ALF+Phred

Phillip San Miguel pmiguel at purdue.edu
Fri Jul 21 09:50:59 EST 2000


    If only... You can export your chromat files from an Alfx in scf
format. Phred can read scf--but it does a very poor job of reading Alfx
scf files. The quality scores are low and they should be low. Phred
makes frequent miscalls. I have examples of reads that the Alfx software
called out to 1000 bases at greater than 99% accuracy (measured by
comparing the read to the consensus sequence for a contig containing
it.) Phred called less than 400 high quality bases.
    It may be possible to alter the running conditions of an Alfx gel to
make the chromats more pleasing to Phred. I don't  know.
    I think the fact is that the pharmacia basecaller for the Alfx is
very good for making calls on Alfx data--Phred is not going to be able
to match it.
    So the question is, why doesn't Pharmacia (/Amersham/Molecular
Dynamics) output phd files? Licor does, CEQ2000 does. My guess is that
Pharmacia is focused on it's MD machine, Megabace. Alfx is more-or-less
orphaned. Too bad, because it is a good machine--do you know of another
machine that can get 1000 base reads on 22cm plates? (Yeah, you have to
run them 12 hrs...)

    How about the GAP4 package though? I saw some mention of Alf gels in
their documentation. Maybe they have a good Alf basecaller that can
assign quality values. Also, there are other commercial packages that
display Alfx format chromats (Sequencher leaps to mind) but I don't
think any of them assign quality scores. Does anyone know?

Phillip San Miguel
Purdue Genomics Core Facility

"[iso-8859-1] Istv=E1n Nagy" wrote:

> We are buying an ALF Express sequencers that we will mainly use for
> fragment analysis (microsatellite polymorphism) and occasionally for
> sequencing.
> I am wondering, whether Phred would be a real alternative of the
> commercial sequencing software from Pharmacia.
>
> Thank you in advance
>
> Istvan
>
> ---


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