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
