Can't run ExonDel.. please help

[duartemolha]

Hi

I was wondering if you could help me because I have done everything on the install instructions but ExonDel still fails.

I have all required perl modules and both R and samtools are on my path.

The command I am using is:

perl  ~/Programs/ExonDel-master/ExonDel.pl -i ExonDel_test.txt -o /home/user/ND-34/ExonDelResults -c /home/user/ND-34/ExonDel_cfg.txt  -g /home/user/ND-34/gene_list.txt

here is the output from the run command

[Mon Nov 30 10:41:59 2015] Only the genes in /home/user/Tasks/ND-34/gene_list.txt will be used
[Mon Nov 30 10:41:59 2015] GC adjustment will not be performed, and the constant cutoffs in config file will be used
[Mon Nov 30 10:41:59 2015] If your bed file has fourth column, it should be gene name and can be matched to genes in home/user/Tasks/ND-34/gene_list.txt
[Mon Nov 30 10:41:59 2015] Loading BED file
Use of uninitialized value $in in open at /home/user/Programs/ExonDel-master/ExonDel.pl line 497, <GENELIST> line 7.

here are the content of each file:

ExonDel_cgf.txt

#reference .bed file
bedfile=/home/user/ND-34/genes.bed
#reference .gtf file
refseq=/home/user/ND-34/refseq_no_chr_prefix.txt
#reference .fa file
reffa=/home/user/ND-34/hg19.fa
#config file for ExonDel software
[perl]
#Minimal percent of covered base pairs for each exon
exon_bp_cover_threshold=0.1
#Minimal percent of covered exons for each gene. 1 means 100%
overall_exon_count_threshold=1
#Minimum mapping quality for an alignment to be used
mapQ=20
#Minimum base quality for a base to be considered
baseQ=20
#where the R bin file is
RBin=R
#where the SAMtools bin file is
samtoolsBin=samtools
#name of the log file
logFileName=ExonDel.log
[R]
#Maximal number of exons for the moving-window in exon deletion detection
maxWinLength=9
#Minimum number of exons for the moving-window in exon deletion detection
minWinLength=1
#Minimum number of exons for a gene to be considered in exon deletion detection
minExonNum=10
#T means True and F means False. If only some genes were used (use -g option), GC adjustment will not be performed no matter what adjustGC below was set (As GC adjustment is based on all genes).
adjustGC=F
#If all genes were used (didn't use -g option), cutoff 1,2 will be determined by cutoffQuantile 1,2 in all genes
cutoffQuantile1=0.01
cutoffQuantile2=0.1
#If only some genes were used (use -g option), cutoff 1,2 will be determined by constant cutoff 1,2 below
cutoff1=2
cutoff2=20

here is the list of genes in gene_list.txt

BRCA1
BRCA2
P53
PTEN
ATM
NF1
ATR

here is the list of genes in genes.bed

10  89623194    89731687    PTEN
11  108093558   108239826   ATM
13  32889616    32973809    BRCA2
17  29421944    29704695    NF1
17  41196311    41277500    BRCA1
3   142168076   142297668   ATR

Here are the first few lines of the refseq_no_chr_prefix.txt (RefSeq UCSC track)
This is exactly the same file downloaded from UCSC, I simply removed the chr prefix because all my bam files do not have the prefix. I made sure all my files are not using the chr prefix, so my fasta reference file also does not have prefix.

#bin    name    chrom   strand  txStart txEnd   cdsStart    cdsEnd  exonCount   exonStarts  exonEnds    score   name2   cdsStartStat    cdsEndStat  exonFrames
0   NM_032291   1   +   66999638    67216822    67000041    67208778    25  66999638,67091529,67098752,67101626,67105459,67108492,67109226,67126195,67133212,67136677,67137626,67138963,67142686,67145360,67147551,67154830,67155872,67161116,67184976,67194946,67199430,67205017,67206340,67206954,67208755,   67000051,67091593,67098777,67101698,67105516,67108547,67109402,67126207,67133224,67136702,67137678,67139049,67142779,67145435,67148052,67154958,67155999,67161176,67185088,67195102,67199563,67205220,67206405,67207119,67216822,   0   SGIP1   cmpl    cmpl    0,1,2,0,0,0,1,0,0,0,1,2,1,1,1,1,0,1,1,2,2,0,2,1,1,
0   NM_001308203    1   +   66999251    67216822    67000041    67208778    22  66999251,66999928,67091529,67098752,67105459,67108492,67109226,67136677,67137626,67138963,67142686,67145360,67154830,67155872,67160121,67184976,67194946,67199430,67205017,67206340,67206954,67208755,  66999355,67000051,67091593,67098777,67105516,67108547,67109402,67136702,67137678,67139049,67142779,67145435,67154958,67155999,67160187,67185088,67195102,67199563,67205220,67206405,67207119,67216822,  0   SGIP1   cmpl    cmpl    -1,0,1,2,0,0,1,0,1,2,1,1,1,0,1,1,2,2,0,2,1,1,
1   NM_001301825    1   +   33547778    33586132    33547850    33585783    9   33547778,33549554,33557650,33558882,33560148,33562307,33563607,33583502,33585644,   33547955,33549728,33557823,33559017,33560314,33562470,33563780,33583717,33586132,   0   AZIN2   cmpl    cmpl    0,0,0,2,2,0,1,0,2,
1   NM_001080397    1   +   8378144 8404227 8378168 8404073 9   8378144,8384365,8385357,8385877,8390268,8395496,8397875,8399552,8403806,    8378246,8384786,8385450,8386102,8390996,8395650,8398052,8399758,8404227,    0   SLC45A1 cmpl    cmpl    0,0,1,1,1,0,1,1,0,

my ExonDel_test contains the list of the bam files I want to process

H521_S2_L001.bam
H926_S3_L001.bam
208210423-ADN-3_S7_L001.bam

[slzhao]

Hello Duarte,

Is there any other information after "Use of uninitialized value $in in
open at /home/OGTIP/duartem/Programs/ExonDel-master/ExonDel.pl line 497,
line 7."? It seems that the program didn't find the bed file.

1. Is the bed file at "/home/user/ND-34/genes.bed"?
2. Would you please try to add a header to the bed file, to see if the
   error message changes? For example:

chr start end gene
10 89623194 89731687 PTEN
11 108093558 108239826 ATM

Thanks!

Best,
Shilin

2015-11-30 4:47 GMT-06:00 Duarte notifications@github.com:

Hi

I was wondering if you could help me because I have done everything on the
install instructions but ExonDel still fails.

I have all required perl modules and both R and samtools are on my path.

The command I am using is:

perl ~/Programs/ExonDel-master/ExonDel.pl -i ExonDel_test.txt -o /home/user/ND-34/ExonDelResults -c /home/user/ND-34/ExonDel_cfg.txt -g /home/user/ND-34/gene_list.txt

here is the output from the run command

[Mon Nov 30 10:41:59 2015] Only the genes in /home/OGTIP/duartem/Tasks/ND-34/Ovarian_gene_list.txt will be used
[Mon Nov 30 10:41:59 2015] GC adjustment will not be performed, and the constant cutoffs in config file will be used
[Mon Nov 30 10:41:59 2015] If your bed file has fourth column, it should be gene name and can be matched to genes in /home/OGTIP/duartem/Tasks/ND-34/Ovarian_gene_list.txt
[Mon Nov 30 10:41:59 2015] Loading BED file
Use of uninitialized value $in in open at /home/OGTIP/duartem/Programs/ExonDel-master/ExonDel.pl line 497, line 7.

here are the content of each file:

ExonDel_cfg.txt
https://github.com/slzhao/ExonDel/files/47168/ExonDel_cfg.txt

here is the list of genes in gene_list.txt

BRCA1
BRCA2
P53
PTEN
ATM
NF1
ATR

here is the list of genes in genes.bed

10 89623194 89731687 PTEN
11 108093558 108239826 ATM
13 32889616 32973809 BRCA2
17 29421944 29704695 NF1
17 41196311 41277500 BRCA1
3 142168076 142297668 ATR

Here are the first few lines of the refseq_no_chr_prefix.txt (RefSeq UCSC
track)
This is exactly the same file downloaded from UCSC, I simply removed the
chr prefix because all my bam files do not have the prefix. I made sure all
my files are not using the chr prefix, so my fasta reference file also does
not have prefix.

#bin name chrom strand txStart txEnd cdsStart cdsEnd exonCount exonStarts exonEnds score name2 cdsStartStat cdsEndStat exonFrames
0 NM_032291 1 + 66999638 67216822 67000041 67208778 25 66999638,67091529,67098752,67101626,67105459,67108492,67109226,67126195,67133212,67136677,67137626,67138963,67142686,67145360,67147551,67154830,67155872,67161116,67184976,67194946,67199430,67205017,67206340,67206954,67208755, 67000051,67091593,67098777,67101698,67105516,67108547,67109402,67126207,67133224,67136702,67137678,67139049,67142779,67145435,67148052,67154958,67155999,67161176,67185088,67195102,67199563,67205220,67206405,67207119,67216822, 0 SGIP1 cmpl cmpl 0,1,2,0,0,0,1,0,0,0,1,2,1,1,1,1,0,1,1,2,2,0,2,1,1,
0 NM_001308203 1 + 66999251 67216822 67000041 67208778 22 66999251,66999928,67091529,67098752,67105459,67108492,67109226,67136677,67137626,67138963,67142686,67145360,67154830,67155872,67160121,67184976,67194946,67199430,67205017,67206340,67206954,67208755, 66999355,67000051,67091593,67098777,67105516,67108547,67109402,67136702,67137678,67139049,67142779,67145435,67154958,67155999,67160187,67185088,67195102,67199563,67205220,67206405,67207119,67216822, 0 SGIP1 cmpl cmpl -1,0,1,2,0,0,1,0,1,2,1,1,1,0,1,1,2,2,0,2,1,1,
1 NM_001301825 1 + 33547778 33586132 33547850 33585783 9 33547778,33549554,33557650,33558882,33560148,33562307,33563607,33583502,33585644, 33547955,33549728,33557823,33559017,33560314,33562470,33563780,33583717,33586132, 0 AZIN2 cmpl cmpl 0,0,0,2,2,0,1,0,2,
1 NM_001080397 1 + 8378144 8404227 8378168 8404073 9 8378144,8384365,8385357,8385877,8390268,8395496,8397875,8399552,8403806, 8378246,8384786,8385450,8386102,8390996,8395650,8398052,8399758,8404227, 0 SLC45A1 cmpl cmpl 0,0,1,1,1,0,1,1,0,

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#10.

[duartemolha]

It is really wierd.. I just tried to debug it and on line 71 when reading the config file... even though it is reading each line correctly the variable $config is not being written... when you get to the end of the look $config is still undef

[duartemolha]

I think I got it... the keyword [perl] needs to be present in the top of the config file!

[duartemolha]

btw on the report.. although I gave as input the UCSC track file containing the exons (the gtf file) , the report outputs some exon deletions that are not even over any of exon.

Also... because this is a target resequencing experiment, not all of the exons of the genes of interest are targeted... is there a way to force the analysis over only my targeted regions?

Many thanks for your continued support

[slzhao]

The bed file is where you should include your target region. Only the
regions in that file will be considered.

Best,
Shilin

2015-11-30 15:45 GMT-06:00 Duarte notifications@github.com:

btw on the report.. although I gave as input the UCSC track file
containing the exons (the gtf file) , the report outputs some exon
deletions that are not even over any of exon.

Also... because this is a target resequencing experiment, not all of the
exons of the genes of interest are targeted... is there a way to force the
analysis over only my targeted regions?

Many thanks for your continued support

—
Reply to this email directly or view it on GitHub
#10 (comment).

[duartemolha]

Unfortunately, this does not seem the case... in my data I have 5 or 6 genes that I have targeted (I have listed them above), however not all of the exons are targeted. For example Exon 1 of NM000059 is not targeted.

My targeted bed file has now been updated so that I am excluding that region, however, this is still what Exon Dell calls as a deletion so I do not believe your software is using only the targeted regions I have given.

In fact besides calling an exon that is not including in the targeted region, it calls some deletions in a region that does not even contain an exon.

In fact, none of the called aberrations can be intersected with the targets.

Here are the regions the software has called a deletion:

10 89623194 89623860 H521
10 89623194 89623860 H926
10 89623194 89623860 ADN
10 89702367 89702526 H521
10 89702367 89702526 H926
10 89702367 89702526 ADN
11 108093558 108093913 H521
11 108093558 108093913 H926
11 108093558 108093913 ADN
13 32889616 32889804 H521
13 32889616 32889804 H926
13 32889616 32889804 ADN
17 41277198 41277340 H521
17 41277198 41277340 H926
17 41277198 41277340 ADN
17 41277287 41277500 H521
17 41277287 41277500 H521
17 41277287 41277500 H926
17 41277287 41277500 H926
17 41277287 41277500 ADN
17 41277287 41277500 ADN
17 41277293 41277468 H521
17 41277293 41277468 H521
17 41277293 41277468 H926
17 41277293 41277468 H926
17 41277293 41277468 ADN
17 41277293 41277468 ADN

Here are the targets inputs

10 89624206 89624326
10 89653763 89653883
10 89685231 89685351
10 89690764 89690884
10 89692709 89693069
10 89711825 89712065
10 89717573 89717813
10 89720628 89720988
10 89725016 89725256
11 108098327 108098678
11 108099857 108100097
11 108106358 108106598
11 108114642 108114882
11 108115454 108115814
11 108117652 108117892
11 108119624 108119864
11 108121373 108121853
11 108122541 108122781
11 108123531 108123651
11 108124473 108124833
11 108126884 108127124
11 108128122 108128361
11 108129697 108129817
11 108137863 108138103
11 108139116 108139356
11 108141771 108141891
11 108141935 108142175
11 108143236 108143356
11 108143394 108143634
11 108150156 108150396
11 108151688 108151928
11 108153401 108153641
11 108154897 108155257
11 108158264 108158504
11 108159647 108159887
11 108160308 108160548
11 108163313 108163553
11 108164001 108164241
11 108165599 108165839
11 108168001 108168121
11 108170406 108170646
11 108172325 108172565
11 108173547 108173787
11 108175370 108175610
11 108178607 108178727
11 108180844 108181084
11 108183121 108183241
11 108186533 108186653
11 108186669 108186909
11 108188053 108188293
11 108190612 108190852
11 108191967 108192207
11 108195974 108196334
11 108196748 108196988
11 108198308 108198548
11 108199736 108199976
11 108200924 108201164
11 108202094 108202333
11 108202565 108202805
11 108203438 108203678
11 108204594 108204714
11 108205645 108205885
11 108206509 108206749
11 108213903 108214143
11 108216432 108216672
11 108217989 108218109
11 108224430 108224670
11 108225509 108225629
11 108235756 108235996
11 108236023 108236263
13 32890571 32890691
13 32893134 32893493
13 32899146 32899386
13 32900202 32900322
13 32900338 32900458
13 32900573 32900813
13 32903544 32903664
13 32904991 32905231
13 32906366 32907566
13 32910347 32915387
13 32918682 32918802
13 32920938 32921058
13 32928971 32929451
13 32930535 32930775
13 32931852 32932092
13 32936625 32936865
13 32937253 32937733
13 32944496 32944736
13 32945044 32945284
13 32950747 32950987
13 32953433 32953673
13 32953848 32954088
13 32954093 32954333
13 32968767 32969127
13 32970988 32971228
13 32972242 32972962
17 7572907 7573027
17 7573860 7574100
17 7576567 7576700
17 7576829 7576949
17 7576966 7577206
17 7577433 7577673
17 7578112 7578582
17 7579271 7579631
17 7579650 7579770
17 7579815 7579935
17 29422297 29422417
17 29482952 29483192
17 29486009 29486129
17 29490179 29490419
17 29496843 29497083
17 29508413 29508533
17 29508705 29508825
17 29509484 29509724
17 29527406 29527646
17 29527996 29528236
17 29528406 29528526
17 29533203 29533443
17 29541416 29541656
17 29545959 29546199
17 29548740 29548979
17 29548996 29549116
17 29550403 29550643
17 29552070 29552310
17 29553397 29553757
17 29554212 29554332
17 29554522 29554642
17 29556022 29556502
17 29556802 29557042
17 29557219 29557459
17 29557841 29557961
17 29559028 29559268
17 29559688 29559928
17 29560005 29560245
17 29562589 29562829
17 29562867 29563107
17 29575949 29576189
17 29579927 29580047
17 29584650 29584770
17 29585321 29585561
17 29586038 29586158
17 29587340 29587580
17 29588682 29588922
17 29592182 29592422
17 29626505 29626625
17 29652813 29653293
17 29654446 29654926
17 29657295 29657535
17 29661832 29662072
17 29663300 29663540
17 29663612 29663972
17 29664373 29664613
17 29664807 29664927
17 29664980 29665220
17 29665652 29665892
17 29667472 29667712
17 29669970 29670210
17 29676083 29676323
17 29677148 29677388
17 29679233 29679473
17 29683419 29683659
17 29683923 29684163
17 29684216 29684456
17 29685449 29685689
17 29685950 29686070
17 29687492 29687732
17 29694209 29694329
17 29700981 29701221
17 29705857 29705993
17 41197636 41197876
17 41199630 41199750
17 41201114 41201234
17 41203047 41203167
17 41209050 41209170
17 41215309 41215429
17 41215869 41215989
17 41219608 41219728
17 41222920 41223280
17 41226323 41226563
17 41228448 41228688
17 41231323 41231443
17 41234386 41234626
17 41242944 41243064
17 41243424 41246904
17 41247840 41247960
17 41249223 41249343
17 41251724 41251964
17 41256103 41256343
17 41256868 41256988
17 41258451 41258571
17 41267709 41267829
17 41276013 41276133
3 142168237 142168477
3 142171777 142171897
3 142171902 142172142
3 142176401 142176641
3 142177755 142177994
3 142178026 142178266
3 142180737 142180977
3 142183890 142184130
3 142185150 142185390
3 142186723 142186963
3 142188114 142188474
3 142188916 142189036
3 142203933 142204173
3 142211943 142212183
3 142215162 142215402
3 142215824 142216064
3 142217407 142217647
3 142218454 142218574
3 142222189 142222309
3 142223942 142224182
3 142226742 142226982
3 142231087 142231327
3 142232291 142232531
3 142234176 142234416
3 142238448 142238688
3 142241506 142241746
3 142242818 142243058
3 142253864 142254104
3 142254936 142255056
3 142257275 142257515
3 142259691 142259931
3 142261492 142261612
3 142266539 142266779
3 142268298 142268538
3 142268939 142269179
3 142272034 142272274
3 142272411 142272882
3 142274670 142275030
3 142275200 142275440
3 142277421 142277661
3 142278068 142278308
3 142279080 142279320
3 142280054 142280294
3 142281032 142281992
3 142284912 142285152
3 142286890 142287010
3 142297457 142297577

[slzhao]

Would you please check the "genesPassQC.bed" file? I think only the regions
in your target bed file will be included in that "genesPassQC.bed" file.
If not, would you please tell me more details in your target bed file
and "genesPassQC.bed" file? thanks~

2015-12-01 4:16 GMT-06:00 Duarte notifications@github.com:

Unfortunately, this does not seem the case... in my data I have 5 or 6
genes that I have targeted (I have listed them above), however not all of
the exons are targeted. For example Exon 1 of NM000059 is not targeted.

My targeted bed file has now been updated so that I am excluding that
region however this is still what Exon Dell calls as a deletion so I do not
beleive your software is using onlt the targeted regions I have given.

—
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#10 (comment).

[duartemolha]

The content of my target file is listed on my last coment

[duartemolha]

genesPassQC.bed.txt

[slzhao]

So this is your genes.bed file?

10 89623194 89731687 PTEN
11 108093558 108239826 ATM
13 32889616 32973809 BRCA2
17 29421944 29704695 NF1
17 41196311 41277500 BRCA1
3 142168076 142297668 ATR

Would you please indicated which region is not in this file, but in
"genesPassQC.bed.txt
https://github.com/slzhao/ExonDel/files/50255/genesPassQC.bed.txt"?

Thanks~

2015-12-02 16:09 GMT-06:00 Duarte notifications@github.com:

genesPassQC.bed.txt
https://github.com/slzhao/ExonDel/files/50255/genesPassQC.bed.txt

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#10 (comment).

[duartemolha]

no... no targets file was that one on my first run ... but you told me that the target file should contain only the targets of my experiment so that is what I put in there
targets.bed.txt

[slzhao]

So would you please provide which region is not in this file, but in "
genesPassQC.bed.txt
https://github.com/slzhao/ExonDel/files/50255/genesPassQC.bed.txt"? So
that I can check.

2015-12-02 16:23 GMT-06:00 Duarte notifications@github.com:

no... no targets file has that one ... but you told me that the target
file should contain only the targets of my experiment so that is what I put
in there
targets.bed.txt
https://github.com/slzhao/ExonDel/files/50273/targets.bed.txt

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#10 (comment).

[duartemolha]

regions_inGenesPassQC_not_in_targets.txt

here is the file of the entries on the GenesPassQC.bed that has not part of my input target bed file

[slzhao]

Thanks! Let me check it.

2015-12-02 16:30 GMT-06:00 Duarte notifications@github.com:

regions_inGenesPassQC_not_in_targets.txt
https://github.com/slzhao/ExonDel/files/50280/regions_inGenesPassQC_not_in_targets.txt

here is the file of the entries on the GenesPassQC.bed that has not part
of my input target bed file

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#10 (comment).

[duartemolha]

any progress? sorry to put pressure ... just checking to see if you found something obviously wrong with what I am doing.
Thanks