Duplex Sequencing Pipeline

A snakemake pipeline for obtaining duplex consensus reads generated using a duplex sequencing protocol (e.g. NanoSeq. The pipeline is similar to the recommend workflow from IDT on processing sequence data with unique molecular identifiers. QC is also performed, both pre- and post-duplex consensus calling.

Installation

The only prerequisite is snakemake. To install snakemake, you will need to install a Conda-based Python3 distribution. For this, Mambaforge is recommended. Once mamba is installed, snakemake can be installed like so:

mamba create -c conda-forge -c bioconda -n snakemake snakemake=7.0.0-0

Now activate the snakemake environment (you'll have to do this every time you want to run the pipeline):

conda activate snakemake

Now clone the repository:

git clone https://github.com/WEHIGenomicsRnD/duplex-seq-pipeline.git
cd duplex-seq-pipeline

Configuration

The configuration file is found under config/config.yaml and the config file for FastQ Screen is found under config/fastq_screen.conf. Please carefully go through these settings. The main settings to consider will be

• read_structure -- ensure that this matches the UMI design of your experiment. Refer to fgbio's ExtractUmisFromBam for details on how to set this parameter.
• umis -- if your UMIs are known (non-random), you can add a path to a UMIs text file (one UMI per line). Specifying a file path for this parameter will trigger a UMI correction step.
• ref -- ensure you have downloaded and specified the correct reference for your data.

Running

Run the pipeline as follows:

conda activate snakemake
snakemake --use-conda --conda-frontend mamba --cores 1

If you want to submit your jobs to the cluster using SLURM, use the following to run the pipeline:

conda activate snakemake
snakemake --use-conda --conda-frontend mamba --profile slurm --jobs 8 --cores 24

The pipeline will generate all results under a results directory. The most relevant directories are:

• results/QC/multiQC -- contains the multiQC report for pre-consensus call reads. Also contains FastQC and FastQ Screen metrics on the raw reads.
• results/QC/consensus/multiQC -- contains the multiQC report generated on reads after duplex consensus calling.
• results/consensus/{sample}__mapped_merged_filtered_clipped.bam -- contains the mapped, filtered and clipped consensus reads. These should be your "final" read alignments for duplex consensus reads.