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
to finish the reaction or use Platinum Genotype from
BRL - it doesn't add extra A.
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
> polymerases, if RXN conditions aren't exactly right tend to throw an A
> end of PCR products if there isn't already one. It's my understanding
> that some groups tend to optimize thier RXNs so the only n+1 product
> thus giving consistant single peaked results. Again, I'm not the
> expert, but play with the idea. Good luck and let me know what you
> if you have time.
>> Alison Brown wrote:
>> > I've been asked a question which I can't answer and I wondered if
> > genotypers out there could help out. We are using the LMSv2 marker
> > ABI to do high through-put genotyping. Several groups have found
> > which give alleles 1 bp apart - the question is how to people deal
> > - are they ignored, or put into normal bins - if so which one, the
> > 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
> can't help but cry. I mean I'd love to be skinny like that but not
> those flies and death and stuff."
> -Mariah Carey, pop singer
--== Sent via Deja.com http://www.deja.com/ ==--
---Share what you know. Learn what you don't.---