Hi,
Would you please assist me, I need to double check.
I use forward and reverse primers to get full length sequence. I amplified 426bp D-loop control region. But my full length sequence giving me 440bp (approximately). So, can I use 440bp (without trim off primers) or should be 426bp for final analysis?
thanks
Dear Habib
if you are using a specific primers, this means that they are part of your sequence and you don't need to remove them from the sequence. if you are using universal or degenerative primers, you should remove them before any further alignment.
this means that you should use only the original bp (in your case 426 bp) for further analysis.
Dear Ahmed,
Thanks for your reply. But I have been getting 440bp as full length sequence (before alignment). or let say after alignment also if i get 440bp or 432bp, then what should i do or can i simply delete the end of sequences (440-426=14bp or 432-426=6bp)?
thanks,
Dear Habib,
You could do your alignment and from there find and delete your forward and reverse primers. I would suggest to delete these sequences (or keep the last 3 nucleotides of primer sequences) so you are sure of your product. If your primers are specific, there is still a small chance you could have a mismatch of your primer with your template, however it is very likely than even if there is any mismatch (Primer-Template) at least the last 3 nucleotides of each primer are definitely rightly aligned. If after deleting your primers your sequence is still longer than expected, then it is fine, you may have an insertion in your template.
Dear Perez,
Thanks for your reply here. Could you please check here my 2 sequences (as an example) and show which part (primer) should trim off. In file, there is forward and reverse sequences and full length together. Full length is about 440bp. I also attached a file of my primers i used. 426bp of D-loop control region was amplified.
You also mentioned that if still there is longer sequences (here more than 426bp) after trim off primers then should do "insertion" or "deletion"? However, let say if less than 426bp, what should i do that case? Should/can I proceed analysis with less than 426bp sequences?
Thanks,
Ahmed, you also could see the file. according to you, as i don't use universal primers. so no need to remove. But how to get 426bp from 440bp. by simply deletion from end of the sequence or? thanks
Ahasan,
I have done a quick alignment of your sequence, deleted the primer sequences except for the last 3 nucleotides of each primer. You have now 405bp sequences. See sequences attached.
426bp is size you would "expect" of your sequence including primer sequences. The product size could change in the event of deletions or insertions in your sample. There is nothing you can do about that.
I deleted most of the primer sequences just to make sure you are having a more accurate sequence because it looks like you could get some variation in this part of the genome.
You could get more sequencing of the same samples to get more accurate sequences.
Best of luck,
Thanks. Could you please highlight the primer sequences before alignment/after alignment from my file which you trimmed off. I really want to see how/which you did. Thanks once again.
and its mean, if full length sequence is more than 426bp (as expected) after trim off primers, then also i can proceed with analysis?
thanks,
and primer seq.should delete before or after all seq. alignment? thanks
You could use the finder application in the toolbar of your alignment programme to find your primer sequence. In my first message I explained what to do.
I don't know the gene you are working with so I am not sure if ~400bp is enough. You will have to check this in the literature for your particular gene.
All the best,
finder toolbar in BioEdit or in which software? I use mtDNA D-loop (control) region. thanks
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