Do you have to sequence both strands?

Steven Coon at umich.edu
Thu Apr 14 16:19:47 EST 1994


In article <Co5xDx.5ru at world.std.com>, vawter at world.std.com (Lisa Vawter)
wrote:

> In article <01HARUDG2ENK005IEC at crcvms.unl.edu>,
> TOM PARSONS <TPARSONS at CRCVMS.UNL.EDU> wrote:
> >Dear Netters;
> >
> >    While I agree in general that compressions require sequence to be determined
> >on both strands to have any confidence that you've got it right, I would
> >like to present a situation where I do not think it is necessary to
> >always sequence both strands.  We are a molecular systematics lab, and we have
> >just finsished sequencing the mitochondrial COII gene from 19 species of
> >black flies.  In all the species, the gene is 690 base pairs in length, 
> >it doesn't have any particularly trecherous regions of compression, and its
> >amino acid sequence is quite conserved across taxa.  If I were to obtain nice
> >looking, completely overlapping sequence from only one strand of the next 
> >black fly species, and the sequence maintained reading frame, aligned 
> >sensibly (conservative replacements) and was 690 base pairs in length, I would
> >deem it a waste of time and (ultimately) taxpayers' money to sequence the other
> >strand. Assuming replicated reading and no ambiguities, the chances that a
> >compression has fooled me, while retaining the above characteristics seem
> >to me to be very low, and the chances that such an error would cause me to
> >draw incorrect phylogenetic conclusions are just as low again.  Sure, it is 
> >possible, but it is also possible that a combination of unlikely artifacts
> >introduces errors into sequence determined from both strands.
> >
> >     I should note that almost all of our sequences have been determined 
> >completely from both strands, .....
>  


I totally agree with this post because it also preferable to have
corroborating evidence that something is correct from more than one
experiment or method. It would be like trying to prove a hypothesis when
you only did one experiment which happened to work. There is also evidence
that some bands are missing in some reactions causing you to read out of
frame which you would catch by sequencing the other strand. I also think it
impossible to unambiguously read any sequence (maybe I have lead a deprived
life) and since one strand should
confirm the other it almost prove it correct. If I sequenced your strand
both ways and found a mistake with your published sequence it would't make
things look very good now would it? I understand the need to be fast but I
think an embarassing mistake is worse than being a little slow. Everyone I
know does both strands eventually to be absolutely sure of what they have.

Good Luck 

Steve Coon
sdcoon at umich.edu
University of Michigan School of Dentistry



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