blast to find gene from an organism based on other organism

Question

[najibveto]

hello i am working on fish and i need to know the sequence of tumor necrosis factor alpha in fathead minnow fish. in nbci , i found just the partial sequence of the gene and i need the full sequence to do my experiment. i looked in ncbi and i found the genome for the fish and the annotation file separately. how can i combine the genome and the annotation? how can i find the gene based on blast and using the gene sequence from relative organism? thanks for your help.

[GenoMax]

Is the gene present/annotated in the annotation file? If so, it should be matter of finding the coordinates in that file. No blast needed. If not, take the sequence from a relative and blast against the genome to identify possible candidates.

[najibveto]

the gene is not present in the annotation file. and the blast of gene from zebrafish (the closed relative to my fish of interest) against the genome didn't give a good result. can you suggest me other methods? which software to use to merge the annotation file and the genome file? thanks for your help.

[GenoMax]

As things generally work, annotation is created using the genome sequence (which comes first). So assuming things were done right, it is possible that the genome sequence as it exists does not have sequence of gene/region you need (another indication is the inability to find anything using a related sequence, though that may be a subjective thing).

You may need to think about an experimental strategy instead of an informatic one. Since you know partial sequence so primer walking/fishing through clone libraries. Designing degenerate primers (based on sequence of the related species) and trying PCR. Find/create an RNAseq dataset to look for the gene you want.

[najibveto]

sorry for my mistake i will write it again correctly.

[GenoMax]

Please use ADD REPLY/ADD COMMENT when responding to existing comments to keep threads logically organized. This should have been placed as a comment against @Michael's post above.

[Michael]

Please don't post screen shots of your console, copy the text from the console and paste into a comment instead!

Answer 1

[Michael]

Try to get the protein sequence of your gene of interest in the closest relative, then use exonerate -model protein2genome with the protein sequence and the genome. Depending on the level of conservation of your protein, this might give you a better gene model than the original gene prediction.

[harold.smith.tarheel]

+1. Exonerate is available here.

[najibveto]

thanks a lot for your help. i tried the suggest program under windows using cygwin but i couldn't do it. can you please help in this procedure? thanks a lot.

[Michael]

What do you mean by i couldn't do it? See also these links: https://www.biostars.org/t/exonerate/ and http://www.ebi.ac.uk/about/vertebrate-genomics/software/exonerate-manual

[najibveto]

hello sorry for my mistake my console log is as follow:

1. $ cd exonerate-2.2.0-cygwin
2. $ cd bin
3. $ exonerate -model protein2genome tnfa.fasta fatheadminnow.fasta
4. -bash: exonerate: command not found

thanks for your help.

[GenoMax]

You either need to provide full paths to all files in question or amend $PATH to include the directories needed here.

For now try: ./exonerate -model protein2genome tnfa.fasta fatheadminnow.fasta

[najibveto]

thanks for your help. i tried it but without results. i provided the full paths as follow but no results.

1. adam@najib /bin/exonerate-2.2.0-cygwin/bin
2. $ ./exonerate -model protein2genome ‪D:\tnfa.fasta ‪D:\GCVQ01.1.fasta

[GenoMax]

Without any further information there is nothing we can help you with. Was there an error or just nothing?

[najibveto]

i did like this and didn't give error but when i checked for results file i couldn't find: 1.adam@najib /bin/exonerate-2.2.0-cygwin/bin

1.$ ./exonerate -model protein2genome ‪D:\tnfa.fasta ‪D:\GCVQ01.1.fasta

1.adam@najib /bin/exonerate-2.2.0-cygwin/bin

is it possible there is error in genome file? because the file format i download was fna from ncbi database.