The final project will ask you to develop and implement a full bioinformatics pipeline for a real-world dataset, focusing on the prinicples of reproducibility and portability we’ve discussed throughout the semester.

You will have your choice of two options, the first being a publication focused on chromatin accessiblity as assayed by ATACseq. You will be asked to generate an end-to-end pipeline for experiment and replicate some of their key figures.

You may also choose to analyze a dataset of your own choosing, provided it meets a few basic requirements:

  1. It must be a dataset that has been published in a peer-reviewed journal
  2. You must have access to the raw data
  3. You must meet with me to discuss your project and get approval before proceeding
    • This is largely to ensure that the work will be feasible and that you will be doing roughly the same amount of work as the ATACseq project.

You will be given a rough outline of the steps your workflow should perform as well as a list of deliverables to generate. When in doubt, you may refer back to the original publication for additional details or guidance on specific steps.

Universal Guidelines

No matter which project you choose, you will be asked to adhere to several guidelines following practices we’ve developed over the semester.

Workflow Manager

  1. You must use Nextflow to generate a working pipeline for your project. You should use the same structure as each of the previous projects.

  2. Each of your processes must be split into separate modules

  3. Your workflow should be driven by either a samplesheet.csv or a directory of files using the Channel.fromFilePairs function. It should download the files as necessary.

Environment Management

You must use either conda or singularity to manage your environments.

  1. If you choose to use conda:
    • Each tool should have its own conda environment and YML file
    • You should pin the most recent version of each tool
  2. If you choose to use singularity:
    • Each tool should have its own singularity image
    • Containers should be from the class repo or another vetted source (biocontainers, etc.)

Github Repository

Your entire workflow must be stored in a github repository provided to you by the GitHub Classroom link for the final project. Your repo should not contain any data or large files, but your entire workflow and analysis code. Any analysis code should be contained within either a Rmarkdown or Jupyter notebook.

Your repo must contain a README.md file with the following information:

  • A description of the source of the data and publication
  • How to run your pipeline
  • The deliverables required for the project

Project 1: ATAC-seq and Differential Chromatin Accessibility

The publication for this project is here: https://pubmed.ncbi.nlm.nih.gov/38829740/

You only need to process the ATACseq data

Your workflow at a high-level should do the following:

  1. Download the data from the publication
  2. Sequence quality control on the raw reads
  3. Adapter trimming (if necessary) and quality filtering
  4. Alignment to the reference genome using ATACseq specific parameters
  5. Remove any alignments aligning to mitochondrial DNA
  6. Correct the alignemnts by shifting +4/-5 for the positive and negative strand respectively 7.OPTIONAL Split your dataset into the nucleosome-free (NFR) and nucleosome-bound (NBR) fractions. If you choose to do this, please justify why and make it clear which fraction you are working with for each subsequent analysis.
  7. Peak calling
  8. Differential chromatin accessibility analysis
  9. Annotation of differentially accessible peaks
  10. Motif finding of differentially accessible peaks
  11. Reproduce figures 6a-6f (it’s only three distinct figures from two conditions)

Deliverables

Your project should consist of:

  1. A Nextflow workflow that implements the above steps in your github classroom repository
  2. A README.md file in your github classroom repository that describes how to run your pipeline
  3. A Jupyter notebook or Rmarkdown file that contains the analysis code
  4. A report that addresses the deliverables outlined below.

The specific deliverables pertaining to the results for this project that should be included in your report are:

  1. A brief discussion (1 paragraph) of the sequencing quality control results and any steps taken to address any identified issues
  2. A brief discussion (1 paragraph) of the alignment statistics, and what they mean for your analysis
  3. You must produce two ATAC-seq specific QC metrics from the following list:
  • TSS enrichment score
  • Fragment Size Distribution
  • Heatmap of signal across TSS split between NFR and NBR
  • Fraction of Reads in Peak (FRiP)

Comment, in no less than a paragraph, on each of your chosen two ATAC-seq QC metrics and what they mean about the success of the experiment.

  1. Report how many differentially accessible regions your pipeline discovered in each of the two conditions.

  2. A figure showing the enrichment results of the differentially accessible regions and a few sentences describing what the enrichment reveals.

  3. A figure showing the motif enrichment results from the differential peaks and a few sentences describing the key motifs found.

  4. Comment on the success of the reproductions of the panels from the original publication. Do you think the results are consistent with the original publication? What do your results show that is different from the original publication?

Project 2: Your Choice

If choosing your own project, your workflow steps will look different and you should determine the steps necessary to generate the results you want. You can of course refer to the methods found in the original publication for guidance.

Deliverables

Your project should consist of:

  1. A Nextflow workflow that implements the above steps in your github classroom repository
  2. A README.md file in your github classroom repository that describes how to run your pipeline
  3. A Jupyter notebook or Rmarkdown file that contains the analysis code
  4. A report that addresses the deliverables outlined in the Project Report Guidelines

The specific deliverables pertaining to the results for this project that should be included in your report are:

  1. A brief discussion (1 paragraph) of the sequencing quality control results and any steps taken to address any identified issues
  2. A brief discussion (1 paragraph) of the alignment statistics, and what they mean for your analysis
  3. You must produce two experiment specific QC metrics
    • Alignment Statistics
    • Sequence Quality Control
    • Experiment-specific metrics (fingerprint plot, sample-sample distance matrix, etc.)

Comment, in no less than a paragraph, on each of your chosen two QC metrics and what they mean about the success of the experiment.

  1. Comment on the success of the reproductions of the panels from the original publication. Do you think the results are consistent with the original publication? What do your results show that is different from the original publication?

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