== Using ngsplot to make stacked heatmaps and profiles of genes or genomic regions (like ChIP-seq peaks) ==

[[https://github.com/shenlab-sinai/ngsplot/wiki | ngsplot]] is a great tool to make heatmaps and profiles of mapped reads (i.e. bam files of RNA-seq, ChIP-seq etc) across genes, transcription starts sites or other regions of interest.
The regions used can be genes included on the ngsplot annotation or custom regions included on a bed file. 

* Here is an example of a ngsplot command using custom regions

{{{
bsub ngs.plot.r -G hg19 -R bed -L 3000 -C config.txt -O Peak_Summits

using this example of the tab delimited config.txt file:

TF1.sorted.bam	  peaks.SUMMIT.bed	"TF1" 
TF2.sorted.bam	  peaks.SUMMIT.bed	"TF2" 
Pol2.sorted.bam   peaks.SUMMIT.bed	"Pol2" 
}}}

* Here is an example of a ngsplot command using gene annotations within the ngsplot package

{{{
# Using transcription start sites (tss) as the features of interest
bsub ngs.plot.r -G hg19 -R tss -L 3000 -C config.txt -O ngsplot_tss

# Using gene bodies as the features of interest
bsub ngs.plot.r -G hg19 -R genebody -L 3000 -C config.txt -O ngsplot_tss
# Note: ngsplot genebody does not include introns when specifying -R genebody
}}}


* For plotting RNA-Seq data with ngsplot, use ''-F rnaseq'' mode/option

ngsplot uses a two-step normalization: i) coverage vectors' length i.e., gene length and ii) library size.
See [[https://github.com/shenlab-sinai/ngsplot/wiki/HowAreYaxisValsCalculated | ngsplot: how the Y-axis values are calculated]].