DiffBind normalization error: invalid argument type (list) - cannot make it work, everything seems correct

Question

[buffealo]

I am trying to conduct peak calling on a publicly available dataset.

I posted before, but no matter what I tried (I followed the manual in variations, ChatGPT, forums, etc.), I could not make DiffBind work for it. This dataset I am trying to analyze is almost identical to the one used in the very beginning of its own documentation.

I have different cell lines, and I want to conduct the analysis by grouping distinct cell lines and obtaining cell line-specific information as well as group-specific information. I tried using different metadata files, which can be seen below.

==> formatted_metadata_diffbind_myc.tsv <==
SampleID    Tissue  Factor  Condition   Treatment   Replicate   bamReads    ControlID   Peaks   PeakCaller
M7_1    mcf7_myc_rep1_peaks.narrowPeak  macs    mcf7_myc_rep1_bowtie_sorted_q20_dupmarked.bam   MCF7in
M7_2    mcf7_myc_rep2_peaks.narrowPeak  macs    mcf7_myc_rep2_bowtie_sorted_q20_dupmarked.bam   MCF7in
M7_3    mcf7_myc_rep3_peaks.narrowPeak  macs    mcf7_myc_rep3_bowtie_sorted_q20_dupmarked.bam   MCF7in
T4_1    t47d_myc_rep1_peaks.narrowPeak  macs    t47d_myc_rep1_bowtie_sorted_q20_dupmarked.bam   T47Din
T4_2    t47d_myc_rep2_peaks.narrowPeak  macs    t47d_myc_rep2_bowtie_sorted_q20_dupmarked.bam   T47Din
T4_3    t47d_myc_rep3_peaks.narrowPeak  macs    t47d_myc_rep3_bowtie_sorted_q20_dupmarked.bam   T47Din
M231_1  mdamb231_myc_rep1_peaks.narrowPeakn macs    mdamb231_myc_rep1_bowtie_sorted_q20_dupmarked.bam   MDAMB231in
M231_2  mdamb231_myc_rep2_peaks.narrowPeakn macs    mdamb231_myc_rep2_bowtie_sorted_q20_dupmarked.bam   MDAMB231in
M231_3  mdamb231_myc_rep3_peaks.narrowPeakn macs    mdamb231_myc_rep3_bowtie_sorted_q20_dupmarked.bam   MDAMB231in
BT_1    bt549_myc_rep1_peaks.narrowPeak macs    bt549_myc_rep1_bowtie_sorted_q20_dupmarked.bam  BT549in
BT_2    bt549_myc_rep2_peaks.narrowPeak macs    bt549_myc_rep2_bowtie_sorted_q20_dupmarked.bam  BT549in
BT_3    BT549   MYC TNBC    Non 3   bt549_myc_rep3_bowtie_sorted_q20_dupmarked.bam  BT549in bt549_myc_rep3_peaks.narrowPeak macs

==> metadata_diffbind_fixed.csv <==
"SampleID","Condition","Replicate","Peaks","bamReads","bamControl"
"M7_1","ER+",1,"mcf7_myc_rep1_peaks.narrowPeak","mcf7_myc_rep1_bowtie_sorted_q20_dupmarked.bam","mcf7_input_bowtie_sorted_q20_dupmarked.bam"
"M7_2","ER+",2,"mcf7_myc_rep2_peaks.narrowPeak","mcf7_myc_rep2_bowtie_sorted_q20_dupmarked.bam","mcf7_input_bowtie_sorted_q20_dupmarked.bam"
"M7_3","ER+",3,"mcf7_myc_rep3_peaks.narrowPeak","mcf7_myc_rep3_bowtie_sorted_q20_dupmarked.bam","mcf7_input_bowtie_sorted_q20_dupmarked.bam"
"T4_1","ER+",1,"t47d_myc_rep1_peaks.narrowPeak","t47d_myc_rep1_bowtie_sorted_q20_dupmarked.bam","t47d_input_bowtie_sorted_q20_dupmarked.bam"
"T4_2","ER+",2,"t47d_myc_rep2_peaks.narrowPeak","t47d_myc_rep2_bowtie_sorted_q20_dupmarked.bam","t47d_input_bowtie_sorted_q20_dupmarked.bam"
"T4_3","ER+",3,"t47d_myc_rep3_peaks.narrowPeak","t47d_myc_rep3_bowtie_sorted_q20_dupmarked.bam","t47d_input_bowtie_sorted_q20_dupmarked.bam"
"M231_1","TNBC",1,"mdamb231_myc_rep1_peaks.narrowPeak","mdamb231_myc_rep1_bowtie_sorted_q20_dupmarked.bam","mdamb231_input_bowtie_sorted_q20_dupmarked.bam"
"M231_2","TNBC",2,"mdamb231_myc_rep2_peaks.narrowPeak","mdamb231_myc_rep2_bowtie_sorted_q20_dupmarked.bam","mdamb231_input_bowtie_sorted_q20_dupmarked.bam"
"M231_3","TNBC",3,"mdamb231_myc_rep3_peaks.narrowPeak","mdamb231_myc_rep3_bowtie_sorted_q20_dupmarked.bam","mdamb231_input_bowtie_sorted_q20_dupmarked.bam"
"BT_1","TNBC",1,"bt549_myc_rep1_peaks.narrowPeak","bt549_myc_rep1_bowtie_sorted_q20_dupmarked.bam","bt549_input_bowtie_sorted_q20_dupmarked.bam"
"BT_2","TNBC",2,"bt549_myc_rep2_peaks.narrowPeak","bt549_myc_rep2_bowtie_sorted_q20_dupmarked.bam","bt549_input_bowtie_sorted_q20_dupmarked.bam"
"BT_3","TNBC",3,"bt549_myc_rep3_peaks.narrowPeak","bt549_myc_rep3_bowtie_sorted_q20_dupmarked.bam","bt549_input_bowtie_sorted_q20_dupmarked.bam"

==> metadata_diffbind_myc.csv <==
SampleID,Tissue,Condition,Replicate,Factor,Treatment,PeakCaller,Peaks,bamReads,bamControl,ControlID
M7_1,MCF7,ER+,1,MYC,Non,macs,mcf7_myc_rep1_peaks.narrowPeak,mcf7_myc_rep1_bowtie_sorted_q20_dupmarked.bam,mcf7_input_bowtie_sorted_q20_dupmarked.bam,MCF7in
M7_2,MCF7,ER+,2,MYC,Non,macs,mcf7_myc_rep2_peaks.narrowPeak,mcf7_myc_rep2_bowtie_sorted_q20_dupmarked.bam,mcf7_input_bowtie_sorted_q20_dupmarked.bam,MCF7in
M7_3,MCF7,ER+,3,MYC,Non,macs,mcf7_myc_rep3_peaks.narrowPeak,mcf7_myc_rep3_bowtie_sorted_q20_dupmarked.bam,mcf7_input_bowtie_sorted_q20_dupmarked.bam,MCF7in
T4_1,T47D,ER+,1,MYC,Non,macs,t47d_myc_rep1_peaks.narrowPeak,t47d_myc_rep1_bowtie_sorted_q20_dupmarked.bam,t47d_input_bowtie_sorted_q20_dupmarked.bam,T47Din
T4_2,T47D,ER+,2,MYC,Non,macs,t47d_myc_rep2_peaks.narrowPeak,t47d_myc_rep2_bowtie_sorted_q20_dupmarked.bam,t47d_input_bowtie_sorted_q20_dupmarked.bam,T47Din
T4_3,T47D,ER+,3,MYC,Non,macs,t47d_myc_rep3_peaks.narrowPeak,t47d_myc_rep3_bowtie_sorted_q20_dupmarked.bam,t47d_input_bowtie_sorted_q20_dupmarked.bam,T47Din
M231_1,MDAMB231,TNBC,1,MYC,Non,macs,mdamb231_myc_rep1_peaks.narrowPeak,mdamb231_myc_rep1_bowtie_sorted_q20_dupmarked.bam,mdamb231_input_bowtie_sorted_q20_dupmarked.bam,MDAMB231in
M231_2,MDAMB231,TNBC,2,MYC,Non,macs,mdamb231_myc_rep2_peaks.narrowPeak,mdamb231_myc_rep2_bowtie_sorted_q20_dupmarked.bam,mdamb231_input_bowtie_sorted_q20_dupmarked.bam,MDAMB231in
M231_3,MDAMB231,TNBC,3,MYC,Non,macs,mdamb231_myc_rep3_peaks.narrowPeak,mdamb231_myc_rep3_bowtie_sorted_q20_dupmarked.bam,mdamb231_input_bowtie_sorted_q20_dupmarked.bam,MDAMB231in
BT_1,BT549,TNBC,1,MYC,Non,macs,bt549_myc_rep1_peaks.narrowPeak,bt549_myc_rep1_bowtie_sorted_q20_dupmarked.bam,bt549_input_bowtie_sorted_q20_dupmarked.bam,BT549in
BT_2,BT549,TNBC,2,MYC,Non,macs,bt549_myc_rep2_peaks.narrowPeak,bt549_myc_rep2_bowtie_sorted_q20_dupmarked.bam,bt549_input_bowtie_sorted_q20_dupmarked.bam,BT549in
BT_3,BT549,TNBC,3,MYC,Non,macs,bt549_myc_rep3_peaks.narrowPeak,bt549_myc_rep3_bowtie_sorted_q20_dupmarked.bam,bt549_input_bowtie_sorted_q20_dupmarked.bam,BT549in

==> myc_metadata.csv <==
SampleID,Tissue,Factor,Condition,Treatment,Replicate,bamReads,ControlID,bamControl,Peaks,PeakCaller
M7_1,MCF7,MYC,ER+,Non,1,mcf7_myc_rep1_bowtie_sorted_q20_dupmarked.bam,MCF7in,mcf7_input_bowtie_sorted_q20_dupmarked.bam,mcf7_myc_rep1_peaks.narrowPeak,raw
M7_2,MCF7,MYC,ER+,Non,2,mcf7_myc_rep2_bowtie_sorted_q20_dupmarked.bam,MCF7in,mcf7_input_bowtie_sorted_q20_dupmarked.bam,mcf7_myc_rep2_peaks.narrowPeak,raw
M7_3,MCF7,MYC,ER+,Non,3,mcf7_myc_rep3_bowtie_sorted_q20_dupmarked.bam,MCF7in,mcf7_input_bowtie_sorted_q20_dupmarked.bam,mcf7_myc_rep3_peaks.narrowPeak,raw
T4_1,T47D,MYC,ER+,Non,1,t47d_myc_rep1_bowtie_sorted_q20_dupmarked.bam,T47Din,t47d_input_bowtie_sorted_q20_dupmarked.bam,t47d_myc_rep1_peaks.narrowPeak,raw
T4_2,T47D,MYC,ER+,Non,2,t47d_myc_rep2_bowtie_sorted_q20_dupmarked.bam,T47Din,t47d_input_bowtie_sorted_q20_dupmarked.bam,t47d_myc_rep2_peaks.narrowPeak,raw
T4_3,T47D,MYC,ER+,Non,3,t47d_myc_rep3_bowtie_sorted_q20_dupmarked.bam,T47Din,t47d_input_bowtie_sorted_q20_dupmarked.bam,t47d_myc_rep3_peaks.narrowPeak,raw
M231_1,MDAMB231,MYC,TNBC,Non,1,mdamb231_myc_rep1_bowtie_sorted_q20_dupmarked.bam,MDAMB231in,mdamb231_input_bowtie_sorted_q20_dupmarked.bam,mdamb231_myc_rep1_peaks.narrowPeak,raw
M231_2,MDAMB231,MYC,TNBC,Non,2,mdamb231_myc_rep2_bowtie_sorted_q20_dupmarked.bam,MDAMB231in,mdamb231_input_bowtie_sorted_q20_dupmarked.bam,mdamb231_myc_rep2_peaks.narrowPeak,raw
M231_3,MDAMB231,MYC,TNBC,Non,3,mdamb231_myc_rep3_bowtie_sorted_q20_dupmarked.bam,MDAMB231in,mdamb231_input_bowtie_sorted_q20_dupmarked.bam,mdamb231_myc_rep3_peaks.narrowPeak,raw
BT_1,BT549,MYC,TNBC,Non,1,bt549_myc_rep1_bowtie_sorted_q20_dupmarked.bam,BT549in,bt549_input_bowtie_sorted_q20_dupmarked.bam,bt549_myc_rep1_peaks.narrowPeak,raw
BT_2,BT549,MYC,TNBC,Non,2,bt549_myc_rep2_bowtie_sorted_q20_dupmarked.bam,BT549in,bt549_input_bowtie_sorted_q20_dupmarked.bam,bt549_myc_rep2_peaks.narrowPeak,raw
BT_3,BT549,MYC,TNBC,Non,3,bt549_myc_rep3_bowtie_sorted_q20_dupmarked.bam,BT549in,bt549_input_bowtie_sorted_q20_dupmarked.bam,bt549_myc_rep3_peaks.narrowPeak,raw

I also tried one with exact paths.

I came until this part:

But I cannot continue with normalizing. I am getting errors such as these:

dbObj <- dba.normalize(dbObj)
Error in sum(sapply(pv$peaks, nrow)) : invalid argument 'type' (list)

I am literally desperate. If you can help me I will more than appreciate. Thank you.

Answer 1

[Rory Stark]

If your peaks are in narrowPeak format, it may help to set the PeakCaller to "narrow".

[buffealo]

Thank you so much. Which metadata format should I proceed with, you think?

[buffealo]

Rory Stark, I tried several variations, and I am getting these errors. These errors also occurred when I tried with the metadata files I wrote above. I have proper index .bai files, and also, my reads are paired-end.

> myc <- dba.analyze("myc_chip_metadata_rory.csv")
Loading sample sheet...
M7_1 MCF7 MYC ER+ Non 1 narrow
M7_2 MCF7 MYC ER+ Non 2 narrow
M7_3 MCF7 MYC ER+ Non 3 narrow
T4_1 T47D MYC ER+ Non 1 narrow
T4_2 T47D MYC ER+ Non 2 narrow
T4_3 T47D MYC ER+ Non 3 narrow
M231_1 MDAMB231 MYC TNBC Non 1 narrow
M231_2 MDAMB231 MYC TNBC Non 2 narrow
M231_3 MDAMB231 MYC TNBC Non 3 narrow
BT_1 BT549 MYC TNBC Non 1 narrow
BT_2 BT549 MYC TNBC Non 2 narrow
BT_3 BT549 MYC TNBC Non 3 narrow
Applying Blacklist/Greylists...
Genome detected: Hsapiens.UCSC.hg38
Applying blacklist...
Blacklist error: Error in h(simpleError(msg, call)): error in evaluating the argument 'i' in selecting a method for function '[': 'q_groups' must be a     CompressedIntegerList object
Unable to apply Blacklist/Greylist.

> myc
12 Samples, 115749 sites in matrix (171059 total):
       ID   Tissue Factor Condition Treatment Replicate Intervals
1    M7_1     MCF7    MYC       ER+       Non         1     79308
2    M7_2     MCF7    MYC       ER+       Non         2     76243
3    M7_3     MCF7    MYC       ER+       Non         3     83461
4    T4_1     T47D    MYC       ER+       Non         1      5966
5    T4_2     T47D    MYC       ER+       Non         2     24230
6    T4_3     T47D    MYC       ER+       Non         3        26
7  M231_1 MDAMB231    MYC      TNBC       Non         1     62073
8  M231_2 MDAMB231    MYC      TNBC       Non         2     80625
9  M231_3 MDAMB231    MYC      TNBC       Non         3     40988
10   BT_1    BT549    MYC      TNBC       Non         1     57094
11   BT_2    BT549    MYC      TNBC       Non         2     50309
12   BT_3    BT549    MYC      TNBC       Non         3     43601

> myc <- dba(sampleSheet = "myc_chip_metadata_rory.csv")
M7_1 MCF7 MYC ER+ Non 1 narrow
M7_2 MCF7 MYC ER+ Non 2 narrow
M7_3 MCF7 MYC ER+ Non 3 narrow
T4_1 T47D MYC ER+ Non 1 narrow
T4_2 T47D MYC ER+ Non 2 narrow
T4_3 T47D MYC ER+ Non 3 narrow
M231_1 MDAMB231 MYC TNBC Non 1 narrow
M231_2 MDAMB231 MYC TNBC Non 2 narrow
M231_3 MDAMB231 MYC TNBC Non 3 narrow
BT_1 BT549 MYC TNBC Non 1 narrow
BT_2 BT549 MYC TNBC Non 2 narrow
BT_3 BT549 MYC TNBC Non 3 narrow

> myc <- dba.count(myc)
Computing summits...
Re-centering peaks...
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Hata: Error processing one or more read files. Check warnings().
In addition: There were 17 warnings (use warnings() to see them)

> warnings()
Warning messages:
1: In mclapply(arglist, fn, ..., mc.preschedule = TRUE, mc.allow.recursive = TRUE) :
  all scheduled cores encountered errors in user code
2:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
3:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
4:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
5:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
6:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
7:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
8:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
9:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
10:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
11:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
12:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
13:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
14:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
15:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
16:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
17:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors



> library(BiocParallel)
> register(SerialParam())
> myc <- dba(sampleSheet = samples)   
M7_1 MCF7 MYC ER+ Non 1 narrow
M7_2 MCF7 MYC ER+ Non 2 narrow
M7_3 MCF7 MYC ER+ Non 3 narrow
T4_1 T47D MYC ER+ Non 1 narrow
T4_2 T47D MYC ER+ Non 2 narrow
T4_3 T47D MYC ER+ Non 3 narrow
M231_1 MDAMB231 MYC TNBC Non 1 narrow
M231_2 MDAMB231 MYC TNBC Non 2 narrow
M231_3 MDAMB231 MYC TNBC Non 3 narrow
BT_1 BT549 MYC TNBC Non 1 narrow
BT_2 BT549 MYC TNBC Non 2 narrow
BT_3 BT549 MYC TNBC Non 3 narrow


> myc <- dba.count(myc)
Computing summits...
Re-centering peaks...
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Reads will be counted as Paired-end.
Hata: Error processing one or more read files. Check warnings().
In addition: There were 17 warnings (use warnings() to see them)

> warnings()
Warning messages:
1: In mclapply(arglist, fn, ..., mc.preschedule = TRUE, mc.allow.recursive = TRUE) :
  all scheduled cores encountered errors in user code
2:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
3:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
4:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
5:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
6:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
7:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
8:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
9:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
10:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
11:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
12:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
13:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
14:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
15:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
16:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors
17:   error in evaluating the argument 'x' in selecting a method for function 'assay': BiocParallel errors