I have sequenced purified amplicons by Sanger sequencing. I want to create a consensus sequence contig for each amplicon. Using the .ab1 files I trimmed the ends of the forward and reverse sequences where scores dropped below 20.
I obtained the reverse compliment of the reverse sequencing and aligned it to the forward with a pairwise global alignment in BioEdit. I expected to have to go back and fill/correct in any mismatches using the chromatograms. However, I feel there are many mismatches throughout the alignment - more than would be expected.
Is this normal? Have I made an error during my editing steps?
Can you attach the original .abi sequence files please?. How big is your amplimer
Paul Rutland Hi, thank you for the response. The amplimer is around 1100 bp. I've attached a forward and reverse .abi files that were obtained from sequencing.
Also to mention, I sent 5 samples for sequencing as a trial run (all for the same gene). I've uploaded the files for one of those samples but I have this issue for all the samples that I have reads for.
Hello Leah,
the first 40 bases roughly are rubbish when sequencing so If we edit out the first 35 bases in both F and R. Then the signal strength is falling and the background rising so let us edit out bases 760-end of the F sequence and also 760 -end of the R sequence..
Now with an 1100 base amplimer we expect a good overlap between the F and R sequences.
But.....if we BLAST the F sequence we get bases 1-755 of the trimmed sequence is the same as bases 60-814 of AMA1. Good so far.
However when we BLAST the R sequence we get 1-441 of trimmed is 1844-1404 of AMA1 and also 508-732 of the trimmed R is the same as 1337-1113 of AMA1. So we have about about 64 bases that do not align that you may want to look into.
Anyway the problem is that bot sequences are very good but getting weaker and you have sequence from 1844-1113 and 814-60 of AMA1 so no actual overlap between the F and R sequences. As both sequences are very good the simplest solution is to accept that you are getting 700 bases of good sequence and design internal sequencing primers which give a strong sequence over the missing 1113-814 part of your sequence so your sequencing primers might be 1-20 forwards,1884-1864 full reverse and maybe 1337-1317 internal reverse and 750-770 internal forwards ( those numbers for example only).This should fill in the sequence gap from 814-1113.
These figures do suggest that your amplimer is,in fact, larger than the 1100bp that you expect and looks more than 1800 bases. You may need to do some checking of your sequence
Paul Rutland Thank you for looking into this in depth. I just double checked and my expected size was actually 1907bp (sorry I've been working with another gene around 1100bp). So in that regard, it seems fine.
Is it not odd for the forward and reverse sequences to have captured different parts of the amplicon?
Good that solves one problem. It is entirely normal that the F and R primers will sequence different ends of the product since the primers are designed to anneal at each end of the pcr product.Nobody gets 1000 bases of good sequencing from Sanger sequencing so your sequences are just too short to meet each other and overlap so you need to have internal sequencing primers such that you sequence bases 1-600,500-1300 and 1200 to 1800 to get good overlaps. You do not need new amplimers just more sequencing primers for the same good quality pcr product that you are already sequencing
Paul Rutland Thank you so much! You've been immensely helpful. This was driving me crazy for a while.
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