This is a follow up question to this post: How can I find out if my gene of interest is duplicated?
Briefly, to identify homologous genes, I performed tBLASTn analyses against my genome of interest using various sequences of a closely related species as queries for the BLAST analyses. Using this method, I found two (and in some cases up to five copies) of my different genes of interest in the target genome. These genes are involved in different organelle functions (i.e., they are not surface antigens) and I want to find out whether these sequences represent duplication events or not. These analyses are being performed on protozoans parasites that belong to the same genus. The query genome is properly annotated, but the target genome is not (it is a whole genome shotgun project that has been assembled at the scaffold level).
Following the advice of @Michael G., I re-analyzed my sequences and blasted them against the entirety of Genbank. If there are two sequences for the same protein (for e.g. protein 3-1 and protein 3-2) and I compare them among themselves they are very similar (over 70% identity at the protein level). When I blast each sequence against the whole protein RefSeq database, the first hit is my query and the rest of the hits correspond to the same protein in other organisms. I believe that these sequences represent duplication events and it is possible that they are paralogues, but more evidence is needed to classify them as such.
However, when there are more than two sequences for the same protein, I found something different. For e.g., say I have three sequences for the same protein: sequence 5-1, sequence 5-2 and sequence 5-3. Sequences 5-2 and 5-3 are very similar (78% identity at the protein level) but when I compare sequences 5-1 and 5-2 I find that they are overlap in the middle, i.e., they are part of the same sequence. Thus, it appears that I don't really have three sequences for this protein but two. Blastp analyses against the whole RefSeq database give the expected result (first hit is my query sequence, the rest of the hits are same sequence in other organisms). So, once again I have what appears to be a duplication event, but in this case one of the sequences appears to be split in two -- could this be because the target genome is not completely assembled? Why could this be happening?
Thank you very much for your help.
Edit: The new results are posted in this thread: Duplicated genes and genome fragmentation - Part 2