GALAXY Workflow for Bacterial Next-Generation Sequencing De Novo Assembly and Annotation

Whole-genome sequencing of prokaryotes is now readily available and affordable on next-generation sequencing platforms. However, the process of de novo assembly can be complicated and tedious for those without a background in computational biology, bioinformatics, or UNIX. Licenses for commercial bioinformatics software may be costly and limited in flexibility. GALAXY is a powerful graphical open-source code-free bioinformatics platform that is freely available on multiple public and private servers. Here, we describe a bacterial de novo assembly workflow using GALAXY. It performs de novo genome assembly using short reads, long reads, or a hybrid method using both short and long reads. Genome annotation, prediction of antimicrobial resistance genes, and multi-locus sequence typing are subsequently performed to characterize the draft genome. Performing genome assembly and annotation on this pipeline allows documentation, parameterization, and sharing, facilitating replication, reuse, and reproducibility of both data and methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Quality check of NGS reads Basic Protocol 2: De novo assembly using Unicycler Basic Protocol 3: Assembly quality check using QUAST and Bandage Basic Protocol 4: Genome annotation using Prokka Basic Protocol 5: Prediction of antimicrobial resistance genes (ARGs) Basic Protocol 6: Multi-locus sequence typing (MLST).