# Comparative Genome Analysis of Illumina, Nanopore, and Hybrid Approaches: A Case Study of the Aquaculture Isolate 160P

## TL;DR

This study compares different DNA sequencing methods for analyzing a bacterial isolate from aquaculture, showing that combining short and long reads improves genome assembly and classification.

## Contribution

The study demonstrates the superiority of hybrid sequencing for bacterial genome analysis and taxonomic classification.

## Key findings

- Hybrid Unicycler assembly provided the highest contiguity with seven contigs and a 4.55 Mb dominant contig.
- 160P is genomically distinct from Aeromonas sobria CECT 4245T based on ANI and dDDH thresholds.
- Hybrid sequencing enhances annotation completeness and taxonomic resolution in bacterial genomics.

## Abstract

In this study, we comparatively assessed short-read (Illumina), long-read (Oxford Nanopore Technologies, ONT), and hybrid (Illumina + ONT) sequencing strategies for bacterial genome analysis using the aquaculture-derived isolate 160P. Genomic DNA was extracted and sequenced on Illumina paired-end and ONT long-read platforms, and de novo assemblies were generated using SPAdes, Canu, Flye, and Unicycler under short-read-only, long-read-only, and hybrid workflows, followed by evaluation with QUAST assembly metrics. Among the tested approaches, the hybrid Unicycler assembly provided the highest contiguity, yielding seven contigs and a dominant 4.55 Mb contig consistent with near-complete chromosomal representation. Downstream analyses included functional genome annotation and in silico screening of antimicrobial resistance determinants (CARD), virulence-associated genes (VFDB), and secondary metabolite biosynthetic gene clusters (antiSMASH). Comparative genomic relatedness based on Average Nucleotide Identity (ANI) and digital DNA–DNA Hybridization (dDDH) indicated that 160P is most closely related to Aeromonas sobria CECT 4245T yet falls below commonly applied species-level thresholds, supporting its placement as a genomically distinct lineage warranting further taxonomic investigation. Collectively, these findings underscore the value of hybrid sequencing for improving assembly continuity, enhancing annotation completeness, and strengthening taxonomic resolution in bacterial pathogen genomics.