I am interested in plotting sequence read coverage around regions/sites of my interest using NGS.PLOT package. I use a single-nucleotide long bed entry-containing bed file and a sequence alignment (bam) file for plotting a heatmap using the following command:
ngs.plot.r -G hg19 -R bed -E my.bed -C sorted.bam -O test_ngsplot_at_bed_entries -T mybed -L 1000 -FL 10 -SS same -D refseq -P 10
The bed file (my.bed) is a 6-column bed file, and it contains roughly 14000 single-nucleotide-long bed entries. An example pattern of the bed entries are given below: chr1 12345 12346 NA 100 + chrX 12345 12346 NA 100 + . .
Unlike the heatmap plotted using the default TSS parameter (for -R flag), for my bed file, I get a weird-looking heatmap wherein the single-ntd-long bed entry is shown as an enlarged region, marked with 5' and 3' ends. The heatmap for TSS is here and for my.bed file is here. Please ignore the title of the image.
I want ngs.plot.r to generate a heatmap similar to the one generated at the default TSS sites. How can I direct ngs.plot to not to expand the bed entry on the plots? Do I have to use organize my bed file differently?
I have checked all the options associated with ngs.plot.r, but I did not find any that would fix solve my problem. Maybe, I am missing something.
Can someone help? Let me know if you need any other information.
Thanks!
DP
I suspect the issue here is caused by the fact that your bed file coordinates are only 1bp long. Looks like its stretching this 1bp region to take up most of the plot. I'm not sure how to fix it but try this: if you want to look at this region +- 1000bp, then edit the bed file to be the coordinates of this larger region, i.e. chr1 11345 13346..., then leave out the -L parameter. Might work?
A nice trick! I will give it a shot.