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EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
http://eurl-listeria.anses.fr
French Agency for Food, Environmental and Occupational Health Safety – Food Safety Laboratory Maisons-Alfort location and Boulogne-sur-Mer location
14, rue Pierre et Marie Curie - F94701 Maisons-Alfort Cedex - Telephone: + 33 149 771 350 - Fax: + 33 149 771 380 Boulevard du Bassin Napoléon - F62200 Boulogne-sur-Mer - Téléphone : +33 321 992 500 – Télécopie : + 33 321 991 725
www.anses.fr - @Anses_fr
Laboratory for Food Safety
Maisons-Alfort location
Introduction to cluster analysis purpose and parameters and Global
Database Management
Benjamin FELIX Joint training course on the use of BioNumerics software to analyse PFGE data
Rome 3-4 July 2017
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
2
Clustering
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Cluster Analysis
PFGE Profiles
Set of band-based profiles
Similarity matrix
Dendrogram/clustering
Coefficient calculation: Algorithm « Dice »
Binary: Band absence or presence
Clustering: Algorithm « UPGMA »
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Dice coefficient: Band-based similarities
Dice:
SD= 2NAB
NA+ NB
SD= 2Ncommon
NTotal
NA
NB
NAB
SD= 2x7
8+9
= 82%
3 bands of difference
A
B
A
82 100
100
72 100 C
B
90
C
…
…
Similarity matrix
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Dice coefficient: Optimization and tolerance
Optimization
1%
Tolerance
1%
Band Matching
Matching?
Optimal settings according to EURL Lm SOP
% < %
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
UPGMA: Unweighted Pair Group Method using Arithmetic average
A
B
A
82 100
100
72 100 C
B
98
C
…
…
Similarity matrix
B+C
C
B
77 100
98
70 100 D
A
95
D
…
…
A
90 100 (82+72)/2
B+C
C
B
95
98
D
A+D
…
A
90 100
B+C+A+D
C
B
73.5
D
…
A
70 80 90 100
73.5
…
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
7
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Database management
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Communication between databases
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
TIF Image
Database exchange
- Permant import of fingerprint, need image
processing, normalization and analysis
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Bundles
Database exchange
- Profile are temporally available in the database
(profiles, sequences and fields) until the software shut
down
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
XML
Database exchange
- Permanent import of data (profiles, sequences and
fields) without any analysis.
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Bundles XML
TIF Image
Database exchange
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Database compatibility
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
15
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Reference system
Reference system included in the gel:
- Salmonella Braenderup H9812 XbaI restriction
profile
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Reference system
Database main reference system included in the
BioNumerics experiment:
- Salmonella Braenderup H9812 XbaI restriction
profile
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Normalization process
Metric =
molecular
size kbp
Percentage of
migration
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Normalization process
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Normalization process
Balance zoom
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Normalization process
Decale
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Normalization process
Distortion
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Normalization process
Normalized gel
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Reference system
Database main reference parameters
Internal reference system
Active zone
Calibration
curve
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
How to proceed when the reference systems are
different ?
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Several reference systems can work together
Re-mapping
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Re-mapping
The re-mapping can be used for bundles and XML
import
XML import implement automatically parallel
reference system
Bundles need re-mapping to be set
beforehand
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Re-mapping
The calibration curve is used to perform the re-
mapping process
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Re-mapping
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Re-mapping is implemented
EURL Lm European Union Reference Laboratory for
Listeria monocytogenes
Re-normalization
- Specific script provided by Applied Maths
- Convert_refsys.BNS
- The script car re-analysed automatically all fingerprint into another
internal reference system
- However this script only to convert
similar reference system e.g. SB01
into SB02
- The script works by batch of 20
gels
- Beware to backup your system
before to proceed
- Beware to verify the output of the
re-normalisation
