Error with own regions

[mpaya]

I'm using a genome that is neither in ensembl nor UCSC, so I made up a -G value and transformed my gff genes into bed format (chrom_name start end gene_name score strand). The documentation does not include many examples for these cases, so I'll indicate here my tries. I have two tissues that I want to plot together. One has duplicates, the other doesn't. The bam files were processed with Picard MarkDuplicates without removal.

1. First, create configuration files to include paths to both tissues specifying that I want all regions plot on each of them.

../../ChIP_leaf/bam_files/2_filt-markdup_C1.bam:../../ChIP_leaf/bam_files/2_filt-markdup_C3.bam	-1	"Leaves"
../..ChIP_FL/bams/2_filt-markdup_FL_C1.bam	-1	"Flowers"

Then, run ngs.plot.r with my made-up genome name, specifying my bed file with -E, the above config file and other parameters. It does not work.

ngs.plot.r -G Bra3.0 -R bed -E gene_lists/Bra_3.0_genes.bed -C config_both.txt -O both -P 6 -FL 300 -IN 1 

Configuring variables...Error in file(file, "rt") : cannot open the connection
Calls: SetupPlotCoord -> ReadBedCoord -> read.table -> file
In addition: Warning messages:
1: In ReadBedCoord(ur) : File name: '-1' does not seem to a correct name 
for bed file.

2: In file(file, "rt") : cannot open file '-1': No such file or directory
Execution halted

2. I modify my config files and indicate my bed file inside instead of '-1'.

ngs.plot.r -G Bra3.0 -R bed  -C config_both.txt -O both -P 6 -FL 300 -IN 1
Configuring variables...Done
Loading R libraries.....Done
Analyze bam files and calculate coverageError in bamFileList(ctg.tbl) : 
  No mix of bam and bam-pair allowed in configuration.
Execution halted

3. Modify the config and duplicate the single file. All runs like normal. I get negative fold changes for the duplicates and a flat line for the doubled file. Heatmaps are blank.

4. Merge bam files (MergeSamFiles tool from Galaxy) of duplicates and run pairs of ChIP:input. Here, leaves (merged files) have significantly higher values than flowers (single sample). Still, no heatmap.

../../ChIP_leaf/bam_files/2_filt-markdup_ChIP.bam:../../ChIP_leaf/bam_files/2_filt-markdup_INPUT.bam	gene_lists/Bra_3.0_genes.bed	"Leaves"
../../ChIP_FL/bams/2_filt-markdup_FL_C1.bam:../../ChIP_FL/bams/2_filt-markdup_FL_I1.bam	gene_lists/Bra_3.0_genes.bed	"Flowers"

ngs.plot.r -G Bra3.0 -R bed -C config_both.txt -O both -P 6 -FL 300 -IN 1
Configuring variables...Done
Loading R libraries.....Done
Analyze bam files and calculate coverageWarning messages:
1: In headerIndexBam(bam.list) :
  Aligner for: ../../ChIP_leaf/bam_files/2_filt-markdup_ChIP.bam cannot be determined. Style of 
standard SAM mapping score will be used. Would you mind submitting an issue 
report to us on Github? This will benefit people using the same aligner.
2: In headerIndexBam(bam.list) :
  Aligner for: ../../ChIP_leaf/bam_files/2_filt-markdup_INPUT.bam cannot be determined. Style of 
standard SAM mapping score will be used. Would you mind submitting an issue 
report to us on Github? This will benefit people using the same aligner.
Warning message:
'isNotPrimaryRead' is deprecated.
Use 'isSecondaryAlignment' instead.
See help("Deprecated") 

My observations.

• On the documentation, the config file would contain '-1' to use the whole genome or gene lists to subset. But it seems not to occur with .bed option. Does a configuration file override -E and -T options? Should subsets of bed files be created for each gene list?
• Should all replicates be merged in a single file before plotting? Since bam-pair does not seem to be meant for duplicates.
• Are results from the duplicated-merged samples comparable to the single sample? Plotting each sample by separate suggests they are different and this result seems reliable.
• Why are my heatmaps empty? It's an issue with document viewer in Linux.

As a suggestion, adding some extra information on the documentation would help avoid these issues.