Hi all,
Are you familiar with Bioedit in the visualization of the sequences? I am trying to read sequences coming from Sanger sequencing method and I was said that the "genuine" sequence is usually that showing clear peaks when visualized in Bioedit. So I should leave out the edges of the sequences that are commonly messy. Do you know why these regions are usually messy? Do you have any further suggestion to give me when I have to decide if a sequence is reliable or not? Why (as a result of the sequencing) do I get two files for the same sequence(one forward and the other one reverse)? I know they have something to do with the primer sequences one provides for the sequencing but I don't understand why I need both.
Thanks
contenance monsieur...
@Silvia:
- yes, you have to leave out those 'messy' parts of the sequence. The poor quality at the beginning of the sequence is one of the many drawbacks of sanger seq. One of the reasons can be, that the Dye-Terminators have not been clompletely removed after the sequencing reaction. Sometimes, the sequences are legible right next to your primer binding site, but you cannot expect that.
- reagrding your second sequence: it is quite usual that one sequences the regions of interest from both directions. The quality of sanger seq is not always as you would hope it was (see above). So, to confirm your results you simply sequence from both directions using a forward and a reverse sequencing primer.
Also (if your seq-primers are not too far apart) the reverse sequence will cover the 'messy' start of your forward sequence where it is illegible. And vice versa.
- Bioedit: if your two sequences do indeed overlap, the reverse sequence should at least in part align with the forward sequence
>load both sequences in bioedit
>mark one of the sequences in the coulumn on the left side
>shift+contr+r (makes it reverse complement)
>mark both sequences (shift-click)
>Sequence>pairwise alignment>align two sequence (allow ends to slide)
>compare and validate
I hope that helps.
P.S.: even if I disagree completely with that other guys manners, I agree with some things he writes: a good book about molecular biology will help you a lot. Hang on! :)
contenance monsieur...
@Silvia:
- yes, you have to leave out those 'messy' parts of the sequence. The poor quality at the beginning of the sequence is one of the many drawbacks of sanger seq. One of the reasons can be, that the Dye-Terminators have not been clompletely removed after the sequencing reaction. Sometimes, the sequences are legible right next to your primer binding site, but you cannot expect that.
- reagrding your second sequence: it is quite usual that one sequences the regions of interest from both directions. The quality of sanger seq is not always as you would hope it was (see above). So, to confirm your results you simply sequence from both directions using a forward and a reverse sequencing primer.
Also (if your seq-primers are not too far apart) the reverse sequence will cover the 'messy' start of your forward sequence where it is illegible. And vice versa.
- Bioedit: if your two sequences do indeed overlap, the reverse sequence should at least in part align with the forward sequence
>load both sequences in bioedit
>mark one of the sequences in the coulumn on the left side
>shift+contr+r (makes it reverse complement)
>mark both sequences (shift-click)
>Sequence>pairwise alignment>align two sequence (allow ends to slide)
>compare and validate
I hope that helps.
P.S.: even if I disagree completely with that other guys manners, I agree with some things he writes: a good book about molecular biology will help you a lot. Hang on! :)
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