Motif Analysis

Written by Mea Casey, PhD

A step-by-step guide for using Pluto's Motif analysis to understand regulatory elements and transcription factors binding to your peaks of interest

Motif analysis enables researchers to identify and investigate sequence patterns that are overrepresented in peaks called from epigenetic experiments. These sequence patterns are the regulatory language that give insight into how and when genes are turned on and off.

In drug discovery, determining underlying sequences for transcription factor binding sites is critical for 1) identifying therapeutic targets, 2) characterizing a drug's mechanism of action, and 3) developing personalized medicine.

Motif analysis is available in the Pluto platform for ATAC-seq, ChIP-seq, and CUT&RUN experiments.

Overview

Motif analysis allows you to determine which transcription factors are binding to your regions of interest, giving you insight into the mechanisms driving regulatory differences across your samples.

Step 1: Set up & run your analysis

Navigate to the analysis catalog in your experiment by clicking on the ‘+ Analysis’ button. Select Motif analysis and click on ‘+ Create Analysis’ to start setting parameters for this analysis. You will have the option to select between Sample peaks, Consensus peaks, or Differential peaks. Your scientific question will drive which of these options you select at first. 

Do my data outputs look the way I expect them to?

If you are interested in identifying transcription factors active in a specific sample (for example, comparing one treatment sample against another as background), select Sample peaks for your Motif analysis. Once selected, you will choose your target sample and optionally a background sample. 

What are the primary binding motifs for transcription factors found across either of my conditions?

If you are interested in identifying transcription factors active across your entire experiment, using peaks that were consistently detected across your samples, select Consensus peaks for your Motif analysis. Once selected, you will have the option to set region size, motif set (e.g., vertebrates, insects, etc.), and choose between GC% or CpG% content bias correction. If repetitive elements are a potential concern with your organism, you may also choose to enable masking before running the analysis.

What transcription factors are driving the regulatory differences between my conditions?

If you are interested in understanding what transcription factors may be driving the differences between your conditions, select Differential peaks. This runs motif analysis on peaks that are significantly more accessible or enriched in one condition versus another, helping you identify the regulatory mechanisms underlying your differential binding results. Once selected, you will choose the specific comparison of interest. In addition to the parameters available for Consensus peaks, you will have added control over thresholding — similar to what is available in other analyses in Pluto.

Step 2: Customize your plot

Once your analysis is done running, you will be able to change your plot type. Typically, Motif analysis results are viewed in a Sequence Logo. However, Pluto also introduces an Enrichment Scatter Plot to streamline the process of going from motif interpretation to hypothesis generation.

Sequence Logo

DNA binding patterns of all motifs are represented as a position-specific probability plot, with taller positions signaling more conserved nucleotides which are critical for transcription factor recognition. In your plot settings, you can toggle between known motifs or de novo motifs. While the nucleotides are typically viewed with the standard color scheme (green, blue, yellow, red), Pluto enables you to apply your own color choices along with adding plot titles and subtitles to make this exportable file publication-ready.

Enrichment Scatter Plot

Interested in viewing multiple motifs at once? Pluto introduces the Enrichment Scatter Plot, which provides an overview of all motifs with log2 enrichment on the x-axis and log10(p-value) on the y-axis. This means that the candidates with the highest enrichment and highest significance are located in the top right. The sequence logo for any dot is available by selecting the point(s) of interest. 

🚀 Ready to uncover the transcription factors driving your biology?

Try it out for yourself by navigating to any ATAC-seq, ChIP-seq, or CUT&RUN experiment in Pluto and selecting Motif analysis in the analysis catalog. Happy analyzing!

For more resources, we encourage you to take a look at our Blog and Knowledge Base. Please reach out to support@pluto.bio if you have additional questions.

As always, our scientific support team is here to help!