Do you have to sequence both strands?

TOM PARSONS TPARSONS at CRCVMS.UNL.EDU
Mon Apr 4 10:58:37 EST 1994


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, due to the way our sequencing primers are laid
out, and the fact that we are sequencing clone PCR fragments.  This is of
course desirable from the standpoint of replication alone.  Because we're 
sequencing cloned products, we need to look at multiple clones per species
(differences between clones due to taq error or "microheteroplasmy" are, in
fact,common), and we select clones in opposite orientations to get both strands.
However, there are small portions of sequence from some of our species that
haven't been sequenced on both strands, but I'm *very* confident of the 
sequence.

     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.

Tom Parsons
University of Nebraska



More information about the Methods mailing list