ALF+Phred

Phillip San Miguel pmiguel at purdue.edu
Fri Jul 28 08:11:21 EST 2000


James Bonfield wrote:

> In <8lg8ll$5g8$1 at mercury.hgmp.mrc.ac.uk> Phillip San Miguel <pmiguel at purdue.edu> writes:
> >     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?
>
> We don't have any base callers - we simply use whatever is in the trace file.
> We do support Alf format data files, but I think recently they created a
> compressed copy which we cannot read (as we don't know what format it is in,
> and intuiting this from a compressed data stream is really hard). Why don't
> people publish their file formats? Anyway, Alf should support SCF output.
>
> The Staden Package makeSCF program (freely downloadable as part of io_lib)
> does have some _very basic_ normalisation functions designed for tidying up
> alf data, but I do not know whether this would aid phred at all.
> [...]

    Yes the Alf will export SCFs. But it appears to export the raw chromatograms rather than
the "processed" chromatograms upon which it presumably bases its own base calling. I think
it possible that were the Alf Express to export its processed chromatograms in SCFs then
phred would do a much better job of calling bases (and assign higher quality values.) The
last version of Alfx software I tested for this was Alfwin 1.1--so version 2 may already do
this. But I doubt it.
    When we first got an Alf express several years ago, Pharmacia (here in the US) had a
dedicated sales and field technical support unit for the Alf express. When the Alfwin
software was first released (it superceded the old, OS2 version of the software), a new
chromat format, ".alx" was included. Our application specialist was angered that Pharmacia
hadn't sent out information on the the format to companies producing support software (like
sequencher) in advance.
    Shortly before the merger of Pharmacia and Amersham I was told that Pharmacia was (at my
request) going to provide phred-like scoring with its base calls in a later version of the
software. But a year later it was no longer being plan. Indeed I got the distinct impression
that the Alf express had been moved from the back burner to the freezer. The only sales or
application specialist who had not fled the company after the first merger had been shifted
to the Megabace division during the second merger (with Molecular Dynamics).
    Yet I did talk to a Pharm/Amer/MD rep at the TIGR meeting last year who was in the Alf
"division". I also talked a representative from the company shortly thereafter on the phone.
But "phred" was not in her vocabulary and I doubt my suggestions about the Alf's exporting
quality values (and the licor does this, as does the Beckman CEQ) went anywhere.
Phillip San Miguel
Purdue Genomics Core Facility




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