How to properly modify genome .gff and .fa to combine 2 loci that are really 1 gene

Question

[michael.nagle]

There are 2 loci in a genome that are separately annotated and were thought to be two separate genes. Experimental evidence from cDNA sequencing now confirms that they are actually one gene. Now I need to modify the .gff and .fa files and re-do bowtie/tophat/cufflinks analysis to find corrected expression values for this combined genes. I have a few questions for this.

1. Column 8 refers to: "frame - One of '0', '1' or '2'. '0' indicates that the first base of the feature is the first base of a codon, '1' that the second base is the first base of a codon, and so on.." What's the best way to determine what this should be? I could do it by eye, but is there software that is best for this?

2. Is there software that I can use to use my CDS sequencing data to generate corrected data for these .gff and .fa files? Is the best way to modify these using a text editor?

Thanks for the help.

Answer 1

[WouterDeCoster]

To partially answer your first question, exons don't necessarily consist of a number of nucleotides dividable by 3. It's perfectly possible that one nucleotide of a codon is in exon 42 and the remaining two nucleotides are in exon 43.

[michael.nagle]

Of course, modified question to ask if this needs to be determined by eye for every exon or if there's software for this.

Answer 2

[Juke34]

You could use this service to look at the longest ORF in each exon. For the first cds piece it's easy it is 0 (If you have your complete gene)

A good way would be to load your two genes in a genome browser allowing manual curation, and you merge your genes manually, then you download the result in gff format.