I am trying to perform the cufflinks/cuffdiff workflow on the Galaxy server. I am comparing expression at 2 times (Day 2 and Day 6) and 2 conditions at each time (P and D). I have 2 biological replicates for each condition at Day 2, 3 replicates for each condition at Day 6.
for example, group labels are:
D2P (2 samples)
D2D (2 samples)
D6P (3 samples)
D6D (3 samples)
I ran Cufflinks v2.1.1 and Cuffdiff v2.1.1 then downloaded the 11 output files for cummerbund analyses. Most of the analyses I carry out in cummerbund provide reasonable output, such as density plots, heat maps, PC analyses. My problem is that I cannot do any cummerbund analyses that demonstrate differences in the replicate samples. For example, I can make a density plot or dendrogram, but if I use replicates='T' or replicates=T I get error messages.
> cuff<-readCufflinks()
> cuff
CuffSet instance with:
4 samples
26911 genes
65068 isoforms
30546 TSS
30084 CDS
161466 promoters
213180 splicing
117294 relCDS
> cd<-csDendro(genes(cuff)) works
> cdr<-csDendro(genes(cuff), replicates='T')
Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near "from": syntax error)
I have rebuilt the database and tried different syntax for replicates but find that any time I try to include replicates I fail. When I run pairwise volcano plots of triplicate samples, I get great looking plots with some genes showing very large (>20 log2(fold change)) and significant (>4 -log10(p)) differences as can be seen in the tabular diff data in excel, but only black "not significant" dots. This makes me think that either cuffdiff is not correctly characterizing the replicates, my db is still somehow incorrect, or I am not using cummerbund correctly.
The last clue is that none of the 11 cuffdiff output files have any notation with replica names. There are no columns labeled with the individual sample bam file names with counts. The online cuffdiff manual suggests there should be 4 read counts files that show replicate data but I have never seen these files.
Any help would be welcome.
mike
for example, group labels are:
D2P (2 samples)
D2D (2 samples)
D6P (3 samples)
D6D (3 samples)
I ran Cufflinks v2.1.1 and Cuffdiff v2.1.1 then downloaded the 11 output files for cummerbund analyses. Most of the analyses I carry out in cummerbund provide reasonable output, such as density plots, heat maps, PC analyses. My problem is that I cannot do any cummerbund analyses that demonstrate differences in the replicate samples. For example, I can make a density plot or dendrogram, but if I use replicates='T' or replicates=T I get error messages.
> cuff<-readCufflinks()
> cuff
CuffSet instance with:
4 samples
26911 genes
65068 isoforms
30546 TSS
30084 CDS
161466 promoters
213180 splicing
117294 relCDS
> cd<-csDendro(genes(cuff)) works
> cdr<-csDendro(genes(cuff), replicates='T')
Error in sqliteExecStatement(con, statement, bind.data) :
RS-DBI driver: (error in statement: near "from": syntax error)
I have rebuilt the database and tried different syntax for replicates but find that any time I try to include replicates I fail. When I run pairwise volcano plots of triplicate samples, I get great looking plots with some genes showing very large (>20 log2(fold change)) and significant (>4 -log10(p)) differences as can be seen in the tabular diff data in excel, but only black "not significant" dots. This makes me think that either cuffdiff is not correctly characterizing the replicates, my db is still somehow incorrect, or I am not using cummerbund correctly.
The last clue is that none of the 11 cuffdiff output files have any notation with replica names. There are no columns labeled with the individual sample bam file names with counts. The online cuffdiff manual suggests there should be 4 read counts files that show replicate data but I have never seen these files.
Any help would be welcome.
mike
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