Prokka: rapid prokaryotic genome annotation

Torsten Seemann [email protected] Victorian Bioinformatics Consortium, AUSTRALIA http://vicbioinformatics.com

This version has been forked from the original repository to include InterProScan as an option to further look for protein domains annotation, as well as provide the EMBL output format. Contact: Laurent Falquet, University of Fribourg, Switzerland. Many thanks to Ivan Topolski and Robin Engler for their support.

##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

This version will come already installed in Vital-IT machines, with all its dependencies. Load the module with

module add UHTS/Analysis/prokka/1.9.1.ips1

But you can also follow these steps if you want to install this version locally:

###Download the archive and extract

Clone this repository or download the zipped version and decompress it. Then add the binary prokka/bin/prokka to your $PATH, that is, add the following line to your $HOME/.bashrc file

export PATH=$PATH:<...>/prokka-1.9.1.ips1

###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.

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

###Test

• Type prokka and it should output it's 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

###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

###EBI submitter

# Add protein domains and use a config file to get a proper EMBL outpu
% prokka --compliant --centre EPFL --outdir PRJNA123456 --locustag TEST \
  --ips --ips_goterms --ips_pathways --embl_header embl_header.txt contigs.fa

###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.
.embl	This is a standard EMBL file derived from the master .gbk. If the input to prokka was a multi-FASTA, then this will be a multi-EMBL, 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.

###EBI submission

To get an .embl file that is compliant with their very strict rules, you will need to specify many of the header attributes manually through a config file. This config file, that you will pass as argument to the --embl_header option, will contain header lines formatted exactly as they are in a complete standard EMBL file (see ftp://ftp.ebi.ac.uk/pub/databases/embl/doc/usrman.txt). If some of the tags are already present in the raw version produced automatically by Prokka, these lines will be replaced by the ones of the config file. If they are not present, these of the config will be added. Rewriting ID and AC lines is not allowed.

Example of config file::

PR   Project:PRJNA0001;
RN   [5]
RP   1-1859
RX   DOI; 10.1007/BF00039495.
RX   PUBMED; 1010101.
RA   Author A, Author B,
RA   Author C, Author D;
RT   "Project title";
RT   "Secondary title";
RL   Plant Mol. Biol. 17(2):209-219(1991).

##Command line options

General:
  --help            This help
  --version         Print version and exit
  --docs            Show full manual/documentation
  --listdb          List all configured databases
  --citation        Print citation for referencing Prokka
  --quiet           No screen output (default OFF)
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 '')
  --embl_header [X] Header lines for EMBL format. (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|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 '')
  --metagenome      Improve gene predictions for highly fragmented genomes (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)
InterProScan options:
  --ips             Run InterProScan to annotate protein domains. (default OFF)
  --ips_appl [X]    Comma-separated list of InterProScan analyses to run (IPS --appl option). (default '0')
  --ips_tempdir [X] Temporary folder for IPS intermediate files. (default '')
  --ips_goterms     Search for GO terms (IPS --goterms option). (default OFF)
  --ips_pathways    Pathways analysis (IPS --pathways options). (default OFF)

##Dependencies

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• BLAST+ Used for similarity searching against protein sequence libraries Camacho C et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009 Dec 15;10:421.

• 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.

• 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.

• InterProScan Annotates protein domains and adds protein GO terms and pathways. Combines different protein signature recognition methods native to the InterPro member databases into one resource with look up of corresponding InterPro and GO annotation. Zdobnov E.M., Apweiler R. InterProScan - an integration platform for the signature-recognition methods in InterPro. Bioinformatics, 17(9):847-8, 2001.

##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.

###InterProScan Databases and lookup service

InterProScan embeds its own versions of the databases it needs, except for Panther models: https://code.google.com/p/interproscan/wiki/HowToDownload#2._Installing_Panther_Models

In this version of prokka, when IPS is activated (--ips option) it runs with "IPR lookup", which either will be very slow through web requests, or requires the installation of a local copy of the service: https://code.google.com/p/interproscan/wiki/LocalLookupService This service is already provided on Vital-IT servers (rserv01).

In general, see here for more about IPS installation and configuration: See https://code.google.com/p/interproscan/wiki/Introduction .

###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

• 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 tge "tbl2asn" stage? It seems that the tbl2asn program from NCBI "expires" after 12 months, and refuses to run. Unfortunately you need to install a newer version which you can download from here.

• 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 it's 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 does Prokka take so long to download? Our server is in Australia, and the international pipes aren't always flowing as well as we'd like. I try to put it on GoogleDrive. Dropbox is no longer possible due to bandwidth quotas. If you are able to mirror Prokka (~2 GB) outside please let me know.

• Why can't I load Prokka .GBK files into Mauve? Mauve is very picky about Genbank files. It does not like long contig names, like those from Velvet or Spades. The simple 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

##Still To Do

• Check for large genomic tracts which are unannotated. Sometimes Prodigal misses big genes.
• Add an example input/output so users can check their copy is producing similar results.
• Output potential homopolymer assembly errors near CDS flanks
• Add the CLUSTERS "PHA" library to Viruses mode.
• Option to include ribosomal binding sites (RBS) as features.
• Check input contigs for runs of Ns, and either complain, or split the file, or additionally create a .AGP scaffold file. (Mitchell Stanton-Cook has done this, need to incorporate)
• Add hyperlinks to tool references in Manual

##Changes

See the ChangeLog.txt file in the doc/ subdirectory of Prokka.

##Bugs

• tbl2asn seems to be removing the "(anti-codon)" part in my tRNA /product values
• tbl2asn putting space in /inference for Infernal

##Citation

Seemann T. Prokka: rapid prokaryotic genome annotation Bioinformatics 2014 Mar 18. [Epub ahead of print] PMID:24642063