ONT_targseq

A comprehensive Nextflow pipeline for analyzing targeted amplicon sequencing data from Oxford Nanopore Technologies (ONT) sequencing platforms.

Overview

This pipeline processes ONT targeted amplicon sequencing data to generate high-quality consensus sequences, taxonomic classifications, phylogenetic analyses, and comprehensive quality control reports.

Pathogens included in the default reference and ABRicate database

Pathogen	Abbreviation	Target Region
Avian Reovirus	ARV	Sigma C
Bovine Respiratory Syncytial Virus	BRSV	F, G, N genes
Fowl Adenovirus	FAdV	Hexon gene
Infectious Bursal Disease Virus	IBDV	VP2 region
Infectious Bronchitis Virus	IBV	S1 region of Spike protein
Influenza A Virus	InfA	HA and NA typing
Porcine Circovirus 3	PCV3	ORF2
Porcine Rotavirus A, B, C	PRV	VP7 and VP4 full length
Porcine Sapovirus	PSaV	VP1 sequence full length

Quick Start

Prerequisites

• Nextflow (≥ 21.04.0), Docker, WSL2 (Windows only)

Basic Usage

nextflow run main.nf \
    --input /path/to/input \
    --out_dir Results \
    --reference /path/to/reference.fasta \
    --kraken_db /path/to/kraken2_db \
    --blastdb_path /path/to/blastdb/nt \
    --blastdb_name nt

Parameters

Required Parameters

Parameter	Description
--input	Path to input directory containing sample subdirectories
--out_dir	Output directory for results
--reference	Path to reference FASTA file. Default reference will contain sequences of pathgens listed above
--kraken_db	Path to Kraken2 database
--blastdb_path	Path to BLAST database directory
--blastdb_name	BLAST database name

Optional Parameters

Parameter	Default	Description
--read_count_threshold	10	Minimum read depth for consensus generation
--trim_barcodes	false	Enable barcode/adapter trimming with Porechop
--medaka_model	r1041_e82_400bps_sup_g615	Medaka model for consensus polishing
--qscore	10	Minimum quality score threshold
--consensus_mode	'simple' or 'bayesian'	samtools option for generating consensus. Default: simple

Input & Output

Input Structure

input_directory/
├── Sample1/
│   └── *.fastq(.gz)
├── Sample2/
│   └── *.fastq(.gz)
└── ...

Required Files

• Reference sequences (FASTA),Kraken2 database, BLAST database

Output Structure

Results organized by analysis type:

• merged/ - Merged FASTQ files per sample
• trimmed/ - Adapter-trimmed reads (if enabled)
• splitbam/ - Split BAM files and initial consensus sequences
• medaka/ - Polished consensus sequences
• multiqc/ - Quality control summary reports
• kraken2/ - Taxonomic classification results
• blast/ - BLAST similarity search results
• mafft/ - Multiple sequence alignments
• iqtree/ - Phylogenetic tree files
• ggtree/ - Tree visualization plots
• igvreports/ - Interactive genome browser reports
• *.html - Final comprehensive HTML reports

Software Dependencies

Tool	Purpose	Description	Citation
nanoq	Quality control	Ultra-fast quality control tool for nanopore reads with comprehensive statistics and filtering capabilities	Steinig & Coin (2022)
Porechop	Adapter trimming	Tool for finding and removing adapters from Oxford Nanopore reads, with support for demultiplexing and chimeric read detection	Wick et al.
minimap2	Sequence alignment	Versatile sequence alignment program for aligning DNA or mRNA sequences against large reference databases	Li (2018)
SAMtools	BAM processing	Suite of programs for interacting with high-throughput sequencing data in SAM/BAM format	Danecek et al. (2021)
Medaka	Consensus polishing	Tool to create consensus sequences and call variants via neural networks from nanopore sequencing data	Oxford Nanopore
Kraken2	Taxonomic classification	System for assigning taxonomic labels to DNA sequences using exact k-mer matches	Wood et al. (2019)
Krona	Taxonomic visualization	Interactive metagenomic visualization tool that displays hierarchical data with multi-layered pie charts	Ondov et al. (2011)
BLAST+	Sequence similarity	Basic Local Alignment Search Tool for finding regions of local similarity between sequences	Camacho et al. (2009)
ABRicate	Sequence screening	Mass screening of contigs for antimicrobial resistance genes and virulence factors	Seemann
MAFFT	Multiple alignment	Multiple sequence alignment program offering various algorithms including progressive, iterative, and structural methods	Katoh & Standley (2013)
IQ-TREE	Phylogenetic analysis	Efficient software for phylogenomic inference with model selection, bootstrap analysis, and tree topology tests	Nguyen et al. (2015)
BEDTools	Genomic intervals	Toolkit for genome arithmetic - intersecting, merging, counting, complementing, and shuffling genomic intervals	Quinlan & Hall (2010)
IGV Reports	Genome visualization	Python application for generating self-contained HTML reports with embedded genome visualizations	Robinson et al. (2023)
R Markdown	Report generation	Dynamic document format combining R code with narrative text to produce elegantly formatted output	Xie et al. (2018)
ORFiPy	ORF prediction	Fast and flexible tool for extracting open reading frames (ORFs) from FASTA files with customizable parameters	Singh & Wurtele (2021)
MultiQC	Quality control	Tool that aggregates results from multiple bioinformatics analyses across many samples into a single report	Ewels et al. (2016)
seqkit	Sequence manipulation	Cross-platform and ultrafast toolkit for FASTA/Q file manipulation with rich functions	Shen et al. (2016)
ggtree	Tree visualization	R package for visualization and annotation of phylogenetic trees with associated data	Yu et al. (2017)