Shalek Lab How Tos

Introduction

• Welcome to the HowTos wiki!
  • This wiki is maintained by the whole lab, please make changes/updates if you see incorrect or outdated information.
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Terra

• How to for new Terra Users
  • What is Terra:
  • What are things I can do on Terra?
  • Basics of Running a Workflow
    • 1. Sign In
    • 2. Create a New Workspace.
    • 3. Add Your Sequence Data and Input CSV File
    • 4. Import a Workflow
    • 5. Configure and Launch a Workflow
    • 6. Advice for Troubleshooting
    • 7. Clean Up
• How do I use Terra Pipelines?
  • Basics
  • Data Model
  • Troubleshooting
• How do I use Terra Notebooks?
  • Do I even need to use Terra Notebooks?
  • What is a Terra compute environment?
  • Creating a basic Seurat v3 environment
  • Moving Data
  • Installing Extra Packages
  • Installing Extra Packages with root permissions
• Drop Seq Tools on Terra
• Output files:
• Troubleshooting
• SeqWell Demultiplexing on Terra
• Workflow
  • About
  • Why run Kallisto-Bustools on the cloud?
  • Writing your sample sheet
  • Configuring the workflow
  • Inputs / Outputs
• Notebook

Data Analysis

• 1. Reading in the data (to R)
• 2. Dropout?
• 3. Filtering
  • 3.1. Why threshold
  • 3.2. What to threshold on
  • 3.3. Examples of bad thresholding (removing biological variation):
• 4. Doublets
• 5. Normalization
  • 5.1. Normalizing pops
• 6. Choosing variable genes
  • 6.1. Choosing variable genes is an iterative process
  • 6.2. If removing genes makes it harder to find some heterogeneity, why remove any at all?
  • 6.3. But it looks like Seurat is filtering on dispersion, not variance?
  • 6.4. Why does Seurat include a threshold for max mean and max dispersion?
• 7. PCA
• 8. Other dimensionality reduction
• 9. Batch Correction
  • 9.1. Which should I use?
• 10. Imputation
• 11. Clustering
• 12. How do I know I have a good clustering?
• 13. What if I am looking for rare cell subsets?
• 14. What if some cells are clearly placed in the wrong clusters?
• 15. Differential Expression
  • 15.1. Some guiding principles:
  • 15.2. Spillover genes
• 16. Cell Ordering (“Pseudotime”)
  • 16.1. Once I have a trajectory what do I do?
• 17. Network Inference
• 18. I have a bunch of genes now what?
  • 18.1. Databases of gene lists and cell types:
• 19. Meta analysis