how to interpret V-plot

[wanluliu]

Hi Alicia,

I'm trying your nucleoATAC (thank you so much for make/maintain nucleoATAC so easy to use for biologist) for some of our ATACseq data from arabidopsis and I have two questions regarding the interpretation of the Vplot from nucleoATAC. The fragment size distribution we got from ATACseq in is somehow hard to see the 'nucleosome bump'.

While I try nucleoATAC, I got really nice V-plot from those exact libraries as shown below.

Since I'm really interested in nucleosome pattern through ATACseq, I'm wondering what would be your interpretation from those data? Do you think we indeed capture some nucleosome signal from our ATACseq library (even we didn't see the nucleosome bump from fragment size distribution?) that we may conclude something from it?

A second question is that we did ATACseq from wildtype and some mutants showed decompaction of heterochromatin (evidence from immunostaining and Hi-C), I saw from the Vplot that the pattern is somewhat 'fuzzy' in the mutant. But I'm not sure whether this is the correct way to interpret the data and if you could give some suggestions that would be awesome!

Looking forward to hear from you!
wanlu

[AliciaSchep]

If the fragment distribution does not show evidence of a nucleosome I would advise against using NucleoATAC or attempting to infer nucleosome positions...

(Re specific question about vplot, the outputs just show the result of the fragment size normalization for the core vplot used by nucleoatac -- meant primarily for QA purposes and not particularly meaningful on their own)

[Baharehh]

hi Alicia,
along the same line, my fragment size distribution looks like a single peak from 50 bp to 350 bp with a peak in ~140 pb. Thus, there is not a secondary peak. we fixed and transposed the cells so i think that caused a little bit of over transposition. However, 50K peaks were reasonably looking good and they were close to TSS. my TSS score was also decent.

question is : what would you advise to use from your program? do you think it is reasonable to look into NFRs? I have ton of 150 bp DNA fragments ( and many peaks around that sizes, if we assume they are correlated).
But I think the nucleosome phasing probably is lost, correct?

Thank you for your kind help
April