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MICROFLUIDICS-BASED AUTOMATED GENOTYPING OF SACCHAROMYCES CEREVISIAE STRAINS BY INTERDELTA SEQUENCE TYPING – AN INTERLABORATORY COMPARISON Ricardo Franco-Duarte1, Ana Catarina Gomes2, Manuel Santos2,3, Bruno de Sousa4, Dorit Schuller1 (1) Molecular and Environmental Research Centre (CBMA) / Department of Biology / University of Minho, Braga, Portugal (2) BIOCANT - Biotechnology Innovation Center, Cantanhede, Portugal (3) CESAM and Biology Department, Aveiro University, Campus Universitário de Santiago, Aveiro (4) Centre for Malaria & Tropical Diseases, Associated Laboratory, Unit of Epidemiology and Biostatistics, Institute of Hygiene and Tropical Medicine - New University of Lisbon, Lisbon, Portugal
Materials and Methods
Introduction
The current approach included 12 genetically diverse S. cerevisiae strains, two different Taq polymerases (commercial and
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High-throughput molecular characterization of microbial isolates requires the application of automated
in-house cloned/prepared) and two different thermal cyclers. PCR amplifications were performed in two laboratories,
Commercial Taq (MBI Fermentas) BioRad Thermal cycler
1
microfluidic electrophoresis. We herein evaluate the factors that affect interlaboratory reproducibility of interdelta
resulting in a total of 384 electrophoretic banding patterns (32 replicates for each strain). From the combinations between
sequence typing for Saccharomyces cerevisiae strain delimitation, using microfluidic electrophoresis (Caliper Lab
strains, Taq polymerase, thermal cycler and laboratory, a total of 60 different groups was obtained. Data were analyzed in
Chip®). This approach is necessary for the constitution of bio-databanks, equitable sharing of genotypic data
terms of the fragment sizes (bp), absolute and relative concentrations of each band (for each band, the absolute value of
among laboratories, for biodiversity conservation and sustainable development of genetic resources.
concentration was divided by the sum of all concentration values for that replicate). Due to the lack of normality
CBMA, University of Minho
Delta sequences are 300 bp regions flanking retrotransposons Ty1 and Ty2 of S. cerevisiae. They occur also as
(Kolmogorov-Smirnov and Shapiro-Wilk tests) and homogeneity of variances (Levene's test), the ANOVA test was not
In-house Taq
separate elements dispersed throughout the genome. About 300 delta elements were described in the genome of
applied. The non-parametric alternative, Kruskal-Wallis one-way analysis of variance was used to test the equality of the
the laboratory strain S288c. Considering the intraspecific variability of delta elements number and locations, a PCR-
medians among the different groups. By rejecting the null hypotheses with a p-value < 0.05, we performed multiple
based method was proposed, relying on the amplification of interdelta regions [1]. The primers amplify DNA
pairwise comparisons using the method proposed by Conover and Iman [3], based on a t-Student distribution to search
regions between neighboring delta sequences, and the PCR reaction therefore produces a mixture of differently
for the origins of the differences, using the following expression: R R j i − n n j i
discriminating power for the analysis of commercial strains [2], but the method was also considered to have a low reproducibility due to the rather low primer annealing temperature (43ºC).
With
t 1−
≥t α 1− 2
8 replicates
CBMA, University of Minho In-house Taq
PLATES
8 replicates
BioRad Thermal cycler
2
Eppendorff Thermal cycler
3
CBMA, University of Minho
4 replictes
Commercial Taq (MBI Fermentas) BioRad Thermal cycler BIOCANT
S 2 (N −1− H N −k
4
) c . 1 + 1 n n i j
In-house Taq
4 replictes
sized fragments, specific for each strain. PCR profile analysis of delta sequences were associated with a good
8 replictess
STRAINS
BioRad Thermal cycler BIOCANT
α the (1-α/2) quantile of a t-Student distribution with (N-k) degrees of freedom, k the number of groups, Hc 2
the value for the test statistic of a Kruskal-Wallis test corrected for ties, and S2 the corresponding variance.
Electrophoretic profile of the 12 strains
Reproducibility of PCR- based interdelta typing for strain comparisons
Comparison between experimental conditions (enzymes, thermal cyclers and laboratories) in each strain, based on the fragment sizes (bp), absolute and relative concentration of each band (Abs conc. And Rel conc.) for each strain. Coloured squares represent statistical significant differences. Each colour is associated with one
left figure shows the unique and very well
pair of comparisons.
and
strain, according to the experimental conditions.
The
majority of the fragments of each strain were
reproducible. Significant differences were observed
bp
regarding DNA concentration for each experimental condition
A - Commercial Taq (MBI Fermentas), BioRad thermal cycler, CBMA laboratory; B - In-house Taq, BioRad thermal cycler, CBMA laboratory; C - In-house Taq, Eppendorff thermal cycler, CBMA laboratory; D - Commercial Taq, BioRad thermal cycler, BIOCANT laboratory; E - In-house Taq, BioRad thermal cycler, BIOCANT laboratory
Abs conc.
Comparison of different experimental conditions for strains delimitation 100% Rel conc.
80% A
60%
C
40%
D E
20% 0% Length (base pairs)
Absolute concentration
Relative concentration
Comparison between the tested conditions in the differentiation of the 12 strains, regarding length of fragment (in base pairs), absolute and relative concentration values. Percentages indicates the differences found between strains when considering each experimental condition. A – Commercial Taq (MBI Fermentas), BioRad thermal cycler, CBMA laboratory; B - In-house Taq, BioRad thermal cycler, CBMA laboratory; C - In-house Taq, Eppendorff thermal cycler, CBMA laboratory; D - Commercial Taq, BioRad thermal cycler, BIOCANT laboratory; E - In-house Taq, BioRad thermal cycler, BIOCANT laboratory
Combination C (In-house Taq, Eppendorff thermal cycler, CBMA laboratory) was able to differentiate almost all strains, in terms of fragment length, being the most suitable combination to strains typing
Combination A, D
Intervals of overall percentage of significant differences between all strains and conditions
B
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Bp
8 -100
0 -100
75 – 100
8 – 50
8 – 58
Abs conc.
0 – 75
0 – 75
16 – 100
0 – 42
8 – 58
Rel. Conc.
0 - 92
0 - 100
83 - 100
0 - 58
25 - 92
The effect of the Taq polymerase on the reproducibility of banding patterns was high in the Biocant laboratory and rather low in CBMA laboratory
(salmon and red
squares) ;
The comparison of fragment sizes showed that the use of different thermal cyclers in laboratory A lead to most reproducible results (green marked box); The comparison of the banding patterns obtained by both laboratories showed a very low reproducibility, that was dependent on the strains analyzed. (dark blue and light blue marked boxes)
The reproducibility of the interdelta sequence typing is best, when fragment sizes are compared. Relative concentration values should not be used, because in replicate analysis of different experimental conditions, distinct numbers of fragments were obtained and affect the ratios of relative concentration.
Conclusions
and E were less capable to separate strains.
The In-house Taq produced better results than the commercial Taq polymerase. Strain patterns in CBMA laboratory were better delimited than the patterns obtained in the in BIOCANT laboratory
Interdelta sequence typing revealed as a very reproducible typing method for multiple replicates of one strain, using the same experimental condition.
Constant banding patterns for each strain in all conditions
Between experimental conditions (combination of different Taq DNA polymerases, thermal cyclers and the accomplishment
The Table shows fragment sizes (bp, average value and standard deviation) that were found in all 32 replicates of each strain R8 97 ± 2,1
R16 96 ± 2,4 134 ± 2
R20
R21 96 ± 2,1
R30 96 ± 2,1
Strain R58 96 ± 2,2
of experiments in different laboratories) the reproducibility was rather low, affecting the number of strains that can be unequivocally distinguished. Highest variability was associated with the use of different Taq polymerases and the
R61 96 ± 1,9
R62 96 ± 2
97 134 161 156 ± 1,7 167 ± 2 157 ± 1,3 188 189 ± 2,1 205 205 ± 1,7 231 232 ± 2 262 262 ± 2,1 285 285 ± 2 320 348 348 ± 8,7 349 ± 4,5 347 ± 4,4 371 425 425 ± 4 425 ± 7 427 ± 5,7 427 ± 3,5 424 ± 3,7 427 ± 3,9 423 ± 3,4 458 453 ± 6,2 462 ± 3,5 486 482 ± 5,8 531 531 ± 13,2 680 721 721 ± 18,5 899 899± 15,6
R81 96 ± 2,1
R88 R101 96 ± 1,9 107 ± 1,8 134 ± 1,9 162 ± 3 186 ± 1,3
accomplishment of the experiments in different laboratories. The effect of the thermal cycler was rather low. For the assembly of results from several laboratories and their comparison for databasing and the equitable sharing of genotypic data among laboratories it is necessary to consider only the fraction of bands that lead to reproducible banding
231 ± 1,5 231 ± 4,4
patterns and to careful standardize the conditions for PCR amplification. 326 ± 3,5 371 ± 3,7 426 ± 3,2
314 ± 4 346 ± 4,4 421 ± 4,8
489 ± 5,3 680 ± 8,7
References [1] Ness, F., Lavalee, F., Dubordieu, D., Aigle, M., and Dulau, L. (1993). Identification of yeast strains using the polymerase chain reaction. J. Sci. Food Agric 62, 8994. [2] Lavallée, F., Salvas, Y., Lamy, S., Thomas, D.Y., Degre, R., and Dulau, L. (1994). PCR and DNA-fingerprinting used as quality-control in the production of wine yeast
For each strain, 3 to 7 bands were apparent in all 32 replicates. Additional bands were found, that were dependent on the experimental conditions. The reproducibility analysis, using only these 3 to 7 bands, would give results around 100%, in terms of fragment sizes.
strains. American Journal of Enology and Viticulture 45, 86-91. [3] Conover, W. J. and Iman, R. L. (1979). On multiple comparison procedures. Technical Report, LA-7677-MS. Los Alamos Scientific Laboratory.
Acknowledgements
This work was funded by the Portuguese Science and Technology Foundation (PTDC/AGR-ALI/103392/2008), the European Community´s Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 232454.
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in house Taq - Commercial Taq (MBI Fermentas) BioRad thermal cycler
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In-houseTaq BioRad thermal cycler
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The right figure shows the 32 replicate profiles of one
Average length (bp) of reproducible fragments
R E S U L T S
Commercial Taq )MBI Fermentas), BioRad thermal cycler
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(LabChip®))
Laboratory comparison (CBMA – Biocant)
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Laboratory comparison (CBMA – Biocant)
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electrophoretic separation by capillary electrophoresis
Taq polymerase comparison Taq polymerase comparison Thermal cycler comparison
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sequences,
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interdelta
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of
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amplification
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distinguished profiles of the twelve strains (PCR-
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The
