Do you have to sequence both strands?
dpp at icbr.ifas.ufl.edu
dpp at icbr.ifas.ufl.edu
Mon Apr 4 21:03:07 EST 1994
In article <01HARUDG2ENK005IEC at crcvms.unl.edu>, TPARSONS at CRCVMS.UNL.EDU
(TOM PARSONS) writes:
> 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
Well, COII is highly conserved and, like all the mt genes, A+T rich.
Therefore, the chances of having compressions and making mistakes in reading
are low. But, try reading a DNA sequence with high G+C content. I will bet
that you will make at least 1 mistake per 100 ntd, even if the sequence is
highly conserved. My experience is: Read Both Strands. People waste more
money (others) by publishing wrong data.
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 hope that it does not become a universal requirement for
> publications that sequence be determined from both strands. It should be
> up to reviewers and editors of a particular manuscript to assess if the
> sequence has been determined with a high degree of confidence. I think science
> would be better served by proceeding to the next step of investigation,
> instead of, e.g., going back to get the second strand of a perfectly clear
> ATGGCAACATGA, when this sequence is completely conserved in all black flies.
> My opinions relating to unpublished sequence submitted directly to GenBank
> are less clear; quality control in GenBank submissions is a larger problem
> than the requirement for sequencing both strands can solve.
Perfect ATGGCAACATGA would make sure your phylogenetic inference is flawless,
upto the power of resolution of the DNA sequence you have selected. Running
another set of reactions would not cost so much if people started
sequencing PCR products directly instead of trying to correct mistakes made by
Taq by sequencing several cloned fragments. Instead of sequencing the same
strand of four clones you can sequence both strands directly and save money
wasted in 2 reactions.
University of Florida
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