Hi,

I'm trying to run DiffBind 3.4.11 on R 4.1.2 on some bed files created using bedtools merge, so they look like:

1   1875658 1934008 EdU_HU_HME1_sgMSL1_R1_peak_54
1   2171957 2261179 EdU_HU_HME1_sgMSL1_R1_peak_55,EdU_HU_HME1_sgMSL1_R1_peak_56
1   2307970 2361163 EdU_HU_HME1_sgMSL1_R1_peak_57,EdU_HU_HME1_sgMSL1_R1_peak_58
1   2413610 2413946 EdU_HU_HME1_sgMSL1_R1_peak_59
1   2424196 2576087 EdU_HU_HME1_sgMSL1_R1_peak_60,EdU_HU_HME1_sgMSL1_R1_peak_61,EdU_HU_HME1_sgMSL1_R1_peak_62,EdU_HU_HME1_sgMSL1_R1_peak_63
1   3497148 3497649 EdU_HU_HME1_sgMSL1_R1_peak_64

My samplesheet looks like:

SampleID,Condition,Replicate,bamReads,ControlID,bamControl,Peaks,PeakCaller
sgMSL1_R1_merged10000,sgMSL1_10000,1,results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R1.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgMSL1_R1_peaks_merged10000.bed,macs
sgMSL1_R1_merged20000,sgMSL1_20000,1,results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R1.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgMSL1_R1_peaks_merged20000.bed,macs
sgMSL1_R2_merged10000,sgMSL1_10000,2,results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R2.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgMSL1_R2_peaks_merged10000.bed,macs
sgMSL1_R2_merged20000,sgMSL1_20000,2,results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R2.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgMSL1_R2_peaks_merged20000.bed,macs
sgTNP2_R1_merged10000,sgTNP2_10000,1,results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R1.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgTNP2_R1_peaks_merged10000.bed,macs
sgTNP2_R1_merged20000,sgTNP2_20000,1,results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R1.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgTNP2_R1_peaks_merged20000.bed,macs
sgTNP2_R2_merged10000,sgTNP2_10000,2,results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R2.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgTNP2_R2_peaks_merged10000.bed,macs
sgTNP2_R2_merged20000,sgTNP2_20000,2,results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R2.mLb.clN.sorted.bam,Input,results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam,results/bedtools_merge/EdU_HU_HME1_sgTNP2_R2_peaks_merged20000.bed,macs

Then I do:

samples <- read.csv('diffbind_samplelist.csv')
dbObj <- dba(sampleSheet=samples,minOverlap=0)

And when I run dbObj <- dba.count(dbObj, summits = F, bUseSummarizeOverlaps=T, bParallel = TRUE) I get:

[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgTNP2_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R2.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/EdU_HU_HME1_sgMSL1_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam.bai
[W::hts_idx_load2] The index file is older than the data file: results/bwa/mergedLibrary/HME1_Input_R1.mLb.clN.sorted.bam.bai
Error in pv$called[, mask] : subscript out of bounds

I have also tried dbObj <- dba.count(dbObj,bUseSummarizeOverlaps=T, bParallel = TRUE) which gives:

Computing summits...
Re-centering peaks...
Error in heights * sapply(called, function(x) x) : non-conformable arrays

Any idea of what's going on? Many thanks in advance.

In the first instance, you need to re-index your .bam files. You can do this using samtools. Alternatively, if you delete all the .bai files, DiffBind will automatically re-index them if the .bai files are missing (so long as you have permissions to write files into the results/bwa/mergedLibrary/ directory).