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Journal of Clinical Microbiology, March 1999, p. 876-877, Vol. 37, No. 3
0095-1137/99/$00.00+0
LETTERS TO THE EDITOR
Critical Observations on Computerized Analysis of Banding
Patterns with Commercial Software Packages
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LETTER |
We read with interest the recently published paper by Gerner-Smidt
et al. (1) critically evaluating two commercial software packages (the BioImage system and the Molecular Analyst Fingerprinting Plus system [MAFP]; Bio-Rad) in relation to their ability to compare discriminate populations of Listeria monocytogenes on the
basis of electrophoretic data. We would like to comment on some aspects of the comparison of banding patterns from different gels and the
critical relevance of the band-linking procedure.
Since electrophoretic mobility of bands is scarcely reproducible in
different experiments, even with the highest degree of standardization
(2), comparison of patterns from different runs can be
performed either by transforming migration data into molecular weights
(BioImage) or by varying the gel picture scale in order to align the
bands of the same internal standard present in all gels (MAFP). In our
opinion, the latter procedure involves linear variation of the lane
dimensions, not necessarily proportional to the actual migration
dynamics of each band in agarose gels.
In order to produce evidence to support our opinion, we ran a 100-bp
ladder (New England Biolabs, Beverley, Mass.) in two lanes on the same
gel for 60 and 90 min. Migration data measured with NIH-Image 1.62 software (National Institutes of Health, Bethesda, Md.) were
transformed (Kaleida Graph 3.08 program; Synergy Software) into
molecular weights by regression analysis using migration data
(distances from the well) and the known molecular weight of the standard.
The normalization procedure suggested by MAFP was reproduced by
graphically aligning the well line and the 100-bp bands of the 60- and
90-min images. The average variability within gels run for the same
time was 1.82% (standard error [SE] = 0.60), while the comparison of
60- and 90-min runs displayed a 9.21% (SE = 1.13) variability.
These data show that the linear modification of gel length suggested by
the manufacturers of the MAFP software, producing a fivefold increase
in the level of error, is not a completely legitimate operation.
However, it is possible to control the accuracy of the alignment by
asking MAFP to calculate and then compare the molecular weights of the
bands of each standard in the merged gels.
Another misleading characteristic of the MAFP software is that
corresponding bands from different lanes of the same gel are linked to
each other by using a tolerance value expressed in pixels. As a matter
of fact, since the relationship between migration (expressed in pixels)
and molecular size (expressed in base pairs) is not necessarily linear,
the same tolerance gives rise to larger molecular weight differences in
the upper part of the gel. This is particularly important in
pulsed-field gel electrophoresis (PFGE) gels, where the regression
curves between migration distances (x axis) and molecular
weights (y axis) exhibit hyperbolic shapes (Fig.
1).
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FIG. 1.
Calibration curve between molecular size (kilobases) and
migration distance (pixels) of a chromosomal DNA resolved in a PFGE gel
of a standard strain.
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The above observations suggest that the application of algorithms based
only on migration distances, besides yielding data which are de facto
inaccessible for comparison to the scientific community, requires
further elaborations by other systems.
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FOOTNOTES |
*
Phone: 39 75 585 6484
Fax: 39 75 585 6470
E-mail: gianlu{at}unipg.it
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REFERENCES |
| 1.
|
Gerner-Smidt, P.,
L. M. Graves,
S. Hunter, and B. Swaminathan.
1998.
Computerized analysis of restriction fragment length polymorphism patterns: comparative evaluation of two commercial software packages.
J. Clin. Microbiol.
36:1318-1323[Abstract/Free Full Text].
|
| 2.
|
van Belkum, A.,
W. van Leeuwen,
M. E. Kaufmann,
B. Cookson,
F. Forey,
J. Etienne,
R. Goering,
F. Tenover,
C. Steward,
F. O'Brien,
W. Grubb,
P. Tassios,
N. Legakis,
A. Morvan,
N. El Solh,
R. de Ryck,
M. Struelens,
S. Salmenlinna,
J. Vuopio-Varkila,
M. Kooistra,
A. Talens,
W. Witte, and H. Verbrugh.
1998.
Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study.
J. Clin. Microbiol.
36:1653-1659[Abstract/Free Full Text].
|
| | | | |
Gianluigi Cardinali*
Alessandro Martini
Dipartimento di Biologia Vegetale
Sez. Microbiologia Applicata
Università degli Studi di Perugia
Borgo 20 Giugno, 74
I-06121 Perugia, Italy
|
| | | | |
Carlo Tascini
Francesco Bistoni
Dipartimento di Medicina Sperimentale
Sez. Microbiologia
Università degli Studi di Perugia
Via del Giochetto
I-06121 Perugia, Italy
|
| |
AUTHORS' REPLY |
The comment by Dr. Cardinali et al. is a note of caution on
normalization of electrophoretic profiles by aligning reference patterns present in all gels to a reference standard by varying the gel
picture scale within each gel and among all gels. Normalization is done
this way by the GelCompar (Molecular Analyst Fingerprinting Plus)
software described in our paper (1). However, Dr. Cardinale et al. make a fundamental mistake in assuming that this shrinking and
stretching process is a linear function in GelCompar. It is not. L. and
P. Vauterin of Applied Maths, Kortrijk, Belgium, who have developed the
software, state that the normalization algorithm is done by using cubic
spline functions, similar but not identical to the functions used to
calculate molecular weights from the migration data. These functions
have a good reputation for faithful interpolation. All reference bands,
but not the wells in the gel which do not take part in the migration
process, are used for normalization.
Dr. Cardinale et al. also find it problematic that linking
corresponding bands from different lanes is done by using a tolerance value expressed in pixels and not in molecular weight. This is actually
one of the advantages of GelCompar over BioImage that is not mentioned
in our paper. Since the physical process of running a gel and capturing
the image is measured entirely in run lengths, the errors on the run
length are much more constant over the gel than errors on the molecular
sizes, which involve a nonlinear transformation of the run length data.
In our paper, the GelCompar normalization showed its robustness in the
first part of the study. The reference standard in one gel was used to
align two distorted gels containing phage 
fragments in the test
lanes. GelCompar performed excellently and was at least as good as the
BioImage software, which does normalization by calculating molecular
weights, at estimating the sizes of the different phage fragments
regardless of the size of the fragments (Table 2 in our paper).
Thus, results obtained by using GelCompar need not be confirmed by
other molecular size-based systems as proposed by Dr. Cardinale et al.
However, like all image analysis programs, GelCompar should be
considered an aid in the analysis of complex banding patterns and the
overall results should always be checked by visual inspection.
| |
REFERENCE |
| 1.
|
Gerner-Smidt, P.,
L. M. Graves,
S. Hunter, and B. Swaminathan.
1998.
Computerized analysis of restriction fragment length polymorphism patterns: comparative evaluation of two commercial software packages.
J. Clin. Microbiol.
36:1318-1323.
|
| | | | |
P. Gerner-Smidt
Department of Gastrointestinal Infections
Statens Seruminstitut
Artillerivej 5
DK-2300 Copenhagen S, Denmark
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| | | | |
L. M. Graves
Susan Hunter
B. Swaminathan
Foodborne and Diarrheal Diseases Branch
Division of Bacterial and Mycotic Diseases
Centers for Disease Control and Prevention
Atlanta, Georgia
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Journal of Clinical Microbiology, March 1999, p. 876-877, Vol. 37, No. 3
0095-1137/99/$00.00+0
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