DiffBind error: No contrasts added. There must be at least two sample groups with at least three replicates.

Question

[hdu2]

Hi, I am doing the differential peaks analysis using DiffBind. My sample has two replicates. And my code is the following:

samples <- read.csv("H101.csv")

dbObj <- dba(sampleSheet=samples)
H101C_H1  H3K27ac Responsive Full-media 1 bed
H101C_H2  H3K27ac Responsive Full-media 2 bed
H101RP_H1  H3K27ac Resistant Full-media 1 bed
H101RP_H2  H3K27ac Resistant Full-media 2 bed

 dbObj

4 Samples, 10143 sites in matrix:
         ID  Factor  Condition  Treatment Replicate Intervals
1  H101C_H1 H3K27ac Responsive Full-media         1      5348
2  H101C_H2 H3K27ac Responsive Full-media         2      5348
3 H101RP_H1 H3K27ac  Resistant Full-media         1      4808
4 H101RP_H2 H3K27ac  Resistant Full-media         2      4808

 dbObj <- dba.blacklist(dbObj, blacklist=DBA_BLACKLIST_HG38)

Genome detected: Hsapiens.UCSC.hg38
Applying blacklist...
Removed: 120 of 20312 intervals.
Counting control reads for greylist...
Building greylist: reads/H101C_Input_chr1_sorted.bam
coverage: 2437116 bp (0.98%)
Building greylist: reads/H101RP_Input_chr1_sorted.bam
coverage: 5649399 bp (2.27%)
H101RP_C: 1003 ranges, 5649399 bases
Master greylist: 1013 ranges, 5724151 bases
Removed: 4054 of 20192 intervals.
Removed: 2084 merged (of 10143) and 2084 (of 10143) consensus.

 dbObj <- dba.count(dbObj, bUseSummarizeOverlaps=TRUE)
Computing summits...
Re-centering peaks...

 dbObj <- dba.contrast(dbObj, reorderMeta=list(Condition="Responsive"))
Warning message:
No contrasts added. There must be at least two sample groups with at least three replicates. 

I also tried the following code and got the same warning message.

dbObj <- dba.contrast(dbObj, categories=DBA_TISSUE, minMembers = 2)

Besides, as some people suggested before, I also tried to do dba.blacklist first before dba.count. But it still didn't work. Then I also extracted all the reads and peaks only from Chr 1, it still didn't work.

Further, I copied all the files and codes to another computer and tried again. Then I got some other random error messages...

The only things worked were those demo files from this package...

Can anyone help me to figure it out? Thank you.

Answer 1

[Rory Stark]

With your sample sheet, the following should work:

dbObj <- dba.contrast(dbObj, minMembers=2, 
                      reorderMeta=list(Condition="Responsive"))

This can be done in a more explicit way in two steps, using the design parameter (not categories) to indicate which metadata values you want to use to set up the contrast:

dbObj <- dba.contrast(dbObj, design="~Condition",
                      reorderMeta=list(Condition="Responsive"))
dbObj <- dba.contrast(minMembers=2)