leenaput/microcredential-nextflow-project

Introduction

leenaput/microcredential-nextflow-project
For the Microcredential Nextflow project, I developed a pipeline to processes nanopore sequencing data from raw FASTQ to alignment, coverage calculation, and QC summary evaluation. A step-by-step outline of how the project was developed can be found here.

Pipeline overview

Pipeline steps:

1. Raw read QC - Generates stats for raw using NanoPlot
2. Read filtering - Quality and length filtering of raw reads using Chopper
3. Filtered reads QC - Generates stats for filtered reads using NanoPlot
4. Read alignment -Maps reads to reference genome using minimap2, sorts and indexes with SAMtools
5. Alignment QC – Computes coverage using SAMtools and generates stats of mapped reads using NanoPlot
6. QC Summary – Evaluates QC values against user-defined thresholds using custom script
7. Final report – Quickly displays QC pass/fail of sample in stdout

Usage

Installation

Clone the repository:

git clone git@github.com:leenput/microcredential-nextflow-project.git

Note: Nextflow should be installed on your system.
If working on VSC, make sure to carry out the following configurations before running the pipeline:

module load Nextflow/24.10.2
export APPTAINER_CACHEDIR=${VSC_SCRATCH}/.apptainer_cache
export APPTAINER_TMPDIR=${VSC_SCRATCH}/.apptainer_tmp

Set parameters

The parameters are included in params.config. Please modify according to your experimental needs.

General parameters

Parameter	Description	Example
--reads	Path to input FASTQ files	./data/*.fastq
--fasta	Reference genome FASTA file	./data/genome.fasta

Please make sure to store your genome sequence file (.fasta) and basecalled ONT reads (.fastq) in the /data workfolder.

For now, you can find the following test data there:

• reference sequence: chr21 of the new human reference genome T2T-CHM13v2
• ONT data: subsampled reads of GIAB sample HG002

Optional parameters

Parameter	Description	Default
--qscore	Minimum quality score (CHOPPER)	7
--minlength	Minimum read length (CHOPPER)	1000
--pct_filtered	% reads passing filtering (QC summary)	80
--pct_mapped	% reads mapped (QC summary)	50
--coverage	Minimum average coverage	10
--filt_n50	Minimum N50 for filtered reads	5000

Run the pipeline

Run the workflow with the following command:

nextflow run main.nf -profile <docker/singularity/conda> 

Output structure

results/
    ├── pipeline_info/
    ├── <sample>/
           ├── filtered_reads/
           ├── alignment/
           ├── quality-control/
                     ├── raw/
                     ├── filtered/
                     ├── mapped/
                     ├── <sample>_QC_summary.txt