EnvGen/prokka

Name: prokka

Owner: Environmental Genomics Group SciLifeLab/KTH Stockholm

Description: Rapid prokaryotic genome annotation

Forked from: tseemann/prokka

Created: 2015-04-29 12:06:26.0

Updated: 2015-04-29 12:08:43.0

Pushed: 2015-04-29 12:08:43.0

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Size: 141713

Language: Perl

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README

Prokka: rapid prokaryotic genome annotation

Torsten Seemann (torsten.seemann@gmail.com) (@torstenseemann)

Contents
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
Download

Download the latest prokka-1.xx.tar.gz archive from http://www.bioinformatics.net.au/software.prokka.shtml

Extract

Choose somewhere to put it, for example in your home directory (no root access required):

% cd $HOME
% tar zxvf prokka-1.xx.tar.gz
% ls prokka-1.xx
Add to PATH

Add the following line to your $HOME/.bashrc file, or to /etc/profile.d/prokka.sh to make it available to all users:

export PATH=$PATH:$HOME/prokka-1.xx/bin
Index the sequence databases
% prokka --setupdb
Install dependencies

Prokka comes with many binaries for Linux and Mac OS X. It will always use your existing installed versions if they exist, but will use the included ones if that fails. For some older systems (eg. Centos 4.x) some of them won't work due to them being dynamically linked against new GLIBC libraries you don't have.

Centos/Fedora/RHEL (RPM)

sudo yum install perl-Time-Piece perl-XML-Simple

Ubuntu/Debian/Mint (APT)

sudo apt-get install libdatetime-perl libxml-simple-perl

You can consult the list of dependencies later in this document.

Choose a rRNA predictor
Option 1 - Don't use one

If Prokka can't find a predictor for rRNA featues (either Barrnap or RNAmmer below) then it simply won't annotate any. Most people don't care that much about them anyway,

Option 2 - Barrnap

This was written by the author of Prokka and is recommended if you prefer speed over absolute accuracy. It uses the new multi-core NHMMER for DNA:DNA profile searches. Download it from http://www.vicbioinformatics.com/software.barrnap.shtml

Option 3 - RNAmmer

RNAmmer was written when HMMER 2.x was the latest release. Since them, HMMER 3.x has been released, and uses the same executable binary names. Prokka needs HMMER3 and RNAmmer (and hence HMMER2) so you need to edit your RNAmmer script to explicitly point your HMMER2 binary instead of using the HMMER3 binary which is more likely to be in your PATH first.

Type which rnammer to find the script, and then edit it with your favourite editor. Find the following lines at the top:

if ( $uname eq "Linux" ) {
#       $HMMSEARCH_BINARY = "/usr/cbs/bio/bin/linux64/hmmsearch";    # OLD
        $HMMSEARCH_BINARY = "/path/to/my/hmmer-2.3.2/bin/hmmsearch"; # NEW (yours)
}

If you are using Mac OS X, you'll also have to change the "Linux" to "Darwin" too. As you can see, I have commented out the original part, and replaced it with the location of my HMMER2 hmmsearch tool, so it doesn't run the HMMER3 one. You need to ensure HMMER3 is in your PATH before the old HMMER2 too.

Test
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
Expert
# It's not just for bacteria, people
% prokka --kingdom Archaea --outdir mydir --genus Pyrococcus --locustag PYCC

# Search for my 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
Genbank submitter
# Register your BioProject and your locus_tag prefix 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
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 annotated sequences, not just CDS. | | .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. |

Command line options
General:
  --help            This help
  --version         Print version and exit
  --docs            Show full manual/documentation
  --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')
  --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)
Dependencies
Databases
The Core (BLAST+) Databases

Prokka uses a variety of databases when trying to assign function to the predicted CDS features. It takes a hierarchial 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 initial core databases are derived from UniProtKB; there is one per “kingdom” supported. To qualify for inclusion, a protein must be (1) from Bacteria (or Archaea or Viruses); (2) not be “Fragment” entries; and (3) 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

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

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
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
LENG  284
FAQ
Still To Do
Changes
Citation

Seemann T.
Prokka: rapid prokaryotic genome annotation
Bioinformatics 2014 Jul 15;30(14):2068-9. PMID:24642063

This work is supported by the National Institutes of Health's National Center for Advancing Translational Sciences, Grant Number U24TR002306. This work is solely the responsibility of the creators and does not necessarily represent the official views of the National Institutes of Health.