Questions on PlotAggregate
See original GitHub issueHello, msbentsen, I have some questions when using TOBIAS for my ATAC-seq data analysis, I read the docs on TOBIAS Github, but still confused. My atac-seq analysis propose is finding different TFBS motifs of A549 cell line between conditionA and conditionB; And my analysis route like:
- after alignment, i use macs2 for peak calling and got
conditionA.narrowpeak
andconditionB.narrowpeak
(they are whole open site of each conditon); - then performed diff-peak analysis, then got peaks of unique open region of each condition,
uniq_open_in_A.bed
anduniq_open_in_B.bed
(they are sub-set of conditionA.narrowpeak and conditionB.narrowpeak); - then use homer for motif enrichment on those unique open regions, and got interested motifs of each condition; till here, i got diff chromatin open regions uniq_open_in_A.bed and uniq_open_in_B.bed and motif on those region;
Next, I use TOBIAS for TF footprint analysis; after ATACorrect --> ScoreBigwig --> BINDetect --> PlotAggregate
, I got my results, seems not the expected one(no obviously footprint on the aggregate plot on most enriched motif). Here are my aggregate plots:
compare to your example pic, it’s quite a different:
Also, I made an aggregate plot between uncorrected and corrected, still not expected(the curve shape of uncorrected and corrected are very same, just one position higher than another on the Y-axis). Here’s the pic:
I tried for couple of days, still cannot get a good plot on each condition.
So I will ask some question based on the information above:
- The result of scorebigwig(footprint.bw) and bindetect(TFBS.bed files) fit my expected result. But why aggregate-plot won’t show a ‘good-shaped’ footprint plot? If aggregate-plot is not good, can I still rely on footprint.bw and TFBS.bed files?
- I have conditionA and B .narrowpaek for the whole peak of two conditions, also uniq_open_A and B .bed for unique open regions(a subset of the whole peak), which kind of peak should I use in:
- ATACorrect --peaks
- ScoreBigwig --regions
- BINDetect --peaks I tried both(whole peak and unique open peak), but cannot get a ‘good’ aggregate plot
- In aggregate plot: the plot curve between corrected.bw and uncorrected.bw, why their shape is so same, and just different position on Y-axis(not like figures in TOBIAS paper and GitHub), does this mean my correction won’t work? Please see pics above; My ATACorrect code is
TOBIAS ATACorrect \
--bam conditionA.bam \
--genome $hg38_path \
--peaks uniq_open_A.bed \
--blacklist $blacklist_path \
--outdir ATACorrect_out \
--cores 20
-
When plotting aggregate footprint, I try to use --smooth 3 or 5, with this param, I can get a ‘better’ footprint plot, does this try make sense? Here is the smoothed pic:
-
in
PlotAggregate
method, What is the difference between these two arguments:--regions
and--whitelist
Hope you can give me some advice, thanks.
Issue Analytics
- State:
- Created 2 years ago
- Comments:10 (5 by maintainers)
Top GitHub Comments
Great, I’m glad we got it solved in the end! I’m closing this issue but feel free to open another in case of other questions.
Hi msbentsen:
I think you are right, it’s my hg38 issue. I changed hg38.fa and performed ATACorrect again, got the clearly Tn5 motif.
Thank you so much for the patience help, and may the force be with you!
Best Chu