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BF528 - Applications in Translational Bioinformatics

Week 4: ChIPseq

Week 4 Overview

For the final week, you will be reading the original paper and interpreting your results in the context of the publication’s results. Specifically, you will be focusing on reproducing the results shown in figure 2 with your own findings. This exercise is not meant to make any assertions as to the ground “truth” but to encourage you think about reproducibility in science.

Reminder

The tasks for this week will largely ask you to re-create the same figures found in the original publication with your own results. Remember that this is not meant to assert that one approach or one set of results are the “ground truth”. In science, we are constantly making assumptions and subjective choices, ideally based on sound logic and past knowledge, that will greatly impact the interpretation of the results we obtain. The purpose of this exercise is to explore the factors that contribute to reproducibility in bioinformatics and develop an understanding of what it means for an experiment or publication to be “reproducible”.

Objectives

  • Read the original publication and focus specifically on the results and discussion for figure 2

  • Reproduce figures 2D, 2E and 2F from the paper

  • Compare other key findings to the original publication

Read the original paper

The original publication has been posted on blackboard. Please read through the paper with a particular focus on the ChIP-Seq experiment presented in figure 2.

Write a methods section

Using the style and guidelines discussed in class, write a methods section that describes the analysis your nextflow pipeline performs.

Overlap your ChIPseq results with the original RNAseq data

In their publication find the link to their GEO submission. Read the methods section of the paper and integrate your called ChIPSeq peaks with the results from their differential expression RNAseq experiment. Use your set of reproducible and filtered peaks, and use the publication’s listed significance thresholds for the RNAseq results.

  1. Create a figure that displays the same information of figure 2F from the original publication using your annotated peaks and the RNAseq results. The figure does not have to be the same style but must convey the same information using your results.

  2. In figures 2D and 2E, the authors identify and highlight two specific genes that were identified in both experiments. Using your list of filtered and reproducible peaks, a genome browser of your choice, and your bigWig files, please re-create these figures with your own results (You do not need to include the RNAseq data, but you should re-create the genomic tracks from your ChIPseq results)

  3. In your provided notebook, please ensure you address the following questions:

  4. Focusing on your results for figure 2F: - Do you observe any differences in the number of overlapping genes from both analyses? - If you do observe a difference, explain at least two factors that may have contributed to these differences. - What is the rationale behind combining these two analyses in this way? What additional conclusions is it supposed to enable you to draw?

  5. Focusing on your results for figures 2D and 2E: - From your annotated peaks, do you observe statistically significant peaks in these same two genes? - How similar do your genomic tracks appear to those in the paper? If you observe any differences, comment briefly on why there may be discrepancies.

Comparing key findings to the original paper

Find the supplementary information for the publication and focus on supplementary figure S2A, S2B, and S2C.

  1. Re-create the table found in supplementary figure S2A. Compare the results with your own findings. Address the following questions:
  • Do you observe differences in the reported number of raw and mapped reads?
  • If so, provide at least two explanations for the discrepancies.
  1. Compare your correlation plot with the one found in supplementary figure S2B.
  • Do you observe any differences in your calculated metrics?
  • What was the author’s takeaway from this figure? What is your conclusion from this figure regarding the success of the experiment?
  1. Create a venn diagram with the same information as found in figure S2C.
  • Do you observe any differences in your results compared to what you see?
  • If so, provide at least two explanations for the discrepancies in the number of called peaks.

Week 4 Detailed Tasks Summary

  • Read the original publication with a particular focus on figure 2

  • Write a methods section for the complete analysis workflow implemented by your pipeline while adhering to the guidelines and style discussed in class

  • Download the original publication’s RNAseq results and apply their listed significance threshold. Use this information to re-create figure 2F.

  • Re-create figures 2D and 2E and ensure you address the listed questions

  • Find supplementary figure S2 and re-create or compare your findings to supplementary figures 2A, 2B and 2C. Ensure you address any listed questions.