Help with cuffdiff and cummeRbund?

[kelseyca]

Hi all!

Sorry to bother with a simple question- I have read through all the cummeRbund posts and tutorials but I seem to be stuck right at the start!

I have ran RNA-seq analyses on galaxy online- tophat, cufflinks, cuffmerge, and cuffdiff. I would now like to visualize results in cummeRbund. I downloaded the cuffdiff files (11 each for two groups) off galaxy and they are in .tabular format. I installed cummeRbund, and ran the following. It does not work. Could the issue be that files should be in .db format? I don't know where the cuffData.db file came from- it appeared before I had even downloaded the cuffdiff files.

> source("http://bioconductor.org/biocLite.R")
> biocLite("cummeRbund")
> getwd()
> setwd("C:/Users/caetano1/Downloads/SEDENTARYDFF")
> list.files()
> library(cummeRbund)
> cuff= readCufflinks (dbFile = "cuffData.db",
+ geneFPKM = "CuffdiffSEDENTARY__gene_FPKM_tracking.tabular",
+ geneDiff = "CuffdiffSEDENTARY__CDS_FPKM_differential_expression_testing.tabular",
+ isoformFPKM = "CuffdiffSEDENTARY__transcript_FPKM_tracking.tabular",
+ isoformDiff = "CuffdiffSEDENTARY__transcript_differential_expression_testing.tabular",
+ TSSFPKM = "CuffdiffSEDENTARY__TSS_groups_FPKM_tracking.tabular",
+ TSSDiff = "CuffdiffSEDENTARY__TSS_groups_differential_expression_testing.tabular",
+ CDSFPKM = "CuffdiffSEDENTARY__CDS_FPKM_tracking.tabular",
+ CDSExpDiff = "CuffdiffSEDENTARY__CDS_FPKM_differential_expression_testing.tabular"",
+ CDSDiff = "CuffdiffSEDENTARY__CDS_overloading_diffential_expression_testing.tabular",
+ promoterFile = "CuffdiffSEDENTARY__promoters_differential_expression_testing.tabular",
+ splicingFile = "CuffdiffSEDENTARY__splicing_differential_expression_testing.tabular",
+ rebuild = T)

I think I'm missing something really obvious here!

Thank you so much!

Kelesy

[muthu545]

Hi kelseyca,

cuffData.db is the database file created by cummeRbund to store all the results from cuffdiff in a easy to access format for commands in cummeRbund in R.

So if you run readCufflinks (dbFile = "cuffData.db",....) command even without loading all the files from cuffdiff into the directory, a default cuffData.db fill will be created.

Hope this helps

Thanks
--
Muthu

[kelseyca]

Hi kelseyca,

cuffData.db is the database file created by cummeRbund to store all the results from cuffdiff in a easy to access format for commands in cummeRbund in R.

So if you run readCufflinks (dbFile = "cuffData.db",....) command even without loading all the files from cuffdiff into the directory, a default cuffData.db fill will be created.

Hope this helps

Thanks
--
Muthu

Muthu,

Thanks for your reply! So, how can I get R to read my cuffdiff files? Are they in the wrong format?

Kelsey

[sazz]

Output files should look like this:

genes.read_group_tracking
genes.fpkm_tracking
genes.count_tracking
gene_exp.diff

I guess, you should delete ".tabular" part and organize them in the right format.

[muthu545]

Kelsey,

As Sazz mentioned, the output files from cuffdiff will not have .tabular file formats.
Please verify your output files from cuffdiff, if it doesnot match names provided in the readCufflinks command, then the files will not be recognized in R.

Simple is to copy all the output files from cuffdiff into a directory and run the following command.

cuff= readCufflinks (dbFile = "cuffData.db",dir="C:/Users/caetano1/Downloads/SEDENTARYDFF",
gtfFile='DIRPATH/gtffile', genome='genomename',rebuild = T)

This command recognizes all the files required to make the directory. You need not specify them individually.

GTF file is needed for some visualization commands in cummeRbund.

Hope this is helpful

Thanks
--
Muthu

[kelseyca]

Kelsey,

As Sazz mentioned, the output files from cuffdiff will not have .tabular file formats.
Please verify your output files from cuffdiff, if it doesnot match names provided in the readCufflinks command, then the files will not be recognized in R.

Simple is to copy all the output files from cuffdiff into a directory and run the following command.

cuff= readCufflinks (dbFile = "cuffData.db",dir="C:/Users/caetano1/Downloads/SEDENTARYDFF",
gtfFile='DIRPATH/gtffile', genome='genomename',rebuild = T)

This command recognizes all the files required to make the directory. You need not specify them individually.

GTF file is needed for some visualization commands in cummeRbund.

Hope this is helpful

Thanks
--
Muthu

Hi Muthu,

One last question. Sorry If I am missing something very obvious here and wasting your time. thank you so much for being so patient and all of your help.

I can not figure out how to export cuffdiff files from galaxy online in any other format than .tabular. I am just clicking "download" under the cuffdiff run. All manuals and FAQ's I have been reading are from running the tuxedo suite offline.

Also, R cannot find the function "readCufflinks".

Kelsey

[kelseyca]

Kelsey,

As Sazz mentioned, the output files from cuffdiff will not have .tabular file formats.
Please verify your output files from cuffdiff, if it doesnot match names provided in the readCufflinks command, then the files will not be recognized in R.

Simple is to copy all the output files from cuffdiff into a directory and run the following command.

cuff= readCufflinks (dbFile = "cuffData.db",dir="C:/Users/caetano1/Downloads/SEDENTARYDFF",
gtfFile='DIRPATH/gtffile', genome='genomename',rebuild = T)

This command recognizes all the files required to make the directory. You need not specify them individually.

GTF file is needed for some visualization commands in cummeRbund.

Hope this is helpful

Thanks
--
Muthu

> cuff= readCufflinks (dbFile = "cuffData.db",dir="C:/Users/caetano1/Downloads/SEDENTARYDFF",
+ gtfFile='DIRPATH/gtffile', genome='genomename',rebuild = T)
Creating database C:/Users/caetano1/Downloads/SEDENTARYDFF/cuffData.db
Reading GTF file
Error in import(FileForFormat(con), ...) :
error in evaluating the argument 'con' in selecting a method for function 'import': Error in FileForFormat(con) : Format 'DIRPATH/gtffile' unsupported
>

[muthu545]

Hi Muthu,

One last question. Sorry If I am missing something very obvious here and wasting your time. thank you so much for being so patient and all of your help.

I can not figure out how to export cuffdiff files from galaxy online in any other format than .tabular. I am just clicking "download" under the cuffdiff run. All manuals and FAQ's I have been reading are from running the tuxedo suite offline.

Also, R cannot find the function "readCufflinks".

Kelsey

Hi Kelsey,

Not a problem.

If that's the case (Galaxy's output is .tabular), then you could rename the files in order to change the .tabular file format, after you download them.

If R cannot find the functions 'readCufflinks', it means you did not load the corresponding library 'cummeRbund' in the current workspace.

Thanks
--
Muthu

[muthu545]

> cuff= readCufflinks (dbFile = "cuffData.db",dir="C:/Users/caetano1/Downloads/SEDENTARYDFF",
+ gtfFile='DIRPATH/gtffile', genome='genomename',rebuild = T)
Creating database C:/Users/caetano1/Downloads/SEDENTARYDFF/cuffData.db
Reading GTF file
Error in import(FileForFormat(con), ...) :
error in evaluating the argument 'con' in selecting a method for function 'import': Error in FileForFormat(con) : Format 'DIRPATH/gtffile' unsupported
>

Kelsey,

Rightnow, its throwing out error because its not able to detect the directory 'DIRPATH' and the gtf file.

I mentioned 'DIRPATH' in order to imply the directory in which you have the .gtf file you used to run cufflinks.
you could copy the XXX.gtf file to the same working directory 'C:/Users/caetano1/Downloads/SEDENTARYDFF' and then replace the DIRPATH/gtffile in the command to 'C:/Users/caetano1/Downloads/SEDENTARYDFF/XXX.gtf' and the 'genomename' to the name of the genome you are working with eg. 'hg19', 'hg18','pt03','mm9','mm10' etc...

Your readcufflinks command should work after this without any error.

thanks
--
Muthu

[kelseyca]

> source("http://bioconductor.org/biocLite.R")
Bioconductor version 2.12 (BiocInstaller 1.10.2), ?biocLite for help
> biocLite("cummeRbund")
BioC_mirror: http://bioconductor.org
Using Bioconductor version 2.12 (BiocInstaller 1.10.2), R version 3.0.1.
Installing package(s) 'cummeRbund'
trying URL 'http://bioconductor.org/packages/2.12/bioc/bin/windows/contrib/3.0/cummeRbund_2.2.0.zip'
Content type 'application/zip' length 2600163 bytes (2.5 Mb)
opened URL
downloaded 2.5 Mb

package ‘cummeRbund’ successfully unpacked and MD5 sums checked

The downloaded binary packages are in
C:\Users\caetano1\AppData\Local\Temp\RtmpQTqdVW\downloaded_packages
Warning message:
installed directory not writable, cannot update packages 'class', 'foreign',
'MASS', 'mgcv', 'nnet', 'spatial'
> getwd()
[1] "\\\\ansci-alpha/Homes/Grads/caetano1/Documents"
> setwd("C:/Users/caetano1/Downloads/SEDENTARYDFF")
> list.files()
[1] "cuffData.db"
[2] "CuffdiffSEDENTARY__CDS_FPKM_differential_expression_testing.tabular"
[3] "CuffdiffSEDENTARY__CDS_FPKM_tracking.tabular"
[4] "CuffdiffSEDENTARY__CDS_overloading_diffential_expression_testing.tabular"
[5] "CuffdiffSEDENTARY__gene_differential_expression_testing.tabular"
[6] "CuffdiffSEDENTARY__gene_FPKM_tracking.tabular"
[7] "CuffdiffSEDENTARY__promoters_differential_expression_testing.tabular"
[8] "CuffdiffSEDENTARY__splicing_differential_expression_testing.tabular"
[9] "CuffdiffSEDENTARY__transcript_differential_expression_testing.tabular"
[10] "CuffdiffSEDENTARY__transcript_FPKM_tracking.tabular"
[11] "CuffdiffSEDENTARY__TSS_groups_differential_expression_testing.tabular"
[12] "CuffdiffSEDENTARY__TSS_groups_FPKM_tracking.tabular"
[13] "mm10.gtf"
> library(cummeRbund)
Loading required package: BiocGenerics
Loading required package: parallel

Attaching package: ‘BiocGenerics’

The following objects are masked from ‘packagearallel’:

clusterApply, clusterApplyLB, clusterCall, clusterEvalQ,
clusterExport, clusterMap, parApply, parCapply, parLapply,
parLapplyLB, parRapply, parSapply, parSapplyLB

The following object is masked from ‘package:stats’:

xtabs

The following objects are masked from ‘package:base’:

anyDuplicated, as.data.frame, cbind, colnames, duplicated, eval,
Filter, Find, get, intersect, lapply, Map, mapply, match, mget,
order, paste, pmax, pmax.int, pmin, pmin.int, Position, rank,
rbind, Reduce, rep.int, rownames, sapply, setdiff, sort, table,
tapply, union, unique, unlist

Loading required package: RSQLite
Loading required package: DBI
Loading required package: ggplot2
Loading required package: reshape2
Loading required package: fastcluster

Attaching package: ‘fastcluster’

The following object is masked from ‘package:stats’:

hclust

Loading required package: rtracklayer
Loading required package: GenomicRanges
Loading required package: IRanges
Loading required package: Gviz
Loading required package: grid

Attaching package: ‘cummeRbund’

The following object is masked from ‘package:GenomicRanges’:

promoters

The following object is masked from ‘package:IRanges’:

promoters

> cuff= readCufflinks (dbFile = "cuffData.db",dir="C:/Users/caetano1/Downloads/SEDENTARYDFF",
+ gtfFile="C:/Users/caetano1/Downloads/SEDENTARYDFF/mm10.gtf", genome='mm10',rebuild = T)
Creating database C:/Users/caetano1/Downloads/SEDENTARYDFF/cuffData.db
Reading GTF file
Error in .parse_attrCol(attrCol, file, colnames) :
Some attributes do not conform to 'tag value' format
>

[jp.]

Please try simple this one.
Note: keep you "diff_out" folder within cuff_data folder
change directory to: cuff_data
> cuff_data<- readCufflinks('diff_out',rebuild=T)