Hi Phillip,
Your recent posting regarding failed lanes on the 3700 prompted me to
respond with our observations. We run 8 96-well plates / day on the
3700-Capillary and 4 96-well plates / day on our 377-Gel machines (awkward
nomenclature, thanks, ABI). Our sequencing technicians prep 800 samples
/ day with an alkaline-lysis/PEG precipitation procedure, and perform
standard PCR sequencing reactions with an isopropanol precipitation
cleanup.
I have been dissatisfied with the quality of the data obtained on the
3700. Our largest problem is the inconsistency of data quality with
runs that give phred Q>20 values ranging from 0 to 554. The average Q>20
for these runs collected over 6 months is 292.8 and the average Trim
length is 362.7.
Most frustrating is the observation that the number of failed lanes is all
over the map. You can download an Excel graph of our data from:
http://chroma.mbt.washington.edu/msg_www/public.links.html and select the
3700 Failed Lanes file. We average 33.77 failed lanes/gel over 6 months,
sigh.
When you reload a sample that failed (anything with a Q>20 </= 100) we do
not see the same pattern of failed lanes (that is, it is not template-
related), and we often see an improvement in quality, although a yucky run
becomes a not-quite-so-yucky run.
Additionally, the 377-gel machine average q>20 = 370.1, and the average
Trim is 504, with only 17.5 failed lanes/gel. While the quality figures
may be related to setting 1/2X sequencing reactions on the 377 and 1/4X
sequencing reactions on the 3700, we get half as many failed lanes on the
377-gels. [Experiments comparing 1/2x and 1/4x reactions more
rigourously are in progress.]
Of those samples which worked, we saw lopsided chromatogram traces where
the signal was very high at the beginning of the reaction, and trailed to
sub-detectable values by the middle of the run. Experiments changing DNA
template concentrations were inconclusive.
On the theory that unincorporated dyes and small molecules remaining in
the reaction plate were being preferentially loaded, a 70% EtOH wash after
an isopropanol precipitation of the sequencing reaction was performed.
Preliminary results indicate an average 2.5X improvement in quality (from
a Q>20 of 80 to 200) over 200 templates each.
We believe something is preventing the entire sample from being loaded
into the capillaries properly. We have tried changing injection time (20,
25, 30, 35, 40, and 45), resuspension buffer (dH20 and/or dFormamide); and
experiments increasing the length of time the samples resuspend are
ongoing. Injection time results are inconclusive, and dFormamide
resuspension results in fewer failed lanes (and higher average qualities).
Can you think of what else we should try? What kinds of things have you
been working on?
Of course, when we change to POP 5, we'll have to do this all over again.
Happy sequencing,
Grace Harrison, Lab Manager,
Department of Molecular Biotechnology
University of Washington, Seattle, WA
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