In article <1g71l0INN4h8 at huon.itd.adelaide.edu.au> ajeffrie at waite.adelaide.edu.au (Alex Jeffries) writes:
>> Fact1: Virus "species" are best described as a population of minor
>sequence varients. The term quasispecies is usually used here.
>> Fact2: PCR amplification has an error rate associated with it. The rate
>is dependent on the type of polymerase used and other factors such as Mg-2+
>concentration and the number of cycles.
>> Question1: When sequencing a virus, where the clones have been
>generated by PCR, how many instances of a particular sequence would have to be
>seen before people here would be happy that they have the "correct" sequence and
>not a PCR artifact.
>
Since a PCR-induced base change is propagated through subsequent cycles,
it is important to collect and compare sequence from independent PCR
amplifications. It would be extremely unlikely to see the same artefact
in independent amplifications.
A problem that we have been thinking about is that, in the absence of cloning,
mutations may be masked or seen as ambiguities, since the starting virus
population is heterogeneous. If we go with cloning before sequencing, there
are extra steps and lots of clones to screen if we are looking for multiple
variants in the same population. Anyone have any novel approaches to these
problems?
-Steve
--
Steve Oberste Internet: oberste at ncifcrf.gov
LCMS, PRI, NCI-FCRDC
PO Box B "Never put off until tomorrow that which
Frederick, MD 21702-1201 you can do the day after tomorrow"
