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-   -   Sequencing the same part: how many possible way? (http://seqanswers.com/forums/showthread.php?t=9866)

IgorFobia 03-04-2011 08:35 AM

Sequencing the same part: how many possible way?
 
Hi,

I don't know a lot about sequencing, my question should be trivial but I cannot find the answer on the web...

Suppose that a genome contains a certain sequence, like AGTACATGG.

My question is: if I sequence this genome, in how many possible ways can this sequence be represented in my data? Does it depend on the sequencing platform?

Here is the list of all the potential possibilities, as far as I can think:

1: "original"
AGTACATGG

2: reverse complement
CCATGTACT

3: complement
TCATGTACC

4: reverse
GGTACATGT

Thank you very much for your help!

fkrueger 03-04-2011 08:53 AM

There is a convention that DNA in biological data is always represented in a 5' to 3' orientation. Thus, you will only come across your original sequence and its reverse complement, but not the other two cases.

IgorFobia 03-04-2011 09:35 AM

Thank you for your very fast answer.

So we know the 5' to 3' orientation even for sequenced (not annotated) reads, don't we?

krobison 03-04-2011 09:40 PM

Yes, the chemistry for all of the sequencing approaches in current use always proceed directionally.

As a historical note, with the original Maxam-Gilbert sequencing method it was possible to read both 5'->3' and 3'->5'. I had the misfortune as a 1st year graduate student to not know this & wonder why an entire cosmid worth of bacterial data had absolutely no open reading frames that looked like anything in the world (indeed, it seemed to be very sparse on long open reading frames). Then it occurred to the person who gave me the data to mention that it needed reversing first.

During the completion of the E.coli genome, Blattner's group at U. Wisonsin discovered one piece of E.coli sequence in Genbank which simply didn't match their data. They checked & indeed the original sequence had been Maxam-Gilbert and was deposited in Genbank backwards (no, I was not the depositor!).


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