NestLink-pipeline

NestLink-pipeline is a pipeline for processing NestLink libraries sequenced by nanopore sequencing. Reads are binned according to their flycodes (UMIs). Accurate consensus sequences are calculated using Medaka. Variants are called with the pipeline, resulting in a flycode assignment table that links protein variants to their respective set of flycodes.

Requirements

Local and cluster execution

• Nextflow (Installation guide), on the cluster it has to be installed in a mamba/ conda environment called nextflow.
• Mamba/ Conda (https://conda-forge.org/)
• mini_align (mini_align.sh placed in ./bin/)

Local execution only

• Podman (https://podman.io/)

Cluster execution only

• Slurm workflow manager
• Singularity

Running the pipeline on the s3it cluster

1. Clone the repository.
2. Edit the params.json file, specify the nanopore reads (bam) and reference sequence.
3. Run the pipeline: sbatch run_NL-pipeline.slurm

Running the pipeline locally

1. Prepare the pipeline as described above.
2. Run the pipeline: bash run_NL-pipeline.sh

Parameters

Parameter	Type	Description
data	String	Path to input BAM file.
reference	String	Path to reference FASTA file.
filter_min_length	Integer	Read filtering minimum length threshold.
filter_max_length	Integer	Read filtering maximum length threshold.
extract_seq_adapter	String	Linked adapter for sequence trimming.
extract_seq_min_length	Integer	Sequence trimming minimum length threshold.
extract_seq_max_length	Integer	Sequence trimming minimum length threshold.
extract_flycode_adapter	String	Linked adapter for flycode extraction.
medaka_dorado_model	String	Dorado model used for basecalling.
flycode_pattern	List(String, String)	Sequences flanking flyodes.
orf1_name	String	Name of ORF 1.
orf1_pattern	List(String, String)	Sequences flanking ORF 1.
orf2_name	String	Name of ORF2 (optional).
orf2_pattern	List(String, String)	Sequences flanking ORF 2 (optional).
outdir	String	Output directory for results.