Differential Expression Pipeline

New snakemake-based version of IMGAG pipeline, developed by Jakob

Install packages from Renv

Before running the pipeline you need to install the R packages. Use this approach:

• Run snakemake pipeline with conda-environments. You will get an error in rule diff_expr. From the error log, take the location of the R conda environment
• Activate the R conda environment
• Start R and install the missing packages

R
renv::install()

Now the dependencies should be installed, you need to repeat this step when the pipeline is run in a new conda environment.

How to use:

1) Setup project

Export metadata from NGSD into the project folder

python3 expression-analysis-smk/scripts/ngsd-project-metadata.py | cut -f1,5,6 > metadata.tsv

If folder name is not the project name, it can be given as parameter

python3 expression-analysis-smk/scripts/ngsd-project-metadata.py  '220100_My_Project` | cut -f1,5,6 > metadata.tsv

Update the group column in the metadata.tsv file and add additional columns for additional factors used in the analysis (age, sex, treatment ..)

Setup the config file

Analysis parameters are set in the config.yml file. Look in the templates/config.yml for default values and available options. Important settings are:

• Reference genome
• File names
• Filter thresholds
• Model and factor names
• Highlighted genes
• Colouring options

Setup the contrasts file

The contrasts file defines the group comparisons calculate in the DGE analysis. Its a tab separated text file with a row for each contrast and the following columns:

• name Appears as name of the sheet in the Excel results
• title Title of Comparison, can be multiple words
• description Subtitle with description of the comparison, free text
• coefficients Defines which groups should be compared. Format: groupwt-groupko The group of the first row is implied and can be omitted. Example: if the group in first row is wt, it would enough to write groupko in the coefficients field.

Run the pipeline

snakemake --profile local -s /mnt/users/ahgrosc1/pipeline/expression-analysis-smk/workflow/Snakefile --cores 5

Available reference builds

• Human build 38 (grch38)
• Human build 37 (grch37)
• Mouse (grcm38)
• Rat (rnor6)
• Chicken (galgal5)
• Worm (wbcel235)
• Fly (bdgp6)
• Macaque (mmul801)

Annotation based on the annotables package.