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Figure 1. Schematic representation of the workflow. Starting from raw reads obtained from NGS platforms, MEGAnnotator2 will perform
read filtering, assembly, quality control of genome sequences, genome alignments, genome comparisons, gene prediction, gene
annotation, and metabolic profiling. Red arrows highlight the mandatory steps of the pipeline.

attributable enzymatic reaction to predicted genes.

As the previous version of the pipeline, MEGAnnotator2 aims to improve every step described above to
enhance the quality of genome sequences and gene annotation by reducing the time and effort required,
thanks to its automated execution. In fact, the user needs only to provide the individual NGS data, and the
MEGAnnotator2 pipeline will manage all steps, ultimately leading to the generation of a GenBank file, thus
leaving the user free to carry out other activities. To enhance the performance of each software package,
MEGAnnotator2 handles the execution of multiple threads as set up in the MEGAnnotator2 parameters.
Accordingly, software parameters can be personalized in the parameter file of MEGAnnotator2 to guide the
execution of each software package by the pipeline without the need to set up the individual programs. The
modular implementation of the pipeline grants flexible execution of the analyses, allowing the user to select
which step to perform and allowing modifying parameters based on the user’s need. For a detailed overview
of the pipeline’s editable parameters, refer to the manual.

The previous version of the software was distributed with distinct virtual machines containing different
databases for gene annotation since many dependencies needed to be installed in the user environment.
Instead, MEGAnnotator2 is provided by an auto installer able to automatically manage the installation of
dependencies, the set-up of the software, and the download of pre-processed databases whose size has been
significantly reduced. The installer file of MEGAnnotator2 can be downloaded at the link http://