How do you identify the specific start and stop locations of mitochondrial genes

[gwilymh]

Hi,

I am currently annotating a mitochondrial genome in Geneious. The genome was assembled using next-generation sequence data from one species, with the published mitochondrial genome of a closely related species used as a reference genome (NCBI reference HQ332445). The assembled and reference genomes are very similar, and the annotations from the reference genome seem to be quite transferable to the newly assembled genome (Geneious has functions for transferring annotations from a reference gene sequence to a consensus gene sequence; including generating translated protein sequences from cds).

I have noticed, however, that 3 or 4 of the mitochondrial coding genes in both the reference mitochondrial genome and the assembled mitochondrial genome do not actually start with start codons (ATG). There are start codons maybe 30-50 bases upstream of the start. I ran a quick BLAST search on the DNA and protein sequences of these genes, and got hits consistent with their current annotations.

Could the gene start positions have been misidentified by the researchers who originally annotated the reference mitochondrial genome? Surely all coding genes have to start with a start codon? Does this mean that the other gene locations on the reference mitochondrial genome could be inaccurate?

Are there any general guidelines/rules of thumb/ best practices for identifying where exactly genes start and stop on the mitochondrial chromosome?

I am still fairly new to annotating genes, so any general or specific suggestions are welcome.

[dsenalik]

I wonder how correctly your reference genome is annotated. Perhaps it was annotated by blast hits, ignoring ORFs.
And was it sequenced with a "homopolymer-susceptible" technology such as 454 or Ion torrent?

Our own question/dilemma in this same situation is whether it is scientifically acceptable/morally correct to adjust these homopolymer "errors" based only on ORF disruption.

e.g. "Well, the assembler says seven "A"s, but if it was eight, the ORF is correct, and 40% of the reads say eight"

But, of course, we should really design primers and check the regions in question, or just do an Illumina run. But, that costs more money. Still, that's what we will probably have to do.

I ramble, but I shall follow this thread with interest.