GFF files derived from Prokka genbank raise errors

[mgalardini]

Hi,

I'm using Roary with a bunch of bacterial genomes; some have been annotated with prokka, some others not. A genbank file is available for all of them. I've converted all the genbank files to gff3 using the bcbio gff writer (https://github.com/chapmanb/bcbb/tree/master/gff), which to the best of my knowledge produces valid GFF3 files.

When running using the prokka generated gff files the program runs smoothly; when running with the gff files derived from the genbank file, the program halts with the following error:

BLAST Database error: No alias or index file found for protein database [/home/user/workspace/Roary/bin/UcWJpjcOru/output_contigs] in search path [/home/user/workspace/Roary/bin::]

Some files are however still produced, like the gene_presence_absence.csv one, even though the genomes columns do not contain the locus_tag but either nothing or the EC_number (see below). A more detailed documentation on the expected GFF format (order of the anotations for instance) would maybe help?

Thanks a lot,
Marco

Example of annotation from prokka:

gnl|Prokka|GENOME02_contig000001   Prodigal:2.6    CDS     42      578     .       +       0       ID=GENOME02_00001;inference=ab initio prediction:Prodigal:2.6;locus_tag=GENOME02_00001;product=hypothetical protein;protein_id=gnl|Prokka|GENOME02_00001

Example annotation from the gff file converted from the prokka genbank file:

GENOME02_contig000001      feature CDS     42      578     .       +       0       codon_start=1;inference=ab initio prediction:Prodigal:2.6;locus_tag=GENOME02_00001;product=hypothetical protein;protein_id=Prokka:GENOME02_00001;transl_table=11;translation=MIAEIFQGGFVVFQQQFSKVHFEAATTHNAHHHDVGGFTAESEGRNLPAAQTQTFREVVQGVSRIFTIFQFEANRRDAFVRATRTDELIRPQFGDFIRQISGNLVRGVLYFGIAFTTEAQEFIVLCNYLTRRAGEVDGKSTNLTTQVVNVEHQFLRQRFFVTPDNPAAAQRSQTEFMA

gene_presence_absence.csv produced from the prokka gff files (6036 lines):

"group_4797","","hypothetical protein","3","3","1","","","","","","GENOME02_00001","GENOME03_01386","GENOME04_00768"

gene_presence_absence.csv produced from the gff files derived from the genbank (2472 lines):

"group_1","","","1","1","1","","","","","","","","EC_number=2.7.2.11"

[andrewjpage]

Hi Marco,
Thanks for reporting the issue. I'm away until Monday so I'm afraid I won't
get a chance to look at it until then.
Regards,
Andrew
On 28 May 2015 11:14, "Marco Galardini" notifications@github.com wrote:

Hi,

I'm using Roary with a bunch of bacterial genomes; some have been
annotated with prokka, some others not. A genbank file is available for all
of them. I've converted all the genbank files to gff3 using the bcbio gff
writer (https://github.com/chapmanb/bcbb/tree/master/gff), which to the
best of my knowledge produces valid GFF3 files.

When running using the prokka generated gff files the program runs
smoothly; when running with the gff files derived from the genbank file,
the program halts with the following error:

BLAST Database error: No alias or index file found for protein database [/home/user/workspace/Roary/bin/UcWJpjcOru/output_contigs] in search path [/home/user/workspace/Roary/bin::]

Some files are however still produced, like the gene_presence_absence.csv
one, even though the genomes columns do not contain the locus_tag but
either nothing or the EC_number (see below). A more detailed documentation
on the expected GFF format (order of the anotations for instance) would
maybe help?

Thanks a lot,
Marco

Example of annotation from prokka:
gnl|Prokka|GENOME02_contig000001 Prodigal:2.6 CDS 42 578 . + 0
ID=GENOME02_00001;inference=ab initio
prediction:Prodigal:2.6;locus_tag=GENOME02_00001;product=hypothetical
protein;protein_id=gnl|Prokka|GENOME02_00001

Example annotation from the gff file converted from the prokka genbank
file:
GENOME02_contig000001 feature CDS 42 578 . + 0 codon_start=1;inference=ab
initio
prediction:Prodigal:2.6;locus_tag=GENOME02_00001;product=hypothetical
protein;protein_id=Prokka:GENOME02_00001;transl_table=11;translation=MIAEIFQGGFVVFQQQFSKVHFEAATTHNAHHHDVGGFTAESEGRNLPAAQTQTFREVVQGVSRIFTIFQFEANRRDAFVRATRTDELIRPQFGDFIRQISGNLVRGVLYFGIAFTTEAQEFIVLCNYLTRRAGEVDGKSTNLTTQVVNVEHQFLRQRFFVTPDNPAAAQRSQTEFMA

gene_presence_absence.csv produced from the prokka gff files (6036 lines):
"group_4797","","hypothetical
protein","3","3","1","","","","","","GENOME02_00001","GENOME03_01386","GENOME04_00768"

gene_presence_absence.csv produced from the gff files derived from the
genbank (2472 lines):
"group_1","","","1","1","1","","","","","","","","EC_number=2.7.2.11"

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#130.

[mgalardini]

Hi,

no problem, I can surely wait until next week; thanks a lot for the amazing work done on this piece of software.

Marco

[andrewjpage]

Hi Marco,
I've just released a new version which I 'hope' will resolve the issue your having (2.3.0). Could you give it a whirl and let me know how you get along?
Andrew

[mgalardini]

Hi Andrew,

absolutely, I'll get back to you tomorrow at the latest, thanks a lot!
Marco

On Mon, Jun 1, 2015 at 4:04 PM andrewjpage notifications@github.com wrote:

Hi Marco,
I've just released a new version which I 'hope' will resolve the issue
your having (2.3.0). Could you give it a whirl and let me know how you get
along?
Andrew

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#130 (comment)
.

[mgalardini]

Hi,

unfortunately I've got a new error before the "usual" one:

2015/06/01 16:40:00 Could not extract any protein sequences from GENOME02.gbk.gff. Does the file contain the assembly as well as the annotation?
2015/06/01 16:40:03 Could not extract any protein sequences from GENOME03.gbk.gff. Does the file contain the assembly as well as the annotation?
2015/06/01 16:40:06 Could not extract any protein sequences from GENOME04.gbk.gff. Does the file contain the assembly as well as the annotation?
BLAST Database error: No alias or index file found for protein database [[...]/test_roary/87V8kzv_9H/output_contigs] in search path [[...]/test_roary::]

I can send you the gff files if you like; they all contain the nucleotide sequences as well.
Marco

[andrewjpage]

That would be great thanks. The email is roary@sanger.ac.uk or
andrewjpage@gmail.com

On 1 June 2015 at 16:45, Marco Galardini notifications@github.com wrote:

Hi,

unfortunately I've got a new error before the "usual" one:

2015/06/01 16:40:00 Could not extract any protein sequences from GENOME02.gbk.gff. Does the file contain the assembly as well as the annotation?
2015/06/01 16:40:03 Could not extract any protein sequences from GENOME03.gbk.gff. Does the file contain the assembly as well as the annotation?
2015/06/01 16:40:06 Could not extract any protein sequences from GENOME04.gbk.gff. Does the file contain the assembly as well as the annotation?
BLAST Database error: No alias or index file found for protein database [[...]/test_roary/87V8kzv_9H/output_contigs] in search path [[...]/test_roary::]

I can send you the gff files if you like; they all contain the nucleotide
sequences as well.
Marco

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#130 (comment)
.

[andrewjpage]

I've updated Roary (v2.3.1) to fall back to using locus_tags when an ID isnt available and the files sent are working now.

[mgalardini]

Thanks, it works like a charm!