Disgruntled POP5 user

Paul Shinn pshinn at mail1.sas.upenn.edu
Sun Jan 30 12:26:34 EST 2000


    We received our Collection 1.1 upgrades 2 weeks ago and I'm testing 
out the POP5 that has been so heralded by PE for it's improved run time 
and longer average read length.  Well, I do like 1.1 better than 1.0 and 
the shorter run time certainly has possibilities.  However, the quality 
of the data I'm getting out of it just doesn't seem up to par with my 
other 3700 using 1.0 and POP6.
    The signal intensity is not as even across the capillary but that
isn't my main beef.  The problem I'm having is that I'm missing bases in 
my sequences.  With POP6 I run the low temperature module and resuspend 
our 1/6 or 1/8 strength BDT reactions in 0.3 mM EDTA.  Beautiful 
results.  I consistently get reads up to 700 bases that match a 
concensus sequence to a degree of 99.99% accuracy.
    I'm doing the same with POP5 and I'm using PE's default POP5 run 
module.  They have changed their injection parameters for POP5 and POP6 
run modules, though.  The missing bases problem is not rampant and 
occurs less than 20% of the plate.  However, I still think it's a 
problem.  If you go to this link, you will see what I am talking about.

http://genome.bio.upenn.edu/sequencing/POP5/

    These are screen captures of sequences from our 3700 using the POP5 
polymer.  There are 3 images.  The samples are from different plates and 
resuspended in 15ul of the following solutions:  HiDI formamide, water, 
0.3mM EDTA (same as in HiDi but no formamide).  They are 1/6 BDT 
reactions using -21 primer off M13.  They are first run plates.
    When I called PE with my results of water and EDTA, they suggested I
try the formamide because that's the protocol they support.  Well, as you
can see that faired no better.  They also said since we're using lower
concentrations of BDT mix, our signal strengths may be low.  The images
also show the relative signal strengths of each peak.  I don't think
they're low.  I have gotten excellent sequence from signals below 20 with
POP6.  With this evidence in their hands, I did not get a satisfactory
answer. 
    Different bottles of POP5 give the same results.  The lot has not 
changed.  The POP5 array views are A LOT dimmer than POP5.  I'm 
wondering if this problem isn't related to the new injection 
parameters.  The injections voltage has been cut nearly in half and the 
injection time is almost twice as long.  There is also a post-injection 
voltage that was not present in 1.0.  I tried a test module with the 
same voltage and injection time as our old module but that didn't do the 
trick.
    There must be labs out there using POP5 or trying to use POP5 before 
us.  What kind of results have you seen?  Were you able to tweak the 
modules any?

							Paul

---
Paul Shinn    
Sequencing Coordinator                                    ,___o
pshinn at neomorph.bio.upenn.edu                            _-\_<,
Arabidopsis thaliana Genome Center                      (*)/'(*)
http://genome.bio.upenn.edu/ATGCUP.html
(215) 573-7256






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