RAST annotation --> Artemis

[Bacteria Genomes]

Hello!
I used RAST to annotate my bacterial genomes and am now having trouble with the output files in Artemis. Artemis seems to be having trouble with the fact that I have multiple contigs and is putting all the genes on the first contig so they all end up on top of each other. If I reduce the annotation file to just the genes in the first contig though it isn't correctly putting the genes in the reading frames so there seems to be multiple problems going on. I have played around with all the different file outputs but they all seem to have problems.

Anyone know how to fix this?

Thanks!

[nickloman]

One option is to join the GenBank entries together with the "union" tool in EMBOSS.

Also the annotation pipeline we developed locally will produce Artemis-ready files http://xbase.ac.uk/annotation/

[maubp]

What file format are you feeding into Artemis? GenBank? GFF3?

[Bacteria Genomes]

I had this problem with GFF3 and GTF files. I have also opened the Genbank and EMBL files from RAST in Artemis but they only show the first contig listed (which for some reason RAST decided should be contig 11...) but these files in Artemis seem to not have all the annotation info that the GFF3 and GTF files have.

[Bacteria Genomes]

One option is to join the GenBank entries together with the "union" tool in EMBOSS.

I looked into this and as far as I could tell it only works for fasta sequences.
Any other suggestions for a similar solution?

[maubp]

I looked into this and as far as I could tell it only works for fasta sequences.

No, it just defaults to output of fasta sequence. Try something like this:

union -sequence cat_three.gb -sformat genbank -outseq union_three.gb -osformat genbank -auto

For more info,

tfm union

OLD: That was the good news. The bad news is that it didn't keep the features (I have EMBOSS:6.3.1), which is probably an enhancement request...

http://lists.open-bio.org/pipermail/emboss/2010-November/004012.html

Would a short Biopython script to merge multiple GenBank records into a single record with the concatenated sequence and all the features be of interest?

NEW: You must also explicitly ask union to keep the features, see below.

[nickloman]

There's a toggle if you want to keep the features, I think it is "-feature Y"

See this script for a worked example:

https://github.com/nickloman/xbase/blob/master/annotation/make-art-file.py

[maubp]

There's a toggle if you want to keep the features, I think it is "-feature Y"

You are right, thanks. e.g.

union -sequence cat_three.gb -sformat genbank -outseq union_three.gb -osformat genbank -feature Y -auto

[Bacteria Genomes]

That got around the problem! Thanks for your help!

[zhangju]

I like to confirm the command to run make-art-file.py script.

<python make-art-file.py small1.art small2.art>

small1.art and small2.art are two genbank files to be concatenated for Artemis load at once.

Is this command right?

[nickloman]

Not exactly.

Concatenate your two Genbank files into a single file, e.g.

cat seq1.gb seq2.gb > both.gb

Then run make-art-file.py with input from the shell, e.g.

python make-art-file.py < both.gb

Hope that helps

[zhangju]

I got lots of syntaxError at "stdin". I copy part of error report and part of input genbank file below. Could you take a look?

(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** NameError: name 'TLFRSINLIPFYSIMEYISGSPAITNALAFANVAGNIVIFIPLGIYLPLFKNDKRAIT' is not defined
(Pdb) *** NameError: name 'NLLFILIVSLFVEITQGLLGIGASDIDDVILNCLGGWIGILGYKFSLFILRDEKIVHT' is not defined
(Pdb) *** SyntaxError: EOL while scanning string literal (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** SyntaxError: invalid syntax (<stdin>, line 1)
(Pdb) *** NameError: name 'IDGHGEDGDEEFISIQDCARKLIEYIDTNCNSQVFAMGGLSIGAQIVTEVLSQREKIT' is not defined
(Pdb) *** NameError: name 'DYAIIESVLVYPIRGTTALTVPVYKLFYGLIKKKWFAGMQAKTLCVPLDMFEQYYQDS' is not defined
(Pdb) *** NameError: name 'LKISRQSLINITLSNGNYNLNECIADTKTKVLIIVGENEVGIMKKSARLLHDKIPGSV' is not defined

Input file

LOCUS Ct_contig00020 105381 bp DNA linear 27-JUL-2011
DEFINITION Clostridium termitidis : Ct_contig00020
ACCESSION unknown
KEYWORDS .
COMMENT -
FEATURES Location/Qualifiers
source 1..105381
/mol_type="genomic DNA"
/db_xref="taxon:29371"
/organism="Clostridium termitidis"
gene 1..558
/locus_tag="Ct_00004390"
/gene_calling_method="Prodigal"
/note="IMG gene_oid=2504589426"
CDS 1..558
/locus_tag="or0446"
/translation="GIMNKRERIKTAFLYGAFICYILLLMKILLLSRISILGLFNNER
TLFRSINLIPFYSIMEYISGSPAITNALAFANVAGNIVIFIPLGIYLPLFKNDKRAIT
NLLFILIVSLFVEITQGLLGIGASDIDDVILNCLGGWIGILGYKFSLFILRDEKIVHT
AITILSVIIGLPVTLYFLFIIKMRF*"
/product="Glycopeptide antibiotics resistance protein"
/note="IMG gene_oid=2504589426"
gene 660..1406
/locus_tag="Ct_00004400"
/gene_calling_method="Prodigal"
/note="IMG gene_oid=2504589427"
CDS 660..1406
/locus_tag="or0447"
/translation="MIFKETPNKQMPTIILLHGGGLSSWSLNSIVEQLQSDFHIITPI
IDGHGEDGDEEFISIQDCARKLIEYIDTNCNSQVFAMGGLSIGAQIVTEVLSQREKIT
DYAIIESVLVYPIRGTTALTVPVYKLFYGLIKKKWFAGMQAKTLCVPLDMFEQYYQDS
LKISRQSLINITLSNGNYNLNECIADTKTKVLIIVGENEVGIMKKSARLLHDKIPGSV
LYTAPGMKHGELSLKYPLKYVDLLKSFFCK*"
/product="hypothetical protein"
/note="IMG gene_oid=2504589427"

[nickloman]

What platform are you running the script / EMBOSS on? It looks a bit like the line format of the file might be wrong. If on UNIX lines should end with the newline character \n.

[zhangju]

I am using Linux Redhat. Is there a way I can attach the original file I am using for your reference. Since I copied from text editor, "\n"s may not show up.

[maubp]

Input file

LOCUS       Ct_contig00020        105381 bp    DNA     linear   27-JUL-2011
DEFINITION  Clostridium termitidis : Ct_contig00020
ACCESSION   unknown
KEYWORDS    .
COMMENT     -
FEATURES             Location/Qualifiers
     source          1..105381
                     /mol_type="genomic DNA"
                     /db_xref="taxon:29371"
                     /organism="Clostridium termitidis"
     gene            1..558
                     /locus_tag="Ct_00004390"
                     /gene_calling_method="Prodigal"
                     /note="IMG gene_oid=2504589426"
     CDS             1..558
                     /locus_tag="or0446"
                     /translation="GIMNKRERIKTAFLYGAFICYILLLMKILLLSRISILGLFNNER
                     TLFRSINLIPFYSIMEYISGSPAITNALAFANVAGNIVIFIPLGIYLPLFKNDKRAIT
                     NLLFILIVSLFVEITQGLLGIGASDIDDVILNCLGGWIGILGYKFSLFILRDEKIVHT
                     AITILSVIIGLPVTLYFLFIIKMRF*"
                     /product="Glycopeptide antibiotics resistance protein"
                     /note="IMG gene_oid=2504589426"
     gene            660..1406
                     /locus_tag="Ct_00004400"
                     /gene_calling_method="Prodigal"
                     /note="IMG gene_oid=2504589427"
     CDS             660..1406
                     /locus_tag="or0447"
                     /translation="MIFKETPNKQMPTIILLHGGGLSSWSLNSIVEQLQSDFHIITPI
                     IDGHGEDGDEEFISIQDCARKLIEYIDTNCNSQVFAMGGLSIGAQIVTEVLSQREKIT
                     DYAIIESVLVYPIRGTTALTVPVYKLFYGLIKKKWFAGMQAKTLCVPLDMFEQYYQDS
                     LKISRQSLINITLSNGNYNLNECIADTKTKVLIIVGENEVGIMKKSARLLHDKIPGSV
                     LYTAPGMKHGELSLKYPLKYVDLLKSFFCK*"
                     /product="hypothetical protein"
                     /note="IMG gene_oid=2504589427"

Using the [ code ] text [ /code ] tags on the forum makes this kind of thing easier to read.

The stop codon is not normally included in the amino acid translation string (although it should be included within the CDS and gene co-ordinates). That probably isn't the problem though given the error messages.