As I understand you have a gap in your alignment. It's normal. But what to do with it depend on what you plan to do with your alignment. If you plan to reconstruct some phylogeny, you can delete in or counting as 1 evolutionary event depend on used model.

Thanks Andrii. I would like to construct phylogenetic tree, phylogenetic analysis (Fst value using Arequine, DnaSP etc ). I normally do phylogenetic analysis, see the population structure of marine fishes. So can i just delete the gap ("----") and work with alignment? 

I didn't get what you mean by "counting as 1 evolutionary event". 

One more query Andrii, for my work, let say my amplified primer sequence was 426bp and after alignment i got 413bp, so can i proceed analysis with 413bp instead of 426bp? is that correct for analysis? 

Thanks, 

Dear Ahasan,

Of course, you can delete the gap in your alignment an continue to work with shorter matrix. But you also must to delete this region from all sequences in alignment.

As for your second question - yes, it is possible to obtain a little bit shorter or longer sequences because of some indels. You always able to check correctness of your product by BLAST in NCBI or inside of genome if it known. If the location and the region is correct, you can use it in phylogeny. But also very important to know how you determined the lenth of your product - is this capillary electrophoresis in sequencer, high resolution gel electrophoresis or simple base counting in obtainedd sequence?

But also note that some regions have a multiple copies inside the genome and some of these copies can be not the same. It is another more complicadet explanation why it can happened. But in this case most likely you'll obtain mutlipe products after PCR.

Best redards

Dear Andrii,

Thanks for your detailed reply. "I must able to delete this regions from all sequences in alignment", do you mean after I get alignment and then trim off primers from both end (forward primer sequence from front and reverse primers from end of aligned sequences)? 

By the way, I explain you how i do sequence alignment and do analysis.

I amplified 4226 bp D-loop (control region) using 2 primers (forward and reverse). After I get both sequences and make sure this is the exact species working with by BLAST in NCBI data base. After than using BioEdit, I took consensus from both forward and reverse sequence. I do "reverse compliment" for reverse sequence before i get the consensus sequence. After that, I do trim off few base/sequences from both end and keep (426bp) from each sample/species sequences. here note that, all 426 bp sequences give me the exact region/species when i BLAST in NCBI with 100% query cover. Finally, i put all sequences from all samples then do multiple alignment and get 426bp (with gaps/"---" but after delete gap also remain 426bp), then construct phylogenetic tree, find Fst value etc. Is that correct methods? 

How i determine the length? Primer designed for 426bp mtDNA D-loop (control region) gene or marker. It was from previous published paper.

Thanks,

Ahasan

Dear Ahasan,

If you obtained full sequence in both directions you are right (I hope for this because the sequences are short), but if not you must make a contig without reverse comlement.

As for your alignment, all will depend on what software you plane to use for phylogeny reconstruction. Some of it can ignore gaps, but some not.  It is not important to cut primer regions in phylogeny reconstruction, because these sites are overwrited and not variable.

thanks Andrii. 

Dear Ahasan, I believe this question, or something pretty similar, has been asked before, so I would search the Q&A for that.  In any case, gaps can be tricky.  My graduate student used simulation to study the effects of gaps in an alignment on phylogenetic accuracy, where she looked at various ways in which the gaps could be treated.  I am attaching the paper here.  Also, I would suggest using MEGA (now version 6) for your phylogenetic reconstruction.  It has different options for gap handling, apart of course, from being an extremely easy program to use.  Good luck.

Dear Sudhindra, Thanks for your answer with paper. I have been using MEGA 6 as well, but i didn't see any option to treat the gap! Could you please tell more details where to find, i mean should i treat gap while put the data for analysis? 

How to find genetic distance? I see there is option in MEGA but only can get pairwise and overall mean distance. 

thanks,

Ahasan 

Hi Ahasan, it appears that you might benefit from reading some of the material under "Resources" in the MEGA website (megasoftware.net).  For using the tool, I'd seriously suggest "A walk through MEGA."  Anyway, distances are typically calculated pairwise.  What other options were you looking for?

Hi Sudhindra,

Thanks for your reply. I will have a look on MEGA resources. Pairwise distance gives a very big excel file, i mean i got lot of pairwise distance. So i asked if there is any distance like mean distance can show only.

thanks,

I suggest POSORT for gap removing from alignment.  

The dashes represent insertions or deletions (together called indels because it is not always easy to know whether the gap(s) in a sequence are due to a deletion event in that taxon or an insertion event in other taxa).  Anyway, although there is evolutionary signal in indels and it is suggested that they be used as such, if you want to delete them for the purposes of phylogenetic analysis, there are multiple ways of doing so.  My student did a simulation study that, among other things, compares them for the purposes of phylogenetic accuracy (see pdf attached).  

A free and user-friendly program to use for exploring sequences, including deleting gaps using multiple methods is MEGA (http://www.megasoftware.net/)