Which DNA sequencer?

Jannik Helweg-Larsen jhelweg at inet.uni2.dk
Mon Jun 28 10:15:47 EST 1999


Some weeks ago I posted this question and got a lot of very helpful replies
in addition to several eager salespeople in the phone next day!

I would like to thank everybody who mailed their opinion- in general it
seems as Li-cor users are quite pleased with this instrument(although the
lack of responses from people using the standard ABI instruments makes me
wonder if this enthusiasm has resemblences to underdog-supporters versus
users of the established standard (e.g like Linux vs. Windows).

Browsing through old postings in this newsgroup it looks like my question
has been asked before, but with few relies posted

I have therefore taken the liberty of posting the replies I got in the
newsgroup (omitting the e-mail adresses- hope this is okay)

We are still contemplating which sequencer and if any body has experience
with the Beckman ceq 2000 system we would very much appreciate informations.

Here are 5 replies:

1.It still a very good question !
At genoscope, we have actually, 10 ABI 377, 1 ABI 310, 1 ABI 3700, 1
MegaBace, 70 LiCor 4200L...

It's not very easy to choose an instrument. That's depends strongly on your
strategy.
You say that you are disappointed by the alf express, but are you sure that
your problems come
from the instrument, or from the fact that you sequence PCR products ?
On all instruments, the PCR products can give bad results, if you have,
says, 20 single repeats
(polyA , polyT..).
If you want to make some assemblages, it's clear that the longer is your
sequence, easier is the
assemblage.
draftly the reads lenght are the following :
LiCor 4200L (1100 bp)> ABI 377 (650 bp)> MegaBace (550 bp)> ABI 310 (500 bp)
> ABI 3700 (450 bp)
The troughtput :
ABI 3700 > MegaBace > ABI 377 > LiCor 4200 L > ABI 310
The price of the instrument :
ABI 3700 > MegaBace > ABI 377 > LiCor 4200 L > ABI 310
The price of the reaction :LiCor 4200 L
ABI 3700 = MegaBace = ABI 377 = ABI 310 >> LiCor 4200 L

The LiCor can do only dye primers, the other can make both reactions, ie Dye
primer and Dye
terminators
I hope this can help you
Sincerely

Herve CRESPEAU
GENOSCOPE

2.Hello Jannik,
We have 6 licor sequencers and are very happy with them.  All six have
two detectors which allows us to sequence from both ends of plasmid
s with 700 nm and 800 nm labled primers.  With 61 cm gels we get over
1000 bp with each primer routinely.  (>greater than 2000 bp per reaction)

The costs are minimal.  We use Epicentres kit which is $225 for 100
reactions.  We use 1/2 volumes so we get 200 reactions per kit. Using
universal primers (i.e. m13 rev or for.) cost about 0.01 each per reaction.
So our cost is as low as ~1.20 for 2000 bp or more.

However, almost all of the sequencing we do is with labled primers.
incorporating a labled dATP with an unlabled primer can be done but it
is tricky.  We have not had much luck with it, but we have not had much
demand so have not optimized it yet.  Labled oligos can be ordered from
li-cor for about $70.  You get enough primer for 1,000 to 1,250 reactions.
If you are going to be sequencing the same sequence over and over its
worth it.  If it is for a single reaction it is very expensive.

We do use unlabled primers that have a M13 forward primer tail for
genotyping and sequencing PCR products.  This way we can use labled M13
forward primer to analyze various sequences.

Hope this helps

Kenneth M. Kaufman, Ph.D.
Scientific Director
Oklahoma City Veterans Administration
Genotyping and DNA Sequencing Center


3.I saw your posting on Bionet Auto-Sequencing inquiring about Li-Cor
machines. One place you might turn to for more information is the
Association of Biomedical Resource Facilities (ABRF). It is a non-profit
organization that promotes good DNA lab practices and the members are from
academia, government and industry. I attended the annual meeting this last
March for the first time and learned a great deal.

One interesting report their "Analysis of the Effects of Different DNA
Sequencing Methods on Sequencing  Quality, Creation of a Quality Control
Resource, and Assessment of the Current State of the Art: Results from the
1998 ABRF DNA Sequence Research Committee Study" at the following link:

http://www.abrf.org/ABRF/ResearchCommittees/dsrcreports/abrfdna2/dsrc98.htm


As for the comparing sequencers, ABRF has done some studies on the matter
and you can find their results by going to the ABRF home page at
http://www.abrf.org and using their search engine to search their site for
Li-Cor references. For instance, there is a comparative graph titled
'Accuracy of Different Machine Types" at

http://www.abrf.org/ABRF/ResearchCommittees/dsrcreports/abrfdna2/Fig2.htm

Anyway, I hope these help. I wish I could give you some first hand input,
but I have never used either machine. I only know about ABRF because I work
at a company that sells DNA sequence analysis software -  you know, trim
the vector, build the contig, do the motiff analysis, translate into the
amino acid sequence, look at the restriction enzyme map., etc. At the
recent ABRF meeting several scientists told me that use our software to
calibrate their machines! The software is called Sequencher and if you have
not decided on an assembly tool yet I would be happy to send you a demo
copy (for the Mac or PC). Just send me your European shipping address and I
will send it right off. Or, if you would like, you can download the Mac
version from our Web Page at www.genecodes.com

Okay, that is all for now. Good luck in your research!

Mike Hennessey
Gene Codes Corp.
----
4.Hi Jannik,

I realise that as an employee of MWG Biotech ( the UK distributor of the
Li-Cor ) I could be seen as a bit biased but being the moderator of this
newsgroup and having some 6 years experience of automated sequencing I
would like to offer an informed opinion.  If you are looking for mutations
in your sequence or need high levels of accuracy for diagnostics the Li
Cor is simply the best system.  I have never been much of a fan of the 310
even before I joined MWG and all I have heard recently is bad things about
it.  The best indicator of its long term use for labs is the number of
310's on the second hand market.  i have answered a few of your comments
in the email as follows.

If you would like answers to any other questions please do not hesitate to
contact me.

Sincerely,

David Cain

On Sat, 5 Jun 1999, Jannik Helweg-Larsen wrote:

> Although I realize this is an old question, please:
>
> Our lab is contemplating buying a new sequencer- probably either ABI 310
or
> Li-cor.
>
> Having used Alph Express for direct cy5 sequencing of PCR products some
time
> I am bit disappointed with this system and would like to hear if anyone
has
> good or bad experience with the Li-cor system or can compare this system
to
> the ABI-system?
>
> Do Li-cor users actually get 900-1000 bp daily??
>
With all sequence data its a case of getting out what you put in, most
labs I work with get in excess of 1KB per direction per clone, PCR
products using tailed primers works well and offers the best way to get
consistantly good template.  My record is 1300 bases from plasmid, 1200
from PCR products and an amazing 1150 bases from a PAC template although
the PAC took a gel to optimise.

> How do the running costs compare?
>
The cost per reaction in sterling is 110 pence for bi-directional
sequencing getting greater than 1KB of data in both directions.  The ABI
costs 450 pence for 1 direction read.

There are also the running costs to consider, the Li-Cor uses little or no
electricity to run.  I have in the past when puched for time run samples
at home ont he weekends ( I am just mad like that ).  I certainly could
not afford to run a 310 at domestic electricity costs.  There are also no
capillaries to replace witht he Li-Cor again a cost saver.

> Our use would be mainly small-scale sequencing, not big genome
> identification.
>
>
Apart from sequencing I have been involved with labs doing the following
techniques very sucessfully on the Li-Cor: AFLP,
microsatelites, quantatative PCR, dosage analysis, mini satelites and a
few interesting developing technologies such as RSCA ( a form of
heteroduplex analysis ) and differential display.

In summary I love the Li-Cor that I work with and would buy one without
hesitation if I needed one for a lab doing sequencing but also wanting to
think about future applications.

5.Dear Jannik,

I set up both Li-Cor and ABI377 prism, in the past three years.  But not ABI
310.
Both Li-cor and ABI 377 can give you a reliable reading length about 700bp
on a daily bases in my hand.  I know ABI310 can only give 400bp because it
uses apillary tube  instead a polyacrylmide gel.
Up to date li-cor's machine can only use dye-labelled primer to do
sequencing reaction and use four lanes per sample (like manuall gel).  ABI's
machine can use both Dye-labelled primer and terminators, which much easy to
use customer's primers, as well as you only need one lane per sample in the
gel.
I think the running cost for both will be quite similar.   I perfer ABI's
machine but you will need a bigger investment at the beginning.
hope this is of any help.  Any further questions please do not hesitate to
cantact me again.
All the best,
Yan Li (Dr.)




Jannik Helweg-Larsen, MD
Department of Infectious Diseases
Hvidovre Hospital, Copenhagen University.
Denmark.




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