[Sequencing] Electropherogram Analysis

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bbs_r
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[Sequencing] Electropherogram Analysis

Post by bbs_r » Sat Apr 01, 2006 11:23 am

Thanks for helping me alot! :lol:
As this is my first time to deal with an electropherogram, I have some trouble with it, which is shown in fig.1.

Again, it is a noob question. Since the machine returns "GNATTTTTGGGNTTACG", but according to the pic, there are some little peaks hidding within the "big" red peak. Do I need to count those little peaks (pointed by red arrows) into the sequence?

Besides, as you can see the letter "T" is returned corresponding to the big red peak (shown by green and blue arrows), how do I read this letter "T"? I mean by the green arrow position or I should take the middle point of the peak (which is the position of blue arrow)? <--- Sorry for my poor English, I hope you got what I mean.

Anyway, I read the sequence as: GCAGATAACGCTT.... What do you think? I mean if you don't mind may you do me an example please? I mean if you were me, and your receive such a picture, what sequence will you predict from it?

Thank you very much!
Attachments
2.GIF
fig. 1 sequence predicted from my PCR product cloned using Invitrogen pCRII vector and TOP10F E.Coli
2.GIF (6.93 KiB) Viewed 2373 times
1.GIF
fig. 2 same as fig.1 but no arrows applied
1.GIF (6.51 KiB) Viewed 2375 times

Trev
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Post by Trev » Sat Apr 01, 2006 12:59 pm

As I remember, the programm computes area under all peaks in current place. In Your case, area under the red peak is bigger then under other peaks. Thats why programm defined "T".
This red peak is big => three "T".
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bbs_r
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Post by bbs_r » Sat Apr 01, 2006 4:44 pm

Thanks for answering. Did you mean I have to ignore the tiny peaks under the big peak? In other words, those tiny peaks are useless?

Thank you!

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Post by Trev » Sat Apr 01, 2006 5:24 pm

In this case - yes, I think.

But I remember some cases, when I was correcting some definded (by computer) nucleotides, because, when quality of peaks is not good and in one place are many same peaks with different color, programm can make a mistakes
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Post by LilKim » Sun Apr 02, 2006 1:32 am

Many years ago... I remember having sequencing problems and lots of background noise.

Someone suggested that I did an alcohol precipitation before I sent my DNA away for sequencing ... and I tried it ... and everything was PERFECT afterwards.

Unfortunately, I don't remember the protocol right now (i'm at a coffee shop) However, if you're interested in the protocol that I used... I can hunt-through my old notebooks to see if I can find it somewhere..

please let me know if you're interested.

- KIM

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Post by bbs_r » Tue Apr 04, 2006 7:08 am

Thanks for your help! Maybe let's come and see the methods of my project. I am opening a new thread now..

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