Hello my Dear,
I have a question about CALR chromatogram analysis. I sequenced and aligned the amplified sequences with the reference sequences but I'm having trouble identifying the anomalies there. The problem being that I can have any sorte of modification (ins/del or del or ins or transversion see all at once). I tried to BLAST the potential pathological sequences but I'm having trouble understanding what comes out of it.
When I align with the normal protein reference sequence I have 20 aa aligned 100% and when I do the same thing with the mutated protein I have 36 aa aligned 100%. The expected protein is 60 aa and the affected portion is exactly where the two proteins align 100%.
A little help would woud not be refused.
Sincerely!
As mentioned earlier by Dr @Paul the. Abi chromatogram will be of help in tackling this question.
Here are an exemple of what i am dealing with hoping you'll be able to see what i missed. Thanks Dr Paul Rutland and Joseph Japhet
Thank you for the sequences Somda Georgina Charlene Soro . The sequencing is good and you are right that when a good sequence becomes 2 sequences overlying each other then the likely cause is an insertion/deletion.
What you do is look at the forward sequence and compare it to the reference sequence. At some point it becomes 2 sequences. You read both sequences then subtract the reference sequence at each position . This leaves the real sequence on the mutated chromosome which you then compare to the reference sequence and you usually find that it matches the reference sequence but at a slightly different position in the reference sequence and this difference defines the insertion or deletion. For instance if your double sequence is at,tc,tc,ga,gg,ga but the known sequence is attggg then the sequence on the mutated chromosome is tccaga and finding this new sequence in the reference sequence defines the indel.
In your case the change looks to me like a deletion of GGA at the position where the forwards and reverse sequences change from being 1 good sequence to 2 overlying sequences
Thank you Dr Paul Rutland, your answer was of great help to me however I wonder how did you discriminate between the 2 overlaying sequences? Are there any tools on Ugene or Bioedit to do it or did you do it manually? And also what reference sequence did you use? The genomic one or the cDNA one?
Sincerely.
Hi Somda Georgina Charlene Soro ,
I BLASTed the unique sequence to get a short online sequence
Homo sapiens voucher GBL49746 calreticulin (CALR) gene, partial cds
Sequence ID: KU639688.1Length: 307Number of Matches: 1
and used this as a reference..
I do not know of any way of doing the analysis in Bioedit or Ugene. I did it by eye....aligning the sequences in Sequencher and subtracting the known expected sequence. I did once come across a paper on decomposition that may be of interest to you so I will attach it
Thanks a lot Dr Paul Rutland I really appreciate your help
Paul Rutland I'm comming back to you cause I couldn't analyse the chromatograms as you did. Can you please send me the document you mentioned ? Thanks
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