PCR:Sequence, or clone and sequence?

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Mon Sep 21 10:17:42 EST 1992


We have a question regarding experiment design.

We are planning to PCR amplify and sequence the transcribed spacer between 
the 18S and 28S rRNA genes from individual mosquitoes.  The purpose is to
compare different populations, which may be subspecies or sibling species.
We are considering two different ways of doing this:

1. Sequence the PCR products directly.  (It is a little tricky getting 
good sequence out of PCR products, but we have had success.)  This will
give us a consensus of the sequence of the hundreds of individual genes
from each mosquito.  Rare variants and PCR artifacts will not be seen.
Common variants will give ambiguities in the sequence.

2. Clone the PCR products and sequence several clones from each mosquito.
This is obviously a lot more work, but it will pick up those rare variants.
Unfortunately, it will also pick up PCR artifacts, and we will not be able
to tell the difference.

You can probably tell that I favor (1), but am I making a mistake?  Is the 
extra information from (2) worth the extra effort and confounding artifacts?

On a similar note, we were told that the groups sequencing human mtDNA were
sequencing each individual SIX times.  Does anyone know if this is true, 
and if so if it is necessary?  I would rather put my resources into six
times as many individuals and accept that the individual data was noisy.
(This sounds like urban folklore to me 8-).

Any chastisement by authentic population geneticists will be cheerfully
accepted.

Andrew Cockburn
USDA



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