Input requested on best DNA Sequencer
The doctor is IN.
some recently acquired grant monies and would like some input from other users
out there as to what features and companies I should look at. Unfortunately,
funds are limiting, so I have to be somewhat thrifty. :)
Please post to this newsgroup or directly. I will compile the replies if there
is enough interest. Thanks.
Dieter Deforce
: I am preparing to buy a sequencing apparatus and power supply with
: Tony
: aar...@desire.wright.edu
We are using the ABI 373 sequencer and we are very
hapy with it . It is sold by Applied Biosystems a division of Perkin Elmer
Drs.Apr. Dieter Deforce Harelbekestraat 72,9000 Gent,Belgium
E-mail : Dieter....@rug.ac.be
tel : 09/221.99.43
fax : 09/220.66.88
Laboratory of Farmaceutical Biotechnology
Peter Gegenheimer
Appended is a comparison of features I've prepared based on my experience.
Our service lab has recently purchased the LI-COR 4000LS sequencer. The lab
which uses it the most, consistently gets 700 to 1000 readable bases per run.
A colleague at another university just told me that, in the course of
evaluating sequencers, they submitted identical samples to ABI, LI-COR, and
Pharmacia. ABI and Pharmacia read about 300 bases; LI-COR read 800 bases.
AUTOMATED DNA SEQUENCER FEATURE COMPARISON
Folks, following is a comparison between the three major brands of automated
DNA sequencer currently on the market, made by ABI, Pharmacia, and LI-COR. I
prepared this when we purchased a LI-COR sequencer for our service lab this
summer (1994). The result s of our investigations concluded that ABI or
LI-COR were the leaders, and that for our needs (a low-budget, multi-user
service lab), the LI-COR was clearly preferable. The following information
will help put this into perspective.
First, only the ABI instrument can run all four sequencing reactions in a
single lane. That means that you CAN get four times as many sequences as the
other machines. (The four reactions don't run in the same ladder, however, so
their electrophoretic mobility must be computer-corrected.) On the other
hand, the ABI will read only about 450-550 bases per run, whereas the LI-COR
will routinely generate 1100 bases of sequence, of which 700 to 900 bases are
accurately readable with operator assistance. (A skilled operator can read
1000 bases at a run.) The C. elegans genome project uses ABI's for routine
sequencing, and Li-Cor's to close gaps. Even Lee Hood's lab is starting to
use Li-Cor for that reason. I have not heard anything good about the
Pharmacia ALF machine, and I was not impressed by its technical specs nor by
the presentation from the sales rep. I have been told that the Pharmacia
machine is not identical to the EMBL instrument upon which its design is
based.
Second, from a technical perspective, the Li-Cor instrument has the most
state-of-the-art features. Although other designs can be adapted for long
reads, Li-Cor has patented the infrared detection technology which gives their
detector a lifetime 10 times greater than that of the others.
Third, when we got our Li-Cor, I didn't expect it to yield good sequence
without several week's tuning-up. I was quite surprised, then, that the same
day it was unboxed and set up, and reactions loaded by our technicians who had
never handled DNA before, we generated over 1000 bases of sequence. 650-700
bases were readable by fully-automatic base-calling (at several hundred bases
per min), and 915 bases were readable with operator assist beyond 700 bases.
Our service lab has found Li-Cor to be very helpful with all stages of use,
from installation to both the chemistry and machine use.
Disclaimer: I have no formal connection with Li-Cor. A former graduate
student of mine is now (after an academic post-doc) an Applications Scientist
in Li-Cor's Biotechnology division. However, I also have high regard for
ABI's scientific and technical abilities.
What follows are the specs we presented to the State Purchasing Department
in Summer 1994.
REQUIRED SPECIFICATIONS, WITH JUSTIFICATION AND COMPARISON OF MANUFACTURER'S
COMPLIANCE:
1. Must be capable of analyzing DNA sequencing reactions in which the
product DNA fragments are labeled with a nonradioactive molecule such as a
fluorescent tag. DNA fragments must be detected in real time without
disassembly of the electrophoresis gel.
* JUSTIFICATION: An instrument which uses radioisotopes is impractical Ä
for safety and regulatory reasons Ä for daily use in the Core Facility.
Nonradioactive analysis methods inherently provide greater resolution, i.e.,
can read greater lengths of DNA, which is a major reason for acquiring an
automated system.
* MEETS SPECS: LI-COR: YES; ABI: YES; PHARM: YES.
2. In totally unassisted mode, must be certified and documented to read over
500 bases of DNA with greater than 99% accuracy. In operator-assisted
semiautomatic mode, must be certified and documented to read over 900 bases,
and up to 1200 bases, of DNA with greater than 99% accuracy.
* JUSTIFICATION: the primary reasons for acquiring an automated sequencer
are to read more bases at once, and with greater accuracy, than manual
methods. The participating users absolutely require the ability to read 800
to 1000 bases in fully automatic mode under ideal conditions, so that under
suboptimal conditions we can still read over 500 bases. The ability to read
1000 to 1200 bases in operator-assisted mode is essential to the economical
operation of the instrument. That is, the more bases that can be read at one
time (in one "run"), the fewer separate runs must be made to cover a large
region of DNA. Since the cost of a sequence determination is based on the
number of separate runs, not on the number of bases read, it is important to
use as few runs as possible.
* MEETS SPECS: LI-COR: YES Ä rated and tested to read at least 800 bases
auto; ca. 1000 bases semi-auto. ABI: no Ä tested to read 550 bases. PHARM:
no Ä rated to read 450 to 500 bases.
3. a) Must be demonstrated to accurately sequence single- or double-stranded
DNA and to sequence directly PCR products. b) Must permit the use of either
fluorescently-labeled primers or fluorescently-labeled nucleotides. c) Must
accommodate DNA sequencing reactions produced by Sequenase and
cycle-sequencing methods.
* JUSTIFICATION: a) A major group of users will be systematics researchers
who can supply only PCR-amplified DNA samples made with a large number of
different primers. b) The sequencer should be able to use the researchers'
own primers, which are not fluorescent, and incorporate fluorescent "tag"
using fluorescently-labeled nucleotides. c) Other users will be supplying
DNAs which are single- or double-stranded, and the sequencing service must be
able to handle all these samples.
* MEETS SPECS: LI-COR: YES*; ABI: YES; PHARM: YES
[Note: LI-COR is reworking their internal labeling protocol, so that for
the moment they guarantee results only with led-labeled primers.]
4. a) Gel electrophoresis chamber must accommodate gels up to 66 cm in
height, for reading sequences farther than 700 bases. b) Must be able to
determine short regions of DNA sequence in 1-2 hr on a short gel as described
above. c) Must accommodate smaller electrophoresis gels between 18 and 25 cm
used for analysis of RNA and proteins.
* JUSTIFICATION: a) The required ability to sequence long regions of DNA
(up to 1200 bp), as set forth in (2), can only be accomplished with long gel
plates. b) However, some users will need only short sequence readings. This
can be done in several ways, but the most economical is to use shorter
electrophoresis gels. These short runs will also be ideal for teaching
purposes. c) In addition, several users will be running DNA and RNA analyses
called "fragment length analyses". These are performed on shorter gels.
* MEETS SPECS: LI-COR: YES Ä accommodates gels up to 66 cm without
modification. ABI: no Ä maximum is 34 cm. PHARM: no Ä maximum is ca. 45
cm (cannot exceed 50 cm).
5. a) Gels must be temperature-controlled with an integral heating device
for precise heating during DNA sequencing runs. b) Electrophoretic field must
be user-settable to continuous or inverse-field modes for maximum resolution
of large DNA fragments.
* JUSTIFICATION: a) Optimal resolution ("detail") in a DNA sequencing run,
and in RNA fragment length analysis, and certain types of DNA fragment length
analysis, requires that the gel be heated to 40ø-60øC. b) Certain types of
fragment-length analyses require good separation of large fragments which can
only be accomplished by field-inversion gel electrophoresis ("FIGE").
* MEETS SPECS: LI-COR: a) YES, b) YES. ABI: a) no Ä uses passive
heat-transfer plate, no heater; b) no. PHARM: a) YES, b) no.
6. Machine as configured must be able to collect data suitable for DNA
fragment analyses including STR (short tandem repeat), SSCP, RAPD, and
comparable protocols.
* JUSTIFICATION: Several important groups of users, ones upon whose support
the funding for this machine was predicated, are doing population genetic
studies which use STR, SSCP, and RAPD analyses. They will need to be able to
run their samples on the sequencer, and then move the data to their own
computers for detailed analysis.
* MEETS SPECS: LI-COR: YES. ABI: no, requires $27,000 software upgrade.
PHARM: YES.
THE FOLLOWING IS A FUNDAMENTAL TECHNICAL REQUIREMENT:
7. Must be usable with gel electrophoresis plates made of standard soda-lime
glass. Replacement cost of longest glass plates must not be over $100. To
accomplish this, DNA detection unit must employ a laser which excites at a
wavelength, such as the near infrared, where interference by plain glass is
negligible.
* JUSTIFICATION: The Core Facility does not have a large budget for
replacement and repair, and can ill-afford any "downtime" resulting from
equipment failure. The glass electrophoresis plates are the component most
likely to break during normal use. Detection systems based on ultraviolet
(UV) lasers require the use of borosilicate or quartz plates, available only
from the system vendor at a cost of ca. $300. In contrast, detection systems
based on infrared (IR) lasers can use plain glass (float glass or soda-lime
glass) plates, which are flat to within 0.01 mm but are inexpensive ($50 to
$80 from vendor in the largest sizes).
* MEETS SPECS: LI-COR: YES, uses soda lime plates costing $50-80. Laser
operates at 785 nm. ABI: no, uses borosilicate plates costing $300. Laser
operates at 488 or 514 nm. PHARM: no, uses a pair of borosilicate plates
costing $155 (front plate) and $900 (rear plate). Laser operates at 488 nm.
THE FOLLOWING IS A FUNDAMENTAL TECHNICAL REQUIREMENT:
8. Detection assembly (laser and detector) must be composed 100% of
solid-state components for long-term reliability. The laser must be solid
state (diode or equivalent); the detector must be solid-state.
* JUSTIFICATION: Conventional gas lasers inherently have a limited lifetime
under continuous operation. Under the usage schedule projected for the Core
Facility, a gas laser would have to be replaced every three years or so. A
solid-state (diode-type) laser, on the other had, has an expected life of
100,000 hours, or 30 years of Core Facility operation. Similarly, solid-state
light detectors have a much greater life expectancy than conventional
photomultiplier tubes.
* MEETS SPECS: LI-COR: YES, YES Ä laser is solid-state, rated for 100,000
hours and warranted for 5 yr. Detectors are photodiodes. ABI: no, no Ä
laser is argon gas; detector is photomultiplier tube. PHARM: no, YES Ä laser
is argon gas; detectors are photodiodes.
9. Detection assembly must be focusable to permit use of gels ranging
between 0.1 and 1.0 mm thick for analytical and preparative work.
* JUSTIFICATION: Ultra-thin gels, 0.1 mm thick, are needed for maximum
accuracy and resolution (greater than 1000 bases) and for high-speed runs. On
the other hand, thicker gels (1.0 mm and up) are needed for preparative RNA
and DNA work, and analysis of larger amounts of material as required by some
of the users.
* MEETS SPECS: LI-COR: YES Ä detection assembly (laser etc.) incorporates
a miniature confocal microscope, adjusts in two dimensions, and is
auto-focussing. ABI: no. PHARM: no.
10. a) Must come equipped with a hard-copy device (printer) which allows
the operator to make routine hard-copy printouts of the raw electrophoresis
data. b) The hard-copy must be a two-dimensional facsimile of the gel itself
(e.g., an autoradiograph-like image or comparable) and c) must be completely
readable in black-and-white.
* JUSTIFICATION: a) Most users have told me they will not accept the
computer's reading of the data unless a printout of the raw data is included
for inspection. b) It is impossible to determine the quality of the data, in
particular to ascertain the absence of "smiling" and other gel artifacts
unless a gel facsimile can be analyzed. Furthermore, for fragment analyses, a
conventional photograph-like gel facsimile image is the easiest and best way
to present and interpret the data. c) For publication of the raw data, or
transmission to other researchers, a black and white image is easier and
cheaper than a color image.
* MEETS SPECS: LI-COR: a) YES. b and c) YES. ABI: a) no, printer costs
$4200 extra. b) no, hard-copy is one-dimensional computer-adjusted
chromatogram of each lane. c) no, printout is in color, cannot be read in
black-and-white. PHARM: a) YES. b) no (same as ABI). c) no (same as ABI).
11. a) Control unit and computer must be capable of simultaneously
acquiring data from one or more electrophoresis units in real time, analyzing
stored data, and transmitting data over a network to end-users. b) A
preemptive, multitasking operating system (IBM OS/2 2.1 or equivalent) is
required to accomplish this.
* JUSTIFICATION: a) The instrument will be operated in the Core Facility
and will serve multiple users from different labs. The more-sophisticated
users will want to be present during the data interpretation phase (called
"operator-assisted" above) to increase the amount of usable data. In
addition, since data files are often too large to save on a floppy disk, they
must be transmitted to the user via Ethernet connection. It is thus
imperative that data interpretation and data transmission be able to occur
simultaneously with data collection. b) Simultaneous operation of multiple
procedures without "losing" data can be accomplished only by a computer
operating system having "preemptive multitasking". The OS/2 operating system
and UNIX are two such systems. We prefer OS/2 for compatibility with
existing software, and because the operators and users are more familiar with
it, and the P.I. can provide technical support.
* MEETS SPECS: LI-COR: a,b) YES Ä Uses IBM's OS/2. Control unit and
computer can actually acquire data from three electrophoresis units
simultaneously. ABI: a,b) no Ä uses Macintosh "System 7" operating system
which is only partially ("cooperatively") multitasking. PHARM: a) not
described. b) YES Ä uses IBM's OS/2.
THE FOLLOWING IS A FUNDAMENTAL TECHNICAL SPECIFICATION:
12. a) The system as provided must allow for replication of the
base-calling software onto one or more remote workstations, and b) the raw
image data from the instrument must be transmissible via the campus network to
these workstations.
* JUSTIFICATION: a) the technicians in the service lab do not have time to
provide more than basic machine operation with automated base-calling;
operator-assisted base-reading is best done by the individual users at their
own locations and on their own time.
* MEETS SPECS: LI-COR: YES, YES -a) basic software license allows ca. 4
remote copies at no extra cost, b) the control computer comes equipped with
network card and TCP/IP software. ABI: no, not stated. PHARM: no, not
stated.
WARRANTY: Finally, Quoted price must include on-site delivery and training.
Minimum warranty must be three years of on-site service on both
electrophoresis unit, power supply, system controller, and computer.
* JUSTIFICATION: The instrument will be operated heavily to serve many
users. Any downtime will adversely affect the Core Facility's ability to
serve its users. More importantly, the Core Facility does not have a large
budget for instrument repair. A multi-year, on-site service contract will
greatly smooth operations.
* MEETS SPECS: LI-COR: a) YES. b) YES/almost Ä 2 years on-site service on
all components except 3 years on-site service on computer and 5 years service
on laser/detector assembly (on-site in first year only). ABI: a) YES. b) no
Ä one year only. PH ARM: a) YES. b) no Ä one year only; 3 months on
computer.
COST FOR SYSTEM AS SPECIFIED:
LI-COR: One each Part Numbers 4000LS; 9940-079; and 4000-52. $79,380.00
(Quote of 4/29/94); complete with system controller and power supply;
multitasking computer; one electrophoresis unit with two gel stands ("Gel
Apparatus"), one 66 cm and one of our choice; two sets of gel plates,
spacers, and combs; two printers (one text, one for gel images); one reagent
kit and one set of DNA primers. 2/3/5 year warranty as described above.
Perkin-Elmer/Applied Biosystems: Quotation #259094 (4/27/94). One each
Model #373A-18; $85,000.00 complete with system controller and power supply;
non-multitasking computer; electrophoresis unit with one short gel stand (34
cm), two sets of gel plates, spacers, and combs; no printer; one reagent kit.
One year warranty. NOTE: This system can do sequencing on a smaller number
of samples than the regular ABI #373A, it cannot do any fragment analysis,
etc. Upgrade to a complete system costs $27,000. ABI also sells a 'long-gel'
system claimed to sequence 500 to 700 bases, for about $120,000, but I have
herd nothing about its abilities in the field.
Pharmacia Biotech: Quotation # 6744-042794-00 (4/27/94). One each Part
number 18-1004-S1; $90,000.00 complete with system controller and power
supply; multitasking computer; one electrophoresis unit with one gel stand
("upper and lower buffer reservoirs") , two sets of gel plates, spacers, and
combs; one plotter (for one-dimensional tracings only); no reagent kit. One
year warranty.
o------------------------------------------------------------------------o
| Peter Gegenheimer | pge...@kuhub.cc.ukans.edu |
| Departments of Biochemistry | voice: 913-864-3939 |
| and of Botany | |
| University of Kansas | FAX : 913-864-5321 |
| 2045 Haworth Hall | "The sleep of reason produces |
| Lawrence KS 66045-2106 | monsters." Goya |
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