Question: motif search with ATACseq
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gravatar for grant.hovhannisyan
4 days ago by
grant.hovhannisyan1.1k wrote:

I have ATACseq data for two yeast species. I have called peaks with MACS2 and did occupancy and affinity analysis of peaks with DiffBind. Now I need to find motifs of TF binding sites in the peaks and compare those motifs between two species.

There is a ton of softs and databases for doing the task, so I am a bit confused on how to start with the analysis. Can anybody share the experience with motif search and motif comparisons, specifically which tools are considered as "best practices" in the field?

Thanks

motif search atacseq • 140 views
ADD COMMENTlink modified 3 days ago by Charles Warden5.4k • written 4 days ago by grant.hovhannisyan1.1k

What exactly are you confused about? You imply you've already done some research on how to do this, so it's difficult to figure out how we could help you. Have you tried any of the "tons of softs and databases" and found them lacking?

ADD REPLYlink written 3 days ago by Friederike2.3k
3
gravatar for Devon Ryan
3 days ago by
Devon Ryan85k
Freiburg, Germany
Devon Ryan85k wrote:

There are basically 2 genres of tools that one can use: those that search for motifs in peaks and those that do footprinting. The former group are well represented by homer and the meme suite (MEME-ChIP in particular). The latter group is mostly represented by wellington. I'm generally not a fan of footprinting with ATAC-seq data, the coverage needed to do it properly is just absurdly high. Given that, one of homer/meme/etc. would be my preference. I generally find homer to be annoying to use, so I personally prefer MEME, but that's more of a personal preference than a best practice.

ADD COMMENTlink written 3 days ago by Devon Ryan85k

Thanks for input Devon! Regarding footprinting, do you think its fine to merge the ATACseq replicates to increase the depth (replicates are very reproducible)? Regarding the motifs, say I have found the motifs in the peaks, what kind of comparison between motifs of different species do you think can be done? Sequence comparison, copy number comparison, others? I am new to this field and want to conceptually understand what makes sense and what does not. Thank you

ADD REPLYlink written 3 days ago by grant.hovhannisyan1.1k

Sure, you can merge replicates. Regarding the comparisons that make sense, that depends on the biological question. Note that comparing across species is rife with issues.

ADD REPLYlink written 3 days ago by Devon Ryan85k
1

I apologize for interrupting another tread, but I would usually encourage the use of replicates.

While Devon is right that the number of reads per ATAC-Seq sample can sometimes be high, if you already have replicates with high-coverage samples, I think you should take advantage of that.

Also, with yeast, getting high coverage should be less of an issue than an organism with a larger genome, such as human or mouse (I am assuming you are studying a species of yeast with limited introns, but I admittedly don't know how the largest yeast genome compares to a vertebrate genome).

ADD REPLYlink written 3 days ago by Charles Warden5.4k

Hi Charles, the genome size is ca 12Mb and the ATACseq reps have around 20mln reads each (the coverage is much higher compared to human genome, though I don't really get what coverage in ATACseq context means). Regarding the replicates, for example I have used them in occupancy analysis with DiffBind, and most of the peaks are shared between the replicates. So I can try maybe merging the reps and focus only on those common peaks.

ADD REPLYlink written 2 days ago by grant.hovhannisyan1.1k

Note that comparing across species is rife with issues.

Yes, especially when they have 30% of genome divergence:)

ADD REPLYlink modified 2 days ago • written 2 days ago by grant.hovhannisyan1.1k
1
gravatar for Charles Warden
3 days ago by
Charles Warden5.4k
Duarte, CA
Charles Warden5.4k wrote:

You might want to take a look at this post: A: How can I find motifs under individual ATAC-peaks?

However, i-cisTarget doesn't have yeast annotations (at least as far as I can tell).

For general peak enrichment, I think the species is also a limitation for Broad-Enrich or GREAT, but perhaps you can look at citations for papers (or the papers themselves) to get some other ideas (but maybe that is a little off target from your motif question).

ADD COMMENTlink written 3 days ago by Charles Warden5.4k

Thank you Charles, very useful! One of the species I analyze is non-model, so I guess indeed there will be limitations. So either I will need to do motif discovery, or search based S. cerevisiae motifs.

ADD REPLYlink written 3 days ago by grant.hovhannisyan1.1k
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