PM4NGS RNASeq Tutorial

• 1. RNA-seq of the lobe-less (lol) mutants in Drosophila melanogaster
  • 1.1. BioProject
  • 1.2. SRA
• 2. GCP Instance Setup
  • 2.1. Create a GCP instance
  • 2.2. Accessing the instance with SSH
  • 2.3. Installing dependencies on the GCP instance with Ubuntu
  • 2.4. Testing the Docker daemon
• 3. Installing PM4NGS
  • 3.1. What is a Python virtual environment?
  • 3.2. Installing PM4NGS
  • 3.3. Testing PM4NGS
• 4. The Jupyter Server
  • 4.1. Accessing the instance with SSH
  • 4.2. Activating pm4ngs_venv virtual environment
  • 4.3. Running the Jupyter Server
  • 4.4. Open the jupyter web interface in your local computer
• 5. PM4NGS for RNA-Seq data analysis
  • 5.1. Sample sheet
  • 5.2. Creating the PM4NGS RNA-Seq project
  • 5.3. Pre-processing QC
  • 5.4. Samples trimming
  • 5.5. Alignment and Quantification
  • 5.6. Differential Gene Expression Analysis
    • 5.6.1. Volcano Plots
    • 5.6.2. Sample correlation
    • 5.6.3. Expression correlation
    • 5.6.4. PCA plots
    • 5.6.5. List of differentially expressed genes
  • 5.7. Gene Ontology Enrichment

Introduction

This tutorial will show how to create and configure a Google Cloud Platform instance for Next Generation Sequencing (NGS) data analysis. PM4NGS is a program manager for NGS data analysis that is designed to generate a standard organizational structure including directory structures for the project, Jupyter notebooks for data management and CWL workflows for the pipeline execution.

Background reading

1. Vera Alvarez R, Pongor LS, Mariño-Ramírez L and Landsman D. PM4NGS, a project management framework for next-generation sequencing data analysis, GigaScience, Volume 10, Issue 1, January 2021, giaa141, https://doi.org/10.1093/gigascience/giaa141

2. Vera Alvarez R, Mariño-Ramírez L and Landsman D. Transcriptome annotation in the cloud: complexity, best practices, and cost, GigaScience, Volume 10, Issue 2, February 2021, giaa163, https://doi.org/10.1093/gigascience/giaa163

Help and Support

For feature requests or bug reports, please open an issue on our GitHub Repository.