Getting started

  1. Create an experiment.csv in the format below, including the header. DNA_F or RNA_F is name of the gzipped fastq of the forward read of the DNA or RNA from the defined condition and replicate. DNA_UMI or RNA_UMI is the corresponding index read with UMIs (excluding sample barcodes), and DNA_R or RNA_R of the reverse read.

    Multiple fastq files can be used for each column by seperating them with ;.

    Right now an UMI have to be used. If you want to use MPRAsnakeflow without an UMI please sitch to MPRAflow or contact us.

    Here is an example of an experiment.csv file and it can be downloaded experiment.csv:

  1. If you would like each insert to be colored based on different user-specified categories, such as positive control, negative control, shuffled control, and putative enhancer. To assess the overall quality, you can create a label.tsv in the format below that maps the name to category as shown here:

insert1_name label1
insert2_name label1
insert3_name label2

The insert names must exactly match the names in the design FASTA file.

  1. Set up the config file

The config file is the heart of MPRAsnakflow. Here different runs can be configured. We recommend to use one config file per MPRA experiment or MPRA roject. But in theory many different experiments can be configured in only one file. It is divided into global (generell settings), assignments (assigment workflow), and experiments (count workflow including variants).

See Config File for more details about the config file. Here is an example running only the count experiments and using a provided assignment file.

  1. Run MPRAsnakeflow

conda activate snakemake
snakemake --configfile config/config.yaml --use-conda -p --cores 4


This will run in local mode using 4 cores. Please submit this command to your cluster’s queue if you would like to run a highly parallelized version.

Be sure that the files, experiment.csv and the config.yaml are correct. All fastq files for the count/experiment part must be in the same folder given by the data_folder option. Please specify your barcode length and umi-length with bc_length and umi_length.

The count files generated by the count workflow, are named: <condition>_<replicate>.merged.config.<config>.tsv.gz and can be found in the results/experiments/<project>/assigned_counts/ folder inside the project folder.