Questions on PlotAggregate

Hello, 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 and conditionB.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 and uniq_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:

1. 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?
2. 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
3. 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

1. 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:

2. in PlotAggregate method, What is the difference between these two arguments: --regions and --whitelist

Hope you can give me some advice, thanks.

[msbentsen]

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.

[hero-outman]

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