BROE@AARDVARK.UCS.UOKNOR.EDU (Bruce Roe) (06/20/91)
J. Walton wrote:
=>
=> My department is considering buying an automated DNA sequencer. Some are
=> enthusiastic ("The Time Has Come") but I've personally heard mainly negative
=> things - too cumbersome, error rates too high, no net savings in time and
=> money, etc.
=> Could I solicit from subscribers to this bulletin board that have
=> had experience with automated DNA sequencing a summary of their experiences
=> and opinions? Information about particular machines would also be very
=> helpful.
=>
=> Thank you very much.
=> Jonathan Walton (21337mgr@msu.bitnet)
=> Plant Research Laboratory
=> Michigan State University
Hi,
Since my laboratory was the initial university test site for the
ABI fluorescent sequencing instrument, and there have been both positive
and negative replies, maybe my 2cents worth will be of interest to others
on the net.
There presently are *only* 2 automated sequencing instruments
worth considering for purchase. The ABI 373 and the LKB/Pharmacia ALF.
Both cost around $100K and both have advantages and disadvantages.
Common Advantages:
-----------------
You never have to read another autoradiograph and enter the
data yourself into the computer.
Pouring gels and loading samples uses the same techniques you
are familar with from radio-labeled sequencing gels.
Common Disadvantages:
--------------------
The data collected is *all* the data, including background,
and thus "@#$%-in ==> @#$%-out" (pardon my vulgarity but you get my point).
The template *must* be pure as can be or else the above is true.
The analysis programs leave alot to be desired because they
really do not take into account local variations from user to user.
(i.e variations in sample quality, variations in gel percentage, sequencing
reaction conditions, etc.)
It takes some doing to optimize the DNA isolation conditions and
the DNA sequencing reaction conditions, unless you have lots of $ and
can afford to buy a DNA sequencing kit every week at $600/100 reactions.
Proof reading and editing the output requires the purchase of
additional programs and computers. We use the MRC programs on a
SPARCstation, not the Vendor supplied programs as they are not as good.
So think about adding another $25K to your purchase price for a SPARC2.
Both may be capable of dye terminator sequencing but the claims
may be over stated by both companies.
Both are capable of sequencing off double stranded template but
again the purity of the template may be a problem.
Specific Advantages of the ABI 373:
----------------------------------
Well built, reliable, rapid and experienced service.
Been around a while and the programs have improved slightly over
the years.
Programs that are used can handle 24 samples and give data out
to 450 nucleotides from the m13 priming site fairly reproducibly, depending
on the quality of the template and reactions (we use Taq Cycle sequencing
reactions which reduces or seems to reduce the effect of contamination of
due to impure template and make our own mixed, buy our own enzyme, make
our own primers).
ABI is addressing the issue of template purity and uniformity
of reaction pipetting and soon will introduce a robotic workstation which
for the present will pipet and perform all incubations for the DNA sequencing
reactions. In the future they may produce a robotic workstation for automated
template DNA isolation.
We have published to papers related to the ABI and automation of both
the isolation and sequencing reactions and a forth- coming issue of "Methods:a
companion to Methods in Enzymology" will be published with additional related
articles.
E.R. Mardis and B.A. Roe. Automated Methods for Single-Stranded DNA Isolation
and Dideoxynucleotide DNA Sequencing Reactions on a Robotic Workstation.
BioTechniques 7, 840-850 (1989).
L.A. Johnston-Dow, E. Mardis, C. Heiner and B.A. Roe. Optimized Methods for
Fluorescent and Radio-labeled DNA Sequencing. BioTechniques 5, 754-765 (1987).
In addition, we are working on a series of programs which may (we
hope) do a better job of interpreting the ABI data than ABI does. These
programs presently are almost done and will run under DOS or MAC OS and
should complement the MRC programs.
Specific Advantages of the Pharmacia ALF:
----------------------------------------
Based on technology from Ansorge's lab at EMBL.
Temperature control of the gel environment is excellent.
Easy to make primers (need only one/sample since ACGT rxns are
loaded in 4 separate lanes).
Specific Disadvantages of the ABI 373:
-------------------------------------
Poor temperature control of the gel during electrophoresis (smiles).
Requires 4 different dyes and thus 4 different primers/sample and
it is difficult to do primer walking. Also, each new primer (based on
sequence) requires a different mobility file for the programs to work
correctly.
Specific Disadvantages of the Pharmacia ALF:
-------------------------------------------
Fewer samples/run than ABI, although the gels are shorter and
run time may be shorter the reality is one run/day and less through-put
with the ALF.
Analysis programs are not as robust as those provided by ABI because
ABI has been producing fluorescent sequencing instruments longer and had
more time to improve their programs than LKB/Pharm. has (although their's
are improving and almost caught up with ABI).
Very few instruments delivered in the US. Many more in Europe.
Thus service and local experience of the tech service folks may not be
as good as ABI (which by the way really isn't that good).
Accuracy:
--------
I have not addressed the issue of accuracy. That is because accuracy
is like beauty "in the eyes of the beholder" and relates back to the purity
of the template and sequencing reactions and gel conditions. Compression
is compression and even humans cannot figure out most compressions accurately
so why expect an instrument programmed by humans to be able to do it.
The bottom line here regarding accuracy is that these two instruments
are at least as good as a human in "accurately" reading the gels in one pass.
Until we figure out how to reproducably obtain really pure template and how to
remove rather than interpet compressions, errors will occur. Most humans read
gels 98-99% accurately over an extended period of time and these two
instruments also give 98-99% accuracy from the priming site to about 450
nucleotides depending upon the above factors.
Bottom line:
-----------
If you've got the money and *really* want to devote a lot of time
for a Ph.D level person to get everything up and running, then I'd recommend
purchasing the ABI 373 along with a SPARC2 and getting the MRC programs
for proof reading. Alternatively, contact both companies and see if they
will demo the instruments for say one month in your lab. This will give
you better first hand experience and you set the conditions for acceptance.
I would *highly* recommend this alternative rather than taking someone's
word for it.
However, don't throw away those radio-labeling sequencing
gel plates, buffer chambers, and power supplies as you may find that
radio-labeled sequencing may be just as quick and efficient, especially
if you can read past 500 nucleotides from a priming site (with multiple
loadings or different percentage gels, etc).
Hope this was informative.
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\ Bruce A. Roe Professor of Chemistry and Biochemistry /
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Disclaimer:
The opinions expressed are my own and as others have stated:
"As I speek for myself and usually only to myself"
Also, I have no commercial ties to the companies mentioned.