Help with DiffBind Generating report-based DBA object... Error: No valid contrasts/methods specified.

Question

[Chris]

Hi all,

Would you please suggest what wrong in this case? I pretty much run the same code as before. Thank you so much.

result <- dba(sampleSheet=samples)
result <- dba.blacklist(result) 
result <- dba.count(result, bParallel=FALSE)
result <- dba.count(result)
result <- dba.normalize(result)
result <- dba.contrast(result, minMembers=2)
result <- dba.analyze(result)
result <- dba.plotProfile(result)

Generating report-based DBA object... Error: No valid contrasts/methods specified.

There is post about this error but I still got error when trying those solutions. Such as the order of the function, the path to the bam and broad peak files in sample sheet.

dba.show(result, bContrasts=TRUE)
     Factor    Group Samples  Group2 Samples2 DB.DESeq2
1 Condition diseased       2 control        2         0

I don't know why the value of DB.DESeq2 in this case was 0 which maybe the reason of the error.

Answer 1

[Rory Stark]

Yes, the plotting function does not handle the case of 0 differentially bound sites gracefully.

It is definitely possible to find zero differentially bound sites below the FDR threshold! Possible reasons for this include:

1. With only two replicates, you may not have enough power to overcome high variance.
2. With a large number of sites being tested, the FDR correction can be enough to move the confidence statistics above the threshold.
3. This could be a true biological result: nothing is changing consistently between these two sample groups

To check intrasample variance, you can specify merge=NULL to the dba.plotProfile() call to see the profile plots for each sample. If the replicates look different, it is probably the first issue. If they look the same, it is probably the third issue. You can also look at the read counts and confidence statistics (such as p-value) using dba.report().

[Chris]

Thanks Rory! May I know how Diffbind can output a bed file (differential_peaks.bed) to use as input for the command findMotifsGenome.pl from HOMER?

Answer 2

[jared.andrews07]

Yes, I presume 0 differentially bound sites found via DESeq2 would throw this error, as there'd be nothing to plot based on the docs:

If an analysis has been completed, the default plot will be based on the results of the first contrast. If the contrast compares two conditions, all of the samples in each condition will be included, with the heatmaps colored separately for samples in each contrast condition.

[Chris]

Even though ms and mf data are similar, Is that possible my code compare ms or mf with themself which make the DB.DESeq2 equal 0? If they are not very different, the value should be small instead of 0, right?