When use please reference: V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers, p.61-78.
To identify protein and RNA genes in bacterial genomic sequences or environmental samples, Softberry developed Fgenesb_annotator pipeline that provides completely automatic, comprehensive annotation of bacterial sequences. The pipeline includes protein, tRNA and rRNA genes identification, finds potential promoters, terminators and operon units.
Predicted genes are annotated based on comparison with known proteins. The package provides options to work with a set of sequences such as scaffolds of bacterial genomes or short reads of DNA extracted from a bacterial community. The final annotation can be presented in GenBank form to be readable by visualization software such as Artemis  and GenomeExplorer (fig. 1 and 2). The gene prediction algorithm is based on Markov chain models of coding regions and translation and termination sites. For annotation of mixed bacterial community, we use special parameters of gene prediction computed based on a large set of known bacterial sequences. Operon models are based on distances between ORFs, frequencies of different genes neighboring each other in known bacterial genomes, and information from predicted potential promoters and terminators. The parameters of gene prediction are automatically trained during initial steps of sequence analysis, so the only input necessary for annotation of a new genome is its sequence. Optionally, parameters from closely related genomes can be used, instead of training new parameters. Bacterial gene/operon prediction and annotation requires, besides Fgenesb_annotator programs and scripts, BLAST, NCBI Non-Redundant database (NR), and a file reconstructed from COG database . RRNA genes are annotated using BLAST similarity with all known bacterial rRNA database. For prediction of tRNA genes, the pipeline uses tRNAscan-SE package .
1. K. Rutherford, J. Parkhill, J. Crook, T. Horsnell, P. Rice, M-A. Rajandream and B. Barrell (2000) Artemis: sequence visualisation and annotation. Bioinformatics 16 (10) 944-945.
2. Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, Rao BS, Kiryutin B, Galperin MY, Fedorova ND, Koonin EV. (2001) The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res. 29, 22-28.
3. Lowe, T.M. & Eddy, S.R. (1997) "tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence", Nucl. Acids Res., 25, 955-964.
General scheme of bacterial genome annotation see here.
All prediction components of FGENESB are extremely fast (minutes per genome). The limiting stage is BLAST annotation, which for E.coli genome takes around 12 hours on a single processor. Using multiple processors and corresponding BLAST would speed up annotation proportionally.
|Fgenesb correctly identified 10-30% more reference genes on the contigs than the Critica-Glimer pipeline in every data set of contigs|
|Fgenesb correctly identified ~70% of reference genes on unassembled reads in all data sets (Fig. 2b) (unassembled reads are typical for bacterial community sequencing) The Critica-Glimmer pipeline exhibited poor results: only 7% genes was accurately predicted|
|Sn (exact prediction)||Sn (exact+overlapping prediction)|
Example of FGENESB output:
Prediction of potential genes in microbial genomes Time: Tue Aug 22 11:21:15 2006 Seq name: gi|15807672|ref|NC_001264.1| Deinococcus radiodurans R1 (partial sequence) Length of sequence - 54865 bp Number of predicted genes - 48, with homology - 48 Number of transcription units - 18, operons - 13 average op.length - 3.3 N Tu/Op Conserved S Start End Score pairs(N/Pv) - TRNA 147 - 222 78.9 # Arg CCG 0 0 + TRNA 315 - 398 63.6 # Leu TAG 0 0 + 5S_RRNA 521 - 637 100.0 # AB001721 [D:2735..2851] + SSU_RRNA 698 - 2181 100.0 # SSU_RRNA ## + LSU_RRNA 2302 - 5345 100.0 # BX248583 [R:613128..616171] + Prom 5304 - 5363 41.4 1 1 Op 1 22/0.000 + CDS 5410 - 6300 498 ## COG1192 ATPases involved … 2 1 Op 2 . + CDS 6297 - 7178 502 ## COG1475 Predicted … + Term 7203 - 7253 9.1 - Term 7191 - 7241 14.2 3 2 Tu 1 . - CDS 7283 - 8746 909 ## COG1012 NAD-dependent … - Prom 8792 - 8851 2.8 4 3 Tu 1 . + CDS 8802 - 9533 302 ## COG2068 Uncharacterized … + Term 9779 - 9818 3.8 - Term 9527 - 9567 9.0 5 4 Op 1 2/0.125 - CDS 9584 - 10762 1005 ## COG1063 Threonine … 6 4 Op 2 . - CDS 10759 - 11457 666 ## COG5637 Predicted integral … - Prom 11697 - 11756 2.4 7 5 Op 1 37/0.000 + CDS 11704 - 12609 872 ## COG1131 ABC-type multidrug … 8 5 Op 2 5/0.000 + CDS 12726 - 13517 812 ## COG0842 ABC-type multidrug … 9 5 Op 3 15/0.000 + CDS 13674 - 14684 1028 ## COG4585 Signal transduction … 10 5 Op 4 . + CDS 14681 - 15316 506 ## COG2197 Response regulator … … 47 18 Op 1 . - CDS 53783 - 54703 431 ## DRA0045 hypothetical … 48 18 Op 2 . - CDS 54700 - 54864 91 ## DRA0046 hypothetical … Predicted protein(s) >gi|15807672|ref|NC_001264.1| GENE 1 5410 - 6300 498 296 aa, chain + ## HITS:3 COG:DRA0001 KEGG:FRAAL2247 NR:6460595 ## COG: DRA0001 COG1192 # Protein_GI_number: 15807673 # Func_class: D Cell cycle control, cell division, chromosome partitioning # Function: ATPases involved in chromosome partitioning # Organism: Deinococcus radiodurans # 37 296 1 260 260 459 100.0 1e-129 ## KEGG: FRAAL2247 # Name: not_defined # Def: chromosome partitioning protein (partial match) [EC:22.214.171.124] # Organism: F.alni # Pathway: not_defined # 48 283 50 291 302 118 35.0 5e-26 ## NR: gi|6460595|gb|AAF12301.1| chromosome partitioning ATPase, putative, ParA family [Deinococcus radiodurans R1]^Agi|15807673|ref|NP_285325.1| chromosome partitioning ATPase, putative, ParA family [Deinococcus radiodurans R1] # 37 296 1 260 260 459 100.0 1e-128 VLKNHLFLRNLIFSVLPVVQHFLTFKEEQSIADLSDMVSAVKTLTVFNHAGGAGKTSLTL NVGYELARGGLRVLLLDLDPQANLTGWLGISGVTREMTVYPVAVDGQPLPSPVKAFGLDV IPAHVSLAVAEGQMMGRVGAQGRLRRALAEVSGDYDVALIDSPPSLGQLAILAALAADQM IVPVPTRQKGLDALPGLQGALTEYREVRPDLTVALYVPTFYDARRRHDQEVLADLKAHLS PLARPVPQREAVWLDSTAQGAPVSEYAPGTPVHADVQRLTADIAAAIGVAYPGENA >gi|15807672|ref|NC_001264.1| GENE 2 6297 - 7178 502 293 aa, chain + ## HITS:3 COG:DRA0002 KEGG:SAR11_0354 NR:12230476 ## COG: DRA0002 COG1475 # Protein_GI_number: 15807674 # Func_class: K Transcription # Function: Predicted transcriptional regulators # Organism: Deinococcus radiodurans # 1 293 1 293 293 478 100.0 1e-135 ## KEGG: SAR11_0354 # Name: parB # Def: chromosome partitioning protein [EC:2.7.7.-] # Organism: P.ubique # Pathway: not_defined # 10 200 12 177 282 107 36.0 7e-23 ## NR: gi|12230476|sp|Q9RZE7|PARB2_DEIRA Probable chromosome 2 partitioning protein parB (Probable chromosome II partitioning protein parB)^Agi|6460594|gb|AAF12300.1| chromosome partitioning protein, ParB family [Deinococcus radiodurans R1]^Agi|15807674|ref|NP_285326.1| chromosome partitioning protein, ParB family [Deinococcus radiodurans R1] # 1 293 1 293 293 478 100.0 1e-133 MTRRRPERRRDLLGLLGETPVDLSQANDIRALPVNELKVGSTQPRRSFDLERLSELAESI RAHGVLQPLLVRSVDGQYEIVAGERRWRAAQLAGLAEVPVVVRQLSNEQARAAALIENLQ RDNLNVIDEVDGKLELIALTLGLEREEARKRLMQLLRAVPGDEHEQLDQVFRSMGETWRT FAKNKLRILNWPQPVLEALRAGLPLTLGSVVASAPPERQAELLKLAQNGASRSQLLQALQ TPSQTSAVTPEHFAKVLSSKRFLSGLDTPTREALDRWLARMPERVRQAIDEQS ...
For each genomic sequence (complete genome, scaffold, read, etc.) the program lists locations of predicted ORFs, rRNAs, tRNAs, promoters and terminators.
ORFs are labeled as CDS and provided with their order number in a sequence and an indicator of whether they are transcribed as a single transcription unit (Tu) or in operons (Op) (of course these are predictions).
If an ORF has a homolog, its short name is provided after a "##" separator (here name of only
one homolog - either from COG, KEGG, or NR - is given; best homologs from all databases are listed in ID lines of predicted proteins, see below).
5 4 Op 2 + CDS 2737 - 3744 871 ## COG0673 Predicted dehydrogenases
is description for predicted gene number 5 in 4th Operon with coordinates 2737 - 3744 in the '+' strand and it is the second gene in operon.
Coding chain for this CDS (+) means a direct chain, (-) means a complementary chain. 871 is a score of gene homology assigned by BLAST, and COG0673 is an ID of its homolog from the COG database.
In other words, first column lists an ordered number of predicted CDS, starting from beginning of a sequence; second column - number of predicted operon/TU, and fourth column - number of gene in an operon (always 1 for a TU).
For some operons, we report supportive evidence related to conservation in relative locations of genes in predicted operon in different bacteria. For example:
3 2 Op 1 4/0.002 + CDS 3193 - 3405 278 ## COG2501 Uncharacterized ACR
Here, in 4/0.002, 4 is a number of observations of this gene being next to one of its neighbors on known bacterial genomes (we call it N-value), while 0.002 is a P-value, an empirical probability of observing N occurrences of genes being adjacent by random chance. P is a very approximate measure. For all P<0.0001, the value in output is 0.000.
At the end of annotation, we also provide protein products of predicted genes in fasta format, with full name of homolog and homology scores according to BLAST.
Information about homologs is given in ID lines of predicted proteins, for example:
>gi|15807672|ref|NC_001264.1| GENE 7 11704 - 12609 872 301 aa, chain + ## HITS:3 COG:DRA0007 KEGG:DRA0007 NR:6460585 ## COG: DRA0007 COG1131 # Protein_GI_number: 15807679 # Func_class: V Defense mechanisms # Function: ABC-type mult idrug transport system, ATPase component # Organism: Deinococcus radiodurans # 1 301 1 301 301 503 100.0 1e-142 ## KEGG: DRA0007 # Name: not_defined # Def: putative ABC-2 type transport system ATP-binding protein # Organism: D.radiodurans # Pathway: ABC transporters - General [PATH:dra02010] # 1 301 1 301 301 503 100.0 1e-142 ## NR: gi|6460585|gb|AAF12291.1| ABC transporter, ATP-binding protein, putative [Deinococcus radiodurans R1]^Agi|15807679|ref|NP_285331.1| ABC transporter, ATP-binding protein, putative [Deinococcus radiodurans R1] # 1 301 1 301 301 503 100.0 1e-141 MITTFEQVSKTYGHVTALSDFNLTLRTGELTALLGPNGAGKSTAIGLLLGLSAPSAGQVR VLGADPRRNDVRARIGAMPQESALPAGLTVREAVTLFASFYPAPLGVDEALALADLGPVA GRRAAQLSGGQKRRLAFALAVVGDPELLLIDEPTTGMDAQSRAAFWEAVTGLRARGRTIL LTTHYLEEAERTADRVVVMNGGRILADDTPQGLRSGVGGARVSFVSDLVQAELERLPGVS AVQVDAAGRADLRTSVPEALLAALIGSGTTFSDLEVRRATLEEAYLQLTGPQDMTAVTRS A
While looking a bit complex for a human eye, it is well suited for parsing by a program.
ID lines of predicted proteins consist of the following parts that are separated from each other by "##" separator:
>gi|15807672|ref|NC_001264.1| GENE 7 11704 - 12609 872 301 aa, chain +
(sequence name, gene number, coordinates of a gene, length of a corresponding protein, chain)
## HITS:3 COG:DRA0007 KEGG:DRA0007 NR:6460585
(shows the number of homologs found in protein databases (takes into account maximum one best homolog per a database), lists homologs IDs in the format DB:ID (e.g., COG:DRA0007);
- for homologs from NR, gi- numbers are given as homologs IDs;
- DB:ns indicates that a protein DB was not searched (e.g., NR:ns);
- DB:no indicates that a protein DB was searched but no homologs were found (e.g., NR:no))
Then, complete ID lines of homologs are given preceded by DB names where they were found by BLAST (e.g., NR:) and followed by statistics from corresponding BLAST outputs.
## COG: DRA0007 COG1131 # Protein_GI_number: 15807679 # Func_class: V Defense mechanisms # Function: ABC-type multidrug transport system, ATPase component # Organism: Deinococcus radiodurans # 1 301 1 301 301 503 100.0 1e-142 ## KEGG: DRA0007 # Name: not_defined # Def: putative ABC-2 type transport system ATP-binding protein # Organism: D.radiodurans # Pathway: ABC transporters - General [PATH:dra02010] # 1 301 1 301 301 503 100.0 1e-142 ## NR: gi|6460585|gb|AAF12291.1| ABC transporter, ATP-binding protein, putative [Deinococcus radiodurans R1]^Agi|15807679|ref|NP_285331.1| ABC transporter, ATP-binding protein, putative [Deinococcus radiodurans R1] # 1 301 1 301 301 503 100.0 1e-141
BLAST parameters of similarity found for predicted protein are shown in the following order:
Start and stop of region of similarity ( 1 301) in predicted protein
Start and stop of region of similarity (1 301) in homolog from a database
Length of homologous protein (301)
BLAST score (503) and Identity (100.0 %)
BLAST Expected value (1e-141)
For other predictions (rRNA, promoters, etc.) we provide only description lines, for example:
- LSU_RRNA 884415 - 887254 98.0 # Leuconostoc oenos S60377
rRNAs are labeled as LSU_RRNA, SSU_RRNA or 5S_RRNA (large subunit, small subunit, and 5S), tRNAs as TRNA, promoters as Prom, and terminators as Term.
Terminator regions (their coordinates and scores) are reported by FindTerm program:
+ Term 492 - 537 -0.9
Promoters (their coordinates and scores) are reported by BPROM program.
Annotation visualization occurs in CGView Viewers automatically. "Manual" visualization process is described here: http://linux1.softberry.com/berry.phtml?topic=index&group=programs&subgroup=cgview