ONT-DMS

ONT-DMS is a Nextflow pipeline for processing barcoded protein variant libraries and NestLink libraries sequenced by nanopore sequencing. Reads are binned according to their barcodes or flycodes (UMIs). Accurate consensus sequences are calculated using Dorado polish. Finally, variants are called with the pipeline, linking barcodes or flycodes with their respective protein variants.

Note

Tested on macOS 15.7 (Mamba), Windows 11 (WSL with Docker), and Ubuntu 22.04 (Mamba, Apptainer).

Requirements

Local execution

• Nextflow (Installation guide).
• Mamba/ Conda (https://conda-forge.org/) or Docker
• Dorado (Installation guide), does not have to be installed when using containers.

Cluster execution

• Slurm workflow manager
• Nextflow, installed in a Mamba/ Conda environment called nextflow.
• Mamba/ Conda or Apptainer
• Dorado

Tip

Add dorado to your PATH e.g. by adding export PATH="$PATH:/home/fackle/data/dorado-1.2.0-linux-x64/bin" to your .bashrc.

Running the pipeline

1. Clone the repository with git clone https://github.com/fabianackle/ONT-DMS.git.
2. Check the nextflow configuration file nextflow.config.
3. Create a params.json file with the parameters listed below, specify the nanopore reads (BAM) and reference sequence, see the examples contained in this repo.
4. Run the pipeline with either ./run_NL-pipeline.sh for local execution or on a cluster with sbatch run_NL-pipeline.slurm.

Tip

The pipeline can also be run directly: nextflow run main.nf -profile conda -params-file params.json.

Parameters

Parameter	Type	Description
data	String	Path to input BAM file(s).†
reference	String	Path to reference FASTA file.
filter_quality	Float	Minimum mean read quality threshold.
filter_min_length	Integer	Read filtering minimum length threshold.
filter_max_length	Integer	Read filtering maximum length threshold.
extract_barcode_adapter	String	Linked cutadapt adapter for barcode extraction.†2
barcode_regex	String	Regular expression matching the barcode.
barcode_min_coverage	Integer	The minimal amount a barcode has to be seen to be considered a high-quality barcode.
polish_bacteria	Boolean	Use a bacterial model for polishing with dorado (optional).†3
barcode_5p	String	5' sequence flanking the barcode.
barcode_3p	String	3' sequence flanking the barcode.
orf_5p	String	5' sequence flanking the ORF.
orf_3p	String	3' sequence flanking the ORF.
translate_barcode	Boolean	Translates barcode, used with flycodes.
outdir	String	Output directory for results.

† for multiple BAM files use *, e.g. data/barcode*.bam. †2 see Linked adapters (combined 5’ and 3’ adapter). †3 do not use if you want to use move tables, as currently there are no models that support both.