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Any Genotypers out there?

Max Myakishev rempel at bigfoot.com
Mon May 17 17:47:31 EST 1999


Alison:

What you have is probably a repeat which is not perfect -
this is a pretty common thing, especially if you genotype a
lot of people and pay attention to 1 bp shifts.

You have 2 options on how to deal with the problem:
1. Number all alleles which you observe.
Theoretically the way of numbering doesn't matter for analysis -
you can pick any numbers you want.

Then you have to be really careful about reading
the peak which migrates one bp.
Make sure the sizing is correct and precise  -
whith poor sizing little shift will screw up your reading.

2. Second option is to merge the split peaks.
You pretend that you don't observe the single base shift and
call the shifted peak by the name of the neighbor.
Just make sure you are consistent in your system of calling.
This way you loose some of the observed information but
don't introduce any mistake in analysis.

Another possibility (which is probably not the case) is that a single bp
shift is due
to adding extra A.  Then you should see intermediate forms.
In this case try to incubate  this marker at 60oC overnight (right after
PCR)
to finish the reaction or use Platinum Genotype from
BRL - it doesn't add extra A.

Max




In article <7gqfep$h6g at net.bio.net>,
  Paul Rasmussen <paul at ib3.gmu.edu> wrote:
> I really don't profess to be any sort of genotyping expert, but I know
some
> polymerases, if RXN conditions aren't exactly right tend to throw an A
onto the
> end of PCR products if there isn't already one.  It's my understanding
in fact
> that some groups tend to optimize thier RXNs so the only n+1 product
appears,
> thus giving consistant single peaked results.  Again, I'm not the
world's
> expert, but play with the idea.  Good luck and let me know what you
figure out
> if you have time.
> Paul
>
> Alison Brown wrote:
>
> > I've been asked a question which I can't answer and I wondered if
any
> > genotypers out there could help out.  We are using the LMSv2 marker
set from
> > ABI to do high through-put genotyping.  Several groups have found
primers
> > which give alleles 1 bp apart - the question is how to people deal
with them
> > - are they ignored, or put into normal bins - if so which one, the
one
> > before or the one after.
> >
> > Alison
>
> --
> Paul Rasmussen
> Research Associate
> Institute for Biosciences, Bioinformatics, and Biotechnology
> George Mason University, Manassas VA
> Phone: (703) 993-8463     Fax: (703) 993-8460
> paul at ib3.gmu.edu              www.ib3.gmu.edu
>
> "Whenever I watch TV and see those poor starving kids all over the
world, I
> can't help but cry. I mean I'd love to be skinny like that but not
with all
> those flies and death and stuff."
> -Mariah Carey, pop singer
>
>


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