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

Question

[estel_ferre]

Good morning, I'm working with ATACseq data for the fist time, and I'm using DiffBind for the Differential Accessibility Analysis, but I have this message error when I make the contrast: No contrasts added. There must be at least two sample groups with at least three replicates. I have two samples of 2 replicates for each sample. I don't know if I can perform this analysis with DiffBind, I would like to know if it's possible and whats the problem, and if its not possible what package I can use instead DiffBind.

This is my script:

GFP_vs_NT<-dba.peakset(NULL,
                        peaks="../bash_results/macs2/ATAC_GFP_1_peaks.xls",
                        peak.caller="macs", sampID="ATAC_GFP_1",
                        condition = "GFP", replicate=1,
                        bamReads ="../bash_results/final_bam/ATAC_GFP_1.BL.bam")

GFP_vs_NT<-dba.peakset(GFP_vs_NT,
                        peaks="../bash_results/macs2/ATAC_GFP_2_peaks.xls",
                        peak.caller="macs", 
                        sampID="ATAC_GFP_2",
                        condition = "GFP", replicate=2, 
                        bamReads = "../bash_results/final_bam/ATAC_GFP_2.BL.bam")

GFP_vs_NT<-dba.peakset(GFP_vs_NT,
                        peaks="../bash_results/macs2/ATAC_NT_1_peaks.xls",
                        peak.caller="macs", sampID="ATAC_NT_1",condition = "NT", replicate=1,
                        bamReads = "../bash_results/final_bam/ATAC_NT_1.BL.bam")

GFP_vs_NT<-dba.peakset(GFP_vs_NT,
                        peaks="../bash_results/macs2/ATAC_NT_2_peaks.xls",
                        peak.caller="macs", sampID="ATAC_NT_2",
                        condition = "NT", replicate=2, 
                        bamReads = "../bash_results/final_bam/ATAC_NT_1.BL.bam")

counts<-dba.count(GFP_vs_NT, 
                  bParallel = TRUE,
                  score=DBA_SCORE_READS)

GFP_vs_NT_counts<-dba.contrast(counts,
                               categories=DBA_CONDITION, 
                               minMembers = 2)

GFP_vs_NT_counts <- dba.contrast(counts,
                                 contrast = c("condition", "replicate"))

GFP_vs_NT_analyzed<-dba.analyze(GFP_vs_NT_counts, 
                                method = DBA_ALL_METHODS)

GFP_vs_NT_DE_peaks <- dba.report(GFP_vs_NT_analyzed)

Answer 1

[jared.andrews07]

You can do this in DiffBind, but you're continuously overwriting your peakset object with each dba.peakset call. Thus, your peakset only has one sample in it (ATAC_NT_2).

Read the vignette to learn how to properly load your data (start on page 6).

[estel_ferre]

Thanks! I saw that but I don't know how to do the samplesheet, I don't have any .csv, my output are bamfiles and peaks, I don't know if I have to do it manually and what I have to put there

[jared.andrews07]

You can put it together manually - the vignette has clear examples of the format. It will contain columns that point to your BAM and peak files for each sample.