(annot-fasta)=
### Annotate from fasta files

As an input file, you can provide a list of .fasta files. 
If you do so, the provided genomes will be annotated using the following tools: 

- [Pyrodigal](https://pyrodigal.readthedocs.io/en/stable/index.html), which is based on Prodigal, to annotate CDSs
- [ARAGORN](http://www.ansikte.se/ARAGORN/) to annotate tRNAs
- [Infernal](http://eddylab.org/infernal/) coupled with HMM of the bacterial and archaeal rRNAs downloaded from [RFAM](https://rfam.xfam.org/) to annotate rRNAs.

To proceed with this stage of the pipeline, you need to create a **genomes.fasta.list** file.
This file should be tab-separated with each line depicting an individual genome and
its pertinent information with the following organization (only the first two columns are mandatory):

- The first column contains a unique genome name
- The second column contains the path to the associated FASTA file
- The following columns contain Contig identifiers present in the associated FASTA file that should be analyzed as being circular.
If you do not have access to the 'circular contig identifiers', you may safely ignore this part as it does not have a big impact on the resulting pangenome.

You can check [this example input file](https://github.com/labgem/PPanGGOLiN/blob/master/testingDataset/genomes.fasta.list).

To run just the annotation step, you can use this minimal command:

```
ppanggolin annotate --fasta genomes.fasta.list
```

#### Use a different genetic code in my annotation step
To annotate the genomes, you can easily change the translation table (or genetic code) used by Pyrodigal just by giving the corresponding number as described [here](https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi).

#### Force the Prodigal procedure
Prodigal can predict gene in [single/normal mode](https://github.com/hyattpd/prodigal/wiki/gene-prediction-modes#normal-mode) 
that includes a training step on your genomes or in [meta/anonymous mode](https://github.com/hyattpd/prodigal/wiki/gene-prediction-modes#anonymous-mode), 
which uses pre-calculated training files.
As recommended in the Prodigal documentation: "Anonymous mode should be used on metagenomic data sets, or on sequences too short to provide good training data."

By default, PPanGGOLiN will determine the best mode based on the contig length.
The procedure can be overridden with the option `-p, --prodigal_procedure`.
The option only accepts **single** or **meta** keywords, corresponding to the Prodigal procedure name.

#### Customize the RNA annotation
If you do not want to predict the RNA (and thus not use Infernal and Aragorn), you can add the `--norna` option to your command.
Otherwise, by default, any CDS overlapping RNA genes will be deleted as they are often false positive calls.
You can prevent this filtering by using the `--allow_overlap` option.

Additionally, the `--kingdom archaea` option can be provided when working with archaea genomes
to specify Infernal's RNA annotation model. 

### Use annotation files for your pangenome

You can provide annotation files in either gff3 files or .gbk/.gbff files, or a mix of them. They should be provided through as a list in a tab-separated file that follows the same format as described for the fasta files. You can check [this example input file](https://github.com/labgem/PPanGGOLiN/blob/master/testingDataset/genomes.gbff.list).

```{note}
Using your own annotation for your genome is highly recommended, particularly if you already
have functional annotations, as they can be added to the pangenome.
```

You can provide them using the following command: 

```
ppanggolin annotate --anno genomes.gbff.list
```

#### How to deal with annotation files without sequences

If your annotation files do not contain the genome sequence, 
you can use both options simultaneously to obtain the gene annotations and gene sequences, as follows: 

```
ppanggolin annotate --anno genomes.gbff.list --fasta genomes.fasta.list
```

#### Take the pseudogenes into account for pangenome analyses

By default, PPanGGOLiN will not take pseudogenes into account. 
However, they could be worth keeping in certain contexts.
It is possible to include pseudogenes in the pangenome by using the `--use_pseudo` option.