The script I had working 2 years ago for DiffBind no longer works. Dba.count seems to reject files it accepted before.
sets_counted_g=dba.count(sets_g)
Computing summits...
Sample: H:/(filepath truncated)/file_sorted.bam125
Re-centering peaks...
Indexing H:/(filepath truncated)/file_sorted.bam
[E::hts_idx_push] NO_COOR reads not in a single block at the end 2 -1
Error in FUN(X[[i]], ...) : failed to build index
file: H:/CH_DATA/ATAC/2020_Second_postprocessing/bam/PoolCH-7_sorted.bam
In addition: Warning message:
Parallel execution unavailable: executing serially.
I thought I'd sorted this file by position, but I went ahead and re-sorted it using
samtools sort file_sorted.bam -o file_sorted_byposition.bam
The result still fails to count:
sets_counted_g=dba.count(sets_g)
Computing summits...
Sample: H:/(filepath truncated)/file_sorted_byposition.bam125
Re-centering peaks...
Error in colnames(result)[4:ncol(result)] <- colnames(classes) :
replacement has length zero
In addition: Warning message:
Parallel execution unavailable: executing serially
Tagged this with both samtools and Diffbind because it looks like a samtools problem, but the script worked as-is 2 years ago for DiffBind. So maybe DiffBind is involved. Picard ValidateSamFile returned no errors on the file.
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corinne_hutfilz •
1 answer
I can verify that this is a bug and not expected behavior. One of those edge cases in R where something that is usually a matrix gets turned into a vector when there is only one column. I'll address this as a bugfix after the Bioconductor 2.13 release. I'll update this issue here when a fix is checked in.
Regarding index files, DiffBind does expect there to be matching index files for each of the bam files. If they are missing, it will attempt to create new ones; if this fails, so will read counting.
I'm not positive but I believe if you have the corresponding .bai (indexed file) for each .bam in the same directory you are pointing to in your Samplesheet, DiffBind will find and use these. Also, you can use samtools index to create these files. But like Rory said, DiffBind creates these regardless.
Yes, for each XX.bam file DiffBind looks for a XX.bam.bai or XX.bai file. If it is missing, it will try to create one so long as the current user has permission to write into the directory.
Hi Rory Stark
I am experiencing the same issue with DiffBind (3.14.0). I am using a previous dataset which only 1 replicate per condition :(
dbObj_1 <- dba(sampleSheet=samples, dir=".")
# This outputs as:
# 1 Samples, 4497 sites in matrix:
# ID Tissue Factor Condition Treatment Replicate Caller Intervals
# 1 Drought_1 Root ER Drought Drought-stress 1 narrow 4497
dbObj <- dba.count(dbObj_1)
# Computing summits...
# Re-centering peaks...
# Error in colnames(result)[4:ncol(result)] <- colnames(classes) :
# replacement has length zero
Was a solution found? or is there a work around here? I have tried adjusting some of the parameters, but I feel like I'm missing something.
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what is the output of
is file_sorted_byposition.bam indexed with
samtools index?Thanks Pierre,
Quickcheck exited without error. I did generate an index for file_sorted_byposition.bam, but DiffBind doesn't call for indexes.
The same error appears when a sampleSheet with only one line is used.
Reproducible from the vignette data:
Is it the expected behaviour?
I can't say I'm surprised, as the package is meant to compare samples, so there's not much point in running it with only 1. You might try posting over on the Bioconductor support forum, as the package dev is active there. They may want to catch that error so that it's more informative.