EMBLmyGFF3 converts an assembly in FASTA format along with associated annotation in GFF3 format into the EMBL flat file format which is the required format for submitting annotated assemblies to ENA.
[ Similarly to prepare your data for submission to NCBI please use Genome Annotation Generator - GAG. ]
! NCBI and ENA are part of INSDC and their data are synchronised every day, so everything submitted in one of this DB will also be accessible in the other.
Based on documentation from:
- http://www.insdc.org/files/feature_table.html
- http://www.ebi.ac.uk/ena/WebFeat/
- ftp://ftp.ebi.ac.uk/pub/databases/embl/doc/FT_current.html#7.1.1
- ftp://ftp.ebi.ac.uk/pub/databases/embl/doc/usrman.txt.
You don't know how to submit to ENA ? Please visit the ENA: Guidelines and Tips
- GFF3 to EMBL conversion tool
Python >=3.9, biopython >=1.78, numpy >=1.22 and the bcbio-gff >=0.6.4 python packages.
In order to install pip please use the following steps:
Mac OS X / LINUX:
Install pip the python package manager:
sudo easy_install pipbiopython and bcbio-gff will be installed automatically with the next steps
conda install -c bioconda emblmygff3pip install git+https://github.com/NBISweden/EMBLmyGFF3.gitor if you do not have administartive rights on your machine
pip install --user git+https://github.com/NBISweden/EMBLmyGFF3.gitClone the repository:
git clone https://github.com/NBISweden/EMBLmyGFF3.gitMove into the folder:
cd EMBLmyGFF3/Install:
python setup.py installor if you do not have administartive rights on your machine:
python setup.py install --userExecuting:
EMBLmyGFF3or
EMBLmyGFF3 -hwill display some help.
pip install git+https://github.com/NBISweden/EMBLmyGFF3.git --upgradeor if you do not have administartive rights on your machine
pip install --user git+https://github.com/NBISweden/EMBLmyGFF3.git --upgradeMove into the repository folder and execute:
git pullpip uninstall EMBLmyGFF3A correct GFF3 file and the genome in FASTA format that has been used to produce the GFF3 file are the mandatory input files. Then, in order to get a valid EMBL flat file suitable for submission you have to fill carefully all mandatory metadata.
/!\ Please be aware that a project ID and an locus tag are mandatory for a submission to ENA. You don't need this information if you don't plan to submit the data (In case you just want an EMBL-like flat file for other purposes). If you don't have yet those information you can add them later by replacing the corresponding fields.
To learn how to obtain a project ID click here.
To learn how to obtain a locus tag click here.
Three examples are provided with the tool and are located in the examples folder. You can try one of the 3 examples by calling EMBLmyGFF3-maker-example or EMBLmyGFF3-augustus-example or EMBLmyGFF3-prokka-example accordingly. This way is convenient when you performed the installation using pip.
EMBLmyGFF3-maker-exampleIf you installed EMBLmyGFF3 using git those example files are located where you have cloned the repository in EMBLmyGFF3/examples/. You could also access these files by downloading the examples folder here. You can then try the examples moving in the examples folder and launching one of the .py or .sh executable like that:
python maker_example.pyor
./maker_example.shEMBLmyGFF3 maker.gff3 maker.faWill prompt you to fill one by one the mandatory information needed to produce a proper EMBL file. Once the software has all the information it needs, it will process the input files and will print the result to STDOUT.
In order to write the result in the desired file use the -o option:
EMBLmyGFF3 maker.gff3 maker.fa -o result.emblMinimum requirement to launch the software and avoid any prompt.
EMBLmyGFF3 maker.gff3 maker.fa \
--topology linear \
--molecule_type 'genomic DNA' \
--transl_table 1 \
--species 'Drosophila melanogaster' \
--locus_tag LOCUSTAG \
--project_id PRJXXXXXXX \
-o result.emblAdding more information than those mandatory (filling the ID).
EMBLmyGFF3 maker.gff3 maker.fa \
--data_class STD \
--topology linear \
--molecule_type "genomic DNA" \
--transl_table 1 \
--species 'Drosophila melanogaster' \
--taxonomy INV \
--locus_tag LOCUSTAG \
--project_id PRJXXXXXXX \
--rg MYGROUP \
-o result.emblAdding more information than those mandatory (filling the ID line + publication and authors information).
EMBLmyGFF3 maker.gff3 maker.fa \
--data_class STD \
--topology linear \
--molecule_type "genomic DNA" \
--transl_table 1 \
--species 'Drosophila melanogaster' \
--taxonomy INV \
--locus_tag LOCUSTAG \
--project_id PRJXXXXXXX \
--rg MYGROUP \
--author 'author for the reference' \
--rt 'reference title' \
--rl 'Some journal' \
-o result.emblYou may prefer to launch the software through a script especially when you want to fill many information, so we provide examples of such scripts in bash (.sh) or python (.py) in the examples folder.
Some parameters are mandatory and some others are not. Here is a list of all parameters available.
You can also find a comprehensive help about the different parameters using the software -h or --help command and even a more advanced help using --ah X or --advanced_help X where X is the parameter you would like to learn more about.
positional arguments:
| Parameter | Description |
|---|---|
| gff_file | Input gff-file. |
| fasta | Input fasta sequence. |
Mandatory Arguments related to the EMBL format to check carrefully:
| Parameter | Description |
|---|---|
| -i, --locus_tag | Locus tag. Used to set up the prefix of the locus_tag qualifier. It has to be registered at ENA prior any submission. More information here. The default is XXX. |
| -p, --project_id | Project ID. Default is 'XXX' (This is used to set up the PR line). |
| -r, --transl_table | Translation table. No default. (This is used to set up the translation table qualifier transl_table of the CDS features.) Please visit NCBI genetic code for more information. |
| -s, --species | Sample species, formatted as 'Genus species' or taxid. No default. (This is used to set up the OS line.) |
| -t, --topology | Sequence topology. No default. (This is used to set up the Topology that is the 3rd token of the ID line.) |
| -m, --molecule_type | Molecule type of the sample. No default value. |
Optional arguments related to the EMBL format:
| Parameter | Description |
|---|---|
| -a , --accession | Bolean. Accession number(s) for the entry. Default value: XXX. The proper value is automatically filled up by ENA during the submission by a unique accession number they will assign. The accession number is used to set up the AC line and the first token of the ID line as well. Please visit this page and this one to learn more about it. Activating the option will set the Accession number with the fasta sequence identifier. |
| -c , --created | Creation time of the original entry. The default value is the date of the day. |
| -d , --data_class | Data class of the sample. Default value 'XXX'. This option is used to set up the 5th token of the ID line. |
| -g , --organelle | Sample organelle. No default value. |
| -k , --keyword | Keywords for the entry. No default value. |
| -l , --classification | Organism classification e.g 'Eukaryota; Opisthokonta; Metazoa'. The default value is the classification found in the NCBI taxonomy DB from the species/taxid given as --species parameter. If none is found, 'Life' will be the default value. |
| -x , --taxonomy | Source taxonomy. Default value 'XXX'. This option is used to set the taxonomic division within ID line (6th token). |
| --de | Description. Default value 'XXX'. |
| --ra , --author | Author for the reference. No default value. |
| --rc | Reference Comment. No default value. |
| --rg | Reference Group, the working groups/consortia that produced the record. Default value 'XXX'. |
| --rl | Reference publishing location. No default value. |
| --rt | Reference Title. No default value. |
| --rx | Reference cross-reference. No default value. |
| Email used to fetch information from NCBI taxonomy database. Default value 'EMBLmyGFF3@tool.org'. | |
| --environmental_sample | Identifies sequences derived by direct molecular isolation from a bulk environmental DNA sample with no reliable identification of the source organism. May be needed when organism belongs to Bacteria. |
| --expose_translations | Copy feature and attribute mapping files to the working directory. They will be used as mapping files instead of the default internal JSON files. You may modify them as it suits you. |
| --force_unknown_features | Force to keep feature types not accepted by EMBL. /!\ Option not suitable for submission purpose. |
| --force_uncomplete_features | Force to keep features whithout all the mandatory qualifiers. /!\ Option not suitable for submission purpose. |
| --interleave_genes | Print gene features with interleaved mRNA and CDS features. |
| --isolate | Individual isolate from which the sequence was obtained. May be needed when organism belongs to Bacteria. |
| --isolation_source | Describes the physical, environmental and/or local geographical source of the biological sample from which the sequence was derived. Mandatory when environmental_sample option used. |
| --keep_duplicates | Do not remove duplicate features during the process. /!\ Option not suitable for submission purpose. |
| --keep_short_sequences | Do not remove short sequences (< 100bp) during the process. /!\ Option not suitable for submission purpose. |
| --locus_numbering_start | Start locus numbering with the provided value. |
| --no_progress | Hide conversion progress counter. |
| --no_wrap_qualifier | By default there is a line wrapping at 80 characters. The cut is at the world level. Activating this option will avoid the line-wrapping for the qualifiers. |
| --translate | Include translation in CDS features. |
| --use_attribute_value_as_locus_tag | Use the value of the defined attribute as locus_tag. |
| --version | Sequence version number. The default value is 1. |
| --strain | Strain from which sequence was obtained. May be needed when organism belongs to Bacteria. |
Optional arguments related to the software:
| Parameter | Description |
|---|---|
| --ah, --advanced_help | Display advanced information of the parameter specified or of all parameters if none specified. |
| -h, --help | Show this help message and exit. |
| -v, --verbose | Increase verbosity. |
| -q, --quiet | Decrease verbosity. |
| --shame | Suppress the shameless plug. |
| -z, --gzip | Gzip output file. |
| --uncompressed_log | Some logs can be compressed for better lisibility, they won't. |
| -o , --output | Output filename. |
The challenge for a correct conversion is the correct mapping between the feature types described in the 3th column as well as the different attribute’s tags of the 9th column of the GFF3 file and the corresponding EMBL features and qualifiers.
If you figure out that a feature type or an attribute's tag is not mapped to the corresponding EMBL features or qualifiers you would like, you will have to modify the corresponding information in the mapping files.
The software will skip the unknown feature types (Non EMBL feature types that are not mapped against an EMBL feature type) and the unknown qualifiers (Non EMBL qualifiers that are not mapped against an EMBL qualifier) and will inform you during the conversion process. If you want to include them within the output, you can add the information needed in the corresponding mapping file.
To access the json mapping files launch the following command:
EMBLmyGFF3 --expose_translationsThe command copy the json mapping files localy. You can then modify them as it suits you. When a json mapping file is present localy, it will be used instead of the default internal one.
The EMBL format accepts 52 different feature types whereas the GFF3 is constrained to be a Sequence Ontology term or accession number (3th column of the GFF3), but nevertheless this constitutes 2278 terms in version 2.5.3 of the Sequence Ontology.
The file handling the proper mapping is translation_gff_feature_to_embl_feature.json
example:
"three_prime_UTR": {
"target": "3'UTR"
}This will map the three_prime_UTR feature type from the 3th column of the GFF3 file to the 3'UTR EMBL feature type. When the feature type from the GFF3 is identical to the EMBL feature no need to specify any target. If a target is needed and you didn't specified it, the tool will throw a warning message during the process.
You can decide which features will be printed in the output using the remove parameter:
"exon": {
"remove": true
}Like that no exon feature will be display in the output.
The embl format accepts 98 different qualifiers where the corresponding attribute tag types in the 9th column of the GFF3 are unlimited. The file handling the proper mapping is translation_gff_attribute_to_embl_qualifier.json
example:
"Dbxref": {
"source description": "A database cross reference.",
"target": "db_xref",
"dev comment": ""
},This will map the Dbxref attribute's tag from the 9th columm of the GFF3 file to the db_xref embl qualifier.
The source (2nd column) as well as the score (6th column) from the GFF3 file can also be handled through the translation_gff_other_to_embl_qualifier.json mapping file.
"source": {
"source description": "The source is a free text qualifier intended to describe the algorithm or
operating procedure that generated this feature. Typically this is the name of
a piece of software, such as Genescan or a database name, such as Genbank. In
effect, the source is used to extend the feature ontology by adding a qualifier
to the type creating a new composite type that is a subclass of the type in the
type column.",
"target": "note",
"prefix": "source:",
"dev comment": "EMBL qualifiers tend to be more specific than this, so very hard to create a good
mapping."
},This will map the source from the 2nd columm of the GFF3 file to the note embl qualifier.
/!\ Please notice the prefix allows to add information dowstream the source value wihtin the qualifier (Upstream information is also possible using suffix).
e.g: The source value is "Prokka":
Within the embl file, instead to get note="Prokka", here we will get note="source:Prokka"
The output can be validated using the ENA flat file validator distributed by EMBL. Please visit http://www.ebi.ac.uk/ena/software/flat-file-validator and/or https://github.com/enasequence/sequencetools for more information.
biopython version There's a bug between bcbio-gff 0.6.4 and biopython 1.68 though, so use biopython 1.67.
If you have several version of biopython or bcbio-gff on your computer it is possible that an incompatible version is used by default which will lead to an execution error. To check the real version used during the execution you can use this command:
python -c "import Bio; from BCBio import GFF; print 'biopython version: '+Bio.__version__; print 'bcbio-gff version: '+GFF.__version__"Duplicated Features
Features that have the same key (feature type) and location as another feature are considered as duplicates and aren't allowed by the EMBL database. So they are remove during the process. If you don't plan to submit the file to ENA and you wish to keep these features, use the --keep_duplicates option.
Norling M, Jareborg N, Dainat J. EMBLmyGFF3: a converter facilitating genome annotation submission to European Nucleotide Archive. BMC Res Notes. 2018 Aug 13;11(1):584. doi: 10.1186/s13104-018-3686-x.
And
Martin Norling1,2, Niclas Jareborg1,3, Jacques Dainat1,2
1National Bioinformatics Infrastructure Sweden (NBIS), SciLifeLab, Uppsala Biomedicinska Centrum (BMC), Husargatan 3, S-751 23 Uppsala, SWEDEN.
2IMBIM - Department of Medical Biochemistry and Microbiology, Box 582, S-751 23 Uppsala, SWEDEN.
3Department of Biochemisty and Biophys-ics, Stockholm University / SciLifeLab, Box 1031, S-171 21 Solna, SWEDEN.