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Old 01-17-2013, 09:53 AM   #41
Frithjof
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Ok, problem solved ...
As mentioned above I first tried the merged.gtf from cuffmerge and also the original .gtf provided to cufflinks. Both giving me errors.
I now re-run the analysis, deleting the old .db first and then recreating the DB with the merged.gtf file from cuffmerge.

Now everything seems to work fine.

Thank you again Loyal for the immediate help

Cheers
Frithjof
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Old 02-18-2013, 09:57 PM   #42
g781
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Hi Loyal,

I have the same problem when ran makeGeneRegionTrack().
I've done with previously solutions but it doesn't seem to work for me.
I am also confused the genome argument. Should I have to tell makeGeneRegionTrack() the exactly name indicating which genome builds to the .gtf annotation?

What information I have are shown below:
diff_out: is a output directory from cuffdiff
merged.gtf: is a gtf file from cuffmerge (I post in the bottom and it has a gene_id in the merged.gtf)

Code:
cuff<-readCufflinks('diff_out',gtfFile='diff_out/merged.gtf',rebuild=T,genome='tb427')
Code:
> myGene
CuffGene instance for gene XLOC_000166 
Short name:	 Tb427.01.710 
Slots:
	 annotation
	 features
	 fpkm
	 repFpkm
	 diff
	 count
	 isoforms	 CuffFeature instance of size 1 
	 TSS		 CuffFeature instance of size 1 
	 CDS		 CuffFeature instance of size 1
Code:
> head(features(myGene))
 [1] seqnames   start      end        width      strand     source    
 [7] type       score      phase      isoform_id gene_id    gene_name 
<0 rows> (or 0-length row.names)
Code:
> genetack<-makeGeneRegionTrack(myGene)
Error in `[.data.frame`(features(object), , featCols) : 
  undefined columns selected
Code:
Tb427_01_v4 Cufflinks exon  228118  229962  . - . gene_id "XLOC_000165"; transcript_id "TCONS_00000307"; exon_number "1"; gene_name "Tb427.01.670"; oId "rna_Tb427.01.670-1"; contained_in "TCONS_00000296"; nearest_ref "rna_Tb427.01.670-1"; class_code "="; tss_id "TSS296"; p_id "P256";
Tb427_01_v4 Cufflinks exon  230768  231499  . - . gene_id "XLOC_000165"; transcript_id "TCONS_00000308"; exon_number "1"; gene_name "Tb427.01.690"; oId "rna_Tb427.01.690-1"; contained_in "TCONS_00000296"; nearest_ref "rna_Tb427.01.690-1"; class_code "="; tss_id "TSS297"; p_id "P257";
Tb427_01_v4 Cufflinks exon  232575  233897  . - . gene_id "XLOC_000165"; transcript_id "TCONS_00000309"; exon_number "1"; gene_name "Tb427.01.700"; oId "rna_Tb427.01.700-1"; contained_in "TCONS_00000296"; nearest_ref "rna_Tb427.01.700-1"; class_code "="; tss_id "TSS285"; p_id "P258";
Tb427_01_v4 Cufflinks exon  234247  235509  . - . gene_id "XLOC_000166"; transcript_id "TCONS_00000310"; exon_number "1"; gene_name "Tb427.01.710"; oId "rna_Tb427.01.710-1"; nearest_ref "rna_Tb427.01.710-1"; class_code "="; tss_id "TSS298"; p_id "P260";
Tb427_01_v4 Cufflinks exon  235715  237370  . - . gene_id "XLOC_000167"; transcript_id "TCONS_00000311"; exon_number "1"; gene_name "Tb427.01.720"; oId "rna_Tb427.01.720-1"; nearest_ref "rna_Tb427.01.720-1"; class_code "="; tss_id "TSS299"; p_id "P261";
Tb427_01_v4 Cufflinks exon  240449  241756  . - . gene_id "XLOC_000169"; transcript_id "TCONS_00000313"; exon_number "1"; gene_name "Tb427.01.740"; oId "rna_Tb427.01.740-1"; nearest_ref "rna_Tb427.01.740-1"; class_code "="; tss_id "TSS301"; p_id "P263";
Many thanks in advance.
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Old 05-08-2013, 03:58 PM   #43
biokari
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I have the same problem!


> sig_gene_unic<-getGenes(cuff,gene_uniq)
Erreur dans sqliteExecStatement(conn, statement, ...) :
RS-DBI driver: (RS_SQLite_exec: could not execute1: cannot start a transaction within a transaction)

Help please
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Old 05-21-2013, 06:45 AM   #44
Steph35
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Unhappy getGenes error

Hi,

I am getting an error when I create gene sets with the last version of cufflinks (2.0.2).When I rebuild the cuffData.db (rebuild=T) as suggested by Loyal, I always get the error «*RS-DBI driver: (error in statement: near ")": syntax error)*»

Any help much appreciated, commands below,
Steph

> cuff_data <- readCufflinks('Diff_out',rebuild=T)
> cuff_data
CuffSet instance with: 3 samples
43025 genes
43025 isoforms
0 TSS
0 CDS
0 promoters
0 splicing
0 relCDS
> data(sampleData)
> myGeneIds<-sampleIDs
> myGeneIds
[1] "XLOC_001363" "XLOC_001297" "XLOC_001339" "XLOC_000132" "XLOC_001265"
[6] "XLOC_000151" "XLOC_001359" "XLOC_000069" "XLOC_000170" "XLOC_000105"
[11] "XLOC_001262" "XLOC_001348" "XLOC_001411" "XLOC_001369" "XLOC_000158"
[16] "XLOC_001370" "XLOC_001263" "XLOC_000115" "XLOC_000089" "XLOC_001240"

> myGenes<-getGenes(cuff_data,myGeneIds)
Getting gene information:
FPKM
Erreur dans sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near ")": syntax error)


> sessionInfo()
R version 2.15.3 (2013-03-01)
Platform: x86_64-unknown-linux-gnu (64-bit)

locale:
[1] LC_CTYPE=fr_FR.UTF-8 LC_NUMERIC=C
[3] LC_TIME=fr_FR.UTF-8 LC_COLLATE=fr_FR.UTF-8
[5] LC_MONETARY=fr_FR.UTF-8 LC_MESSAGES=fr_FR.UTF-8
[7] LC_PAPER=C LC_NAME=C
[9] LC_ADDRESS=C LC_TELEPHONE=C
[11] LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C

attached base packages:
[1] grid stats graphics grDevices utils datasets methods
[8] base

other attached packages:
[1] cummeRbund_2.0.0 Gviz_1.2.1 rtracklayer_1.18.2
[4] GenomicRanges_1.10.7 IRanges_1.16.6 fastcluster_1.1.9
[7] reshape2_1.2.2 ggplot2_0.9.3.1 RSQLite_0.11.2
[10] DBI_0.2-5 BiocGenerics_0.4.0

loaded via a namespace (and not attached):
[1] AnnotationDbi_1.20.7 Biobase_2.18.0 biomaRt_2.14.0
[4] Biostrings_2.26.3 biovizBase_1.6.2 bitops_1.0-5
[7] BSgenome_1.26.1 cluster_1.14.3 colorspace_1.2-1
[10] dichromat_2.0-0 digest_0.6.3 GenomicFeatures_1.10.2
[13] gtable_0.1.2 Hmisc_3.10-1 labeling_0.1
[16] lattice_0.20-14 MASS_7.3-23 munsell_0.4
[19] parallel_2.15.3 plyr_1.8 proto_0.3-10
[22] RColorBrewer_1.0-5 RCurl_1.95-4.1 Rsamtools_1.10.2
[25] scales_0.2.3 stats4_2.15.3 stringr_0.6.2
[28] tools_2.15.3 XML_3.96-0.2 zlibbioc_1.4.0
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Old 06-18-2013, 04:23 AM   #45
Charitra
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Dear All
I got problem with cummeRbund.... my RNA-seq data has fusion gene HLA-DQB1. When I conducted diff analysis (cufflinks), cummeRbund can not show this gene. I tried HLA-DQB1, HLADQB1, HLADQB, HLA, DQB1, DQB etc... it says:
> mygene<- getGene(cuff_data,'HLA-DQB1')
No gene set returned (Gene might not be in database?)
Error in validObject(.Object) : invalid class “CuffGene” object: FALS

However, I can find other genes with no problem with same commands...

Is anybody out there has somthing to help me ?
Thanks in advance.
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Old 08-13-2013, 03:22 PM   #46
fraise13
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Default Error in sqliteExecStatement(con, statement, bind.data) :

Hi All,

I seem to be having similar problems as everyone else and I have looked through many of the posts without finding the solution. However, I am new to R and RNA-seq in general.

Here is what I am getting in RStudio:
> cuff
CuffSet instance with:
4 samples
19856 genes
24928 isoforms
21518 TSS
23593 CDS
119136 promoters
129108 splicing
119136 relCDS
> disp<-dispersionPlot(gene(cuff))
Error in dispersionPlot(gene(cuff)) :
error in evaluating the argument 'object' in selecting a method for function 'dispersionPlot': Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘gene’ for signature ‘"CuffSet"’
> disp<-dispersionPlot(genes(cuff))
> disp
Error in `$<-.data.frame`(`*tmp*`, "SCALE_X", value = 1L) :
replacement has 1 row, data has 0
In addition: Warning message:
In max(panels$ROW) : no non-missing arguments to max; returning -Inf
> genes.scv<-fpkmSCVPlot(genes(cuff))
Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near ")": syntax error)
> dens<-csDensity(genes(cuff))
> dens
Warning messages:
1: Removed 1414 rows containing non-finite values (stat_density).
2: Removed 2084 rows containing non-finite values (stat_density).
3: Removed 1457 rows containing non-finite values (stat_density).
4: Removed 1150 rows containing non-finite values (stat_density).

In particular, this error: Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near ")": syntax error)
seems to be common with everyone. Has anyone figured out the cause and how to fix it?

I did add the gtfFile and genome arguments as suggested.

I would really appreciate any help. I am using v. 0.97551 of RStudio and Cuffdiff v. 2.1.1 on Galaxy.

Thanks,
Fres
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Old 09-05-2013, 09:44 AM   #47
adiallo
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Thumbs up

Hello,
I had the same problem. I think you are using a version of Cufflinks < 2.0.0. And cummeRbund 2.0 needs Cufflinks >= 2.0.0.
So you need to redo all the cuff part with a newer version. Or use cummRBund 1.0.

Cheers ,
Alpha
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Old 09-05-2013, 11:01 PM   #48
fraise13
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Thank you Alpha. I will look into that. I also found that part of the problem may be that cummeRbund requires some files (read_groups.info) that cuffdiff in Galaxy does not include in the output. Galaxy may add an option to include these files in the future.
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Old 10-18-2013, 01:28 AM   #49
IBseq
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Dear all,
any suggestion about this issue I have?

I run the following commands:
> library (cummeRbund)
Loading required package: RSQLite
Loading required package: DBI
Loading required package: ggplot2
Loading required package: reshape2
> cuff<-readCufflinks ()
> cuff
CuffSet instance with:
2 samples
24302 genes
42262 isoforms
28828 TSS
25037 CDS
24302 promoters
28828 splicing
0 relCDS
> iso.diff <- diffData(isoforms(cuff))
> iso.diff.top <- iso.diff[order(iso.diff$q_value),][1:20,]
> iso.diff.top
isoform_id sample_1 sample_2 status value_1 value_2
19598 TCONS_00019647 q1 q2 OK 0.00142518 63.8030
23003 TCONS_00023056 q1 q2 OK 0.01873000 267.5380
16085 TCONS_00016126 q1 q2 OK 0.00828880 53.7744
20375 TCONS_00020425 q1 q2 OK 0.01018440 41.0838
14718 TCONS_00014757 q1 q2 OK 0.01255110 52.7526
38909 TCONS_00038994 q1 q2 OK 0.00506554 18.4579
9364 TCONS_00009383 q1 q2 OK 0.01624700 41.5996
21567 TCONS_00021617 q1 q2 OK 0.02047770 43.5210
21617 TCONS_00021668 q1 q2 OK 0.07737150 173.5400
12908 TCONS_00012945 q1 q2 OK 0.01454510 29.1976
2916 TCONS_00002919 q1 q2 OK 0.02808040 52.5176
11888 TCONS_00011918 q1 q2 OK 0.01283370 23.4124
24967 TCONS_00025024 q1 q2 OK 0.07871780 93.7237
36578 TCONS_00036659 q1 q2 OK 0.15108000 179.3100
18827 TCONS_00018874 q1 q2 OK 0.19685300 402.2040
26748 TCONS_00026809 q1 q2 OK 0.07271350 66.3874
24573 TCONS_00024628 q1 q2 OK 0.01203420 20.7750
5805 TCONS_00005818 q1 q2 OK 0.00198928 2.9240
35808 TCONS_00035884 q1 q2 OK 0.10710700 108.1710
26864 TCONS_00026926 q1 q2 OK 0.05353970 86.0766
log2_fold_change test_stat p_value q_value significant
19598 15.45020 -7.91793 2.44249e-15 5.34124e-11 yes
23003 13.80210 -7.39372 1.42775e-13 1.56110e-09 yes
16085 12.66350 -7.33093 2.28484e-13 1.66550e-09 yes
20375 11.97800 -7.27264 3.52607e-13 1.92770e-09 yes
14718 12.03720 -7.23914 4.51417e-13 1.97432e-09 yes
38909 11.83120 -7.17187 7.39853e-13 2.69652e-09 yes
9364 11.32220 -6.84004 7.91722e-12 2.47334e-08 yes
21567 11.05340 -6.73898 1.59504e-11 3.87558e-08 yes
21617 11.13120 -6.75063 1.47207e-11 3.87558e-08 yes
12908 10.97110 -6.67956 2.39653e-11 5.24073e-08 yes
2916 10.86900 -6.61070 3.82498e-11 6.62383e-08 yes
11888 10.83310 -6.59542 4.24061e-11 6.62383e-08 yes
24967 10.21750 -6.60852 3.88183e-11 6.62383e-08 yes
36578 10.21290 -6.59690 4.19833e-11 6.62383e-08 yes
18827 10.99660 -6.54690 5.87441e-11 8.56411e-08 yes
26748 9.83447 -6.44433 1.16116e-10 1.58701e-07 yes
24573 10.75350 -6.42860 1.28785e-10 1.65663e-07 yes
5805 10.52150 -6.40323 1.52120e-10 1.84809e-07 yes
35808 9.98005 -6.39398 1.61620e-10 1.86017e-07 yes
26864 10.65080 -6.35030 2.14901e-10 2.34972e-07 yes


however, these top 20 include transcripts that start with NM_00000 and NR_00000

what I want is to select only the NM_ as these are proteins/transcript, NR_ if I am not wrong is the gene (here cummerbund only gives TCONS, but I have double checked themn with the NM/NR nomenclature and I know they are mixed up as it is listing by fold change...)

How do I command this on cummerbund?

thanks,
ibseq
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Old 10-22-2013, 08:17 PM   #50
tokitky
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Hi all,

I have a similar problem.

>genediffID <- read.table("genediffID.txt",header= F, sep="/t")
> myGenes <-getGenes(cuff,genediffID)
Error in sqliteExecStatement(conn, statement, ...) :
RS-DBI driver: (RS_SQLite_exec: could not execute1: cannot start a transaction within a transaction)

genediffID was orgninally a column vector but row vector format seemed to be appropriate.

I solved the problem as follows.

>gendiffID <- t(genediffID)
> myGenes <-getGenes(cuff,genediffID)

I also had a similar diffrent problem.

>genediffID2 <- read.table("genediffID2.txt",header= F, sep="/t")
> myGenes2 <-getGenes(cuff,genediffID2)

Error in sqliteExecStatement(conn, statement, bind.data) :
RS-DBI driver: (expired SQLiteConnection)

At this time, cuff was broken by some reason.
So I made cuff again.

>cuff <- readCufflinks("input folder")
>genediffID2 <- read.table("genediffID2.txt",header= F, sep="/t")
> myGenes2 <-getGenes(cuff,genediffID2)

then, it worked.
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Old 10-24-2013, 05:37 AM   #51
IBseq
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dear cummerbund users,
i am trying to run the following command:

library(cummeRbund)
cuff <- readCufflinks()
....

iso.diff <- diffData(isoforms(cuff))
iso.diff.top <- iso.diff[order(iso.diff$q_value),][1:20,]

which works fine, however I would like to run it not through all my samples but a list I have selected (a family of proteins i have extracted and only the significant ones).

I made this table with the isoforms of interest but I don't know how to input the command....any suggestions? I saved this table where all the cuffdiff outputs are and called it myisoforms_exp.diff (thus similar to the original file)

I did not understand how we create a gene set, so I thought this way might work..


thanks,
ibseq
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Old 11-13-2013, 08:46 AM   #52
nilesh.gardi2688
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Default Heatmap in cummeRbund package

How to alter the scale of heatmap in cummerBund? heatMidpoint option in csHeatmap is seems to be non functional.
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Old 12-09-2013, 12:07 PM   #53
adiallo
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Hello
Use :
myGeneIds <- scan("C://heat//tcf4_ensg.txt",what="character")
myGenes<-getGenes(cuff,myGeneIds)

h<-csHeatmap(myGenes,cluster='both')

Cheers
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Old 12-24-2013, 08:19 PM   #54
nancysky
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Default Same error as yours

Hi Fres,

I'm having exactly the same error right now. Can I ask how did you solve the problem? Finding a higher version of cufflinks? How could you find it? I only see one version of cufflinks provided by the server, and that one is older. I can see I have something in cuff but just cannot let the anticipated disp.

Thanks a lot!!!
Nancy





Quote:
Originally Posted by fraise13 View Post
Hi All,

I seem to be having similar problems as everyone else and I have looked through many of the posts without finding the solution. However, I am new to R and RNA-seq in general.

Here is what I am getting in RStudio:
> cuff
CuffSet instance with:
4 samples
19856 genes
24928 isoforms
21518 TSS
23593 CDS
119136 promoters
129108 splicing
119136 relCDS
> disp<-dispersionPlot(gene(cuff))
Error in dispersionPlot(gene(cuff)) :
error in evaluating the argument 'object' in selecting a method for function 'dispersionPlot': Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘gene’ for signature ‘"CuffSet"’
> disp<-dispersionPlot(genes(cuff))
> disp
Error in `$<-.data.frame`(`*tmp*`, "SCALE_X", value = 1L) :
replacement has 1 row, data has 0
In addition: Warning message:
In max(panels$ROW) : no non-missing arguments to max; returning -Inf
> genes.scv<-fpkmSCVPlot(genes(cuff))
Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near ")": syntax error)
> dens<-csDensity(genes(cuff))
> dens
Warning messages:
1: Removed 1414 rows containing non-finite values (stat_density).
2: Removed 2084 rows containing non-finite values (stat_density).
3: Removed 1457 rows containing non-finite values (stat_density).
4: Removed 1150 rows containing non-finite values (stat_density).

In particular, this error: Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near ")": syntax error)
seems to be common with everyone. Has anyone figured out the cause and how to fix it?

I did add the gtfFile and genome arguments as suggested.

I would really appreciate any help. I am using v. 0.97551 of RStudio and Cuffdiff v. 2.1.1 on Galaxy.

Thanks,
Fres
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Old 09-15-2014, 04:10 PM   #55
Gonza
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Hi all,

i am getting this error and cannot figure put how to fix it. Please let me know if you have some tips.
Thans

cuff<-readCufflinks()
cuff

Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: database disk image is malformed)
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Old 09-16-2014, 06:12 AM   #56
adiallo
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Hello Gonza,
What version of the cufflinks package did you use.
Did all the steps went well, without mistakes until the cuffdiff command ?

Here is an example of code that works for me:
library(cummeRbund)

cuff_71_69 <- readCufflinks('_cuffdiff_results_path',rebuild=T)

#===============================================================================================================================================
# To assess the distributions of FPKM scores across samples, you can use the csDensity plot.
dens<-csDensity(genes(cuff_71_69))
dens

dens<-csDensity(genes(cuff_71_69),logMode=TRUE, pseudocount=0.0001)
dens


#===============================================================================================================================================
# Boxplots can be visualized using the csBoxplot method.
b<-csBoxplot(genes(cuff_71_69))
b

Cheers,
Alpha
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Old 09-16-2014, 08:31 AM   #57
Gonza
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Thanks for your reply.
I used cufflinks v2.2.1 and all the steps went well (in fact, in some else computer I was able to upload cuffdiff to R, for some reason in mine does not work - and i have the latest R installed).
Will try your code though.
G
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Old 10-23-2014, 10:14 AM   #58
skdhanraj
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Hi CummeRbund users,

This might be simple question but can anybody explain these warning messages after readCufflinks() command:


Warning messages:
1: attributes are not identical across measure variables; they will be dropped
.
.
8: attributes are not identical across measure variables; they will be dropped

Are these attributes from merged.gtf file?

Thanks,
SK
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Old 12-01-2014, 02:06 PM   #59
SrCardgage
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Quote:
Originally Posted by skdhanraj View Post

Warning messages:
1: attributes are not identical across measure variables; they will be dropped
.
.
8: attributes are not identical across measure variables; they will be dropped

Are these attributes from merged.gtf file?

Thanks,
SK
I am seeing these warnings as well. Has some file not been created correctly?

I am not looking for novel genes/transcripts. So, I went directly from tophat (with --no-novel-juncs flag) to cuffdiff. I doubt this would make a difference, but cannot be sure.
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Old 12-01-2014, 02:44 PM   #60
Gonza
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Hi All,

After a lot of struggle I finally found out what the problem was. In my case, the genes.gtf filed used had some # symbols. When importing cuffdiff to R, the lines containing # made it fail. I created a new genes.gtf without # and it worked.

$sed -r 's/#//g' genes.gtf > new_genes.gtf

That it was my problem, I do not know how 'universal' of a problem that is. But it may be a good idea to check your gtf file.

######################################################################################################

BTW, I am assuming you already did the upgrade:

>source("http://www.bioconductor.org/biocLite.R")
>biocLite("BiocUpgrade")

Then, install "cummeRbund"
>biocLite("cummeRbund")

Last, install or reinstall "RSQLite"
>biocLite("RSQLite")
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