FgenesB-Annotator

Description

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 [1] 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 [2]. RRNA genes are annotated using BLAST similarity with all known bacterial rRNA database. For prediction of tRNA genes, the pipeline uses tRNAscan-SE package [3].

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.

Fig.1. Bacterial Genome Explorer to work with annotations and comparison of genomes.

The package includes options to work with a set of sequences such as scaffolds of bacterial genomes, or short sequencing reads extracted from bacterial communities. For community sequence annotation, we developed ABsplit program that separates archaebacterial and eubacterial sequences (available separately). Final annotation can be presented in GenBank format to be readable by visualization software such as Artemis or Softberry Bacterial Genome Explorer (fig. 1 and 2, GenBank parser is available separately).

Fig.2. Comparison of two bacterial genomes view of Genome Explorer.

Main Steps of FGENESB annotation.

Many steps are optional and can be switched ON/OFF in configuration file.

STEP 1. Finds all potential ribosomal RNA genes using BLAST against bacterial and/or archaeal rRNA databases, and masks detected rRNA genes.

STEP 2. Predicts tRNA genes using tRNAscan-SE program (Washington University) and masks detected tRNA genes.

STEP 3. Initial predictions of long ORFs that are used as a starting point for calculating parameters for gene prediction. Iterates until stabilizes. Generates parameters such as 5th-order in-frame Markov chains for coding regions, 2nd-order Markov models for region around start codon and upstream RBS site, stop codon and probability distributions of ORF lengths.

STEP 4. Predicts operons based only on distances between predicted genes.

STEP 5. Runs BLAST for predicted proteins against COG database, cog.pro.

STEP 6. Finds conserved operonic pairs from blast output through cog data.

STEP 7. Uses information about conservation of neighboring gene pairs in known genomes to improve operon prediction.

STEP 8. Runs BLAST for predicted proteins against KEGG database.

STEP 9. Runs BLAST for predicted proteins against NR database.

STEP 10. Adds names of homologs from COG/KEGG/NR (found through BLAST) to annotation file (file with prediction results).

STEP 11. Predicts potential promoters (BPROM program) or terminators (BTERM) in upstream and downstream regions, correspondingly, of predicted genes. BTERM is the program predicting bacterial-independent terminators with energy scoring based on discriminant function of hairpin elements.

STEP 12. Refines operon predictions using predicted promoters and terminators as additional evidences.

FGENESB gene prediction engine is one of the most accurate prokaryotic gene finders available: see Table 1 for its comparison with two other popular gene prediction programs.

Table 1. Comparison of three popular bacterial gene finders. Accuracy estimate was done on a set of difficult short genes that was previously used for evaluating other bacterial gene finders (http://opal.biology.gatech.edu/GeneMark/genemarks.cgi). First set (51set) has 51 genes with at least 10 strong similarities to known proteins. Then 72set has 72 genes with at least two strong similarities, and 123set has 123 genes with at least one protein homolog. Here are the prediction results on these three sets for GeneMarkS and Glimmer (calculated by Besemer et al. (2001) Nucl. Acids Res. 29:2607-2618) and FGENESB gene prediction engine (calculated by Softberry).

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.

Explanation of Fgenesb_annotator output

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:2.7.10.2] # 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

...

Example of FGENESB output in GenBank format (scripts run_tgb.pl, togenbank.pl):


     gene            complement(147..222)
                     /gene="Arg CCG"
     tRNA            complement(147..222)
                     /gene="Arg CCG"
                     /product="tRNA-Arg"
                     /note="Arg CCG 0 0"
     gene            315..398
                     /gene="Leu TAG"
     tRNA            315..398
                     /gene="Leu TAG"
                     /product="tRNA-Leu"
                     /note="Leu TAG 0 0"
     gene            521..637
                     /gene="AB001721 [D:2735..2851]"
     rRNA            521..637
                     /gene="AB001721 [D:2735..2851]"
                     /product="5S ribosomal RNA"
                     /note="AB001721 [D:2735..2851]"
     gene            698..2181
                     /gene="SSU_RRNA"
     rRNA            698..2181
                     /gene="SSU_RRNA"
                     /product="16S ribosomal RNA"
                     /note="SSU_RRNA"
     gene            2302..5345
                     /gene="BX248583 [R:613128..616171]"
     rRNA            2302..5345
                     /gene="BX248583 [R:613128..616171]"
                     /product="23S ribosomal RNA"
                     /note="BX248583 [R:613128..616171]"
     promoter        5304..5363
     CDS             5410..6300
                     /function="ATPases involved in chromosome partitioning"
                     /note="Operon 1 Gene 1 COG1192 ATPases involved in
                     chromosome partitioning"
                     /translation="VLKNHLFLRNLIFSVLPVVQHFLTFKEEQSIADLSDMVSAVKTL
                     TVFNHAGGAGKTSLTLNVGYELARGGLRVLLLDLDPQANLTGWLGISGVTREMTVYPV
                     AVDGQPLPSPVKAFGLDVIPAHVSLAVAEGQMMGRVGAQGRLRRALAEVSGDYDVALI
                     DSPPSLGQLAILAALAADQMIVPVPTRQKGLDALPGLQGALTEYREVRPDLTVALYVP
                     TFYDARRRHDQEVLADLKAHLSPLARPVPQREAVWLDSTAQGAPVSEYAPGTPVHADV
                     QRLTADIAAAIGVAYPGENA"
                     /transl_table=11
     CDS             6297..7178
                     /function="Predicted transcriptional regulators"
                     /note="Operon 1 Gene 2 COG1475 Predicted transcriptional
                     regulators"
                     /translation="MTRRRPERRRDLLGLLGETPVDLSQANDIRALPVNELKVGSTQP
                     RRSFDLERLSELAESIRAHGVLQPLLVRSVDGQYEIVAGERRWRAAQLAGLAEVPVVV
                     RQLSNEQARAAALIENLQRDNLNVIDEVDGKLELIALTLGLEREEARKRLMQLLRAVP
                     GDEHEQLDQVFRSMGETWRTFAKNKLRILNWPQPVLEALRAGLPLTLGSVVASAPPER
                     QAELLKLAQNGASRSQLLQALQTPSQTSAVTPEHFAKVLSSKRFLSGLDTPTREALDR
                     WLARMPERVRQAIDEQS"
                     /transl_table=11
     terminator      7203..7253
     terminator      complement(7191..7241)
     CDS             complement(7283..8746)
                     /function="NAD-dependent aldehyde dehydrogenases"
                     /note="Operon 2 Gene 1 COG1012 NAD-dependent aldehyde
                     dehydrogenases"
                     /translation="MTTTDLRTTYSSVTRSQAYFDGEWRNAPRNFEVRHPGNGEVIGE
                     VADCTPTDARQAIDAAEVALREWRQVNPYERGKILRRWHDLMFEHKEELAQLMTLEMG
                     KPISETRGEVHYAASFIEWCAEEAGRIAGERINLRFPHKRGLTISEPVGIVYAVTPWN
                     FPAGMITRKAAPALAAGCVMILKPAELSPMTALYLTELWLKAGGPANTFQVLPTNDAS
                     ALTQPFMNDSRVRKLTFTGSTEVGRLLYQQAAGTIKRVSLELGGHAPFLVFDDADLER
                     AASEVVASKFRNSGQTCVCTNRVYVQRGVAEEFIRLLTEKTAALQLGDPFDEATQVGP
                     VVEQAGLDKVQRQVQDALTKGAQATTGGQVSSGLFFQPTVLVDVAPDSLILREETFGP
                     VAPVTIFDTEEEGLRLANDSEYGLAAYAYTRDLGRAFRIAEGLEYGIVGINDGLPSSA
                     APHVPFGGMKNSGVGREGGHWGLEEYLETKFVSLGLS"
                     /transl_table=11
     promoter        complement(8792..8851)

...

BASE COUNT     11009 a   16099 c   16880 g   10877 t
ORIGIN
        1 tctttgctcg ccatacccaa agtctacacg ctgattttca cgtttccaga ccctgccctc
       61 tcgctactca gctctccaag tttgctcgct tgatgaatga tcaaatcttt taaagataaa
      121 agccatgcgt gaggctagat caacccttgt gcccccggca ggattcgaac ctgcggcctt
...
    54841 gtcgcccagt tgaatggctc gccac
//

Example of FGENESB output in Sequin format:


>Feature test_seq
222     147     gene
                        locus_tag       C8J_0001
222     147     tRNA
                        product tRNA-Arg
                        inference       profile:tRNAscan-SE:1.23
315     398     gene
                        locus_tag       C8J_0002
315     398     tRNA
                        product tRNA-Leu
                        inference       profile:tRNAscan-SE:1.23
521     637     gene
                        locus_tag       C8J_0003
521     637     rRNA
                        product 5S ribosomal RNA
698     2181    gene
                        locus_tag       C8J_0004
698     2181    rRNA
                        product 16S ribosomal RNA
2302    5345    gene
                        locus_tag       C8J_0005
2302    5345    rRNA
                        product 23S ribosomal RNA
5304    6300    gene
                        locus_tag       C8J_0006
5304    5363    promoter
5410    6300    CDS
                        product hypothetical protein
                        note    similar to D.radiodurans chromosome partitioning ATPase …
                        protein_id      gnl|bbsrc|C8J_0006
                        inference       ab initio prediction:Fgenesb:2.0
6297    7253    gene
                        locus_tag       C8J_0007
6297    7178    CDS
                        product chromosome partitioning protein, ParB family
                        protein_id      gnl|bbsrc|C8J_0007
                        inference       ab initio prediction:Fgenesb:2.0
7203    7253    terminator
8851    7191    gene
                        locus_tag       C8J_0008
7241    7191    terminator
8746    7283    CDS
                        product succinate-semialdehyde dehydrogenase
                        EC_number       1.2.1.16
                        protein_id      gnl|bbsrc|C8J_0008
                        inference       ab initio prediction:Fgenesb:2.0
8851    8792    promoter
...

Description of Fgenesb_annotator output fields:

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).
For example:


  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); notes:
- 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.