How to make a TSS enrichment plot in ATAC-seq analysis?

Question

[Dan]

How to make a TSS enrichment plot in ATAC-seq analysis, which shows the TSS enrichment of nucleosome-free fragments, mono-nucleosome fragments, and di-nucleosome fragments?

I see a tutorial (https://rockefelleruniversity.github.io/RU_ATAC_Workshop.html) about TSS enrichment plot, which uses soGGi::plotRegion function, I am wondering what is meaning of score in the plot? How should I normalize the score value based on the total reads?

Thanks

[sp]

Hi Dan,

I was wondering if you found out how to make the same graph with NFR and NBR regions with different curves. I was able to recreate the plot using plotProfile (Deeptools), but I can't seem to find an option to highlight those regions in computeMatrix or plotProfile. Unsure as to how to proceed. My image is below for reference:

Answer 1

[Trivas]

Not sure what the Score is actually quantifying, but you can do a simple min-max normalization to force the score values to be between 0 and 1 (ie fraction of signal). This looks like a glorified wrapped of the rtracklayer and GViz packages.

Truthfully, I found using the Deeptools suite really easy. Customization might be a little trickier, but it was super fast and the raw data is output so you can bring that into R and plot using ggplot if that's more your speed.

[Dan]

I sorted bam files to get the open region reads with insert size less than 100bp, then use bamCoverage --normalizeUsing RPGC to get the .bw file and use macs2 callpeak to get the .bed file, then run computeMatrix and plotHeatmap of Deeptools to get the TSS enrichment plot. Is this correct? What is the meaning of the score here? Thanks.