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I was experimenting Prokka and RAST annotation tools. So, I took a well-annotated swinepox virus genome from genebank (NCBI Reference Sequence: NC_003389.1).

I ran those sequences on Prokka and RAST Seed server at the same time. I can see that only a few (may be around 1%) of the genes were annotated. Most of them were predicted as hypothetical protein. And the results were comparable between Prokka and RAST.

I would assume that these tools look for similar sequences in NCBI and find the best-match protein. But looks like that is not the case.

I am looking for prediction tool(s) that will:

  • find a well annotated swinepox virus genome in Genbank to predict most of the proteins.
  • improve on the prediction of Prokka and Rast to assign functions to otherwise hypothetical protein

Would prediction tools such as Genemark or other genome annotation tools be beneficial.

Alternatively have I misunderstood the concept of annotation?

Assistance would be very welcome.

I have attached the image for comparison. Left one the Swinepox genome in .gb format and right one is the same genome annotated with Prokka.

Image : sorry for the low quality screenshot

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  • $\begingroup$ Please, provide links to the annotation servers along with all options you've specified. $\endgroup$
    – Eli Korvigo
    Commented Jul 20, 2018 at 19:47
  • $\begingroup$ I used Galaxy server. I chose viruses under kingdom. All the other parameters were kept as default. $\endgroup$
    – LoveVirology
    Commented Jul 21, 2018 at 19:29
  • $\begingroup$ Prokka works well on bacterial genomes. I quesiton whether it will be useful here. $\endgroup$
    – M__ ♦
    Commented Dec 23, 2018 at 22:48

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Both of the annotation tools you used are designed for prokaryotic genome annotation. I would not expect them to work very well for viruses.

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  • $\begingroup$ You can choose between Bacteria, Archea or Viruses when using prokka. So, I think it should work for viruses too. $\endgroup$
    – LoveVirology
    Commented Jul 21, 2018 at 19:33
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    $\begingroup$ @LRJoshi viruses (especially small ones) tend to have overlapping and nested ORFs: something a standard prokaryotic/eukaryotic gene detector (e.g. Glimmer, Genemark, Prodigal) is unlikely to account for. $\endgroup$
    – Eli Korvigo
    Commented Jul 21, 2018 at 19:47
  • $\begingroup$ Prokka can do viruses, but you may want to change which translation table it uses to get better blast results $\endgroup$
    – Joe Healey
    Commented Dec 24, 2018 at 10:47
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I was able to get good annotation using prokka by using following commands in prokka.

prokka --proteins reference.gb --outdir annotation  --prefix myprotein contigs.fa

All the reading frames were annotated. Actually the ones that are hypothetical in reference genomes are also annotated.

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  • $\begingroup$ I definitely agree that Prokka could be repurposed and think this is a good response. $\endgroup$
    – M__ ♦
    Commented Mar 8, 2022 at 16:02
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If you are looking for another tool, you could try eggnog mapper: it is an annotation tool based on orthology assignments. The EggNOG database happens to have a section for viral Orthologous Groups so you might obtain better results with it.

Note that emapper annotates coding sequences, not whole genomes. You need an extra step to predict CDS, for instance using Prodigal.

Basic usage with the optimized database for viral models:

prodigal -i my.genome.fna -o my.genes -a my.proteins.faa

then

python emapper.py -i my.proteins.faa --output polb_viruses  -d viruses
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  • $\begingroup$ Interesting, do you have a source on this? The documentation of eggnog-mapper mentions "the annotation of novel genomes". I am currently using it to annotate MAGs, so I'd rather know if I am doing something wrong. $\endgroup$
    – Nils Giordano
    Commented Jul 24, 2018 at 8:34
  • $\begingroup$ Eggnog resulted in empty file. Maybe it doesn't work for individual genomes as mentioned by @EliKorvigo $\endgroup$
    – LoveVirology
    Commented Jul 26, 2018 at 2:15
  • $\begingroup$ eggnog-mapper takes predicted proteins as input, not whole genomes. You should not use it on raw metagenomes either. You need an extra step to predict CDS with the tool of your choice (e.g. Prodigal). prodigal -i my.genome.fna -o my.genes -a my.proteins.faa then feed my.proteins.faa to eggnog-mapper. $\endgroup$
    – Nils Giordano
    Commented Jul 26, 2018 at 6:48
  • $\begingroup$ My wording has been quite a bit too harsh, I guess. I have only applied eggnog to metagenomes and metatranscriptomes, because in these cases it's not practical to use group-specific homology databases and HMMs. Eggnog's authors have even emphasised this point in their paper. On the other hand, when you do have a clean assembly, you can use a hierarchical group-specific annotator, such as Prokka. Of course, it doesn't mean one can't use eggnog for genomes, hence I find it necessary to apologise for my previous statement. $\endgroup$
    – Eli Korvigo
    Commented Jul 26, 2018 at 19:49

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