Saturation Mutagenesis-Reinforced Functional Assays (SMuRF) is an accessible workflow for deep mutational scanning (DMS) studies. The manuscript is currently available on BioRxiv. This workflow involves a 2-way extension PCR-based saturation mutagenesis and high-throughput functional assays that can be evaluated with short-read Next-generation sequencing.

We have organized scripts used for SMuRF across three repositories:

• Balthazar repository is used to create the oligo library needed for the saturation mutagenesis as well as perform QC for the resulting construct.
• Gagarmel repository (this repository) is used to process the raw data from NGS to quantify the enrichment of the variants.
• Azrael repository is used to generate and analyze the functional scores, and plot the results.

Requirements

• Java 1.8
• Python (tested on v3.8.6)
• Cromwell (tested on v56)
• samtools (tested on v1.16)
• bwa (tested on v0.7.17)

Python libraries required

pysam (test on v0.16.0.1)

Step 1: Loading required libraries

Tested on McCleary cluster at Yale

module load Java/1.8.345
module load Python/3.8.6-GCCcore-10.2.0
module load SAMtools/1.16-GCCcore-10.2.0
module load BWA/0.7.17-GCC-10.2.0
module load Pysam/0.16.0.1-GCC-10.2.0

Step 2: Preparation of input files

• Change the output directory paths in the cromwell.options file
• Change the paths in large1.json and fkrp.json to reference the absolute paths for the scripts and reference_files folders.
• Change the input files to reference the demultiplexed FASTQ files and the block demarcations

Step 3: Submit the job to the Slurm job manager

sbatch launch_cromwell.sh

Output files will be located in the directory defined in the cromwell.options file