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https://github.com/tseemann/prokka

:zap: :aquarius: Rapid prokaryotic genome annotation
https://github.com/tseemann/prokka

bacterial-genomes functional-assignment gene-finding genome-annotation

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:zap: :aquarius: Rapid prokaryotic genome annotation

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# Prokka: rapid prokaryotic genome annotation



## Introduction



Whole genome annotation is the process of identifying features of interest

in a set of genomic DNA sequences, and labelling them with useful

information. Prokka is a software tool to annotate bacterial, archaeal and

viral genomes quickly and produce standards-compliant output files.



## Installation



### Bioconda

If you use [Conda](https://conda.io/docs/install/quick.html)

you can use the [Bioconda channel](https://bioconda.github.io/):

```

conda install -c conda-forge -c bioconda -c defaults prokka

```



### Brew

If you are using the [MacOS Brew](http://brew.sh/) 

or [LinuxBrew](http://brew.sh/linuxbrew/) packaging system:

```

brew install brewsci/bio/prokka

```



### Docker

Maintained by https://hub.docker.com/u/staphb

```



docker pull staphb/prokka:latest

docker run staphb/prokka:latest prokka -h

```



### Singularity

```

singularity build prokka.sif docker://staphb/prokka:latest

singularity exec prokka.sif prokka -h

```



### Ubuntu/Debian/Mint

```

sudo apt-get install libdatetime-perl libxml-simple-perl libdigest-md5-perl git default-jre bioperl

sudo cpan Bio::Perl

git clone https://github.com/tseemann/prokka.git $HOME/prokka

$HOME/prokka/bin/prokka --setupdb

```



### Centos/Fedora/RHEL

```

sudo yum install git perl-Time-Piece perl-XML-Simple perl-Digest-MD5 perl-App-cpanminus git java perl-CPAN perl-Module-Build

sudo cpanm Bio::Perl

git clone https://github.com/tseemann/prokka.git $HOME/prokka

$HOME/prokka/bin/prokka --setupdb

```



### MacOS

```

sudo cpan Time::Piece XML::Simple Digest::MD5 Bio::Perl

git clone https://github.com/tseemann/prokka.git $HOME/prokka

$HOME/prokka/bin/prokka --setupdb

```



## Test



* Type `prokka` and it should output its help screen.

* Type `prokka --version` and you should see an output like `prokka 1.x`

* Type `prokka --listdb` and it will show you what databases it has installed to use.



## Invoking Prokka



### Beginner

```

# Vanilla (but with free toppings)

% prokka contigs.fa



# Look for a folder called PROKKA_yyyymmdd (today's date) and look at stats

% cat PROKKA_yyyymmdd/*.txt

```



### Moderate

```

# Choose the names of the output files

% prokka --outdir mydir --prefix mygenome contigs.fa



# Visualize it in Artemis

% art mydir/mygenome.gff

```



### Specialist

```

# Have curated genomes I want to use to annotate from

% prokka --proteins MG1655.gbk --outdir mutant --prefix K12_mut contigs.fa



# Look at tabular features

% less -S mutant/K12_mut.tsv

```



### Expert

```

# It's not just for bacteria, people

% prokka --kingdom Archaea --outdir mydir --genus Pyrococcus --locustag PYCC



# Search for your favourite gene

% exonerate --bestn 1 zetatoxin.fasta mydir/PYCC_06072012.faa | less

```



### Wizard

```

# Watch and learn

% prokka --outdir mydir --locustag EHEC --proteins NewToxins.faa --evalue 0.001 --gram neg --addgenes contigs.fa



# Check to see if anything went really wrong

% less mydir/EHEC_06072012.err



# Add final details using Sequin

% sequin mydir/EHEC_0607201.sqn

```



### NCBI Genbank submitter

```

# Register your BioProject (e.g. PRJNA123456) and your locus_tag prefix (e.g. EHEC) first!

% prokka --compliant --centre UoN --outdir PRJNA123456 --locustag EHEC --prefix EHEC-Chr1 contigs.fa



# Check to see if anything went really wrong

% less PRJNA123456/EHEC-Chr1.err



# Add final details using Sequin

% sequin PRJNA123456/EHEC-Chr1.sqn

```



### European Nucleotide Archive (ENA) submitter



```

# Register your BioProject (e.g. PRJEB12345) and your locus_tag (e.g. EHEC) prefix first!

% prokka --compliant --centre UoN --outdir PRJEB12345 --locustag EHEC --prefix EHEC-Chr1 contigs.fa



# Check to see if anything went really wrong

% less PRJNA123456/EHEC-Chr1.err



# Install and run Sanger Pathogen group's Prokka GFF3 to EMBL converter

# available from https://github.com/sanger-pathogens/gff3toembl

# Find the closest NCBI taxonomy id (e.g. 562 for Escherichia coli)

% gff3_to_embl -i "Submitter, A." \

    -m "Escherichia coli EHEC annotated using Prokka." \

    -g linear -c PROK -n 11 -f PRJEB12345/EHEC-Chr1.embl \

    "Escherichia coli" 562 PRJEB12345 "Escherichia coli strain EHEC" PRJEB12345/EHEC-Chr1.gff



# Download and run the latest EMBL validator prior to submitting the EMBL flat file

# from http://central.maven.org/maven2/uk/ac/ebi/ena/sequence/embl-api-validator/

# which at the time of writing is v1.1.129

% curl -L -O http://central.maven.org/maven2/uk/ac/ebi/ena/sequence/embl-api-validator/1.1.129/embl-api-validator-1.1.129.jar

% java -jar embl-api-validator-1.1.129.jar -r PRJEB12345/EHEC-Chr1.embl



# Compress the file ready to upload to ENA, and calculate MD5 checksum

% gzip PRJEB12345/EHEC-Chr1.embl

% md5sum PRJEB12345/EHEC-Chr1.embl.gz

```



### Crazy Person

```

# No stinking Perl script is going to control me

% prokka \

        --outdir $HOME/genomes/Ec_POO247 --force \

        --prefix Ec_POO247 --addgenes --locustag ECPOOp \

        --increment 10 --gffver 2 --centre CDC  --compliant \

        --genus Escherichia --species coli --strain POO247 --plasmid pECPOO247 \

        --kingdom Bacteria --gcode 11 --usegenus \

        --proteins /opt/prokka/db/trusted/Ecocyc-17.6 \

        --evalue 1e-9 --rfam \

        plasmid-closed.fna

```



## Output Files



| Extension | Description |

| --------- | ----------- |

| .gff | This is the master annotation in GFF3 format, containing both sequences and annotations. It can be viewed directly in Artemis or IGV. |

| .gbk | This is a standard Genbank file derived from the master .gff. If the input to prokka was a multi-FASTA, then this will be a multi-Genbank, with one record for each sequence. |

| .fna | Nucleotide FASTA file of the input contig sequences. |

| .faa | Protein FASTA file of the translated CDS sequences. |

| .ffn | Nucleotide FASTA file of all the prediction transcripts (CDS, rRNA, tRNA, tmRNA, misc_RNA) |

| .sqn | An ASN1 format "Sequin" file for submission to Genbank. It needs to be edited to set the correct taxonomy, authors, related publication etc. |

| .fsa | Nucleotide FASTA file of the input contig sequences, used by "tbl2asn" to create the .sqn file. It is mostly the same as the .fna file, but with extra Sequin tags in the sequence description lines. |

| .tbl | Feature Table file, used by "tbl2asn" to create the .sqn file. |

| .err | Unacceptable annotations - the NCBI discrepancy report. |

| .log | Contains all the output that Prokka produced during its run. This is a record of what settings you used, even if the --quiet option was enabled. |

| .txt | Statistics relating to the annotated features found. |

| .tsv | Tab-separated file of all features: locus_tag,ftype,len_bp,gene,EC_number,COG,product |



## Command line options



    General:

      --help            This help

      --version         Print version and exit

      --citation        Print citation for referencing Prokka

      --quiet           No screen output (default OFF)

      --debug           Debug mode: keep all temporary files (default OFF)

    Setup:

      --listdb          List all configured databases

      --setupdb         Index all installed databases

      --cleandb         Remove all database indices

      --depends         List all software dependencies

    Outputs:

      --outdir [X]      Output folder [auto] (default '')

      --force           Force overwriting existing output folder (default OFF)

      --prefix [X]      Filename output prefix [auto] (default '')

      --addgenes        Add 'gene' features for each 'CDS' feature (default OFF)

      --locustag [X]    Locus tag prefix (default 'PROKKA')

      --increment [N]   Locus tag counter increment (default '1')

      --gffver [N]      GFF version (default '3')

      --compliant       Force Genbank/ENA/DDJB compliance: --genes --mincontiglen 200 --centre XXX (default OFF)

      --centre [X]      Sequencing centre ID. (default '')

    Organism details:

      --genus [X]       Genus name (default 'Genus')

      --species [X]     Species name (default 'species')

      --strain [X]      Strain name (default 'strain')

      --plasmid [X]     Plasmid name or identifier (default '')

    Annotations:

      --kingdom [X]     Annotation mode: Archaea|Bacteria|Mitochondria|Viruses (default 'Bacteria')

      --gcode [N]       Genetic code / Translation table (set if --kingdom is set) (default '0')

      --prodigaltf [X]  Prodigal training file (default '')

      --gram [X]        Gram: -/neg +/pos (default '')

      --usegenus        Use genus-specific BLAST databases (needs --genus) (default OFF)

      --proteins [X]    Fasta file of trusted proteins to first annotate from (default '')

      --hmms [X]        Trusted HMM to first annotate from (default '')

      --metagenome      Improve gene predictions for highly fragmented genomes (default OFF)

      --rawproduct      Do not clean up /product annotation (default OFF)

    Computation:

      --fast            Fast mode - skip CDS /product searching (default OFF)

      --cpus [N]        Number of CPUs to use [0=all] (default '8')

      --mincontiglen [N] Minimum contig size [NCBI needs 200] (default '1')

      --evalue [n.n]    Similarity e-value cut-off (default '1e-06')

      --rfam            Enable searching for ncRNAs with Infernal+Rfam (SLOW!) (default '0')

      --norrna          Don't run rRNA search (default OFF)

      --notrna          Don't run tRNA search (default OFF)

      --rnammer         Prefer RNAmmer over Barrnap for rRNA prediction (default OFF)



### Option: --proteins



The `--proteins` option is recommended when you have good quality reference genomes

and want to ensure gene naming is consistent. Some species use specific terminology

which will be often lost if you rely on the default Swiss-Prot database included

with Prokka.



If you have Genbank or Protein FASTA file(s) that you want to annotate genes from

as the first priority, use the `--proteins myfile.gbk`. Please make sure it has a

recognisable file extension like `.gb` or `.gbk` or auto-detect will fail.   The

use of Genbank is recommended over FASTA, because it will provide `/gene` 

and `/EC_number` annotations that a typical `.faa` file will not provide, unless

you have specially formatted it for Prokka.



### Option: --prodigaltf



Instead of letting `prodigal` train its gene model on the contigs you

provide, you can pre-train it on some good closed reference genomes first

using the `prodigal -t` option. Once you've done that, provide `prokka`

the training file using the `--prodgialtf` option.



### Option: --rawproduct



Prokka annotates proteins by using sequence similarity to other proteins in its database,

or the databases the user provides via `--proteins`. By default, Prokka tries to "cleans" the

`/product` names to ensure they are compliant with Genbank/ENA conventions. 

Some of the main things it does is:



* set vague names to `hypothetical protein`

* consistifies terms like `possible`, `probable`, `predicted`, ... to `putative`

* removes EC, COG and locus_tag identifiers 



Full details can be found in the `cleanup_product()` function in the `prokka` script.

If you feel your annotations are being ruined, try using the `--rawproduct` option, 

and please [file an issue](https://github.com/tseemann/prokka/issues/) if you find

an example of where it is "behaving badly" and I will fix it.



## Databases



### The Core (BLAST+) Databases



Prokka uses a variety of databases when trying to assign function to the

predicted CDS features.  It takes a hierarchical approach to make it fast.  

A small, core set of well characterized proteins are first searched using

BLAST+.  This combination of small database and fast search typically

completes about 70% of the workload.  Then a series of slower but more

sensitive HMM databases are searched using HMMER3.



The three core databases, applied in order, are:



1. [ISfinder](https://isfinder.biotoul.fr/):

Only the tranposase (protein) sequences; the whole transposon is not annotated.



2. [NCBI Bacterial Antimicrobial Resistance Reference Gene Database](https://www.ncbi.nlm.nih.gov/bioproject/313047):

Antimicrobial resistance genes curated by NCBI.



3. [UniProtKB (SwissProt)](https://www.uniprot.org/uniprot/?query=reviewed:yes): 

For each `--kingdom` we include curated proteins with evidence that

(i) from Bacteria (or Archaea or Viruses);

(ii) not be "Fragment" entries;

and (iii) have an evidence level ("PE") of 2 or lower, which

corresponds to experimental mRNA or proteomics evidence.



#### Making a Core Databases



If you want to modify these core databases, the included script

`prokka-uniprot_to_fasta_db`, along with the official `uniprot_sprot.dat`,

can be used to generate a new database to put in `/opt/prokka/db/kingdom/`. 

If you add new ones, the command `prokka --listdb` will show you whether it

has been detected properly.



#### The Genus Databases



:warning: This is no longer recommended. Please use `--proteins` instead.



If you enable `--usegenus` and also provide a Genus via `--genus` then it

will first use a BLAST database which is Genus specific.  Prokka comes with

a set of databases for the most common Bacterial genera; type prokka

`--listdb` to see what they are.



#### Adding a Genus Databases



If you have a set of Genbank files and want to create a new Genus database,

Prokka comes with a tool called `prokka-genbank_to_fasta_db` to help.  For

example, if you had four annotated "Coccus" genomes, you could do the

following:



```

% prokka-genbank_to_fasta_db Coccus1.gbk Coccus2.gbk Coccus3.gbk Coccus4.gbk > Coccus.faa

% cd-hit -i Coccus.faa -o Coccus -T 0 -M 0 -g 1 -s 0.8 -c 0.9

% rm -fv Coccus.faa Coccus.bak.clstr Coccus.clstr

% makeblastdb -dbtype prot -in Coccus

% mv Coccus.p* /path/to/prokka/db/genus/

```

 

### The HMM Databases



Prokka comes with a bunch of HMM libraries for HMMER3. They are mostly

Bacteria-specific.  They are searched after the core and genus databases. 

You can add more simply by putting them in `/opt/prokka/db/hmm`.  Type

`prokka --listdb` to confirm they are recognised.



### FASTA database format



Prokka understands two annotation tag formats, a plain one and a detailed one.



The plain one is a standard FASTA-like line with the ID after the `>` sign, and the protein `/product` 

after the ID (the "description" part of the line):

```

>SeqID product

```



The detailed one consists of a special encoded three-part description line. The parts are the `/EC_number`, 

the `/gene` code, then the `/product` - and they are separated by a special "~~~" sequence:

```

>SeqID EC_number~~~gene~~~product~~~COG

```



Here are some examples. Note that not all parts need to be present, but the "~~~" should still be there:

```

>YP_492693.1 2.1.1.48~~~ermC~~~rRNA adenine N-6-methyltransferase~~~COG1234

MNEKNIKHSQNFITSKHNIDKIMTNIRLNEHDNIFEIGSGKGHFTLELVQRCNFVTAIEI

DHKLCKTTENKLVDHDNFQVLNKDILQFKFPKNQSYKIFGNIPYNISTDIIRKIVF*

>YP_492697.1 ~~~traB~~~transfer complex protein TraB~~~

MIKKFSLTTVYVAFLSIVLSNITLGAENPGPKIEQGLQQVQTFLTGLIVAVGICAGVWIV

LKKLPGIDDPMVKNEMFRGVGMVLAGVAVGAALVWLVPWVYNLFQ*

>YP_492694.1 ~~~~~~transposase~~~

MNYFRYKQFNKDVITVAVGYYLRYALSYRDISEILRGRGVNVHHSTVYRWVQEYAPILYQ

QSINTAKNTLKGIECIYALYKKNRRSLQIYGFSPCHEISIMLAS*

```



The same description lines apply to HMM models, except the "NAME" and "DESC" fields are used:

```

NAME  PRK00001

ACC   PRK00001

DESC  2.1.1.48~~~ermC~~~rRNA adenine N-6-methyltransferase~~~COG1234

LENG  284

```    

    

## FAQ



* __Where does the name "Prokka" come from?__  

Prokka is a contraction of "prokaryotic annotation".  It's also relatively

unique within Google, and also rhymes with a native Australian marsupial

called the quokka.



* __Can I annotate by eukaryote genome with Prokka?__  

No.  Prokka is specifically designed for Bacteria, Archaea and Viruses.  It

can't handle multi-exon gene models; I would recommend using MAKER 2 for

that purpose.



* __Why does Prokka keeps on crashing when it gets to the "tbl2asn" stage?__  

It seems that the tbl2asn program from NCBI "expires" after 6-12 months, and

refuses to run.  Unfortunately you need to install a newer version which you

can download from [here](http://www.ncbi.nlm.nih.gov/genbank/tbl2asn2/).



* __The hmmscan step seems to hang and do nothing?__      

The problem here is GNU Parallel.  It seems the Debian package for hmmer has

modified it to require the `--gnu` option to behave in the 'default' way. 

There is no clear reason for this.  The only way to restore normal behaviour

is to edit the prokka script and change `parallel` to `parallel --gnu`.



* __Why does prokka fail when it gets to hmmscan?__  

Unfortunately HMMER keeps changing its database format, and they aren't

upward compatible.  If you upgraded HMMER (from 3.0 to 3.1 say) then you

need to "re-press" the files.  This can be done as follows:

```

cd /path/to/prokka/db/hmm

mkdir new

for D in *.hmm ; do hmmconvert $D > new/$D ; done

cd new

for D in *.hmm ; do hmmpress $D ; done

mv * ..

rmdir new

```



* __Why can't I load Prokka .GBK files into Mauve?__  

Mauve uses BioJava to parse GenBank files, and it is very picky about Genbank files. 

It does not like long contig names,

like those from Velvet or Spades. One solution is to use `--centre XXX` 

in Prokka and it will rename all your contigs to be NCBI (and Mauve)

compliant. It does not like the ACCESSION and VERSION strings that Prokka

produces via the "tbl2asn" tool. The following Unix command will fix them:

`egrep -v '^(ACCESSION|VERSION)' prokka.gbk > mauve.gbk`



* __How can I make my GFF not have the contig sequences in it?__

```

sed '/^##FASTA/Q' prokka.gff > nosequence.gff

```



## Bugs



Submit problems or requests to the [Issue Tracker](https://github.com/tseemann/prokka/issues).



## Changes



* Read the [release notes](https://github.com/tseemann/prokka/releases)

* Read the [ChangeLog.txt](https://raw.githubusercontent.com/tseemann/prokka/master/doc/ChangeLog.txt)

* Look at the [Github commits](https://github.com/tseemann/prokka/commits/master)



## Citation



Seemann T.  

*Prokka: rapid prokaryotic genome annotation*  

**Bioinformatics** 2014 Jul 15;30(14):2068-9.

[PMID:24642063](http://www.ncbi.nlm.nih.gov/pubmed/24642063)  



## Dependencies



### Mandatory



* __BioPerl__  

Used for input/output of various file formats  

_Stajich et al, The Bioperl toolkit: Perl modules for the life sciences. Genome Res. 2002 Oct;12(10):1611-8._



* __GNU Parallel__  

A shell tool for executing jobs in parallel using one or more computers  

_O. Tange, GNU Parallel - The Command-Line Power Tool, ;login: The USENIX Magazine, Feb 2011:42-47._



* __BLAST+__  

Used for similarity searching against protein sequence libraries  

_Camacho C et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009 Dec 15;10:421._



* __Prodigal__  

Finds protein-coding features (CDS)  

_Hyatt D et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010 Mar 8;11:119._



* __TBL2ASN__

Prepare sequence records for Genbank submission

[Tbl2asn home page](https://www.ncbi.nlm.nih.gov/genbank/tbl2asn2/)



### Recommended



* __Aragorn__  

Finds transfer RNA features (tRNA)  

_Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004 Jan 2;32(1):11-6._



* __Barrnap__  

Used to predict ribosomal RNA features (rRNA). My licence-free replacement for RNAmmmer.  

_Manuscript under preparation._



* __HMMER3__  

Used for similarity searching against protein family profiles  

_Finn RD et al. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011 Jul;39(Web Server issue):W29-37._



### Optional



* __minced__  

Finds CRISPR arrays

[Minced home page](https://github.com/ctSkennerton/minced)



* __RNAmmer__  

Finds ribosomal RNA features (rRNA)  

_Lagesen K et al. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35(9):3100-8._



* __SignalP__  

Finds signal peptide features in CDS (sig_peptide)  

_Petersen TN et al. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011 Sep 29;8(10):785-6._



* __Infernal__  

Used for similarity searching against ncRNA family profiles  

_D. L. Kolbe, S. R. Eddy. Fast Filtering for RNA Homology Search. Bioinformatics, 27:3102-3109, 2011._



# Licence



[GPL v3](https://raw.githubusercontent.com/tseemann/prokka/master/doc/LICENSE.Prokka)



## Author



* Torsten Seemann

* Web: https://tseemann.github.io/

* Twitter: [@torstenseemann](https://twitter.com/torstenseemann)

* Blog: [The Genome Factory](https://thegenomefactory.blogspot.com/)