Loss of resolution on 3700, and failed lanes

Carrie Sougnez carrie at genome.wi.mit.edu
Thu Mar 2 17:03:11 EST 2000

Hello Grace,
    I am from the MIT/Whitehead Institute Genome Center.  We also run 8 plates
a day (really 2 - 384well plates/day) on the 3700.  We run 123 instruments at
a time.   We too see a wide range of high and low quality data on the 3700.
We can usually attribute low quality data to a internal problem in the
instrument.  For example plumbing, camera, and laser alignment problems can
all cause low quality data.  What types of maintenance protocols are you using
to keep your instruments performing?

We also see discrepancy in data quality between the left and right side of the
array  (a difference of 100 phred 20 bases from left to right).    This
correlates to the signal intensity from left to right, but there is no fix for
this yet... we have had limited success in changing the temperature of the
cuvette during run modules.

As for your resusupension solution, you may want to try 0.5mM EDTA.  We tested
water, formamide, 12.5% pyrillidinone, and EDTA.  The 0.5mM EDTA gives us the
longest read lengths.

Carrie Sougnez
Coordinator, Sequence Detection
Whitehead Institute Center for Genome Research

"G. Harrison" wrote:

> 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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