>To: autoseq@net.bio.net
>Newsgroups: bionet.genome.autosequencing
>Date: Thu, 02 Mar 2000 17:03:11 -0500
>From: Carrie Sougnez <carrie@genome.wi.mit.edu>
>Subject: Re: Loss of resolution on 3700, and failed lanes
>Sender: owner-autoseq@hgmp.mrc.ac.uk
>
>
>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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