtered out all motifs!!! Job finished
1 answer
It seems that there is something wrong with gene id conversion.
You may try this:
use input file with gene names in TAIR format and add option -noconvert. In this way ids will not be converted.
I ran analysis with small list of randomly chosen genes:
AT3G44150
AT4G36350
AT5G48910
ATCG00170
AT1G46336
AT2G05410
AT2G44255
AT3G42713
AT4G08460
AT1G12667
From the lines of Perl program output it seems that analysis worked:
Autonormalization: 2-mers (16 total)
AA 13.45% 12.71% 1.058
CA 4.91% 5.76% 0.851
GA 6.09% 5.95% 1.023
TA 10.08% 9.08% 1.110
AC 5.07% 5.19% 0.977
CC 2.43% 2.95% 0.823
GC 2.46% 2.41% 1.020
TC 6.09% 5.95% 1.023
AG 6.50% 5.52% 1.176
CG 1.57% 2.25% 0.700
GG 2.43% 2.95% 0.823
TG 4.91% 5.76% 0.851
AT 9.00% 10.08% 0.893
CT 6.50% 5.52% 1.176
GT 5.07% 5.19% 0.977
TT 13.45% 12.71% 1.058
Autonormalization: 3-mers (64 total)
Progress: Step6 - Gene Ontology Enrichment Analysis
Progress: Step7 - Known motif enrichment
Reading input files...
24167 total sequences read
506 motifs loaded
Cache length = 11180
Using hypergeometric scoring
Checking enrichment of 506 motif(s)
Also:
Progress: Step8 - De novo motif finding (HOMER)
Scanning input files...
Parsing sequences...
|0% 50% 100%|
================================================================================
Total number of Oligos: 32874
Autoadjustment for sequence coverage in background: 1.09x
Oligos: 32874 of 34497 max
Tree : 66660 of 172485 max
Optimizing memory usage...
Cache length = 11180
Using hypergeometric scoring
Global Optimization Phase: Looking for enriched oligos with up to 1 mismatches...
Screening oligos 32874 (allowing 0 mismatches):
|0% 50% 100%|
================================================================================
94.03% skipped, 5.97% checked (1962 of 32874), of those checked:
94.03% not in target, 0.00% increased p-value, 0.00% high p-value
Screening oligos 32874 (allowing 1 mismatches):
|0% 50% 100%|
================================================================================
94.03% skipped, 5.97% checked (1962 of 32874), of those checked:
0.00% not in target, 5.67% increased p-value, 1.08% high p-value
Reading input files...
24167 total sequences read
Cache length = 11180
Using hypergeometric scoring
The result with 10 bp length motifs was saved in homerMotifs.motifs10 file and the first lines of the file looks like this:
>AAGCTTWGAA 1-AAGCTTWGAA 10.724573 -10.744085 0 T:3.0(37.50%),B:371.1(0.73%),P:1e-4 Tpos:271.0,Tstd:74.6,Bpos:164.2,Bstd:141.4,StrandBias:-10.0,Multiplicity:1.00
0.997 0.001 0.001 0.001
0.719 0.001 0.001 0.279
0.001 0.001 0.997 0.001
0.001 0.763 0.001 0.235
0.001 0.001 0.001 0.997
0.001 0.001 0.001 0.997
0.517 0.001 0.001 0.481
0.199 0.231 0.558 0.012
0.722 0.276 0.001 0.001
0.997 0.001 0.001 0.001
>ACTGTTTTAA 2-ACTGTTTTAA 7.180883 -9.252319 0 T:3.0(37.50%),B:615.9(1.21%),P:1e-4 Tpos:242.5,Tstd:55.4,Bpos:167.1,Bstd:148.2,StrandBias:0.0,Multiplicity:1.33
0.997 0.001 0.001 0.001
0.001 0.997 0.001 0.001
0.001 0.001 0.001 0.997
0.001 0.001 0.997 0.001
0.189 0.001 0.001 0.809
0.001 0.001 0.001 0.997
0.001 0.189 0.001 0.809
0.001 0.189 0.001 0.809
0.997 0.001 0.001 0.001
0.809 0.001 0.189 0.001
As I am not used in working with homer check if this makes sense. Besides you can write to cbenner@ucsd.edu email as suggested on homer website
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